Cyanoarene-aniline compounds for the treatment of skin diseases

Abstract

Provided herein are compounds, pharmaceutical compositions comprising the compounds, methods of preparing the compounds, and methods of using the compounds and compositions in treating a disease or condition in a subject, wherein the subject is in need of a MEK inhibitor, wherein the compound is a compound according to formula (I): wherein R 1 , R 2 , R 2a , R 3a , R 3 and X as described in this article. #imgabs0#

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 62 / 769,871, filed on November 20, 2018, the entire contents of which are incorporated herein for all purposes. Background of the Invention

[0004] Neurofibromatosis type 1 (NF1) occurs in approximately 1:3500 newborns and is one of the most common autosomal dominant monogenic diseases affecting human neurological function. Clinically, NF1 disease is characterized by the presence of benign peripheral nerve tumors, called neurofibromas, involving Schwann cells with biallelic mutations in the NF1 gene, as well as other neoplastic and non-neoplastic manifestations. See Jousma et al. Pediatr. Blood Cancer 62:1709-1716, 2015. NF1 is associated with several skin diseases, including cutaneous neurofibromas; plexiform neurofibromas; café au laitspot; and freckles of the axilla and groin. Cutaneous neurofibromas occur in more than 95% of NF1 patients and can appear anywhere on the body, causing itching, irritation, infection, physical pain, and disfigurement. Moreover, cutaneous neurofibromas are associated with social isolation and anxiety.

[0005] NF1 is caused by one or more germline mutations in NF1, a gene that inactivates the RAS pathway. Because the NF1 gene encodes a Ras-GAP protein, loss of NF1 results in high Ras-GTP. Therefore, research on NF1 has focused on detecting inhibitors in the Ras signaling pathway, including the Ras-MAPK cascade. See Jousma et al. Pediatr. Blood Cancer 62: 1709-1716, 2015. Four different MAPK cascades have been identified and named based on their MAPK modules. See Akinleye et al. Journal of Hematology & Oncology 6: 27, 2013. MEK proteins belong to a family of enzymes that are located upstream of their respective specific MAPK targets in the four MAP kinase signaling pathways. Two of these MEK proteins (MEK1 and MEK2) are closely related and participate in this signaling pathway cascade. Inhibitors of MEK1 and MEK2 have been shown to effectively inhibit MEK signal transduction downstream of Ras, thus providing a strong theoretical basis for targeting MEK to treat NF1. See Rice et al. Medicinal Chemistry Letters 3: 416-421, 2012.

[0006] Currently available MEK inhibitors are designed to have oral bioavailability for systemic delivery and are associated with significant side effects, including decreased left ventricular ejection fraction, elevated creatine phosphokinase, pneumonia, renal failure, diarrhea, infection, urticaria, and maculopapular rash, all of which are dose limiting or require permanent discontinuation. In addition, clinical trials have shown that long-term high-dose use of MEK inhibitors can produce side effects. See Huang et al. Journal of Ocular Pharmacology and Therapeutics (J. Ocul. Pharmacol. Ther.) 25:519–530, 2009. For example, PD0325901 (a MEK inhibitor in clinical trials) exhibits neurological side effects associated with ataxia, confusion, and syncope. In addition, some other side effects have been observed with systemic exposure to MEK inhibitors, including: acneiform rash, elevated CPK, nausea, vomiting, diarrhea, abdominal pain, and fatigue. Therefore, there is a need for MEK therapies for NF1-related cutaneous neurofibromas that limit these serious side effects. Summary of the invention

[0007] In one aspect, provided herein are compounds of formula (I):

[0008]

[0009] or a stereoisomer thereof, a mixture of stereoisomers, and / or a pharmaceutically acceptable salt thereof,

[0010] in:

[0011] X is -CR 3b or N;

[0012] R 1 For-OR 4 、-NR 5 R 5a 、-N(OR 5b )R 5a or unsubstituted or replaced by one or two R 6 substituted N-linked heterocycloalkyl;

[0013] R 2 is halogen, C1-C6 alkyl, -S-C1-C6 alkyl, C3-C8 cycloalkyl, C2-C6 alkenyl, or C2-C6 alkynyl;

[0014] R 2a is halogen or C1-C6 alkyl;

[0015] R 3 , R 3a and R 3bEach is independently hydrogen, halogen, C1-C6 alkyl, C2-C6 alkenyl or C2-C6 alkynyl;

[0016] R 4 , R 5 and R 5b Each is independently hydrogen, C1-C6 alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkyl-C1-C6 alkyl, C1-C6 hydroxyalkyl, C1-C6 alkoxy-C1-C6 alkyl, amino-C1-C6 alkyl, C1-C6 alkylamino-C1-C6 alkyl, di(C1-C6 alkyl)amino-C1-C6 alkyl, heterocycloalkyl, heterocycloalkyl-C1-C6 alkyl, or R 7 -C(O)-C1-C6 alkyl, wherein each of the C3-C8 cycloalkyl and heterocycloalkyl groups is unsubstituted or substituted with 1 to 6 R 6 replaced by;

[0017] R 5a is hydrogen or C1-C6 alkyl;

[0018] Each R 6 independently halogen, hydroxy, oxo, C1-C6 alkyl, C3-C8 cycloalkyl, C1-C6 alkoxy, C1-C6-hydroxyalkyl, C1-C6 haloalkyl, amino, C1-C6 alkylamino, di(C1-C6 alkyl)amino, amino-C1-C6 alkyl, C1-C6 alkylamino-C1-C6 alkyl, or di(C1-C6 alkyl)amino-C1-C6 alkyl;

[0019] R 7 is hydroxy, C1-C6 alkoxy, amino, C1-C6 alkylamino, di(C1-C6 alkyl)amino, hydroxyamino or N-C1-C6 alkylhydroxyamino; and

[0020] Provided that the compound is not ethyl 6-cyano-2-((2-fluoro-4-iodophenyl)amino)-5-methylnicotinate.

[0021] In a second aspect, provided herein is a pharmaceutical composition comprising a compound of formula (I) and a pharmaceutically acceptable carrier.

[0022] In a third aspect, provided herein is a method for treating a MEK inhibitor-responsive disorder, a MEK inhibitor-responsive skin disorder, a MEK-mediated disorder or disease, or a MEK-mediated skin disorder, the method comprising administering to a patient in need thereof a therapeutically effective amount of a compound of formula (I) or a composition of a compound of formula (I), thereby treating the disorder or disease.

[0023] BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Synthetic Scheme 1 for preparing compounds of formula (Ia) is shown.

[0025] Figure 2 Synthetic Scheme 2 for preparing compounds of formula (Ia) is shown.

[0026] Figure 3 Synthetic Scheme 3 is shown for the preparation of compounds of formula (Ib).

[0027] Figure 4 Inhibition of pERK in human cutaneous neurofibroma explants following treatment with compound 1.030 using the protocol described in Example 47 is shown.

[0028] Figure 5 Inhibition of pERK in human cutaneous neurofibroma explants following treatment with compound 1.030 (at a concentration of 500 nM) using the protocol described in Example 47 is shown. DETAILED DESCRIPTION OF THE INVENTION

[0030] I.General

[0031] Provided herein are compounds of formula (I), pharmaceutical compositions comprising compounds of formula (I), and methods of using these compounds or compositions to treat MEK inhibitor-responsive conditions or diseases, MEK inhibitor-responsive skin conditions or diseases, MEK-mediated conditions or diseases, or MEK-mediated dermal conditions or diseases.

[0032] II. Definitions

[0033] Abbreviations used herein have their conventional meanings in the chemical and biological fields.

[0034] Where substituents are designated by their conventional chemical formula (written from left to right), they equally include substituents resulting from the structure written from right to left, for example -CH2O- is intended to include -OCH2-.

[0035] "Alkyl" refers to a straight or branched chain, saturated, aliphatic group having a specified number of carbon atoms (i.e., C1-C6 means 1 to 6 carbons). Alkyl groups may include any number of carbons, such as C1-C2, C1-C3, C1-C4, C1-C5, C1-C6, C1-C7, C1-C8, C1-C9, C1-C 10, C2-C3, C2-C4, C2-C5, C2-C6, C3-C4, C3-C5, C3-C6, C4-C5, C4-C6 and C5-C6. For example, C1-C6 alkyl includes, but is not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, hexyl, etc. Alkyl may also refer to alkyl groups having up to 20 carbon atoms, such as, but not limited to, heptyl, octyl, nonyl, decyl, etc.

[0036] The term "alkylene" refers to a straight or branched chain saturated, aliphatic group having a specified number of carbon atoms (i.e., C1-C6 means 1 to 6 carbons) and connecting at least two other groups, i.e., divalent hydrocarbon groups. The two parts connected to the alkylene group can be connected to the same atom or different atoms of the alkylene group. For example, a straight chain alkylene group can be a divalent group -(CH2) n -, wherein n is 1, 2, 3, 4, 5 or 6. Representative alkylene groups include, but are not limited to, methylene, ethylene, propylene, isopropylene, butylene, isobutylene, sec-butylene, pentylene and hexylene.

[0037] "Alkenyl" refers to a straight or branched hydrocarbon having at least 2 carbon atoms and at least one double bond and having a specified number of carbon atoms (i.e., C2-C6 means 2 to 6 carbons). Alkenyl groups may include any number of carbons, such as C2, C2-C3, C2-C4, C2-C5, C2-C6, C2-C7, C2-C8, C2-C9, C2-C 10 , C3, C3-C4, C3-C5, C3-C6, C4, C4-C5, C4-C5, C5, C5-C6 and C6. The alkenyl group may have any suitable number of double bonds, including but not limited to 1, 2, 3, 4, 5 or more. Examples of alkenyl groups include but are not limited to: vinyl (ethenyl), propenyl, isopropenyl, 1-butenyl, 2-butenyl, isobutenyl, butadienyl, 1-pentenyl, 2-pentenyl, isopentenyl, 1,3-pentadienyl, 1,4-pentadienyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 1,3-hexadienyl, 1,4-hexadienyl, 1,5-hexadienyl, 2,4-hexadienyl, or 1,3,5-hexatrienyl.

[0038] "Alkynyl" refers to a straight or branched hydrocarbon having at least 2 carbon atoms and at least one triple bond and having a specified number of carbon atoms (i.e., C2-C6 means 2 to 6 carbons). Alkynyl groups may include any number of carbons, such as C2, C2-C3, C2-C4, C2-C5, C2-C6, C2-C7, C2-C8, C2-C9, C2-C 10, C3, C3-C4, C3-C5, C3-C6, C4, C4-C5, C4-C6, C5, C5-C6 and C6. Examples of alkynyl include, but are not limited to, ethynyl, propynyl, 1-butynyl, 2-butynyl, butadiynyl, 1-pentynyl, 2-pentynyl, isopentenyl, 1,3-pentadiynyl, 1,4-pentadiynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 1,3-hexadiynyl, 1,4-hexadiynyl, 1,5-hexadiynyl, 2,4-hexadiynyl or 1,3,5-hexatriynyl.

[0039] "Cycloalkyl" refers to a saturated or partially unsaturated, monocyclic, fused bicyclic or bridged polycyclic ring assembly containing 3-12 ring atoms or the specified number of atoms. Cycloalkyl may include any number of carbon atoms, such as C3-C6, C4-C6, C5-C6, C3-C8, C4-C8, C5-C8, C6-C8, C3-C9, C3-C 10 、C3-C 11 and C3-C 12 . Saturated monocyclic cycloalkyl rings include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cyclooctyl. Saturated bicyclic and polycyclic cycloalkyl rings include, for example, norbornane, [2.2.2] bicyclooctane, decahydronaphthalene, and adamantane. Cycloalkyl groups may also be partially unsaturated, having one or more double or triple bonds in the ring. Representative partially unsaturated cycloalkyl groups include, but are not limited to, cyclobutenyl, cyclopentenyl, cyclohexenyl, cyclohexadienyl (1,3- and 1,4-isomers), cycloheptenyl, cycloheptadienyl, cyclooctenyl, cyclooctadienyl (1,3-, 1,4-, and 1,5-isomers), norbornene, and norbornadiene. When the cycloalkyl group is a saturated monocyclic C3-C8 cycloalkyl group, exemplary groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl.

[0040] "Cycloalkylalkyl" refers to a group having an alkyl component and a cycloalkyl component, wherein the alkyl component connects the cycloalkyl component to the point of attachment. The alkyl component is as defined above, except that the alkyl component is at least divalent (alkylene) to connect to the cycloalkyl component and to the point of attachment. The alkyl component may include any number of carbons, such as C1-C6, C1-C2, C1-C3, C1-C4, C1-C5, C2-C3, C2-C4, C2-C5, C2-C6, C3-C4, C3-C5, C3-C6, C4-C5, C4-C6 and C5-C6. The cycloalkyl component is as defined above. Exemplary cycloalkyl-alkyls include, but are not limited to, cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl and cyclohexylmethyl.

[0041] "Alkoxy" refers to an alkyl group with an oxygen atom connecting the alkyl group to the point of attachment: alkyl-O-. The alkoxy group can have any suitable number of carbon atoms, such as C1-C6. Alkoxy groups include, for example, methoxy, ethoxy, propoxy, iso-propoxy, butoxy, 2-butoxy, iso-butoxy, sec-butoxy, tert-butoxy, pentyloxy, hexyloxy, and the like.

[0042] "Hydroxyalkyl" refers to an alkyl group as described above, wherein at least one hydrogen atom is replaced by a hydroxyl group. As for the alkyl group, the hydroxyalkyl group may have any suitable number of carbon atoms, such as C1-C6. Exemplary hydroxyalkyl groups include, but are not limited to, hydroxymethyl, hydroxyethyl (wherein the hydroxyl group is at the 1- or 2-position), hydroxypropyl (wherein the hydroxyl group is at the 1-, 2- or 3-position), hydroxybutyl (wherein the hydroxyl group is at the 1-, 2-, 3- or 4-position), hydroxypentyl (wherein the hydroxyl group is at the 1-, 2-, 3-, 4- or 5-position), hydroxyhexyl (wherein the hydroxyl group is at the 1-, 2-, 3-, 4-, 5- or 6-position), 1,2-dihydroxyethyl, and the like.

[0043] "Alkoxyalkyl" refers to a group having an alkyl component and an alkoxy component, wherein the alkyl component connects the alkoxy component to the point of attachment. The alkyl component is as defined above, except that the alkyl component is at least divalent (alkylene) to connect to the alkoxy component and to the point of attachment. The alkyl component may include any number of carbons, such as C1-C2, C1-C3, C1-C4, C1-C5, C1-C6, C2-C3, C2-C4, C2-C5, C2-C6, C3-C4, C3-C5, C3-C6, C4-C5, C4-C6, and C5-C6. The alkoxy component is as defined above. Examples of alkoxy-alkyl include, but are not limited to, 2-ethoxy-ethyl and methoxymethyl.

[0044] "Halogen" or "halo" refers to fluorine, chlorine, bromine or iodine.

[0045] "Haloalkyl" refers to an alkyl group as defined above in which some or all of the hydrogen atoms are replaced by halogen atoms. As with alkyl groups, haloalkyl groups may have any suitable number of carbon atoms, such as C1-C6. For example, haloalkyl groups include: trifluoromethyl, fluoromethyl, 2,2,2-trifluoroethyl, and the like. In some cases, the term "perfluoro" may be used to define a compound or group in which all hydrogens are replaced by fluorine. For example, perfluoromethyl refers to 1,1,1-trifluoromethyl.

[0046] As used herein, "amino" refers to -NH2 unless otherwise indicated.

[0047] Unless otherwise indicated, as used herein, "alkylamino" refers to a -NHR group, wherein R is an alkyl group as defined herein, or an N-oxide derivative thereof. In some embodiments, the alkylamino group is a C1-C6-alkyl-amino group. In some embodiments, the C1-C6-alkyl-amino group is a methylamino group, an ethylamino group, a n-, i-, t-propylamino group, a n-, i-, t-butylamino group, or a methylamino-N-oxide group, and the like.

[0048] Unless otherwise indicated, as used herein, "dialkylamino" refers to a NR'R group, wherein R and R' are alkyl as defined herein, or an N-oxide derivative thereof. In some embodiments, dialkylamino is di(C1-C6)alkyl-amino. In some embodiments, di(C1-C6)alkyl-amino is dimethylamino, methylethylamino, diethylamino, or dimethylamino-N-oxide, and the like.

[0049] Unless otherwise indicated, as used herein, "aminoalkyl" refers to an alkyl group substituted with one or two NH2. In some embodiments, the aminoalkyl group is amino-C1-C6-alkyl.

[0050] Unless otherwise indicated, as used herein, "alkylaminoalkyl" refers to an alkyl group substituted with one or two -NH(alkyl) groups. In some embodiments, the alkylaminoalkyl group is C1-C6-alkyl-amino-C1-C6-alkyl.

[0051] Unless otherwise indicated, as used herein, "dialkylaminoalkyl" refers to an alkyl group substituted with one or two -N(alkyl)2 groups. In some embodiments, the dialkylaminoalkyl group is di(C1-C6alkyl)amino-C1-C6alkyl.

[0052] As used herein, unless otherwise specified, "hydroxyamino" refers to -NHOH.

[0053] Unless otherwise indicated, as used herein, "N-alkylhydroxyamino" refers to the substitution of the hydrogen of -NHOH with an alkyl group as defined herein. In some embodiments, N-alkylhydroxyamino is N-C1-C6 alkyl-hydroxyamino. In some embodiments, N-C1-C6 alkyl-hydroxyamino is N-methylhydroxyamino, N-ethylhydroxyamino, N-(normal, iso-propyl)-hydroxyamino or N-(normal, iso-tert-butyl)hydroxyamino, etc.

[0054] "Heterocycloalkyl" refers to a saturated ring system having 3 to 12 ring members and 1 to 4 heteroatoms selected from N, O and S. The heteroatoms may also be oxidized, such as, but not limited to, -S(O)- and -S(O)2-. Heterocycloalkyl may include any number of ring atoms, for example, 3 to 6, 4 to 6, 5 to 6, 3 to 8, 4 to 8, 5 to 8, 6 to 8, 3 to 9, 3 to 10, 3 to 11 or 3 to 12 ring members. Any suitable number of heteroatoms may be included in the heterocycloalkyl, for example, 1, 2, 3 or 4, or 1 to 2, 1 to 3, 1 to 4, 2 to 3, 2 to 4 or 3 to 4. The heterocycloalkyl radicals may include, for example, aziridinyl, azetidinyl, pyrrolidinyl, piperidinyl, azepanyl, azocanyl, quinuclidinyl, pyrazolidinyl, imidazolidinyl, piperazinyl (1,2-, 1,3- and 1,4-isomers), oxiranyl, oxetanyl, tetrahydrofuranyl, oxanyl (tetrahydropyranyl), oxepanyl, thioethane, thietanyl, thiolanyl (tetrahydrothiophenyl), thianyl (tetrahydrothiopyranyl), oxazolidinyl, isoxazolidinyl, thiazolidinyl, isothiazolidinyl, dioxolanyl, dithiolanyl, morpholinyl, thiomorpholinyl, dioxanyl or dithianyl. Heterocycloalkyl can also be fused to an aromatic or non-aromatic ring system to form a member including but not limited to indoline. Heterocycloalkyl can be unsubstituted or substituted. For example, heterocycloalkyl can be substituted by C1-C6 alkyl or oxo (=O) and the like.

[0055] The heterocycloalkyl group may be attached at any position on the ring. For example, aziridinyl can be 1- or 2-aziridinyl, azetidinyl can be 1- or 2-azetidinyl, pyrrolidinyl can be 1-, 2- or 3-pyrrolidinyl, piperidinyl can be 1-, 2-, 3- or 4-piperidinyl, pyrazolidinyl can be 1-, 2-, 3- or 4-pyrazolidinyl, imidazolidinyl can be 1-, 2-, 3- or 4-imidazolidinyl, piperazinyl can be 1-, 2-, 3- or 4-piperazinyl, tetrahydrofuranyl can be 1- or 2-tetrahydrofuranyl, oxazolidinyl can be 2-, 3-, 4- or 5-oxazolidinyl, isoxazolidinyl can be 2-, 3-, 4- or 5-isoxazolidinyl, thiazolidinyl can be 2-, 3-, 4- or 5-thiazolidinyl, isothiazolidinyl can be 2-, 3-, 4- or 5-isothiazolidinyl, and morpholinyl can be 2-, 3- or 4-morpholinyl.

[0056] "N-linked heterocycloalkyl" or "nitrogen-linked heterocycloalkyl" refers to a heterocycloalkyl group that is linked through the N-position on the ring. For example, an N-linked aziridinyl group is aziridin-1-yl, an N-linked azetidinyl group is azetidin-1-yl, an N-linked pyrrolidinyl group is pyrrolidin-1-yl, an N-linked piperidinyl group is piperidin-1-yl, an N-linked pyrazolidinyl group is pyrazolidin-1-yl or pyrazolidin-2-yl, an N-linked imidazolidinyl group can be imidazolidin-1-yl or imidazolidin-3-yl, an N-linked piperazinyl group is piperazin-1-yl or piperazin-4-yl, an N-linked oxazolidinyl group is oxazolidin-3-yl, an N-linked isoxazolidinyl group is isoxazolidin-2-yl, an N-linked thiazolidinyl group is thiazolidin-3-yl, an N-linked isothiazolidinyl group is isothiazolidin-2-yl, and an N-linked morpholinyl group is 4-morpholinyl.

[0057] When the heterocycloalkyl group contains 3 to 8 ring members and 1 to 3 heteroatoms, representative members include, but are not limited to: pyrrolidinyl, piperidinyl, tetrahydrofuranyl, oxanyl, tetrahydrothiophenyl, thiacyclohexyl, pyrazolidinyl, imidazolidinyl, piperazinyl, oxazolidinyl, isoxazolidinyl, thiazolidinyl, isothiazolidinyl, morpholinyl, thiomorpholinyl, dioxanyl and dithianyl. The heterocycloalkyl group can also form a ring with 5 to 6 ring members and 1 to 2 heteroatoms, and representative members include, but are not limited to: pyrrolidinyl, piperidinyl, tetrahydrofuranyl, tetrahydrothiophenyl, pyrazolidinyl, imidazolidinyl, piperazinyl, oxazolidinyl, isoxazolidinyl, thiazolidinyl, isothiazolidinyl and morpholinyl.

[0058] "Protective groups" refer to compounds that render a functional group chemically inert to a particular set of reaction conditions, but are removable in subsequent synthetic steps to restore the functional group to its original state. Such protecting groups are well known to those of ordinary skill in the art and are included in "Protective groups in Organic Synthesis", 4th edition, TW Greene and PGM Wuts, John Wiley & Co., New York, 2006, the entire contents of which are incorporated herein by reference.

[0059] "Salt" refers to an acid salt or basic salt of a compound of the invention. Illustrative examples of pharmaceutically acceptable salts are salts of inorganic acids (hydrochloric acid, hydrobromic acid, phosphoric acid, etc.), organic acids (acetic acid, propionic acid, glutamic acid, citric acid, etc.), quaternary ammonium (methyl iodide, ethyl iodide, etc.) salts. It should be understood that pharmaceutically acceptable salts are non-toxic. Additional information about suitable pharmaceutically acceptable salts can be found in Remington's Pharmaceutical Sciences, 17th edition, Mark Publishing Company, Easton, Pennsylvania, 1985, which is incorporated herein by reference.

[0060] Pharmaceutically acceptable salts of the acidic compounds of the invention are salts formed with bases, i.e. cationic salts, such as alkali and alkaline earth metal salts, such as sodium, lithium, potassium, calcium, magnesium and ammonium salts, such as ammonium, trimethyl-ammonium, diethylammonium and tris-(hydroxymethyl)-methyl-ammonium salts.

[0061] Similarly, acid addition salts, for example of inorganic acids, organic carboxylic acids and organic sulfonic acids, for example hydrochloric acid, methanesulfonic acid, maleic acid, may also provide a basic group (eg pyridyl) which forms part of the structure.

[0062] The neutral forms of the compounds can be regenerated by contacting the salt with a base or acid and isolating the parent compound in the conventional manner. The parent form of the compound differs from its various salt forms in certain physical properties, such as solubility in polar solvents, but for the purposes of the present invention the salts are otherwise equivalent to the parent form of the compound.

[0063] "Isomers" refer to compounds with the same chemical formula but distinguishable in structure. Certain compounds of the invention possess asymmetric carbon atoms (optical centers) or double bonds; racemates, diastereomers, geometric isomers and individual isomers are intended to be included within the scope of the invention.

[0064] "Tautomer" refers to one of two or more structural isomers that exist in equilibrium and are readily converted from one to the other.

[0065] "Solvate" refers to a compound provided herein or a salt thereof, which further includes a stoichiometric or non-stoichiometric amount of a solvent bound by non-covalent intermolecular forces. When the solvent is water, the solvate is a hydrate.

[0066] "Hydrate" refers to a compound that is complexed with at least one water molecule. The compounds of the present invention may be complexed with 1-10 water molecules.

[0067] With respect to a composition, "substantially free" or "substantially absent" of a stereoisomer means that the composition comprises at least 85 or 90% by weight, in some embodiments at least 95%, 98%, 99% or 100% by weight of the specified stereoisomer of the compound. In some embodiments, the methods and compounds provided herein are substantially free of stereoisomers.

[0068] With respect to a composition, "isolated" refers to a composition comprising at least 85%, 90%, 95%, 98%, 99% to 100% by weight of a particular compound, with the remainder comprising other chemical forms or stereoisomers.

[0069] As used herein, "composition" refers to a product containing specified ingredients in specified amounts, as well as any product based directly or indirectly on the manufacture of a combination containing the specified ingredients in specified amounts. "Pharmaceutically acceptable" means that the carrier, diluent or excipient must be compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.

[0070] "Pharmaceutically acceptable excipients" refers to substances that aid in the administration of an active agent to a subject and in its absorption by a subject. Pharmaceutical excipients that can be used in the present invention include, but are not limited to, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavoring agents, and pigments. Those skilled in the art will recognize that other pharmaceutical excipients can be used in the present invention.

