Attorney Docket No.: P-381-WO/71TD-385423-WO TRIAZOLO INDAZOLE COMPOUNDS AS JAK INHIBITORS CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Patent Application No.63/633,204, filed April 12, 2024, which is hereby incorporated by reference in its entirety. 5 FIELD Provided herein are compounds useful as Janus kinase (JAK) inhibitors. Also provided herein are pharmaceutical compositions comprising such compounds and methods of using such compounds to treat, e.g., inflammatory and fibrotic diseases, including respiratory diseases, autoimmune diseases, and cancers. 10 BACKGROUND The JAK family comprises four members: JAK1, JAK2, JAK3, and tyrosine kinase 2 (TYK2). Binding of cytokine to a JAK-dependent cytokine receptor induces receptor dimerization, which results in phosphorylation of tyrosine residues on the JAK kinase, effecting JAK activation. Phosphorylated JAKs, in turn, bind and phosphorylate various STAT proteins, which dimerize, internalize in the cell 15 nucleus, and directly modulate gene transcription, leading, among other effects, to the downstream effects associated with inflammatory disease. The JAKs usually associate with cytokine receptors in pairs as homodimers or heterodimers. Specific cytokines are associated with specific JAK pairings. Selective modulation of individual JAKs is a promising therapy. For example, selective inhibition of JAK1 is expected to be of therapeutic benefit for a range of inflammatory conditions, such 20 as rheumatoid arthritis (RA), and noninflammatory diseases driven by JAK1-mediated signal transduction. Further, JAK1 has also been involved in hyperproliferative disorders since it was found to be mutated in acute myeloid leukemia, acute lymphoblastic leukemia, and in uterine leiomyosarcoma. Accordingly, a selective JAK1 inhibitor is an attractive therapeutic agent for a number of diseases. It is contemplated that safe therapy should minimize inhibition of JAK2 as JAK2 plays an integral role in the 25 erythropoietin (EPO) signaling pathway. It also has been found that selectivity for JAK1 over JAK2 can widen the therapeutic index for JAK1-mediated effects relative to JAK2-dependent anemia. SUMMARY Provided herein are compounds of formula (I): 30 ein.
nd as described herein, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. 1
Attorney Docket No.: P-381-WO/71TD-385423-WO Also provided herein is a method of treating an inflammatory disease (for example, a respiratory disease, such as asthma) and lung rejection, in a mammal, the method comprising administering to the mammal (or human) a compound as described herein or a pharmaceutically acceptable salt thereof. The present disclosure also provides a compound as described herein, or a pharmaceutically 5 acceptable salt thereof, for use in therapy (for example, medical therapy, such as treating a respiratory disease) as well as the use of such compound in the manufacture of a formulation or medicament for treating an inflammatory disease (such as a respiratory disease) in a mammal (e.g., a human). DETAILED DESCRIPTION Definitions 10 When describing this disclosure including its various aspects and embodiments, the following terms have the following meanings, unless otherwise indicated. The term “alkyl” means a monovalent saturated hydrocarbon group which may be linear or branched or combinations thereof. Unless otherwise defined, such alkyl groups typically contain from 1 to 10 carbon atoms. Representative alkyl groups include, by way of example, methyl (Me), ethyl (Et), n-15 propyl (n-Pr) or (nPr), isopropyl (i-Pr) or (iPr), n-butyl (n-Bu) or (nBu), sec-butyl, isobutyl, tert-butyl (t- Bu) or (tBu), n-pentyl, n-hexyl, 2,2-dimethylpropyl, 2-methylbutyl, 3-methylbutyl, 2-ethylbutyl, 2,2- dimethylpentyl, 2-propylpentyl, and the like. When a specific number of carbon atoms are intended for a particular term, the number of carbon atoms is shown preceding the term. For example, the term “C1-3 alkyl” means an alkyl group having 20 from 1 to 3 carbon atoms wherein the carbon atoms are in any chemically-acceptable configuration, including linear or branched configurations. “Alkoxy” refers to the group “alkyl-O-”. Examples of alkoxy groups include, e.g., methoxy, ethoxy, n-propoxy, iso-propoxy, n-butoxy, tert-butoxy, sec-butoxy, n-pentoxy, n-hexoxy and 1,2- dimethylbutoxy. 25 The term “cycloalkyl” means a monovalent saturated or partially unsaturated carbocyclic group which may be monocyclic or multicyclic. Unless otherwise defined, such cycloalkyl groups typically contain from 3 to 10 carbon atoms. Representative cycloalkyl groups include, by way of example, cyclopropyl (cPr), cyclobutyl (cBu), cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, adamantyl, and the like. 30 In some embodiments, the compounds of this disclosure are capable of forming acid and/or base salts by virtue of the presence of amino and/or carboxyl groups or groups similar thereto. Base addition salts can be prepared from inorganic and organic bases. Salts derived from inorganic bases include, by way of example only, sodium, potassium, lithium, ammonium, calcium and magnesium salts. Salts derived from organic bases include, but are not limited to, salts of primary, secondary and tertiary 35 amines. Specific examples of suitable amines include, by way of example only, isopropylamine, trimethyl amine, diethyl amine, tri(iso-propyl) amine, tri(n-propyl) amine, ethanolamine, 2- dimethylaminoethanol, tromethamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, 2
Attorney Docket No.: P-381-WO/71TD-385423-WO choline, betaine, ethylenediamine, glucosamine, N-alkylglucamines, theobromine, purines, piperazine, piperidine, morpholine, N-ethylpiperidine, and the like. Acid addition salts may be prepared from inorganic and organic acids. Salts derived from inorganic acids include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like. Salts derived from organic acids include 5 acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluene-sulfonic acid, salicylic acid, and the like. The term “pharmaceutically acceptable salt” means a salt that is acceptable for administration to a patient or a mammal, such as a human (e.g., salts having acceptable mammalian safety for a given 10 dosage regime). Representative pharmaceutically acceptable salts include salts of acetic, ascorbic, benzenesulfonic, benzoic, camphorsulfonic, citric, ethanesulfonic, edisylic, fumaric, gentisic, gluconic, glucoronic, glutamic, hippuric, hydrobromic, hydrochloric, isethionic, lactic, lactobionic, maleic, malic, mandelic, methanesulfonic, mucic, naphthalenesulfonic, naphthalene-1,5-disulfonic, naphthalene-2,6- disulfonic, nicotinic, nitric, orotic, pamoic, pantothenic, phosphoric, succinic, sulfuric, tartaric, p- 15 toluenesulfonic and xinafoic acid, and the like. The term “therapeutically effective amount” means an amount sufficient to effect treatment when administered to a patient in need of treatment. The term “treating” or “treatment” means ameliorating or suppressing the medical condition, disease or disorder being treated (e.g., a respiratory disease) in a patient (particularly a human); or 20 alleviating the symptoms of the medical condition, disease or disorder. The term “hydroxy protecting group” refers to a chemical moiety which is added to, and later removed from, a hydroxy functionality to obtain chemoselectivity in a subsequent chemical reaction. Exemplary protecting groups, as well as the methods for deprotection, include, but are not limited to, acetyl (Ac) (removed by acid or base), benzoyl (Bz) (removed by acid or base), benzyl (Bn) 25 (removed by hydrogenolysis), β-methoxyethoxymethyl ether (MEM) (removed by acid), dimethoxytrityl or [bis-(4-methoxyphenyl)phenylmethyl] (DMT) (removed by weak acid), methoxymethyl ether (MOM) (removed by acid), methoxytrityl or [(4-methoxyphenyl)diphenylmethyl] (MMT) (removed by acid and hydrogenolysis), p-methoxybenzyl ether (PMB) (removed by acid, hydrogenolysis, or oxidation), methylthiomethyl ether (removed by acid), pivaloyl (Piv) (removed by acid, base or reductant agents), 30 tetrahydropyranyl (THP) (removed by acid), tetrahydrofuran (THF) (removed by acid), trityl (triphenylmethyl, Tr) (removed by acid and hydrogenolysis), silyl ether (e.g., trimethylsilyl (TMS), tert- butyldimethylsilyl (TBDMS), tri-iso-propylsilyloxymethyl (TOM), and triisopropylsilyl (TIPS) ethers) (removed by acid or fluoride ion, such as NaF, TBAF (tetra-n-butylammonium fluoride, HF-Py, or HF- NEt3)), methyl ethers (removed by cleavage such as TMSI in dichloromethane or acetonitrile or 35 chloroform, or BBr3 in DCM), ethoxyethyl ethers (EE) (removed by 1N hydrochloric acid). The term “amine protecting group” refers to a chemical moiety which is added to, and later removed from, an amine functionality to obtain chemoselectivity in a subsequent chemical reaction. Exemplary protecting groups, as well as the methods for deprotection, include, but are not limited to, 9- 3
Attorney Docket No.: P-381-WO/71TD-385423-WO fluorenylmethoxycarbonyl (Fmoc) (removed by base), tert-butyloxycarbonyl (Boc) (removed by strong acid), carboxybenzyl (Cbz) (removed by hydrogenolysis), acetyl (Ac) (removed by base), benzyl (Bn) (removed by hydrogenolysis), benzoyl (Bz) (removed by base), carbamate (removed by acid and mild heating), p-methoxybenzyl (PMB) (removed by hydrogenolysis), 3,4-dimethoxybenzyl (DMPM) 5 (removed by hydrogenolysis), p-methoxyphenyl (PMP) (removed by ammonium cerium(IV) nitrate (CAN)), tosyl (Ts) (removed by concentrated acid and strong reducing agents), and 2,2,2- trichloroethoxycarbonyl (Troc) (removed by Zn insertion in the presence of acetic acid). Provided herein are compounds of formula (I’): I’) 10 or a pharmaceutically acceptab R1 and R1a
are each independently H or F, provided that at least one of R1 and R1a is H; , or 15
R4 is C1-C3 alkyl optionally substituted with hydroxy or C1-C3 alkoxy; R5 is C1-C3 alkyl; R5a and R5b are each independently C1-C3 alkyl; R6 is H; C1-C3 alkyl optionally substituted with C1-C3 alkoxy or cyano; or C3-5 cycloalkyl; and 20 R7 is H, F, or hydroxy. In some embodiments, R1 is H. In some embodiments, R1a is H. In some embodiments, R1 is F. In some embodiments, R1a is F. In some embodiments, R5 is C1-C2 alkyl. In some embodiments, R5 is -CH3. In some embodiments, R5 is -CH2CH3. 4
Attorney Docket No.: P-381-WO/71TD-385423-WO In some embodiments, R5a is -CH3, and R5b is -CH3. In some embodiments, R4 is -CH3, R5a is -CH3, and R5b is -CH3. In some embodiments, R6 is H; C1-C2 alkyl optionally substituted with C1-C2 alkoxy or cyano; or C3-5 cycloalkyl. 5 In some embodiments, R7 is H. In some embodiments, R7 is F or hydroxy. Also provided herein are compounds of formula (I): 10
R4 is C1-C3 alkyl optionally substituted with hydroxy or C1-C3 alkoxy; R5 is -CH2CH3; 15 R6 is C1-C3 alkyl optionally substituted with C1-C3 alkoxy; or R6 is C3-5 cycloalkyl. In some embodiments, R1 is H or F; ;
R4 is C1-C3 alkyl optionally substituted with hydroxy; 20 R6 is C1-C3 alkyl optionally substituted with C1-C3 alkoxy; or R6 is C3-5 cycloalkyl; provided tha , then R1 is H.
5
Attorney Docket No.: P-381-WO/71TD-385423-WO In some embodiments, R3 is C1-C2alkyl optionally substituted with hydroxy or C1-C2alkoxy. In some embodiments, R3 is C1-C2alkyl optionally substituted with hydroxy. In some embodiments, R3 is C1-C2 alkyl optionally substituted with C1-C2 alkoxy. In some embodiments, R3 is C1-C2 alkyl optionally substituted with methoxy. In some embodiments, R3 is -CH3, 5 -CH2CH3, or -CH2-O-CH3. In some embodiments, R4 is C1-C3 alkyl optionally substituted with hydroxy. In some embodiments, R4 is C1-C3 alkyl optionally substituted with C1-C3 alkoxy. In some embodiments, R4 is C1-C2 alkyl optionally substituted with hydroxy or C1-C3 alkoxy. In some embodiments, R4 is C1-C2 alkyl optionally substituted with hydroxy or C1-C2 alkoxy. In some 10 embodiments, R4 is C1-C2 alkyl optionally substituted with hydroxy or methoxy. In some embodiments, R4 is -CH3, -CH2CH3, -CH2CH2-OH, or -CH2CH2-O-CH3. In some embodiments, R4 is -CH3 or -CH2CH3. In some embodiments, R4 is -CH3. In some embodiments, R6 is C1-C3 alkyl optionally substituted with C1-C2 alkoxy or cyclopropyl. In some embodiments, R6 is C3-5 cycloalkyl. In some embodiments, R6 is cyclopropyl. 15 In some embodiments, R6 is C1-C3 alkyl optionally substituted with C1-C2 alkoxy. In some embodiments, R6 is C1-C3 alkyl optionally substituted with methoxy. In some embodiments, R6 is C1-C3 alkyl. In some embodiments, R6 is -CH3, -CH2CH3, -CH(CH3)2, -CH2CH2-O-CH3, or cyclopropyl. In some embodiments, R6 is -CH3, -CH2CH3, or -CH(CH3)2. In some embodiments, R6 is -CH3. 20 In some embodiments, R1 is H. In some embodiments, R1 is F. In some embodiments, R3 is -CH3, -CH2CH3, or -CH2-O-CH3; and R4 is -CH3, -CH2CH3, or -CH2CH2-OH. In some embodiments, R3 is -CH3, -CH2CH3, or -CH2-O-CH3; and R4 is -CH3. , 25
6
Attorney Docket No.: P-381-WO/71TD-385423-WO , , 5
7
Attorney Docket No.: P-381-WO/71TD-385423-WO , r
5 acceptable salt thereof. Table 1 Compound Structure No.
8
Attorney Docket No.: P-381-WO/71TD-385423-WO Compound Structure No.
9
Attorney Docket No.: P-381-WO/71TD-385423-WO Compound Structure No.
10
Attorney Docket No.: P-381-WO/71TD-385423-WO Compound Structure No. Some em
armaceutically acceptable salt thereof. Table 2 Compound Structure No.
