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  • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
  • C07D403/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a chain containing hetero atoms as chain links
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  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
  • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
  • A61K31/445—Non condensed piperidines, e.g. piperocaine
  • A61K31/4523—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems
  • A61K31/4545—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems containing a six-membered ring with nitrogen as a ring hetero atom, e.g. pipamperone, anabasine
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  • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
  • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
  • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
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  • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
  • A61K31/5375—1,4-Oxazines, e.g. morpholine
  • A61K31/5377—1,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol
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  • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
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  • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
  • A61K31/553—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole having at least one nitrogen and one oxygen as ring hetero atoms, e.g. loxapine, staurosporine
• • A—HUMAN NECESSITIES
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  • C07D231/02—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings not condensed with other rings
  • C07D231/10—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
  • C07D231/14—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
  • C07D231/38—Nitrogen atoms
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  • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
  • C07D401/04—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings directly linked by a ring-member-to-ring-member bond
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  • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
  • C07D403/04—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings directly linked by a ring-member-to-ring-member bond
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  • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
  • C07D405/14—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing three or more hetero rings
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  • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
  • C07D417/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing three or more hetero rings
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  • C07D451/00—Heterocyclic compounds containing 8-azabicyclo [3.2.1] octane, 9-azabicyclo [3.3.1] nonane, or 3-oxa-9-azatricyclo [3.3.1.0<2,4>] nonane ring systems, e.g. tropane or granatane alkaloids, scopolamine; Cyclic acetals thereof
  • C07D451/02—Heterocyclic compounds containing 8-azabicyclo [3.2.1] octane, 9-azabicyclo [3.3.1] nonane, or 3-oxa-9-azatricyclo [3.3.1.0<2,4>] nonane ring systems, e.g. tropane or granatane alkaloids, scopolamine; Cyclic acetals thereof containing not further condensed 8-azabicyclo [3.2.1] octane or 3-oxa-9-azatricyclo [3.3.1.0<2,4>] nonane ring systems, e.g. tropane; Cyclic acetals thereof
• • A—HUMAN NECESSITIES
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  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
  • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
  • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
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  • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
  • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
  • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

• Autophagy (literally meaning“self eating”) is a process that enables cells to recycle cellular organelles, proteins, stored lipids, glucagon, and other materials for the purpose of generating nutrients under periods of stress. These cellular contents are recycled by engulfment in vesicles called autophagosomes. Autophagosomes subsequently merge with lysosomes that degrade the autophagosomal contents for recycling of nutrients to the cell.
• Tumor cells are prone to activate autophagy, as these cells have a high metabolic demand, experience cellular stress, and frequently are in hypoxic environments with limited blood flow and nutrient supply.
• chemotherapy and targeted therapies have been shown to induce autophagy as a treatment resistance mechanism, and combination of autophagy inhibition (by genetic loss of function mutations in autophagy genes or by pharmacologic means) with chemotherapeutic regimens has been shown to suppress tumor growth and trigger tumor cell apoptosis to a greater extent than single agent chemotherapy.
• Mutant Ras proteins drive approximately 30 percent of all human cancers - including 95 percent of pancreatic cancers and 45 percent of colorectal cancers, and treatment of these mutant Ras cancers is currently an area of high unmet medical need. Mutant Ras cancers are highly proliferative and depend on basal levels of autophagy for survival, suggesting that inhibition of autophagy in these“autophagy addicted” cancers is a viable therapeutic approach. [0004] Currently, the most widely used autophagy inhibitors are chloroquine and hydroxychloroquine, which are well-known anti-malarial agents.
• ULK1 kinase is the initiating protein of autophagy and is a serine/threonine kinase. The ULK1 kinase complex is activated in response to cellular stress including nutrient deprivation and energy depletion.
• Nutrient deprivation activates ULK kinase activity through inhibition of mTORCl, and energy depletion activates ULK kinase activity through activation by AMP-activated protein kinase AMPK.
• kinase dead mutants of ULK kinase block initiation of canonical autophagy, suggesting that small molecule inhibitors of ULK kinase activity would be able to block autophagy.
• ULKl inhibits autophagy in cancer cells, relieving FOX3 A turn-over and upregulation of the pro-apoptotic protein PUMA.
• ULKl kinase activity has been shown to be required for Bcl-2-L-13 mediated mitophagy (autophagy of damaged mitochondria).
• ULKl and ULK2 kinases have also been demonstrated to rewire cancer cell glucose metabolism. ULK inhibitors may also find utility in blocking these noncanonical protumoral activities of ULK.
• Autophagy is also upregulated in host cells and tissues in cancer.
• Autophagy in pancreatic tissue stellate cells was demonstrated to support tumor growth.
• Pancreatic stellate cells were shown to support pancreatic cancer tumor metabolism through autophagic alanine secretion.
• Inhibition of host tissue autophagy was demonstrated to lead to a depletion in circulating arginine (a required amino acid for tumor metabolism and growth) through liver - mediated increases in arginase secretion.
• Activation of ULKl kinase was also shown to inactivate the STING pathway in immune cells through inhibitory phosphorylation of STING, mediating a negative feedback mechanism for limiting an innate immune cell response mediated by interferons.
• Mutant Ras cancers are addicted to autophagy. In pancreatic cancer, mutant Ras signals predominantly through the MAPKAP pathway. Mutant Ras activates RAF kinases, which in turn activate MEK kinases, which finally activate ERK kinases: mutant Ras - RAF - MEK - ERK. Despite mutant Ras signaling through the MAPKAP pathway, inhibitors of this pathway have provided no or little clinical benefit in clinical trials when used as single agents. It has been recently reported that inhibition of the MAPKAP pathway induces autophagy as a compensatory survival mechanism. When MEK inhibitors were combined with the autophagy inhibitor hydroxychloroquine, there was synergistic activity leading to regression of a number of mutant Ras or mutant BRAF cancers.
• LRRK2 kinase Mutations in the gene encoding LRRK2 kinase are causative of Parkinson’s disease. LRRK2 point mutations are found in both familial (inherited) as well as sporadic Parkinson’s disease patients. The most common mutation of LRRK2 in Parkinson’s disease is LRRK2 G2019S. These mutations in LRRK2 are gain-of-function mutations that cause overactivation of LRRK2 signaling. Ongoing autophagy is a process that is used by brain neuronal cells to maintain health and homeostasis.
• LRRK2 activity suppresses autophagy, and the LRRK2 G2019S gain-of-function mutant even moreso suppresses autophagy and has been linked to aggressive forms of Parkinson’s disease.
• Increased LRRK2 kinase activity has also been linked to immunoinflammatory diseases including colitis and Crohn’s disease and inflammatory bowel disease.
• LRRK2 is present in antigen-presenting cells including dendritic cells.
• LRRK2 activity has been shown to be important in Dectin-1 mediated innate immune responses, including an activation of the NFkB pathway and increased TNF-alpha production in dendritic cells of patients with Crohn’s disease.
• Inhibitors of LRRK2 are sought for the treatment of neurodegenerative diseases including Parkinson’s disease, and also are sought for the treatment of gastrointestinal diseases including Crohn’s disease, ulcerative colitis, and inflammatory bowel disease.
• Described herein are compounds that are inhibitors of autophagy, pharmaceutical compositions, and their use as agents in the treatment of disorders such as cancer, processes for their preparation, and pharmaceutical compositions containing them as an active ingredient.
• Such pharmaceutical compositions may comprise the compound as the sole active agent or in combination with other active agents in the presence of a pharmaceutically acceptable excipient.
• the described compounds are inhibitors of ULK kinase activity, including ULK1 and ULK2 activity.
• alkyl refers to a saturated straight or branched hydrocarbon.
• exemplary alkyl groups include, but are not limited to, straight or branched hydrocarbons of 1-6, 1-4, 1-3, or 1-2 carbon atoms, referred to herein as Ci-C 6 alkyl, Ci-C4alkyl, Ci-C3alkyl, and Ci-C2alkyl, respectively.
• Exemplary alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, 2-methyl- 1 -butyl, 3 -methyl -2 -butyl, 2-methyl-l-pentyl, 3- methyl-1 -pentyl, 4-methyl- 1 -pentyl, 2-methyl-2-pentyl, 3 -methyl -2-pentyl, 4-methyl-2-pentyl, 2,2-dimethyl- 1 -butyl, 3, 3 -dimethyl- 1 -butyl, 2-ethyl- 1 -butyl, butyl, isobutyl, t-butyl, pentyl, isopentyl, neopentyl, hexyl, etc.
• alkenyl refers to an unsaturated straight or branched hydrocarbon having at least one carbon-carbon double bond.
• alkenyl groups include, but are not limited to, a straight or branched group of 2-6 or 3-4 carbon atoms, referred to herein as C2-C6alkenyl, and C3-C4alkenyl, respectively.
• alkenyl groups include, but are not limited to, vinyl, allyl, butenyl, pentenyl, etc.
• alkoxy refers to a straight or branched alkyl group attached to oxygen (alkyl-O-).
• exemplary alkoxy groups include, but are not limited to, alkoxy groups of 1-6 or 2-6 carbon atoms, referred to herein as Ci-C 6 alkoxy, and C2-C6alkoxy, respectively.
• exemplary alkoxy groups include, but are not limited to methoxy, ethoxy, isopropoxy, etc.
• alkoxyalkyl refers to a straight or branched alkyl group attached to oxygen, attached to a second straight or branched alkyl group (alkyl-O-alkyl-).
• Exemplary alkoxyalkyl groups include, but are not limited to, alkoxyalkyl groups in which each of the alkyl groups independently contains 1-6 carbon atoms, referred to herein as Ci-C 6 alkoxy- Ci-C 6 alkyl and Ci-C6alkoxy-C2-C6alkyl.
• Exemplary alkoxyalkyl groups include, but are not limited to methoxymethyl, 2-m ethoxy ethyl, 1 -methoxy ethyl, 2-methoxypropyl, ethoxymethyl, 2- isopropoxy ethyl etc.
• alkynyl refers to an unsaturated straight or branched hydrocarbon having at least one carbon-carbon triple bond.
• exemplary alkynyl groups include, but are not limited to, straight or branched groups of 2-6, or 3-6 carbon atoms, referred to herein as C2-C6alkynyl, and C3-C6alkynyl, respectively.
• exemplary alkynyl groups include, but are not limited to, ethynyl, propynyl, butynyl, pentynyl, hexynyl, methylpropynyl, etc.
• cycloalkyl or a“carbocyclic group” as used herein refers to a saturated or partially unsaturated hydrocarbon group of, for example, 3-6, or 4-6 carbons, referred to herein as C3-C6cycloalkyl or C4-C6cycloalkyl, respectively.
• exemplary cycloalkyl groups include, but are not limited to, cyclohexyl, cyclopentyl, cyclopentenyl, cyclobutyl or cyclopropyl.
• cycloalkoxy refers to a cycloalkyl group attached to oxygen (cycloalkyl-O-).
• exemplary cycloalkoxy groups include, but are not limited to, cycloalkoxy groups of 3-6 carbon atoms, referred to herein as C3-6cycloalkoxy groups.
• Exemplary cycloalkoxy groups include, but are not limited to, cyclopropoxy, cyclobutoxy, cyclopentoxy, cyclohexyloxy, etc.
• halo or“halogen” as used herein refer to F, Cl, Br, or I.
• heteroaryl refers to a monocyclic aromatic 5 or 6 membered ring system containing one or more heteroatoms, for example one to three
• heteroaryl ring may be linked to the adjacent radical though carbon or nitrogen.
• heteroaryl rings include but are not limited to furan, thiophene, pyrrole, thiazole, oxazole, isothiazole, isoxazole, imidazole, pyrazole, triazole, pyridine or pyrimidine etc.
• heterocyclyl or“heterocyclic group” are art-recognized and refer to saturated or partially unsaturated, 4-10 membered ring structures, including monocyclic, bridged or fused rings, and whose ring structures include one to three heteroatoms, such as nitrogen, oxygen, and sulfur. Where possible, heterocyclyl rings may be linked to the adjacent radical through carbon or nitrogen. Examples of heterocyclyl groups include, but are not limited to, pyrrolidine, piperidine, morpholine, thiomorpholine, piperazine, oxetane, azetidine,
• lactam refers to cyclic amides of amino carboxylic acids, having a l-azacycloalkan-2-one structure, or analogues having unsaturation or heteroatoms replacing one or more carbon atoms of the ring.
• alpha-lactam refers to a lactam comprised of a 3-membered ring.
• a “beta-lactam” refers to a lactam comprised of a 4- membered ring.
• a "gamma-lactam” refers to a lactam comprised of a 5-membered ring.
• a “delta-lactam” refers to a lactam comprised of a 6-membered ring.
• epsilon-lactam refers to a lactam comprised of a 7-membered ring.
• A“combination therapy” is a treatment that includes the administration of two or more therapeutic agents, e.g ., a compound of Formula I and a MAPKAP pathway inhibitor, to a patient in need thereof.
• “Individual,”“patient,” or“subject” are used interchangeably and include any animal, including mammals, preferably mice, rats, other rodents, rabbits, dogs, cats, swine, cattle, sheep, horses, or primates, and most preferably humans.
• the compounds described herein can be administered to a mammal, such as a human, but can also be administered to other mammals such as an animal in need of veterinary treatment, e.g, domestic animals (e.g, dogs, cats, and the like), farm animals (e.g, cows, sheep, pigs, horses, and the like) and laboratory animals (e.g, rats, mice, guinea pigs, and the like).
• A“MAPKAP pathway inhibitor” is an inhibitor of the MAP kinase signaling pathway.
• Inhibitors of this pathway include Ras inhibitors (e.g. AMG-510 or MRTX 849), RAF inhibitors (e.g. dabrafenib, vemurafenib, or LY3009120), MEK inhibitors (e.g. trametinib, binimetinib, selumetinib, or cobimetinib), and ERK inhibitors (e.g. ulixertinib SCH772984, or LY3214996).