[0071] “IC 50 ” refers to the amount, concentration or dose of a particular test compound that achieves a 50% inhibition of a maximum response in an assay measuring that response.

[0072] "Inhibit," "inhibits," and "inhibitor" refer to a compound that inhibits a particular effect or function or a method that inhibits a particular effect or function.

[0073] "Administering" refers to administering to a subject orally, as a suppository, topical contact, parenteral, intravenous, intraperitoneal, intramuscular, intralesional, intranasal or subcutaneous, intrathecally, or implanting a sustained-release device, such as a mini-osmotic pump.

[0074] "Treatment," "therapeutic," and "therapy" refer to any indication of success in treating or ameliorating an injury, pathology, or condition, including any objective or subjective parameter, such as alleviation of symptoms; remission; diminishing or making the injury, pathology, or condition more tolerable to the patient; slowing the rate of degeneration or decline; making the endpoint of decline less debilitating; improving the patient's physical or mental well-being. Treatment or amelioration of symptoms may be based on objective or subjective parameters; including the results of a physical examination, neuropsychiatric examination, and / or psychiatric evaluation.

[0075] "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 provided herein. Non-limiting examples include humans, other mammals, cattle, rats, mice, dogs, monkeys, goats, sheep, cows, deer, and other non-mammals. In some embodiments, the patient is a human.

[0076] "Therapeutically effective amount" refers to the amount of a compound or pharmaceutical composition that can be used to treat or improve an identified disease or condition, or to show a detectable therapeutic or inhibitory effect. The exact amount will depend on the purpose of the treatment and can be determined by those skilled in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (Vol. 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).

[0077] The present disclosure provides "soft" MEK inhibitors, compositions comprising "soft" MEK inhibitors, and methods of treating and / or preventing skin disorders (e.g., MEK inhibitor-responsive skin diseases or MEK-mediated skin diseases, such as dermal rasopathy, such as skin diseases associated with neurofibromatosis type 1 (NF1), such as cutaneous neurofibroma, subcutaneous neurofibroma, and superficial plexiform neurofibroma) with MEK inhibitors (e.g., "soft" MEK inhibitors). For example, the methods described herein provide for local or non-systemic administration, such as topical, intradermal, or intralesional administration of a MEK inhibitor, e.g., a "soft" MEK inhibitor, such as a "soft" MEK inhibitor, whereby the side effects exhibited by systemic exposure (such as the known side effects exhibited by MEK inhibitors designed for systemic administration) are significantly reduced.

[0078] In some embodiments, "soft MEK inhibitors" are compounds that inhibit MEK1 and / or 2 and are characterized by predictable and controllable metabolism / degradation into non-toxic and less biologically active or inactive (i.e., do not inhibit or inhibit MEK1 and / or 2 to a lesser extent) products after achieving a therapeutic effect in the skin.

[0079] "Hard MEK inhibitors" refer to MEK inhibitors known in the art. In some embodiments, hard MEK inhibitors are designed for oral bioavailability. This is necessary to deliver therapeutically effective levels of MEK inhibitors to surrounding lesions using systemic delivery. Hard MEK inhibitors include, for example, PD0325901; PD184161; SMK-17; AS703026 (Pimasertib, MSC1936369); RO-4987655; Selumetinib (AZD6244, ARRY142886); Binimetinib (MEK162, ARRY-162, ARRY-438162); Refametinib; Cobimetinib (GDC-0973, XL518); GDC-0623; AZD8330 (ARRY-424704); CI-1040 (PD184352); PD198306; and PD318088.

[0080] Although not wishing to be bound by theory, it is believed that soft MEK inhibitors, for example, "soft" MEK inhibitors as described herein, are more unstable than known "hard" MEK inhibitors. Due to their inherent metabolic instability, for example, for degradation that reaches the body circulation, "soft" MEK inhibitors, for example, "soft" MEK inhibitors such as described herein, have skin activity, but have low systemic exposure after local or non-systemic (such as topical, intradermal or intralesional) administration because they are rapidly degraded when exposed to plasma or blood or liver metabolic enzymes. Unlike "soft" MEK inhibitors, known MEK inhibitors have been designed for oral bioavailability in the past, which requires good stability in plasma or blood and good stability to liver metabolism required for systemic delivery at therapeutically effective levels, and are more likely to produce undesirable side effects and higher toxicity. Therefore, "soft" MEK inhibitors, such as soft MEK inhibitors such as described herein, have less systemic toxicity.

[0081] "A", "an" or "one", when used herein for a substituent group or "substituent group", means at least one. For example, where a compound is substituted with "one" alkyl or aryl group, the compound is optionally substituted with at least one alkyl and / or at least one aryl group, wherein each alkyl and / or aryl group is optionally different. In another embodiment, where a compound is substituted with "one" substituent group, the compound is substituted with at least one substituent group, wherein each substituent group is optionally different.

[0082] III. Compounds

[0083] In one aspect, provided herein are compounds of formula (I):

[0084]

[0085] or a stereoisomer thereof, a mixture of stereoisomers, and / or a pharmaceutically acceptable salt thereof,

[0086] in:

[0087] X is -CR 3b or N;

[0088] R 1 For-OR 4 、-NR 5 R 5a 、-N(OR 5b )R 5a or unsubstituted or replaced by one or two R 6 Substituted N-linked heterocycloalkyl

[0089] R 2 is halogen, C1-C6 alkyl, -S-C1-C6 alkyl, C3-C8 cycloalkyl, C2-C6 alkenyl, or C2-C6 alkynyl;

[0090] R 2a is halogen or C1-C6 alkyl;

[0091] R 3 , R 3a and R 3b Each is independently hydrogen, halogen, C1-C6 alkyl, C2-C6 alkenyl or C2-C6 alkynyl;

[0092] R 4 , R 5 and R 5b Each is independently hydrogen, C1-C6 alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkyl-C1-C6 alkyl, C1-C6 hydroxyalkyl, C1-C6 alkoxy-C1-C6 alkyl, amino-C1-C6 alkyl, C1-C6 alkylamino-C1-C6 alkyl, di(C1-C6 alkyl)amino-C1-C6 alkyl, heterocycloalkyl, heterocycloalkyl-C1-C6 alkyl, or R 7 -C(O)-C1-C6 alkyl, wherein each of the C3-C8 cycloalkyl and heterocycloalkyl groups is unsubstituted or substituted with 1 to 6 R 6 replaced by;

[0093] R 5a is hydrogen or C1-C6 alkyl;

[0094] Each R 6independently halogen, hydroxy, oxo, C1-C6 alkyl, C3-C8 cycloalkyl, C1-C6 alkoxy, C1-C6-hydroxyalkyl, C1-C6 haloalkyl, amino, C1-C6 alkylamino, di(C1-C6 alkyl)amino, amino-C1-C6 alkyl, C1-C6 alkylamino-C1-C6 alkyl, or di(C1-C6 alkyl)amino-C1-C6 alkyl;

[0095] R 7 is hydroxy, C1-C6 alkoxy, amino, C1-C6 alkylamino, di(C1-C6 alkyl)amino, hydroxyamino or N-C1-C6 alkylhydroxyamino; and

[0096] Provided that the compound is not ethyl 6-cyano-2-((2-fluoro-4-iodophenyl)amino)-5-methylnicotinate.

[0097] In some embodiments, R 2 , R 4 , R 5 , R 5b and R 6 In some embodiments, the cycloalkyl group described in is each a saturated monocyclic C3-C8 cycloalkyl group. In some embodiments, the C3-C8 cycloalkyl group alone or as a part of a C3-C8 cycloalkyl-C1-C6 alkyl group is a cyclopropyl or cyclobutyl group. In some embodiments, the C3-C8 cycloalkyl group alone or as a part of a C3-C8 cycloalkyl-C1-C6 alkyl group is unsubstituted. In some embodiments, the C3-C8 cycloalkyl group alone or as a part of a C3-C8 cycloalkyl-C1-C6 alkyl group is substituted by 1 to 6 R6, and R 6 As defined and described herein.

[0098] Regarding R as one or more substituents of C3-C8 cycloalkyl 6 In some embodiments, each R 6 R is independently halogen, hydroxy, oxo, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 hydroxyalkyl, C1-C6 haloalkyl, amino, C1-C6 alkylamino, di(C1-C6 alkyl)amino, amino-C1-C6 alkyl, C1-C6 alkylamino-C1-C6 alkyl, or di(C1-C6 alkyl)amino-C1-C6 alkyl. In some embodiments, each R 6 R is independently halogen, hydroxy, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 hydroxyalkyl, C1-C6 haloalkyl, amino, C1-C6 alkylamino, or di(C1-C6 alkyl)amino. 6R is independently halogen, hydroxy, C1-C6 alkyl, C1-C6 alkoxy, or amino. 6 is independently hydroxyl or C1-C6 alkyl. 6 are independently hydroxy or amino.

[0099] In some embodiments, R 1 , R 4 , R 5 and R 5b In some embodiments, the heterocycloalkyl group is a 3- to 8-membered heterocycloalkyl group having 1 to 3 N, O, or S heteroatoms. In some embodiments, the heterocycloalkyl group is a 3- to 6-membered heterocycloalkyl group having 1 to 2 N or O heteroatoms. In some embodiments, alone or as part of a heterocycloalkyl-C1-C6 alkyl group, the heterocycloalkyl group is unsubstituted. In some embodiments, alone or as part of a heterocycloalkyl-C1-C6 alkyl group, the heterocycloalkyl group is substituted by 1 to 6 R 6 Replaced by R 6 As defined and described herein. In some embodiments, the N-linked heterocycloalkyl is replaced by one or two R 6 replaced, and R 6 As defined and described herein.

[0100] With respect to R as one or more substituents of heterocycloalkyl or N-linked heterocycloalkyl 6 In some embodiments, each R 6 R is independently halogen, hydroxy, oxo, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 hydroxyalkyl, C1-C6 haloalkyl, amino, C1-C6 alkylamino, di(C1-C6 alkyl)amino, amino-C1-C6 alkyl, C1-C6 alkylamino-C1-C6 alkyl, or di(C1-C6 alkyl)amino-C1-C6 alkyl. In some embodiments, each R 6 R is independently halogen, hydroxy, oxo, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 hydroxyalkyl, C1-C6 haloalkyl, amino, C1-C6 alkylamino, or di(C1-C6 alkyl)amino. 6 R is independently halogen, hydroxy, oxo, C1-C6 alkyl, C1-C6 alkoxy, or amino. 6 is independently hydroxyl or C1-C6 alkyl. 6 is independently hydroxy, oxo, or amino. 6 are independently hydroxy or amino.

[0101] In some embodiments, X is -CR3b , and the compound is represented by formula (Ia):

[0102]

[0103] Among them, R 1 , R 2 , R 2a , R 3 , R 3a and R 3b As defined in any aspect or embodiment described herein.

[0104] In some embodiments of Formula (Ia), R 3a and R 3b Each is independently hydrogen, halogen or C1-C6 alkyl.

[0105] In some embodiments of Formula (Ia), R 3 is hydrogen, halogen, or C1-C6 alkyl. 3 In some embodiments, R 3 In some embodiments, R 3 is fluorine, chlorine, bromine or iodine. 3 In some embodiments, R 3 In some embodiments, R3 is methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl or hexyl. 3 It is methyl.

[0106] In some embodiments of Formula (Ia), R 3a is hydrogen, halogen, or C1-C6 alkyl. 3a In some embodiments, R 3a In some embodiments, R 3a is fluorine, chlorine, bromine or iodine. 3a In some embodiments, R 3a is C1-C6 alkyl. 3a is methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl or hexyl. 3a It is methyl.

[0107] In some embodiments of Formula (Ia), R 3b is hydrogen, halogen, or C1-C6 alkyl. 3b In some embodiments, R3b In some embodiments, R 3b is fluorine, chlorine, bromine or iodine. 3b In some embodiments, R 3b is C1-C6 alkyl. 3b is methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl or hexyl. 3b It is methyl.

[0108] In some embodiments of Formula (Ia), R 3 , R 3a and R 3b In some embodiments of Formula (Ia), R 3 and R 3a Each is hydrogen and R 3b In some embodiments of Formula (Ia), R 3 and R 3a Each is hydrogen and R 3b For fluorine.

[0109] Returning to formula (I), in some embodiments, X is N, and the compound is represented by formula (Ib):

[0110]

[0111] Among them, R 1 , R 2 , R 2a , R 3 and R 3a As defined in any aspect or embodiment described herein.

[0112] In some embodiments of Formula (Ib), R 3 and R 3a Each is independently hydrogen, halogen or C1-C6 alkyl.

[0113] In some embodiments of Formula (Ib), R 3 is hydrogen, halogen, or C1-C6 alkyl. 3 In some embodiments, R 3 In some embodiments, R 3 is fluorine, chlorine, bromine or iodine. 3 In some embodiments, R 3 is C1-C6 alkyl. 3 It is methyl.

[0114] In some embodiments of Formula (Ib), R 3a is hydrogen, halogen, or C1-C6 alkyl. 3a In some embodiments, R 3a In some embodiments, R 3a is fluorine, chlorine, bromine or iodine. 3a In some embodiments, R 3a is C1-C6 alkyl. 3a It is methyl.

[0115] In some embodiments of Formula (Ib), R 3 and R 3a Each is independently hydrogen or halogen. In some embodiments, R 3 and R 3a In some embodiments, R 3 and R 3a One of R is hydrogen and the other is halogen. 3 and R 3a One is hydrogen and the other is fluorine.

[0116] With respect to any of Formula (I), (Ia) and (Ib), in some embodiments, R 2 is halogen, C1-C6 alkyl, -S-C1-C6 alkyl, C3-C8 cycloalkyl, C2-C6 alkenyl, or C2-C6 alkynyl. 2 is halogen or C1-C6 alkyl. 2 It is halogen, –SCH3, –CH3, C2-C3 alkenyl or C2-C3 alkynyl.

[0117] In some embodiments of any of Formulas (I), (Ia), and (Ib), R2 is halogen. 2 In some embodiments, R 2 In some embodiments, R 2 In some embodiments, R 2 For bromine.

[0118] In some embodiments of any of Formulas (I), (Ia), and (Ib), R 2 is C1-C6 alkyl. 2 is C1-C3 alkyl. 2 It is methyl.

[0119] In some embodiments of any of Formulas (I), (Ia), and (Ib), R2 In some embodiments, R 2 In some embodiments, R 2 It is -SCH3.

[0120] In some embodiments of any of Formulas (I), (Ia), and (Ib), R 2 is a C3-C8 cycloalkyl group. 2 It is cyclopropyl.

[0121] In some embodiments of any of Formulas (I), (Ia), and (Ib), R 2 In some embodiments, R 2 In some embodiments, R 2 is vinyl (ethenyl), propenyl, ethacryl, 1-butenyl, 2-butenyl, isobutenyl, or butadienyl. 2 For vinyl.

[0122] In some embodiments of any of Formulas (I), (Ia), and (Ib), R 2 is C2-C6 alkynyl. 2 is C2-C3 alkynyl. 2 is ethynyl or propynyl. In some embodiments, R 2 It is ethynyl.

[0123] In some embodiments of any of Formulas (I), (Ia), and (Ib), R 2a is halogen or C1-C3 alkyl. 2a is halogen or CH3. In some embodiments, R 2a is fluorine or CH3. In some embodiments, R 2a is iodine or CH3. In some embodiments, R 2a is chlorine or CH3. In some embodiments, R 2a It is bromine or CH3.

[0124] In some embodiments of any of Formulas (I), (Ia), and (Ib), R 2a In some embodiments, R 2a In some embodiments, R 2a In some embodiments, R 2a In some embodiments, R 2a For bromine.

[0125] In some embodiments of any of Formulas (I), (Ia), and (Ib), R 2a is C1-C6 alkyl. 2a is C1-C3 alkyl. 2a For CH3.

[0126] In some embodiments of any of Formulas (I), (Ia), and (Ib), R 2 and R 2a In some embodiments, R 2 is halogen and R 2a is C1-C6 alkyl. 2 is a C1-C6 alkyl group and R 2a In some embodiments, R 2 is -S-C1-C6 alkyl and R 2a In some embodiments, R 2 is –SCH3 and R 2a In some embodiments, R 2 is a C3-C8 cycloalkyl group and R 2a In some embodiments, R 2 is cyclopropyl and R 2a In some embodiments, R 2 is a C2-C6 alkenyl group and R 2a In some embodiments, R 2 is a C2-C6 alkynyl group and R 2a In some embodiments, R 2 is ethynyl and R 2a In some embodiments, R 2 and R 2a Each is independently fluorine, chlorine, bromine or iodine. 2 is iodine and R 2a In some embodiments, R 2 is halogen and R 2a is -CH3. In some embodiments, R 2 is bromine and R 2a is -CH3. In some embodiments, R 2 is iodine and R 2a is -CH3. In some embodiments, R 2 is –SCH3 and R 2a In some embodiments, R 2 is ethynyl and R 2a For fluorine.

[0127] With respect to any of Formula (I), (Ia) and (Ib), in some embodiments, R 1 For-OR 4 、-NR 5 R 5a , or-N(OR 5b )R 5a .

[0128] In some embodiments of any of Formulas (I), (Ia), and (Ib), R1 is -OR 4 In some embodiments, R 4 In some embodiments, R 4 is C1-C6 alkyl. 4 is unsubstituted or substituted with 1 to 6 R 6 substituted C3-C8 cycloalkyl, and R 6 As defined and described herein. In some embodiments, R 4 is unsubstituted or substituted with 1 to 6 R 6 substituted C3-C8 cycloalkyl, and each R 6 is independently hydroxyl or C1-C6 alkyl. 4 is C3-C8 cycloalkyl-C1-C6 alkyl, wherein the C3-C8 cycloalkyl is unsubstituted or substituted by 1 to 6 R 6 replaced, and R 6 As defined and described herein. In some embodiments, R 4 is C3-C8 cycloalkyl-C1-C6 alkyl, wherein the C3-C8 cycloalkyl is unsubstituted or substituted with 1 to 6 R 6 replaced, and each R 6 is independently hydroxyl or C1-C6 alkyl. 4 is cyclopropyl, cyclobutyl, cyclopropyl-C1-C6 alkyl, or cyclobutyl-C1-C6 alkyl; and each of the cyclopropyl and cyclobutyl groups is unsubstituted or substituted by 1 to 6 R 6 replaced, and R 6 As defined and described herein. In some embodiments, R 4 is cyclopropyl, cyclobutyl, cyclopropyl-C1-C6 alkyl, or cyclobutyl-C1-C6 alkyl; and each of the cyclopropyl and cyclobutyl is unsubstituted or substituted with 1 to 6 R 6 Substituted and each R 6 is independently hydroxyl or C1-C6 alkyl. 4 is C1-C6 hydroxyalkyl. 4is C1-C6 alkoxy-C1-C6 alkyl. 4 is amino-C1-C6 alkyl. 4 is C1-C6 alkylamino-C1-C6 alkyl. 4 is di(C1-C6 alkyl)amino-C1-C6 alkyl. 4 is a heterocycloalkyl group which is unsubstituted or substituted by 1 to 6 R6, and R 6 As defined and described herein. In some embodiments, R 4 is unsubstituted or substituted with 1 to 6 R 6 substituted heterocycloalkyl, and each R 6 is independently hydroxyl or C1-C6 alkyl. 4 is heterocycloalkyl-C1-C6alkyl, wherein the heterocycloalkyl is unsubstituted or substituted with 1 to 6 R 6 replaced, and R 6 As defined and described herein. In some embodiments, R 4 is heterocycloalkyl-C1-C6alkyl, wherein the heterocycloalkyl is unsubstituted or substituted with 1 to 6 R 6 replaced, and each R 6 is independently hydroxyl or C1-C6 alkyl. 4 is oxetanyl, azetidinyl, pyrrolidinyl, piperidinyl, oxetanyl-C1-C6 alkyl, azetidinyl-C1-C6 alkyl, pyrrolidinyl-C1-C6 alkyl, piperidinyl-C1-C6 alkyl, or 2,2-dimethyl-1,3-dioxolan-4-yl-C1-C6 alkyl. In some embodiments, R 4 For R 7 -C(O)-C1-C6 alkyl; and R 7 is hydroxy, C1-C6 alkoxy, amino, C1-C6 alkylamino, di(C1-C6 alkyl)amino, hydroxyamino or N-C1-C6 alkylhydroxyamino. 4 For R 7 -C(O)-C1-C6 alkyl; and R 7 It is hydroxy, C1-C6 alkoxy, amino or hydroxyamino.

[0129] In some embodiments of any of Formulas (I), (Ia), and (Ib), R 1 Selected from the group consisting of: -OH,

[0130]

[0131] In some embodiments of any of Formulas (I), (Ia), and (Ib), R1 is -OR 5 In some embodiments, R 5 In some embodiments, R 5 is C1-C6 alkyl. 5 is unsubstituted or substituted with 1 to 6 R 6 substituted C3-C8 cycloalkyl, and R 6 As defined and described herein. In some embodiments, R 5 is unsubstituted or substituted with 1 to 6 R 6 substituted C3-C8 cycloalkyl, and each R 6 is independently hydroxyl or C1-C6 alkyl. 5 is C3-C8 cycloalkyl-C1-C6 alkyl, wherein the C3-C8 cycloalkyl is unsubstituted or substituted by 1 to 6 R 6 replaced, and R 6 As defined and described herein. In some embodiments, R 5 is C3-C8 cycloalkyl-C1-C6 alkyl, wherein the C3-C8 cycloalkyl is unsubstituted or substituted with 1 to 6 R 6 replaced, and each R 6 is independently hydroxyl or C1-C6 alkyl. 5 is cyclopropyl, cyclobutyl, cyclopropyl-C1-C6 alkyl, or cyclobutyl-C1-C6 alkyl; and each of the cyclopropyl and cyclobutyl groups is unsubstituted or substituted by 1 to 6 R 6 replaced, and R 6 As defined and described herein. In some embodiments, R 5 is cyclopropyl, cyclobutyl, cyclopropyl-C1-C6 alkyl, or cyclobutyl-C1-C6 alkyl; and each of the cyclopropyl and cyclobutyl is unsubstituted or substituted with 1 to 6 R 6 Substituted and each R 6 is independently hydroxyl or C1-C6 alkyl. 5 is C1-C6 hydroxyalkyl. 5 is C1-C6 alkoxy-C1-C6 alkyl. 5 is amino-C1-C6 alkyl. 5 is C1-C6 alkylamino-C1-C6 alkyl. 5 is di(C1-C6 alkyl)amino-C1-C6 alkyl. 5 is unsubstituted or substituted with 1 to 6 R6 substituted heterocycloalkyl, and R 6 As defined and described herein. In some embodiments, R 5 is unsubstituted or substituted with 1 to 6 R 6 substituted heterocycloalkyl, and each R 6 is independently hydroxyl or C1-C6 alkyl. 5 is heterocycloalkyl-C1-C6alkyl, wherein the heterocycloalkyl is unsubstituted or substituted with 1 to 6 R 6 replaced, and R 6 As defined and described herein. In some embodiments, R 5 is heterocycloalkyl-C1-C6alkyl, wherein the heterocycloalkyl is unsubstituted or substituted with 1 to 6 R 6 replaced, and each R 6 is independently hydroxyl or C1-C6 alkyl. 5 is oxetanyl, azetidinyl, pyrrolidinyl, piperidinyl, oxetanyl-C1-C6 alkyl, azetidinyl-C1-C6 alkyl, pyrrolidinyl-C1-C6 alkyl, piperidinyl-C1-C6 alkyl, or 2,2-dimethyl-1,3-dioxolan-4-yl-C1-C6 alkyl. In some embodiments, R 5 For R 7 -C(O)-C1-C6 alkyl; and R 7 is hydroxy, C1-C6 alkoxy, amino, C1-C6 alkylamino, di(C1-C6 alkyl)amino, hydroxyamino or N-C1-C6 alkylhydroxyamino. 5 For R 7 -C(O)-C1-C6 alkyl; and R 7 It is hydroxy, C1-C6 alkoxy, amino or hydroxyamino.

[0132] In some embodiments of any of Formulas (I), (Ia), and (Ib), R 1 For-NR 5 R 5a And R 5 Selected from the group consisting of hydrogen,

[0133]

[0134] In some embodiments of any of Formulas (I), (Ia), and (Ib), R1 is -N(OR 5b )R 5a In some embodiments, R 5b In some embodiments, R 5bis C1-C6 alkyl. 5b is unsubstituted or substituted with 1 to 6 R 6 Substituted C3-C8 cycloalkyl, and R 6 As defined and described herein. In some embodiments, R 5b is unsubstituted or substituted with 1 to 6 R 6 substituted C3-C8 cycloalkyl, and each R 6 is independently hydroxyl or C1-C6 alkyl. 5b is C3-C8 cycloalkyl-C1-C6 alkyl, wherein the C3-C8 cycloalkyl is unsubstituted or substituted with 1 to 6 R 6 Substituted and R 6 As defined and described herein. In some embodiments, R 5b is C3-C8 cycloalkyl-C1-C6 alkyl, wherein the C3-C8 cycloalkyl is unsubstituted or substituted with 1 to 6 R 6 Substituted and each R 6 is independently hydroxyl or C1-C6 alkyl. 5b is cyclopropyl, cyclobutyl, cyclopropyl-C1-C6 alkyl, or cyclobutyl-C1-C6 alkyl; and each of the cyclopropyl and cyclobutyl groups is unsubstituted or substituted by 1 to 6 R 6 replaced, and R 6 As defined and described herein. In some embodiments, R 5b is cyclopropyl, cyclobutyl, cyclopropyl-C1-C6 alkyl, or cyclobutyl-C1-C6 alkyl; and each of the cyclopropyl and cyclobutyl is unsubstituted or substituted with 1 to 6 R 6 Substituted and each R 6 is independently hydroxyl or C1-C6 alkyl. 5b is C1-C6 hydroxyalkyl. 5b is C1-C6 alkoxy-C1-C6 alkyl. 5b is amino-C1-C6 alkyl. 5b is C1-C6 alkylamino-C1-C6 alkyl. 5b is di(C1-C6 alkyl)amino-C1-C6 alkyl. 5b is unsubstituted or substituted with 1 to 6 R 6 Substituted heterocycloalkyl and R 6 As defined and described herein. In some embodiments, R 5b is unsubstituted or substituted with 1 to 6 R 6 substituted heterocycloalkyl, and each R6 is independently hydroxyl or C1-C6 alkyl. 5b is a heterocycloalkyl-C1-C6 alkyl group, wherein the heterocycloalkyl group is unsubstituted or substituted with 1 to 6 R 6 Substituted and R 6 As defined and described herein. In some embodiments, R 5b is a heterocycloalkyl-C1-C6 alkyl group, wherein the heterocycloalkyl group is unsubstituted or substituted with 1 to 6 R 6 Substituted and each R 6 is independently hydroxyl or C1-C6 alkyl. 5b is oxetanyl, azetidinyl, pyrrolidinyl, piperidinyl, oxetanyl-C1-C6 alkyl, azetidinyl-C1-C6 alkyl, pyrrolidinyl-C1-C6 alkyl, piperidinyl-C1-C6 alkyl, or 2,2-dimethyl-1,3-dioxolan-4-yl-C1-C6 alkyl. In some embodiments, R 5b For R 7 -C(O)-C1-C6 alkyl; and R 7 is hydroxy, C1-C6 alkoxy, amino, C1-C6 alkylamino, di(C1-C6 alkyl)amino, hydroxyamino or N-C1-C6 alkylhydroxyamino. 5b For R 7 -C(O)-C1-C6 alkyl; and R 7 It is hydroxy, C1-C6 alkoxy, amino or hydroxyamino.