11
Attorney Docket No.: P-381-WO/71TD-385423-WO Compound Structure No. 31
12
Attorney Docket No.: P-381-WO/71TD-385423-WO Compound Structure No. 40 Chemical implemented in
ChemDraw software (PerkinElmer, Inc., Cambridge, MA). Furthermore, the triazole portion of the compounds of the present disclosure exists in tautomeric forms. It will be understood that although structures are shown, or named, in a particular form, the 5 present disclosure also includes the tautomer thereof. The compounds of the present disclosure may contain one or more chiral centers and therefore, such compounds (and intermediates thereof) can exist as racemic mixtures; pure stereoisomers (i.e., enantiomers or diastereomers); stereoisomer-enriched mixtures and the like. Chiral compounds shown or named herein without a defined stereochemistry at a chiral center are intended to include any or all 10 possible stereoisomer variations at the undefined stereocenter unless otherwise indicated. The depiction or naming of a particular stereoisomer means the indicated stereocenter has the designated stereochemistry with the understanding that minor amounts of other stereoisomers may also be present unless otherwise indicated, provided that the utility of the depicted or named compound is not eliminated by the presence of another stereoisomer. 13
Attorney Docket No.: P-381-WO/71TD-385423-WO The compounds of the present disclosure may also contain several basic groups (e.g., amino groups) and therefore, such compounds can exist as the free base or in various salt forms, such a mono- protonated salt form, a di-protonated salt form, a tri-protonated salt form, etc or mixtures thereof. All such forms are included within the scope of this disclosure, unless otherwise indicated. 5 This disclosure also includes isotopically-labeled compounds of the present disclosure (such as isotopically-labeled compounds of formula (I’), i.e., compounds of formula (I’) where one or more atoms has been replaced or enriched with an atom having the same atomic number but an atomic mass different from the atomic mass that predominates in nature). Examples of isotopes that may be incorporated into a compound of the present disclosure, such as compounds of formula (I’) include, but are not limited to, 10 2H, 3H, 11C, 13C, 14C, 13N, 15N, 15O, 17O, and 18O. Some embodiments provide for compounds of the present disclosure, such as compounds of formula (I’), enriched in tritium or carbon-14, which compounds can be used, for example, in tissue distribution studies. In some embodiments, provided herein are compounds of the present disclosure, such as those of formula (I’), enriched in deuterium especially at a site of metabolism, which compounds are expected to have greater metabolic stability. In 15 some embodiments, provided herein are compounds of the present disclosure enriched in a positron emitting isotope, such as 11C, 15O and 13N, which compounds can be used, for example, in Positron Emission Tomography (PET) studies. General Synthetic Procedures Compounds of the present disclosure, and intermediates thereof, can be prepared according to the 20 following general methods and procedures. The substituents and variables (e.g., R1, R1a, R2, etc.) used in the following schemes have the same meanings as those defined elsewhere herein unless otherwise indicated. Additionally, compounds having an acidic or basic atom or functional group may be used or may be produced as a salt unless otherwise indicated (in some cases, the use of a salt in a particular reaction will require conversion of the salt to a non-salt form, e.g., a free base, using routine procedures 25 before conducting the reaction). Although a particular embodiment of the present disclosure may be shown or described in the following procedures, those skilled in the art will recognize that other embodiments or aspects of the present disclosure can also be prepared using such procedures or by using other methods, reagents, and starting materials known to those skilled in the art (including those with different protecting groups 30 known in the art). In particular, it will be appreciated that compounds of the present disclosure may be prepared by a variety of process routes in which reactants are combined in different orders to provide different intermediates en route to producing final products. General methods for preparing final compounds of the present disclosure are illustrated in the following schemes. Accordingly, some embodiments provide for methods of preparing a compound as 35 described herein. Aldehyde Intermediates 1, 2, 3, and 4 can be prepared as shown in the Example section. In some embodiments, compounds of the present disclosure may be synthesized according to the synthetic 14
Attorney Docket No.: P-381-WO/71TD-385423-WO scheme shown in Method A. In some embodiments, R1, R1a, and R2 are as described herein; PG1 is benzyl (Bn) or trimethylsilylethoxymethyl (SEM); PG2 is tetrahydropyranyl (THP); and PG3 is SEM. Method A 5 4)
undergoes reductive amination (such as under conditions as described herein) to provide intermediate A- 1, which is then subjected to deprotection conditions (such as those described herein) to provide compounds of formula (I’). In some embodiments, compounds of the present disclosure may be synthesized according to the 10 synthetic scheme shown in Method B. In some embodiments, R1, R1a, and R2 are as described herein; PG1 is Bn or SEM; PG2 is THP; PG3 is SEM; and PG4 is an amine protecting group (e.g., attached to a nitrogen atom of R2), such as t-butyloxycarbonyl (BOC). Method B 15
reductive amination (such as under conditions as described herein) to provide intermediate A-2, which is then subjected to standard deprotection conditions (such as those described herein). In some embodiments, compound A-2 is first subjected to deprotection conditions to cleave PG1, followed by deprotection conditions to cleave PG2, PG3, and PG4. In some embodiments, PG2, PG3, and PG4 can be 20 removed in one step or subsequent steps (e.g., in any order). After deprotection, the resulting intermediate may be further derivatized at an amine group of R2 via reductive amination (such as those described herein) to achieve compounds of formula (I’). In some embodiments, PG1 is a hydroxy protecting group. In some embodiments, PG2, PG3, and PG4 are independently an amine protecting group. 25 Pharmaceutical Compositions The compounds of the present disclosure and pharmaceutically acceptable salts thereof are typically used in the form of a pharmaceutical composition or formulation. Such pharmaceutical compositions may advantageously be administered to a patient by inhalation. In addition, pharmaceutical 15
Attorney Docket No.: P-381-WO/71TD-385423-WO compositions may be administered by any acceptable route of administration including, but not limited to, oral, rectal, nasal, topical (including transdermal) and parenteral modes of administration. Accordingly, in some embodiments, the disclosure is directed to a pharmaceutical composition comprising a pharmaceutically acceptable carrier or excipient and a compound of formula (I’) or (I) 5 where, as defined above, “compound of formula (I’)” means a compound of formula (I’) or a pharmaceutically acceptable salt thereof, and “compound of formula (I)” means a compound of formula (I) or a pharmaceutically acceptable salt thereof. Optionally, such pharmaceutical compositions may contain other therapeutic and/or formulating agents if desired. In some embodiments, such pharmaceutical compositions further comprise one or more other therapeutic agents. In some 10 embodiments, the one or more other therapeutic agents are useful for treating a respiratory disease in a mammal (e.g. a human). When discussing compositions and uses thereof, or “compound of the present disclosure” may also be referred to herein as the “active agent”. As used herein, the term “compound of the present disclosure” is intended to include all compounds encompassed by formula (I’) and (I) as well as the 15 species embodied in formula (I’) and (I), and compounds as described herein, and pharmaceutically acceptable salts thereof. The pharmaceutical compositions of the present disclosure typically contain a therapeutically effective amount of a compound of the present disclosure. Those skilled in the art will recognize, however, that a pharmaceutical composition may contain more than a therapeutically effective amount, 20 i.e., bulk compositions, or less than a therapeutically effective amount, i.e., individual unit doses designed for multiple administration to achieve a therapeutically effective amount. Typically, such pharmaceutical compositions will contain from about 0.01 to about 95% by weight of the active agent; including, for example, from about 0.05 to about 30% by weight; and from about 0.1 % to about 10% by weight of the active agent. In some embodiments, pharmaceutical 25 compositions contain from 0.1 mg to 100 mg of the active agent; including, for example, from 1 mg to 20 mg of the active agent including, for example, from 1 mg to 10 mg of the active agent. Any conventional carrier or excipient may be used in the pharmaceutical compositions of the present disclosure. The choice of a particular carrier or excipient, or combinations of carriers or excipients, will depend on the mode of administration being used to treat a particular patient or type of 30 medical condition or disease state. In this regard, the preparation of a suitable pharmaceutical composition for a particular mode of administration is well within the scope of those skilled in the pharmaceutical arts. Additionally, the carriers or excipients used in the pharmaceutical compositions of the present disclosure are commercially available. By way of further illustration, conventional formulation techniques are described in Remington: The Science and Practice of Pharmacy, 20th Edition, 35 Lippincott Williams & White, Baltimore, Maryland (2000); and H.C. Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, 7th Edition, Lippincott Williams & White, Baltimore, Maryland (1999). 16
Attorney Docket No.: P-381-WO/71TD-385423-WO Representative examples of materials which can serve as pharmaceutically acceptable carriers include, but are not limited to, the following: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, such as microcrystalline cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; 5 gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer’s solution; ethyl alcohol; phosphate buffer solutions; 10 and other non-toxic compatible substances employed in pharmaceutical compositions. Pharmaceutical compositions are typically prepared by thoroughly and intimately mixing or blending the active agent with a pharmaceutically acceptable carrier and one or more optional ingredients. The resulting uniformly blended mixture can then be shaped or loaded into tablets, capsules, pills and the like using conventional procedures and equipment. 15 In some embodiments, the pharmaceutical composition is suitable for inhaled administration. Pharmaceutical compositions for inhaled administration are typically in the form of an aerosol or a powder. Such compositions are generally administered using inhaler delivery devices, such as a dry powder inhaler (DPI), a metered-dose inhaler (MDI), a nebulizer inhaler, or a similar delivery device. In a particular embodiment, the pharmaceutical composition is administered by inhalation using 20 a dry powder inhaler. Such dry powder inhalers typically administer the pharmaceutical composition as a free-flowing powder that is dispersed in a patient’s airstream during inspiration. To achieve a free- flowing powder composition, the therapeutic agent is typically formulated with a suitable excipient such as lactose, starch, mannitol, dextrose, polylactic acid (PLA), polylactide-co-glycolide (PLGA) or combinations thereof. Typically, the therapeutic agent is micronized and combined with a suitable 25 carrier to form a composition suitable for inhalation. A representative pharmaceutical composition for use in a dry powder inhaler comprises lactose and a compound of the present disclosure in micronized form. Such a dry powder composition can be made, for example, by combining dry milled lactose with the therapeutic agent and then dry blending the components. The composition is then typically loaded into a dry powder dispenser, or into inhalation 30 cartridges or capsules for use with a dry powder delivery device. Dry powder inhaler delivery devices suitable for administering therapeutic agents by inhalation are described in the art and examples of such devices are commercially available. For example, representative dry powder inhaler delivery devices or products include Aeolizer (Novartis); Airmax (IVAX); ClickHaler (Innovata Biomed); Diskhaler (GlaxoSmithKline); Diskus/Accuhaler 35 (GlaxoSmithKline); Ellipta (GlaxoSmithKline); Easyhaler (Orion Pharma); Eclipse (Aventis); FlowCaps (Hovione); Handihaler (Boehringer Ingelheim); Pulvinal (Chiesi); Rotahaler (GlaxoSmithKline); SkyeHaler/Certihaler (SkyePharma); Twisthaler (Schering-Plough); Turbuhaler (AstraZeneca); Ultrahaler (Aventis); and the like. 17
Attorney Docket No.: P-381-WO/71TD-385423-WO In another particular embodiment, the pharmaceutical composition is administered by inhalation using a metered-dose inhaler. Such metered-dose inhalers typically discharge a measured amount of a therapeutic agent using a compressed propellant gas. Accordingly, pharmaceutical compositions administered using a metered-dose inhaler typically comprise a solution or suspension of the therapeutic 5 agent in a liquefied propellant. Any suitable liquefied propellant may be employed including hydrofluoroalkanes (HFAs), such as 1,1,1,2-tetrafluoroethane (HFA 134a) and 1,1,1,2,3,3,3-heptafluoro- n-propane, (HFA 227); and chlorofluorocarbons, such as CCl3F. In some embodiments, the propellant is one or more hydrofluoroalkanes. In some embodiments, the hydrofluoroalkane formulation contains a co-solvent, such as ethanol or pentane, and/or a surfactant, such as sorbitan trioleate, oleic acid, lecithin, 10 and glycerin. A representative pharmaceutical composition for use in a metered-dose inhaler comprises from about 0.01% to about 5% by weight of a compound of the present disclosure; from about 0% to about 20% by weight ethanol; and from about 0% to about 5% by weight surfactant; with the remainder being an HFA propellant. Such compositions are typically prepared by adding chilled or pressurized 15 hydrofluoroalkane to a suitable container containing the therapeutic agent, ethanol (if present) and the surfactant (if present). To prepare a suspension, the therapeutic agent is micronized and then combined with the propellant. The composition is then loaded into an aerosol canister, which typically forms a portion of a metered-dose inhaler device. Metered-dose inhaler devices suitable for administering therapeutic agents by inhalation are 20 described in the art and examples of such devices are commercially available. For example, representative metered-dose inhaler devices or products include AeroBid Inhaler System (Forest Pharmaceuticals); Atrovent Inhalation Aerosol (Boehringer Ingelheim); Flovent (GlaxoSmithKline); Maxair Inhaler (3M); Proventil Inhaler (Schering); Serevent Inhalation Aerosol (GlaxoSmithKline); and the like. 25 In some embodiments, the pharmaceutical composition is administered by inhalation using a nebulizer inhaler. Such nebulizer devices typically produce a stream of high velocity air that causes the pharmaceutical composition to spray as a mist that is carried into the patient's respiratory tract. Accordingly, when formulated for use in a nebulizer inhaler, the therapeutic agent can be dissolved in a suitable carrier to form a solution. Alternatively, the therapeutic agent can be micronized or nanomilled 30 and combined with a suitable carrier to form a suspension. A representative pharmaceutical composition for use in a nebulizer inhaler comprises a solution or suspension comprising from about 0.05 µg/mL to about 20 mg/mL of a compound of the present disclosure and excipients compatible with nebulized formulations. In some embodiments, the solution has a pH of about 3 to about 8. 35 Nebulizer devices suitable for administering therapeutic agents by inhalation are described in the art and examples of such devices are commercially available. For example, representative nebulizer devices or products include the Respimat Softmist Inhaler (Boehringer Ingelheim); the AERx Pulmonary Delivery System (Aradigm Corp.); the PARI LC Plus Reusable Nebulizer (Pari GmbH); and the like. 18