• Ras inhibitors e.g. AMG-510 or MRTX 849
• RAF inhibitors e.g. dabrafenib, vemurafenib, or LY3009120
• MEK inhibitors e.g. trametinib, binimetinib, selumetinib, or cobimetinib
• “Pharmaceutically or pharmacologically acceptable” include molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, or a human, as appropriate.
• preparations should meet sterility, pyrogenicity, and general safety and purity standards as required by FDA Office of Biologies standards.
• compositions may also contain other active compounds providing supplemental, additional, or enhanced therapeutic functions.
• composition refers to a composition comprising at least one compound as disclosed herein formulated together with one or more pharmaceutically acceptable carriers.
• salt(s) refers to salts of acidic or basic groups that may be present in compounds used in the compositions.
• Compounds included in the present compositions that are basic in nature are capable of forming a wide variety of salts with various inorganic and organic acids.
• the acids that may be used to prepare pharmaceutically acceptable acid addition salts of such basic compounds are those that form non-toxic acid addition salts, i.e., salts containing pharmacologically acceptable anions, including, but not limited to, malate, oxalate, chloride, bromide, iodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate, isonicotinate, acetate, lactate, salicylate, citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, / oluenesulfonate and pamoate (i.e., 1 , 1 ,
• Compounds included in the present compositions that are acidic in nature are capable of forming base salts with various pharmacologically acceptable cations.
• Examples of such salts include alkali metal or alkaline earth metal salts, particularly calcium, magnesium, sodium, lithium, zinc, potassium, and iron salts.
• Compounds included in the present compositions that include a basic or acidic moiety may also form pharmaceutically acceptable salts with various amino acids.
• the compounds of the disclosure may contain both acidic and basic groups; for example, one amino and one carboxylic acid group. In such a case, the compound can exist as an acid addition salt, a zwitterion, or a base salt.
• the compounds of the disclosure may contain one or more chiral centers and, therefore, exist as stereoisomers.
• the term“stereoisomers” when used herein consist of all enantiomers or diastereomers. These compounds may be designated by the symbols“(+),”
• the term“therapeutically effective amount” means the amount of the subject compound that will elicit the biological or medical response of a tissue, system or animal, (e.g. mammal or human) that is being sought by the researcher, veterinarian, medical doctor or other clinician.
• the compounds described herein are administered in therapeutically effective amounts to treat a disorder.
• Treating includes any effect, e.g., lessening, reducing, modulating, or eliminating, that results in the improvement of the condition, disease, disorder and the like.
• the disclosure also embraces isotopically labeled compounds which are identical to those recited herein, except that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature.
• isotopes that can be incorporated into compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine and chlorine, such as 2 H, 3 ⁇ 4, 13 C, 14 C, 15 N, 18 0, 17 0, 31 P, 32 P, 35 S, 18 F, and 36 C1, respectively.
• a compound of the disclosure may have one or more H atoms replaced with deuterium.
• Individual enantiomers and diasteriomers of compounds of the present invention can be prepared synthetically from commercially available starting materials that contain asymmetric or stereogenic centers, or by preparation of racemic mixtures followed by resolution methods well known to those of ordinary skill in the art.
• Stereoselective syntheses a chemical or enzymatic reaction in which a single reactant forms an unequal mixture of stereoisomers during the creation of a new stereocenter or during the transformation of a pre-existing one, are well known in the art.
• Stereoselective syntheses encompass both enantio- and diastereoselective transformations, and may involve the use of chiral auxiliaries. For examples, see Carreira and Kvaemo, Classics in Stereoselective Synthesis , Wiley-VCH: Weinheim, 2009.
• Z is selected from the group consisting of a 4 membered lactam ring bound through the nitrogen atom or a 6-10 membered lactam ring bound through the nitrogen atom, wherein a lactam ring atom may optionally be oxygen or NR 6 when the lactam ring is a 6-10 membered ring and an available carbon atom on 4 membered lactam ring or a 6-10 membered lactam is optionally substituted by R 36 .
• Z is selected from the group consisting of a 4-10 membered lactam ring bound through the nitrogen atom, wherein any available carbon atom on a 4 -10 membered lactam is optionally substituted by R 36 , and wherein a lactam ring atom may optionally be oxygen or NR 6 when the lactam ring is a 6-10 membered ring.
• W is N.
• A is selected from the group consisting of pyrazolyl, triazolyl, thiazolyl, and oxazolyl.
• Z is selected from:
• V is selected from the group consisting of oxygen, C(R 34 ) 2 , and NR 6
• each occurrence of R 34 is independently selected from H and R 36
• each occurrence of R 36 is independently selected from Ci-C6alkyl and C 3 -C 6 cycloalkyl and wherein each Ci-C6alkyl and C 3 -C 6 cycloalkyl may be optionally substituted by one or more independent occurrences of fluorine, or two R 36 are joined together with the carbon to which they are attached to form a C 3 -C 6 cycloalkyl
• q is 0, 1, 2, or 3
• r is 2 or 4.
• Z is selected from:
• V is selected from the group consisting of oxygen, C(R 34 ) 2 , and NR 6 ; each occurrence of R 34 is independently selected from H and R 36 , wherein each occurrence of R 36 is independently selected from Ci-C6alkyl and C 3 -C 6 cycloalkyl and wherein each Ci-C6alkyl and C 3 -C 6 cycloalkyl may be optionally substituted by one or more independent occurrences of fluorine, or two R 36 are joined together with the carbon to which they are attached to form a C 3 -C 6 cycloalkyl; q is 0, 1, 2, or 3; and r is 2 or 3.
• Z is selected from the group consisting of:
• Z is selected from:
• V is selected from the group consisting of oxygen, CH2, and NR 6 ; q is 0, 1, 2, or 3; and r is 2 or 3.
• Z is selected from the group consisting of:
• R 4 is D.
• R 4 is selected from the group consisting of:
• R 4 is selected from the group consisting of:
• R 4 is B.
• R 4 is selected from the group consisting of:
• u is 1 or 2.
• R 4 is selected from the group consisting of:
• R 4 is selected from the group consisting of:
• m is 0. In some embodiments, m is 1. In some embodiments, m is 2. In some embodiments, m is 3.
• L is a direct bond to the A ring, i.e. where m is 0.
• L is selected from the group consisting of -CH2-, -
• L is -CH2-. In some embodiments, L is - CH2CH2-. In some embodiments, L is -CH2CH2CH2-.
• R 1 is selected from the group consisting of halogen, Ci- Csalkyl, and Cs-Cscycloalkyl, wherein Ci-Csalkyl or Cs-Cscycloalkyl may be optionally substituted with one, two, or three independent occurrences of fluorine.
• R 1 is CF 3 .
• R 1 is halogen.
• R 1 is bromo.
• R 1 is cyclopropyl.
• R 1 is CF2H.
• R 2 is selected from the group consisting of Ci-Csalkyl, H, and C3-C4cycloalkyl, wherein Ci-Csalkyl or C3-C4cycloalkyl may be optionally substituted with one, two, or three independent occurrences of fluorine.
• R 2 is selected from the group consisting of Ci-C2alkyl and C3-C4cycloalkyl. In some embodiments, R 2 is halogen. In some embodiments, R 2 is selected from the group consisting of chloro and bromo. In some embodiments, R 2 is bromo. In some embodiments, R 2 is chloro.
• n 3.
• the compound is represented by:
• A-l is selected from the group consisting of:
• R 1 is selected from the group consisting of halogen, cyano, Ci-Csalkyl, and Cs-Cscycloalkyl, wherein each Ci-Csalkyl and Cs-Cscycloalkyl may be optionally substituted by one, two or three independent occurrences of fluorine;
• R 2 is selected from the group consisting of halogen, Ci- Cialkyl and C 3 -C 4 cycloalkyl, wherein each Ci-C 2 alkyl and C 3 -C 4 cycloalkyl may be optionally substituted by one, two or three independent occurrences of fluorine;
• R 4 is selected from the group consisting of:
• each occurrence of R 7 is independently selected from the group consisting of H, C 1 -C 6 alkyl, C 3 - C6cycloalkyl, wherein each Ci-C6alkyl and C 3 -C 6 cycloalkyl may be optionally substituted by one or more independent occurrences of fluorine, or two R 7 are joined together with the atom to which they are attached to form oxo; each occurrence of R 6 and R 9 is independently selected from the group consisting of H, Ci-Cealkyl, and C 3 -C 6 cycloalkyl, wherein each Ci-C6alkyl and C 3 -C 6 cycloalkyl may be optionally substituted by one or more independent occurrences of fluorine; Z is selected from the group consisting of:
• each occurrence of R 34 is independently selected from H and R 36 , wherein each occurrence of R 36 is independently selected from Ci-C 6 alkyl and C3-C6cycloalkyl and wherein each Ci-C 6 alkyl and C3-C6cycloalkyl may be optionally substituted by one or more independent occurrences of fluorine, or two R 36 are joined together with the carbon to which they are attached to form a C3- C 6 cycloalkyl; L is -(C(R 10 ) 2 ) m -; each occurrence of R 10 is independently selected from the group consisting of H, Ci-C3alkyl, and C3-C5cycloalkyl, wherein each Ci-C3alkyl and C3-C5cycloalkyl may be optionally substituted by one or more independent occurrences of fluorine, or two R 10 are joined together with the carbon to which they are attached to form a C3-C5cycloalkyl; m is 0, 1,
• n 2, 3, or 4; provided that: when m is 0, R 4 is C-linked to the pyrazole ring, when m is 1, R 4 is Cdinked to L, then and when m is 2 or 3, R 4 is N-linked or Cdinked to L.
• A-l is:
• n 3.
• n is 0. In some embodiments, m is 1. In some
• n is 2. In some embodiments, m is 3.
• L is a direct bond
• L is selected from the group consisting of -CH2-, -
• L is -CH2-. In some embodiments, L is - CH2CH2-. In some embodiments, L is -CH2CH2CH2-.
• Z is selected from the group consisting of:
• Z is selected from the group consisting of:
• R 1 is selected from the group consisting of halogen, Ci- Csalkyl, and Cs-Cscycloalkyl, wherein Ci-Csalkyl may be optionally substituted with one, two, or three occurrences of fluorine.
• R 1 is CF 3 .
• R 1 is CF2H.
• R 1 is halogen.
• R 1 is bromo.
• R 1 is cyclopropyl.
• R 2 is selected from the group consisting of H, C3- Cscycloalkyl, Ci-Csalkyl, halogen, CN, Ci-Csalkenyl, and C2-Csalknyl, wherein Ci-Csalkyl and C3-C5cycloalkyl may be optionally substituted with one, two, or three independent occurrences of fluorine.
• R 2 is selected from the group consisting of Ci-2alkyl and C3- 4cycloalkyl.
• R 2 is selected from the group consisting of chloro and bromo.
• R 2 is selected from the group consisting of Ci-Csalkyl, H, and C3-C4cycloalkyl. In some embodiments, R 2 is selected from the group consisting of Ci- C2alkyl and C3-C4cycloalkyl. In some embodiments, R 2 is selected from the group consisting of chloro and bromo.
• R 4 is:
• the compound is represented by a formula selected from the group consisting of:
• each occurrence of R 1 is bromo; each occurrence of R 2 is independently selected from Ci-C 2 alkyl, C 3 -C 4 cycloalkyl, bromo, and chloro; each occurrence of R 9 is selected from H and Ci-C 3 alkyl; each occurrence of R 34 is independently selected from the group consisting of H and Ci-C 3 alkyl; and n is 3.
• each occurrence of R 1 is bromo; each occurrence of R 2 is independently selected from Ci-C 2 alkyl and C 3 -C 4 cycloalkyl; each occurrence of R 9 is selected from H and Ci-C 3 alkyl; each R 34 is H; and n is 3.
• each occurrence of R 1 is CF 3 ; each occurrence of R 2 is independently selected from Ci-C 2 alkyl, C 3 -C 4 cycloalkyl, bromo, and chloro; each occurrence of R 9 is independently selected from H and Ci-C 3 alkyl; each occurrence of R 34 is independently selected from the group consisting of H and Ci-C 3 alkyl; and n is 3.
• each occurrence of R 1 is CHF 2 ; each occurrence of R 2 is independently selected from Ci-C 2 alkyl, C 3 -C 4 cycloalkyl, bromo, and chloro; each occurrence of R 9 is selected from H and Ci-C 3 alkyl; each occurrence of R 34 is independently selected from the group consisting of H and Ci-C 3 alkyl; and n is 3.
• each occurrence of R 1 is CHF 2 ; each occurrence of R 2 is independently selected from Ci-C 2 alkyl and C 3 -C 4 cycloalkyl; each occurrence of R 9 is selected from H and Ci-C 3 alkyl; each R 34 is H; and n is 3.
• the compound is represented by Formula IIA. l as defined above. In some embodiments, the compound is represented Formula IIA.3 as defined above. In some embodiments, the compound is represented a formula selected from the group consisting of Formula IIA.3, Formula IIA.4, Formula IIA.5, Formula IIA.6, and Formula IIA.7 as defined above.
• the compound is represented by a formula selected from the group consisting of:
• each occurrence of R 1 is CF 3 ; each occurrence of R 2 is independently selected from Ci-C 2 alkyl, C 3 -C 4 cycloalkyl, bromo, and chloro; each occurrence of R 6 is independently selected from Ci-C 3 alkyl and C 3 - C 4 cycloalkyl; each occurrence of R 9 is independently selected from H and Ci-C 3 alkyl; and n is 3.
• each occurrence of R 1 is CF 2 H; each occurrence of R 2 is independently selected from Ci-C 2 alkyl, C 3 -C 4 cycloalkyl, bromo, and chloro; each occurrence of R 6 is independently selected from Ci-C 3 alkyl and C 3 -C 4 cycloalkyl; each occurrence of R 9 is independently selected from H and Ci-C 3 alkyl; and n is 3.