[0135] In some embodiments of any of Formulas (I), (Ia), and (Ib), R 1 -N(OR 5b )R 5a AND-OR 5b Selected from the group consisting of: -OH,

[0136]

[0137] In some embodiments of any of Formulas (I), (Ia), and (Ib), R 5a In some embodiments, R 5a is C1-C6 alkyl. 5a is C1-C4 alkyl. 5a is methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, or tert-butyl. 5a It is methyl.

[0138] In some embodiments of any of Formulas (I), (Ia), and (Ib), R 1 is unsubstituted or replaced by one or two R 6 Substituted N-linked heterocycloalkyl, wherein R 6 As defined and described herein. In some embodiments, the N-connected heterocycloalkyl is an N-connected azetidinyl, an N-connected pyrrolidinyl, an N-connected isoxazolidinyl, an N-connected piperidinyl, or an N-connected morpholinyl. In some embodiments, the N-connected heterocycloalkyl is an N-connected azetidinyl. In some embodiments, the N-connected heterocycloalkyl is an N-connected pyrrolidinyl. In some embodiments, the N-connected heterocycloalkyl is an N-connected isoxazolidinyl. In some embodiments, the N-connected heterocycloalkyl is an N-connected piperidinyl. In some embodiments, the N-connected heterocycloalkyl is an N-connected morpholinyl. In some embodiments, R 1 is unsubstituted or replaced by one or two R 6 A substituted N-linked azetidinyl group wherein R 6 As defined and described herein. In some embodiments, R 1 is unsubstituted or replaced by one or two R 6 Substituted N-linked pyrrolidinyl, wherein R 6 As defined and described herein. In some embodiments, R 1 is unsubstituted or replaced by one or two R 6 Substituted N-linked piperidinyl, wherein R 6 As defined and described herein. In some embodiments, R 1 is unsubstituted or replaced by one or two R 6 Substituted N-linked isoxazolidinyl, wherein R 6 As defined and described herein. In some embodiments, R 1 is unsubstituted or replaced by one or two R 6 Substituted N-linked morpholinyl, wherein R 6 As defined and described herein.

[0139] Regarding R as one or two substituents of N-linked heterocycloalkyl 6 In some embodiments, each R 6 are independently hydroxy, oxo or amino. 6 In some embodiments, R 6 In some embodiments, R 6 In some embodiments, R 6 One of them is a hydroxyl group and the other R 6 It is amino.

[0140] In some embodiments of any of Formulas (I), (Ia), and (Ib), R 1 is an N-linked heterocycloalkyl group which is unsubstituted or substituted with hydroxy, oxo or amino. 1 is an N-linked azetidinyl group which is unsubstituted or substituted with hydroxy, oxo or amino. 1 is an N-linked pyrrolidinyl group which is unsubstituted or substituted with hydroxy, oxo or amino. 1 is an N-linked piperidinyl group which is unsubstituted or substituted with hydroxy, oxo or amino. 1 is an N-linked isoxazolidinyl group which is unsubstituted or substituted with hydroxy, oxo or amino. 1 is an N-linked morpholinyl group which is unsubstituted or substituted with hydroxy, oxo or amino.

[0141] In some embodiments, the compound of formula (I) or (Ia) is represented by any of the following formulae:

[0142]

[0143]

[0144]

[0145] Among them, R 2 , R 2a , R 3 , R 3a and R 3b As defined in any aspect or embodiment described herein.

[0146] In some embodiments of the above structure having Formula (Ia), R 2 is iodine and R 2a In some embodiments of the above structures, R 2 is iodine and R 2a In some embodiments of the above structures, R 2 is ethynyl and R 2a In some embodiments of the above structures, R 2 is ethynyl and R 2a In some embodiments of the above structures, R 2 -SCH3 and R 2a In some embodiments of the above structures, R 2 -SCH3 and R 2a It is methyl.

[0147] In some embodiments of the above structure having Formula (Ia), R 3 , R 3a and R 3b In some embodiments of the above structures, R 3 and R 3a Each is hydrogen and R 3b In some embodiments of the above structures, R 3 and R 3a Each is hydrogen and R 3b For fluorine.

[0148] In some embodiments, the compound of formula (I) or (Ib) is represented by any of the following formulae:

[0149]

[0150]

[0151]

[0152] Among them, R 2 , R 2a , R 3 and R 3a As defined in any aspect or embodiment described herein.

[0153] In some embodiments of the above structure having Formula (Ib), R 2 is iodine and R 2a In some embodiments of the above structures, R 2 is iodine and R 2a In some embodiments of the above structures, R 2 is ethynyl and R 2a In some embodiments of the above structures, R 2 is ethynyl and R 2a In some embodiments of the above structures, R 2 -SCH3 and R 2a In some embodiments of the above structures, R 2 -SCH3 and R 2a It is methyl.

[0154] In some embodiments of the above structure having Formula (Ib), R 3 and R 3a Each is hydrogen.

[0155] Illustrative compounds of formula (I) are listed in Table 1.

[0156] Table 1: Compounds of formula (I)

[0157]

[0158]

[0159]

[0160]

[0161]

[0162] Additional compounds of formula (I) are listed in Table 2.

[0163] Table 2: Additional compounds of formula (I)

[0164]

[0165]

[0166]

[0167] In some embodiments, the compound of formula (I) or (Ia) is selected from the group consisting of:

[0168]

[0169]

[0170] In one embodiment, the compound of formula (I) or (Ib) is not ethyl 6-cyano-2-((2-fluoro-4-iodophenyl)amino)-5-methylnicotinate.

[0171] Other forms of compounds

[0172] The compounds of the present invention may exist as salts. The present invention includes such salts. Examples of applicable salt forms include: hydrochlorides, hydrobromides, sulfates, mesylates, nitrates, maleates, acetates, citrates, fumarates, tartrates (e.g., (+)-tartrates, (-)-tartrates or mixtures thereof, including racemic mixtures), succinates, benzoates, and salts with amino acids such as glutamic acid. These salts can be prepared by methods known to those skilled in the art. Also included are base addition salts, such as sodium, potassium, calcium, ammonium, organic amino or magnesium salts, or similar salts. When the compounds of the present invention contain relatively basic functional groups, acid addition salts can be obtained by contacting the neutral forms of these compounds with a sufficient amount of the desired acid (without solvent or in a suitable inert solvent). Examples of acceptable acid addition salts include those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, monohydrogencarbonic acid, phosphoric acid, monohydrogenphosphoric acid, dihydrogenphosphoric acid, sulfuric acid, monohydrogensulfuric acid, hydroiodic acid or phosphorous acid, and salts derived from organic acids such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid, methanesulfonic acid, etc. Also included are salts of amino acids such as arginine, and salts of organic acids such as glucuronic acid or galacturonic acid. Certain specific compounds of the present invention contain both basic and acidic functional groups, such that the compounds can be converted into base or acid addition salts.

[0173] Other salts include acid salts or base salts of the compounds used in the methods of the present invention. Illustrative examples of pharmaceutically acceptable salts are salts of inorganic acids (hydrochloric acid, hydrobromic acid, phosphoric acid, etc.), organic acids (acetic acid, propionic acid, glutamic acid, citric acid, etc.) and quaternary ammonium (methyl iodide, ethyl iodide, etc.) salts. It should be understood that pharmaceutically acceptable salts are non-toxic. Additional information about suitable pharmaceutically acceptable salts can be found in Remington's Pharmaceutical Sciences, 17th edition, Mark Publishing Company, Easton, Pennsylvania, 1985, which is incorporated herein by reference.

[0174] Pharmaceutically acceptable salts include salts of active compounds prepared with relatively nontoxic acids or bases, depending on the specific substituents present on the compounds described herein. When the compounds of the present invention contain relatively acidic functional groups, base addition salts can be obtained by contacting the neutral forms of these compounds with a sufficient amount of the desired base (without solvent or in a suitable inert solvent). Examples of pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amino or magnesium salts, or similar salts. When the compounds of the present invention contain relatively basic functional groups, acid addition salts can be obtained by contacting the neutral forms of these compounds with a sufficient amount of the desired acid (without solvent or in a suitable inert solvent). Examples of pharmaceutically acceptable acid addition salts include those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, monohydrogencarbonic acid, phosphoric acid, monohydrogenphosphoric acid, dihydrogenphosphoric acid, sulfuric acid, monohydrogensulfuric acid, hydroiodic acid or phosphorous acid, and salts derived from relatively non-toxic organic acids such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid, methanesulfonic acid, etc. Also included are salts of amino acids such as arginine, and organic acids such as glucuronic acid or galacturonic acid (see, e.g., Berge et al., "Pharmaceutical Salts", Journal of Pharmaceutical Science, 1977, 66, 1-19). Certain specific compounds of the present invention contain both basic and acidic functional groups, allowing the compounds to be converted into base or acid addition salts.

[0175] The neutral forms of the compounds are preferably regenerated by contacting the salt with a base or acid and isolating the parent compound in the conventional manner. The parent form of the compound differs from the various salt forms in certain physical properties, such as solubility in polar solvents.

[0176] Some compounds of the present invention can exist in non-solvated form as well as solvated form, including hydrated form. Typically, the solvated form is equivalent to the non-solvated form and is included within the scope of the present invention. Some compounds of the present invention can exist in polycrystalline or amorphous form. Typically, all physical forms are equivalent for the purposes contemplated by the present invention and are intended to be included within the scope of the present invention.

[0177] Certain compounds of the invention possess asymmetric carbon atoms (optical centers) or double bonds; enantiomers, racemates, diastereomers, tautomers, geometric isomers, stereoisomeric forms (which may be defined in terms of absolute stereochemistry as (R)- or (S)- or for amino acids as (D)- or (L)-) and individual isomers are included within the scope of the invention. Compounds of the invention do not include those known in the prior art that are too unstable to synthesize and / or separate. The invention is intended to include compounds in racemic and optically pure form. Optically active (R)- and (S)- or (D)- and (L)- isomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques.

[0178] Isomers include compounds that have the same number and kind of atoms, and therefore the same molecular weight, but differ in the structural arrangement or configuration of the atoms.

[0179] It will be clear to those skilled in the art that certain compounds of the present invention may exist in tautomeric forms, all of which are within the scope of the present invention. Tautomers refer to one of two or more structural isomers that exist in equilibrium and are easily converted from one isomeric form to another.

[0180] Unless otherwise stated, structures depicted herein are also meant to include all stereochemical forms of the structures; that is, the R and S configurations for each asymmetric center. Therefore, single stereochemical isomers as well as enantiomeric and diastereomeric mixtures of the present compounds are within the scope of the invention.

[0181] Unless otherwise indicated, the compounds of the present invention may also contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute these compounds. For example, the compounds of the present invention may be labeled with radioactive or stable isotopes, such as deuterium ( 2 H), tritium ( 3 H), iodine-125 ( 125 I), fluorine-18( 18 F), nitrogen-15 ( 15 N), oxygen-17 ( 17 O), oxygen-18 ( 18 O), carbon-13 ( 13 C) or carbon-14 ( 14 C) All isotopic variations of the compounds of the present invention, whether radioactive or not, are encompassed within the scope of the present invention.

[0182] In addition to salt forms, the present invention provides compounds in the form of prodrugs. Prodrugs of compounds described herein are those compounds that are susceptible to chemical changes under physiological conditions to provide compounds of the present invention. In addition, prodrugs can be converted into compounds of the present invention by chemical or biochemical methods in an ex vivo environment. For example, when the prodrug is placed in a transdermal patch reservoir with a suitable enzyme or chemical reagent, the prodrug will slowly convert into the compound of the present invention.

[0183] IV. Composition

[0184] In another aspect, provided herein is a pharmaceutical composition comprising a compound of formula (I) and a pharmaceutically acceptable carrier.

[0185] The compounds provided herein can be formulated into pharmaceutical compositions using methods available in the art and disclosed herein. Any compound disclosed herein can be provided in an appropriate pharmaceutical composition and administered by an appropriate route of administration.

[0186] Administration of the compounds described herein to a subject may be local or non-systemic, such as topical, subcutaneous, intradermal, or intralesional. In some embodiments, the compound may be administered by topical administration. In some embodiments, the compound may be administered by intradermal administration. In some embodiments, the compound may be administered by intralesional administration (e.g., by intralesional injection).

[0187] The methods provided herein include administering to a patient in need thereof a pharmaceutical composition containing at least one compound as described herein, including a compound of formula (I) (if appropriate in salt form), alone or in combination with one or more compatible and pharmaceutically acceptable carriers (e.g., diluents or adjuvants), or in combination with another agent for treating a MEK inhibitor-responsive condition or disease, a MEK inhibitor-responsive skin condition or disease, a MEK-mediated condition or disease, or a MEK-mediated skin condition or disease.

[0188] In some embodiments, the second agent can be formulated or packaged with the compound provided herein. Of course, the second agent will be formulated with the compound provided herein only when, according to the judgment of those skilled in the art, such a co-formulation should not interfere with the activity or administration method of either formulation. In some embodiments, the compound provided herein and the second agent are formulated separately. For the convenience of practitioners in this field, they can be packaged together, or packaged separately.

[0189] In clinical practice, the active agents provided herein can be administered by any conventional route, particularly topically, subcutaneously, intradermally, intralesionally, orally, parenterally, rectally, or by inhalation (e.g., in the form of an aerosol). In some embodiments, the compounds provided herein are administered topically, subcutaneously, intradermally, or intralesionally. In some embodiments, the compounds provided herein are administered topically. In some embodiments, the compounds provided herein are administered intradermally. In some embodiments, the compounds provided herein are administered intralesionally.

[0190] As solid compositions for oral administration, tablets, pills, hard gelatin capsules, powders or granules can be used. In these compositions, the active product is mixed with one or more inert diluents or adjuvants (eg sucrose, lactose or starch).

[0191] These compositions may contain substances other than diluents, for example lubricants, such as magnesium stearate, or coatings for controlled release.

[0192] As liquid compositions for oral administration, pharmaceutically acceptable solutions, suspensions, emulsions, syrups and elixirs containing inert diluents (such as water or liquid paraffin) can be used. These compositions may also contain substances other than diluents, and in some embodiments, contain wetting, sweetening or flavoring products.

[0193] As compositions for topical application, 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, butane 1,3 diol, isopropyl myristate, isopropyl palmitate, mineral oil and mixtures thereof).

[0194] Compositions for parenteral, intralesional or intradermal administration can be emulsions or sterile solutions. As solvent or carrier, propylene glycol, polyethylene glycol, vegetable oil (particularly olive oil), or injectable organic esters can be used, in some embodiments, ethyl oleate. These compositions can also contain adjuvants, particularly wetting agents, isotonic agents, emulsifiers, dispersants and stabilizers. In some embodiments, sterilization can be carried out in several ways, using a bacterial filter, by radiation or by heating. They can also be prepared in the form of sterile solid compositions, which can be dissolved in sterile water or any other injectable sterile medium when used.

[0195] Compositions for rectal administration are suppositories or rectal capsules which contain, in addition to the active ingredient, excipients such as cocoa butter, semi-synthetic glycerides or polyethylene glycols.

[0196] The composition can also be an aerosol. For use in the form of a liquid aerosol, the composition can be a stable sterile solution or a solid composition dissolved in unheated sterile water, saline or any other pharmaceutically acceptable carrier when used. In order to use in the form of a dry aerosol intended for direct inhalation, the active ingredient is finely divided and combined with a water-soluble solid (in some embodiments, dextran, mannitol or lactose) diluent or carrier.

[0197] In some embodiments, the compositions provided herein are pharmaceutical compositions or single unit dosage forms. The pharmaceutical compositions and single unit dosage forms provided herein include one or more preventive or therapeutic agents (e.g., compounds or other preventive or therapeutic agents provided herein) in a preventive or therapeutically effective amount, and one or more typical pharmaceutically acceptable carriers or excipients. In some embodiments, the term "pharmaceutically acceptable" refers to those approved by a regulatory agency of a federal or state government or listed in the U.S. Pharmacopoeia or other generally recognized pharmacopoeias for animals, more specifically for humans. The term "carrier" includes diluents, adjuvants (e.g., Freund's adjuvant (complete and incomplete)), excipients or administration carriers. Such drug carriers can be sterile liquids, such as water and oil, including those of petroleum, animal, plant or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. When the pharmaceutical composition is administered intravenously, water can be used as a carrier. Saline solutions and aqueous glucose solutions and aqueous glycerol solutions can also be used as liquid carriers, particularly liquid carriers for injectable solutions. Examples of suitable pharmaceutical carriers are described in Remington: The Science and Practice of Pharmacy, 22nd Edition (September 15, 2012).

[0198] Typical pharmaceutical compositions and dosage forms include one or more excipients. Suitable excipients are well known to those skilled in the art of pharmacy, and in some embodiments, suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talcum, sodium chloride, skim milk powder, glycerine, propylene glycol, water, ethanol, etc. Whether a particular excipient is suitable for incorporation into a pharmaceutical composition or dosage form depends on multiple factors well known in the art, including but not limited to the mode in which the dosage form is administered to an object and the specific active ingredient in the dosage form. If desired, compositions or single unit dosage forms can also contain a small amount of wetting agent or emulsifier, or pH buffer.

[0199] The lactose-free compositions provided herein can include excipients known in the art and, in some embodiments, listed in the U.S. Pharmacopoeia (USP 36-NF 31 S2). Typically, the lactose-free compositions include active ingredients, binders / fillers, and pharmaceutical phase capacity and pharmaceutically acceptable amounts of lubricants. Exemplary lactose-free dosage forms include active ingredients, microcrystalline cellulose, pregelatinized starch, and magnesium stearate.

[0200] Also contemplated herein are anhydrous pharmaceutical compositions and dosage forms comprising active ingredients, since water can promote the degradation of certain compounds. For example, the addition of water (e.g., 5%) is widely accepted in the pharmaceutical field as a method of simulating long-term storage to determine properties such as the shelf life or stability of a formulation over time. See, for example, Jens T. Carstensen, Drug Stability: Principles and Practice, Second Edition, Marcel Dekker, New York, 1995, pp. 379-80. In fact, water and heat accelerate the decomposition of certain compounds. Therefore, the effect of water on the formulation is very important because moisture and / or humidity are typically encountered during the manufacture, handling, packaging, storage, shipment, and use of the formulation.

[0201] Anhydrous pharmaceutical compositions and dosage forms provided herein can be prepared using anhydrous or low moisture ingredients and low moisture or low humidity conditions. Pharmaceutical compositions and dosage forms comprising lactose and at least one active ingredient comprising a primary or secondary amine can be anhydrous if substantial contact with moisture and / or humidity during manufacturing, packaging, and / or storage is expected.

[0202] Anhydrous pharmaceutical compositions should be prepared and stored to maintain their anhydrous nature. Therefore, anhydrous compositions can be packaged using materials known to prevent exposure to water so that they can be included in suitable formulation packages. In some embodiments, suitable packaging includes, but is not limited to, hermetically sealed foils, plastics, unit dose containers (such as bottles), blister packs, and strip packs.

[0203] Also provided are pharmaceutical compositions and dosage forms comprising one or more compounds that reduce the rate at which an active ingredient decomposes.Such compounds, referred to herein as "stabilizers," include, but are not limited to, antioxidants such as ascorbic acid, pH buffers, or salt buffers.

[0204] The pharmaceutical composition and separate unit dosage form can be in the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations, etc. Oral formulations can include standard carriers, such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. These compositions and dosage forms will contain a preventive or therapeutically effective amount of a prophylactic or therapeutic agent, in some embodiments, in a purified form, together with an appropriate amount of a carrier, so as to provide a form of an appropriate subject for administration. The preparation should be compatible with the mode of administration. In some embodiments, the pharmaceutical composition or separate unit dosage form is sterile and is administered to a subject in a suitable form, in some embodiments, an animal subject (such as a mammalian subject), in some embodiments, a human.

[0205] A pharmaceutical composition is formulated to be compatible with its intended route of administration. In some embodiments, the route of administration includes, but is not limited to, parenteral administration, such as intravenous, intradermal, subcutaneous, intramuscular, subcutaneous, oral, oral, sublingual, inhalation, intranasal, transdermal, topical, transmucosal, intratumoral, intrasynovial and rectal administration. In some embodiments, the route of administration is intradermal administration, topical administration or intralesional administration. In some embodiments, the route of administration is non-systemic administration. In some embodiments, the composition is formulated according to conventional methods as a pharmaceutical composition suitable for intravenous, subcutaneous, intramuscular, oral, intranasal or topical administration to people. In some embodiments, the pharmaceutical composition is formulated according to conventional methods for subcutaneous administration to people. Typically, the composition for intravenous administration is a solution in a sterile isotonic aqueous buffer. If necessary, the composition may also include a solubilizer, and a local anesthetic (such as lidocaine (lignocamne)) to relieve pain at the injection site.

[0206] In some embodiments, dosage forms include, but are not limited to, tablets; caplets; capsules, such as soft elastic gelatin capsules; cachets; lozenges; troches; dispersions; suppositories; ointments; poultices; pastes; powders; dressings; creams; plasters; solutions; patches; aerosols (such as nasal sprays or inhalers); gels; liquid dosage forms suitable for oral or mucosal administration to a subject, including suspensions (such as 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 (such as crystalline or amorphous solids) that can be reconstituted to provide a liquid dosage form suitable for parenteral administration to a patient.

[0207] The composition, shape and type of dosage forms provided herein generally vary according to their use. In some embodiments, the dosage form for the initial treatment of MEK inhibitor responsiveness or disease, MEK inhibitor responsiveness skin disorder or disease, MEK-mediated disorder or disease, or MEK-mediated skin disorder or disease may contain a larger amount of one or more active ingredients contained therein than the dosage form for maintaining the treatment of the same disorder or disease. Similarly, compared with the oral dosage form for the treatment of the same disease or disorder, the parenteral dosage form may contain a smaller amount of one or more active ingredients contained therein. These and other ways of the specific dosage forms covered herein are different from each other, which are obvious to those skilled in the art. See, for example, Remington: The Science and Practice of Pharmacy; Pharmaceutical Press; 22nd edition (September 15, 2012).

[0208] Typically, the components of the composition are provided separately or mixed together in unit dosage form, in some embodiments, as a lyophilized powder or anhydrous concentrate dried in a hermetically sealed container (such as an ampoule or a sachet) indicating the active dose. When the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. In the case of a composition administered by injection, an ampoule of sterile water for injection or normal saline can be provided so that the components can be mixed before administration.

[0209] Typical dosage forms include a compound provided herein, or a pharmaceutically acceptable salt, solvate or hydrate thereof, in the range of about 0.1 mg to about 1000 mg per day, administered in a single dose once a day in the morning, or in divided doses throughout the day with food. Specific dosage forms may have about 0.1, 0.2, 0.3, 0.4, 0.5, 1.0, 2.0, 2.5, 5.0, 10.0, 15.0, 20.0, 25.0, 50.0, 100, 200, 250, 500 or 1000 mg of active compound.

[0210] Oral dosage form

[0211] Pharmaceutical compositions suitable for oral administration can be presented in discrete dosage forms, such as, but not limited to, tablets (such as chewable tablets), sachets, capsules, and liquids (such as flavored syrups). Such dosage forms contain a predetermined amount of active ingredient and can be prepared by pharmaceutical methods known to those skilled in the art. See, generally, Remington: The Science and Practice of Pharmacy; Pharmaceutical Press; 22nd Edition (September 15, 2012).

[0212] In some embodiments, the oral dosage form is solid and prepared with anhydrous ingredients in an anhydrous environment, as described in detail herein. However, the scope of the compositions provided herein extends beyond anhydrous solid oral dosage forms. Therefore, other forms are described herein.

[0213] Typical oral dosage forms are prepared by combining active ingredients with at least one excipient in a closely mixed form according to conventional pharmaceutical compounding techniques. Excipients can take various forms, depending on the formulation form required for administration. In some embodiments, excipients suitable for 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 solid oral dosage forms (such as powders, tablets, capsules and capsules) include but are not limited to starch, sugar, microcrystalline cellulose, diluents, granulating agents, lubricants, binders and disintegrants.

[0214] Tablets and capsules are the most advantageous oral unit dosage forms due to ease of administration, in which solid excipients can be used. If necessary, tablets can be coated with standard aqueous or non-aqueous techniques. Such dosage forms can be prepared by any pharmaceutical method. In general, pharmaceutical compositions and dosage forms are prepared by uniformly and intimately mixing the active ingredient with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product into a desired presentation form.

