Patent Application
20250206745
Autoimmune diseases are associated with the overproduction of proinflammatory factors. One of them is interleukin-1 (IL-1), produced by activated macrophages, monocytes, fibroblasts, and other components of the innate immune system like dendritic cells. IL-1 is involved in a variety of cellular activities, including cell proliferation, differentiation and apoptosis (Masters, S. L., et. al., Annu. Rev. Immunol. 2009. 27:621-68).
In humans, 22 NLR proteins are divided into four NLR subfamilies according to their N-terminal domains. NLRA contains a CARD-AT domain, NLRB (NAIP) contains a BIR domain, NLRC (including NOD1 and NOD2) contains a CARD domain, and NLRP contains a pyrin domain. Multiple NLR family members are associated with inflammasome formation.
Although inflammasome activation appears to have evolved as an important component of host immunity to pathogens, the NLRP3 inflammasome is unique in its ability to activate in response to endogenous sterile danger signals. Many such sterile signals have been elucidated, and their formation is associated with specific disease states. For example, uric acid crystals found in gout patients are effective triggers of NLRP3 activation. Similarly, cholesterol crystals found in atherosclerotic patients can also promote NLRP3 activation. Recognition of the role of sterile danger signals as NLRP3 activators led to IL-1 and IL-18 being implicated in a diverse range of pathophysiological indications including metabolic, physiologic, inflammatory, hematologic and immunologic disorders.
The disclosure arises from a need to provide further compounds for the specific modulation of NLRP3-dependent cellular processes. In particular, compounds with improved physicochemical, pharmacological and pharmaceutical properties to existing compounds are desirable.
In some aspects, the present disclosure relates to a compound of Formula (I):
or a prodrug, solvate, or pharmaceutically acceptable salt thereof, wherein:
In some aspects, the present disclosure provides a compound obtainable by, or obtained by, a method for preparing a compound as described herein.
In some embodiments, the present disclosure provides a pharmaceutical composition comprising a compound described herein and one or more pharmaceutically acceptable carriers or excipients.
In some aspects, the present disclosure provides an intermediate as described herein, being suitable for use in a method for preparing a compound as described herein.
In some aspects, the present disclosure provides a method of inhibiting inflammasome (e.g., the NLRP3 inflammasome) activity (e.g., in vitro or in vivo), comprising contacting a cell with an effective amount of a compound of the present disclosure.
In some aspects, the present disclosure provides a method of treating or preventing a disease or disorder disclosed herein in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound of the present disclosure or a pharmaceutical composition of the present disclosure.
In some aspects, the present disclosure provides a compound of the present disclosure for use in inhibiting inflammasome (e.g., the NLRP3 inflammasome) activity (e.g., in vitro or in vivo).
In some aspects, the present disclosure provides a compound of the present disclosure or a pharmaceutically acceptable salt thereof for use in treating or preventing a disease or disorder disclosed herein.
In some aspects, the present disclosure provides use of a compound of the present disclosure in the manufacture of a medicament for inhibiting inflammasome (e.g., the NLRP3 inflammasome) activity (e.g., in vitro or in vivo).
In some aspects, the present disclosure provides use of a compound of the present disclosure in the manufacture of a medicament for treating or preventing a disease or disorder disclosed herein.
In some aspects, the present disclosure provides a method of preparing a compound of the present disclosure.
In some aspects, the present disclosure provides a method of preparing a compound, comprising one or more steps described herein.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In the specification, the singular forms also include the plural unless the context clearly dictates otherwise. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. All publications, patent applications, patents and other references mentioned herein are incorporated by reference. The references cited herein are not admitted to be prior art to the claimed invention. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting. In the case of conflict between the chemical structures and names of the compounds disclosed herein, the chemical structures will control.
Other features and advantages of the disclosure will be apparent from the following detailed description and claims.
Autoimmune diseases are associated with the overproduction of proinflammatory factors. One of them is interleukin-1 (IL-1), produced by activated macrophages, monocytes, fibroblasts, and other components of the innate immune system like dendritic cells, involved in a variety of cellular activities, including cell proliferation, differentiation and apoptosis (Masters, S. L. et al., Annu. Rev. Immunol. 2009. 27:621-68).
Autoimmune diseases are associated with the overproduction of proinflammatory factors. One of them is interleukin-1 (IL-1), produced by activated macrophages, monocytes, fibroblasts, and other components of the innate immune system like dendritic cells, involved in a variety of cellular activities, including cell proliferation, differentiation, and apoptosis (Masters, S. L., et al.