Attorney Docket No.: P-381-WO/71TD-385423-WO In yet another aspect, the pharmaceutical compositions of the present disclosure may alternatively be prepared in a dosage form intended for oral administration. Suitable pharmaceutical compositions for oral administration may be in the form of capsules, tablets, pills, lozenges, cachets, dragees, powders, granules; or as a solution or a suspension in an aqueous or non-aqueous liquid; or as an 5 oil-in-water or water-in-oil liquid emulsion; or as an elixir or syrup; and the like; each containing a predetermined amount of a compound of the present disclosure as an active ingredient. When intended for oral administration in a solid dosage form, the pharmaceutical compositions of the present disclosure will typically comprise the active agent and one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate. Optionally or alternatively, such solid 10 dosage forms may also comprise: fillers or extenders, binders, humectants, solution retarding agents, absorption accelerators, wetting agents, absorbents, lubricants, coloring agents, and buffering agents. Release agents, wetting agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the pharmaceutical compositions of the present disclosure. Alternative formulations may also include controlled release formulations, liquid dosage forms 15 for oral administration, transdermal patches, and parenteral formulations. Conventional excipients and methods of preparation of such alternative formulations are described, for example, in the reference by Remington, supra. The following non-limiting examples illustrate representative pharmaceutical compositions of the present disclosure. 20 Dry Powder Composition A micronized compound of formula (I’) or (I) (1 g) is blended with milled lactose (25 g). This blended mixture is then loaded into individual blisters of a peelable blister pack in an amount sufficient to provide between about 0.1 mg to about 4 mg of the compound of formula (I’) or (I) per dose. The contents of the blisters are administered using a dry powder inhaler. 25 Dry Powder Composition A micronized compound of formula (I’) or (I) (1 g) is blended with milled lactose (20 g) to form a bulk composition having a weight ratio of compound to milled lactose of 1:20. The blended composition is packed into a dry powder inhalation device capable of delivering between about 0.1 mg to about 4 mg of the compound of formula (I’) or (I) per dose. 30 Metered-Dose Inhaler Composition A micronized compound of formula (I’) or (I) (10 g) is dispersed in a solution prepared by dissolving lecithin (0.2 g) in demineralized water (200 mL). The resulting suspension is spray dried and then micronized to form a micronized composition comprising particles having a mean diameter less than about 1.5 μm. The micronized composition is then loaded into metered-dose inhaler cartridges 35 containing pressurized 1,1,1,2-tetrafluoroethane in an amount sufficient to provide about 0.1 mg to about 4 mg of the compound of formula (I’) or (I) per dose when administered by the metered dose inhaler. 19
Attorney Docket No.: P-381-WO/71TD-385423-WO Nebulizer Composition A compound of formula (I’) or (I) (25 mg) is dissolved in a solution containing 1.5-2.5 equivalents of hydrochloric acid, followed by addition of sodium hydroxide to adjust the pH to 3.5 to 5.5 and 3% by weight of glycerol. The solution is stirred well until all the components are dissolved. The 5 solution is administered using a nebulizer device that provides about 0.1 mg to about 4 mg of the compound of formula (I’) or (I) per dose. Utility The compounds of the present disclosure are Janus kinase (JAK) inhibitors. The JAK inhibitors of the present disclosure have been designed for the treatment of inflammatory and fibrotic diseases, 10 including inflammatory and fibrotic diseases of the respiratory tract. In some embodiments, the compounds have been designed to enable delivery of a potent anti-cytokine agent directly to the site of action of respiratory disease in the lung while limiting systemic exposure. Limiting systemic exposure can be achieved by increasing selectivity for lung tissue, by having a permeability to enhance retention in lung tissue or a combination thereof. 15 As shown in Assays 1-2 and Tables 4 and 5, the compounds of the present disclosure have been shown to be potent inhibitors of JAK1 and/or JAK2. It is contemplated that the compounds of the present disclosure can selectively modulate a particular JAK over other JAK enzymes. It is further contemplated that the compounds of the present disclosure advantageously can selectively inhibit JAK1 as compared to JAK2 and are thus useful as therapeutic agents. 20 In some embodiments, the compounds of the present disclosure exhibit a JAK1/2 selectivity, as calculated as described herein, of at least about 0.7. In some embodiments, the compounds of the present disclosure exhibit a JAK1/2 selectivity of at least about 0.8. In some embodiments, the compounds of the present disclosure exhibit a JAK1/2 selectivity of at least about 0.9. In some embodiments, the compounds of the present disclosure exhibit a JAK1/2 selectivity of at least about 1. 25 The anti-inflammatory activity of JAK inhibitors has been robustly demonstrated in preclinical models of asthma. Cytokines implicated in asthma inflammation which signal through the JAK-STAT pathway include IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-11, IL-13, IL-23, IL-31, IL-27, thymic stromal lymphopoietin (TSLP), interferon-γ (IFNγ) and granulocyte-macrophage colony-stimulating factor (GM- CSF). Accordingly, the compounds of the present disclosure are expected to be useful for the treatment 30 of inflammatory respiratory disorders, such as asthma. Inflammation and fibrosis of the lung is characteristic of other respiratory diseases in addition to asthma such as chronic obstructive pulmonary disease (COPD), cystic fibrosis (CF), pneumonitis, interstitial lung diseases (including idiopathic pulmonary fibrosis), acute lung injury, acute respiratory distress syndrome, bronchitis, emphysema, bronchiolitis obliterans, and sarcoidosis. The present compounds, therefore, are also expected to be 35 useful for the treatment of chronic obstructive pulmonary disease, cystic fibrosis, pneumonitis, interstitial lung diseases (including idiopathic pulmonary fibrosis), acute lung injury, acute respiratory distress syndrome, bronchitis, emphysema, bronchiolitis obliterans, and sarcoidosis. Further, Asthma endotypes 20
Attorney Docket No.: P-381-WO/71TD-385423-WO may be broadly regarded as type 2 (T2) high or T2-low. Based on their mechanism of action, the compounds of the disclosure have the potential to treat both endotypes, T2-high and T2-low. The compounds of the present disclosure possess biological activity involved in the inhibition of cytokines associated with inflammation. Therefore, the compounds of the present disclosure are expected 5 to be useful for the treatment of certain specific respiratory diseases, as detailed below. Eosinophilic airway inflammation is a characteristic feature of diseases collectively termed eosinophilic lung diseases. Eosinophilic diseases have been associated with IL-4, IL-13 and IL-5 signaling. Eosinophilic lung diseases include infections (especially helminthic infections), drug-induced pneumonitis (induced, for example, by therapeutic drugs such as antibiotics, phenytoin, or l-tryptophan), 10 fungal-induced pneumonitis (e.g., allergic bronchopulmonary aspergillosis), hypersensitivity pneumonitis and eosinophilic granulomatosis with polyangiitis (formerly known as Churg-Strauss syndrome). Eosinophilic lung diseases of unknown etiology include idiopathic acute eosinophilic pneumoni, idiopathic chronic eosinophilic pneumonia, hypereosinophilic syndrome, and Löffler syndrome. A polymorphism in the IL-6 gene has been associated with elevated IL-6 levels and an increased 15 risk of developing pulmonary arterial hypertension (PAH). Corroborating the role of IL-6 in PAH, inhibition of the IL-6 receptor chain gp130 ameliorated the disease in a rat model of PAH. Cytokines such as IFNγ, IL-12 and IL-6 have been implicated in a range of non-allergic lung diseases such as sarcoidosis, and lymphangioleiomyomatosis. Bronchiectasis and infiltrative pulmonary diseases are diseases associated with chronic 20 neutrophilic inflammation. Pathological T cell activation is critical in the etiology of multiple respiratory diseases. Autoreactive T cells play a role in bronchiolitis obliterans organizing pneumonia (also termed COS). Similar to COS the etiology of lung transplant rejections is linked to an aberrant T cell activation of the recipients T cells by the transplanted donor lung. Lung transplant rejections may occur early as Primary 25 Graft Dysfunction (PGD), organizing pneumonia (OP), acute rejection (AR) or lymphocytic bronchiolitis (LB) or they may occur years after lung transplantation as Chronic Lung Allograft Dysfunction (CLAD). CLAD was previously known as bronchiolitis obliterans (BO) but now is considered a syndrome that can have different pathological manifestations including BO, restrictive CLAD (rCLAD or RAS) and neutrophilic allograft dysfunction. Chronic lung allograft dysfunction (CLAD) is a major challenge in 30 long-term management of lung transplant recipients as it causes a transplanted lung to progressively lose functionality. CLAD is poorly responsive to treatment and therefore, there remains a need for effective compounds capable of preventing or treating this condition. Several JAK-dependent cytokines such as IFNγ and IL-5 are up-regulated in CLAD and lung transplant rejection. Moreover, high lung levels of CXCR3 chemokines such as CXCL9 and CXCL10 which are downstream of JAK-dependent IFN 35 signaling, are linked to worse outcomes in lung transplant patients. Systemic JAK inhibition has been shown to be effective in kidney transplant rejection. Therefore, JAK inhibitors have the potential to be effective in treating or preventing lung transplant rejection and CLAD. Similar T cell activation events as described as the basis for lung transplant rejection also are considered the main driver of lung graft- 21
Attorney Docket No.: P-381-WO/71TD-385423-WO versus-host disease (GVHD) which can occur post hematopoietic stem cell transplants. Similar to CLAD, lung GVHD is a chronic progressive condition with extremely poor outcomes and no treatments are currently approved. A retrospective, multicenter survey study of 95 patients with steroid-refractory acute or chronic GVHD who received the systemic JAK inhibitor ruxolitinib as salvage therapy demonstrated 5 complete or partial response to ruxolitinib in the majority of patients including those with lung GVHD. As systemic JAK inhibition is associated with serious adverse events and a small therapeutic index, the need remains for an inhaled lung-directed, non-systemic JAK inhibitor to prevent and/or treat lung transplant rejection or lung GVHD. The compounds of the present disclosure have the characteristics required to meet this need. 10 Therefore, provided herein is a method of treating or preventing lung transplant rejection in a human in need thereof comprising administering to the human a compound of the present disclosure, or a pharmaceutically acceptable salt thereof, as described herein, or a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a compound of the present disclosure, or a pharmaceutically acceptable salt thereof, as described herein. In some embodiments, the lung transplant 15 rejection is selected from the group consisting of primary graft dysfunction, organizing pneumonia, acute rejection, lymphocytic bronchiolitis, and chronic lung allograft dysfunction. In some embodiments, the lung transplant rejection is acute lung transplant rejection. In some embodiments, the lung transplant rejection is chronic lung allograft dysfunction. In some embodiments, the lung transplant rejection is selected from the group consisting of bronchiolitis obliterans, restrictive chronic lung allograft 20 dysfunction, and neutrophilic allograft dysfunction. More recently, immune-checkpoint inhibitor induced pneumonitis, another T cell mediated lung disease emerged with the increased use of immune-checkpoint inhibitors. In cancer patients treated with these T cell stimulating agents, fatal pneumonitis can develop. The compounds of the disclosure possess biological activity allowing inhibition of IFNγ secretion. 25 In some embodiments, therefore, the present disclosure provides a method of treating a respiratory disease in a mammal (e.g., a human), the method comprising administering to the mammal (or human) a compound of the present disclosure, or a pharmaceutically acceptable salt thereof, as described herein, or a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a compound of the present disclosure, or a pharmaceutically acceptable salt thereof, as described herein. 30 In some embodiments, the respiratory disease is asthma, chronic obstructive pulmonary disease (COPD), cystic fibrosis, pneumonitis, cystic fibrosis (CF), pneumonitis, interstitial lung diseases (including idiopathic pulmonary fibrosis), acute lung injury, acute respiratory distress syndrome, bronchitis, emphysema, bronchiolitis obliterans, or sarcoidosis. In some embodiments, the respiratory disease is asthma or chronic obstructive pulmonary disease. In some embodiments, the Asthma is T2- 35 high Asthma. In some embodiments, the Asthma is T2-low Asthma. In some embodiments, the respiratory disease is a lung infection, an eosinophilic disease, a helminthic infection, pulmonary arterial hypertension, lymphangioleiomyomatosis, bronchiectasis, an infiltrative pulmonary disease, drug-induced pneumonitis, fungal induced pneumonitis, allergic 22