[0215] In some embodiments, tablets can be prepared by compression or molding. Compressed tablets can be prepared by compressing the active ingredient in a free-flowing form (such as powder or granules) optionally mixed with an excipient in a suitable machine. Molded tablets can be made by molding a mixture of powdered compounds moistened with an inert liquid diluent in a suitable machine.

[0216] In some embodiments, excipients that can be used for oral dosage forms include, but are not limited to, binders, fillers, disintegrants, and lubricants. Binders suitable for pharmaceutical compositions and dosage forms include, but are not limited to, corn starch, potato starch or other starches, gelatin, natural and synthetic gums such as gum arabic, sodium alginate, alginic acid, other alginates, powdered gum astragalus, guar gum, cellulose and its derivatives (such as ethyl cellulose, cellulose acetate, carboxymethyl cellulose calcium, sodium carboxymethyl cellulose), polyvinyl pyrrolidone, methyl cellulose, pregelatinized starch, hydroxypropyl methyl cellulose, (such as No. 2208, 2906, 2910), microcrystalline cellulose and mixtures thereof.

[0217] In some embodiments, fillers suitable for use in pharmaceutical compositions and dosage forms disclosed herein include, but are not limited to, talc, calcium carbonate (such as granules or powders), microcrystalline cellulose, powdered cellulose, dextrose (dextrates), kaolin, mannitol, silicic acid, sorbitol, starch, pregelatinized starch, and mixtures thereof. The binder or filler in the pharmaceutical composition is typically present in about 50-about 99% by weight of the pharmaceutical composition or dosage form.

[0218] In some embodiments, suitable forms of microcrystalline cellulose include, but are not limited to, materials sold as AVICEL PH 101, AVICEL PH 103, AVICEL RC 581, AVICEL PH 105 (available from FMC Corporation, U.S. Viscosity Division, AVICEL Sales, Marcus Hook, PA), and mixtures thereof. A particular binder is a mixture of microcrystalline cellulose and sodium carboxymethylcellulose, sold as AVICEL RC 581. Suitable anhydrous or low moisture excipients or additives include AVICEL PH 103, AVICEL RC 581, AVICEL PH 105, and mixtures thereof. TM and starch 1500LM.

[0219] The disintegrant used in the composition is to disintegrate when the tablet is exposed to an aqueous environment. Tablets containing too much disintegrant may disintegrate in storage, while tablets containing too little disintegrant may not disintegrate at the desired rate or under the desired conditions. Therefore, a sufficient amount of disintegrant (neither too much nor too little to avoid adversely changing the release of the active ingredient) should be used to form a solid oral dosage form. The amount of disintegrant used varies according to the type of preparation, and it is easy for a person of ordinary skill in the art to distinguish. Typical pharmaceutical compositions contain about 0.5 to about 15% by weight of disintegrant, particularly about 1 to about 5% by weight of disintegrant.

[0220] Disintegrants that can be used in pharmaceutical compositions and dosage forms include, but are not limited to, agar, alginic acid, calcium carbonate, microcrystalline cellulose, croscarmellose sodium, crospovidone, polacrilin potassium, sodium starch glycolate, potato or tapioca starch, pregelatinized starch, other starches, clays, other alginics, other celluloses, gums, and mixtures thereof.

[0221] Lubricants that can be used for pharmaceutical compositions and dosage forms include, but are not limited to, calcium stearate, magnesium stearate, mineral oil, light mineral oil, glycerol, sorbitol, mannitol, polyethylene glycol, other glycols, stearic acid, sodium lauryl sulfate, talc, hydrogenated vegetable oils (such as peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil, and soybean oil), zinc stearate, ethyl oleate, ethyl laurate, agar, and mixtures thereof. In some embodiments, additional lubricants include micropowder (syloid) silica gel (AEROSIL 200, manufactured by WR Grace, Baltimore, Maryland), a coagulated aerosol of synthetic silica (sold by Degussa, Plano, Texas), CAB O SIL (a pyrogenic silica product sold by Cabot, Boston, Massachusetts), and mixtures thereof. If all are used, lubricants are generally used in an amount of less than about 1% by weight of the pharmaceutical composition or dosage form into which they are incorporated.

[0222] Delayed release dosage form

[0223] Active ingredients such as compounds provided herein can be administered by controlled release means or delivery devices known to those of ordinary skill in the art. In some embodiments, but not limited to those described in the following U.S. Patent Nos.: 3,845,770; 3,916,899; 3,536,809; 3,598,123; 4,008,719; 5,674,533; 5,059,595; 5,591,767; 5,120,548; 5,073,543; 5,639,476; 5,354,556; 5,639,480; 5,733,566; 5,739,108; 5,891 ,474; 5,922,356; 5,972,891; 5,980,945; 5,993,855; 6,045,830; 6,087,324; 6,113,943; 6,197,350; 6,248,363; 6,264,970; 6,267,981; 6,376,461; 6,419,961; 6,589,548; 6,613,358; and 6,699,500; each of which is incorporated herein by reference in its entirety. This dosage form can be used to provide slow or controlled release of one or more active ingredients. In some embodiments, hydropropyl methylcellulose, other polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, liposomes, microspheres, or combinations thereof are used to provide desired release profiles in varying proportions. Suitable controlled-release formulations known to those of ordinary skill in the art, including those described herein, can be readily selected for use with the active ingredients provided herein. Thus, included herein are individual unit dosage forms suitable for oral administration, such as, but not limited to, tablets, capsules, gel caps, and sachets suitable for controlled release.

[0224] All controlled-release pharmaceutical products have a common goal, namely to improve drug therapy relative to their corresponding non-controlled release drugs. Ideally, the use of optimally designed controlled-release formulations in medical treatment is characterized by using the least amount of raw material in the shortest time to cure or control a disease or condition. The advantages of controlled-release formulations include extended activity of the drug, reduced dosage frequency, and increased patient compliance. In addition, controlled-release formulations can be used to affect the time of onset of action or other characteristics, such as blood levels of the drug, and thus can affect the occurrence of side (such as adverse) effects.

[0225] Most controlled-release formulations are designed to initially release a certain amount of drug (active ingredient) to rapidly produce the desired therapeutic effect, and gradually and continuously release additional amounts of drug to maintain that level of therapeutic or preventive effect over an extended period of time. In order to maintain this constant level of drug in the body, the drug must be released from the dosage form at a rate that will replace the amount of drug being metabolized and excreted from the body. Controlled release of active ingredients can be stimulated by various physiological factors, including but not limited to pH, temperature, enzymes, water or other physiological diseases or conditions or compounds.

[0226] In some embodiments, the drug can be administered using intravenous infusion, implantable osmotic pumps, transdermal patches, liposomes, or other modes of administration. In some embodiments, a pump can 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 some embodiments, a polymer material can be used. In some embodiments, a controlled release system can be placed in a patient at an appropriate location determined by a person skilled in the art, i.e., only a small portion of the systemic dose is required (see, e.g., Goodson, Medical Applications of Controlled Release, Vol. 2, pp. 115-138 (1984)). Other controlled-release systems are described in the review by Langer (Science 249: 1527-1533 (1990)). The active ingredient can be dispersed in a solid 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 rubber, polydimethylsiloxane, silicone carbonate copolymers, hydrophilic polymers such as hydrogels of acrylic acid and methacrylate, collagen, cross-linked polyvinyl alcohol, and cross-linked partially hydrolyzed polyvinyl acetate, which are not The composition is soluble in body fluids such as polyethylene, polypropylene, ethylene / propylene copolymers, ethylene / ethyl acrylate copolymers, ethylene / vinyl acetate copolymers, silicone rubber, polydimethylsiloxane, chloroprene rubber, chlorinated polyethylene, polyvinyl chloride, vinyl chloride and vinyl acetate, vinylidene chloride, ethylene and propylene, ionomer polyethylene terephthalate, butyl rubber epichlorohydrin rubber, ethylene / vinyl alcohol copolymers, ethylene / vinyl acetate / vinyl alcohol terpolymers and ethylene / vinyloxyethanol. The active ingredient then diffuses through the outer polymer film in a release rate controlling step. The percentage of active ingredient in this parenteral composition is highly dependent on its characteristics and the needs of the patient.

[0227] Parenteral Dosage Forms

[0228] In some embodiments, parenteral dosage forms are provided. In some embodiments, parenteral dosage forms can be administered to a subject by various routes, including but not limited to subcutaneous, intravenous (including push injection), intramuscular and intra-arterial. In some embodiments, parenteral dosage forms can be administered to a subject by various routes, including but not limited to topical, intradermal or intralesional. Because their administration usually bypasses the natural defense of the subject to pollutants, parenteral dosage forms are usually sterile or can be disinfected before being administered to a subject. In some embodiments, parenteral dosage forms include but are not limited to, preparing solutions for injection, dry products that can be dissolved or suspended in pharmaceutically acceptable injection carriers, suspensions for injection and emulsions.

[0229] Suitable carriers that can be used to provide 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, ethanol, polyethylene glycol, and polypropylene glycol; and non-aqueous carriers such as, but not limited to, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate.

[0230] Compounds that increase the solubility of one or more of the active ingredients disclosed herein can also be incorporated into parenteral dosage forms.

[0231] Transdermal, topical and mucosal dosage forms

[0232] Transdermal, topical and mucosal dosage forms are also provided. Transdermal, topical and mucosal dosage forms include, but are not limited to, ophthalmic solutions, sprays, aerosols, creams, lotions, ointments, gels, solutions, emulsions, suspensions or other forms known to those skilled in the art. See, for example, Remington: The Science and Practice of Pharmacy; Pharmaceutical Press; 22nd edition (September 15, 2012); and Introduction to Pharmaceutical Dosage Forms, 4th edition, Lea & Febiger, Philadelphia (1985). Dosage forms suitable for treating oral mucosal tissues can be formulated as mouthwashes or oral gels. In addition, transdermal dosage forms include "reservoir-type" or "matrix-type" patches that can be applied to the skin and worn for a specific period of time to allow the penetration of the desired amount of active ingredients.

[0233] The term "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition or vehicle involved in carrying or transporting any subject composition or its components, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material. Each carrier must be "acceptable" in the sense of being compatible with the subject composition and its components and not harmful to the patient. Suitable carriers (such as excipients and diluents) and other materials that can be used to provide transdermal, topical and mucosal dosage forms included herein are well known to those skilled in the pharmaceutical art and depend on the specific tissue to which a given pharmaceutical composition or dosage form will be applied. In view of this fact, typical carriers include, but are not limited to, water, acetone, ethanol, ethylene glycol, propylene glycol, butane 1,3 diol, isopropyl myristate, isopropyl palmitate, mineral oil and mixtures thereof to form non-toxic and pharmaceutically acceptable lotions, tinctures, creams, emulsions, gels or ointments. In some embodiments, the materials that can be used as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) astragalus powder; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository wax; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn starch, etc. Rice oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerol, sorbitol, mannitol, polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethanol; (20) phosphate buffer solution; and (21) other non-toxic compatible substances used in pharmaceutical formulations. If desired, moisturizers or humectants can also be added to the pharmaceutical compositions and dosage forms. Examples of such additional ingredients are well known in the art. See, for example, Remington: The Science and Practice of Pharmacy; Pharmaceutical Press; 22nd edition (September 15, 2012).

[0234] Depending on the specific tissue to be treated, additional components may be used before, in combination with, or after treatment with the provided active ingredient. In some embodiments, a penetration enhancer may be used to assist in delivering the active ingredient to the tissue. Suitable penetration enhancers include, but are not limited to: acetone; various alcohols, such as ethanol, oleyl alcohol, and tetrahydrofuran methanol; alkyl sulfoxides, such as dimethyl sulfoxide; dimethylacetamide; dimethylformamide; polyethylene glycol; pyrrolidones, such as polyvinyl pyrrolidone; Kollidon grades (Povidone, Polyvidone); urea; and various water-soluble or insoluble sugar esters such as Tween 80 (Polysorbate 80) and Span 60 (Sorbitol Monostearate).

[0235] The pH value of pharmaceutical composition or dosage form can also be regulated, or the pH value of the tissue to which pharmaceutical composition or dosage form is applied, to improve the delivery of one or more active components. Similarly, the polarity of the solvent carrier, its ionic strength or tension (tonicity) can be regulated to improve delivery. Compounds such as stearate can also be added to pharmaceutical composition or dosage form, to advantageously change the hydrophilicity or lipophilicity of one or more active components, thereby improving delivery. In this respect, stearate can be used as the lipid carrier, emulsifier or surfactant of preparation and as delivery enhancement or penetration enhancer. Different salts, hydrates or solvates of active component can be used to further regulate the property of the resulting composition.

[0236] Dosage and unit dosage form

[0237] In human therapeutics, the physician will determine the posology he deems most appropriate, depending on the prophylactic or therapeutic treatment, and on the age, weight, stage of the condition or disease, and other specific factors specific to the subject to be treated. In some embodiments, the dosage is about 1 to about 1000 mg per day for an adult, or about 5 to about 250 mg per day for an adult, or about 10 to 50 mg per day. In some embodiments, the dosage is about 5 to about 400 mg per day for each adult, or 25 to 200 mg per day. In some embodiments, dosage rates of about 50 to about 500 mg per day are also contemplated.

[0238] In other aspects, there is provided a method for treating a disease or condition in a subject in need and / or a MEK inhibitor responsive condition or disease in a subject, a MEK inhibitor responsive skin condition or disease, a MEK mediated skin condition or disease, or a MEK mediated skin condition or disease by administering a therapeutic or preventive effective amount of a compound or a pharmaceutically acceptable salt thereof to a subject in need. The amount of a compound or composition having a therapeutic or preventive effect in treating a disease or one or more symptoms thereof will vary with the nature and severity of the disease or condition and the route of administration of the active ingredient. Frequency and dosage will also vary according to the specific factors of each subject, depending on the severity of a specific therapy (such as a therapeutic agent or preventive agent), a condition, a disease or condition, a route of administration, and the age, body, body weight, reaction, and past medical history of the subject. The effective dose can be inferred from a dose-response curve derived from an in vitro or animal model test system.

[0239] In some embodiments, exemplary dosages of compositions include milligrams or micrograms of active compound per kilogram of subject or sample weight (e.g., about 10 micrograms per kilogram to about 50 milligrams per kilogram, about 100 micrograms per kilogram to about 25 milligrams per kilogram, or about 100 micrograms per kilogram to about 10 milligrams per kilogram). For compositions provided herein, in some embodiments, the dosage administered to a subject is 0.140 mg / kg to 3 mg / kg of the subject's body weight, based on the weight of the active compound. In some embodiments, the dose administered to a subject is between 0.20 mg / kg and 2.00 mg / kg, between 0.30 mg / kg and 1.50 mg / kg, between 1 mg / kg and 100 mg / kg, between 5 mg / kg and 50 mg / kg, between 10 mg / kg and 50 mg / kg, between 20 mg / kg and 50 mg / kg, between 15 mg / kg and 40 mg / kg, between 15 mg / kg and 35 mg / kg, between 15 mg / kg and 30 mg / kg, between 25 mg / kg and 35 mg / kg, between 10 mg / kg and 30 mg / kg, between 10 mg / kg and 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.

[0240] In some embodiments, the recommended daily dose range of the composition for the diseases or conditions described herein provided herein is within the range of about 0.1 mg to about 1000 mg per day, given as a single dose once a day or divided doses throughout the day. In some embodiments, the daily dose is administered twice a day in equal divided doses. In some embodiments, the daily dose range should be about 10 mg to about 200 mg per day, in some embodiments, about 10 mg to about 150 mg per day, and in other embodiments, about 25 mg to about 100 mg per day. In some cases, it may be necessary to use a dose of the active ingredient outside the range disclosed herein, which will be apparent to those of ordinary skill in the art. In addition, it should be noted that the clinician or treating physician will know how and when to interrupt, adjust or terminate treatment in conjunction with patient response.

[0241] Different therapeutically effective amounts may be applicable to different diseases and conditions, as those of ordinary skill in the art readily know. Similarly, amounts sufficient to prevent, control, treat or improve such conditions, but not sufficient to cause or to reduce the adverse effects associated with the compositions provided herein, are also included in the dosage amounts and dosage frequency tables described herein. In addition, when multiple doses of the compositions provided herein are administered to an object, not all doses need to be the same. In some embodiments, the dose given to an object can be increased to improve the preventive or therapeutic effect of the composition, or can be reduced to reduce one or more side effects that a particular object is experiencing.

[0242] In some embodiments, the dosage of the compositions provided herein administered to prevent, treat, manage, or improve a disorder or one or more symptoms thereof in a subject is 0.1 mg / kg, 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 10 mg / kg, or 15 mg / kg or more of the subject's body weight, based on the weight of the active compound. In some embodiments, the dosage of the composition or the composition provided herein for administration to prevent, treat, control or ameliorate a disease or one or more symptoms thereof in a subject is a unit dose of 0.1 mg to 200 mg, 0.1 mg to 100 mg, 0.1 mg to 50 mg, 0.1 mg to 25 mg, 0.1 mg to 20 mg, 0.1 mg to 15 mg, 0.1 mg to 10 mg, 0.1 mg to 7.5 mg, 0.1 mg to 5 mg, 0.1 to 2.5 mg, 0.25 mg to 20 mg, 0.25 to 15 mg, 0.25 to 12 mg, 0.25 to 10 mg, 0.25 mg to 7.5 mg, 0.25 mg to 5 mg, 0.5 mg to 2.5 mg, 1 mg to 20 mg, 1 mg to 15 mg, 1 mg to 12 mg, 1 mg to 10 mg, 1 mg to 7.5 mg, 1 mg to 5 mg, or 1 mg to 2.5 mg.

[0243] In some embodiments, treatment or prevention can be started 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 can be, for example, about 60 to about 400 mg per day, or about 100 to about 200 mg per day, for 1 day to 5 weeks. The loading dose can be followed by one or more maintenance doses. In some embodiments, each maintenance dose is independently about 10 mg to about 200 mg per day, about 25 mg and about 150 mg per day, or about 25 and about 80 mg per day. The maintenance dose can be administered daily, either as a single dose or as divided doses.

[0244] In some embodiments, the dosage of compound or composition provided herein can be applied to achieve the steady-state concentration of active ingredient in the patient's blood or serum. Steady-state concentration can be measured according to the available technology of technicians or can be determined based on the patient's physical characteristics (such as height, weight and age). In some embodiments, a sufficient amount of compound or composition provided herein is applied to achieve a steady-state concentration of about 300 to about 4000ng / mL, about 400 to about 1600ng / mL or about 600 to about 1200ng / mL in the patient's blood or serum. In some embodiments, a loading dose can be applied to obtain a steady-state blood or serum concentration of about 1200 to about 8000ng / mL or about 2000 to about 4000ng / mL in 1 to 5 days. In some embodiments, a maintenance dose can be applied to obtain a steady-state concentration of about 300 to about 4000ng / mL, about 400 to about 1600ng / mL or about 600 to about 1200ng / mL in the patient's blood or serum.

[0245] In some embodiments, the same composition can be administered repeatedly, and the administration can be separated by at least 1 day, 2 days, 3 days, 5 days, 10 days, 15 days, 30 days, 45 days, 2 months, 75 days, 3 months or 6 months. In some embodiments, the same preventive or therapeutic agent can be administered repeatedly, and the administration can be separated by at least 1 day, 2 days, 3 days, 5 days, 10 days, 15 days, 30 days, 45 days, 2 months, 75 days, 3 months or 6 months.

[0246] In some aspects, provided herein is a unit dose comprising a compound or a pharmaceutically acceptable salt thereof in a form suitable for administration. These forms are described in detail herein. In some embodiments, the unit dose comprises an active ingredient of 1 to 1000 mg, 5 to 250 mg, or 10 to 50 mg. In a specific embodiment, the unit dose comprises an active ingredient of about 1, 5, 10, 25, 50, 100, 125, 250, 500, or 1000 mg. Such a unit dose can be prepared according to techniques familiar to those skilled in the art.

[0247] The dosage may vary within a range depending on the dosage form employed and the route of administration utilized. For any compound, the therapeutically effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a certain level in the skin with lesions such as cutaneous neurofibromas, subcutaneous neurofibromas, or superficial plexiform neurofibromas, including IC values ​​determined in cell culture. 50 (i.e., the concentration of the test compound that achieves a half-maximal inhibition of symptoms). Such information can be used to more accurately determine useful doses in humans. In addition, levels in plasma can be measured, for example, by high performance liquid chromatography to determine systemic exposure.

[0248] It will also be understood that the specific dosage and treatment regimen for any particular patient will depend on a variety of factors, including the activity of the specific compound used, age, size of lesions, number of lesions, general health, sex, diet, time of administration, drug combination, and the judgment of the treating physician and the severity of the specific disease being treated. The amount of a soft MEK inhibitor (such as a soft MEK inhibitor described herein) in the composition will also depend on the specific soft MEK inhibitor in the composition.

[0249] In some embodiments, the topical dose is about 0.01 μg / cm 2 , about 0.05μg / cm 2 , about 0.1μg / cm 2 , about 0.15μg / cm 2 , about 0.2μg / cm 2 , about 0.3μg / cm 2 , about 0.4μg / cm 2 , about 0.5μg / cm 2 , about 0.6μg / cm 2 , about 0.7μg / cm 2 , about 0.8μg / cm 2 , or about 0.9 μg / cm 2 ; or about 0.01-0.03 μg / cm 2 , about 0.03-0.05μg / cm 2, about 0.05-0.1μg / cm 2 , about 0.1-0.3μg / cm 2 , about 0.3-0.5μg / cm 2 , about 0.5-0.8μg / cm 2 , about 0.8-1.0μg / cm 2 , about 1-10μg / cm 2 , about 10-20μg / cm 2 , about 20-30μg / cm 2 , about 30-40μg / cm 2 , about 40-50μg / cm 2 , about 50-60μg / cm 2 , about 60-70μg / cm 2 , about 70-80μg / cm 2 , about 80-90μg / cm 2 , about 90-100 μg / cm 2 , about 100-125μg / cm 2 , about 125-150μg / cm 2 , about 150-175μg / cm 2 , about 175-200 μg / cm 2 , about 200-250μg / cm 2 , about 250-300μg / cm 2 , about 300-350μg / cm 2 , about 350-400μg / cm 2 , about 400-450μg / cm 2 , about 450-500μg / cm 2 , about 500-550μg / cm 2 , about 550-600μg / cm 2 , about 600-650μg / cm 2 , about 650-700μg / cm 2 , about 700-750μg / cm 2 , about 750-800μg / cm 2 , about 800-850μg / cm 2 , about 850-900μg / cm 2 , about 900-950μg / cm 2 or about 950-1000 μg / cm 2 within the range.

[0250] In some embodiments, the topical dose is about 0.5-1.0 mg / cm 21.0-1.5mg / cm 2 1.5-2.0mg / cm 2 2.5-2.5mg / cm 2 3.0-3.5mg / cm 2 , 3.5-5.0mg / cm 2 , 5.0-7.5mg / cm 2 , 7.5-10mg / cm 2 , 1-10mg / cm 2 , about 10-20mg / cm 2 , about 20-30mg / cm 2 , about 30-40mg / cm 2 , about 40-50mg / cm 2 , about 50-60mg / cm 2 , about 60-70mg / cm 2 , about 70-80mg / cm 2 , about 80-90mg / cm 2 , about 90-100mg / cm 2 , about 100-125mg / cm 2 , about 125-150mg / cm 2 , about 150-175mg / cm 2 , about 175-200mg / cm 2 , about 200-250mg / cm 2 , about 250-300mg / cm 2 , about 300-350mg / cm 2 , about 350-400mg / cm 2 , about 400-450mg / cm 2 , about 450-500mg / cm 2 , about 500-550mg / cm 2 , about 550-600mg / cm 2 , about 600-650mg / cm 2 , about 650-700mg / cm 2 , about 700-750mg / cm 2 , about 750-800mg / cm 2 , about 800-850mg / cm 2 , about 850-900mg / cm 2 , about 900-950mg / cm 2 or about 950-1000mg / cm 2 within the range.

[0251] V. Kit

[0252] Also provided is a kit for treating a MEK inhibitor responsive condition or disease, a MEK inhibitor responsive skin condition or disease, a MEK-mediated condition or disease, or a MEK-mediated skin condition or disease in a method for treating a MEK inhibitor responsive condition or disease, a MEK inhibitor responsive skin condition or disease, a MEK-mediated condition or disease, or a MEK-mediated skin condition or disease in a subject in need thereof; or for a MEK inhibitor responsive condition or disease, a MEK inhibitor responsive skin condition or disease, a MEK-mediated condition or disease, or a MEK-mediated skin condition or disease. The kit may include a compound or composition provided herein, a second agent or composition, and instructions, wherein the instructions provide information about the usage of treating a MEK inhibitor responsive condition or disease, a MEK inhibitor responsive skin condition or disease, a MEK-mediated condition or disease, or a MEK-mediated skin condition or disease to a medical service staff. The instructions may be provided in a printed form, or in the form of an electronic medium such as a floppy disk, a CD, or a DVD, or in the form of a website address from which the instructions may be obtained. The unit dose of the compound or composition or the second agent or composition provided herein may include such a dose, that is, when administered to a subject, the treatment or prevention effective plasma level of the compound or composition may be maintained in the subject for at least 1 day. In some embodiments, the compound or composition can be provided as a sterile aqueous pharmaceutical composition or a dry powder (eg, lyophilized) composition.

[0253] In some embodiments, suitable packaging is provided. As used herein, "packaging" includes solid substrates or materials that are generally used in systems and can keep the compound provided herein and / or the second medicament suitable for administration within fixed limits. These materials include glass and plastic (such as polyethylene, polypropylene and polycarbonate) bottles, vials, paper, plastics and plastic foil laminated envelopes, etc. If electron beam sterilization technology is used, packaging should have a sufficiently low density to allow the contents therein to be sterilized.