Annu. Rev. Immunol. 2009. 27:621-68). Cytokines from the IL-1 family are highly active and, as important mediators of inflammation, primarily associated with acute and chronic inflammation (Sims, J. et al., Nature Reviews Immunology 10, 89-102 (February 2010)). The overproduction of IL-1 is considered to be a mediator of some autoimmune and autoinflammatory diseases. Autoinflammatory diseases are characterised by recurrent and unprovoked inflammation in the absence of autoantibodies, infection, or antigen-specific T lymphocytes.
Proinflammatory cytokines of the IL-1 superfamily include IL-1α, IL-1β, IL-18, and IL-36α, β, λ and are produced in response to pathogens and other cellular stressors as part of a host innate immune response. Unlike many other secreted cytokines, which are processed and released via the standard cellular secretory apparatus consisting of the endoplasmic reticulum and Golgi apparatus, IL-1 family members lack leader sequences required for endoplasmic reticulum entry and thus are retained intracellularly following translation. In addition, IL-1β, IL-18, and IL-36α, β, λ are synthesised as procytokines that require proteolytic activation to become optimal ligands for binding to their cognate receptors on target cells.
In the case of IL-1α, IL-1β and IL-18, it is now appreciated that a multimeric protein complex known as an inflammasome is responsible for activating the proforms of IL-1β and IL-18 and for release of these cytokines extracellularly. An inflammasome complex typically consists of a sensor molecule, such as an NLR (Nucleotide-Oligerimisation Domain (NOD)-like receptor), an adaptor molecule ASC (Apoptosis-associated speck-like protein containing a CARD (Caspase Recruitment Domain)) and procaspase-1. In response to a variety of “danger signals”, including pathogen-associated molecule patterns (PAMPs) and danger associated molecular patterns (DAMPs), subunits of an inflammasome oligomerise to form a supramolecular structure within the cell. PAMPs include molecules such as peptidoglycan, viral DNA or RNA and bacterial DNA or RNA. DAMPs, on the other hand, consist of a wide range of endogenous or exogenous sterile triggers including monosodium urate crystals, silica, alum, asbestos, fatty acids, ceramides, cholesterol crystals and aggregates of beta-amyloid peptide. Assembly of an inflammasome platform facilitates autocatalysis of procaspase-1 yielding a highly active cysteine protease responsible for activation and release of pro-IL-1β and pro-IL-18. Thus, release of these highly inflammatory cytokines is achieved only in response to inflammasome sensors detecting and responding to specific molecular danger signals.
In humans, 22 NLR proteins are divided into four NLR subfamilies according to their N-terminal domains. NLRA contains a CARD-AT domain, NLRB (NAIP) contains a BIR domain, NLRC (including NOD1 and NOD2) contains a CARD domain, and NLRP contains a pyrin domain. Multiple NLR family members are associated with inflammasome formation including NLRP1, NLRP3, NLRP6, NLRP7, NLRP12 and NLRC4 (IPAF).
Two other structurally distinct inflammasome structures containing a PYHIN domain (pyrin and HIN domain containing protein) namely Absent in Melanoma 2 (AIM2) and IFNλ-inducible protein 16 (IFI16) (Latz et al., Nat Rev Immunol 2013 13(6) 397-311) serve as intracellular DNA sensors. Pyrin (encoded by the MEFV gene) represents another type of inflammasome platform associated with proIL-1β activation (Chae et al., Immunity 34, 755-768, 2011).
Requiring assembly of an inflammasome platform to achieve activation and release of IL-1β and IL-18 from monocytes and macrophages ensures their production is carefully orchestrated via a 2-step process. First, the cell must encounter a priming ligand (such as the TLR4 receptor ligand LPS, or an inflammatory cytokine such as TNFα) which leads to NFkB dependent transcription of NLRP3, pro-IL-1β and pro-IL-18. The newly translated procytokines remain intracellular and inactive unless producing cells encounter a second signal leading to activation of an inflammasome scaffold and maturation of procaspase-1.
In addition to proteolytic activation of pro-IL-1β and pro-IL-18, active caspase-1 also triggers a form of inflammatory cell death known as pyroptosis through cleavage of gasdermin-D. Pyroptosis allows the mature forms of IL-1β and IL-18 to be externalised along with release of alarmin molecules (compounds that promote inflammation and activate innate and adaptive immunity) such as high mobility group box 1 protein (HMGB1), IL-33, and IL-1α.