• the compound is represented by a formula selected from the group consisting of:
• each occurrence of R 1 is bromo; each occurrence of R 2 is independently selected from Ci-C 2 alkyl, C 3 -C 4 cycloalkyl, bromo, and chloro ; each occurrence of R 9 is selected from H and Ci-C 3 alkyl; each occurrence of R 34 is independently selected from the group consisting of H and Ci-C 3 alkyl; and n is 3.
• each occurrence of R 1 is CF 3 ; each occurrence of R 2 is independently selected from Ci-C 2 alkyl, C 3 -C 4 cycloalkyl, bromo, and chloro; each occurrence of R 9 is independently selected from H and Ci-C 3 alkyl; each occurrence of R 34 is independently selected from the group consisting of H and Ci-C 3 alkyl; and n is 3.
• each occurrence of R 1 is CF 3 ; each occurrence of R 2 is independently selected from Ci-C 2 alkyl and C 3 -C 4 cycloalkyl; each occurrence of R 9 is independently selected from H and Ci-C 3 alkyl; each R 34 is H; and n is 3.
• each occurrence of R 1 is CHF 2 ; each occurrence of R 2 is independently selected from Ci-C 2 alkyl, C 3 -C 4 cycloalkyl, bromo, and chloro; each occurrence of R 9 is selected from H and Ci-C 3 alkyl; each occurrence of R 34 is independently selected from the group consisting of H and Ci-C 3 alkyk; and n is 3.
• each occurrence of R 1 is CHF 2 ; each occurrence of R 2 is independently selected from Ci-C 2 alkyl and C 3 -C 4 cycloalkyl; each occurrence of R 9 is selected from H and Ci-C 3 alkyl; each R 34 is H; and n is 3.
• the compound is represented by a formula selected from the group consisting of:
• each occurrence of R 1 is CF 3 ; each occurrence of R 2 is independently selected from Ci-C 2 alkyl, C 3 -C 4 cycloalkyl, bromo, and chloro; each occurrence of R 6 is independently selected from the group consisting of Ci-C 3 alkyl and C 3 -C 4 cycloalkyl; each occurrence of R 9 is independently selected from H and Ci-C 3 alkyl; and n is 3.
• each occurrence of R 1 is CF 2 H; each occurrence of R 2 is independently selected from Ci-C 2 alkyl, C 3 -C 4 cycloalkyl, bromo, and chloro; each occurrence of R 6 is independently selected from the group consisting of Ci-C 3 alkyl and C 3 -C 4 cycloalkyl; each occurrence of R 9 is independently selected from H and Ci-C 3 alkyl; and n is 3.
• the compound is represented by a formula selected from the group consisting of:
• each occurrence of R 1 is bromo; each occurrence of R 2 is independently selected from Ci-C 2 alkyl, C 3 -C 4 cycloalkyl, bromo, and chloro; each occurrence of R 9 is selected from H and Ci-C 3 alkyl; each occurrence of R 34 is independently selected from the group consisting of H and Ci-C 3 alkyl; and n is 3.
• each occurrence of R 1 is bromo; each occurrence of R 2 is independently selected from Ci-C 2 alkyl and C 3 -C 4 cycloalkyl; each occurrence of R 9 is selected from H and Ci-C 3 alkyl; each R 34 is H; and n is 3.
• each occurrence of R 1 is CF 3 ; each occurrence of R 2 is independently selected from Ci-C 2 alkyl, C 3 -C 4 cycloalkyl, bromo, and chloro; each occurrence of R 9 is independently selected from H and Ci-C 3 alkyl; each occurrence of R 34 is independently selected from the group consisting of H and Ci-C 3 alkyl; and n is 3.
• each occurrence of R 1 is CF 3 ; each occurrence of R 2 is independently selected from Ci-C 2 alkyl and C 3 -C 4 cycloalkyl; each occurrence of R 9 is independently selected from H and Ci-C 3 alkyl; each R 34 is H; and n is 3.
• each occurrence of R 1 is CHF 2 ; each occurrence of R 2 is independently selected from Ci-C 2 alkyl, C 3 -C 4 cycloalkyl, bromo, and chloro; each occurrence of R 9 is selected from H and Ci-C 3 alkyl; each occurrence of R 34 is independently selected from the group consisting of H and Ci-C 3 alkyk; and n is 3.
• each occurrence of R 1 is CHF 2 ; each occurrence of R 2 is independently selected from Ci-C 2 alkyl and C 3 -C 4 cycloalkyl; each occurrence of R 9 is selected from H and Ci-C 3 alkyl; each R 34 is H; and n is 3.
• the compound is represented by Formula IIA.19 as defined above. In some embodiments, the compound is represented by Formula IIA.21 as defined above.
• the compound is represented by a formula selected from:
• each occurrence of R 1 is CF 3 ; each occurrence of R 2 is independently selected from Ci-C 2 alkyl C 3 - C 4 cycloalkyl, bromo, and chloro; each occurrence of R 6 is independently selected from Ci- C 3 alkyl and C 3 -C 4 cycloalkyl; R 9 is independently selected from H and Ci-C 3 alkyl; and n is 3.
• each occurrence of R 1 is CF 2 H; each occurrence of R 2 is independently selected from Ci-C 2 alkyl, C 3 -C 4 cycloalkyl, bromo, and chloro; each occurrence of R 6 is independently selected from Ci-C 3 alkyl and C 3 -C 4 cycloalkyl; each occurrence of R 9 is independently selected from H and Ci-C 3 alkyl; and n is 3.
• the compound is represented by:
• A- 1 is selected from the group consisting of:
• R 1 is selected from the group consisting of halogen, cyano, Ci-Csalkyl, and Cs-Cscycloalkyl, wherein each Ci-Csalkyl and Cs-Cscycloalkyl may be optionally substituted by one, two or three independent occurrences of fluorine;
• R 2 is selected from Ci-C 2 alkyl, C 3 -C 4 cycloalkyl, and halogen;
• R 4 is selected from the group consisting of:
• each occurrence of R 7 is independently selected from the group consisting of H, C 1 -C 6 alkyl, C 3 - C6cycloalkyl, wherein each Ci-C6alkyl and C 3 -C 6 cycloalkyl may be optionally substituted by one or more independent occurrences of fluorine, or two R 7 are joined together with the atom to which they are attached to form oxo; each occurrence of R 6 and R 9 is independently selected from the group consisting of H, Ci-Cealkyl, and C 3 -C 6 cycloalkyl, wherein each Ci-C6alkyl and C 3 -C 6 cycloalkyl may be optionally substituted by one or more independent occurrences of fluorine; Z is selected from the group consisting of:
• each occurrence of R 34 is independently selected from H and R 36 , wherein each occurrence of R 36 is independently selected from Ci-C 6 alkyl and C 3 -C 6 cycloalkyl and wherein each Ci-C 6 alkyl and C 3 -C 6 cycloalkyl may be optionally substituted by one or more independent occurrences of fluorine, or two R 36 are joined together with the carbon to which they are attached to form a C 3 - C 6 cycloalkyl; L is -(C(R 10 ) 2 ) m -; each occurrence of R 10 is independently selected from the group consisting of H, Ci-C 3 alkyl, and C 3 -C 5 cycloalkyl, wherein each Ci-C 3 alkyl and C 3 -C 5 cycloalkyl may be optionally substituted by one or more independent occurrences of fluorine, or two R 10 are joined together with the carbon to which they are attached to form a C 3 -C 5 cycloalkyl
• n 2, 3, or 4; provided that: when m is 0, R 4 is C-linked to the pyrazole ring, when m is 1, R 4 is Cdinked to L, then and when m is 2 or 3, R 4 is N-linked or Cdinked to L.
• A-l is:
• n 3.
• n is 0. In some embodiments, m is 1. In some
• n is 2. In some embodiments, m is 3.
• L is a direct bond
• L is selected from the group consisting of -CH 2 -, -
• L is -CH 2 -. In some embodiments, L is - CH 2 CH 2 -. In some embodiments, L is -CH 2 CH 2 CH 2 -. In some embodiments, L is -CH 2 CH 2 CH 2 -.
• Z is selected from the group consisting of:
• Z is selected from the group consisting of:
• R 1 is selected from the group consisting of halogen, Ci- Csalkyl, and Cs-Cscycloalkyl, wherein Ci-Csalkyl or Cs-Cscycloalkyl may be optionally substituted with one, two, or three occurrences of fluorine.
• R 1 is CF 3 .
• R 1 is CF2H.
• R 1 is halogen.
• R 1 is bromo.
• R 1 is cyclopropyl.
• R 2 is selected from the group consisting of H, C3- Cscycloalkyl, Ci-Csalkyl, halogen, CN, Ci-Csalkenyl, and C2-Csalknyl, wherein Ci-Csalkyl or C3-C5cycloalkyl may be optionally substituted with one, two, or three independent occurrences of fluorine.
• R 2 is selected from Ci-2alkyl and C3-4cycloalkyl.
• R 2 is selected from chloro and bromo.
• R 2 is selected from the group consisting of Ci-Csalkyl, H, and C3-C4cycloalkyl. In some embodiments, R 2 is selected from Ci-C2alkyl and C3- C4cycloalkyl. In some embodiments, R 2 is selected from chloro and bromo.
• R 4 is:
• the compound is represented by a formula selected from the group consisting of:
• each occurrence of R 1 is bromo; each occurrence of R 2 is independently selected from Ci-C 2 alkyl, C 3 -C 4 cycloalkyl, bromo, and chloro ; each occurrence of R 9 is selected from H and Ci-C 3 alkyl; each occurrence of R 34 is independently selected from the group consisting of H and Ci-C 3 alkyl; and n is 3.
• each occurrence of R 1 is CF 3 ; each occurrence of R 2 is independently selected from Ci-C 2 alkyl, C 3 -C 4 cycloalkyl, bromo, and chloro; each occurrence of R 9 is independently selected from H and Ci-C 3 alkyl; each occurrence of R 34 is independently selected from the group consisting of H and Ci-C 3 alkyl; and n is 3.
• each occurrence of R 1 is CF 3 ; each occurrence of R 2 is independently selected from Ci-C 2 alkyl and C 3 -C 4 cycloalkyl; each occurrence of R 9 is independently selected from H and Ci-C 3 alkyl; each R 34 is H; and n is 3.
• each occurrence of R 1 is CHF 2 ; each occurrence of R 2 is independently selected from Ci-C 2 alkyl, C 3 -C 4 cycloalkyl, bromo, and chloro; each occurrence of R 9 is selected from H and Ci-C 3 alkyl; each occurrence of R 34 is independently selected from the group consisting of H and Ci-C 3 alkyk; and n is 3.
• each occurrence of R 1 is CHF 2 ; each occurrence of R 2 is independently selected from Ci-C 2 alkyl and C 3 -C 4 cycloalkyl; each occurrence of R 9 is selected from H and Ci-C 3 alkyl; each R 34 is H; and n is 3.
• the compound is represented by a formula selected from the group consisting of:
• each occurrence of R 1 is CF 3 ; each occurrence of R 2 is independently selected from Ci-C 2 alkyl, C 3 -C 4 cycloalkyl, bromo, and chloro; each occurrence of R 6 is independently selected from Ci-C 3 alkyl and C 3 - C 4 cycloalkyl; each occurrence of R 9 is independently selected from H and Ci-C 3 alkyl; and n is 3.
• each occurrence of R 1 is CF 2 H; each occurrence of R 2 is independently selected from Ci-C 2 alkyl, C 3 -C 4 cycloalkyl, bromo, and chloro; each occurrence of R 6 is independently selected from Ci-C 3 alkyl and C 3 -C 4 cycloalkyl; each occurrence of R 9 is independently selected from H and Ci-C 3 alkyl; and n is 3.
• the compound is represented by a formula selected from the group consisting of:
• each occurrence of R 1 is bromo; each occurrence of R 2 is independently selected from Ci-C 2 alkyl, C 3 -C 4 cycloalkyl, bromo, and chloro; each occurrence of R 9 is selected from H and Ci-C 3 alkyl; each occurrence of R 34 is independently selected from the group consisting of H and Ci-C 3 alkyl; and n is 3.
• each occurrence of R 1 is bromo; each occurrence of R 2 is independently selected from Ci-C 2 alkyl and C 3 -C 4 cycloalkyl; each occurrence of R 9 is selected from H and Ci-C 3 alkyl; each R 34 is H; and n is 3.
• each occurrence of R 1 is CF 3 ; each occurrence of R 2 is independently selected from Ci-C 2 alkyl, C 3 -C 4 cycloalkyl, bromo, and chloro; each occurrence of R 9 is independently selected from H and Ci-C 3 alkyl; each occurrence of R 34 is independently selected from the group consisting of H and Ci-C 3 alkyl; and n is 3.
• each occurrence of R 1 is CF 3 ; each occurrence of R 2 is independently selected from Ci-C 2 alkyl and C 3 -C 4 cycloalkyl; each occurrence of R 9 is independently selected from H and Ci-C 3 alkyl; each R 34 is H; and n is 3.
• each occurrence of R 1 is CHF 2 ; each occurrence of R 2 is independently selected from Ci-C 2 alkyl, C 3 -C 4 cycloalkyl, bromo, and chloro; each occurrence of R 9 is selected from H and Ci-C 3 alkyl; each occurrence of R 34 is independently selected from the group consisting of H and Ci-C 3 alkyk; and n is 3.
• each occurrence of R 1 is CHF 2 ; each occurrence of R 2 is independently selected from Ci-C 2 alkyl and C 3 -C 4 cycloalkyl; each occurrence of R 9 is selected from H and Ci-C 3 alkyl; each R 34 is H; and n is 3.