[0254] VI. Methods

[0255] In a third aspect, a method for treating a disease or condition in a subject in need thereof is provided herein, and the disease or condition is a MEK inhibitor responsive condition or disease in the subject, a MEK inhibitor responsive skin condition or disease, a MEK mediated condition or disease, or a MEK mediated skin condition or disease. The method comprises administering to the subject a therapeutically or prophylactically effective amount of a compound disclosed herein, such as a compound of formula (I) and a compound in Table 1 and Table 2, including a single enantiomer thereof, a mixture of enantiomeric pairs, a single diastereomer, a mixture of diastereomers, a single stereoisomer, a mixture of stereoisomers, or a tautomeric form; or a pharmaceutically acceptable salt, solvate, prodrug, phosphate, or active metabolite thereof.

[0256] In some embodiments, the method comprises administering to the subject a therapeutically effective amount of a compound of formula (I) and a compound in Table 1 and Table 2, including a single enantiomer, a mixture of enantiomer pairs, a single diastereomer, a mixture of diastereomers, a single stereoisomer, a mixture of stereoisomers, or a tautomeric form thereof; or a pharmaceutically acceptable salt or solvate thereof. In some embodiments, the method comprises administering to the subject a therapeutically effective amount of a compound of formula (I) and a compound in Table 1, including a single enantiomer, a mixture of enantiomer pairs, a single diastereomer, a mixture of diastereomers, a single stereoisomer, a mixture of stereoisomers, or a tautomeric form thereof; or a pharmaceutically acceptable salt or solvate thereof.

[0257] In some embodiments, the MEK inhibitor-responsive skin disorder or MEK-mediated skin disorder is selected from the group consisting of cutaneous RASopathy, neurofibromatosis type 1, cutaneous neurofibroma, subcutaneous neurofibroma, and superficial plexiform neurofibroma.

[0258] In some embodiments, the MEK inhibitor-responsive skin disorder or MEK-mediated skin disorder is neurofibromatosis type 1.

[0259] In some embodiments, administration comprises contacting the soft MEK inhibitor with the skin, mucous membrane, vagina, penis, larynx, vulva, cervix or anus of the subject by topical or non-systemic application of the soft MEK inhibitor (e.g., topical, intradermal, or intralesional application) or by suppository application of the soft MEK inhibitor.

[0260] In some embodiments, a tumor associated with neurofibromatosis type 1 (NF1), such as a cutaneous neurofibroma, a subcutaneous neurofibroma, or a superficial plexiform neurofibroma, is reduced, such as by at least about 15% (e.g., about 15% to about 60%) in size or total volume relative to a reference standard, thereby treating the subject. In some embodiments, the reference standard is, for example, an untreated control tumor size or total volume from the same subject or a different subject.

[0261] In some embodiments, the size or total tumor volume of a tumor associated with neurofibromatosis type 1 (NF1), such as a cutaneous neurofibroma, a subcutaneous neurofibroma, or a superficial plexiform neurofibroma, is reduced by at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60% relative to a reference standard. In some embodiments, the reference standard is an untreated control tumor size or total volume, for example from the same subject or a different subject.

[0262] In some embodiments, the method comprises evaluating the subject using magnetic resonance imaging (MRI) or optical imaging, for example, to evaluate the volume of a tumor obtained from the subject before, during, and / or after treatment.

[0263] Neurofibromatosis type 1 (NF1): In some embodiments, the skin condition is associated with NF1. NF1, also known as von Recklinghausen Neurofibromatosis or peripheral neurofibromatosis, occurs in approximately 1:3000 newborns and is the most prevalent genetic disease and one of the most common neurocutaneous diseases. NF1 is caused by a deficiency of neurofibromin, which leads to overactivation of various cell signaling pathways (such as Ras and Rho) and is associated with several skin conditions, including cutaneous neurofibromas (DF); subcutaneous neurofibromas; superficial plexiform neurofibromas (PF); cutaneous neurofibromas (CF); café-au-lait spots; and freckles of the armpits and groin. More than 95% of NF1 patients develop DF. DF can appear anywhere on the body, and 88% of NF1 patients over the age of 40 have more than 100 DF. DF can cause severe physical pain, disfigurement, and social anxiety. Facial DF can produce significant social anxiety issues and pain in affected individuals. DF (also known as cutaneous neurofibroma or discrete neurofibroma) grows from small nerves in or under the skin and appears as a small bump, typically beginning during adolescence. Current treatment options for DF are limited to surgical excision and CO2 laser removal, both of which cause scarring and are not preventive.

[0264] Other cutaneous RAS diseases: In some embodiments, the skin disease is associated with increased activation of Ras. In some embodiments, the cutaneous RAS disease is selected from the group consisting of psoriasis, keratocanthoma (KA), hyperkeratosis, papilloma, Noonan syndrome (NS), cardiofacial skin syndrome (CFC), Krustylo syndrome (facial skin skeletal syndrome or FCS syndrome), ocular ectodermal syndrome, cafe au lait spots, and multiple lentigines syndrome (formerly known as leopard syndrome).

[0265] In some or any embodiments, the disease to be reduced, ameliorated, treated or prevented is not cancer (eg, melanoma).

[0266] In some embodiments, the disease to be reduced, ameliorated, treated or prevented is cancer, cutaneous RASopathy, skin diseases associated with neurofibromatosis type 1, cutaneous neurofibroma, subcutaneous neurofibroma or superficial plexiform neurofibroma, psoriasis, keratoacanthoma (KA), hyperkeratosis, papilloma, Noonan syndrome (NS), cardiofacial skin syndrome (CFC), Costillo syndrome (faciocutaneous skeletal syndrome or FCS syndrome), ocular ectodermal syndrome, café au lait spots, and multiple lentigo syndrome (formerly known as leopard skin syndrome).

[0267] In some embodiments, the disease to be reduced, improved, treated or prevented is cancer. In some embodiments, the disease to be reduced, improved, treated or prevented is selected from the group consisting of basal cell carcinoma, squamous cell carcinoma, actinic keratosis, Kaposi's sarcoma, cutaneous lymphoma, cervical cancer, HPV-related squamous cell carcinoma and melanoma.

[0268] In some embodiments, the disease to be reduced, ameliorated, treated or prevented is a cutaneous RASopathy, a skin disease associated with neurofibromatosis type 1, cutaneous neurofibroma, subcutaneous neurofibroma or superficial plexiform neurofibroma, psoriasis, keratoacanthoma (KA), hyperkeratosis, papilloma, Noonan syndrome (NS), cardiofacial skin syndrome (CFC), Costillo syndrome (faciocutaneous skeletal syndrome or FCS syndrome), ocular ectodermal syndrome, café au lait spots, and multiple lentigo syndrome (formerly known as leopard skin syndrome).

[0269] In some embodiments, the compounds described herein are used to reduce a MEK inhibitor-responsive condition or disease, a MEK inhibitor-responsive skin condition or disease, a MEK-mediated condition or disease, or a MEK-mediated skin condition or disease in a subject in need thereof.

[0270] In some embodiments, the compounds described herein are used to alleviate a MEK inhibitor-responsive condition or disease, a MEK inhibitor-responsive skin condition or disease, a MEK-mediated condition or disease, or a MEK-mediated skin condition or disease in a subject in need thereof.

[0271] In some embodiments, the compounds described herein are used to prevent a MEK inhibitor-responsive disorder or disease, a MEK inhibitor-responsive skin disorder or disease, a MEK-mediated disorder or disease, or a MEK-mediated skin disorder or disease in a subject in need thereof.

[0272] In some embodiments, the compounds described herein are used to treat a MEK inhibitor-responsive disorder or disease, a MEK inhibitor-responsive skin disorder or disease, a MEK-mediated disorder or disease, or a MEK-mediated skin disorder or disease in a subject in need thereof.

[0273] Analytical methods

[0274] The efficacy of a compound in treating a MEK inhibitor-responsive condition or disease, a MEK inhibitor-responsive skin condition or disease, a MEK-mediated condition or disease, or a MEK-mediated skin condition or disease in a subject in need thereof can be determined according to any assay known to those of skill in the art. Exemplary assays are provided elsewhere herein.

[0275] VII. Combination therapy

[0276] In some embodiments, the compounds and compositions provided herein can be used for treating a MEK inhibitor-responsive condition or disease, a MEK inhibitor-responsive skin condition or disease, a MEK-mediated condition or disease, or a MEK-mediated skin condition or disease in a subject in need thereof, the method comprising further administering a second agent effective for treating the skin condition or disease. The second agent can be any agent known to those skilled in the art to be effective for treating a skin condition or disease, including agents currently approved by the U.S. Food and Drug Administration or other similar agencies in countries outside the United States.

[0277] In some embodiments, the compounds provided herein are administered in combination with one second agent. In further embodiments, the compounds provided herein are administered in combination with two second agents. In still further embodiments, the compounds provided herein are administered in combination with two or more second agents.

[0278] In some embodiments, the method includes the steps of administering to a subject in need thereof an amount of a compound effective to treat a MEK inhibitor responsive condition or disease, a MEK inhibitor responsive skin condition or disease, a MEK mediated condition or disease, or a MEK mediated skin condition or disease, in combination with a second agent effective to treat or prevent a MEK inhibitor responsive condition or disease, a MEK inhibitor responsive skin condition or disease, a MEK mediated condition or disease, or a MEK mediated skin condition or disease in a subject in need thereof. The compound can be any compound described herein, and the second agent can be any second agent described in the art or herein. In some embodiments, the compound is in the form of a pharmaceutical composition or dosage form, as described elsewhere herein.

[0279] As used herein, the term "combination" includes the use of more than one therapy (e.g., one or more preventive and / or therapeutic agents). The use of the term "combination" does not limit the order in which treatment (e.g., preventive and / or therapeutic agents) are given to objects with diseases. The first therapy (e.g., preventive or therapeutic agents such as compounds provided herein) can be administered to objects with diseases before the second agent (e.g., a preventive or therapeutic agent) is administered (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks ago), while administering the second agent or after administering the second agent (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks later).

[0280] As used herein, the term "synergistic" includes a combination of a compound provided herein and another therapy (such as a preventive or therapeutic agent) that has been or is being used to prevent, control or treat a disease, which is more effective than the additive effect of these therapies. The synergistic effect of a combination therapy (such as a combination of a preventive or therapeutic agent) allows the use of one or more therapies at lower doses and / or less frequent administration of the therapy to subjects suffering from the condition. The use of a lower dose of therapy (such as a preventive or therapeutic agent) and / or less frequent administration of the therapy reduces the toxicity associated with administering the therapy to the subject without reducing the efficacy of the therapy in preventing or treating the disease). In addition, a synergistic effect can result in improved efficacy of the agent in preventing or treating a condition. Finally, a synergistic therapy (such as a combination of preventive or therapeutic agents) can avoid or reduce adverse or undesirable side effects associated with the use of any one therapy alone.

[0281] The active compounds provided herein can be combined or administered alternately with another therapeutic agent, particularly a drug that is effective in treating a MEK inhibitor-responsive condition or disease, a MEK inhibitor-responsive skin condition or disease, a MEK-mediated condition or disease, or a MEK-mediated skin condition or disease in a subject in need thereof. In a combined therapy, effective doses of two or more agents are administered together, while in an alternating or sequential step therapy, effective doses of each agent are administered continuously or sequentially. The dose administered depends on the absorption, inactivation, and excretion rate of the drug and other factors known to those skilled in the art. It should be noted that the dose value will also vary with the severity of the MEK inhibitor-responsive condition or disease, MEK inhibitor-responsive skin condition or disease, MEK-mediated condition or disease, or MEK-mediated skin condition or disease condition to be alleviated. It should be further understood that for any particular subject, a specific dosage regimen and schedule should be adjusted over time according to individual needs and the professional judgment of the person managing or supervising the application of the composition.

[0282] In some embodiments, the dosage of the second agent used in the combination therapy is provided herein. In some embodiments, the dosage used in the combination therapy provided herein is lower than the dosage that has been or is currently used to treat MEK inhibitor responsiveness or disease, MEK inhibitor responsiveness skin disorder or disease, MEK mediated disorder or disease, or MEK mediated skin disorder or disease. The recommended dose of the second agent can be obtained from the knowledge of those skilled in the art. For those second agents approved for clinical use, the recommended dose is in, for example, Hardma et al., ed., 1996, Goodman & Gilman The Pharmacological Basis Of Therapeutics, Ninth Edition, Mc-Graw-Hill, New York; Physician's Desk Reference (PDR) 57th Edition, 2003, Medical Economics Co., Inc., Montville, New Jersey; All of which are incorporated herein by reference.

[0283] The present disclosure provides a combination therapy by administering the soft MEK inhibitor described herein together with one or more additional agents. In some embodiments, the one or more additional agents are selected from:

[0284] acne medications (such as Accutane, azelaic acid, benzoyl peroxide, salicylic acid);

[0285] Analgesics (e.g., acetaminophen, capsaicin), for example Cox2 inhibitors such as celecoxib);

[0286] Anesthetics (such as benzocaine, benzocaine / menthol, dibucaine, diperodon, lidocaine, lidocaine / prilocaine, pramoxine);

[0287] Anti-infective drugs (such as Crotamiton);

[0288] Antipruritic drugs (eg, ammonium lactate, benzocaine, ascomycin macrolactams such as pimecrolimus);

[0289] Antipruritic drugs / 5HT3 receptor antagonists (such as ondansetron);

[0290] Antibiotics (such as clindamycin, doxycycline, erythromycin, tetracycline);

[0291] Antiemetics with anti-sympathetic nervous system physiological effects (such as diphenhydramine);

[0292] Antifibrotic drugs (such as collagenase, pirfenidone);

[0293] Antihistamines (such as triprolidine Fexofenadine ( D-12, -24), Astepro / Astelin nasal spray (Azalastine) Hydroxyzine hydrochloride Diphenhydramine hydrochloride Brompheniramine ( Cold and allergic reaction elixirs), (Cetirizine), (Chlorpheniramine), Descoratadine ( D-12, D-24), loratadine ( D-12, D-24 and ), Dimenhydrinate Diphenhydramine ( allergy, ), Doxylamine Strengthen nighttime cold medicine), cyproheptadine Promethazine Acrivastine Clemastine Doxylamine Levoceterizine

[0294] Mast cell stabilizers (eg, β2-adrenergic agonists, cromolyn, cromolyn sodium, Ketotifen, methylxanthines, omalizumab, pemirolast, quercetin, ketotifen );

[0295] Anti-inflammatory drugs (eg, NSAIDs (eg, aspirin, choline, magnesium salicylate, diclofenac potassium) Diclofenac sodium ( XR), diclofenac sodium and misoprostol Diflunisal Etodolac XL), Fenoprofen Calcium Flurbiprofen Ibuprofen IB Indomethacin ( SR), ketoprofen ( KT, ), magnesium salicylate (Arthrita, Choose, Doan'pill, Magan, Mobidin, Mobogesic, meclofenamic acid sodium Mefenamic acid (Mefenamic acid ), Meloxicam Nabumetone Naproxen Naproxen Sodium Oxaprozin Piroxicam Rofecoxib Salsalate (Amigesic, Anaflex 750, Disalcid, Marthritic, Mono-Gesic, Salflex, Salsitab), sodium salicylate, sulindac Tolmetin Sodium Valdecoxib );

[0296] Receptor tyrosine kinase inhibitors (such as sunitinib);

[0297] Alkylating agents (eg, dacarbazine, carboplatin);

[0298] CDK4 / 6 inhibitors (such as LEE011);

[0299] PKC inhibitors (such as AEB071);

[0300] MAPK inhibitors (such as RAS inhibitors / farnesyl transferase inhibitors (such as Tipifarnib), Raf kinase inhibitors (such as sorafenib (BAY43-9006, Nexavar), Vemurafenib, Dabrafenib, LGX818, TAK-632, MLN2480, PLX-4720), ERK inhibitors (such as SCH772984, VTX11e);

[0301] PI3K inhibitors (such as LY294002);

[0302] AKT inhibitors (such as MK 2206);

[0303] PI3K / Akt inhibitors (such as buparlisib, cixutumumab);

[0304] mTOR inhibitors (eg, topical rapamycin, RAD001 (everolimus / rapamycin), temsirolimus, sirolimus);

[0305] Tyrosine kinase inhibitors (eg, imatinib Cabozantinib (tyrosine kinase c-Met and VEGFR2 inhibitor), nilotinib

[0306] VEGF inhibitors (such as ranibizumab) Cediranib);

[0307] Immune response modifiers (eg, topical imiquimod, interferon, PEG interferon);

[0308] Calcium channel blockers (such as Avocil (Mederma) / 15% verapamil, vitamin D alone, doxycycline injection);

[0309] Statins (such as lovastatin, methotrexate, vinblastine, pregabalin, temozolomide, PLX3397);

[0310] HDAC inhibitors (such as AR-42);

[0311] HSP-90 inhibitors (such as glatiramer (Ganetespib));

[0312] Retinoids (such as adapalene, isotretinoin, tazarotene, tretinoin);

[0313] Steroids (e.g., alclomethasone, amcinonide, betamethasone, betamethasone dipropionate, augmented betamethasone dipropionate, budesonide, clobetasol propionate, cortisone, desonide, dexamethasone, diflorasone diacetate, fluocinoloneacetonide, fluocinonide, flurandrenolide, fluticasone propionate, halobetasol propionate, propionate), Halocinonide, hydrocortisone, hydrocortisone butyrate, hydrocortisone valerate, methylprednisolone, mometasone, mometasone furoate, prednicarbate, prednisolone, prednisone, triamcinolone acetonide);

[0314] Topical calcineurin inhibitors (such as pimecrolimus (Aninda) Cream 1%, Novartis, tacrolimus (Protopic ointment, Astellas); and

[0315] Non-drug interventions (such as photodynamic therapy (Levulan Kerastick topical + phototherapy), electrodessication (ED), YAG laser).

[0316] In various embodiments, the therapy (e.g., a compound and a second agent as provided herein) is administered at least 5 minutes apart, at least 30 minutes apart, at least 1 hour apart, about 1 hour apart, about 1 hour apart, about 2 hours apart, about 3 hours apart, about 4 hours apart, about 4 hours apart, about 5 hours apart, about 6 hours apart, about 7 hours apart, about 8 hours apart, about 9 hours apart, about 10 hours apart, about 10 hours apart, about 11 hours apart, about 12 hours apart, about 12 hours apart, 18 hours apart, 24 hours apart, 36 hours apart, 36 hours apart, 48 hours apart, 52 hours apart, 60 hours apart, 72 hours apart, 84 hours apart, 84 hours apart, 96 hours apart, or 6 hours apart. In various embodiments, the therapy is administered no more than 24 hours apart or no more than 48 hours apart. In some embodiments, two or more therapies are administered in the same patient visit. In some embodiments, a compound provided herein and a second agent are administered simultaneously.

[0317] In some embodiments, the compound provided herein and the second agent are administered about 2 to 4 days apart, about 4 to 6 days apart, about 1 week apart, about 1 to 2 weeks apart, or more than 2 weeks apart.

[0318] In some embodiments, the same agent can be administered repeatedly, and the administration can be separated by at least 1 day, 2 days, 3 days, 5 days, 10 days, 15 days, 30 days, 45 days, 2 months, 75 days, 3 months, or 6 months. In some embodiments, the same agent can be administered repeatedly, and the administration can be separated by at least 1 day, 2 days, 3 days, 5 days, 10 days, 15 days, 30 days, 45 days, 2 months, 75 days, 3 months, or 6 months.

[0319] In some embodiments, provided herein are compounds and second agents administered to patients in order and at intervals, and in some embodiments, administered to mammals, such as humans, so that provided herein are compounds that can act with other agents to provide increased benefits than when administered in other ways. In some embodiments, the second active agent can be administered simultaneously or in any order in sequence at different times in a timely manner; however, if not administered simultaneously, they should be administered close enough in time to provide the desired treatment or preventive effect. In some embodiments, provided herein are compounds and second active agents that exert their effects at overlapping times. Each second active agent can be administered separately in any appropriate form and by any appropriate route. In some embodiments, provided herein are compounds administered before, simultaneously, or after administration of the second active agent.

[0320] In some embodiments, provided herein is a compound and a second agent administered to a patient in a cycle. Cyclic therapy includes administering a first agent (such as a first preventive or therapeutic agent) for a period of time, followed by administration of a second agent and / or a third agent (such as a second and / or third preventive or therapeutic agent) for a period of time and repeating the order of administration. Cyclic therapy can reduce the development of resistance to one or more therapies, avoid or reduce the side effects of a therapy, and / or improve the efficacy of treatment.

[0321] In some embodiments, provided herein is a compound and a second active agent with a cycle of less than about 3 weeks, about once every two weeks, about once every 10 days, or about once a week. A cycle can include administering a compound and a second agent provided herein by an infusion of about 90 minutes per cycle, about 1 hour per cycle, and about 45 minutes per cycle. Each cycle can include a pause of at least 1 week, a pause of at least 2 weeks, and a pause of at least 3 weeks. The number of cycles of administration is about 1 to about 12 cycles, more typically about 2 to about 10 cycles, and more typically about 2 to about 8 cycles.

[0322] In some embodiments, the patient receives treatment of different courses at the same time, i.e., a single dose of a second agent is administered separately at a certain time interval, so that the compound provided herein can act together with the second active agent. In some embodiments, a component can be administered once a week in combination with other components that can be administered once every two weeks or every three weeks. In other words, even if it is not administered at the same time or on the same day, the administration method is also carried out simultaneously.

[0323] The second agent may be additive or synergistic with the compound provided herein. In some embodiments, the compound provided herein is administered simultaneously with one or more second agents in the same pharmaceutical composition. In some embodiments, the compound provided herein is administered simultaneously with one or more second agents in a separate pharmaceutical composition. In some embodiments, the compound provided herein is administered before or after the use of the second agent. It is also contemplated that the compound provided herein and the second agent are administered by the same or different routes of administration (such as oral and parenteral). In some embodiments, when the compound provided herein is administered simultaneously with a second agent that potentially produces adverse side effects including but not limited to toxicity, the second active agent may advantageously be administered at a dose lower than the threshold value that causes adverse side effects.

[0324] VIII. Examples

[0325] General synthetic method

[0326] Compounds provided herein can be prepared, separated or obtained by any method apparent to those skilled in the art. Compounds provided herein can be prepared according to the exemplary preparation schemes provided below. Reaction conditions, steps and reactants not provided in the exemplary preparation schemes are apparent to those skilled in the art and are known. As used herein, the symbols and conventions used in these processes, schemes and embodiments, whether or not specific abbreviations are specifically defined, are consistent with the symbols and conventions used in contemporary scientific literature (e.g., Journal of the American Chemical Society or Journal of Biological Chemistry). Specifically, but not limited to, the following abbreviations may be used in the examples and throughout the specification: g (gram); mg (milligram); mL (milliliter); μL (microliter); mM (millimole); μM (micromolar); Hz (hertz); MHz (megahertz); mmol (millimole); hr or hrs (hours); min (minutes); MS (mass spectrometry); ESI (electrospray ionization); TLC (thin layer chromatography); HPLC (high performance liquid chromatography); THF (tetrahydrofuran); CDCl3 (deuterated chloroform); AcOH (acetic acid); DCM (dichloromethane); DMSO (dimethyl sulfoxide); DMSO-d6 (deuterated dimethyl sulfoxide); EtOAc (ethyl acetate); MeOH (methanol); Tces (2,2,2-trichloroethoxysulfonyl); -Si(tert-Bu)(Ph)2 and -Si t BuPh2 (tert-butyl-diphenylsilyl); and BOC (tert-butyloxycarbonyl).

[0327] For all the following examples, standard processing and purification methods known to those skilled in the art can be used. Unless otherwise stated, all temperatures are expressed in ° C (degrees Celsius). Unless otherwise stated, all reactions are carried out at room temperature. The synthetic methods shown herein are intended to illustrate applicable chemistry by using specific examples and do not indicate the scope of the present disclosure.

[0328] The compound of formula (Ia) can be Figure 1 The method is shown in Scheme 1, wherein R 1 For-OR 4 、-NR 5 R 5a 、-N(OR 5b )R 5a , or unsubstituted or replaced by one or two R 6 substituted N-linked heterocycloalkyl; and X, R 2 , R 2a , R 3 , R 3a , R 4 , R 5 , R 5a , R 5b and R6 As defined in any aspect, embodiment or claim described herein. In some embodiments, R 3 and R 3a Each is hydrogen.

[0329] Compounds of formula (103) may be prepared by methods apparent to those skilled in the art starting with commercially available or conventionally available acids of formula (101) and commercially available or conventionally available anilines of formula (102). Acid compounds of formula (103) may be activated with a variety of reagents apparent to those skilled in the art to produce a compound having a suitable leaving group attached to the carbonyl of the C(O)OH acid group, such as an acid chloride produced by reaction of (103) with thionyl chloride or an active ester produced by reaction of (103) with a reagent such as EDCI or HOBt. The acid chloride or active ester may then be reacted with a molecule of formula R 4 OH, HNR 5 R 5a 、HN(OR 5b )R 5a , R a -H (where R a is unsubstituted or replaced by one or two R 6 A compound of formula (Ia) is reacted with a substituted N-linked heterocycloalkyl) or a suitably protected form thereof to prepare a compound of formula (Ia).

[0330] The compound of formula (Ia) can be Figure 2 The method is shown in Scheme 2, wherein R 1 For-OR 4 、-NR 5 R 5a 、-N(OR 5b )R 5a , or unsubstituted or replaced by one or two R 6 Substituted N-linked heterocycloalkyl; R 2 is iodine; R 3b is fluorine; and R 2a , R 3 , R 3a , R 4 , R 5 , R 5a , R 5b and R 6 As defined in any aspect, embodiment or claim described herein. In some embodiments, R 3 and R 3a Each is hydrogen.

[0331] Compounds of formula (106) may be prepared by methods apparent to one skilled in the art starting with a commercially available or conventionally available acid of formula (104) and a commercially available or conventionally available aniline of formula (105). Compounds of formula (106) are then treated with Zn(CN)2 in the presence of a catalyst such as Pd(PPh3)4. Compounds of formula (108) are prepared by treating (107) in the presence of silver trifluoroacetate or, alternatively, with iodine monochloride. Acid compounds of formula (108) may be activated with a variety of reagents apparent to one skilled in the art to produce a compound having a suitable leaving group attached to the carbonyl of the C(O)OH acid group, such as an acyl chloride produced by reacting (108) with thionyl chloride or an active ester produced by reacting (108) with a reagent such as EDCI or HOBt. The acyl chloride or active ester may then be reacted with a precipitate of formula R 4 OH, HNR 5 R 5a 、HN(OR 5b )R 5a , R a -H (where R a is unsubstituted or replaced by one or two R 6 substituted N-linked heterocycloalkyl) or a suitable protected form thereof to prepare a compound of formula (Ia), wherein R 2 is iodine and R 3 For fluorine.