Although inflammasome activation appears to have evolved as an important component of host immunity to pathogens, the NLRP3 inflammasome is unique in its ability activate in response to endogenous and exogenous sterile danger signals. Many such sterile signals have been elucidated, and their formation is associated with specific disease states. For example, uric acid crystals found in gout patients are effective triggers of NLRP3 activation. Similarly, cholesterol crystals found in atherosclerotic patients can also promote NLRP3 activation. Recognition of the role of sterile danger signals as NLRP3 activators led to IL-1β and IL-18 being implicated in a diverse range of pathophysiological indications including metabolic, physiologic, inflammatory, hematologic and immunologic disorders.
A link to human disease is best exemplified by discovery that mutations in the NLRP3 gene which lead to gain-of-function confer a range of autoinflammatory conditions collectively known as cryopyrin-associated periodic syndromes (CAPS) including familial cold autoinflammatory syndrome (FCAS), Muckle-Wells syndrome (MWS) and Neonatal onset multisystem inflammatory disease (NOMID) (Hoffman et al., Nat. Genet. 29(3) (2001) 301-305). Likewise, sterile mediator-induced activation of NLRP3 has been implicated in a wide range of disorders including joint degeneration (gout, rheumatoid arthritis, osteoarthritis), cardiometabolic (type 2 diabetes, atherosclerosis, hypertension), Central Nervous System (Parkinson's disease, Alzheimer's disease, multiple sclerosis, amyotrophic lateral sclerosis, or Huntington's disease), gastrointestinal (Crohn's disease, ulcerative colitis, Irritable Bowel Disease (IBD)), lung (chronic obstructive pulmonary disease (COPD), asthma, idiopathic pulmonary fibrosis) and liver (fibrosis, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis (NASH)). It is further believed that NLRP3 activation promotes kidney inflammation and thus contributes to chronic kidney disease (CKD).
Current treatment options for diseases where IL-1 is implicated as a contributor to pathogenesis include the IL-1 receptor antagonist anakinra, an Fc-containing fusion construct of the extracellular domains of the IL-1 receptor and IL-1 receptor accessory protein (rilonacept) and the anti-IL-1β monoclonal antibody canakinumab. For example, canakinumab is licensed for CAPS, Tumor Necrosis Factor Receptor Associated Periodic Syndrome (TRAPS), Hyperimmunoglobulin D Syndrome (HIDS)/Mevalonate Kinase Deficiency (MKD), Familial Mediterranean Fever (FMF) and gout.
Some small molecules have been reported to inhibit function of the NLRP3 inflammasome. Glyburide, for example, is a specific inhibitor of NLRP3 activation, albeit at micromolar concentrations which are unlikely attainable in vivo. Non-specific agents such as parthenolide, Bay 11-7082, and 3,4-methylenedioxy-β-nitrostyrene are reported to impair NLRP3 activation but are expected to possess limited therapeutic utility due to their sharing of a common structural feature consisting of an olefin activated by substitution with an electron withdrawing group; this can lead to undesirable formation of covalent adducts with protein-bearing thiol groups. A number of natural products, for example β-hydroxybutyrate, sulforaphane, quercetin, and salvianolic acid, also are reported to suppress NLRP3 activation. Likewise, numerous effectors/modulators of other molecular targets have been reported to impair NLRP3 activation including agonists of the G-protein coupled receptor TGR5, an inhibitor of sodium-glucose co-transport epigliflozin, the dopamine receptor antagonist A-68930, the serotonin reuptake inhibitor fluoxetine, fenamate non-steroidal anti-inflammatory drugs, and the 0-adrenergic receptor blocker nebivolol. Utility of these molecules as therapeutics for the chronic treatment of NLRP3-dependent inflammatory disorders remains to be established.
The disclosure relates to compounds useful for the modulation of NLRP3-dependent cellular processes. In particular, compounds with improved physicochemical, pharmacological and pharmaceutical properties to existing NLRP3-modulating compounds are desired.
In some aspects, the present disclosure relates to a compound of Formula (I):
or a prodrug, solvate, or pharmaceutically acceptable salt thereof, wherein:
It is understood that, for a compound of Formula (I), X, R1, R2, R3, L, R3a, Ra, and Rb can each be, where applicable, selected from the groups described herein, and any group described herein for any of X, R1, R2, R3, L, R3a, Ra, and Rb can be combined, where applicable, with any group described herein for one or more of the remainder of X, R1, R2, R3, L, R3a, Ra, and Rb.
In some embodiments, X is CH.
In some embodiments, X is N.
In some embodiments, R1 is halo.
In some embodiments, R1 is F, Cl, Br, or I.
In some embodiments, R1 is F. In some embodiments, R1 is Cl. In some embodiments, R1 is Br. In some embodiments, R1 is I.