• the compound is represented by a formula selected from the group consisting of:
• each occurrence of R 1 is CF3; each occurrence of R 2 is independently selected from Ci-C2alkyl, C3-C4cycloalkyl, bromo, and chloro; each occurrence of R 6 is independently selected from the group consisting of Ci-C3alkyl and C3-C4cycloalkyl; each occurrence of R 9 is independently selected from H and Ci-C3alkyl; and n is 3.
• the compound is represented by a formula selected from the group consisting of:
• each occurrence of R 1 is bromo; each occurrence of R 2 is independently selected from Ci-C 2 alkyl, C 3 -C 4 cycloalkyl, bromo, and chloro; each occurrence of R 9 is selected from H and Ci-C 3 alkyl; each occurrence of R 34 is independently selected from the group consisting of H and Ci-C 3 alkyl; and n is 3.
• each occurrence of R 1 is bromo; each occurrence of R 2 is independently selected from Ci-C 2 alkyl and C 3 -C 4 cycloalkyl; each occurrence of R 9 is selected from H and Ci-C 3 alkyl; each R 34 is H; and n is 3.
• each occurrence of R 1 is CF 3 ; each occurrence of R 2 is independently selected from Ci-C 2 alkyl, C 3 -C 4 cycloalkyl, bromo, and chloro; each occurrence of R 9 is independently selected from H and Ci-C 3 alkyl; each occurrence of R 34 is independently selected from the group consisting of H and Ci-C 3 alkyl; and n is 3.
• each occurrence of R 1 is CF 3 ; each occurrence of R 2 is independently selected from Ci-C 2 alkyl and C 3 -C 4 cycloalkyl; each occurrence of R 9 is independently selected from H and Ci-C 3 alkyl; each R 34 is H; and n is 3.
• each occurrence of R 1 is CHF 2 ; each occurrence of R 2 is independently selected from Ci-C 2 alkyl, C 3 -C 4 cycloalkyl, bromo, and chloro; each occurrence of R 9 is selected from H and Ci-C 3 alkyl; each occurrence of R 34 is independently selected from the group consisting of H and Ci-C 3 alkyl; and n is 3.
• each occurrence of R 1 is CHF 2 ; each occurrence of R 2 is independently selected from Ci-C 2 alkyl and C 3 -C 4 cycloalkyl; each occurrence of R 9 is selected from H and Ci-C 3 alkyl; each R 34 is H; and n is 3.
• the compound is represented by a formula selected from:
• the compound is represented by a formula selected from the group consisting of: l-(3-((5-cyclopropyl-2-((l-(l-methylpiperidin-4-yl)-lH-pyrazol-4- yl)amino)pyrimidin-4-yl)amino)propyl)piperidin-2-one, 1 -(3 -((5 -chloro-2-(( 1 -( 1 - methylpiperidin-4-yl)-lH-pyrazol-4-yl)amino)pyrimidin-4-yl)amino)propyl)piperidin-2-one, 1- (3-((5-chloro-2-((l-(l-(cyclopropylmethyl)piperidin-4-yl)-lH-pyrazol-4-yl)amino)pyrimidin-4- yl)amino)propyl)piperidin-2-one, l-(3-(3-(5-cycloprop
• Compounds described herein can act as inhibitors of autophagy useful in the treatment of a disorder in a patient in need thereof.
• the disorder for example, can be a tumor, e.g., a solid tumor.
• the disorder may also be cancer.
• Exemplary disorders also include gastrointestinal stromal tumors, esophageal cancer, gastric cancer, melanomas, gliomas, glioblastomas, ovarian cancer, bladder cancer, pancreatic cancer, prostate cancer, lung cancers, breast cancers, renal cancers, hepatic cancers, osteosarcomas, multiple myelomas, cervical carcinomas, cancers that are metastatic to bone, papillary thyroid carcinoma, non-small cell lung cancer, and colorectal cancers,
• a cancer treated by the methods described herein may be a metastatic cancer.
• the compounds described herein are useful for the treatment of cancers caused by RAS mutation.
• the cancer is caused by a KRAS mutation.
• the cancer has additional mutations in tumor suppressor proteins, including mutations in TP53, PTEN, CDN2A/INK4A, pl6, or STAG2. In some embodiments, these additional mutations occur in one or more of TP53, PTEN, CDN2A/INK4A, pi 6, or STAG2.
• the cancer is pancreatic ductal adenocarcinoma.
• the cancer is lung cancer. In some embodiments, the cancer is colorectal.
• determination of cellular inhibition of autophagy by compounds described herein is determined by monitoring of autophagic flux, for instance by monitoring inhibition of autophagy-mediated clearance of mCherry/GFP-LC3 fusion protein. In some embodiments, determination of cellular inhibition of autophagy by compounds described herein is determined by monitoring of accumulation of autophagic proteins such as p62 or LC-3. In some embodiments, determination of cellular inhibition of autophagy by compounds described herein is determined by decreased clearance of luciferase-tagged LC3 protein. In some embodiments, determination of cellular inhibition of autophagy by compounds described herein is determined by monitoring decreases in cellular autophagosomes, for instance by measurement of fluorescent puncta with the autophagosome marker Cyto-ID.
• cellular inhibition of ULK kinase by compounds described herein is determined by inhibition of phosphorylation of cellular ULK substrates including ATG13, ATG14, Beclin 1, or STING either in tumor cells or in non-tumor host tissues. In some embodiments, cellular inhibition of ULK kinase by compounds described herein is determined in host tissues including immune cells.
• in vivo inhibition of autophagy by compounds described herein is determined by inhibition of phosphorylation of cellular ULK substrates including ATG13, ATG14, Beclin 1, or STING either in tumor cells or in non-tumor host tissues.
• in vivo inhibition of ULK kinase by compounds described herein is determined in host tissues including immune cells.
• the in vivo inhibition of autophagic flux by compounds described herein can be used as a pharmacodynamic model for monitoring the kinetics and extent of such ULK inhibition.
• tin vivo inhibition of ULK kinase by compounds described herein is determined in pancreatic cancer-bearing animals.
• in vivo inhibition of ULK kinase by compounds described herein is determined in lung cancer-bearing animals. In some embodiments, in vivo inhibition of ULK kinase is determined in colorectal cancer- bearing animals. In some embodiments, in vivo inhibition of autophagy by compounds described herein is determined by inhibition of autophagic flux in tumor cells, or in non-tumor host tissues by monitoring inhibition of autophagosome formation, or by accumulation of autophagic proteins such as p62 or LC-III. In some embodiments, in vivo inhibition of autophagy is determined in host tissues including immune cells. In some embodiments, the in vivo inhibition of autophagic flux can be used as a pharmacodynamic model for monitoring the kinetics and extent of such ULK inhibition.
• inhibition of autophagy and anti-tumor activity by compounds described herein are evaluated in xenograft studies utilizing human RAS mutant cell lines in immunocompromised mice, for instance in SCID or nude mice.
• inhibition of autophagy and anti-tumor activity by compounds described herein are evaluated in xenograft studies utilizing human RAS mutant patient-derived tumor xenografts (PDXs) in immunocompromised mice, for instance in SCID or nude mice.
• PDXs patient-derived tumor xenografts
• xenograft studies include evaluation of compounds described herein in pancreatic cancer models.
• inhibition of autophagy and anti-tumor activity by compounds described herein are evaluated in syngeneic murine genetically engineered models (GEMs) of mutant RAS cancers.
• GEMs murine genetically engineered models
• inhibition of autophagy and anti-tumor activity by compounds described herein are evaluated in the murine GEM syngeneic orthotopic pancreatic cancer model known as the KPC model (LSL-Kras G12D/+ ;LSL-Trp53 R172H/+ ;Pdx-l-Cre) or variants of the KPC model.
• compounds described herein will be evaluated in xenograft or GEM cancer models in combination with a MEK inhibitor. In some embodiments, compounds described herein will be evaluated in xenograft or GEM cancer models in
• compounds described herein will be evaluated in xenograft or GEM cancer models in combination with an ERK inhibitor. In some embodiments, compounds described herein will be evaluated in xenograft or GEM cancer models in combination with a RAS G12C direct inhibitor.
• inhibition of autophagy and anti-tumor activity by compounds described herein is evaluated in immunocompetent murine cancer models to assess an immunomodulatory component to the mechanism of action of ULK inhibitors.
• the immunocompetent murine model is the murine GEM syngeneic orthotopic pancreatic cancer model known as the KPC model (LSL-Kras G12D/+ ;LSL-Trp53 R172H/+ ;Pdx-l- Cre) or variants of the KPC model.
• immunomodulatory properties of compounds described herein are evaluated in combination with a MEK inhibitor.
• immunomodulatory properties of compounds described herein are evaluated in combination with a RAF inhibitor.
• immunomodulatory properties of compounds described herein are evaluated in combination with an ERK inhibitor. In some embodiments, immunomodulatory properties of compounds described herein are evaluated in combination with a RAS G12C direct inhibitor. [000139] In some embodiments, the immunomodulatory component of ULK inhibition is an enhanced innate immune response. In some embodiments, the immunomodulatory
• the component of ULK inhibition is an enhanced adaptive immune response.
• the immunomodulatory component of ULK inhibition is an enhanced activity of antigen- presenting cells.
• the immunomodulatory component of ULK inhibition is an enhanced anti-tumor activity of myeloid cells including macrophages.
• the immunomodulatory component of ULK inhibition is an enhanced anti -tumor activity of Natural Killer cells. In some embodiments, the immunomodulatory component of ULK inhibition is an enhanced activity of effector T Cells, including cytotoxic T Cells.
• a method of treating a disorder described herein that includes: administering a therapeutically effective amount of compound described herein in a patient in need thereof, and during or after the course of administration (e.g., at discrete time points, such as one week, two weeks, or on month after initial
• detecting comprises contacting a sample obtained from the patient
• a contemplated method comprises optionally contacting a sample obtained from the patient (including but not limited to a tumor, blood, saliva, or tissue) prior to administration of the compound with a phospho-ATG13 antibody ELISA assay, and comparing the level of phospho-ATG13 in the sample obtained prior to administration with the level of phospho-ATG13 in the sample obtained during or after the course of
• the phospho-ATG13 is p-S318ATG13.
• a method of treating a disorder described herein that includes: administering a therapeutically effective amount of compound described herein in a patient in need thereof, and during or after the course of administration (e.g., at discrete time points, such as one week, two weeks, or on month after initial
• detecting comprises contacting a sample obtained from the patient
• a contemplated method comprises optionally contacting a sample obtained from the patient (including but not limited to a tumor, blood, saliva, or tissue) prior to administration of the compound with a phospho-ATG14 antibody ELISA assay, and comparing the level of phospho-ATG14 in the sample obtained prior to administration with the level of phospho-ATG14 in the sample obtained during or after the course of
• the phospho-ATG14 is p-ATG14 Ser29.
• a method of treating a disorder described herein that includes: administering a therapeutically effective amount of compound described herein in a patient in need thereof, and during or after the course of administration (e.g., at discrete time points, such as one week, two weeks, or on month after initial
• detecting comprises contacting a sample obtained from the patient
• a contemplated method comprises optionally contacting a sample obtained from the patient (including but not limited to a tumor, blood, saliva, or tissue) prior to administration of the compound with a p62 antibody ELISA assay, and comparing the level of p62 in the sample obtained prior to administration with the level of p62 in the sample obtained during or after the course of administration.
• a method of treating a disorder described herein that includes: administering a therapeutically effective amount of compound described herein in a patient in need thereof, and during or after the course of administration (e.g., at discrete time points, such as one week, two weeks, or on month after initial
• detecting comprises contacting a sample obtained from the patient
• a contemplated method comprises optionally contacting a sample obtained from the patient (including but not limited to a tumor, blood, saliva, or tissue) prior to administration of the compound with a pBeclin antibody ELISA assay, and comparing the level of pBeclin in the sample obtained prior to administration with the level of pBeclin in the sample obtained during or after the course of administration.
• the compounds provided herein may be administered to patients (animals and humans) in need of such treatment in dosages that will provide optimal pharmaceutical efficacy. It will be appreciated that the dose required for use in any particular application will vary from patient to patient, not only with the particular compound or composition selected, but also with the route of administration, the nature of the condition being treated, the age and condition of the patient, concurrent medication or special diets then being followed by the patient, and other factors which those skilled in the art will recognize, with the appropriate dosage ultimately being at the discretion of the attendant physician.
• a compound provided herein may be administered orally, subcutaneously, topically, parenterally, by inhalation spray or rectally in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and vehicles.
• Parenteral administration may include subcutaneous injections, intravenous or intramuscular injections or infusion techniques.
• Treatment can be continued for as long or as short a period as desired.
• the compositions may be administered on a regimen of, for example, one to four or more times per day.
• a suitable treatment period can be, for example, at least about one week, at least about two weeks, at least about one month, at least about six months, at least about 1 year, or indefinitely.
• a treatment period can terminate when a desired result is achieved. .
• Compounds described herein can be administered in combination with one or more additional therapeutic agents to treat a disorder described herein, such as cancer.
• a pharmaceutical composition comprising a compound described herein, e.g. , a compound of Formula I as defined herein, one or more additional therapeutic agents, and a pharmaceutically acceptable excipient.
• a compound of Formula I as defined herein and one additional therapeutic agent is administered.
• a compound of Formula I as defined herein and two additional therapeutic agents are administered.
• a compound of Formula I as defined herein and three additional therapeutic agents are administered.
• Combination therapy can be achieved by administering two or more therapeutic agents, each of which is formulated and administered separately.
• a compound of Formula I as defined herein and an additional therapeutic agent can be formulated and administered separately.
• Combination therapy can also be achieved by administering two or more therapeutic agents in a single formulation, for example a pharmaceutical composition comprising a compound of Formula I as one therapeutic agent and one or more additional therapeutic agents such as a MAPKAP pathway inhibitor or chemotherapeutic agent.
• a compound of Formula I as defined herein and an additional therapeutic agent can be administered in a single formulation.