[0332] The compound of formula (Ib) can be Figure 3 The method is shown in Scheme 3, wherein R 1 For-OR 4 、-NR 5 R 5a 、-N(OR 5b )R 5a , or unsubstituted or replaced by one or two R 6 Substituted N-linked heterocycloalkyl; R 2 is iodine; R 3b is fluorine; and R 2a , R 3 , R 3a , R 4 , R 5 , R 5a , R 5b and R are as defined in any aspect, embodiment or claim described herein. In some embodiments, R 3 and R 3a In some embodiments, R 3 is hydrogen and R 3a For fluorine.

[0333] Starting from commercially available or conventionally available acid of formula (109) and commercially available or conventionally available aniline of formula (105), compounds of formula (110) can be prepared by methods that are obvious to those skilled in the art. Alternatively, compounds of formula (110) can be converted to methyl esters such as those shown in formula (111) by methods known in the art. Compounds of formula (110) or (111) are then treated with Zn(CN)2 in the presence of a catalyst such as Pd(PPh3)4. Compounds of formula (114) or (115) are prepared by treating compounds of formula (110) or (111) in the presence of silver trifluoroacetate or, alternatively, with iodine monochloride. In the case of compounds of formula (115), the methyl ester is then hydrolyzed to the corresponding acid. Acid compounds of formula (114) can be activated with a variety of reagents apparent to those skilled in the art to produce compounds having a suitable leaving group attached to the carbonyl group of the C(O)OH acid group, such as an acid chloride produced by reaction of (114) with thionyl chloride or an active ester produced by reaction of (114) with a reagent such as EDCI or HOBt. The acid chloride or active ester can then be reacted with a compound of formula R 4 OH, HNR 5 R 5a 、HN(OR 5b )R 5a , R a -H (where R a is unsubstituted or replaced by one or two R 6 A compound of formula (Ib) is reacted with a substituted N-linked heterocycloalkyl) or a suitably protected form thereof to prepare a compound of formula (Ib).

[0334] The compounds in Table 1 and Table 2 were prepared using the exemplary preparative schemes provided herein and methods known to one of ordinary skill in the art.

[0335] Example 1: 4-cyano-2-((2-fluoro-4-iodophenyl)amino)benzoic acid

[0336]

[0337] A solution of 4-cyano-2-fluorobenzoic acid (3.0 g, 18.1 mmol) in THF (30 mL) stirred at -78 °C in N2 was treated with LDA (2.0 M in THF, 27.2 mL, 54.5 mmol) (dropwise). After 20 min, a solution of 2-fluoro-4-iodoaniline (12.9 g, 54.5 mmol) in dry THF (15 mL) was added dropwise, the reaction mixture was continued to stir and allowed to warm to room temperature. After 16 h, the reaction mixture was concentrated in vacuo, acidified with 1 M HCl and extracted twice with Et2O. The combined organic phases were washed with brine, dried over Na2SO4, filtered and concentrated in vacuo. The crude residue was purified by trituration in boiling DCM to give the product as a yellow solid (1.57 g, 22.7%). m / z 381.1 [MH] - . 1 H NMR(400MHz,DMSO-d6)δppm 13.91(s,1H),9.74(s,1H),8.04(d,J=8.2Hz,1H),7.77(dd,J=10.3,2.0Hz,1H ),7.58(dt,J=8.5,1.3Hz,1H),7.42–7.33(m,2H),7.24(dd,J=8.2,1.6Hz,1H)

[0338] Example 2: 4-Cyano-2-((2-fluoro-4-iodophenyl)amino)benzoic acid

[0339]

[0340] A microwave vial was charged with 4-cyano-2-((2-fluoro-4-iodophenyl)amino)benzoic acid (80% pure) (0.19 g, 0.4 mmol), 3-hydroxyazetidine hydrochloride (0.07 g, 0.6 mmol), HATU (0.25 g, 0.6 mmol) and diisopropylethylamine (140 μL, 0.8 mmol) and N,N-dimethylformamide (6 mL). The reaction mixture was stirred at room temperature overnight. The reaction was quenched with water and extracted with ethyl acetate. The organics were washed with water, brine and dried over sodium sulfate. The solvent was evaporated. The residue was purified by flash chromatography (12 g silica gel, 0-5% methanol in dichloromethane) and then by reverse phase HPLC (30-95% acetonitrile / water) to give the product as a light yellow solid (71 mg, 41%). m / z 438.1 [M+1] + . 1H NMR (300MHz, DMSO-d6): δ8.97 (s, 1H), 7.69-7.65 (dd, J = 1.2Hz and 0.9Hz,1H),7.55-7.47(m,2H),7.40(s,1H),7.28-7.18(m,2H),5.80(s,1H),4.45(s,1 H),4.35(t,J=7.6Hz,1H),4.21(t,J=8.9Hz,1H),4.00-3.98(m,1H),3.76-3.73(m,1H).

[0341] The following compounds can be prepared as described in Example 2 by replacing 3-hydroxyazetidine hydrochloride with the appropriate amine which is either commercially available or prepared using conditions known to one of ordinary skill in the art.

[0342]

[0343]

[0344]

[0345] Example 16: N-(azetidin-3-ylmethoxy)-4-cyano-2-((2-fluoro-4-iodophenyl)amino)benzamide

[0346]

[0347] Step 1: 3-(((4-cyano-2-((2-fluoro-4-iodophenyl)amino)benzamido)oxy)methyl)azacyclic tert-Butyl-1-carboxylate

[0348] A microwave vial was charged with 4-cyano-2-((2-fluoro-4-iodophenyl)amino)benzoic acid (0.100 g, 0.3 mmol), tert-butyl 3-((aminooxy)methyl)azetidine-1-carboxylate (60% pure) (0.13 g, 0.4 mmol), HATU (0.15 g, 0.4 mmol) and diisopropylethylamine (135 μL, 0.8 mmol) in N,N-dimethylformamide (3 mL). The reaction mixture was stirred at room temperature overnight. The reaction was quenched with water and extracted with ethyl acetate. The organics were washed with water, brine and dried over sodium sulfate. The solvent was evaporated. The residue was purified by flash chromatography (12 g silica gel, 0-70% ethyl acetate in hexanes). The product fractions were collected and the solvent was removed to give a yellow oil (77 mg, 52%). m / z 565.2 [M-1] - .

[0349] Step 2: N-(azetidin-3-ylmethoxy)-4-cyano-2-((2-fluoro-4-iodophenyl)amino)benzamide

[0350] A round bottom flask was charged with tert-butyl 3-(((4-cyano-2-((2-fluoro-4-iodophenyl)amino)benzamido)oxy)-1-carboxylate (0.077 g, 0.1 mmol) in dichloromethane (6 mL) and hydrogen chloride (4.0 M in 1,4-dioxane, 0.510 ml, 2.0 mmol) was added dropwise. The reaction mixture was stirred at room temperature overnight. The reaction mixture was concentrated in vacuo. The residue was purified by reverse phase HPLC ((10-95% acetonitrile / water (0.1% TFA)). The product fractions were collected, washed with sodium bicarbonate and dried in vacuo to give the product as a yellow solid. m / z 461.7 [M+1] + . 1 H NMR (300MHz, CDCl3): δ8.41 (s, 1H), 7.67 (d, J = 8.1Hz, 1H), 7.50-7.42 (m, 2H), 7.35 (s, 1H), 7.12-7.03 ( m,1H),4.16-4.10(m,1H),4.06-4.00(m,1H),3.94-3.90(m,1H),3.73-3.68(m,1H),2.89-2.79(m,3H).

[0351] Example 17: 4-Cyano-2-((2-fluoro-4-iodophenyl)amino)-N-hydroxybenzamide

[0352]

[0353] A solution of hydroxylamine hydrochloride (333 mg, 0.52 mmol) in dry DMF (2.4 mL) and dry acetonitrile (2.4 mL) stirred at room temperature was treated with Et3N (1.33 mL, 9.6 mmol). After 1 h, the suspension was diluted with DCM (2.4 mL), cooled to 0 °C and a solution of 4-cyano-2-((2-fluoro-4-iodophenyl)amino)benzoyl chloride (209 mg, 0.52 mmol) in dry THF (2.4 mL) was added. After 1 h, the reaction mixture was diluted with EtOAc, quenched with saturated aqueous NH4Cl, partitioned, and the aqueous phase was extracted with EtOAc. The combined organic phases were washed with brine, dried over Na2SO4, filtered and concentrated in vacuo. The crude material was purified by preparative HPLC to give the product as a yellow solid (37 mg, 17.8%). m / z 396.1 [MH] - . 1H NMR (400MHz, DMSO-d6): δppm 11.60(s,1H),9.46(s,1H),9.41(s,1H),7.72(dd,J=10.5,2.0Hz,1H),7 .64(d,J=8.0Hz,1H),7.56–7.49(m,1H),7.46(s,1H),7.37–7.28(m,2H).

[0354] Example 18: 2-((4-cyano-2-((2-fluoro-4-iodophenyl)amino)benzamido)oxy)ethan-1-amine 2,2,2-trifluoroacetate

[0355] Step 1: (2-((4-cyano-2-((2-fluoro-4-iodophenyl)amino)benzamido)oxy)ethyl)amino Tert-butyl ester

[0356]

[0357] A solution of 4-cyano-2-((2-fluoro-4-iodophenyl)amino)benzoic acid (150 mg, 0.39 mmol) and HATU (298 mg, 0.78 mmol) in dry DMF (3.9 mL) was treated with dry DIPEA (0.13 mL, 0.78 mmol). The reaction mixture was stirred at 50 °C for 30 min, cooled to room temperature and a solution of tert-butyl (2-(aminooxy)ethyl)carbamate (103 mg, 0.58 mmol) in dry DMF (0.5 mL) was added. After 16 h, the reaction mixture was diluted with EtOAc, quenched with saturated H2O, partitioned, and the aqueous phase was extracted with EtOAc. The combined organic phases were washed with brine, dried over Na2SO4, filtered and concentrated in vacuo. The crude residue was purified by flash column chromatography (silica gel, 1-40% EtOAC in hexane) to give the product as a yellow oil (87 mg, 41%). UPLC-MS (acidic method, 2 min): rt 1.27 min. m / z 539.1 [MH] - . 1 H NMR (400MHz, CDCl3): δppm 10.56(s,1H),9.45(s,1H),7.59(d,J=8.1Hz,1H),7.57–7.46(m,2H),7.32(s,1H),7. 16–7.07(m,2H),5.06(s,1H),4.01(t,J=4.9Hz,2H),3.54–3.45(m,2H),1.49(s,9H).

[0358] Step 2: 2-((4-cyano-2-((2-fluoro-4-iodophenyl)amino)benzamido)oxy)ethan-1-amine 2,2,2- Trifluoroacetate

[0359]

[0360] A solution of (2-((4-cyano-2-((2-fluoro-4-iodophenyl)amino)benzamido)oxy)ethyl)-carbamic acid tert-butyl ester (67 mg, 0.12 mmol) in dioxane (1.0 mL) stirred at room temperature was treated with 4N HCl in dioxane (62 μl, 0.24 mmol). After 1 h, another portion of 4N HCl in dioxane (62 μl, 0.24 mmol) was added, and similarly after 16 h. After 48 h, another portion of 4N HCl in dioxane (0.25 mL, 1 mmol) was added. After 48 h, the reaction mixture was concentrated in vacuo. The crude material was purified by preparative HPLC to give the product as a trifluoroacetate salt as a yellow solid (21.1 mg, 32%). m / z 441.1 [M+H] + . 1 H NMR (400MHz, DMSO-d6): δppm 7.79–7.69(m,2H),7.54(dd,J=8.3,1.9Hz,1H),7.45(d,J=1.5Hz,1H),7.35(dd,J=8 .1,1.5Hz,1H),7.29(t,J=8.6Hz,1H),4.07(t,J=5.1Hz,2H),3.08(t,J=5.2Hz,2H).

[0361] Example 19: Methyl 2-((4-cyano-2-((2-fluoro-4-iodophenyl)amino)benzamido)oxy)acetate

[0362]

[0363] A solution of 4-cyano-2-((2-fluoro-4-iodophenyl)amino)benzoic acid (500 mg, 1.30 mmol) and HATU (995 mg, 2.61 mmol) in dry DMF (13.0 mL) was treated with dry DIPEA (0.45 mL, 2.61 mmol). The reaction mixture was stirred at 50 °C for 30 min, cooled to room temperature and a solution of methyl 2-(aminooxy)acetate hydrochloride (270 mg, 1.96 mmol) in dry DMF (1.0 mL) was added, followed by dry DIPEA (0.34 mL, 2.0 mmol). After 48 h, the reaction mixture was diluted with EtOAc, quenched with saturated H2O, partitioned, and the aqueous phase was extracted with EtOAc. The combined organic phases were washed with brine, dried over Na2SO4, filtered and concentrated in vacuo. The crude residue was purified by flash column chromatography (silica gel, 10-41% EtOAC in hexanes) to give the product as a yellow solid (373 mg, 61%). m / z 468.1 [MH] - . 1H NMR (400MHz, DMSO-d6): δppm12.29(s,1H),9.16(s,1H),7.76–7.60(m,2H),7.52(dt ,J=8.4,1.4Hz,1H),7.41(s,1H),7.35–7.19(m,2H),4.72–4.55(m,2H),3.70(s,3H).

[0364] Example 20: 2-((4-cyano-2-((2-fluoro-4-iodophenyl)amino)benzamido)oxy)acetic acid

[0365]

[0366] A stirred solution of methyl 2-((4-cyano-2-((2-fluoro-4-iodophenyl)amino)benzamido)oxy)acetate (306 mg, 0.64 mmol) in THF (1.93 mL), MeOH (0.64 mL) and H2O (1.28 mL) at room temperature was treated with 1M LiOH (0.64 mL, 0.64 mmol). After 24 h, another portion of 1M LiOH (0.64 mL, 0.64 mmol) was added and stirring continued. After 24 h, the reaction mixture was diluted with saturated aqueous NaHCO3, partitioned with EtOAc, the aqueous phase was acidified with 1M HCl and extracted with EtOAc. The organic phase was washed with 1M HCl and brine, dried over NaHCO3, filtered and concentrated in vacuo to give the product as a yellow solid (238 mg, 81%). m / z 454.0 [MH] - . 1 H NMR (400MHz, DMSO-d6): δppm 13.00(s,1H),12.30(s,1H),9.21(s,1H),7.72(dd,J=10.4,1.9Hz,1H),7.68(s,1H) ,7.53(dt,J=8.5,1.4Hz,1H),7.40(d,J=1.6Hz,1H),7.34–7.24(m,2H),4.52(s,2H).

[0367] Example 21: N-(2-amino-2-oxoethoxy)-4-cyano-2-((2-fluoro-4-iodophenyl)amino)benzamide

[0368]

[0369] Step 1: 2-((4-cyano-2-((2-fluoro-4-iodophenyl)amino)benzamido)oxy)acetyl chloride

[0370]

[0371] A solution of 2-((4-cyano-2-((2-fluoro-4-iodophenyl)amino)benzamido)oxy)acetic acid (181 mg, 0.39 mmol) in dioxane (1.5 mL) stirred at room temperature under N2 was treated with SOCl2 (0.3 ml, 3.9 mmol). After 24 h, the reaction mixture was concentrated in vacuo and azeotroped with dry toluene (3×5 mL). The resulting orange solid was used in the subsequent reaction without further purification. m / z 468.1[M+H] - (Detected as the corresponding methyl ester after quenching an aliquot of the mixture with 10% pyridine in MeOH).

[0372] Step 2: N-(2-amino-2-oxoethoxy)-4-cyano-2-((2-fluoro-4-iodophenyl)amino)benzamide

[0373]

[0374] A solution of 2-((4-cyano-2-((2-fluoro-4-iodophenyl)amino)benzamido)oxy)acetyl chloride (188 mg, 0.39 mmol) in dry dioxane (0.9 mL) stirred at 0 °C under N2 was treated with 0.5M NH3 in dioxane (0.91 mL). After 48 h, the reaction mixture was diluted with EtOAc, quenched with 1M HCl, partitioned and the organic phase was washed with brine, dried over Na2SO4, filtered and concentrated in vacuo. The crude material was purified by preparative HPLC to give the product as a yellow solid (49.7 mg, 28%). m / z 453.0 [MH] - . 1 H NMR (400MHz, DMSO-d6): δppm 12.29(br s,1H),9.40(br s,1H),7.74–7.69(m,2H),7.54–7.48(m,1H),7.40(s,1H),7.34–7.24(m,2H),4.34(s,2H).

[0375] Example 22: Methyl 3-((4-cyano-2-((2-fluoro-4-iodophenyl)amino)benzamido)oxy)propanoate

[0376]

[0377] A stirred solution of 4-cyano-2-((2-fluoro-4-iodophenyl)amino)benzoic acid (500 mg, 1.30 mmol) and HATU (995 mg, 2.61 mmol) in dry DMF (13.0 mL) at room temperature was treated with dry DIPEA (0.45 mL, 2.61 mmol). The reaction mixture was stirred at 50 °C for 30 min, cooled to room temperature and a suspension of methyl 3-(aminooxy)propanoate hydrochloride (305 mg, 1.96 mmol) in dry acetonitrile (2.9 mL) and dry DMF (2.9 mL) was added, followed by dry DIPEA (0.45 mL, 2.6 mmol). After 16 h, the reaction mixture was diluted with EtOAc, quenched with saturated H2O, partitioned, and the aqueous phase was extracted with EtOAc. The combined organic phases were washed with brine, dried over Na2SO4, filtered and concentrated in vacuo. The crude residue was purified by flash column chromatography (silica gel, 20-50% EtOAC in hexanes) to give the product as a yellow solid (369 mg, 59%). m / z 482.0 [MH] - . 1 H NMR (400MHz, DMSO-d6): δppm 12.05(s,1H),9.29(s,1H),7.72(dd,J=10.5,1.9Hz,1H),7.66(s,1H),7.53(d,J=8.6Hz,1H) ,7.42(d,J=1.7Hz,1H),7.36–7.25(m,2H),4.14(m,2H),3.61(s,3H),2.70(t,J=6.6Hz,2H).

[0378] Example 23: 3-((4-cyano-2-((2-fluoro-4-iodophenyl)amino)benzamido)oxy)propanoic acid

[0379]

[0380] A stirred solution of methyl 3-((4-cyano-2-((2-fluoro-4-iodophenyl)amino)benzamido)oxy)propanoate (321 mg, 0.66 mmol) in THF (4.0 mL), MeOH (1.33 mL) and H2O (2.65 mL) at room temperature was treated with 1M LiOH (1.33 mL, 1.33 mmol). After 3 h, another portion of 1M LiOH (0.32 mL, 0.32 mmol) was added and stirring continued. After 3 h, the reaction mixture was diluted with saturated aqueous NaHCO3, partitioned with EtOAc, the aqueous phase was acidified and extracted with EtOAc. The first EtOAc extract was washed with 1M HCl and brine, dried over NaHCO3, filtered and concentrated in vacuo to give the product as a yellow solid (203 mg, 65.5% yield, 79% purity). The second EtOAc extract was washed with brine, dried over NaHCO3, filtered and concentrated in vacuo to give the product as a yellow solid (46 mg, 14.8% yield, 100% purity). m / z 468.1 [MH] - . 1 HNMR (400MHz, DMSO-d6): δppm 12.37(m,2H),9.29(s,1H),7.76–7.62(m,2H),7.53(dd,J=8.1,1.9Hz,1H),7 .42(d,J=1.7Hz,1H),7.35–7.24(m,2H),4.12(s,2H),2.61(t,J=6.1Hz,2H).

[0381] Example 24: 3-((2-fluoro-4-iodophenyl)amino)-4-(3-oxoisoxazolidine-2-carbonyl)benzonitrile

[0382]

[0383] A solution of 2-((4-cyano-2-((2-fluoro-4-iodophenyl)amino)benzamido)oxy)propanoic acid (203 mg, 0.43 mmol) in dioxane (1.7 mL) stirred at room temperature under N2 was treated with SOCl2 (0.32 ml, 4.3 mmol). After 24 h, the reaction mixture was concentrated in vacuo and azeotroped with dry toluene (3×5 mL). The resulting orange solid was used in the next step without further purification. m / z 450.0 [M+H] - .

[0384] Example 25: N-(3-amino-3-oxopropoxy)-4-cyano-2-((2-fluoro-4-iodophenyl)amino)benzamide

[0385]

[0386] A solution of 3-((2-fluoro-4-iodophenyl)amino)-4-(3-oxoisoxazolidine-2-carbonyl)benzonitrile (195 mg, 0.43 mmol) in dry dioxane (1.03 mL) stirred at 0 °C under N2 was treated with 0.5 M NH3 in dioxane (0.99 mL, 0.49 mmol). After 30 min, the reaction mixture was diluted with EtOAc, quenched with 1 M HCl, partitioned and the organic phase was washed with brine, dried over Na2SO4, filtered and concentrated in vacuo. The crude material was purified by preparative HPLC to give the product as a yellow solid (22.2 mg, 11%). m / z 467.1 [MH] - . 1 H NMR (400MHz, DMSO-d6): δppm 12.04 (br s, 1H), 9.47 (br s,1H),7.71(dd,J=10.4,2.0Hz,2H),7.52(d,J=8.0Hz,1H),7.41(s,1H),7.33–7.26(m,2H),6.90(br s, 2H), 4.09 (t, J = 5.1Hz, 2H), 2.44 (t, J = 6.3Hz, 2H).

[0387] Example 26: 4-cyano-2-((2-fluoro-4-iodophenyl)amino)benzoic acid-3-hydroxycyclobutyl ester

[0388]

[0389] Step 1: 4-Cyano-2-((2-fluoro-4-iodophenyl)amino)benzoyl chloride (general preparation)

[0390]

[0391] A suspension of 4-cyano-2-((2-fluoro-4-iodophenyl)amino)benzoic acid (200 mg, 0.52 mmol) in dioxane (1.61 mL) stirred at room temperature was treated with SOCl2 (0.32 ml, 5.23 mmol). The reaction mixture was further stirred at 50 °C under N2. After 48 h, the reaction mixture was concentrated in vacuo and azeotroped with dry toluene (2×5 mL). The resulting crude material in residual toluene was used in the next step without further purification.

[0392] Step 2: 4-Cyano-2-((2-fluoro-4-iodophenyl)amino)benzoic acid-3-hydroxycyclobutyl ester

[0393] To a solution of cyclobutane-1,3-diol (159 mg, 1.8 mmol) and Et3N (0.16 mL, 1.1 mmol) in dry THF (1.0 mL) stirred at 0°C under N2 was added a solution of 4-cyano-2-((2-fluoro-4-iodophenyl)amino)benzoyl chloride (157 mg, 0.39 mmol) in dry THF (1.3 mL). After 1 h, the reaction mixture was diluted with EtOAc, quenched with saturated aqueous NH4Cl, partitioned, and the aqueous phase was re-extracted with EtOAc. The combined organic phases were washed with brine, dried over Na2SO4, filtered, and concentrated in vacuo. The crude residue was purified by flash column chromatography (silica gel, 5-31% EtOAC in hexanes) to give the product as a yellow solid (43.7 mg, 24.7%). m / z 451.1 [MH] - . 1 H NMR (400MHz, DMSO-d6): δppm 9.36(s,1H),8.06(d,J=8.2Hz,1H),7.76(dd,J=10.3,1.9Hz,1H),7.58(dt,J=8.4,1.3Hz,1H),7.38–7.30(m,2H),7.27(dd,J =8.2,1.6Hz,1H),5.26(d,J=6.5Hz,1H),4.74(p,J=7.3Hz,1H),3.88(h,J=6.9Hz,1H),2.86–2.70(m,2H),2.12–1.98(m,2H).

[0394] Example 27: 3-Hydroxy-2,2,4,4-tetramethylcyclobutyl 4-cyano-2-((2-fluoro-4-iodophenyl)amino)benzoate

[0395]

[0396] To a solution of 2,2,4,4-tetramethylcyclobutane-1,3-diol (520 mg, 3.6 mmol) and Et3N (0.32 mL, 2.3 mmol) in dry THF (2.0 mL) stirred at 0°C under N2 was added a solution of 4-cyano-2-((2-fluoro-4-iodophenyl)amino)benzoyl chloride (314 mg, 0.78 mmol) in dry THF (2.7 mL). After 48 h, the reaction mixture was diluted with EtOAc, quenched with saturated aqueous NH4Cl, partitioned, and the aqueous phase was re-extracted with EtOAc. The combined organic phases were washed with brine, dried over Na2SO4, filtered, and concentrated in vacuo. The crude residue was purified by flash column chromatography (silica gel, 0-50% EtOAC in hexanes) to give the product as a yellow solid (26.6 mg, 6.7%). m / z 506.9 [MH] - .1 H NMR (400MHz, DMSO-d6): δppm (Note: amixture of diastereoisomers 2:1)9.31(s,0.35H),9.29(s,0.67H),8.10(d,J=8.2Hz,0.37H),8.06(d,J=8.2Hz,0.63H) ,7.77(dd,J=10.3,1.9Hz,1H),7.59(dd,J=7.8,1.9Hz,1H),7.39–7.34(m,2H),7.34–7.27 (m,1H),5.01(t,J=4.8Hz,1H),4.52(s,0.35H),4.41(d,J=0.9Hz,0.67H),3.53(d,J=4.8H z,0.36H),3.37(d,J=4.8Hz,0.66H),1.18(s,4H),1.09(s,2H),1.07(s,2H),1.00(s,4H).