Attorney Docket No.: P-381-WO/71TD-385423-WO bronchopulmonary aspergillosis, hypersensitivity pneumonitis, eosinophilic granulomatosis with polyangiitis, idiopathic acute eosinophilic pneumonia, idiopathic chronic eosinophilic pneumonia, hypereosinophilic syndrome, Löffler syndrome, bronchiolitis obliterans organizing pneumonia, acute and chronic lung transplant rejections (including PGD, OP, LB, AR and CLAD, BO, restrictive CLAD and 5 neutrophilic allograft dysfunction), lung graft-versus-host disease, or immune-checkpoint-inhibitor induced pneumonitis. The present disclosure further provides a method of treating asthma in a mammal (e.g. a human), the method comprising administering to the mammal (or human) a compound of the present disclosure, or a pharmaceutically acceptable salt thereof, as described herein, or a pharmaceutical composition 10 comprising a pharmaceutically acceptable carrier and a compound of the present disclosure, or a pharmaceutically acceptable salt thereof, as described herein. When used to treat asthma (or other diseases or disorders described herein), the compounds of the present disclosure, or pharmaceutically acceptable salts thereof, described herein will typically be administered in a single daily dose or in multiple doses per day, although other forms of administration 15 may be used. The amount of active agent administered per dose or the total amount administered per day will typically be determined by a physician, in the light of the relevant circumstances, including the condition to be treated, the chosen route of administration, the actual compound administered and its relative activity, the age, weight, and response of the individual patient, the severity of the patient's symptoms, and the like. 20 The present disclosure further provides a method of treating a respiratory disease (including but not limited to a disease described herein) in a mammal (e.g. a human), the method comprising administering to the mammal (or human), a compound of the present disclosure, or a pharmaceutically acceptable salt thereof, as described herein, or a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a compound of the present disclosure, or a pharmaceutically 25 acceptable salt thereof, as described herein. In some embodiments, the respiratory disease is selected from the group consisting of asthma, chronic obstructive pulmonary disease, cystic fibrosis, pneumonitis, idiopathic pulmonary fibrosis, acute lung injury, acute respiratory distress syndrome, bronchitis, emphysema, bronchiolitis obliterans, sarcoidosis, an eosinophilic disease, a helminthic infection, pulmonary arterial hypertension, 30 lymphangioleiomyomatosis, bronchiectasis, an infiltrative pulmonary disease, drug-induced pneumonitis, fungal induced pneumonitis, allergic bronchopulmonary aspergillosis, hypersensitivity pneumonitis, eosinophilic granulomatosis with polyangiitis, idiopathic acute eosinophilic pneumonia, idiopathic chronic eosinophilic pneumonia, hypereosinophilic syndrome, Löffler syndrome, bronchiolitis obliterans organizing pneumonia, lung graft-versus-host disease, COVID-19, SARS, MERS, chronic rhinosinusitis35 with or without nasal polyps, nasal polyposis, sinusitis with nasal polyps, rhinitis, and immune- checkpoint-inhibitor induced pneumonitis. When used to treat a respiratory disease (including but not limited to a disease described herein), the compounds of the present disclosure, or pharmaceutically acceptable salts thereof, described herein 23
Attorney Docket No.: P-381-WO/71TD-385423-WO will typically be administered in a single daily dose or in multiple doses per day, although other forms of administration may be used. The amount of active agent administered per dose or the total amount administered per day will typically be determined by a physician, in the light of the relevant circumstances, including the condition to be treated, the chosen route of administration, the actual 5 compound administered and its relative activity, the age, weight, and response of the individual patient, the severity of the patient's symptoms, and the like. Human coronavirus is a common respiratory pathogen and typically induces mild upper respiratory disease. The two highly pathogenic viruses, Severe Acute Respiratory Syndrome associated- Coronavirus (SARS-CoV-1) and Middle East Respiratory Syndrome-associated Coronavirus (MERS- 10 CoV), caused severe respiratory syndromes resulting in more than 10% and 35% mortality, respectively. A subgroup of patients with COVID-19 appears to have a hyperinflammatory “cytokine storm” resulting in acute lung injury and acute respiratory distress syndrome (ARDS). This cytokine storm may also spill over into the systemic circulation and produce sepsis and ultimately, multi-organ dysfunction syndrome. The dysregulated cytokine signaling that appears in COVID-19 is characterized by increased expression 15 of interferons (IFNs), interleukins (ILs), and chemokines, resulting in ALI and associated mortality. Monoclonal antibodies directed against IL-6 (tocilizumab) appear to be effective in treating patients with ALI from COVID-19. Infection with mouse adapted strains of the 2003 SARS-CoV-1 and 2012 MERS- CoV, as well as a transgenic mouse expressing the human SARS-CoV-1 receptor hACE2 infected with human SARS-CoV-1, demonstrate elevations of JAK-dependent cytokines, such as IFNγ, IL-6, and IL- 20 12, and downstream chemokines, such as chemokine (C-C motif) ligand 10 (CCL10), CCL2, and CCL7. JAK inhibitors have also been shown to be beneficial in mouse models of lipopolysaccharide-or ganciclovir-induced ALI. Finally, based on the results of clinical trials, baricitinib, a JAK inhibitor, received an emergency use authorization (EUA) in combination with remdesivir, for the treatment of COVID-19 in patients requiring supplemental oxygen, invasive mechanical ventilation, or extracorporeal 25 membrane oxygenation Therefore, compounds of the present disclosure, or pharmaceutically acceptable salts thereof described herein could be uniquely suited to dampen the cytokine storm associated with COVID-19. By delivering to the lung and avoiding systemic immunosuppression, additional infections that lead to worsened mortality may also be avoided. This is particularly true in those patients requiring ventilatory 30 support. Therefore, the present disclosure provides a method of treating a mammal (or patient) infected with a coronavirus such as SARS-CoV-1, SARS-CoV-2, and MERS-CoV, or the symptoms thereof, the method comprising administering to the mammal (or patient) a compound of the present disclosure, or a pharmaceutically acceptable salt thereof, as described herein, or a pharmaceutical composition 35 comprising a pharmaceutically acceptable carrier and a compound of the present disclosure, or a pharmaceutically acceptable salt thereof, as described herein. The present disclosure also provides a method of treating ALI and/or ARDS in a mammal (or a patient) caused by a coronavirus infection (such as SARS-CoV-1, SARS-CoV-2, and MERS-CoV), the method comprising administering to the mammal 24
Attorney Docket No.: P-381-WO/71TD-385423-WO (or patient) a compound of the present disclosure, or a pharmaceutically acceptable salt thereof, as described herein, or a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a compound of the present disclosure, or a pharmaceutically acceptable salt thereof, as described herein. The mechanism of action of JAK inhibitors has been linked to the treatment of nasal 5 inflammatory diseases. Further, Dupilumab, which acts by blocking the IL-4 and IL-13 signaling pathways, has been approved for the treatment of chronic rhinosinusitis with nasal polyps. Therefore, also provided herein is a method of treating nasal inflammatory diseases in a mammal (e.g. a human), the method comprising administering to the mammal (or human) a compound of the present disclosure, or a pharmaceutically acceptable salt thereof, as described herein, or a pharmaceutical 10 composition comprising a pharmaceutically acceptable carrier and a compound of the present disclosure, or a pharmaceutically acceptable salt thereof, as described herein. In some embodiments, the nasal inflammatory disease is selected from the group consisting of chronic rhinosinusitis with or without nasal polyps, nasal polyposis, sinusitis with nasal polyps, and rhinitis (non-allergic, allergic, perenial, and vasomotor rhinitis). 15 As JAK inhibitors, the compounds of the present disclosure may also be useful for a variety of other diseases. The compounds of the present disclosure may be useful for a variety of gastrointestinal inflammatory indications that include, but are not limited to, inflammatory bowel disease, ulcerative colitis (proctosigmoiditis, pancolitis, ulcerative proctitis and left-sided colitis), Crohn’s disease, collagenous colitis, lymphocytic colitis, Behcet’s disease, celiac disease, immune checkpoint inhibitor 20 induced colitis, ileitis, eosinophilic esophagitis, graft versus host disease-related colitis, and infectious colitis. Ulcerative colitis, Crohn’s disease, collagenous colitis, lymphocytic colitis, eosinophilic esophagitis, graft versus host disease-related colitis, infectious colitis, Behcet’s disease, celiac disease, immune checkpoint inhibitor induced colitis (e.g., CTLA-4 inhibitor-induced colitis; PD-1- or PD-L1- inhibitor-induced colitis), and ileitis are characterized by elevation of certain pro-inflammatory cytokine 25 levels. As many pro-inflammatory cytokines signal via JAK activation, compounds described in this disclosure may be able to alleviate the inflammation and provide symptom relief. In particular, the compounds of the present disclosure may be useful for the induction and maintenance of remission of ulcerative colitis, and for the treatment of Crohn’s disease, immune checkpoint inhibitor induced colitis, and the gastrointestinal adverse effects in graft versus host disease. In some embodiments, therefore, the 30 present disclosure provides a method of treating a gastrointestinal inflammatory disease in a mammal (e.g., a human), the method comprising administering to the mammal a compound of the present disclosure, or a pharmaceutically acceptable salt thereof, as described herein, or a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a compound of the present disclosure, or a pharmaceutically acceptable salt thereof, as described herein. 35 Atopic dermatitis and other inflammatory skin diseases have been associated with elevation of proinflammatory cytokines that rely on the JAK-STAT pathway. Therefore, the compounds of the present disclosure, or a pharmaceutically acceptable salt thereof, may be beneficial in a number of dermal inflammatory or pruritic conditions that include, but are not limited to atopic dermatitis, alopecia areata, 25
Attorney Docket No.: P-381-WO/71TD-385423-WO vitiligo, psoriasis, dermatomyositis, cutaneous T cell lymphoma and subtypes (Sezary syndrome, mycosis fungoides, pagetoid reticulosis, granulomatous slack skin, lymphomatoid papulosis, pityriasis lichenoides chronica, pityriasis lichenoides et varioliformis acuta, CD30+ cutaneous T-cell lymphoma, secondary cutaneous CD30+ large cell lymphoma, non-mycosis fungoides CD30− cutaneous large T-cell 5 lymphoma, pleomorphic T-cell lymphoma, Lennert lymphoma, subcutaneous T-cell lymphoma, angiocentric lymphoma, blastic NK-cell lymphoma), prurigo nodularis, lichen planus, primary localized cutaneous amyloidosis, bullous pemphigoid, skin manifestations of graft versus host disease, pemphigoid, discoid lupus, granuloma annulare, lichen simplex chronicus, vulvar/scrotal/perianal pruritus, lichen sclerosus, post herpetic neuralgia itch, lichen planopilaris, and foliculitis decalvans. In 10 particular, atopic dermatitis, alopecia areata, vitiligo, prurigo nodularis, lichen planus, primary localized cutaneous amyloidosis, bullous pemphigoid, and dermal manifestations of graft versus host disease are characterized by elevation of certain cytokines that signal via JAK activation. Accordingly, compounds of the present disclosure, or a pharmaceutically acceptable salt thereof, may be able to alleviate associated dermal inflammation or pruritus driven by these cytokines. In particular, compounds of the 15 present disclosure, or a pharmaceutically acceptable salt thereof, may be expected to be useful for the treatment of atopic dermatitis and other inflammatory skin diseases. In some embodiments, therefore, the present disclosure provides a method of treating an inflammatory skin disease in a mammal (e.g., a human), the method comprising applying a pharmaceutical composition comprising a compound of the present disclosure, or a pharmaceutically acceptable salt thereof, as described herein and a 20 pharmaceutical carrier to the skin of the mammal. In some embodiments, the inflammatory skin disease is atopic dermatitis. Many ocular diseases have been shown to be associated with elevations of proinflammatory cytokines that rely on the JAK-STAT pathway. The compounds of the present disclosure, or a pharmaceutically acceptable salt thereof, therefore, may be useful for the treatment of a number of ocular 25 diseases that include, but are not limited to, uveitis, diabetic retinopathy, diabetic macular edema, dry eye disease, age-related macular degeneration, and atopic keratoconjunctivitis. In particular, uveitis, diabetic retinopathy, diabetic macular edema, dry eye disease, and age-related macular degeneration are characterized by elevation of certain pro-inflammatory cytokines that signal via the JAK-STAT pathway. Accordingly, compounds of the present disclosure, or a pharmaceutically acceptable salt thereof, may be 30 able to alleviate the associated ocular inflammation and reverse disease progression or provide symptom relief. In some embodiments, therefore, the present disclosure provides a method of treating an ocular disease in a mammal (e.g. a human), the method comprising administering a pharmaceutical composition comprising a compound of the present disclosure, or a pharmaceutically acceptable salt thereof, as described herein and a pharmaceutical carrier to the eye of the mammal (or human). In some 35 embodiments, the ocular disease is uveitis, diabetic retinopathy, diabetic macular edema, dry eye disease, age-related macular degeneration, or atopic keratoconjunctivitis. In some embodiments, the method comprises administering a compound of the present disclosure, or a pharmaceutically acceptable salt thereof, as described herein by intravitreal injection. A compound of the present disclosure, or a 26