• Other combinations are also encompassed by combination therapy. While the two or more agents in the combination therapy can be administered simultaneously, they need not be.
• administration of a first agent can precede administration of a second agent (or combination of agents) by minutes, hours, days, or weeks.
• the two or more agents can be administered within minutes of each other or within 1, 2, 3, 6, 9, 12, 15, 18, or 24 hours of each other or within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14 days of each other or within 2, 3, 4, 5, 6, 7, 8, 9, or weeks of each other. In some cases, even longer intervals are possible. While in many cases it is desirable that the two or more agents used in a combination therapy be present in within the patient's body at the same time, this need not be so.
• Combination therapy can also include two or more administrations of one or more of the agents used in the combination using different sequencing of the component agents. For example, if agent X and agent Y are used in a combination, one could administer them
• the one or more additional therapeutic agents that may be administered in combination with a compound provided herein can be a MAPKAP pathway inhibitor.
• MAPKAP pathway inhibitors include, for example, MEK inhibitors, ERK inhibitors, RAF inhibitors, and Ras inhibitors.
• Exemplary MEK inhibitors include, but are not limited to, trametinib,
• Exemplary ERK inhibitors include, but are not limited to, include, but are not limited to, ulixertinib, SCH772984, LY3214996, ravoxertinib, VX-l le, and pharmaceutically acceptable salts thereof.
• Exemplary RAF inhibitors include, but are not limited to, LY3009120, LXH254, RAF709, dabrafenib, vemurafenib, and pharmaceutically acceptable salts thereof.
• Exemplary Ras inhibitors include, but are not limited to, AMG-510, MRTX849, and pharmaceutically acceptable salts thereof.
• the compounds described herein may be administered in combination with other therapeutic agents known to treat cancers.
• Such other therapeutic agents include radiation therapy, anti-tubulin agents, DNA alkylating agents, DNA synthesis-inhibiting agents, DNA intercalating agents, anti-estrogen agents, anti -androgens, steroids, anti-EGFR agents, kinase inhibitors, mTOR inhibitors, PI3 kinase inhibitors, cyclin-dependent kinase inhibitors, CD4/CD6 kinase inhibitors, topoisomerase inhibitors, Histone Deacetylase (HD AC) inhibitors, DNA methylation inhibitors, anti-HER2 agents, anti -angiogenic agents, proteasome inhibitors, thalidomide, lenalidomide, antibody-drug-conjugates (ADCs), immunotherapeutic agents including immunomodulating agents, targeted therapeutic agents cancer vaccines, and CAR-T cell therapy.
• other therapeutic agents include radiation therapy, anti-tubulin agents, DNA alkylating
• the additional therapeutic agents can be chemotherapeutic agents including but not limited to an anti-tubulin agents (for example, paclitaxel, paclitaxel protein-bound particles for injectable suspension including nab-paclitaxel, eribulin, docetaxel, ixabepilone, vincristine, auristatins, or maytansinoids), vinorelbine, DNA-alkylating agents (including cisplatin, carboplatin, oxaliplatin, cyclophosphamide, ifosfamide, temozolomide), DNA intercalating agents or DNA topoisomerase inhibitors (including anthracyclines such as doxorubicin, pegylated liposomal doxorubicin, daunorubicin, idarubicin, mitoxantrone, or epirubicin, camptothecins such as topotecan, irinotecan, or
• the additional therapeutic agents can be kinase inhibitors including but not limited to erlotinib, gefitinib, neratinib, afatinib, osimertinib, lapatanib, crizotinib, brigatinib, ceritinib, alectinib, lorlatinib, everolimus, temsirolimus, abemaciclib, LEE011, palbociclib, cabozantinib, sunitinib, pazopanib, sorafenib, regorafenib, sunitinib, axitinib, dasatinib, imatinib, nilotinib, idelalisib, ibrutinib, BLU-667, Loxo 292, larotrectinib, and quizartinib, anti-estrogen agents including but not limited to tam
• topoisom erase I inhibitors including but not limited to irinotecan, camptothecin, exatecan, and topotecan
• topoisomerase II inhibitors including but not limited to anthracyclines, etoposide, etoposide phosphate, and mitoxantrone
• Histone Deacetylase (HD AC) inhibitors including but not limited to vorinostat, romidepsin, panobinostat, valproic acid, and belinostat
• DNA methylation inhibitors including but not limited to DZNep and 5-aza-2'-deoxycytidine
• proteasome inhibitors including but not limited to bortezomib and carfilzomib, thalidomide, lenalidomide, pomalidomide
• biological agents including but not limited to trastuzumab, ado- trastuzumab, pertuzumab, cetuximab, panitumumab,
• the additional therapeutic agents can be any therapeutic agents. [000153] In some embodiments, the additional therapeutic agents can be any therapeutic agents.
• immunomodulatory agents including but not limited to anti-PD-lor anti-PDL-1 therapeutics including pembrolizumab, nivolumab, atezolizumab, durvalumab, BMS-936559, or avelumab, anti-TIM3 (anti-HAVcr2) therapeutics including but not limited to TSR-022 or MBG453, anti- LAG3 therapeutics including but not limited to relatlimab, LAG525, or TSR-033, anti-4-lBB (anti-CD37, anti-TNFRSF9), CD40 agonist therapeutics including but not limited to SGN-40, CP-870,893 or R07009789, anti-CD47 therapeutics including but not limited to Hu5F9-G4, anti- CD20 therapeutics, anti-CD38 therapeutics, STING agonists including but not limited to ADU- S100, MK-1454, ASA404, or amidobenzimidazoles, anthracyclines including but not limited to doxorubicin or mitox
• the additional therapeutic agent is selected from a luteinizing hormone-releasing hormone (LHRH) analog, including goserelin and leuprolide.
• LHRH luteinizing hormone-releasing hormone
• the additional therapeutic agent is selected from the group consisting of selected from the group consisting of everolimus, trabectedin, abraxane, TLK 286, AV-299, DN-101, pazopanib, GSK690693, RTA 744, ON 0910.Na, AZD 6244 (ARRY- 142886), AMN-107, TKI-258, GSK461364, AZD 1152, enzastaurin, vandetanib, ARQ-197, MK-0457, MLN8054, PHA-739358, R-763, AT-9263, pemetrexed, erlotinib, dasatanib, nilotinib, decatanib, panitumumab, amrubicin, oregovomab, Le
• CC 8490 cilengitide, gimatecan, IL13-PE38QQR, INO 1001, IPdRi KRX-0402, lucanthone, LY 317615, neuradiab, vitespan, Rta 744, Sdx 102, talampanel, atrasentan, Xr 311, romidepsin, ADS- 100380, sunitinib, 5-fluorouracil, vorinostat, etoposide, gemcitabine, doxorubicin, irinotecan, liposomal doxorubicin, 5'-deoxy-5-fluorouridine, vincristine, temozolomide, ZK- 304709, seliciclib; PD0325901, AZD-6244, capecitabine, L-Glutamic acid, N-[4-[2-(2-amino- 4,7-dihydro-4-oxo-lH-pyrrolo[2,3-
• compositions comprising compounds as disclosed herein formulated together with a pharmaceutically acceptable carrier.
• present disclosure provides pharmaceutical compositions comprising compounds as disclosed herein formulated together with one or more
• compositions include those suitable for oral, rectal, topical, buccal, parenteral (e.g., subcutaneous, intramuscular, intradermal, or intravenous) rectal, vaginal, or aerosol administration, although the most suitable form of administration in any given case will depend on the degree and severity of the condition being treated and on the nature of the particular compound being used.
• parenteral e.g., subcutaneous, intramuscular, intradermal, or intravenous rectal, vaginal, or aerosol administration
• disclosed compositions may be formulated as a unit dose, and/or may be formulated for oral or subcutaneous administration.
• compositions may be used in the form of a
• compositions for example, in solid, semisolid or liquid form, which contains one or more of the compound described herein, as an active ingredient, in admixture with an organic or inorganic carrier or excipient suitable for external, enteral or parenteral applications.
• the active ingredient may be compounded, for example, with the usual non-toxic, pharmaceutically acceptable carriers for tablets, pellets, capsules, suppositories, solutions, emulsions, suspensions, and any other form suitable for use.
• the active object compound is included in the
• composition in an amount sufficient to produce the desired effect upon the process or condition of the disease.
• the principal active ingredient may be mixed with a pharmaceutical carrier, e.g., conventional tableting ingredients such as corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, di calcium phosphate or gums, and other pharmaceutical diluents, e.g., water, to form a solid preformulation composition containing a homogeneous mixture of a compound provided herein, or a non-toxic
• a pharmaceutical carrier e.g., conventional tableting ingredients such as corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, di calcium phosphate or gums, and other pharmaceutical diluents, e.g., water, to form a solid preformulation composition containing a homogeneous mixture of a compound provided herein, or a non-toxic
• compositions as homogeneous, it is meant that the active ingredient is dispersed evenly throughout the composition so that the composition may be readily subdivided into equally effective unit dosage forms such as tablets, pills and capsules.
• solid dosage forms for oral administration capsules, tablets, pills, dragees, powders, granules and the like
• the subject composition is mixed with one or more
• pharmaceutically acceptable carriers such as sodium citrate or dicalcium phosphate, and/or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as, for example, acetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such a tal
• compositions may also comprise buffering agents.
• Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.
• a tablet may be made by compression or molding, optionally with one or more accessory ingredients.
• Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent.
• Molded tablets may be made by molding in a suitable machine a mixture of the subject composition moistened with an inert liquid diluent. Tablets, and other solid dosage forms, such as dragees, capsules, pills and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the
• compositions for inhalation or insufflation include solutions and suspensions in pharmaceutically acceptable, aqueous or organic solvents, or mixtures thereof, and powders.
• Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs.
• the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, cyclodextrins and mixtures thereof.
• inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate
• Suspensions in addition to the subject composition, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.
• suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.
• Formulations for rectal or vaginal administration may be presented as a suppository, which may be prepared by mixing a subject composition with one or more suitable non-irritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the body cavity and release the active agent.
• suitable non-irritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the body cavity and release the active agent.
• Dosage forms for transdermal administration of a subject composition include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants.
• the active component may be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants which may be required.
• the ointments, pastes, creams and gels may contain, in addition to a subject composition, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.
• excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.
• Powders and sprays may contain, in addition to a subject composition, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances.
• Sprays may additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.
• compositions and compounds of the present disclosure may alternatively be administered by aerosol. This is accomplished by preparing an aqueous aerosol, liposomal preparation or solid particles containing the compound.
• a non-aqueous (e.g., fluorocarbon propellant) suspension could be used.
• Sonic nebulizers may be used because they minimize exposing the agent to shear, which may result in degradation of the compounds contained in the subject compositions.
• an aqueous aerosol is made by formulating an aqueous solution or suspension of a subject composition together with conventional pharmaceutically acceptable carriers and stabilizers.
• the carriers and stabilizers vary with the requirements of the particular subject composition, but typically include non-ionic surfactants (Tweens, Pluronics, or polyethylene glycol), innocuous proteins like serum albumin, sorbitan esters, oleic acid, lecithin, amino acids such as glycine, buffers, salts, sugars or sugar alcohols. Aerosols generally are prepared from isotonic solutions.
• compositions of the present disclosure suitable for parenteral administration comprise a subject composition in combination with one or more
• sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.
• aqueous and non-aqueous carriers examples include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate and
• cyclodextrins Proper fluidity may be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
• enteral pharmaceutical formulations including a disclosed compound and an enteric material; and a pharmaceutically acceptable carrier or excipient thereof.
• Enteric materials refer to polymers that are substantially insoluble in the acidic environment of the stomach, and that are predominantly soluble in intestinal fluids at specific pHs.
• the small intestine is the part of the gastrointestinal tract (gut) between the stomach and the large intestine, and includes the duodenum, jejunum, and ileum.
• the pH of the duodenum is about 5.5
• the pH of the jejunum is about 6.5
• the pH of the distal ileum is about 7.5.
• enteric materials are not soluble, for example, until a pH of about 5.0, of about 5.2, of about 5.4, of about 5.6, of about 5.8, of about 6.0, of about 6.2, of about 6.4, of about 6.6, of about 6.8, of about 7.0, of about 7.2, of about 7.4, of about 7.6, of about 7.8, of about 8.0, of about 8.2, of about 8.4, of about 8.6, of about 8.8, of about 9.0, of about 9.2, of about 9.4, of about 9.6, of about 9.8, or of about 10.0.
• Exemplary enteric materials include cellulose acetate phthalate (CAP), hydroxypropyl methylcellulose phthalate (HPMCP), polyvinyl acetate phthalate (PVAP), hydroxypropyl methylcellulose acetate succinate (HPMCAS), cellulose acetate trimellitate, hydroxypropyl methylcellulose succinate, cellulose acetate succinate, cellulose acetate hexahydrophthalate, cellulose propionate phthalate, cellulose acetate maleate, cellulose acetate butyrate, cellulose acetate propionate, copolymer of methylmethacrylic acid and methyl methacrylate, copolymer of methyl acrylate, methylmethacrylate and methacrylic acid, copolymer of methylvinyl ether and maleic anhydride (Gantrez ES series), ethyl methyacrylate-methylmethacrylate-chlorotrimethylammonium ethyl acrylate copolymer, natural resins
• kits for use by a e.g. a consumer in need of treatment of cancer include a suitable dosage form such as those described above and instructions describing the method of using such dosage form to mediate, reduce or prevent inflammation.
• the instructions would direct the consumer or medical personnel to administer the dosage form according to administration modes known to those skilled in the art.
• kits could advantageously be packaged and sold in single or multiple kit units.
• An example of such a kit is a so-called blister pack.
• Blister packs are well known in the packaging industry and are being widely used for the packaging of pharmaceutical unit dosage forms (tablets, capsules, and the like). Blister packs generally consist of a sheet of relatively stiff material covered with a foil of a preferably transparent plastic material.