[0397] Example 28: 2,3-dihydroxycyclopropyl 4-cyano-2-((2-fluoro-4-iodophenyl)amino)benzoate

[0398]

[0399] To a solution of 1,2,3-propanetriol (332 mg, 3.6 mmol) and Et3N (0.32 mL, 2.3 mmol) in dry THF (2.7 mL) stirred at 0°C under N2 was added a solution of 4-cyano-2-((2-fluoro-4-iodophenyl)amino)benzoyl chloride (314 mg, 0.78 mmol) in dry THF (2.0 mL). After 16 h, the reaction mixture was diluted with EtOAc, quenched with saturated aqueous NH4Cl, partitioned, and the aqueous phase was re-extracted with EtOAc. The combined organic phases were washed with brine, dried over Na2SO4, filtered, and concentrated in vacuo. A portion of the crude material (198 mg) was purified by preparative HPLC to give the product as a clear yellow solid (34 mg, 9.5%). m / z 455.0 [MH] - . 1H NMR (400MHz, DMSO-d6): δppm 9.33(s,1H),8.12(d,J=8.2Hz,1H),7.77(dd,J=10.3,1.9Hz,1H),7.59(dt,J=8.4,1.4Hz,1H),7.39–7.30(m,2H),7.29(dd,J=8.2,1.6Hz,1H), 5.12(d,J=5.3Hz,1H),4.75(t,J=5.7Hz,1H),4.37(dd,J=11.1,3.9Hz,1H),4.23(dd,J=11.1,6.2Hz,1H),3.88–3.76(m,1H),3.53–3.38(m,2H).

[0400] Example 29: 4-Cyano-3-fluoro-2-((2-fluoro-4-iodophenyl)amino)benzoic acid

[0401]

[0402] Step 1: 2-Fluoro-4-(trimethylsilyl)aniline

[0403] A round bottom flask was charged with 4-bromo-2-fluoroaniline (1.00 g, 5.3 mmol) and anhydrous tetrahydrofuran (8 mL), cooled to -78 °C and a 2.5 M solution of nBuLi in hexane (8 ml, 20 mmol) was added dropwise, keeping the internal temperature below -60 °C. The reaction mixture was treated dropwise with trimethylsilyl chloride (2.26 ml, 17.4 mmol.), keeping the internal temperature below -60 °C. The reaction mixture was warmed to 0 °C for one hour. The reaction mixture was poured into ice-cold 2 M hydrochloric acid and stirred vigorously for 10 minutes. The organic layer was separated and the aqueous layer was extracted with ethyl acetate. The combined organics were dried over magnesium sulfate and evaporated to dryness to give the product as a colorless oil (0.58 g, 54%). m / z 184.2 [M+1] + .

[0404] Step 2: 4-Bromo-3-fluoro-2-((2-fluoro-4-(trimethylsilyl)phenyl)amino)benzoic acid

[0405] To a stirred tetrahydrofuran (6 mL) solution of 2-fluoro-4-(trimethylsilyl)aniline (1.0 g, 5.1 mmol) was added lithium diisopropylamide (2.0 M in THF / heptane / ethylbenzene, 2.5 ml, 5.1 mmol) at -78 °C. The resulting suspension was vigorously stirred for 10 minutes, after which a solution of 4-bromo-2,3-difluorobenzoic acid (0.400 g, 1.7 mmol) in tetrahydrofuran (5 mL) was added. The cold bath was subsequently removed and the reaction mixture was stirred overnight at room temperature. The mixture was concentrated and the concentrate was treated with 3M hydrochloric acid (10 mL). The resulting suspension was extracted with ethyl acetate. The combined organics were dried over sodium sulfate. The solvent was removed in vacuo. Hexane was added to the residue. The precipitated beige solid (382 mg) was washed with hexane and a few drops of ethyl acetate and separated by filtration. m / z 400.1 [M + ]. 1 H NMR (300MHz, DMSO-d6): δ9.06(s,1H),7.70-7.66(m,1H),7.38-7.33(m,1H) ,7.27(d,J=11.7Hz,1H),7.20-7.17(m,1H),6.93-6.86(m,1H),0.22(s,9H).

[0406] Step 3: 4-Cyano-3-fluoro-2-((2-fluoro-4-(trimethylsilyl)phenyl)amino)benzoic acid

[0407] A microwave vial was charged with 4-bromo-3-fluoro-2-((2-fluoro-4-(trimethylsilyl)phenyl)amino)benzoic acid (0.180 g, 0.4 mmol), zinc cyanide (0.05 g, 0.4 mmol) and tetrakis(triphenylphosphine)palladium(0) (0.05 g, 0.05 mmol) in N,N-dimethylformamide under argon. The reaction mixture was stirred at 90 °C overnight. The reaction was quenched with water and extracted with ethyl acetate. The organics were washed with water, brine and dried over sodium sulfate. The solvent was evaporated. The residue was purified by flash chromatography (12 g silica gel, 0-70% ethyl acetate in hexanes) to give the product as a yellow solid (50 mg, 32%). 1 H NMR (300MHz, CDCl3): δ8.95 (s, 1H), 7.93 (d, J = 8.4Hz, 1H), 7.24-7.21 (m, 2H), 7.19-7.08 (m, 1H), 7.00-6.93 (m, 1H), 0.28 (s, 9H).

[0408] Step 4: 4-Cyano-3-fluoro-2-((2-fluoro-4-iodophenyl)amino)benzoic acid

[0409] A round bottom flask was charged with 4-cyano-3-fluoro-2-((2-fluoro-4-(trimethylsilyl)phenyl)amino)benzoic acid (0.49 g, 1.4 mmol) in anhydrous methanol (6 mL) and anhydrous dichloromethane (6 mL). Silver trifluoroacetate (0.66 g, 3.0 mmol) was added. The reaction mixture was cooled to 0°C. Iodine (0.72 g, 2.8 mmol) was then added in one portion and the reaction mixture was stirred at 0°C for 2 hours. The reaction mixture was filtered through celite and the solvent was removed. The residue was treated with saturated sodium thiosulfate and extracted with ethyl acetate. The combined organics were dried over sodium sulfate. The solvent was removed in vacuo. Dichloromethane was added to the residue to precipitate a solid. The solid was collected by filtration and dried to give the product as a beige solid (200 mg, 35%). 1 H NMR (300MHz, DMSO-d6): δ7.83-7.81(m,1H),7.59-7.54(dd,J=1.8 and 1.8Hz,1H),7.39-7.36(m,2H),6.74-6.66(m,1H).

[0410] Example 30: 4-Cyano-3-fluoro-2-((2-fluoro-4-iodophenyl)amino)-N-(2-hydroxyethoxy)benzamide

[0411]

[0412] A microwave vial was charged with 4-cyano-3-fluoro-2-((2-fluoro-4-iodophenyl)amino)benzoic acid (0.100 g, 0.2 mmol), 2-((aminooxy)ethanol (0.03 g, 0.4 mmol), HATU (0.14 g, 0.4 mmol) and diisopropylethylamine (86 μL, 0.5 mmol) and N,N-dimethylformamide (3 mL). The reaction mixture was stirred at room temperature overnight. The reaction was quenched with water and extracted with ethyl acetate. The organics were washed with water, brine and dried over sodium sulfate. The solvent was evaporated. The residue was purified by flash chromatography (12 g silica gel, 0-5% methanol in dichloromethane). The product fractions were collected and the solvent was removed. The residue was further purified by reverse phase HPLC (20-80% acetonitrile / water) to give the product as a light yellow solid. m / z 458.0 [M-1] -.1HNMR (300MHz, DMSO-d6): δ11.93(s,1H),8.44(s,1H),7.67-7.62(m,1H),7.56(d,J=9.6Hz,1H),7. 47-7.44(m,1H),7.35(d,J=8.4Hz,1H),6.70-6.63(m,1H),3.79(t,J=4.4Hz,2H),3.55-3.53(m,2H).

[0413] The following compounds can be prepared as described in Example 30 by replacing 2-(aminooxy)ethanol with the appropriate amine which is either commercially available or prepared using conditions known to one of ordinary skill in the art.

[0414]

[0415] Example 32: 6-cyano-2-((2-fluoro-4-iodophenyl)amino)nicotinate

[0416]

[0417] Step 1: 6-Chloro-2-((2-fluoro-4-(trimethylsilyl)phenyl)amino)nicotinic acid

[0418]

[0419] LiHMDS (1M in THF, 100 mL, 100 mmol) was added to a solution of 2-fluoro-4-(trimethylsilyl)aniline (9.2 g, 50 mmol) in dry THF (40 mL) stirred at -78 ° C. The reaction mixture was stirred at -78 ° C for 1 h, followed by a solution of 2,6-dichloroisonicotinic acid (8 g, 41.7 mmol) in dry THF (31 mL). The reaction mixture was stirred at -78 ° C for 1 h and then gradually warmed to room temperature. The reaction mixture was quenched with saturated NH4Cl aqueous solution (100 mL) at 0 ° C, extracted with EtOAc (200 mL), acidified with 1M HCl and distributed. The aqueous phase was extracted with EtOAc (2 x 100 mL), the organic phase was washed with brine (100 mL), dried over Na2SO4, and concentrated in vacuo. The crude material was purified by trituration with methanol to give the product as a yellow solid (10.6 g, 75%). m / z 339.1 / 341.1[M+H] + . 1H NMR (400MHz, DMSO-d6): δppm 13.94(s,1H),10.79(d,J=2.9Hz,1H),8.41(t,J=8.1Hz,1H),8.27(d,J=8.1Hz,1H),7.37(m,2H),7.00(d,J=8.1Hz,1H),0.26(s,9H).

[0420] Step 2: 6-Chloro-2-((2-fluoro-4-(trimethylsilyl)phenyl)amino)nicotinate

[0421]

[0422] A solution of 6-chloro-2-((2-fluoro-4-(trimethylsilyl)phenyl)amino)nicotinic acid (0.5 g, 1.476 mmol) in DCM (7.4 mL) stirred at room temperature was treated with DIPEA (0.26 mmol, 1.476 mmol). After 10 min, the reaction mixture was cooled to 0 °C and treated with DMF (0.03 mL) and oxalyl chloride (0.12 mL, 1.476 mmol) and then warmed to room temperature. The reaction mixture was stirred for 30 minutes and then slowly added to a solution of DIPEA (0.26 mL, 1.476 mmol) in MeOH (7.4 mL) stirred at 0 °C and then warmed to room temperature. After 15 minutes, the reaction mixture was concentrated in vacuo. The residue was dissolved in EtOAc (20 mL), washed with saturated aqueous NaHCO3 (10 mL), H2O (10 mL), brine (10 mL), dried over Na2SO4 and concentrated in vacuo to give the product as a brown solid (0.45 g, 87%). m / z 353.1 / 355.1 [M+H] + . 1 H NMR (400MHz, DMSO-d6): δppm 10.48(d,J=3.0Hz,1H),8.36(t,J=7.9Hz,1H),8.29(d,J=8.2Hz,1H),7.43–7.33(m,2H),7.02(d,J=8.2Hz,1H),3.91(s,3H),0.26(s,9H).

[0423] Step 3: 6-cyano-2-((2-fluoro-4-(trimethylsilyl)phenyl)amino)nicotinate

[0424]

[0425] A degassed solution of methyl 6-chloro-2-((2-fluoro-4-(trimethylsilyl)phenyl)amino)nicotinate (0.4 g, 1.13 mmol), zinc cyanide (0.11 g, 0.96 mmol) and tetrakis(triphenylphosphine)palladium(0) (0.13 g, 0.11 mmol) in NMP (3.5 mL) was heated in a microwave oven at 190 °C for 15 min. The reaction mixture was filtered, dissolved with EtOAc (20 mL), washed with saturated aqueous NaHCO3 (10 mL), H2O (10 mL), brine (10 mL), dried over Na2SO4 and concentrated in vacuo. The crude material was purified by flash column chromatography (silica gel, 0-15% EtOAc in hexanes) to give the product as a yellow solid (0.133 g, 43%). m / z 344.1 [M+H] + . 1 H NMR (400MHz, DMSO-d6): δ10.43(d,J=2.8Hz,1H),8.46(d,J=7.8Hz,1H),8.32(t,J =8.0Hz,1H),7.54(d,J=7.8Hz,1H),7.45–7.36(m,2H),3.94(s,3H),0.26(s,9H).

[0426] Step 4: 6-cyano-2-((2-fluoro-4-iodophenyl)amino)nicotinate

[0427]

[0428] A suspension of silver tetrafluoroborate (85 mg, 0.437 mmol) in DCM (0.5 mL) was stirred at -50 °C in the dark for 5 min, then a solution of methyl 6-cyano-2-((2-fluoro-4-(trimethylsilyl)phenyl)amino)nicotinate (50 mg, 0.146 mmol) in DCM (1 mL) was added dropwise. After 15 min, the reaction mixture was treated with iodine monochloride (26 mg, 0.161 mmol) in DCM (0.3 mL). The reaction mixture was stirred for 15 min, and another portion of iodine monochloride (7 mg, 0.044 mmol) in DCM (0.1 mL) was added. After 15 min, the reaction mixture was quenched with saturated aqueous Na2S2O3 solution (1 mL), extracted with EtOAc (3×5 mL), the organic phase was washed with brine (5 mL), dried over Na2SO4 and the solvent removed in vacuo to give the product as a yellow solid (53 mg, 91%). m / z 398.0[M+H] + . 1H NMR (400MHz, DMSO-d6): δppm 10.37(d,J=2.8Hz,1H),8.46(d,J=7.8Hz,1H),8.11(t,J=8.6Hz,1H),7.76(dd,J= 10.6, 2.0Hz, 1H), 7.64 (dd, J=8.6, 1.7Hz, 1H), 7.55 (d, J=7.8Hz, 1H), 3.94 (s, 3H).

[0429] Example 33: 6-Cyano-2-((2-fluoro-4-iodophenyl)amino)nicotinic acid

[0430]

[0431] A solution of 6-cyano-2-((2-fluoro-4-iodophenyl)amino)nicotinic acid methyl ester (0.42 g, 1.06 mmol) in THF (3.2 mL), MeOH (1.05 mL) and H2O (2.1 mL) stirred at room temperature was treated with 1M LiOH aqueous solution (1.06 mL, 1.06 mmol). After 30 min, another 1M LiOH aqueous solution (1.06 mL, 1.06 mmol) was added. After 30 min, the reaction mixture was concentrated in vacuo and the residue was distributed between EtOAc (10 mL) and H2O (10 mL). The aqueous phase was acidified to pH 3 with 3M HCl aqueous solution and extracted with EtOAc (3 x 10 mL). The combined organic layers were washed with brine (10 mL), dried over Na2SO4, and concentrated in vacuo to give the product as a yellow solid (0.40 g quantitative). m / z 382.0 [MH] - . 1 H NMR (400MHz, DMSO-d6): δppm 8.42 (d, J = 7.7Hz, 1H), 8.21 (t, J = 8.5Hz, 1H), 7.73 (dd, J = 10.8, 1.9Hz, 1H), 7.62 (d, J = 8.6Hz, 1H), 7.52 (d, J = 7.7Hz, 1H).

[0432] Example 34: 6-Cyano-N-ethoxy-2-((2-fluoro-4-iodophenyl)amino)nicotinamide

[0433]

[0434] To a suspension of 6-cyano-2-((2-fluoro-4-iodophenyl)amino)nicotinic acid (100 mg, 0.26 mmol) in dry DMF (2.5 mL) was added HATU (200 mg, 0.52 mmol) and DIPEA (0.13 mL, 0.65 mmol), and the reaction mixture was heated at 50 °C for 30 min. The reaction mixture was then cooled to room temperature and a solution of ethoxylamine hydrochloride (38 mg, 0.39 mmol) pretreated with DIPEA (0.05 mL, 0.39 mmol) for 5 min in dry DMF (0.5 mL) was added. After 1 h, the reaction mixture was concentrated in vacuo, and the residue was dissolved in EtOAc (5 mL) and washed with H2O (5 mL) and brine (5 mL). The organic phase was dried over Na2SO4 and concentrated in vacuo. The crude material was purified by flash column chromatography (silica gel, 0-37% EtOAc in hexanes) followed by trituration with diethyl ether to give the product as a yellow solid (7 mg, 6%). UPLC-MS (acidic method, 2 min): rt 1.26 min, m / z 425.1 [MH] - . 1 H NMR (400MHz, DMSO-d6): δppm 12.18(s,1H),10.89(s,1H),8.16(d,J=7.7Hz,2H),7.71(dd,J=10.7,2.0Hz,1H),7.60(d t,J=8.6,1.5Hz,1H),7.55(d,J=7.8Hz,1H),4.00(d,J=7.1Hz,2H),1.24(t,J=7.0Hz,3H).

[0435] Example 35: 6-Cyano-2-((2-fluoro-4-iodophenyl)amino)-N-(2-hydroxyethoxy)nicotinamide

[0436]

[0437] To a stirred solution of 6-cyano-2-((2-fluoro-4-iodophenyl)amino)nicotinic acid (100 mg, 0.261 mmol) and HATU (198.5 mg, 0.522 mmol) in DMF (3 mL) at room temperature was added DIPEA (91 μl, 0.522 mmol) dropwise and the reaction was monitored until the HATU-activated acid was complete. After 2 h, 2-(aminooxy)ethan-1-ol (30.2 mg, 0.395 mmol) was added to the reaction mixture and it was stirred at room temperature for 45 min. The reaction mixture was quenched with H2O (30 mL) and extracted with EtOAc (4×20 mL). The combined organic phases were washed with brine (2×20 mL), dried over Na2SO4, and concentrated in vacuo. The crude product was purified by preparative HPLC to give the product as a yellow solid (55.2 mg, 48%). m / z 443.0[M+H] + . 1 H NMR (400MHz, DMSO-d6): δppm12.29(br s,1H),10.73(s,1H),8.16(d,J=7.8Hz,1H),8.14(t,J=8.5Hz,1H),7.71(dd,J =10.7,2.0Hz,1H),7.60(dt,J=8.6,1.3Hz,1H),7.55(d,J=7.8Hz,1H),4.79(br s,1H),4.00(t,J=4.8Hz,2H),3.66(t,J=4.9Hz,2H).

[0438] Example 36: 6-cyano-5-fluoro-2-((2-fluoro-4-iodophenyl)amino)nicotinate

[0439]

[0440] Step 1: 6-Chloro-5-fluoro-2-((2-fluoro-4-(trimethylsilyl)phenyl)amino)nicotinic acid

[0441]

[0442] A solution of 2-fluoro-4-(trimethylsilyl)aniline (8.4 g, 45.7 mmol) in dry THF (34 mL) stirred at -78 °C was treated with LiHMDS (1 M in THF, 91.2 mL, 91.2 mmol). The reaction mixture was stirred at -78 °C for 1 h, followed by the addition of a solution of 2,6-dichloro-5-fluoro-isonicotinic acid (8 g, 38 mmol) in dry THF (30 mL). The reaction mixture was stirred at -78 °C for 1 h, then gradually warmed to room temperature and stirred for 1 h. The reaction mixture was cooled to 0 °C and quenched with saturated aqueous NH4Cl, then diluted with EtOAc (200 mL), acidified to pH 3 with 1 M HCl, partitioned and the aqueous phase extracted with EtOAc (2×100 mL). The organic phase was washed with brine (100 mL), dried over Na2SO4, and concentrated in vacuo. The crude material was purified by trituration with methanol to give the product as a yellow solid (7.5 g, 55%). m / z 357.0 / 359.0 [M+H] + . 1 H NMR (400MHz, DMSO-d6) δppm 10.69 (s, 1H), 8.36 (t, J = 8.0Hz, 1H), 8.28 (d, J = 8.5Hz, 1H), 7.41–7.32 (m, 2H), 0.25 (s, 9H).

[0443] Step 2: 6-Chloro-5-fluoro-2-((2-fluoro-4-(trimethylsilyl)phenyl)amino)nicotinate

[0444]

[0445] A stirred suspension of 6-chloro-5-fluoro-2-((2-fluoro-4-(trimethylsilyl)phenyl)amino)nicotinic acid (2.4 g, 6.726 mmol) and DMF (0.32 mL) in DCM (32 mL) at 0°C was treated with oxalyl chloride (2.85 mL, 33.63 mmol) and then stirred at reflux for 1 h. The reaction mixture was concentrated in vacuo, azeotroped with toluene (3×25 mL), and the residue was then treated with ice-cold methanol (32 mL). The resulting suspension was stirred at reflux. After 2 h, the reaction mixture was cooled and the formed precipitate was filtered under reduced pressure. The collected precipitate was washed with ice-cold methanol (3×5 mL) and dried in vacuo to give the product as a yellow solid (2.18 g, 88%). m / z 371.1 / 373.1[M+H] + . 1 H NMR (400MHz, DMSO-d6) δppm 10.33 (d, J = 3.0Hz, 1H), 8.37–8.25 (m, 2H), 7.44–7.33 (m, 2H), 3.92 (s, 3H), 0.26 (s, 9H).

[0446] Step 3: 6-cyano-5-fluoro-2-((2-fluoro-4-(trimethylsilyl)phenyl)amino)nicotinate

[0447]

[0448] A degassed solution of methyl 6-chloro-5-fluoro-2-((2-fluoro-4-(trimethylsilyl)phenyl)amino)nicotinate (0.5 g, 1.35 mmol), zinc cyanide (0.14 g, 1.215 mmol) and tetrakis(triphenylphosphine)palladium(0) (0.23 g, 0.20 mmol) in NMP (4 mL) was heated in a microwave oven at 150 °C for 15 min. The reaction mixture was filtered, diluted with EtOAc (20 mL), washed with saturated aqueous NaHCO3 solution (20 mL) and partitioned. The aqueous phase was extracted with EtOAc (3 x 10 mL) and the combined organic phases were washed with H2O (20 mL), brine (20 mL), dried over Na2SO4, and concentrated in vacuo. The crude material was purified by flash column chromatography (silica gel, 0-15% EtOAC in hexanes) to give the product as a yellow solid (0.425 g, 87%). m / z 362.1[M+H] + . 1 H NMR (400MHz, DMSO-d6) δppm 10.29(d,J=2.8Hz,1H),8.51(d,J=8.7Hz,1H),8.28(t,J=8.0Hz,1H),7.46–7.37(m,2H),3.96(s,3H),0.27(s,9H).

[0449] Step 4: 6-cyano-5-fluoro-2-((2-fluoro-4-iodophenyl)amino)nicotinate

[0450]

[0451] A suspension of silver tetrafluoroborate (0.69 g, 3.531 mmol) in DCM (4.2 mL) was stirred at -50 °C in the dark for 10 min, then a solution of methyl 6-cyano-5-fluoro-2-((2-fluoro-4-(trimethylsilyl)phenyl)amino)nicotinate (0.425 g, 1.177 mmol) in DCM (8.5 mL) was added dropwise. After 30 min, the reaction mixture was treated with iodine monochloride (0.21 g, 1.295 mmol) in DCM (2.5 mL). The reaction mixture was stirred for 30 min, and another portion of iodine monochloride (0.21 g, 1.295 mmol) in DCM (2.5 mL) was added. After 30 min, the reaction mixture was quenched with saturated aqueous Na2S2O3 solution (10 mL), extracted with EtOAc (3×25 mL), the organic phase was washed with brine (25 mL), dried over Na2SO4 and the solvent was removed in vacuo to give the product as a yellow solid (0.36 g, 76%). m / z 416.4 [M+H] + . 1 H NMR (400MHz, DMSO-d6): δppm 10.22(s,1H),8.50(d,J=8.8Hz,1H),8.05(t,J=8.6Hz,1H),7.76(dd,J=10.6,2.0Hz,1H),7.66–7.60(m,1H),3.95(s,3H).

[0452] Example 37: 6-Cyano-5-fluoro-2-((2-fluoro-4-iodophenyl)amino)nicotinic acid

[0453]

[0454] A suspension of methyl 6-cyano-5-fluoro-2-((2-fluoro-4-iodophenyl)amino)nicotinate (0.36 g, 0.898 mmol) in THF (2.7 mL), MeOH (0.9 mL) and H2O (1.8 mL) stirred at room temperature was treated with 1M aqueous LiOH solution (0.9 mL, 0.898 mmol). After 30 min, the reaction mixture was concentrated in vacuo and the residue was partitioned between EtOAc (10 mL) and H2O (10 mL). The aqueous phase was acidified to pH 3 with 3M aqueous HCl and extracted with EtOAc (3 x 10 mL). The combined organic layers were washed with brine (10 mL), dried over Na2SO4, and concentrated in vacuo to give the product as a brown solid (0.30 g, 86%). m / z 400.0 [MH] - . 1H NMR (400MHz, DMSO-d6): δppm 10.70 (s, 1H), 8.43 (d, J = 8.7Hz, 1H), 8.13 (t, J = 8.7Hz, 1H), 7.72 (dd, J = 10.7, 1.9Hz, 1H), 7.61 (dt, = 8.6, 1.6Hz, 1H).

[0455] Example 38: MEK inhibition assay-1

[0456] The following method can be used to determine biochemical activity. The MEK1 inhibitory activity of the compound was tested using the following method. See Anastassiadis T et al. Comprehensive assay of kinase catalytic activity reveals features of kinase inhibitor selectivity. Nat Biotechnol., 2011, 29(11), 1039-45.

[0457] Reagents:

[0458] Reaction buffer: 20 mM Hepes (pH 7.5), 10 mM MgCl2, 1 mM EGTA, 0.02% Brij35, 0.02 mg / ml BSA, 0.1 mM Na3VO4, 2 mM DTT, 1% DMSO

[0459] Enzyme: MEK1, Invitrogen Cat# PV3303

[0460] N-terminally His-tagged recombinant human full-length protein expressed in insect cells. Activated by RAF1 in vitro. MW = 49.2 kDa, GenBank accession number NP_002746.

[0461] Substrate: 5 μM ERK2(K52R),

[0462] The kinase lethal mutant (GenBank accession number NM_0011949), aa2-358 with an N-terminal His6 tag, MW = 43.63 kDa, was expressed in E. coli.