Attorney Docket No.: P-381-WO/71TD-385423-WO pharmaceutically acceptable salt thereof, as described herein may also be used in combination with one or more compounds useful to ocular diseases. The compounds of the present disclosure, or a pharmaceutically acceptable salt thereof, may also be useful to treat other diseases such as other inflammatory diseases, autoimmune diseases or cancers. 5 The compounds of the present disclosure, or a pharmaceutically acceptable salt thereof, may be useful to treat one or more of cytokine release syndrome (CRS), arthritis, rheumatoid arthritis, juvenile rheumatoid arthritis, transplant rejection, xerophthalmia, psoriatic arthritis, diabetes, insulin dependent diabetes, motor neuron disease, myelodysplastic syndrome, pain, sarcopenia, cachexia, septic shock, systemic lupus erythematosus, leukemia, chronic lymphocytic leukemia, chronic myelocytic leukemia, acute 10 lymphoblastic leukemia, acute myelogenous leukemia, ankylosing spondylitis, myelofibrosis, B-cell lymphoma, hepatocellular carcinoma, Hodgkins disease, breast cancer, Multiple myeloma, melanoma, non-Hodgkin lymphoma, non-small-cell lung cancer, ovarian clear cell carcinoma, ovary tumor, pancreas tumor, polycythemia vera, Sjoegrens syndrome, soft tissue sarcoma, sarcoma, splenomegaly, T-cell lymphoma, and thalassemia major. 15 Combination Therapy Compounds of the present disclosure, or a pharmaceutically acceptable salt thereof, described herein may be used in combination with one or more agents which act by the same mechanism or by different mechanisms to treat a disease. The different agents may be administered sequentially or simultaneously, in separate compositions or in the same composition. Useful classes of agents for 20 combination therapy include, but are not limited to, a beta 2 adrenoceptor agonist, a muscarinic receptor antagonist, a glucocorticoid agonist, a G-protein coupled receptor-44 antagonist, a leukotriene D4 antagonist, a muscarinic M3 receptor antagonist, a histamine H1 receptor antagonist, an immunoglobulin E antagonist, a PDE 4 inhibitor, an IL-4 antagonist, a muscarinic M1 receptor antagonist, a histamine receptor antagonist, an IL-13 antagonist, an IL-5 antagonist, a 5-lipoxygenase inhibitor, a beta 25 adrenoceptor agonist, a CCR3 chemokine antagonist, a CFTR stimulator, an immunoglobulin modulator, an interleukin 33 ligand inhibitor, a PDE 3 inhibitor, a phosphoinositide-3 kinase delta inhibitor, a thromboxane A2 antagonist, an elastase inhibitor, a Kit tyrosine kinase inhibitor, a leukotriene E4 antagonist, a leukotriene antagonist, a PGD2 antagonist, a TNF alpha ligand inhibitor, a TNF binding agent, a complement cascade inhibitor, an eotaxin ligand inhibitor, a glutathione reductase inhibitor, an 30 histamine H4 receptor antagonist, an IL-6 antagonist, an IL2 gene stimulator, an immunoglobulin gamma Fc receptor IIB modulator, an interferon gamma ligand, an interleukin 13 ligand inhibitor, an interleukin 17 ligand inhibitor, a L-Selectin antagonist, a leukocyte elastase inhibitor, a leukotriene C4 antagonist, a Leukotriene C4 synthase inhibitor, a membrane copper amine oxidase inhibitor, a metalloprotease-12 inhibitor, a metalloprotease-9 inhibitor, a mite allergen modulator, a muscarinic receptor modulator, a 35 nicotinic acetylcholine receptor agonist, a nuclear factor kappa B inhibitor, a p-Selectin antagonist, a PDE 5 inhibitor, a PDGF receptor antagonist, a phosphoinositide-3 kinase gamma inhibitor, a TLR-7 agonist, a TNF antagonist, an Abl tyrosine kinase inhibitor, an acetylcholine receptor antagonist, an 27
Attorney Docket No.: P-381-WO/71TD-385423-WO acidic mammalian chitinase inhibitor, an ACTH receptor agonist, an actin polymerization modulator, an adenosine A1 receptor antagonist, an adenylate cyclase stimulator, an adrenoceptor antagonist, an adrenocorticotrophic hormone ligand, an alcohol dehydrogenase 5 inhibitor, an alpha 1 antitrypsin stimulator, an alpha 1 proteinase inhibitor, an androgen receptor modulator, an angiotensin converting 5 enzyme 2 stimulator, an ANP agonist, a Bcr protein inhibitor, a beta 1 adrenoceptor antagonist, a beta 2 adrenoceptor antagonist, a beta 2 adrenoceptor modulator, a beta amyloid modulator, a BMP10 gene inhibitor, a BMP15 gene inhibitor, a calcium channel inhibitor, a cathepsin G inhibitor, a CCL26 gene inhibitor, a CCR3 chemokine modulator, a CCR4 chemokine antagonist, a cell adhesion molecule inhibitor, a chaperonin stimulator, a chitinase inhibitor, a collagen I antagonist, a complement C3 10 inhibitor, a CSF-1 antagonist, a CXCR2 chemokine antagonist, a cytokine receptor common beta chain modulator, a cytotoxic T-lymphocyte protein-4 stimulator, a deoxyribonuclease I stimulator, a deoxyribonuclease stimulator, a dipeptidyl peptidase I inhibitor, a DNA gyrase inhibitor, a DP prostanoid receptor modulator, an E-Selectin antagonist, an EGFR family tyrosine kinase receptor inhibitor, an elastin modulator, an Endothelin ET-A antagonist, an Endothelin ET-B antagonist, an epoxide hydrolase 15 inhibitor, a FGF3 receptor antagonist, a Fyn tyrosine kinase inhibitor, a GATA 3 transcription factor inhibitor, a Glucosylceramidase modulator, a Glutamate receptor modulator, a GM-CSF ligand inhibitor, a Guanylate cyclase stimulator, a H+ K+ ATPase inhibitor, an hemoglobin modulator, an Heparin agonist, an Histone deacetylase inhibitor, an Histone deacetylase-2 stimulator, an HMG CoA reductase inhibitor, an I-kappa B kinase beta inhibitor, an ICAM1 gene inhibitor, an IL-17 antagonist, an IL-17 20 receptor modulator, an IL-23 antagonist, an IL-4 receptor modulator, an Immunoglobulin G modulator, an Immunoglobulin G1 agonist, an Immunoglobulin G1 modulator, an Immunoglobulin epsilon Fc receptor IA antagonist, an Immunoglobulin gamma Fc receptor IIB antagonist, an Immunoglobulin kappa modulator, an Insulin sensitizer, an Interferon beta ligand, an Interleukin 1 like receptor antagonist, an Interleukin 18 ligand inhibitor, an Interleukin receptor 17A antagonist, an Interleukin-1 beta ligand 25 inhibitor, an Interleukin-5 ligand inhibitor, an Interleukin-6 ligand inhibitor, a KCNA voltage-gated potassium channel-3 inhibitor, a Kit ligand inhibitor, a Laminin-5 agonist, a Leukotriene CysLT1 receptor antagonist, a Leukotriene CysLT2 receptor antagonist, a LOXL2 gene inhibitor, a Lyn tyrosine kinase inhibitor, a MARCKS protein inhibitor, a MDR associated protein 4 inhibitor, a Metalloprotease-2 modulator, a Metalloprotease-9 modulator, a Mineralocorticoid receptor antagonist, a Muscarinic M2 30 receptor antagonist, a Muscarinic M4 receptor antagonist, a Muscarinic M5 receptor antagonist, a Natriuretic peptide receptor A agonist, a Natural killer cell receptor modulator, a Nicotinic ACh receptor alpha 7 subunit stimulator, a NK cell receptor modulator, a Nuclear factor kappa B modulator, an opioid growth factor receptor agonist, a P-Glycoprotein inhibitor, a P2X3 purinoceptor antagonist, a p38 MAP kinase inhibitor, a Peptidase 1 modulator, a phospholipase A2 inhibitor, a phospholipase C inhibitor, a 35 plasminogen activator inhibitor 1 inhibitor, a platelet activating factor receptor antagonist, a PPAR gamma agonist, a prostacyclin agonist, a protein tyrosine kinase inhibitor, a SH2 domain inositol phosphatase 1 stimulator, a signal transduction inhibitor, a sodium channel inhibitor, a STAT-3 modulator, a Stem cell antigen-1 inhibitor, a superoxide dismutase modulator, a T cell surface 28
Attorney Docket No.: P-381-WO/71TD-385423-WO glycoprotein CD28 inhibitor, a T-cell surface glycoprotein CD8 inhibitor, a TGF beta agonist, a TGF beta antagonist, a thromboxane synthetase inhibitor, a thymic stromal lymphoprotein ligand inhibitor, a thymosin agonist, a thymosin beta 4 ligand, a TLR-8 agonist, a TLR-9 agonist, a TLR9 gene stimulator, a Topoisomerase IV inhibitor, a Troponin I fast skeletal muscle stimulator, a Troponin T fast skeletal 5 muscle stimulator, a Type I IL-1 receptor antagonist, a Type II TNF receptor modulator, an ion channel modulator, a uteroglobin stimulator, and a VIP agonist. Also provided, herein, is a pharmaceutical composition comprising a compound of the present disclosure, or a pharmaceutically acceptable salt thereof, as described herein and one or more other therapeutic agents. The therapeutic agent may be selected from the agents described above. In some 10 embodiments, the pharmaceutical composition is suitable for delivery to the lungs. In some embodiments, the pharmaceutical composition is suitable for inhaled or nebulized administration. In some embodiments, the pharmaceutical composition is a dry powder or a liquid composition. Further, the present disclosure provides a method of treating a disease or disorder in a mammal (e.g., a human) comprising administering to the mammal (or human) a compound of the present 15 disclosure, or a pharmaceutically acceptable salt thereof, as described herein and one or more other therapeutic agents. When used in combination therapy, the agents may be formulated in a single pharmaceutical composition, or the agents may be provided in separate compositions that are administered simultaneously or at separate times, by the same or by different routes of administration. Such 20 compositions can be packaged separately or may be packaged together as a kit. The two or more therapeutic agents in the kit may be administered by the same route of administration or by different routes of administration. EXAMPLES The following synthetic and biological examples are offered to illustrate the disclosure and are 25 not to be construed in any way as limiting the scope of the disclosure. In the examples below, the following abbreviations have the following meanings unless otherwise indicated. Abbreviations not defined below have their generally accepted meanings. Abbreviations ACN = Acetonitrile AcOH = Acetic acid Boc2O = di-tert-butyl dicarbonate BSA = bovine serum albumin, Fraction V d = day(s) DCM = Dichloromethane DHP = Dihydropyran DIPEA or DIEA = N,N-diisopropylethylamine DMF = N,N-dimethyforamide DMP = Dess–Martin periodinane DPPF = 1,1’-bis(diphenylphosphino)ferrocene 29
Attorney Docket No.: P-381-WO/71TD-385423-WO EDTA = ethylenediaminetetraacetic acid EGTA = ethylene glycol-bis(β-aminoethyl ether)-N,N,N’,N’-tetraacetic acid EtOAc = Ethyl Acetate g = Grams h = Hour or hours HEPES = 4-(2-hyrdroxyethyl)-1-piperazine ethanesulfonic acid IPA Isopropyl alcohol LDA = lithium diisopropylamide MeOH = Methanol mg = Milligrams min = Minute or minutes mL = Milliliters mL = Millilitre mmol = Millimoles NaH = Sodium hydride NBS = N-Bromosuccinimide NIS = N-Iodosuccinimide Pd(dppf)Cl2 = [1,1′-bis(diphenylphosphino)ferrocene]-dichloropalladium(II) Pd(PPh3)4 = tetrakis(triphenylphosphine)palladium(0) Pd/C = palladium on activated carbon, 10% loading PTSA = p-Toluenesulfonic acid rt or RT = Room temperature, ambient, about 25°C SEM-Cl = 2-(Trimethylsilyl)ethoxymethyl chloride SiO2 = silicon dioxide or silica STAB = Sodium triacetoxyborohydride THF = Tetrahydrofuran TEA = Triethylamine TLC = Thin Layer Chromatography THP = tetrahydropyran Unless noted otherwise, all materials, such as reagents, starting materials and solvents, were purchased from commercial suppliers, such as Sigma-Aldrich, Fluka Riedel-de Haën, and the like, and were used without further purification. 5 Reactions were run under nitrogen atmosphere, unless noted otherwise. The progress of reactions was monitored by thin layer chromatography (TLC), analytical high-performance liquid chromatography (anal. HPLC), and mass spectrometry, the details of which are given in specific examples. Reactions were worked up as described specifically in each preparation; commonly, reaction mixtures were purified by extraction and other purification methods such as temperature- and solvent- 10 dependent crystallization, and precipitation. In addition, reaction mixtures were routinely purified by preparative HPLC, typically using Microsorb C18 and Microsorb BDS column packings and conventional eluents. Progress of reactions was typically monitored by liquid chromatography mass spectrometry (LCMS). Characterization of isomers was typically done by Nuclear Overhauser effect spectroscopy (NOE). Characterization of reaction products was routinely carried out by mass 30
Attorney Docket No.: P-381-WO/71TD-385423-WO spectrometry and/or 1H-NMR spectroscopy. For NMR measurement, samples were dissolved in deuterated solvent (CD3OD, CDCl3, or DMSO-d6), and 1H-NMR spectra were acquired with a Varian Gemini 2000 instrument (400 MHz) under standard observation conditions or a Bruker 400 MHz, Avance II spectrometer with a 5mm DUL (Dual) 13C probe. Mass spectrometric identification of 5 compounds was typically conducted using an electrospray ionization method (ESMS) with an Applied Biosystems (Foster City, CA) model API 150 EX instrument or an Agilent (Palo Alto, CA) model 1200 LC/MSD instrument. Synthesis of Intermediates Preparation of 2-(4-(benzyloxy)-2-ethylphenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane 10
sodium hydride (5.90 g, 147 mmol) at 0°C portion wise. The reaction was stirred at 0°C for 30 min, and then benzyl bromide (16.0 mL, 135 mmol) was added dropwise at 0°C. The reaction was allowed to stir at RT for 2h and then carefully poured into ice cold water (1L) and extracted with ethyl acetate (2 x 100 15 mL). The combined organic layers were washed with brine, dried over anhydrous Na2SO4 and concentrated under reduced pressure. The crude residue was purified by silica gel column chromatography (5% ethyl acetate/heptanes) to afford 1-(benzyloxy)-3-ethylbenzene (25 g, 96% yield). 1H NMR (400 MHz, CDCl3) δ 7.48-7.31 (m, 5H), 7.22 (t, J = 7.6 Hz, 1H), 6.88-6.80 (m, 3H), 5.07 (s, 2H), 2.47 (q, J = 7.6 Hz, 2H), 1.23 (t, J = 7.6 Hz, 3H). 20 (b) 1-(benzyloxy)-3-ethylbenzene (25 g, 118 mmol) was dissolved in ACN (250 mL) and cooled to 0°C. NBS (21 g, 118 mmol) was added portionwise over a period of 30 minutes. The resulting reaction mixture was stirred at RT for 2h, and then poured into ice cold water and extracted in EtOAc (2 x 200 mL). The combined organic layers were washed with water, dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The crude residue was purified by silica gel column 25 chromatography (100% heptanes) to afford 4-(benzyloxy)-1-bromo-2-ethylbenzene (31 g, 91 % yield). (c) To a stirred solution of 4-(benzyloxy)-1-bromo-2-ethylbenzene (20 g, 68.7 mmol) in 1,4- dioxane (200 mL) was added bis(pinacolato)diboron (20.9 g, 82.4 mmol) and potassium acetate (13.5 g, 137 mmol). The reaction mixture was degassed with nitrogen for 10 minutes. PdCl2(dppf) (502 mg, 0.69 mmol) was added, and the reaction mixture was stirred and heated at 110°C overnight. The reaction mixture 30 was filtered through a Celite bed, rinsing with EtOAc (2 x 200 mL). The combined filtrate was washed with water (500 mL), brine (500 mL), dried over anhydrous Na2SO4 and concentrated under reduced pressure. The crude residue was purified by silica gel column chromatography (10% ethyl acetate/heptanes) to afford 2-[4-(benzyloxy)-2-ethylphenyl]-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (12 g, 52 % yield). 31
Attorney Docket No.: P-381-WO/71TD-385423-WO Preparation of 6-(4-(benzyloxy)-2-ethylphenyl)-1-(tetrahydro-2H-pyran-2-yl)-3-(trimethylstannyl)- 1H-indazole