• the packaging process recesses are formed in the plastic foil.
• the recesses have the size and shape of the tablets or capsules to be packed.
• the tablets or capsules are placed in the recesses and the sheet of relatively stiff material is sealed against the plastic foil at the face of the foil which is opposite from the direction in which the recesses were formed.
• the tablets or capsules are sealed in the recesses between the plastic foil and the sheet.
• the strength of the sheet is such that the tablets or capsules can be removed from the blister pack by manually applying pressure on the recesses whereby an opening is formed in the sheet at the place of the recess. The tablet or capsule can then be removed via said opening.
• a memory aid on the kit, e.g., in the form of numbers next to the tablets or capsules whereby the numbers correspond with the days of the regimen which the tablets or capsules so specified should be ingested.
• a memory aid is a calendar printed on the card, e.g., as follows "First Week, Monday, Tuesday, . . . etc. . . . Second Week, Monday, Tuesday, . . . " etc.
• a “daily dose” can be a single tablet or capsule or several pills or capsules to be taken on a given day.
• a daily dose of a first compound can consist of one tablet or capsule while a daily dose of the second compound can consist of several tablets or capsules and vice versa.
• the memory aid should reflect this.
• R 4 -L-substituted pyrazoles B-I are prepared by direct alkylation of pyrazole.
• pyrazoles B-I are regioselectively nitrated to provide nitro- pyrazoles C-I by standard conditions familiar to those skilled in the art.
• hydrogenation of nitro-pyrazoles C-I employing a hydrogenation catalyst, such as palladium or nickel, provides pyrazole amines D-I.
• examples of R 4 include heterocyclyl with suitable optional substituents as exemplified by the tables of interemediates below and NR 6 R 9 , where each of R 6 and R 9 can independently be an alkyl group, examples of L include -(CH2) m - where m can be 0,
• R 4 is C-linked to the pyrazole, when m is 1, R 4 is C-linked to L, and when m is 2 or 3, R 4 is N-linked or C-linked to L.
• A-I-2 (wherein LG is an appropriate leaving group needed in the subsequent cyclization/pyrazole-forming reaction).
• Reaction of A-I-2 with hydrazine 4-1 (readily available to those skilled in the art) in the presence of acid provides predominately either B-I-l or its regioisomer B-I-2.
• the regiochemistry of cyclization is controlled under conditions familiar to one skilled in the art (such as temperature and solvent).
• Conditions for the synthesis of B-I-l include where LG is OEt, in a protic solvent (such as ethanol) at low temperature (-10 °C to RT) in the presence of acid.
• Conditions for the synthesis of B-I-2 include where LG is NMe2, in a protic solvent (such as ethanol) at reflux in the presence of acid.
• esters B-I-l or B-I-2 are converted to the corresponding acids C-I-l or C-I-2, using standard conditions known to those skilled in the art.
• Either C-I-l or C-I-2 is converted to the corresponding amines D-I-l or D-I-2 by Curtius rearrangement.
• examples of LG include OMe, OEt, and N(CEL)2
• examples of R 2 include alkyl and cycloalkyl, where alkyl and cycloalkyl can be optionally fluorinated
• examples of R 4 include heterocyclyl with suitable optional substituents as exemplified by the tables of interemediates below and NR 6 R 9 , where each of R 6 and R 9 can independently be an alkyl group
• examples of L include -(CH2) m - where m can be 0, 1, 2, or 3 and when m is 0, R 4 is C-linked to the pyrazole, when m is 1, R 4 is C-linked to L, and when m is 2 or 3, R 4 is N-linked or C-linked to L.
• Scheme 3 illustrates the general preparation of amines D-I-3.
• Pyrazole esters B-I-3 are prepared by reaction of hydrazines 4-1 (readily available to those skilled in art) with intermediates A-I-3.
• the R 2 and R 3 moieties are varied independently such that the R 3 is the same, or different to R 2 .
• Esters B-I-3 are converted to the corresponding acids C-I-3 using standard conditions known to those skilled in the art.
• Acids C-I-3 are converted to amines D-I-3 under standard Curtius rearrangement conditions known to those skilled in the art.
• examples of R 2 and R 3 can independently include alkyl and cycloalkyl, where alkyl and cycloalkyl can be optionally fluorinated
• examples of R 4 include heterocyclyl with suitable optional substituents as exemplified by the tables of interemediates below and NR 6 R 9 , where each of R 6 and R 9 can independently be an alkyl group
• examples of L include -(CH2) m - where m can be 0, 1, 2, or 3 and when m is 0, R 4 is C-linked to the pyrazole, when m is 1, R 4 is C-linked to L, and when m is 2 or 3, R 4 is N-linked or C-linked to L.
• hydrazines 4-1 that are not commercially available are readily prepared by the two methods shown in Scheme 4.
• One method involves the diazotization of amines 4-2 followed by reduction using conditions familiar to those skilled in the art, for example by the treatment with Sn(II)Cb in the presence of a proton source.
• hydrazines 4-1 are available from the corresponding N-tert-butoxycarbonylhydrazines 4-3 by acid-catalyzed removal of the tert-butoxycarbonyl group.
• the conversion of carbamates 4-3 to hydrazines 4-1 are also accomplished in situ within a reaction sequence.
• carbamates 4-3 are surrogates for hydrazines 4-1 in all schemes in which the hydrazines 4-1 is normally used in the presence of an acid.
• the N-tert-butoxycarbonylhydrazines 4-3 can be prepared by reductive amination with commercially available aldehydes or ketones 4-4 and tert-butyl hydrazinecarboxylate.
• examples of R 4 include heterocyclyl with suitable optional substituents as exemplified by the tables of interemediates below and NR 6 R 9 , where each of R 6 and R 9 can independently be an alkyl group, examples of L include -(C(R 10 )2) m - where an example of R 10 is H and where m can be 0, 1, 2, or 3, and when m is 0, R 4 is C-linked to a nitrogen, when m is 1, R 4 is C-linked to L, and when m is 2 or 3, R 4 is N-linked or C-linked to L.
• general pyrazol e-amines D-I-4 or D-I-5 are prepared by the two methods shown in Scheme 5.
• examples of R 4 -L-linked 5-1 or 5-2 are shown..
• One method involves alkylation of B-I-4 (readily available to those skilled in art) with commercially available 5-1 to provide nitro-pyrazoles C-I-4 and/or C-I-5 in the presence of base (e.g. potassium carbonate, cesium carbonate or sodium hydride) and a polar aprotic solvent (dimethyl sulfoxide, dimethylformamide, tetrahydrofuran or the like), at temperatures between ambient and 150 °C.
• base e.g. potassium carbonate, cesium carbonate or sodium hydride
• a polar aprotic solvent dimethyl sulfoxide, dimethylformamide, tetrahydrofuran or the like
• D-I-4 or D-I-5 involves a Mitsunobu reaction of B-I-4 with commercially available 5-2 to provide the nitro-pyrazoles C-I-4 and C-I-5.
• C-I-4 and C-I-5 are separated by SFC purification, crystallization or chromatography.
• a hydrogenation catalyst such as palladium or nickel or mild reducing conditions such as zinc or iron and ammonium chloride provides pyrazole-amines D-I-4 and D-I-5.
• examples of LG include Cl and Br
• examples of R 2 include alkyl, cycloalkyl, alkoxy, halogen, and CN, where alkyl, cycloalkyl, or alkoxy can be optionally fluorinated
• examples of R 4 include heterocyclyl with suitable optional substituents as exemplified by the tables of interemediates below, C(0)NR 6 R 9 , and NR 6 R 9 , where each of R 6 and R 9 can independently be H or an alkyl group
• examples of L include -(CFb - where m can be 0, 1, 2, or 3, and when m is 0, R 4 is C-linked to the pyrazole, when m is 1, R 4 is C-linked to L, and when m is 2 or 3, R 4 is N-linked or C-linked to L.
• general pyrazol e-amines D-I-6 and D-I-7 are prepared as shown in Scheme 6.
• examples of R 4 -L-linked 6-1 are shown.
• Alkylation of B-I-4 (readily available to those skilled in the art) with 6-1 provide a mixture of nitro-pyrazoles B-I-5 and B-I-6.
• These two isomers B-I-5 and B-I-6 are separated by SFC purification, crystallization or chromatography.
• Each isomer B-I-5 or B-I-6 are activated using MsCl or TsCl to provide B-I- 7 or B-I-8 (R is Ms or Ts), respectively.
• examples of LG include Cl and Br
• examples of R include mesylate and tosylate
• examples of R 2 include alkyl, cycloalkyl, alkoxy, halogen, and CN, where alkyl, cycloalkyl, or alkoxy can be optionally fluorinated
• examples of R 4 include heterocyclyl with suitable optional substituents as exemplified by the tables of interemediates below and NR 6 R 9 , where each of R 6 and R 9 can independently be an alkyl group
• examples of L include -(CH2) m - where m is 2 or 3, and R 4 is N-linked.
• Scheme 7 illustrates the synthesis of general amines D-I-8 and D-I-9.
• condensation of commercially available aldehydes with glyoxal in the presence of ammonium hydroxide provides R 2 -substituted imidazoles A-I-4.
• examples of R 4 -L-linked 5- 1 or 5-2 are shown.
• Nitration of imidazoles A-I-4 by conditions known to those skilled in the art e.g., nitric acid/concentrated sulfuric acid at temperatures ranging from 0 °C to 100 °C
• B-I-9 are converted to a mixture of C-I-8 and C-I-9 by alkylation (5-1) or Mitsunobu reaction (5-2).
• substituted nitro-imidazoles C-I-8 and C-I-9 are available by either alkylation (5-1) or Mitsunobu reaction (5-2) from A-I-4 to afford B-I-10.
• Subsequent nitration of B-I-10 then provides C-I-8 and C-I-9.
• These two regioisomers C-I-8 and C-I-9 can be separated by SFC purification, crystallization or chromatography.
• examples of LG include Cl and Br
• examples of R 2 include alkyl and cycloalkyl, where alkyl and cycloalkyl can be optionally fluorinated
• examples of R 4 include heterocyclyl with suitable optional substituents as exemplified by the tables of interemediates below and NR 6 R 9 , where each of R 6 and R 9 can independently be an alkyl group
• examples of L include -(CH2) m - where m can be 2 or 3
• L is N-linked to the imidazole ring.
• substituted thiazole amines D-I- 10 can be prepared from appropriately substituted thiazoles A-I-5 (readily available to those skilled in the art). This method has been described in W02006072436, the contents of which are hereby incorporated by reference in their entireties. Nitration of bromothiazoles A-I-5 using fuming nitric acid or nitric acid with sulfuric acid afford bromo-nitrothiazoles B-I-ll.
• the bromo functionality on B-I-ll can be displaced by various amines R 4 -H using conditions familiar to those skilled in the art such as Buchwald, Ullmann or nucleophilic aromatic substitution reactions in the presence of TEA or K 2 CO 3 to furnish C-I-10 where R 4 is N-linked.
• a Suzuki reaction of B-I-ll with commercially available or synthetic boronates T including but not limited to other boron salts is used to provide C-I-10 where R 4 is C-linked.
• a hydrogenation catalyst such as palladium or nickel, or mild reducing conditions such as zinc or iron and ammonium chloride provides the corresponding thiazole-amines D-I-10.
• examples of R 2 include alkyl, cycloalkyl, alkoxy, halogen, and CN, where alkyl, cycloalkyl, or alkoxy can be optionally fluorinated and examples of R 4 include heterocyclyl with suitable optional substituents as exemplified by the tables of interemediates below and NR 6 R 9 , where each of R 6 and R 9 can independently be an alkyl group.
• substituted thiazole amines D-I- 11 can be prepared from appropriately substituted thiazoles A-I-5 (readily available to those skilled in the art). This method has been described in WO2009158373, WO2016135163, and WO2011075515, the contents of which are hereby incorporated by reference in their entireties. Nitration of bromothiazoles A-I-5 using fuming nitric acid or nitric acid with sulfuric acid afford bromo-nitrothiazoles B-I-ll.
• the bromo functionality on B-I-ll can be displaced by various linker (L) synthons using conditions familiar to those skilled in the art such as carbonylation (C- I-lla), Negishi (zinc mediated coupling conditions, (C-I-llb)), Sonogashira or Heck coupling reaction following by appropriate reduction to furnish C-C linked C-I-llc.
• C-I-lla, C-I-llb and C-I-llc can be reduced to primary alcohols, followed by mesylation or tosylation to form C-I- 12. Nucleophilic substitution of C-I-12 with different nucelophiles R 4 -H furnishes C-I-13.
• examples of LG include OTs and OMs
• examples of R include methyl and ethyl
• examples of R 2 include alkyl, cycloalkyl, alkoxy, halogen, and CN, where alkyl, cycloalkyl, or alkoxy can be optionally fluorinated
• examples of R 4 include heterocyclyl with suitable optional substituents as exemplified by the tables of interemediates below and NR 6 R 9 , where each of R 6 and R 9 can independently be an alkyl group
• examples of L include -(CH2) m - where m can be 1, 2, or 3.
• examples of R 2 can be alkyl, cycloalkyl, alkoxy, halogen, or CN, where alkyl, cycloalkyl, or alkoxy can be optionally fluorinated, and examples of R 4 include heterocyclyl with suitable optional substituents as exemplified by the tables of interemediates below and NR 6 R 9 , where each of R 6 and R 9 can independently be an alkyl group.
• LG OMs or OTs
• substituted thiazole amines D- 1-13 can be prepared from appropriately substituted thiazoles A-I-6 (readily available to those skilled in the art). This method has been described in WO2009158373, WO2016135163, and WO2011075515, the contents of which are hereby incorporated by reference in their entireties. Nitration of bromothiazoles A-I-6 using fuming nitric acid or nitric acid with sulfuric acid afford bromo-nitrothiazoles B-I-12.