In some embodiments, R1 is —CN.
In some embodiments, R1 is C1-C6 alkyl.
In some embodiments, R1 is methyl. In some embodiments, R1 is ethyl. In some embodiments, R1 is propyl. In some embodiments, R1 is butyl. In some embodiments, R1 is pentyl.
In some embodiments, R1 is hexyl. In some embodiments, R1 is isopropyl. In some embodiments, R1 is isobutyl. In some embodiments, R1 is isopentyl. In some embodiments, R1 is isohexyl. In some embodiments, R1 is secbutyl. In some embodiments, R1 is secpentyl. In some embodiments, R1 is sechexyl. In some embodiments, R1 is tertbutyl.
In some embodiments, R1 is C1-C6 haloalkyl.
In some embodiments, R1 is halomethyl. In some embodiments, R1 is haloethyl. In some embodiments, R1 is halopropyl. In some embodiments, R1 is halobutyl. In some embodiments, R1 is halopentyl. In some embodiments, R1 is halohexyl.
In some embodiments, R1 is —CF3.
In some embodiments, R1 is Cl, —CN, methyl, or —CF3.
In some embodiments, R2 is H.
In some embodiments, R2 is C1-C6 alkyl.
In some embodiments, R2 is methyl. In some embodiments, R2 is ethyl. In some embodiments, R2 is propyl. In some embodiments, R2 is butyl. In some embodiments, R2 is pentyl.
In some embodiments, R2 is hexyl. In some embodiments, R2 is isopropyl. In some embodiments, R2 is isobutyl. In some embodiments, R2 is isopentyl. In some embodiments, R2 is isohexyl. In some embodiments, R2 is secbutyl. In some embodiments, R2 is secpentyl. In some embodiments, R2 is sechexyl. In some embodiments, R2 is tertbutyl.
In some embodiments, R2 is H or methyl.
In some embodiments, R3 is H, —(C1-C3 alkylene)-N(Ra)(Rb), —(C1-C3 alkylene)-C(O)N(Ra)(Rb), —(C1-C3 alkylene)-N(Ra)C(O)(Rb), —(C1-C3 alkylene)-C(O)(C1-C6 alkyl), C1-C6 alkyl, C1-C6 alkoxyl, or -L-4- to 10-membered heterocyclyl.
In some embodiments, R3 is —(C1-C3 alkylene)-N(Ra)(Rb), —(C1-C3 alkylene)-C(O)N(Ra)(Rb), —(C1-C3 alkylene)-N(Ra)C(O)(Rb), —(C1-C3 alkylene)-C(O)(C1-C6 alkyl), C1-C6 alkyl, C1-C6 alkoxyl, or -L-4- to 10-membered heterocyclyl, wherein the heterocyclyl is optionally substituted with one or more R3a.
In some embodiments, R3 is —(C1-C3 alkylene)-N(Ra)(Rb), —(C1-C3 alkylene)-C(O)N(Ra)(Rb), —(C1-C3 alkylene)-N(Ra)C(O)(Rb), —(C1-C3 alkylene)-C(O)(C1-C6 alkyl), C1-C6 alkyl, C1-C6 alkoxyl, or -L-4- to 10-membered heterocyclyl.
In some embodiments, R3 is H.
In some embodiments, R3 is —(C1-C3 alkylene)-N(Ra)(Rb), —(C1-C3 alkylene)-C(O)N(Ra)(Rb), —(C1-C3 alkylene)-N(Ra)C(O)(Rb), or —(C1-C3 alkylene)-C(O)(C1-C6 alkyl).
In some embodiments, R3 is —(C1-C3 alkylene)-N(Ra)(Rb).
In some embodiments, R3 is —(C1 alkylene)-N(Ra)(Rb). In some embodiments, R3 is —(C2 alkylene)-N(Ra)(Rb). In some embodiments, R3 is —(C3 alkylene)-N(R)(Rb).
In some embodiments, R3 is —(C1-C3 alkylene)-C(O)N(Ra)(Rb).
In some embodiments, R3 is —(C1 alkylene)-C(O)N(Ra)(Rb). In some embodiments, R3 is —(C2 alkylene)-C(O)N(Ra)(Rb). In some embodiments, R3 is —(C3 alkylene)-C(O)N(R)(Rb).
In some embodiments, R3 is —(C1-C3 alkylene)-N(Ra)C(O)(Rb).
In some embodiments, R3 is —(C1 alkylene)-N(Ra)C(O)(Rb). In some embodiments, R3 is —(C2 alkylene)-N(Ra)C(O)(Rb). In some embodiments, R3 is —(C3 alkylene)-N(Ra)C(O)(Rb).