5 mmol) in DCM (300 mL) at 0oC was added dropwise 3,4-dihydropyran (46.4 mL, 508 mmol). The reaction was allowed to stir at RT for 16 h. The reaction mixture was then diluted with DCM (200 mL) and washed with water (2 x 200 mL), saturated NaHCO3 solution (50 mL) and brine (50 mL). The organic layer was dried over anhydrous Na2SO4 and concentrated under reduced pressure. The crude residue was purified by silica gel column chromatography (10% ethyl acetate/heptanes) to afford 6- 10 bromo-1-(tetrahydro-2H-pyran-2-yl)-1H-indazole (56 g, 78% yield). (b) To a stirred solution of 6-bromo-1-(tetrahydro-2H-pyran-2-yl)-1H-indazole (20 g, 71.1 mmol) and 2-(4-(benzyloxy)-2-ethylphenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (26.5 g, 78.2 mmol) in dioxane (150 mL): H2O (20 mL) was added Na2CO3 (15.1 g, 142 mmol). The reaction mixture was purged with argon for 10 min and PdCl2(dppf). DCM (5.81 g, 7.11 mmol) was added. The reaction was 15 allowed to stir at 100°C for 16 h, then filtered through a Celite bed, rinsing with EtOAc (2 x 200 mL). The combined filtrate was washed with water (2 x 100 mL) and brine (100 mL). The organic layer was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The crude residue was purified by silica gel column chromatography (12% EtOAc/heptanes) to afford 6-(4-(benzyloxy)-2- ethylphenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-indazole (23 g, 78% yield). 20 (c) To a stirred solution of 6-(4-(benzyloxy)-2-ethylphenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H- indazole (43 g, 104 mmol) in methanol (215 mL) at 0°C was added 6N HCl (60 mL) dropwise. The resulting solution was warmed to RT and then heated at 90°C for 16 h. The reaction mixture was concentrated under reduced pressure, neutralized slowly with sat. NaHCO3 (200mL) at 0°C and extracted with EtOAc (2 x 300mL). The combined organic layers were washed with brine (100mL), dried over25 anhydrous Na2SO4, filtered and concentrated under reduced pressure. The crude product 6-(4- 32
Attorney Docket No.: P-381-WO/71TD-385423-WO (benzyloxy)-2-ethylphenyl)-1H-indazole (36 g, purity 72%) was taken to next step without purification. m/z: [M+H]+ calcd for C22H21N2O 329.17, found 329.24. (d) To a stirred solution of 6-(4-(benzyloxy)-2-ethylphenyl)-1H-indazole (36 g, 110 mmol) in DMF (120 mL) at 0°C was added NIS (49.3 g, 219 mmol) portionwise. The reaction mixture was stirred 5 at room temperature for 2h, diluted with water (200 mL) and extracted with ethyl acetate (3 x 100 mL). The combined organic layers were washed with sodium thiosulfate solution (200 mL) and brine (200 mL). The organic layer was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to afford crude 6-(4-(benzyloxy)-2-ethylphenyl)-3-iodo-1H-indazole (46 g), which was used in the next step without purification. 10 (e) To a stirred solution of 6-(4-(benzyloxy)-2-ethylphenyl)-3-iodo-1H-indazole (46 g, 101 mmol) in DCM (250 mL) and PTSA (1.74 g, 10.1 mmol) at 0oC was added 3,4-dihydropyran (13.9 mL, 152 mmol) dropwise. The reaction was allowed to stir at room temperature for 16 h. The reaction was then diluted with DCM (400 mL) and washed with saturated NaHCO3 (100 mL) followed by brine (100 mL). The organic layer was dried over anhydrous Na2SO4 and concentrated under reduced pressure. The15 crude residue was purified by silica gel column chromatography (15% EtOAc/heptanes) to afford 6-(4- (benzyloxy)-2-ethylphenyl)-3-iodo-1-(tetrahydro-2H-pyran-2-yl)-1H-indazole (40 g, 74% yield). m/z: [M+H]+ calcd for C27H28IN2O2539.12, found 539.20. (f) A stirred solution of 6-(4-(benzyloxy)-2-ethylphenyl)-3-iodo-1H-indazole (20 g, 37.1 mmol) in toluene (200 mL) was purged with argon for 10 min at room temperature.1,1,1,2,2,2- 20 hexamethyldistannane (9.31 mL, 44.6 mmol) was added, followed by Pd(PPh3)4 (4.29 g, 3.71 mmol). The reaction mixture was heated at 110°C for 2h, and then filtered through Celite pad and washed with EtOAc (2 x 200 mL). The combined filtrate was concentrated under reduced pressure and the crude residue purified through neutral alumina column chromatography (1% EtOAc/heptanes) to afford 6-(4- (benzyloxy)-2-ethylphenyl)-1-(tetrahydro-2H-pyran-2-yl)-3-(trimethylstannyl)-1H-indazole (15.5 g, 73% 25 yield). m/z: [M+H]+ calcd for C30H37N2O2Sn 577.19, found 577.18. Preparation of 5-bromo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-1,2,4-triazol-3-yl)methanol (a) M
solved in DMF (40 mL) at 0°C and 60% NaH (1.16 g, 29.12 mmol) was added to it portionwise. The reaction mixture 30 was stirred for 10 min and SEM-Cl (5.16 mL, 29.12 mmol) was then added dropwise. The reaction mixture was allowed to warm to room temperature. After 1 h, the reaction mixture was poured into ice cold water. The aqueous layer was extracted with ethyl acetate (3 x 100 mL) and the combined organic layers were dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The crude residue was purified by silica gel flash column chromatography (15% ethyl acetate/heptanes) to afford 5- 33
Attorney Docket No.: P-381-WO/71TD-385423-WO bromo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-1,2,4-triazole-3-carboxylate (5.1 g, 6:4 mixture of SEM regio-isomers, 62% yield). (b) To a stirred solution of (5-bromo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-1,2,4-triazole-3- carboxylate (5.0 g, 14.92 mmol) in 1:1 THF:MeOH (50 mL) at 0°C was added sodium borohydride 5 (675 mg, 17.91) and the reaction was stirred at room temperature for 2 h. MeOH (50.0 mL) was added and the reaction mixture was allowed to stir for 30 min, then concentrated under reduced pressure. The residue was diluted with water and extracted with EtOAc (3 x 100 mL). The combined organic layers were dried over anhydrous Na2SO4 and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (20% ethyl acetate/heptanes) to afford (5-bromo-1-((2- 10 (trimethylsilyl)ethoxy)methyl)-1H-1,2,4-triazol-3-yl)methanol (4.3 g, 7:3 mixture of SEM regio-isomers, 94% yield). m/z: [M+H]+ calcd for C9H12BrN3O2Si 308.04, found 308.10. Preparation of 6-(4-(benzyloxy)-2-ethylphenyl)-7-fluoro-1-(tetrahydro-2H-pyran-2-yl)-3- (trimethylstannyl)-1H-indazole 15
(150 mL) was added hydrazine hydrate (10.5 mL, 339 mmol) dropwise at RT. The reaction was allowed to stir at 160oC for 6 h and then 130oC for 16 h. The reaction mixture was cooled to RT and concentrated under reduced pressure to afford an off-white solid which was triturated with pentane (500 mL) and filtered through a sintered funnel to afford 6-bromo-7-fluoro-1H-indazole (12.0 g, 82% yield). m/z: [M-H]- calcd 20 for C7H4BrFN 212.95, found 212.90. (b) 6-bromo-7-fluoro-1H-indazole (12 g, 56.07 mmol) was dissolved in DCM (300 mL) and PTSA (2.13 g, 11.21 mmol).3,4-dihydropyran (15.35 mL, 168.21 mmol) was added portionwise and the 34
Attorney Docket No.: P-381-WO/71TD-385423-WO reaction was then allowed to stir at RT for 16 h. The solution was diluted with DCM (200 mL) and washed with water (2 x 200 mL), saturated NaHCO3 solution (50 mL) and brine (50 mL). The organic layer was dried over anhydrous Na2SO4 and concentrated under reduced pressure. The crude residue was purified by silica gel flash column chromatography (10% ethyl acetate/heptanes) to afford 6-bromo-7- 5 fluoro-1-(tetrahydro-2H-pyran-2-yl)-1H-indazole (11.2 g, 67% yield). m/z: [M+H] + calcd for C12H13BrFN2O 299.02, found 298.90. (c) To a stirred solution of 6-bromo-7-fluoro-1-(tetrahydro-2H-pyran-2-yl)-1H-indazole (9.1 g, 30.43 mmol) and 2-(4-(benzyloxy)-2-ethylphenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (12.3 g, 36.52 mmol) in dioxane (100 mL): H2O (20 mL) was added Na2CO3 (6.45 g, 60.86 mmol). The flask was 10 purged with argon for 10 min and PdCl2(dppf). DCM (2.48 g, 3.04 mmol) was added. The reaction mixture was heated at 110°C for 16 h, then filtered through a Celite bed, washing with ethyl acetate (2 x 200 mL). The filtrate was washed with water (2 x 100 mL) and brine (100 mL). The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure, and the crude residue was purified by silica gel flash column chromatography (15-20% EtOAc/heptanes) to afford 6-(4- 15 (benzyloxy)-2-ethylphenyl)-7-fluoro-1-(tetrahydro-2H-pyran-2-yl)-1H-indazole (7.1 g, 54% yield). (m/z): [M+H] + calcd for C27H28FN2O2431.21, found 431.05. (d) To a stirred solution of 6-(4-(benzyloxy)-2-ethylphenyl)-7-fluoro-1-(tetrahydro-2H-pyran-2- yl)-1H-indazole (7.1 g, 16.51 mmol) in methanol (50 mL) in THF (50 mL) was added 4 M HCl in dioxane (70 mL) dropwise at 0°C. The resulting solution was warmed to RT and heated at 70°C for 16 h. 20 The solution was concentrated under reduced pressure and neutralized slowly at 0°C with saturated NaHCO3 (100 mL) and further extracted with EtOAc (2 x 100mL). The combined organic layer was further washed with brine (100mL), dried over anhydrous Na2SO4 and filtered. The filtrate was concentrated under reduced pressure to afford 6-(4-(benzyloxy)-2-ethylphenyl)-7-fluoro-1H-indazole (5.2 g, 91% yield). (m/z): [M+H] + calcd for C22H20FN2O 347.16, found 347.15. 25 (e) To a stirred solution of 6-(4-(benzyloxy)-2-ethylphenyl)-7-fluoro-1H-indazole (4.0 g, 11.5 mmol) in DMF (40 mL) at 0°C was added NIS (5.2 g, 23.1 mmol) portionwise. The reaction mixture was stirred at room temperature for 2h, then was diluted with water (50 mL) and extracted with ethyl acetate (3 x 30 mL). The combined organic layer was washed with sodium thiosulfate solution (50 mL), brine (50 mL), dried over anhydrous Na2SO4, filtered and concentrated. The crude residue was purified by30 silica gel flash column chromatography (20-25% EtOAc/heptanes) to afford 6-(4-(benzyloxy)-2- ethylphenyl)-7-fluoro-3-iodo-1H-indazole (4.1 g, 75% yield). (m/z): [M+H] + calcd for C22H19FIN2O 473.05, found 472.95. (f) To a stirred solution of 6-(4-(benzyloxy)-2-ethylphenyl)-7-fluoro-3-iodo-1H-indazole (4.0 g, 8.47 mmol) in DCM (50 mL) was added PTSA (322 mg, 1.69 mmol) followed by 3,4-DHP (3.9 mL, 42.3 35 mmol) dropwise at 0oC. The solution was allowed to stir at RT for 16 h, then diluted with DCM (100 mL), washed with sat. NaHCO3 (50 mL) and brine (50 mL). The organic layer was separated and dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The crude residue was purified by silica gel flash column chromatography (10-15% EtOAc/heptanes) to afford 6-(4- 35
Attorney Docket No.: P-381-WO/71TD-385423-WO (benzyloxy)-2-ethylphenyl)-7-fluoro-3-iodo-1-(tetrahydro-2H-pyran-2-yl)-1H-indazole (4.0 g, 77% yield). (m/z): [M-H] - calcd for C27H25FIN2O2555.09, found 554.94. (g) A stirred solution of 6-(4-(benzyloxy)-2-ethylphenyl)-7-fluoro-3-iodo-1-(tetrahydro-2H- pyran-2-yl)-1H-indazole (2.0 g, 3.59 mmol) in toluene (20 mL) was purged with argon for 10 min, and 5 1,1,1,2,2,2-hexamethyldistannane (0.9 mL, 4.31 mmol) was added to it followed by Pd(PPh3)4 (415 mg, 0.35 mmol) at room temperature. The reaction mixture was heated at 110°C for 2 h, allowed to cool, filtered through Celite pad and washed with EtOAc (2 x 20 mL). The filtrate was concentrated under reduced pressure and the crude residue was purified by column chromatography using neutral alumina (2% EtOAc/heptanes) to afford 6-(4-(benzyloxy)-2-ethylphenyl)-7-fluoro-1-(tetrahydro-2H-pyran-2-yl)- 10 3-(trimethylstannyl)-1H-indazole (1.1 g, 52% yield). (m/z): [M+H] + calcd for C30H35FN2O2Sn 594.17, found 594.75. Preparation of 5-(6-(4-(benzyloxy)-2-ethylphenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-3-yl)- 1-((2-(trimethylsilyl)ethoxy)methyl)-1H-1,2,4-triazole-3-carbaldehyde, Aldehyde Intermediate 1: 15 (a) To
-1,2,4-triazol-3- yl)methanol (2 g, 6.51 mmol) in toluene (30 mL) at room temperature was added 6-(4-(benzyloxy)-2- ethylphenyl)-1-(tetrahydro-2H-pyran-2-yl)-3-(trimethylstannyl)-1H-indazole (4.5 g, 7.78 mmol). The reaction mixture was purged with N2 gas for 10 min and CuI (248 mg, 1.29 mmol) was added, followed by Pd(PPh3)4 (748 mg, 0.64 mmol). The reaction mixture was heated at 120oC for 4 h, then filtered 20 through a Celite pad, washing with EtOAc (3 x 20 mL). The filtrate was concentrated under reduced pressure, and the crude residue purified using silica gel column chromatography (20% ethyl acetate/heptanes increasing to 30% ethyl acetate/heptanes) to afford (5-(6-(4-(benzyloxy)-2-ethylphenyl)- 1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-3-yl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-1,2,4-triazol-3- yl)methanol (1.3 g, mixture of SEM regioisomers, 31% yield). (m/z): [M+H]+ calcd for C36H46N5O4Si 25 640.33, found 640.45. (b) To a stirred solution of (5-(6-(4-(benzyloxy)-2-ethylphenyl)-1-(tetrahydro-2H-pyran-2-yl)- 1H-indazol-3-yl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-1,2,4-triazol-3-yl)methanol (1.1 g, 1.72 mmol) 36
Attorney Docket No.: P-381-WO/71TD-385423-WO in DCM (30 mL) at room temperature was added DMP (1.46 g, 3.44 mmol). The reaction mixture was stirred at room temperature for 16 h, then poured into saturated NaHCO3 solution (100 mL). The mixture was then filtered through a Celite pad, washing with DCM (3 x 50 mL). The filtrate was extracted with DCM (2 x 100 mL), and the combined organic layers were dried over anhydrous Na2SO4, filtered and 5 concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (15% ethyl acetate/heptanes) to afford 5-(6-(4-(benzyloxy)-2-ethylphenyl)-1- (tetrahydro-2H-pyran-2-yl)-1H-indazol-3-yl)-1-((2-(trimethylsilyl)ethoxy) methyl)-1H-1,2,4-triazole-3- carbaldehyde (610 mg, 55% yield). (m/z): [M+H]+ calcd for C36H44N5O4Si 638.32, found 638.50. Preparation of 5-(6-(4-(benzyloxy)-2-ethylphenyl)-7-fluoro-1-(tetrahydro-2H-pyran-2-yl)-1H- 10 indazol-3-yl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-1,2,4-triazole-3-carbaldehyde, Aldehyde Intermediate 2: (a) A
(trimethylsilyl)ethoxy)methyl)-1H-1,2,4-triazol-3-yl)methanol (500 mg, 1.62 mmol) and 6-(4- 15 (benzyloxy)-2-ethylphenyl)-7-fluoro-1-(tetrahydro-2H-pyran-2-yl)-3-(trimethylstannyl)-1H-indazole (1.06 g, 1.78 mmol) in toluene (10 mL). The reaction mixture was purged with nitrogen for 5 min and tetrakis(triphenylphosphine)palladium(0) (187 mg, 0.162 mmol) and copper(I) iodide (61 mg, 0.324 mmol) were added. The pressure tube was sealed and the reaction mixture was stirred at 120oC for 16 h, then cooled to RT and diluted with EtOAc. The mixture was filtered through a Celite pad, and the residue 20 washed with EtOAc (2 x 25 mL). The filtrate was further diluted with ethyl acetate (20 mL) and washed with water (2 x 20 mL) followed by brine (20 mL). The organic layer was separated and dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The crude residue was purified by silica gel column chromatography (30% EtOAc/heptanes) to afford (5-(6-(4-(benzyloxy)-2-ethylphenyl)- 7-fluoro-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-3-yl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-1,2,4- 25 triazol-3-yl)methanol (300 mg, 23% yield). (m/z): [M+H] + calcd for C36H45FN5O4658.32, found 658.10. (b) To a stirred solution of (5-(6-(4-(benzyloxy)-2-ethylphenyl)-7-fluoro-1-(tetrahydro-2H- pyran-2-yl)-1H-indazol-3-yl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-1,2,4-triazol-3-yl)methanol (350 mg, 0.532 mmol) in DCM (10 mL) was added DMP (451 mg, 1.06 mmol) at room temperature and the 37