• the bromo functionality on B-I-12 can be displaced by various R 4 groups using conditions familiar to those skilled in the art such as carbonylation (C-I-15a), Negishi (zinc mediated coupling conditions (C-I-15b), Sonogashira or Heck coupling reaction followed by reduction to furnish C-C linked C-I-15c.
• C-I-15a, C-I-15b and C-I-15c can be reduced to primary alcohols, followed by mesylation or tosylation to form C-I-16.
• Nucleophilic substitution of C-I-16 with different nucleophiles R 4 -H furnishes C-I-17.
• reduction of C-I-17 in presence of hydrogen catalyst such as palladium or nickel or mild reducing conditions such as zinc or iron and ammonium chloride provides corresponding thiazole-amines D-I-13.
• examples of LG include OTs and OMs
• examples of R include methyl and ethyl
• examples of R 2 include alkyl, cycloalkyl, alkoxy, halogen, and CN, where alkyl, cycloalkyl, or alkoxy can be optionally fluorinated
• examples of R 4 include heterocyclyl with suitable optional substituents as exemplified by the tables of interemediates below and NR 6 R 9 , where each of R 6 and R 9 can independently be an alkyl group
• examples of L include -(CH2) m - where m can be 1, 2, or 3.
• Scheme 12 describes the synthesis of substituted oxazoles D-I-14 as reported in WO2014078378, the content of which are hereby incorporated by reference in its entirety.
• A-I-7 react with R 2 substituted aminoalkyl nitriles 12-1 (readily available to those skilled in the art) to furnish A-I-8.
• A-I-8 are converted oxazole-amines D-I-14 under acidic conditions such as acetic acid, sulfuric acid or hydrochloric acid.
• examples of R 2 include alkyl, and cycloalkyl, where alkyl, cycloalkyl, or alkoxy can be optionally fluorinated
• examples of R 4 include heterocyclyl with suitable optional substituents as exemplified by the tables of interemediates below and NR 6 R 9 , where each of R 6 and R 9 can independently be an alkyl group
• examples of L include - (CH2) m - where m can be 0, 1, 2, or 3.
• examples of R 2 include alkyl, cycloalkyl, and alkoxy, where alkyl, cycloalkyl, or alkoxy can be optionally fluorinated, and examples of R 4 include heterocyclyl with suitable optional substituents as exemplified by the tables of interemediates below and NR 6 R 9 , where each of R 6 and R 9 can independently be an alkyl group, and examples of L include - (CH2) m - where m can be 0, 1, 2, or 3.
• the oxazole-amines D-I-16 can be prpared from 2-halo oxazoles C-I-20 as described in WO2012033195, the contents of which are hereby incorporated by reference in their entireties.
• Reaction of C-I-20 with various amines R 4 - H under Buchwald coupling conditions provide 2-aminoalkyl substituted oxazoles C-I-21 where R 4 is N-linked.
• a Suzuki reaction of C-I-20 with commercially available or synthetic boronates T including but not limited to other boron salts are used to provide C-I-21 where R 4 is C-linked.
• Hydrolysis of oxazole-esters C-I-21 furnish carboxylic acids which can be converted into oxazole-amines D-I-16 under Curtius rearrangement using sodium azide or DPPA.
• examples of R 2 can be alkyl, cycloalkyl, or alkoxy, where alkyl, cycloalkyl, or alkoxy can be optionally fluorinated, and examples of R 4 include heterocyclyl with suitable optional substituents as exemplified by the tables of interemediates below and NR 6 R 9 , where each of R 6 and R 9 can independently be an alkyl group, where each of R 6 and R 9 can independently be an alkyl group.
• Triazoles B-I-14 are prepared from dinitro-esters A-I-10 by reaction with aldehydes 15-1 (readily available to those skilled in the art) using the procedure described in Asian J. of Chem, 2014, 26, 4744 and Hanneng Cailliao, 2008, 16, 49, the contents of which are hereby incorporated by reference in their entireties.
• B-I-14 may be prepared by nitration of A-I-ll.
• B-I-14 are converted to a mixture of C-I-22 and C-I-23 by alkylation (5-1) or Mitsunobu reaction (5-2). These two regioisomers C-I-22 and C-I-23 can be separated by SFC purification, crystallization or chromatography. Reduction of nitro-triazoles C-I-22 or C-I-23 in the presence of a hydrogenation catalyst, such as palladium or nickel, or mild reducing conditions such as zinc or iron and ammonium chloride provides the corresponding triazole-amines D-I-17 and D-I-18, respectively.
• a hydrogenation catalyst such as palladium or nickel
• mild reducing conditions such as zinc or iron and ammonium chloride
• examples of R 2 can be alkyl, cycloalkyl, alkoxy, halogen, or CN, where alkyl, cycloalkyl, or alkoxy can be optionally fluorinated
• examples of R 4 include heterocyclyl with suitable optional substituents as exemplified by the tables of interemediates below and NR 6 R 9 , where each of R 6 and R 9 can independently be an alkyl group
• examples of L include -(CH2) m - where m can be 2 or 3.
• Scheme 16 illustrates the general preparation of boronic acid/boronic ester T, which are not commercially available. These compounds can be readily prepared from substituted carboxylic acids.
• the carboxylic acids can be activated by 2-hydroxyisoindoline-l,3- dione in the presence of coupling reagent (e.g. DCI or Et3N/HATU) to afford Q.
• coupling reagent e.g. DCI or Et3N/HATU
• examples of R 4 include alkyl, cycloalkyl, and heterocyclyl with suitable optional substituents as exemplified by the tables of interemediates below and examples of L include -(CH2) m - where m can be 0, 1, 2, or 3, and when m is 0, R 4 is C-linked to the boronate ester, when m is 1, then R 4 is C-linked to L, and when m is 2 or 3, then R 4 is N-linked or C-linked to L.
• Scheme 17 illustrates the general preparation of L-I-l and L-I-2.
• 2,4-dichloro-5-iodopyrimidine reacts with TMSCHF2 in a solvent such as NMP or DMF in the presence of Cul and CsF to produce difluoromethylpyrimidine L-II-1
• Difluoromethylpyrimidine L-II-1 can be converted to methylthiorpyrimidine L-I-l by treatment with sodium thiomethoxide and zinc chloride in diethyl ether at a temperature lower than 10 °C (WO2012110773, the content of which is hereby incorporated by reference in its entirety).
• trifluoromethylpyrimidine L-I-2 can be prepared from the commercially available 2,4-dichloro-5-(trifluoromethyl)pyrimidine, L-II-2.
• R 1 can be cycloalkyl.
• Treatment of commercially available 5-bromo-2-chloro-4- (methylthio)pyrimidine with boronic esters/boronic acids/trifluoroborates in the presence of a palladium catalyst (Suzuki coupling) afford R 1 -substituted thiopyrimidines L-III (t 0).
• R 1 in this scheme examples include cycloalkyl such as cyclopropyl.
• Scheme 19 illustrates the general preparation of intermediates H (I-XIII) by a widely known method.
• the Boc group of G is a protecting group that is removed upon exposure to acid, for example HC1 or TFA.
• H-I through H-XIII are prepared by this method.
• q can be 0, 1, 2, or 3, r can be 2, 3, or 4, V can be C(R 34 )2, O, or NR 6 , where R 6 is alkyl, n can be 2, 3, or 4, each R 34 can, independently, be H, Ci-C 6 alkyl, or two R 34 can be taken together to form a cycloalkyl.
• lactam H-XIV can be prepared from DBU via one step process as illustrated in Scheme 20.
• DBU is hydrolyzed by potassium hydroxide in a solution of methanol and water at ambient temperature to provide H-XIV.
• Scheme 21 illustrates the general preparation of key intermediates J and K.
• Key intermediates J can be prepared from H (either free base or HC1 salt) and methylthiopyrimidine L-I in the presence of an organic base (e. g. triethylamine or DIEA) with optional heating to provide key intermediates J.
• key intermediates K can be prepared from H with either L-II or L-III.
• q can be 0, 1, 2, or 3, r can be 2, 3, or 4, V can be C(R 34 )2, O, or NR 6 , where R 6 is alkyl, n can be 2, 3, or 4, each R 34 can, independently, be H, Ci-C 6 alkyl, or two R 34 can be taken together to form a cycloalkyl group.
• Scheme 22 illustrates the general preparation of key pyridine intermediates M.
• an aprotic solvent e. g. DME, DMF, DMSO, or NMP
• R 1 can be Br, Cl, alkyl optionally substituted by one or more fluorine atoms, or cycloalkyl
• q can be 0, 1, 2, or 3
• r can be 2, 3, or 4
• V can be C(R 34 )2, O, or NR 6 , where R 6 is alkyl
• n can be 2, 3, or 4
• each R 34 can, independently, be H, Ci-C 6 alkyl, or two R 34 can be taken together to form a cycloalkyl.
• Scheme 23 illustrates general preparations of compounds of Formula I-A from substituted D-I.
• the preparation of Formula I-A can be accomplished from key intermediates K and J.
• the nucleophilic substitution reaction of K with amines D-I is typically performed in a polar solvent at temperatures ranging from ambient temp to 150 °C, in some embodiments with microwave heating, optionally in the presence of an acid for example 4 N HC1 in 1,4-dioxane to provide compounds of Formula I-A.
• Compounds D-I which are not commercially available, can be readily prepared (see schemes 1-16).
• the sulfoxides react with amines D-I by a nucleophilic substitution reaction, typically performed in a polar solvent at temperatures ranging from ambient temp to 150 °C, in some embodiments with microwave heating, optionally in the presence of an acid for example 4 N HC1 in 1,4-dioxane or pTSA to afford compounds of Formula I-A.
• Formula I-A contains a protecting group such as a Boc group
• this protecting group can be deprotected under acidic conditions to provide Formula I-A (free amine or salt).
• Further treatment of Formula I-A (free base or salt) with sodium cyanoborohydride or sodium triacetoxyborohydride and an aldehyde or ketone in the presence of a catalytic amount of acetic acid in polar solvents such as MeOH (reductive amination conditions) afford N-substituted Formula I-A.
• the free amine (or salt) can be treated with commercially available acyl chloride or sulfonyl chloride to afford N-substituted Formula I-A.
• Scheme 24 illustrates the general preparation of compounds of Formula I-B.
• the preparation of Formula I-B can be accomplished by a Buchwald-Hartwig coupling reaction with D-I and M. Many amines D-I which are not commercially available can be readily prepared (see schemes 1-16).
• reductive alkylation, acylation and sulfonylation can be performed to provide Formula I-B after deprotection of Formula I-B that contains a protecting group such as a Boc group.
• the combined organic extract was washed with brine (150 mL), dried over anhydrous NaiSCL, and filtered under reduced pressure.
• the crude was purified by silica gel column chromatography (40 to 50 % EtOAc/hexane, 15 CV’s) to obtain tert-butyl (3 -(5 -oxo- 1,4- oxazepan-4-yl)propyl carbamate (12 g, 50 % yield) as yellow liquid.
• the product was dissolved in DCM (50 mL) and treated with 4 N HC1 in 1,4-dioxane (4 eq).
• reaction mixture was diluted with water (200 mL) and extracted with EtOAc (3 x 300 mL). The combined organic extracts were washed with brine (200 mL), dried over anhydrous Na 2 SC> 4 , filtered and concentrated under reduced pressure.
• reaction mixture was stirred at rt for 16 h.
• the reaction mixture was quenched with sat’d NaHCCh solution (40 mL) and stirred for few minutes. Layers were separated and the aqueous layer was extracted with EtOAc (2 x 30 mL) and combined organics were washed with brine, dried over anhydrous Na 2 S0 4 , filtered, concentrated under reduced pressure.
• Table G Exemplary Compounds that can be prepared by General Methods K through N.
• Assays were conducted in 384-well plates (100 uL final volume) using 19 nM ULK1 (Eurofms CAT# 14-959), 0.25 mg/mL myelin basic protein, 1.5 units pyruvate kinase, 2.1 units lactate dehydrogenase, 1 mM phosphoenol pyruvate, 0.28 mM NADH and 1 mM ATP in assay buffer (100 mM Tris, pH 7.5, 15 mM MgCk, 0.5 mM DTT, 0.1 % octyl-glucoside, 0.002% (w/v) BSA, and 0.002% Triton X-100).
• Inhibition of ULK1 was measured by adding serial diluted test compound (final assay concentration of 1% DMSO). A decrease in absorption at 340 nm was monitored continuously for 6 hours at 30 °C on a multi-mode microplate reader (BioTek). The reaction rate was calculated using the 2-3 h time frame. The reaction rate at each concentration of compound was converted to percent inhibition using controls (i.e. reaction with no test compound and reaction with a known inhibitor) and IC50 values were calculated by fitting a four-parameter sigmoidal curve to the data using Prism (GraphPad software).
• ULK1 protein sequence (residues 1-314 with N-terminal His tag; SEQ. ID NO: 1)
• EILK1 kinase Activity of EILK1 kinase was determined spectroscopically using a coupled pyruvate kinase/lactate dehydrogenase assay that continuously monitors the ATP hydrolysis- dependent oxidation of NADH (e.g., Schindler et al. Science (2000) 289: 1938-1942).
• Assays were conducted in 384-well plates (100 uL final volume) using 0.1 nM EILK1 (from Beryllium), 0.075 mM peptide substrate (YANWLAASIYLDGKKK (SEQ ID NO: 5)), 1.5 units pyruvate kinase, 2.1 units lactate dehydrogenase, 1 mM phosphoenol pyruvate, 0.28 mM NADH and 1 mM ATP in assay buffer (100 mM Tris, pH 7.5, 15 mM MgCh, 0.5 mM DTT, 0.004% (w/v) BSA, and 0.004% Triton X-100).