In some embodiments, R3 is —(C1-C3 alkylene)-C(O)(C1-C6 alkyl).
In some embodiments, R3 is —(C1 alkylene)-C(O)(C1-C6 alkyl). In some embodiments, R3 is —(C2 alkylene)-C(O)(C1-C6 alkyl). In some embodiments, R3 is —(C3 alkylene)-C(O)(C1-C6 alkyl).
In some embodiments, R3 is C1-C6 alkyl or C1-C6 alkoxyl.
In some embodiments, R3 is C1-C6 alkyl.
In some embodiments, R3 is methyl. In some embodiments, R3 is ethyl. In some embodiments, R3 is propyl. In some embodiments, R3 is butyl. In some embodiments, R3 is pentyl.
In some embodiments, R3 is hexyl. In some embodiments, R3 is isopropyl. In some embodiments, R3 is isobutyl. In some embodiments, R3 is isopentyl. In some embodiments, R3 is isohexyl. In some embodiments, R3 is secbutyl. In some embodiments, R3 is secpentyl. In some embodiments, R3 is sechexyl. In some embodiments, R3 is tertbutyl.
In some embodiments, R3 is C1-C6 alkoxyl.
In some embodiments, R3 is methoxyl. In some embodiments, R3 is ethoxyl. In some embodiments, R3 is propoxyl. In some embodiments, R3 is butoxyl. In some embodiments, R3 is pentoxyl. In some embodiments, R3 is hexoxyl.
In some embodiments, R3 is L-4- to 10-membered heterocyclyl optionally substituted with one or more R3a.
In some embodiments, R3 is L-4- to 10-membered heterocyclyl substituted with one or more R3a.
In some embodiments, R3 is L-4- to 10-membered heterocyclyl.
In some embodiments, R3 is L-4-membered heterocyclyl optionally substituted with one or more R3a.
In some embodiments, R3 is L-4-membered heterocyclyl substituted with one or more R3a.
In some embodiments, R3 is L-4-membered heterocyclyl.
In some embodiments, R3 is L-5-membered heterocyclyl optionally substituted with one or more R3a.
In some embodiments, R3 is L-5-membered heterocyclyl substituted with one or more R3a.
In some embodiments, R3 is L-5-membered heterocyclyl.
In some embodiments, R3 is L-6-membered heterocyclyl optionally substituted with one or more R3a.
In some embodiments, R3 is L-6-membered heterocyclyl substituted with one or more R3a.
In some embodiments, R3 is L-6-membered heterocyclyl.
In some embodiments, R3 is L-7-membered heterocyclyl optionally substituted with one or more R3a.
In some embodiments, R3 is L-7-membered heterocyclyl substituted with one or more R3a.
In some embodiments, R3 is L-7-membered heterocyclyl.
In some embodiments, R3 is L-8-membered heterocyclyl optionally substituted with one or more R3a.
In some embodiments, R3 is L-8-membered heterocyclyl substituted with one or more R3a.
In some embodiments, R3 is L-8-membered heterocyclyl.
In some embodiments, R3 is L-9-membered heterocyclyl optionally substituted with one or more R3a.
In some embodiments, R3 is L-9-membered heterocyclyl substituted with one or more R3a.
In some embodiments, R3 is L-9-membered heterocyclyl.
In some embodiments, R3 is L-10-membered heterocyclyl optionally substituted with one or more R3a.
In some embodiments, R3 is L-10-membered heterocyclyl substituted with one or more R3a.
In some embodiments, R3 is L-10-membered heterocyclyl.
In some embodiments, L is a covalent bond.
In some embodiments, L is —CH2—, —(CH2)2—, —C(═O)—, or —CH2—C(═O)—.
In some embodiments, L is —CH2—. In some embodiments, L is —(CH2)2—. In some embodiments, L is —C(═O)—. In some embodiments, L is —CH2—C(═O)—.
In some embodiments R3 is H methyl
In some embodiments, R3a is halo, oxo, —CN, or —OH.
In some embodiments, R3a is halo.
In some embodiments, R3a is F, Cl, Br, or I.
In some embodiments, R3a is F. In some embodiments, R3a is Cl. In some embodiments, R3a is Br. In some embodiments, R3a is I.
In some embodiments, R3a is oxo.
In some embodiments, R3a is —CN.
In some embodiments, R3a is —OH.
In some embodiments, R3a is —C(═O)H, —C(O)(C1-C6 alkyl), —C(O)2(C1-C6 alkyl),- or S(O)2(C1-C6 alkyl).