Attorney Docket No.: P-381-WO/71TD-385423-WO reaction was stirred for 16 h. The reaction mixture was then poured into saturated NaHCO3 solution (20 mL), filtered through Celite bed, and washed with DCM (3 x 20 mL). The filtrate was diluted with DCM (30 mL), washed with brine, dried over anhydrous Na2SO4 and filtered. The filtrate was concentrated under reduced pressure and purified by silica gel column chromatography (20-25% ethyl 5 acetate/heptanes) to afford 5-(6-(4-(benzyloxy)-2-ethylphenyl)-7-fluoro-1-(tetrahydro-2H-pyran-2-yl)- 1H-indazol-3-yl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-1,2,4-triazole-3-carbaldehyde (200 mg, 57% yield). (m/z): [M+H] + calcd for C36H43FN5O4656.31, found 656.15. Preparation of 5-(6-(2-ethyl-4-((2-(trimethylsilyl)ethoxy)methoxy)phenyl)-1-(tetrahydro-2H-pyran- 2-yl)-1H-indazol-3-yl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-1,2,4-triazole-3-carbaldehyde, 10 Aldehyde Intermediate 3:
mmol) in dry DMF (50 mL) was added NaH (60% w/w, 1.16 g, 29.1 mmol) at 0oC under a nitrogen atmosphere. The suspension was allowed to stir at the same temp for 15 min. SEM-Cl (6.46 mL, 36.4 mmol) was added 15 dropwise and the resulting mixture was stirred at RT for 2 h. The reaction was carefully quenched with ice-cold water (100 mL) and extracted with ethyl acetate (3 x 100 mL). The combined organics were dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The crude residue was purified by silica gel column chromatography (15-20% ethyl acetate/heptanes) to afford methyl 5-bromo-1-((2- (trimethylsilyl)ethoxy)methyl)-1H-1,2,4-triazole-3-carboxylate (6.1 g, mixture of SEM-regioisomers, 74% 20 yield). 38
Attorney Docket No.: P-381-WO/71TD-385423-WO (b) Methyl 5-bromo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-1,2,4-triazole-3-carboxylate (5.0 g, 14.88 mmol) and 6-(4-(benzyloxy)-2-ethylphenyl)-7-fluoro-1-(tetrahydro-2H-pyran-2-yl)-3- (trimethylstannyl)-1H-indazole (9.41 g, 16.36 mmol) were dissolved in toluene (50 mL) and nitrogen was purged through the suspension for 5 min. Tetrakis(triphenylphosphine)palladium(0) (1.71 g, 1.48 mmol) 5 and copper(I) iodide (566 mg, 2.97mmol) were added to the mixture. The reaction was heated at 120oC under vigorous stirring for 2 h then allowed to cool. The reaction mixture was filtered through a Celite bed, and the residue was washed with EtOAc (200 mL). The filtrate was diluted with ethyl acetate, washed with water (2 x 100 mL) followed by brine (100 mL). The organic layer was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to get the crude material which was10 purified by silica gel column chromatography (25-30% EtOAc/heptanes) to afford methyl 5-(6-(4- (benzyloxy)-2-ethylphenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-3-yl)-1-((2- (trimethylsilyl)ethoxy)methyl)-1H-1,2,4-triazole-3-carboxylate (5.4 g, mixture of SEM-regioisomers). (m/z): [M+H] + calcd for C37H46N5O5Si 668.33, found 668.20. (c) To a stirred solution of methyl 5-(6-(4-(benzyloxy)-2-ethylphenyl)-1-(tetrahydro-2H-pyran-2- 15 yl)-1H-indazol-3-yl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-1,2,4-triazole-3-carboxylate (2.6 g, 3.89 mmol) in MeOH (70 mL) was added 10% Pd/C (200 mg, 10% w/w) at room temperature and the reaction was subjected to hydrogenation under balloon pressure at RT overnight. After 16 h, the reaction mixture was filtered through a Celite bed, washing with 10% MeOH in DCM (3 x 100 mL). The filtrate was concentrated under reduced pressure to obtain the desired compound 5-(6-(2-ethyl-4-hydroxyphenyl)-1-20 (tetrahydro-2H-pyran-2-yl)-1H-indazol-3-yl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-1,2,4-triazole-3- carboxylate (2.0 g,75% yield). (m/z): [M+H] + calcd for C30H44N5O5Si 578.28, found 578.15. (d) 5-(6-(2-ethyl-4-hydroxyphenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-3-yl)-1-((2- (trimethylsilyl)ethoxy)methyl)-1H-1,2,4-triazole-3-carboxylate (2.0 g, 3.46 mmol) was dissolved in dry DMF (20 mL) at 0oC under a nitrogen atmosphere. Sodium hydride (60% w/w, 166 mg, 4.15 mmol) was 25 added, and the resulting reaction mixture was allowed to stir for 15 min. SEM-Cl (0.96 mL, 5.19 mmol) was added dropwise at 0oC and stirring was continued at RT for 1 h. The reaction was carefully quenched with ice-cold water (50 mL) and extracted with ethyl acetate (3 x 50 mL). The combined organic layer was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The crude residue was purified by silica gel column chromatography (15-20% ethyl acetate/heptanes) to afford 5-(6-(2-30 ethyl-4-((2-(trimethylsilyl)ethoxy)methoxy)phenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-3-yl)-1- ((2-(trimethylsilyl)ethoxy)methyl)-1H-1,2,4-triazole-3-carboxylate (1.3 g, 44% yield). (m/z): [M+H] + calcd for C36H54N5O6Si2 708.36, found 708.45. (e) To a stirred solution of 5-(6-(2-ethyl-4-((2-(trimethylsilyl)ethoxy)methoxy)phenyl)-1- (tetrahydro-2H-pyran-2-yl)-1H-indazol-3-yl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-1,2,4-triazole-3- 35 carboxylate (1.3 g, 1.84 mmol) in tetrahydrofuran (10 mL):methanol (10 mL) was added NaBH4 (486 mg, 12.9 mmol) portionwise at 0oC. The resulting reaction mixture was stirred at RT for 2 h. The solution was concentrated under reduced pressure, and the crude residue diluted with water (50 mL) and extracted with EtOAc (3 x 100 mL). The combined organic layers were dried over anhydrous Na2SO4, filtered and 39
Attorney Docket No.: P-381-WO/71TD-385423-WO concentrated under reduced pressure. The crude residue was obtained was purified by silica gel column chromatography (15-20% ethyl acetate/ heptanes) to afford (5-(6-(2-ethyl-4-((2- (trimethylsilyl)ethoxy)methoxy)phenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-3-yl)-1-((2- (trimethylsilyl)ethoxy)methyl)-1H-1,2,4-triazol-3-yl)methanol (1.1 g, 81% yield). (m/z): [M+H] + calcd 5 for C35H54N5O5Si2680.37, found 680.15. (f) To a stirred solution of ((5-(6-(2-ethyl-4-((2-(trimethylsilyl)ethoxy)methoxy)phenyl)-1- (tetrahydro-2H-pyran-2-yl)-1H-indazol-3-yl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-1,2,4-triazol-3- yl)methanol (1.1 g, 1.62 mmol) in dichloromethane (35 mL) at room temperature was added DMP (2.06 g, 4.85 mmol) and the reaction mixture was stirred for 30 min. The reaction was then poured into a 10 saturated NaHCO3 solution (100 mL) under stirring and the resulting suspension filtered through a Celite bed, washing with DCM (3 x 50 mL). The filtrate was extracted with DCM (2 x 50 mL) and the combined organic layers were dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography (10-15% ethyl acetate/heptanes) to afford 5-(6-(2-ethyl-4-((2-15 (trimethylsilyl)ethoxy)methoxy)phenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-3-yl)-1-((2- (trimethylsilyl)ethoxy)methyl)-1H-1,2,4-triazole-3-carbaldehyde (910 mg, 86% yield). (m/z): [M+H] + calcd for C35H52N5O5Si2678.35, found 678.40. 40
Attorney Docket No.: P-381-WO/71TD-385423-WO Preparation of 5-(6-(4-(benzyloxy)-2-ethylphenyl)-4-fluoro-1-(tetrahydro-2H-pyran-2-yl)-1H- indazol-3-yl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-1,2,4-triazole-3-carbaldehyde, Aldehyde Intermediate 4: 5 (
(50.0 g, 226.24 mmol) in dioxane (300 mL). The resulting solution was cooled to 0oC and hydrazine monohydrate (32.9 mL, 678.72mmol) was added slowly. The reaction mixture was allowed to stir at rt overnight. To this solution, TEA (158 mL, 1131 mmol) was added and stirring was continued at 120°C under sealed condition for 2h. The reaction was concentrated, and the resulting residue was diluted with water and 10 extracted using ethyl acetate (2 x 200 mL). The combined extracts were concentrated under reduced pressure and the crude residue triturated with pentane, filtered through a sintered funnel, and dried under high vacuum to obtain 6-bromo-4-fluoro-1H-indazole (48.0 g, 82% yield).1H NMR (400 MHz, DMSO- d6) δ 13.52 (br s, 1H), 8.22 (s, 1H), 7.65 (s, 1H), 7.41 (m, 1H), 7.15 (d, J = 9.4 Hz, 1H). m/z: [M+H]- calcd for C7H3BrFN2212.95, found 213.0. 15 (b) 6-bromo-4-fluoro-1H-indazole (48 g, 223.25 mmol) was dissolved in DCM (1 L) and PTSA (7.61 g, 44.65 mmol).3,4-dihydropyran (59 mL, 669.73 mmol) was added slowly, and the reaction was allowed to stir at RT for 16 h. The reaction mixture was then washed with water (2 x 500 mL), followed by saturated NaHCO3 solution (400 mL) and brine (400 mL). The organic layer was dried over anhydrous Na2SO4 and concentrated under reduced pressure. The crude residue was purified by silica gel 41
Attorney Docket No.: P-381-WO/71TD-385423-WO column chromatography (10% ethyl acetate/heptanes) to afford as 6-bromo-4-fluoro-1-(tetrahydro-2H- pyran-2-yl)-1H-indazole (40.0 g, 60% yield).1H NMR (400 MHz, DMSO-d6) δ 8.27 (s, 1H), 7.95 (s, 1H), 7.26 (d, J = 9.5 Hz, 1H), 5.90 (d, J = 7.9 Hz, 1H), 3.87 (m, 1H), 3.77 (m, 1H), 2.30 (m, 1H), 1.99 (m, 2H), 1.73 (m, 1H), 1.58 (m ,2H). 5 (c) To a stirred solution of 6-bromo-4-fluoro-1-(tetrahydro-2H-pyran-2-yl)-1H-indazole (8.0 g, 26.7 mmol) and 2-(4-(benzyloxy)-2-ethylphenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (10.9 g, 32.1 mmol) in dioxane (160 mL): H2O (40 mL) was added Na2CO3 (5.67 g, 53.5 mmol). The reaction mixture was purged with argon for 10 min and PdCl2(dppf). DCM (2.18 g, 2.67 mmol) was added. The reaction mixture was heated at 110°C for 16h, then allowed to cool. The reaction contents were filtered through a 10 Celite bed, and the residue washed with ethyl acetate (2 x 200 mL). The filtrate was diluted with ethyl acetate, then washed with cold water (2 x 100 mL) followed by brine (100 mL). The organic layer was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The crude residue was purified by silica gel column chromatography (8-10% EtOAc/heptanes) to afford 6-(4-(benzyloxy)-2- ethylphenyl)-4-fluoro-1-(tetrahydro-2H-pyran-2-yl)-1H-indazole (7.3 g, 63.4% yield). (m/z) [M+H] + 15 calcd for C27H28FN2O2431.21, found 431.10. (d) To a stirred solution of 6-(4-(benzyloxy)-2-ethylphenyl)-4-fluoro-1-(tetrahydro-2H-pyran-2- yl)-1H-indazole (7.3 g, 17.0 mmol) in methanol (50 mL): THF (50 mL) at 0°C was added 6N HCl in dioxane (35 mL) dropwise. The resulting solution was warmed to room temperature then heated at 70°C for 16h. The reaction mixture was allowed to cool, concentrated under reduced pressure and then 20 neutralized slowly at 0°C with saturated NaHCO3 (100 mL) and extracted with EtOAc (2 x 100mL). The combined organic layers were further washed with brine (100mL), dried over anhydrous Na2SO4 and concentrated under reduced pressure to afford 6-(4-(benzyloxy)-2-ethylphenyl)-4-fluoro-1H-indazole (4.8 g, 82% yield). (m/z) [M+H] + calcd for C22H20FN2O 347.16, found 347.05. (e) To a stirred solution 6-(4-(benzyloxy)-2-ethylphenyl)-4-fluoro-1H-indazole (4.0 g, 11.5 25 mmol) in DMF (40 mL) at 0°C was added NIS (6.5 g, 28.9 mmol) portionwise. The reaction mixture was stirred at room temperature for 3h, then diluted with water (50 mL) and extracted with ethyl acetate (3 x 30 mL). The combined organic layers were washed with sodium thiosulfate solution (50 mL) followed by brine (50 mL) and dried over anhydrous Na2SO4. The filtrate was concentrated under reduced pressure to afford 6-(4-(benzyloxy)-2-ethylphenyl)-4-fluoro-3-iodo-1H-indazole (4.8 g, 88% yield). (m/z) [M+H] - 30 calcd for C22H17FIN2O 471.04, found 471.05. (f) To a stirred solution of compound 6-(4-(benzyloxy)-2-ethylphenyl)-4-fluoro-3-iodo-1H- indazole (4.8 g, 10.2 mmol) in DCM (50 mL) at 0oC was added PTSA (387 mg, 2.03 mmol) followed by 3,4-DHP (2.32 mL, 25.4 mmol) dropwise. The reaction mixture was allowed to stir at room temperature for 16 h and then was diluted with DCM (100 mL) and washed with saturated NaHCO3 (50 mL) followed 35 by brine (50 mL). The organic layer was separated and dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The crude residue was purified by silica gel column chromatography (10-12% EtOAc/heptanes) to obtain 6-(4-(benzyloxy)-2-ethylphenyl)-4-fluoro-3-iodo-1- 42
Attorney Docket No.: P-381-WO/71TD-385423-WO (tetrahydro-2H-pyran-2-yl)-1H-indazole (3.3 g, 58% yield). (m/z) [M+H] +calcd for C27H27FIN2O2 557.11, found 557.10. (g) A stirred solution of 6-(4-(benzyloxy)-2-ethylphenyl)-4-fluoro-3-iodo-
o-2H- pyran-2-yl)-1H-indazole (3.3 g, 5.96 mmol) in toluene (35 mL) was purged with argon for 10 min. 5 1,1,1,2,2,2-hexamethyldistannane (1.49 mL, 7.12 mmol) was added, followed by Pd(PPh3)4 (685 mg, 0.59 mmol). The reaction mixture was heated at 110°C for 2h, allowed to cool, filtered through Celite pad and washed with EtOAc (2 x 50 mL). The filtrate was concentrated under reduced pressure and the crude residue purified through column chromatography over neutral alumina (1% EtOAc/heptanes) to afford 6-(4-(benzyloxy)-2-ethylphenyl)-4-fluoro-1-(tetrahydro-2H-pyran-2-yl)-3-(trimethylstannyl)-1H- 10 indazole (2.8 g, 79% yield). (m/z) [M+H] +calcd for C30H36FN2O2Sn 595.18, found 595.05. (h) An Ace pressure tube was charged with (5-bromo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H- 1,2,4-triazol-3-yl)methanol (500 mg, 1.62 mmol) and 6-(4-(benzyloxy)-2-ethylphenyl)-4-fluoro-1- (tetrahydro-2H-pyran-2-yl)-3-(trimethylstannyl)-1H-indazole (1.15 g, 1.95 mmol) in toluene (10 mL). The reaction mixture was purged with nitrogen for 5 min. Tetrakis(triphenylphosphine)palladium(0) (187 15 mg, 0.162 mmol) and Copper(I) iodide (61 mg, 0.324 mmol) were added. The pressure tube was sealed and the reaction mixture was stirred at 120oC for 16h. The reaction was allowed to cool to room temperature, diluted with EtOAc, filtered through a Celite pad and the residue washed with EtOAc (2 x 25 mL). The filtrate was further diluted with ethyl acetate (20 mL) and washed with cold water (2 x 20 mL) followed by brine (20 mL). The organic layer was separated and dried over anhydrous Na2SO4, 20 filtered and concentrated under reduced pressure. The crude residue was purified by silica gel column chromatography (25-30% EtOAc/heptanes) to afford ((5-(6-(4-(benzyloxy)-2-ethylphenyl)-4-fluoro-1- (tetrahydro-2H-pyran-2-yl)-1H-indazol-3-yl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-1,2,4-triazol-3- yl)methanol (320 mg, 23% yield). (m/z) [M+H] +calcd for C36H45FN5O4Si 658.32, found 658.25. (i) To a stirred solution of (5-(6-(4-(benzyloxy)-2-ethylphenyl)-4-fluoro-1-(tetrahydro-2H-pyran- 25 2-yl)-1H-indazol-3-yl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-1,2,4-triazol-3-yl)methanol (700 mg, 1.06 mmol) in dichloromethane (15 mL) was added DMP (677 mg, 1.6 mmol) at room temperature. The reaction mixture was stirred for 2h then poured into saturated NaHCO3 solution (20 mL) and filtered through Celite bed, washing with DCM (3 x 20 mL). The filtrate was diluted with DCM (30 mL), washed with brine, dried over sodium sulfate and concentrated under reduced pressure. The crude residue30 was purified by silica gel column chromatography (15-20% ethyl acetate/heptanes) to afford 5-(6-(4- (benzyloxy)-2-ethylphenyl)-4-fluoro-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-3-yl)-1-((2- (trimethylsilyl)ethoxy)methyl)-1H-1,2,4-triazole-3-carbaldehyde. (m/z) [M+H] + calcd for C36H43FN5O4Si 656.31, found 656.25. 43