• Inhibition of ULK1 was measured by adding serial diluted test compound (final assay concentration of 1% DMSO). A decrease in absorption at 340 nm was monitored continuously for 6 hours at 30 °C on a multi-mode microplate reader (BioTek). The reaction rate was calculated using the 2-3 h time frame. The reaction rate at each concentration of compound was converted to percent inhibition using controls (i.e. reaction with no test compound and reaction with a known inhibitor) and IC 50 values were calculated using software routines in Prism (GraphPad software).
• ULK1 protein sequence (residues 1-283; SEQ. ID NO: 2)
• EILK2 kinase Activity of EILK2 kinase was determined spectroscopically using a coupled pyruvate kinase/lactate dehydrogenase assay that continuously monitors the ATP hydrolysis- dependent oxidation of NADH (e.g., Schindler et al. Science (2000) 289: 1938-1942).
• Assays were conducted in 384-well plates (100 uL final volume) using 9.7 nM ULK2 (Eurofms CAT# 14-772), 0.25 mg/mL myelin basic protein, 1.5 units pyruvate kinase, 2.1 units lactate dehydrogenase, 1 mM phosphoenol pyruvate, 0.28 mM NADH and 1 mM ATP in assay buffer (100 mM Tris, pH 7.5, 15 mM MgCh, 0.5 mM DTT, 0.1 % octyl-glucoside, 0.002% (w/v) BSA, and 0.002% Triton X-100).
• Inhibition of ULK2 was measured by adding serial diluted test compound (final assay concentration of 1% DMSO). A decrease in absorption at 340 nm was monitored continuously for 6 hours at 30 °C on a multi-mode microplate reader (BioTek). The reaction rate was calculated using the 2-3 h time frame. The reaction rate at each concentration of compound was converted to percent inhibition using controls (i.e. reaction with no test compound and reaction with a known inhibitor) and IC50 values were calculated by fitting a four-parameter sigmoidal curve to the data using Prism (GraphPad software).
• ULK2 protein sequence (residues 1-306 with N-terminal GST and His tag; SEQ. ID NO: 3)
• Example 177 Cellular inhibition of ULK kinase substrate ATG13 protein
• A549 (KRAS mutant) human lung cancer cells (6,000 cells/well) were added to a
• 10 pL of media containing trametinib or DMSO as a control was added to wells.
• the final concentration of trametinib in wells was 250 nM.
• a dose response of a test compound (0.6 pL per well) was added.
• DMSO 0.6 pL was added to control wells.
• the plate was briefly shaken to mix wells and then incubated at 37 °C overnight. The next day, the media was aspirated and cells were washed with Dulbecco’s Phosphate Buffered Saline (Gibco). Cells were lysed using MPER lysis buffer (Pierce, Rockford, IL) containing Halt Phosphatase and Protease Inhibitors (Pierce, Rockford, IL) and Phosphatase inhibitor cocktail 2 (Sigma, St. Louis, MO) at 4 °C for 10 minutes with shaking.
• MPER lysis buffer Pierce, Rockford, IL
• Halt Phosphatase and Protease Inhibitors Pieris, IL
• Phosphatase inhibitor cocktail 2 Sigma, St. Louis, MO
• Example 178 pATG13 levels of mutant KRas MiaPaCa-2 cells after treatment with ULK inhibitors in combination with Trametinib
• MiaPaCa-2 human pancreatic cancer cells (10000 cells/well) were added to a 384- well tissue-culture treated plate in 50 pL of pre-warmed DMEM medium supplemented with 10% characterized fetal bovine serum (Invitrogen, Carlsbad, CA), 100 units/mL penicillin G, 100 pg/mL streptomycin, and 2.5% Horse Serum and allowed to grow overnight at 37 °C, 5% C02, and 95% humidity. The following day, 10 pL of media containing trametinib or DMSO as a control was added to wells. The final concentration of trametinib in wells was 250 nM.
• a dose response of a test compound (0.6 pL per well) was added.
• DMSO (0.6 pL) was added to control wells.
• the plate was briefly shaken to mix wells and then incubated at 37 °C overnight. The next day, the media was aspirated and cells were washed with Dulbecco’s Phosphate Buffered Saline (Gibco).
• Cells were lysed using MPER lysis buffer (Pierce, Rockford, IL) containing Halt Phosphatase and Protease Inhibitors (Pierce, Rockford, IL) and Phosphatase inhibitor cocktail 2 (Sigma, St. Louis, MO) at 4 °C for 10 minutes with shaking.
• Biotinylated pS318-ATG13 antibody (Rockland Immunochemicals Cat#600-401-C49) was diluted in assay diluent and added to each well and incubated at room temperature for 1 hour. Plate wells were washed with ELISA wash buffer. Streptavidin linked to horseradish peroxidase (Thermo Fisher Cat#21140) was diluted in assay diluent and added to each well and incubated at room temperature for 1 hour. Plate wells were washed with ELISA wash buffer. High sensitivity TMB substrate (Biolegend Cat#421101) was added to each well and incubated at room temperature for 20 minutes. The reaction was stopped with 2N Sulfuric Acid.
• the plate was analyzed at on a plate reader measuring absorbance at 450 nm and 540 nm (background). Signal was calculated by first subtracting the background absorbance at 540 nm from the absorbance at 450 nm for each well. Next, the background corrected absorbance at 450 nm from blank wells was subtracted from test wells. Data was compared to control wells to determine % ATG13 phosphorylation. GraphPad Prism was used to calculate IC50 values.
• Example 179 pATG13 levels of mutant KRas HCT-116 cells after treatment with ULK inhibitors in combination with Trametinib
• HCT-116 human colon cancer cells (10000 cells/well) were added to a 384-well tissue-culture treated plate in 50 pL of pre-warmed DMEM medium supplemented with 10% characterized fetal bovine serum (Invitrogen, Carlsbad, CA), 100 units/mL penicillin G, and 100 pg/mL streptomycin and allowed to grow overnight at 37 °C, 5% C02, and 95% humidity.
• 10 pL of media containing trametinib or DMSO as a control was added to wells.
• the final concentration of trametinib in wells was 250 nM.
• a dose response of a test compound (0.6 pL per well) was added.
• DMSO 0.6 pL
• DMSO 0.6 pL
• the plate was briefly shaken to mix wells and then incubated at 37 °C overnight.
• the media was aspirated and cells were washed with Dulbecco’s Phosphate Buffered Saline (Gibco).
• Cells were lysed using MPER lysis buffer (Pierce, Rockford, IL) containing Halt Phosphatase and Protease Inhibitors (Pierce, Rockford, IL) and Phosphatase inhibitor cocktail 2 (Sigma, St. Louis, MO) at 4 °C for 10 minutes with shaking.
• Biotinylated pS318-ATG13 antibody (Rockland Immunochemicals Cat#600-401-C49) was diluted in assay diluent and added to each well and incubated at room temperature for 1 hour. Plate wells were washed with ELISA wash buffer. Streptavidin linked to horseradish peroxidase (Thermo Fisher Cat#21140) was diluted in assay diluent and added to each well and incubated at room temperature for 1 hour. Plate wells were washed with ELISA wash buffer. High sensitivity TMB substrate (Biolegend Cat#421101) was added to each well and incubated at room temperature for 20 minutes. The reaction was stopped with 2N Sulfuric Acid.
• the plate was analyzed at on a plate reader measuring absorbance at 450 nm and 540 nm (background). Signal was calculated by first subtracting the background absorbance at 540 nm from the absorbance at 450 nm for each well. Next, the background corrected absorbance at 450 nm from blank wells was subtracted from test wells. Data was compared to control wells to determine % ATG13 phosphorylation. GraphPad Prism was used to calculate IC50 values.
• Example 180 pATG13 levels of mutant BRAF A375 cells after treatment with ULK inhibitors in combination with Trametinib [000255] A375 human malignant melanoma cancer cells (20000 cells/well) were added to a
• 96-well tissue-culture treated plate in 100 pL of pre-warmed DMEM medium supplemented with 10% characterized fetal bovine serum (Invitrogen, Carlsbad, CA), 100 units/mL penicillin G, and 100 pg/mL streptomycin and allowed to grow overnight at 37 °C, 5% C02, and 95% humidity.
• 100 pL of media containing trametinib or DMSO as a control was added to wells.
• the final concentration of trametinib in wells was 250 nM.
• a dose response of a test compound (0.5 pL per well) was added.
• DMSO 0.5 pL was added to control wells.
• the plate was briefly shaken to mix wells and then incubated at 37 °C overnight. The next day, the media was aspirated and cells were washed with Dulbecco’s Phosphate Buffered Saline (Gibco). Cells were lysed using MPER lysis buffer (Pierce, Rockford, IL) containing Halt Phosphatase and Protease Inhibitors (Pierce, Rockford, IL) and Phosphatase inhibitor cocktail 2 (Sigma, St. Louis, MO) at 4 °C for 10 minutes with shaking.
• MPER lysis buffer Pierce, Rockford, IL
• Halt Phosphatase and Protease Inhibitors Pieris, IL
• Phosphatase inhibitor cocktail 2 Sigma, St. Louis, MO
• Cellular levels of phospho-Serine 318 ATG13 were measured via an ELISA method.
• Total ATG13 Antibody (Cell Signaling Cat#13273) was used to coat the wells.
• the plate was incubated at 4 °C overnight and washed with ELISA wash buffer (Biolegend Cat#421601 ) The wells were then blocked with assay diluent (Biolegend Cat#421203) for 1 hour at room temperature. Plate wells were washed with ELISA wash buffer. Cell lysate was added to wells and incubated at room temperature for 2 hours. Plate wells were washed with ELISA wash buffer.
• Biotinylated pS318-ATG13 antibody (Rockland Immunochemicals Cat#600-401-C49) was diluted in assay diluent and added to each well and incubated at room temperature for 1 hour. Plate wells were washed with ELISA wash buffer. Streptavidin linked to horseradish peroxidase (Thermo Fisher Cat#21140) was diluted in assay diluent and added to each well and incubated at room temperature for 1 hour. Plate wells were washed with ELISA wash buffer. High sensitivity TMB substrate (Biolegend Cat#421101) was added to each well and incubated at room temperature for 20 minutes. The reaction was stopped with 2N Sulfuric Acid.
• the plate was analyzed at on a plate reader measuring absorbance at 450 nm and 540 nm (background). Signal was calculated by first subtracting the background absorbance at 540 nm from the absorbance at 450 nm for each well. Next, the background corrected absorbance at 450 nm from blank wells was subtracted from test wells. Data was compared to control wells to determine % ATG13 phosphorylation. GraphPad Prism was used to calculate IC50 values.
• T24 human urinary bladder cancer cells (25000 cells/well) were added to a 96- well tissue-culture treated plate in 100 pL of pre-warmed DMEM medium supplemented with 10% characterized fetal bovine serum (Invitrogen, Carlsbad, CA), 100 units/mL penicillin G, and 100 pg/mL streptomycin and allowed to grow overnight at 37 °C, 5% C02, and 95% humidity.
• 100 pL of media containing trametinib or DMSO as a control was added to wells.
• the final concentration of trametinib in wells was 250 nM.
• a dose response of a test compound (0.5 pL per well) was added.
• DMSO 0.5 pL
• DMSO 0.5 pL
• the plate was briefly shaken to mix wells and then incubated at 37 °C overnight.
• the media was aspirated and cells were washed with Dulbecco’s Phosphate Buffered Saline (Gibco).
• Cells were lysed using MPER lysis buffer (Pierce, Rockford, IL) containing Halt Phosphatase and Protease Inhibitors (Pierce, Rockford, IL) and Phosphatase inhibitor cocktail 2 (Sigma, St. Louis, MO) at 4 °C for 10 minutes with shaking.
• Assays were conducted in 384-well plates (100 uL final volume) using 26.4 nM LRRK2 (Thermo Fisher), 0.1 mM peptide substrate (RLGRDKYKTLRQIRQ (SEQ ID NO: 6)), 1.5 units pyruvate kinase, 2.1 units lactate dehydrogenase, 1 mM phosphoenol pyruvate, 0.28 mM NADH and 1 mM ATP in assay buffer (100 mM Tris, pH 7.5, 15 mM MgCh, 0.5 mM DTT, 0.004% (w/v) BSA, and 0.004% Triton X-100).
• Inhibition of LRRK2 was measured by adding serial diluted test compound (final assay concentration of 1% DMSO). A decrease in absorption at 340 nm was monitored continuously for 6 hours at 30 °C on a multi-mode microplate reader (BioTek). The reaction rate was calculated using the 2-3 h time frame. The reaction rate at each concentration of compound was converted to percent inhibition using controls (i.e. reaction with no test compound and reaction with a known inhibitor) and IC 50 values were calculated using software routines in Prism (GraphPad software).
• LRRK2 protein sequence (residues 970-2528; SEQ. ID NO. 4)
• Example 183 Evaluation of ULK inhibitors in pancreatic ductal adenocarcinoma (PD AC) in vitro and in vivo
• ULK inhibitors will be evaluated in PD AC flux assays, and the IC50 of the compounds in a panel of multiple PD AC cell lines, including cells derived from primary tumors of a Trp53 lox/+ , LSL-Kras G12D , Rosa-rtTA LSL , p48Cre + ) will be determined using a clonogenicity 2D assay and a 3D organoid assay, in the absence or the presence of trametinib.
• the therapeutic efficacy of ULK inhibitors in PD AC models will be evaluated by (i) assessing the tumor kinetics of PD AC subcutaneously; (ii) assessing the tumor kinetics of PD AC (KPC implanted C57 black mice) orthotopically in the pancreas in syngeneic models; (iii) assessing tumor growth kinetics in syngeneic models with ULK inhibitors and MEK inhibitors; (iv) assessing the compounds in the PD AC autochthonous model; (v) assessing histological changes in the tumor microenvironment; (vi) assessing the changes in the immune cell infiltrates in the tumors upon inhibition by ULK inhibitors; (vii) assessing the efficacy of ULK inhibitors in combination with immune checkpoint blockade.

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