In some embodiments, R3a is —C(═O)H.
In some embodiments, R3a is —C(O)(C1-C6 alkyl).
In some embodiments, R3a is —C(O)(methyl). In some embodiments, R3a is —C(O)(ethyl). In some embodiments, R3a is —C(O)(propyl). In some embodiments, R3a is —C(O)(butyl). In some embodiments, R3a is —C(O)(pentyl). In some embodiments, R3a is —C(O)(hexyl).
In some embodiments, R3a is —C(O)2(C1-C6 alkyl).
In some embodiments, R3a is —C(O)2(methyl). In some embodiments, R3a is —C(O)2(ethyl).
In some embodiments, R3a is —C(O)2(propyl). In some embodiments, R3a is —C(O)2(butyl). In some embodiments, R3a is —C(O)2(pentyl). In some embodiments, R3a is —C(O)2(hexyl).
In some embodiments, R3a is —S(O)2(C1-C6 alkyl).
In some embodiments, R3a is —S(O)2(methyl). In some embodiments, R3a is —S(O)2(ethyl).
In some embodiments, R3a is —S(O)2(propyl). In some embodiments, R3a is —S(O)2(butyl). In some embodiments, R3a is —S(O)2(pentyl). In some embodiments, R3a is —S(O)2(hexyl).
In some embodiments, R3a is C1-C6 alkyl, —C1-C6 alkyl-CN, C1-C6 alkoxyl, C1-C6 haloalkyl, or 4- to 10-membered heterocyclyl.
In some embodiments, R3a is C1-C6 alkyl.
In some embodiments, R3a is methyl. In some embodiments, R3a is ethyl. In some embodiments, R3a is propyl. In some embodiments, R3a is butyl. In some embodiments, R3a is pentyl. In some embodiments, R3a is hexyl. In some embodiments, R3a is isopropyl. In some embodiments, R3a is isobutyl. In some embodiments, R3a is isopentyl. In some embodiments, R3a is isohexyl. In some embodiments, R3a is secbutyl. In some embodiments, R3a is secpentyl. In some embodiments, R3a is sechexyl. In some embodiments, R3a is tertbutyl.
In some embodiments, R3a is —C1-C6 alkyl-CN.
In some embodiments, R3a is -methyl-CN. In some embodiments, R3a is -ethyl-CN. In some embodiments, R3a is -propyl-CN. In some embodiments, R3a is -butyl-CN. In some embodiments, R3a is -pentyl-CN. In some embodiments, R3a is -hexyl-CN.
In some embodiments, R3a is C1-C6 alkoxyl.
In some embodiments, R3a is methoxyl. In some embodiments, R3a is ethoxyl. In some embodiments, R3a is propoxyl. In some embodiments, R3a is butoxyl. In some embodiments, R3a is pentoxyl. In some embodiments, R3a is hexoxyl.
In some embodiments, R3a is C1-C6 haloalkyl.
In some embodiments, R3a is halomethyl. In some embodiments, R3a is haloethyl. In some embodiments, R3a is halopropyl. In some embodiments, R3a is halobutyl. In some embodiments, R3a is halopentyl. In some embodiments, R3a is halohexyl.
In some embodiments, R3a is 4- to 10-membered heterocyclyl.
In some embodiments, R3a is 4-membered heterocyclyl. In some embodiments, R3a is 5-membered heterocyclyl. In some embodiments, R3a is 6-membered heterocyclyl. In some embodiments, R3a is 7-membered heterocyclyl. In some embodiments, R3a is 8-membered heterocyclyl. In some embodiments, R3a is 9-membered heterocyclyl. In some embodiments, R3a is 10-membered heterocyclyl.
In some embodiments, R3a is oxetanyl.
In some embodiments, R3a is oxo, —CN, —OH, fluoro, methyl, —C(═O)H, —CH2—CF3, —CH2—CN, —CH2—CF2H, —CH2—CH2—OCH3, —C(O)(CH3), —C(O)OCH3, —SO2CH3, or oxetanyl.
In some embodiments, each Ra and Rb independently is H, C1-C6 alkyl, or 4- to 10-membered heterocyclyl.
In some embodiments, Ra is H, C1-C6 alkyl, or 4- to 10-membered heterocyclyl, wherein the heterocyclyl is optionally substituted with one or more C1-C6 alkyl.
In some embodiments, Ra is H, C1-C6 alkyl or 4- to 10-membered heterocyclyl.
In some embodiments, Ra is C1-C6 alkyl or 4- to 10-membered heterocyclyl, wherein the heterocyclyl is optionally substituted with one or more C1-C6 alkyl.