[0463] Prepare substrate in fresh reaction buffer. Deliver kinase into substrate solution and mix gently. Deliver test compounds in 100% DMSO to kinase reaction mixture by acoustic technology (Echo 550; nanoliter range) and incubate at room temperature for 20 minutes. 33P-ATP was delivered to the reaction mixture to initiate the reaction. The reaction mixture was incubated at room temperature for 2 hours. The kinase activity was detected by the P81 filter binding method.

[0464] Example 39: MEK inhibition assay-2

[0465] Compounds were tested for MEK1 inhibitory activity using the following procedure (protocol available at thermofisher.com / content / dam / LifeTech / migration / files / drug-discovery / pdfs.par.60256.file.dat / 20130430%20ssbk%20customer%20protocol%20and%20assay%20conditions.pdf). The Z′-LYTE biochemical assay (ThermoFisher) employs a fluorescence-based coupled enzyme format and is based on the differential sensitivity of phosphorylated and non-phosphorylated peptides to proteolytic cleavage.

[0466] Test compounds in 100% DMSO were screened in wells with 1% DMSO (final).For a 10 point titration, 3-fold serial dilutions were performed starting from a starting concentration of 30 μM.

[0467] The peptide / kinase, MAP2K1 (MEK1) / inactive MAPK1 (ERK2) / Ser / Thr03, mixture ("peptide / kinase mixture") was diluted to 2X working concentration in the following buffer ("kinase buffer"): 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgCl2, 1 mM EGTA. The final 10 μL kinase reaction included 0.06-0.25 ng MAP2K1 (MEK1), 105 ng inactive MAPK1 (ERK2) and 2 μM Ser / Thr 03 in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgCl2, 1 mM EGTA. After incubation for 1 hour, 5 μL of 1:1024 diluted developing reagent A (available from Invitrogen, catalog number PV3295) was added.

[0468] ATP solution was diluted to 4X working concentration in kinase buffer (50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgCl2, 1 mM EGTA). ATP Km apparent value was determined previously using Z'-LYTE assay. Development reagent was diluted in development buffer (available from Invitrogen, catalog number P3127).

[0469] Assay protocol: 2.5 μL of 4X test compound or 100 nL of 100X test compound plus 2.4 μL of kinase buffer, 5 μL of 2X peptide / kinase mix, 2.5 μL of 4X ATP solution were added to the plate and placed on a shaker for 30 seconds. The kinase reaction was carried out at room temperature for 60 minutes before adding 5 μL of developer solution and the mixture was shaken on a shaker for 30 seconds. The reaction mixture was incubated at room temperature for 60 minutes. Fluorescence was measured using a plate reader and the data analyzed.

[0470] The maximum emission ratio is established by a 0% phosphorylation control (100% inhibition control), which does not contain ATP and therefore does not exhibit kinase activity. This control produces 100% cleavage peptides in the development reaction. A 100% phosphorylation control consisting of a synthetic phosphorylated peptide with the same sequence as the peptide substrate is designed to calculate the percentage of phosphorylation. This control produces a very low percentage of cleavage peptides in the development reaction. The 0% phosphorylation and 100% phosphorylation controls allow calculation of the percentage of phosphorylation obtained in a particular reaction well. The control wells do not include any kinase inhibitors.

[0471] A 0% inhibition control containing active kinase was used to establish the minimum emission ratio in the screen. This control was designed to produce 10-50% phosphorylated peptides in the kinase reaction. Cascade analysis can produce up to 70% phosphorylated peptides.

[0472] Run a known inhibitor control standard curve (10-point titration) for each individual kinase on the same plate as the kinase to ensure that the kinase is within the expected IC determined previously. 50 The inner range is suppressed.

[0473] Prepare the following controls for each concentration of test compound analyzed. Establish development reaction interference by comparing test compound control wells without ATP to 0% phosphorylation controls (which contain no test compound). The expected value for a non-interfering compound should be 100%. Any values ​​outside of 90% to 110% are marked. Test compound fluorescence interference is determined by comparing test compound control wells without kinase / peptide mixture (zero peptide control) to 0% inhibition controls. The expected value for a non-fluorescent compound should be 0%. Any values ​​> 20% are marked.

[0474] The data in Table A were calculated. XLfit of IDBS was used. The dose response curves were fit to the 205 model (sigmoidal dose response model). If the bottom of the curve did not fit between -20% and 20% inhibition, it was set to 0% inhibition. If the top of the curve did not fit between 70% and 130% inhibition, it was set to 100% inhibition.

[0475] Table A

[0476]

[0477] FI = Fluorescence intensity

[0478] C 100％ = Average coumarin emission signal of 100% Phos control

[0479] C 0％ = Average coumarin emission signal of 0% Phos control

[0480] F 100％ = 100% average fluorescence emission signal of Phos control

[0481] F 0％ = Mean fluorescence emission signal of 0% Fhos control

[0482] DRI = Development Reaction = Disturbance

[0483] TCFI = Test Compound Fluorescence Interference

[0484] Table 3 lists the MEK1 inhibition analysis results of the selected compounds according to the above steps. A represents IC 50 Less than or equal to 150nM, B means IC 50 Greater than 150 nM and less than or equal to 1.5 μM, and C represents IC 50 Greater than 1.5μM.

[0485] Table 3: MEK1 inhibition analysis results

[0486]

[0487]

[0488] Example 40: Cell-based assay-1

[0489] Preparation of cell lines for testing soft MEK inhibitors in NF1-related cell proliferation assays can be found in Basu et al., Nature 356:713-715, 1992; and DeClue et al., Cell 69:265-273, 1992. In addition, exemplary in vitro and in vivo models for determining the efficacy of the soft MEK inhibitors described herein can be found in U.S. Pat. Nos. 8,211,875 and 8,487,004, the entire contents of which are incorporated by reference.

[0490] Example 41: Cell-based assay-2

[0491] Alternatively, cell-based activity was determined using the following method. Test compounds were dissolved in DMSO as 10 nM stock solutions. Cell titer - 2.0 Luminescent cell viability assay reagents were purchased from Promega (Madison, Wisconsin). A375 and HCT116 cell lines were purchased from the American Type Culture Collection (Manassas, Virginia). For A375 cells, the cell culture medium was DMEM + 10% FBS. The cell culture medium is listed in the table below. For HCT116 cells, the cell culture medium was McCoy's 5A + 10% FBS. All culture media were supplemented with 100 μg / mL penicillin and 100 μg / mL streptomycin. Cultures were maintained at 37°C, a humidified atmosphere of 5% CO2 and 95% air.

[0492] Test compounds were diluted in DMSO solution, starting from 10mM, 10 doses and 3-fold dilutions were performed in the source plate. 25nL of each test compound was delivered from the source plate to each well of a 384-well cell culture plate (T = final) by Echo550. 25 μL of culture medium containing 2000 A375 or HCT116 cells was added to each well of the cell culture plate (T = 0 and T = final) in duplicate. 25 μL of cell titer Glo2.0 reagent was added to each well of the cell culture plate (T = 0). The contents were mixed on an orbital shaker for 2 minutes and incubated at room temperature for 15 minutes to stabilize the luminescent signal. Luminescence was recorded with an Envision 2104 multi-label plate reader (Perkin Elmer, Santa Clara, California). The number of viable cells in the culture medium was determined based on the ATP present in each culture well quantitatively. The cells in the cell culture plate (T = final) were incubated with compounds at 37 ° C, 5% CO2 for 72 hours. 25 μL of Cell Titer Glo2.0 reagent was added to each well. The contents were mixed on an orbital shaker for 2 minutes and incubated at room temperature for 15 minutes to stabilize the luminescent signal. Luminescence was recorded using an Envision 2104 multi-label plate reader (PerkinElmer, Santa Clara, California). The number of viable cells in the culture medium was determined based on the quantification of ATP present in each culture well. GI was plotted using the GraphPad Prism4 program based on the sigmoidal dose-response equation Y=Bottom+(Top-Bottom) / (1+10^((LogEC50-X)*HillSlope)) 50 All parameters in the equation were calculated using GraphPad Prism 4 program. 50 According to [(T i -T z ) / (CT z )]*100=50, where T i T = data of the cell line containing the test compound at the end; T zA is the data of the cell line without compound at T = 0 h; C is the data of the cell line with control compound staurosporine (Sigma-Aldrich) at T = 72 h. 50 Serves 10 X where X is calculated using Excel using a curve fitting equation at Y=50.

[0493] Example 42: S9 stability analysis

[0494] The metabolic stability of a compound in human skin can be assessed by evaluating the disappearance rate of the compound in human S9 skin fragments. Similarly, the metabolic stability of a compound in human liver can be assessed by evaluating the disappearance rate of the compound in human S9 liver fractions. The following protocol was used to assess the differences between skin and liver metabolism.

[0495] The analysis was performed at 37°C in a 96-well microtiter plate. The reaction mixture (25 μL) contained a final concentration of 1 μM test compound, 2 mg / mL liver or skin protein, and 1 mM NADPH in a buffer (100 mM potassium phosphate, pH 7.4 buffer, containing 1 mM EDTA and 3 mM MgCl2). At each time point (0, 15, 30, and 60 minutes), 150 μL of a quenching solution (100% acetonitrile, containing 0.1% formic acid) containing an internal standard was transferred to each well. In addition to the zero-minute control, a mixture containing the same components except NADPH was prepared as a negative control. Verapamil or testosterone was included as a positive control to verify the performance of the analysis. The plate was sealed and centrifuged at 4000 rpm for 15 minutes at 4°C. The supernatant was transferred to a new plate for LC / MS / MS analysis.

[0496] All samples were analyzed by LC / MS / MS using an AB Sciex API 4000 instrument coupled to a Shimadzu LC-20AD LC pump system. The samples were separated and analyzed using a Waters Atlantis T3 dC18 reverse phase HPLC column (20 mm x 2.1 mm) at a flow rate of 0.5 mL / min. The mobile phases included 0.1% formic acid in water (solvent A) and 0.1% formic acid in 100% acetonitrile (solvent B).

[0497] The extent of metabolism was calculated as the disappearance of the test compound compared to a 0 minute control reaction incubation. The initial rate was calculated for the compound concentration and used to determine t 1 / 2 The intrinsic clearance, CL int =(0.693)(1 / t 1 / 2 (min)) (mL incubation / mg of S9 protein).

[0498] Example 43: Microsome Stability Analysis

[0499] Human liver microsomes can be used to assess the metabolic stability of test compounds to predict intrinsic clearance. Human liver microsomes were obtained from Corning Gentest.

[0500] The analysis was performed at 37°C in a 96-well microtiter plate. The reaction mixture (25 μL) contained a final concentration of 1 μM test compound, 0.5 mg / mL liver microsomal protein, and 1 mM NADPH in a buffer (100 mM potassium phosphate, pH 7.4 buffer, containing 3 mM MgCl2). At each time point (0, 15, 30, and 60 minutes), 150 μL of a quenching solution (100% acetonitrile, containing 0.1% formic acid) containing an internal standard was transferred to each well. Verapamil was included as a positive control to verify the performance of the analysis. The plate was sealed and centrifuged at 4000 rpm for 15 minutes at 4°C. The supernatant was transferred to a new plate for LC / MS / MS analysis.

[0501] All samples were analyzed by LC / MS / MS using an AB Sciex API 4000 instrument coupled to a Shimadzu LC-20AD LC pump system. The samples were separated and analyzed at a flow rate of 0.5 mL / min using a Waters Atlantis T3 dC18 reversed phase HPLC column (20 mm x 2.1 mm). The mobile phase consisted of 0.1% formic acid in water (solvent A) and 0.1% formic acid in 100% acetonitrile (solvent B).

[0502] The extent of metabolism was calculated as the disappearance of the test compound compared to the 0 min incubation. The initial rate was used to calculate the compound and to determine t 1 / 2 The intrinsic clearance, CL int =(0.693)(1 / t 1 / 2 (min)) (g liver / kg body weight) (mL incubation / mg microsomal protein) (45 mg microsomal protein / g liver weight).

[0503] Example 44: Test results of the test compound

[0504] The following applies to Table 4 below.

[0505] Assay 1 was the biochemical MEK IC as described in Example 38 50 (nM) analysis. A1 indicates IC 50 Less than or equal to 150nM, B1 means IC 50 Greater than 150 nM and less than or equal to 1.5 μM.

[0506] Analysis 2 was based on A375 (BRAF) GI as described in Example 41.50 (nM) for cell analysis. A2 indicates IC 50 Less than or equal to 500nM, B2 means IC 50 Greater than 500 nM and less than or equal to 1.5 μM.

[0507] Assay 3 was performed as described in Example 41 based on the HCT116 (Kras) GI 50 (nM) for cell analysis. A3 indicates IC 50 Less than or equal to 750nM, B3 means IC 50 Greater than 750 nM and less than or equal to 2 μM.

[0508] Assay 4 is a liver S9 half-life stability assay as described in Example 42. A4 indicates a half-life greater than 50 minutes and less than or equal to 200 minutes, and B4 indicates a half-life less than or equal to 50 minutes.

[0509] Table 4: Test results of the tested compounds

[0510]

[0511] Example 45: In vivo model

[0512] Research steps Topical formulations of the compounds described herein and vehicle were applied to the skin of nude mice in duplicate. The skin was biopsied at discrete time intervals and split in half, half frozen in liquid nitrogen and half formalin fixed and paraffin embedded. Proteins were isolated for Western blot analysis to analyze p-ERK levels. Immunostaining for p-ERK was performed on FFPE sections for cell-specific analysis of p-ERK levels. Other analyses included H&E staining to investigate skin integrity.

[0513] The compounds were evaluated in terms of inhibition of p-ERK, a downstream biomarker of RAS / MAPK signaling in mouse skin. In addition, proliferation in mouse skin, apoptosis in mouse skin, and histological integrity of mouse skin were also evaluated.

[0514] Mouse : Before the study began, 129 mice aged 8 weeks obtained from Jackson Laboratory were shaved. About 21 mice were used for the study. The compound was applied to the hairless dorsal skin of the mice, and skin biopsies were obtained using 6 mm punch biopsies before treatment, 24 hours, 72 hours, and 96 hours at 12 hour intervals.

[0515] Western blot analysis: For immunoblotting, epidermal skin was snap frozen in liquid nitrogen immediately after biopsy. Epidermis was lysed in lysis buffer and run on western blot. Antibodies used for immunoblotting included rabbit anti-phospho-p44 / 42 MAPK (1:3000, Cell Signaling) and rabbit anti-p44 / 42 MAPK (1:3000, Cell Signaling), mouse anti-actin (1:5000, Sigma-Aldrich), donkey anti-mouse IgG conjugated to horseradish peroxidase (HRP; 1:40000, Amersham Biosciences) and goat anti-rabbit IgG conjugated to HRP (1:40000, Jackson ImmunoResearch).

[0516] Immunohistochemistry was performed on 5 μm paraffin sections. Antigen retrieval was performed using standard protocols with enzyme treatment (1:1000). The antibody used was rabbit p-ERK (Cell Signaling, 4307S, 1:100). Bonded polymer optimized anti-rabbit HRP detection agent (Leica Biosystems) was used according to the manufacturer's protocol. The sections were then counterstained with hematoxylin, dehydrated, and covered with TissueTek-Prisma and coverslipper (Sakura).

[0517] Histological analysis : H&E was performed on 5 μM paraffin sections and tissues were examined to assess cytotoxicity, inflammation, or other changes in mouse skin integrity.

[0518] Exogenous RAS activation in mouse skin : Experiments will be performed on untreated mouse skin. Additionally, the skin was pretreated with TPA to increase p-ERK levels. TPA-induced RAS / MAPK activation was performed using 12.5uG of TPA in 100μL of acetone to nude mouse skin for 96 hours. Studies were performed 48 hours after TPA exposure.

[0519] T-tests were used to assess differences in p-ERK and Ki-67 in samples treated with topical MEK1 inhibitors compared to vehicle controls.

[0520] Example 46: In vivo model

[0521] The compounds described herein are tested in a NF1 mouse model (e.g., a genetically modified mouse model of NF1, a model of human dermal neurofibromas xenografted into nude mice, or both). flox / floxThe Dhh-Cre mouse model described by Jousma et al. Pediatr. Blood Cancer 62:1709-1716 was used in this study. Magnetic resonance imaging (MRI) and volumetry were used to measure tumor volume.

[0522] Example 47: Human Dermal Neurofibroma Explant Protocol

[0523] Dermal neurofibromas (or cutaneous neurofibromas) are benign tumors that develop in individuals affected by neurofibromatosis type 1 (NF1), a rare genetic disease caused by mutations in the NF1 gene that lead to downstream activation of the RAS / MAPK pathway. Recent studies have shown that inhibition of MEK1 using a systemic MEK inhibitor can suppress neurofibromas and other NF-1-related tumors in mouse models. See, e.g., New Engl J Med 2016, 375; 26; J Clin Invest. 2013, 123(1), 340–347; and Pediatr Blood Cancer 2015, 62(10), 1709–1716. This study established an in vitro neurofibroma explant model.

[0524] Purpose : The primary objective is to evaluate the efficacy of compounds described herein or topically formulated compounds described herein in inhibiting p-ERK, a downstream biomarker of RAS / MAPK signaling in neurofibroma explants. A secondary objective is to evaluate the permeability of neurofibroma explants treated with compounds described herein (topical application of the compound).

[0525] Solution-1:

[0526] Sample Collection and Eligibility Primary dermal neurofibromas or cutaneous neurofibromas were obtained from patients with a clinical or genetic diagnosis of NF1. Discarded human neurofibroma samples were obtained from the Stanford Surgery Clinic using approved human subjects protocols (Stanford IRB #18325). Specimens were identified under the guidance of the principal investigator and placed in cell proliferation medium (DMEM / F12 with penicillin / streptomycin (0.1%); amphotericin B (40 μg / mL); B27 (without vitamin A).

[0527] Patients were enrolled in the study if they were older than 18 years; had not been receiving chemotherapy at the time of biopsy; and had a clinical and / or genetic diagnosis of NF1 based on the presence of two of the following:

[0528] 1. Six or more café-au-lait macules with a diameter greater than 5 mm in prepubertal individuals and a maximum diameter greater than 15 mm in postpubertal individuals.

[0529] 2. Two or more neurofibromas of any type or one plexiform neurofibroma.

[0530] 3. Freckles in the armpit or groin area.

[0531] 4. Two or more Lisch nodules (iris hamartomas).

[0532] 5. Optic nerve glioma.

[0533] 6. Unique bony lesions, such as sphenoid dysplasia or cortical thinning of long bones, with or without pseudoarthrosis.

[0534] 7. First-degree relative (parent, sibling, or offspring) with NF-1 according to the above criteria

[0535] Research steps : The specimens were primary, untreated neurofibromas, at least 6 mm in size; the specimens were excised by shave, punch biopsy, or elliptical excision; the specimens were histologically diagnosed as dermal neurofibromas or cutaneous neurofibromas. The specimens were identified under the guidance of the principal investigator.

[0536] The samples were cut into 2 mm fragments and placed in 24-well plates containing cell proliferation medium (DMEM / F12 with penicillin / streptomycin (0.1%); amphotericin B (40 μg / mL); B27 (without vitamin A)) and immersed in the medium containing the drug. For topical gel application, the samples were placed in 96-well plates with the epidermal surface exposed to air.

[0537] Western blot analysis : For immunoblotting, whole skin biopsies were lysed in lysis buffer and subjected to Western blot analysis. Antibodies used for immunoblotting included rabbit anti-phospho-p44 / 42 MAPK (1:3000, Cell Signaling) and rabbit anti-p44 / 42 MAPK (1:3000, Cell Signaling), rabbit anti-phospho-Mek1 / 2 (1:3000, Cell Signaling), mouse anti-actin (1:5000, Sigma-Aldrich), donkey anti-mouse IgG conjugated to horseradish peroxidase (HRP; 1:40000, Amersham Biosciences), and goat anti-rabbit IgG conjugated to HRP (1:40000, Jackson ImmunoResearch).

[0538] Immunohistochemistry was performed on 5 μm paraffin sections. Antigen retrieval was performed using standard protocols with enzyme treatment (1:10 00). The antibody used was rabbit p-ERK (Cell Signaling, 4307S, 1:100). A bonded polymer optimized anti-rabbit HRP detector (Leica Biosystems) was used according to the manufacturer's protocol. The sections were then counterstained with hematoxylin, dehydrated, and covered with a TissueTek-Prisma and coverslipper (Sakura).

[0539] Data analysis : Semi-quantitative Western blot was used to assess differences in p-ERK in samples treated with compounds described herein compared to vehicle controls.

[0540] Research Administration : This study was conducted under the supervision of the IRB, and informed consent and HIPAA authorization were obtained from the patients.

[0541] Solution-2:

[0542] Explant protocol Human skin neurofibroma explant samples were collected in DMEM / F-12 (Thermo Fisher, CAT#11320033), supplemented with 1×B27 supplement (Thermo Fisher, CAT#17504044), 2.5 μg / ml amphotericin B (Thermo Fisher, CAT#15290018) and 50 units / ml penicillin-50 μg / ml streptomycin (Thermo Fisher, CAT#15070063) and incubated in the same medium for subsequent processing. The specimens were cut into small cubes containing epidermis and dermis. The tissue was partially immersed in the culture medium of a 384-well plate, and the epidermis was exposed to air.

[0543] The tissue was completely immersed in the culture medium of the 48-well plate, and 5 μl of the compound dissolved in DMSO was added to 200 μL of culture medium in each well. After incubation at 37°C and 5% CO2 for 4 h, the tissue was harvested and half of the specimen was snap-frozen in liquid nitrogen for western blot analysis. The other half of the specimen was fixed in 10% formalin for 24 h and then transferred to 70% ethanol for western blot analysis.

[0544] The skin samples were thawed on ice and weighed. Then 10 volumes (10 μL per mg tissue) of lysis buffer (RIPA buffer + 0.5 mM EDTA + 1× stop protease and phosphatase inhibitor cocktail) were added to each sample. The samples were cut into small pieces and homogenized with an asonicating probe on ice. The homogenized samples were centrifuged at 12000 rpm for 10 minutes at 4°C. The supernatant was transferred to a new tube and stored at -80°C until Western blot analysis.

[0545] Western blot analysis :The lysate was thawed on ice and the protein concentration was determined by BCA protein assay kit using bovine serum albumin (BSA) as standard. All samples were diluted with lysis buffer to achieve the same final concentration. 10-20 μg of total protein was loaded into each well and separated on NuPAGE 4-12% Bis-Tris gel (Thermo Fisher) in 1×NuPAGE MES SDS running buffer (Thermo Fisher, cat# NP0002). The protein was then transferred to a PVDF membrane. The membrane was then blocked for 1 hour in 1×TBST (Tris buffered saline + 0.1% Tween 20) containing 5% skim milk (for total ERK) or 5% BSA (for phospho-ERK and α-tubulin). The following primary antibodies were used (diluted in the same blocking solution): 1:3000 monoclonal rabbit anti-phospho-p44 / 42MAPK (Erk1 / 2) (Thr202 / Tyr204) antibody (Cell Signaling Technology, Cat#4370L), 1:3000 monoclonal rabbit anti-p44 / 42MAPK (Erk1 / 2) antibody (Cell Signaling Technology, Cat#4695S), 1:3000-1:4000 monoclonal mouse anti-α-tubulin (DM1A) antibody (Cell Signaling Technology, Cat#3873S). The membrane was incubated with the primary antibodies overnight at 4°C and then washed three times with 1×TBST. Secondary antibodies goat anti-rabbit IgG (H + L), HRP (Thermo Fisher, Cat # 31460) and peroxidase-conjugated affinipure goat anti-mouse IgG (H + L) (Jackson ImmunoResearch, Cat # 115-035-062) were diluted at the same concentration as the primary antibody in 1× TBST containing 2% skim milk (for total ERK) or 2% BSA (for phospho-ERK) and a-tubulin and incubated at room temperature for 1-3 hours. After washing three times with 1× TBST, the blot was developed with WesternBright ECL HRP substrate (Advansta, cat # K12045-D50).

[0546] Immunohistochemistry Antigen retrieval was performed by enzyme treatment. Sections were blocked with 10% normal goat serum and then incubated in 1:100 diluted rabbit monoclonal antibody for phosphorylated-p44 / 42 MAPK (ERK1 / 2) (Cell Signaling Technology) or mouse anti-Ki-67 (Pharmingen) at room temperature for 60 minutes. Detection was performed using a peroxidase-conjugated anti-rabbit system (Leica Biosystems).

[0547] Data analysis : Semi-quantitative Western blot was used to assess differences in p-ERK in samples treated with compounds described herein compared to vehicle controls.

[0548] Figure 4 Inhibition of pERK in human cutaneous neurofibroma (cNF) explants after 4 hours of treatment with compound 1.030 (at concentrations of 5 nM, 50 nM and 500 nM) is demonstrated.

[0549] Figure 5 Inhibition of pERK in human cutaneous neurofibroma (cNF) explants following treatment with compound 1.030 (at a concentration of 500 nM) is demonstrated.

[0550] Although the foregoing invention has been described in detail by way of illustration and example for the purpose of clear understanding, it will be appreciated by those skilled in the art that certain changes and modifications may be implemented within the scope of the appended claims. In addition, each reference provided herein is incorporated by reference in its entirety to the same extent as each reference is incorporated by reference individually. In the event of a conflict between the present application and the references provided herein, the present application shall prevail.

Claims

1. A compound selected from the group consisting of: or a pharmaceutically acceptable salt thereof.

2. The compound according to claim 1, characterized in that As shown below: or a pharmaceutically acceptable salt thereof.

3. The compound according to claim 1, characterized in that As shown below: or a pharmaceutically acceptable salt thereof.

4. A pharmaceutical composition comprising the compound according to any one of claims 1 to 3 and a pharmaceutically acceptable carrier.

5. Use of the compound of claim 3 in the preparation of a medicament for treating a MEK inhibitor-responsive skin disorder or a MEK-mediated skin disorder characterized by elevated p-ERK levels; wherein, The MEK inhibitor-responsive skin disorder or MEK-mediated skin disorder is neurofibromatosis type 1.

Images