Attorney Docket No.: P-381-WO/71TD-385423-WO Preparation of 3-ethyl-4-(3-(3-((4-methylpiperazin-1-yl)methyl)-1H-1,2,4-triazol-5-yl)-1H-indazol- 6-yl)phenol (Compound No.5): METHOD A (a yran-2-yl)-1H-
5 indazol-3-yl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-1,2,4-triazole-3-carbaldehyde (Aldehyde Intermediate 1) (140 mg, 0.219 mmol) and 1-methylpiperazine (50 µL, 0.439 mmol) in methanol (6 mL) was added acetic acid (0.25 mL, 4.38 mmol). The reaction mixture was stirred at room temperature for 1 h. Sodium cyanoborohydride (39.4 mg, 0.658 mmol) was added and stirring continued for 16 h. The reaction mixture was then concentrated under reduced pressure and the crude residue was washed and 10 triturated with n-pentane (25 mL). The product was used in the next step without purification (160 mg, 47% yield). (m/z): [M+H]+ calcd for C41H56N7O3Si 722.42, found 722.80. (b) To a stirred solution of 6-(4-(benzyloxy)-2-ethylphenyl)-3-(3-((4-methylpiperazin-1- yl)methyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-1,2,4-triazol-5-yl)-1-(tetrahydro-2H-pyran-2-yl)-1H- indazole (160 mg, 0.22 mmol) in methanol (3 mL, 74.1 mmol) was added 8N HCl (1.6 mL) dropwise at 15 0oC. The reaction mixture was heated at 80oC for 36 h and then concentrated under reduced pressure. The crude residue was triturated with n-pentane and then purified by reverse phase HPLC to afford the TFA salt of the title compound (12 mg, 7% yield).1H NMR (400 MHz, MeOD) δ 8.70 (dd, J = 8.4, 0.8 Hz, 1H), 7.44 (s, 1H), 7.19 (dd, J = 8.4, 1.2 Hz, 1H), 7.07 (d, J = 8.0 Hz, 1H), 6.78 (d, J = 2.4 Hz, 1H), 6.69 (dd, J = 8.0, 2.4 Hz, 1H), 3.95 (s, 2H), 3.78-3.21 (broad m, 6H), 2.90 (s, 3H), 2.67-2.59 (broad m, 2H), 20 2.57 (q, J = 7.6 Hz, 2H), 1.07 (t, J = 7.6 Hz, 3H). (m/z): [M+H]+ calcd for C23H28N7O 418.24, found 418.25. 44
Attorney Docket No.: P-381-WO/71TD-385423-WO Preparation of 3-ethyl-4-(3-(3-((8-methyl-3,8-diazabicyclo[3.2.1]octan-3-yl)methyl)-1H-1,2,4- triazol-5-yl)-1H-indazol-6-yl)phenol (Compound No.4): METHOD A ( , 0.188 5
mmol) in 1,2 -dichloroethane (4 mL) at room temperature was added triethylamine (66 µL, 0.470 mmol) and the reaction mixture was stirred for 30 min. The solution was cooled to 0oC and 5-(6-(4- (benzyloxy)-2-ethylphenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-3-yl)-1-((2- (trimethylsilyl)ethoxy)methyl)-1H-1,2,4-triazole-3-carbaldehyde (Aldehyde Intermediate 1) (120 mg, 0.188 mmol) was added, followed by AcOH (43 µL, 0.75 mmol) and STAB (80 mg, 0.376 mmol). The 10 reaction was stirred at room temperature for 16 h and then concentrated under reduced pressure. The crude product was triturated with n-pentane (25 mL) and used in the next step without purification (105 mg). (m/z): [M+H]+ calcd for C43H58N7O3Si 748.44, found 748.30. (b) To a stirred solution of 6-(4-(benzyloxy)-2-ethylphenyl)-3-(3-((8-methyl-3,8- diazabicyclo[3.2.1]octan-3-yl)methyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-1,2,4-triazol-5-yl)-1- 15 (tetrahydro-2H-pyran-2-yl)-1H-indazole (80 mg, 0.107 mmol) in methanol (4 mL) at 0oC was added 6N HCl (0.8 mL). The reaction was stirred at 80oC for 2 days and then concentrated under reduced pressure. The residue was triturated with n-pentane and then purified by reverse phase HPLC to afford the TFA salt of 3-ethyl-4-(3-(3-((8-methyl-3,8-diazabicyclo[3.2.1]octan-3-yl)methyl)-1H-1,2,4-triazol-5-yl)-1H- indazol-6-yl)phenol (22 mg, 26% yield).1H NMR (400 MHz, DMSO-d6/CF3CO2D) δ 8.25 (d, J = 8.4, 20 1H), 7.47 (s, 1H), 7.20 (d, J = 9.2, 1H), 7.04 (d, J = 8.0 Hz, 1H), 6.75 (d, J = 2.4 Hz, 1H), 6.68 (dd, J = 8.4, 2.4 Hz, 1H), 4.40 (br s, 2H), 4.09 (br s, 2H), 3.59 (d, J = 12.8 Hz, 2H), 3.39 (d, J = 12.8 Hz, 2H), 2.69 (br s, 3H), 2.46 (d, J = 4.0 Hz, 2H), 2.26-2.16 (br m, 4H), 1.00 (t, J = 7.6 Hz, 3H). (m/z): [M+H]+ calcd for C25H30N7O 444.25, found 444.35. 45
Attorney Docket No.: P-381-WO/71TD-385423-WO Preparation of 3-ethyl-4-(3-(3-((4-ethylpiperazin-1-yl)methyl)-1H-1,2,4-triazol-5-yl)-1H- indazol-6-yl)phenol (Compound No.1): METHOD B (a)
pyran-2-yl)-1H- 5 indazol-3-yl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-1,2,4-triazole-3-carbaldehyde (Aldehyde Intermediate 1) (440 mg, 0.690 mmol) and tert-butyl piperazine-1-carboxylate (385 mg, 2.072 mmol) in methanol (15 mL) was added acetic acid (0.78 mL, 13.8 mmol) at RT, and the reaction mixture was allowed to stir at for 1 h. Sodium cyanoborohydride (440 mg, 2.07 mmol) was added to this solution and stirring was continued overnight. The reaction mixture was concentrated under reduced pressure and the 10 resulting crude was purified by silica gel column chromatography (50% ethyl acetate/heptanes) to afford tert-butyl 4-((5-(6-(4-(benzyloxy)-2-ethylphenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-3-yl)-1-((2- (trimethylsilyl)ethoxy)methyl)-1H-1,2,4-triazol-3-yl)methyl)piperazine-1-carboxylate (380 mg, 68% yield). (m/z): [M+H]+ calcd for C45H61N7O5Si 807.45, found 808.35. (b) tert-butyl 4-((5-(6-(4-(benzyloxy)-2-ethylphenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol- 15 3-yl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-1,2,4-triazol-3-yl)methyl)piperazine-1-carboxylate (380 mg, 0.47 mmol) was dissolved in IPA (3 mL):THF (3 mL). Nitrogen was purged through the solution and 10% Pd/C (80 mg, 20% w/w) was added. The reaction was subjected to hydrogenation under balloon pressure at RT for 16 h. The mixture was filtered through a Celite bed and washed with 10% MeOH in DCM (3 x 20 mL). The filtrate was concentrated under reduced pressure to obtain the desired compound20 4-((5-(6-(2-ethyl-4-hydroxyphenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-3-yl)-1-((2- 46
Attorney Docket No.: P-381-WO/71TD-385423-WO (trimethylsilyl)ethoxy)methyl)-1H-1,2,4-triazol-3-yl)methyl)piperazine-1-carboxylate (280 mg, 83% yield). (m/z): [M+H]+ calcd for C38H55N7O5Si 717.40, found 718.35. (c) To a stirred solution of 4-((5-(6-(2-ethyl-4-hydroxyphenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H- indazol-3-yl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-1,2,4-triazol-3-yl)methyl)piperazine-1-carboxylate 5 (280 mg, 0.39 mmol) in methanol (5 mL) was added 6N HCl (3 mL) dropwise at 0oC. The reaction was heated to 80oC for 16 h, and then concentrated under reduced pressure. The resulting brown viscous liquid was triturated with n-pentane to obtain 3-ethyl-4-(3-(3-(piperazin-1-ylmethyl)-1H-1,2,4-triazol-5- yl)-1H-indazol-6-yl)phenol as a crude HCl salt (220 mg) that was used without further purification. (m/z): [M+H]+ calcd for C22H25N7O 403.21, found 404.35. 10 (d) To a stirred solution of 3-ethyl-4-(3-(3-(piperazin-1-ylmethyl)-1H-1,2,4-triazol-5-yl)-1H- indazol-6-yl)phenol HCl salt (100 mg, 0.248 mmol) in methanol (5 mL) was added acetaldehyde (33 mg, 0.744 mmol) followed by acetic acid (0.28 mL, 4.96 mmol). The reaction mixture was stirred for 1 h at room temperature. Sodium cyanoborohydride (158 mg, 0.74 mmol) was then added and stirring was continued for 16 h. The reaction mixture was then concentrated under reduced pressure to obtain a brown15 viscous liquid that was purified by reverse phase HPLC to afford the TFA salt of 3-ethyl-4-(3-(3-((4- ethylpiperazin-1-yl)methyl)-1H-1,2,4-triazol-5-yl)-1H-indazol-6-yl)phenol (23 mg, 12% yield).1H NMR (400 MHz, DMSO-d6/D2O) δ 8.23 (d, J = 8.4 Hz, 1H), 7.42 (s, 1H), 7.15 (dd, J = 8.4, 0.8 Hz, 1H), 7.04 (d, J = 8.0 Hz, 1H), 6.73 (d, J = 2.4 Hz, 1H), 6.67 (dd, J = 8.0, 2.4 Hz, 1H), 3.89 (s, 2H), 3.45 (br s, 2H), 3.20-2.90 (br m, 6h), 2.66-2.46 (br m, 4H), 1.18 (t, J = 7.6 Hz, 3H). (m/z): [M+H]+ calcd for C24H29N7O 20 431.24, found 432.4. Preparation of 3-ethyl-4-(7-fluoro-3-(3-((4-methylpiperazin-1-yl)methyl)-1H-1,2,4-triazol-5-yl)-1H- indazol-6-yl)phenol (Compound No.16): METHOD A (a) T
ydro-2H-pyran- 25 2-yl)-1H-indazol-3-yl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-1,2,4-triazole-3-carbaldehyde (Aldehyde Intermediate 2) (180 mg, 0.274 mmol) in methanol (5 mL) was added acetic acid (0.34 mL, 5.49 mmol) followed by 1-methylpiperazine (82.5 mg, 0.823 mmol), and the reaction mixture was stirred for 15 min. Sodium cyanoborohydride (52 mg, 0.823 mmol) was then added and the reaction continued for 16 h at RT. The solution was concentrated under reduced pressure and the residue triturated with diethyl ether to 47
Attorney Docket No.: P-381-WO/71TD-385423-WO afford 6-(4-(benzyloxy)-2-ethylphenyl)-7-fluoro-3-(3-((4-methylpiperazin-1-yl)methyl)-1-((2- (trimethylsilyl)ethoxy)methyl)-1H-1,2,4-triazol-5-yl)-1-(tetrahydro-2H-pyran-2-yl)-1H-indazole (210 mg, used without further purification). (m/z): [M+H]+ calcd for C41H55FN7O3Si 740.41, found 740.10. (b) To a stirred solution of 6-(4-(benzyloxy)-2-ethylphenyl)-7-fluoro-3-(3-((4-methylpiperazin-1- 5 yl)methyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-1,2,4-triazol-5-yl)-1-(tetrahydro-2H-pyran-2-yl)-1H- indazole (210 mg) in methanol (5 mL) was added 8N HCl (2.0 mL) dropwise at 0oC. The reaction was then heated to 80oC for 16 h then allowed to cool. The reaction mixture was concentrated under reduced pressure to a brown viscous liquid. The residue was washed and triturated with n-pentane and purified by reverse phase HPLC to afford the TFA salt of 3-ethyl-4-(7-fluoro-3-(3-((4-methylpiperazin-1-yl)methyl)- 10 1H-1,2,4-triazol-5-yl)-1H-indazol-6-yl)phenol (51 mg, yield 23%). (m/z): [M+H]+ calcd for C23H27FN7O 436.23, found 436.35. The compounds in Table 3 were prepared using similar synthetic methods and the appropriate reactants. Table 3 Compound Aldehyde Synthetic M Calculated Found I i ethod M+H + M+H +
48
Attorney Docket No.: P-381-WO/71TD-385423-WO Compound Aldehyde Synthetic Method Calculated Found No. Intermediate [M+H]+ [M+H]+
49
Attorney Docket No.: P-381-WO/71TD-385423-WO Biological Assays The compounds of the present disclosure have been characterized in one or more of the following biological assays. Assay 1: Biochemical JAK Kinase Assays 5 A panel of LanthaScreen JAK biochemical assays (JAK1 and 2) were carried in a common kinase reaction buffer (50 mM HEPES, pH 7.5, 0.01% Brij-35, 10 mM MgCl2, and 1 mM EGTA). Recombinant GST-tagged JAK enzymes and a GFP-tagged STAT1 peptide substrate were obtained from Life Technologies. Serially diluted compounds were pre-incubated with each of the two JAK enzymes and the 10 substrate in white 384-well microplates (Corning) at ambient temperature for 1h. ATP was subsequently added to initiate the kinase reactions in 10 µL total volume, with 1% DMSO. The final enzyme concentrations for JAK1 and 2 are 4.2 nM and 0.1 nM, respectively; the corresponding Km ATP concentrations used are 25 µM and 3 µM; while the substrate concentration is 200 nM for all assays. Kinase reactions were allowed to proceed for 1 hour at ambient temperature before a 10 μL preparation 15 of EDTA (10mM final concentration) and Tb-anti-pSTAT1 (pTyr701) antibody (Life Technologies, 2 nM final concentration) in TR-FRET dilution buffer (Life Technologies) was added. The plates were allowed to incubate at ambient temperature for 1h before being read on the EnVision reader (Perkin Elmer). Emission ratio signals (520nm/495nm) were recorded and utilized to calculate the percent inhibition values based on DMSO and background controls. 20 For dose-response analysis, percent inhibition data were plotted vs. compound concentrations, and IC50 values were determined from a 4-parameter robust fit model with the Prism software (GraphPad Software). Results were expressed as pIC50 (negative logarithm of IC50) and subsequently converted to pKi (negative logarithm of dissociation constant, Ki) using the Cheng-Prusoff equation. Test compounds having a lower Ki value or higher pKi value in the JAK assays show greater 25 inhibition of JAK activity. Assay 2: Cellular JAKI Potency Assay The JAKI cellular potency assay was carried out by measuring inhibition of interleukin-13 (IL- 13, R&D Systems) induced STAT6 phosphorylation in BEAS-2B human lung epithelial cells (ATCC). BEAS-2B cells were grown at 37°C in a 5% CO2 humidified incubator in 50% DMEM/50% F-12 30 medium (Life Technologies) supplemented with 10% FBS (Hyclone), 100 U/mL penicillin, 100 µg/mL streptomycin (Life Technologies), and 2 mM GlutaMAX (Life Technologies). On day 1 of the assay, cells were seeded at a 7,500 cells/well density in white poly-D-lysine-coated 384-well plates (Corning) with 25µL medium and were allowed to adhere overnight in the incubator. On day 2 of the assay, the medium was removed and replaced with 12 µL of assay buffer (Hank's Balanced Salt Solution/HBSS, 35 25mM HEPES, and 1 mg/ml bovine serum albumin/BSA) containing dose-responses of test compounds. Compounds were serially diluted in DMSO and then diluted another 1000-fold in media to bring the final DMSO concentration to 0.1%. Cells were incubated with test compounds at 37°C for 1 h and followed by the addition of 12 μL of pre-warmed IL-13 (80 ng/mL in assay buffer) for stimulation. After incubating 50
Attorney Docket No.: P-381-WO/71TD-385423-WO at 37°C for 30 min, the assay buffer (containing compound and IL-13) was removed, and 10 μL of cell lysis buffer (25 mM HEPES, 0.1 % SDS, 1 % NP-40, 5 mM MgCl2, 1.3 mM EDTA, 1 mM EGTA, supplemented with Complete Ultra mini protease inhibitors and PhosSTOP from Roche Diagnostics). The plates were shaken at ambient temperature for 30min before the addition of detection reagents. 5 Levels of pSTAT6 were measured using the AlphaLISA SureFire Ultra pSTAT6 (Tyr641) assay kit from PerkinElmer. For dose-response analysis, percent inhibition data were plotted vs. compound concentrations, and IC50 values were determined from a 4-parameter robust fit model with the Graphpad Prism software. Results are expressed as the negative logarithm of the IC50 value, pIC50. Test compounds having a lower IC50 value or higher pIC50 value in this assay show greater 10 inhibition of IL-13 induced STAT6 phosphorylation. In Vitro Assay Results The compounds were tested in the BEAS-2B cellular potency assay and JAK1 and JAK2 assays described above. JAK1/2 selectivity was derived by subtracting JAK2 pKi from JAK1 pKi. Table 4 Compound JAK1 JAK2 JAK1/2 BEAS2B IL13 No
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Attorney Docket No.: P-381-WO/71TD-385423-WO Compound JAK1 JAK2 JAK1/2 BEAS2B IL13 No. (pKi) (pKi) Selectivity pSTAT6 pIC50
hibit advantageous properties over their imidazole-containing analogs. Data provided in Table 5 were obtained as described above. 5 Table 5 BEAS2B JAK1 JAK2 JAK1/2 IL13 6
52
Attorney Docket No.: P-381-WO/71TD-385423-WO While the present disclosure has been described with reference to specific aspects or embodiments thereof, it will be understood by those of ordinary skilled in the art that various changes can be made or equivalents can be substituted without departing from the true spirit and scope of the disclosure. Additionally, to the extent permitted by applicable patent statutes and regulations, all 5 publications, patents and patent applications cited herein are hereby incorporated by reference in their entirety to the same extent as if each document had been individually incorporated by reference herein. 53