In some embodiments, Ra is C1-C6 alkyl or 4- to 10-membered heterocyclyl.
In some embodiments, Ra is H.
In some embodiments, Ra is C1-C6 alkyl.
In some embodiments, Ra is methyl. In some embodiments, Ra is ethyl. In some embodiments, Ra is propyl. In some embodiments, Ra is butyl. In some embodiments, Ra is pentyl.
In some embodiments, Ra is hexyl. In some embodiments, Ra is isopropyl. In some embodiments, Ra is isobutyl. In some embodiments, Ra is isopentyl. In some embodiments, Ra is isohexyl. In some embodiments, Ra is secbutyl. In some embodiments, Ra is secpentyl. In some embodiments, Ra is sechexyl. In some embodiments, Ra is tertbutyl.
In some embodiments, Ra is 4- to 10-membered heterocyclyl optionally substituted with one or more C1-C6 alkyl.
In some embodiments, Ra is 4- to 10-membered heterocyclyl substituted with one or more C1-C6 alkyl.
In some embodiments, Ra is 4- to 10-membered heterocyclyl.
In some embodiments, Ra is 4-membered heterocyclyl optionally substituted with one or more C1-C6 alkyl.
In some embodiments, Ra is 4-membered heterocyclyl substituted with one or more C1-C6 alkyl.
In some embodiments, Ra is 4-membered heterocyclyl.
In some embodiments, Ra is 5-membered heterocyclyl optionally substituted with one or more C1-C6 alkyl.
In some embodiments, Ra is 5-membered heterocyclyl substituted with one or more C1-C6 alkyl.
In some embodiments, Ra is 5-membered heterocyclyl.
In some embodiments, Ra is 6-membered heterocyclyl optionally substituted with one or more C1-C6 alkyl.
In some embodiments, Ra is 6-membered heterocyclyl substituted with one or more C1-C6 alkyl.
In some embodiments, Ra is 6-membered heterocyclyl.
In some embodiments, Ra is 7-membered heterocyclyl optionally substituted with one or more C1-C6 alkyl.
In some embodiments, Ra is 7-membered heterocyclyl substituted with one or more C1-C6 alkyl.
In some embodiments, Ra is 7-membered heterocyclyl.
In some embodiments, Ra is 8-membered heterocyclyl optionally substituted with one or more C1-C6 alkyl.
In some embodiments, Ra is 8-membered heterocyclyl substituted with one or more C1-C6 alkyl.
In some embodiments, Ra is 8-membered heterocyclyl.
In some embodiments, Ra is 9-membered heterocyclyl optionally substituted with one or more C1-C6 alkyl.
In some embodiments, Ra is 9-membered heterocyclyl substituted with one or more C1-C6 alkyl.
In some embodiments, Ra is 9-membered heterocyclyl.
In some embodiments, Ra is 10-membered heterocyclyl optionally substituted with one or more C1-C6 alkyl.
In some embodiments, Ra is 10-membered heterocyclyl substituted with one or more C1-C6 alkyl.
In some embodiments, Ra is 10-membered heterocyclyl.
In some embodiments, Ra is oxetanyl optionally substituted with one or more C1-C6 alkyl.
In some embodiments, Ra is oxetanyl substituted with one or more C1-C6 alkyl.
In some embodiments, Ra is oxetanyl.
In some embodiments, Ra is tetrahydrofuranyl optionally substituted with one or more C1-C6 alkyl.
In some embodiments, Ra is tetrahydrofuranyl substituted with one or more C1-C6 alkyl.
In some embodiments, Ra is tetrahydrofuranyl.
In some embodiments, Ra is pyrrolidine optionally substituted with one or more C1-C6 alkyl.
In some embodiments, Ra is pyrrolidine substituted with one or more C1-C6 alkyl.
In some embodiments, Ra is pyrrolidine.
In some embodiments, Ra is oxetanyl. In some embodiments, Ra is tetrahydrofuranyl. In some embodiments, Ra is 1-methylpyrrolidine.
In some embodiments, Ra is H, methyl, oxetanyl, tetrahydrofuranyl, or 1-methylpyrrolidine.
In some embodiments, Rb is H, C1-C6 alkyl, or 4- to 10-membered heterocyclyl, wherein the heterocyclyl is optionally substituted with one or more C1-C6 alkyl.
In some embodiments, Rb is H, C1-C6 alkyl or 4- to 10-membered heterocyclyl.
In some embodiments, Rb is C1-C6 alkyl or 4- to 10-membered heterocyclyl, wherein the heterocyclyl is optionally substituted with one or more C1-C6 alkyl.
In some embodiments, Rb is C1-C6 alkyl or 4- to 10-membered heterocyclyl.
In some embodiments, Rb is H.
In some embodiments, Rb is C1-C6 alkyl.
In some embodiments, Rb is methyl. In some embodiments, Rb is ethyl. In some embodiments, Rb is propyl. In some embodiments, Rb is butyl. In some embodiments, Rb is pentyl.
In some embodiments, Rb is hexyl. In some embodiments, Rb is isopropyl. In some embodiments, Rb is isobutyl. In some embodiments, Rb is isopentyl. In some embodiments, Rb is isohexyl. In some embodiments, Rb is secbutyl. In some embodiments, Rb is secpentyl. In some embodiments, Rb is sechexyl. In some embodiments, Rb is tertbutyl.
In some embodiments, Rb is 4- to 10-membered heterocyclyl optionally substituted with one or more C1-C6 alkyl.
In some embodiments, Rb is 4- to 10-membered heterocyclyl substituted with one or more C1-C6 alkyl.
In some embodiments, Rb is 4- to 10-membered heterocyclyl.
In some embodiments, Rb is 4-membered heterocyclyl optionally substituted with one or more C1-C6 alkyl.
In some embodiments, Rb is 4-membered heterocyclyl substituted with one or more C1-C6 alkyl.
In some embodiments, Rb is 4-membered heterocyclyl.
In some embodiments, Rb is 5-membered heterocyclyl optionally substituted with one or more C1-C6 alkyl.
In some embodiments, Rb is 5-membered heterocyclyl substituted with one or more C1-C6 alkyl.
In some embodiments, Rb is 5-membered heterocyclyl.
In some embodiments, Rb is 6-membered heterocyclyl optionally substituted with one or more C1-C6 alkyl.
In some embodiments, Rb is 6-membered heterocyclyl substituted with one or more C1-C6 alkyl.
In some embodiments, Rb is 6-membered heterocyclyl.
In some embodiments, Rb is 7-membered heterocyclyl optionally substituted with one or more C1-C6 alkyl.
In some embodiments, Rb is 7-membered heterocyclyl substituted with one or more C1-C6 alkyl.
In some embodiments, Rb is 7-membered heterocyclyl.
In some embodiments, Rb is 8-membered heterocyclyl optionally substituted with one or more C1-C6 alkyl.
In some embodiments, Rb is 8-membered heterocyclyl substituted with one or more C1-C6 alkyl.
In some embodiments, Rb is 8-membered heterocyclyl.
In some embodiments, Rb is 9-membered heterocyclyl optionally substituted with one or more C1-C6 alkyl.
In some embodiments, Rb is 9-membered heterocyclyl substituted with one or more C1-C6 alkyl.
In some embodiments, Rb is 9-membered heterocyclyl.
In some embodiments, Rb is 10-membered heterocyclyl optionally substituted with one or more C1-C6 alkyl.
In some embodiments, Rb is 10-membered heterocyclyl substituted with one or more C1-C6 alkyl.
In some embodiments, Rb is 10-membered heterocyclyl.
In some embodiments, Rb is oxetanyl optionally substituted with one or more C1-C6 alkyl.
In some embodiments, Rb is oxetanyl substituted with one or more C1-C6 alkyl.
In some embodiments, Rb is oxetanyl.
In some embodiments, Rb is tetrahydrofuranyl optionally substituted with one or more C1-C6 alkyl.
In some embodiments, Rb is tetrahydrofuranyl substituted with one or more C1-C6 alkyl.
In some embodiments, Rb is tetrahydrofuranyl.
In some embodiments, Rb is pyrrolidine optionally substituted with one or more C1-C6 alkyl.
In some embodiments, Rb is pyrrolidine substituted with one or more C1-C6 alkyl.
In some embodiments, Rb is pyrrolidine.
In some embodiments, Rb is oxetanyl. In some embodiments, Rb is tetrahydrofuranyl. In some embodiments, Rb is 1-methylpyrrolidine.
In some embodiments, Rb is H, methyl, oxetanyl, tetrahydrofuranyl, or 1-methylpyrrolidine.
In some embodiments, each Ra and Rb independently is H, methyl, oxetanyl, tetrahydrofuranyl, or 1-methylpyrrolidine.
In some embodiments, X is N; R1 is —CN, halo, or C1-C6 alkyl; R2 is C1-C6 alkyl; R3 is —(C1-C3 alkylene)-C(O)N(Ra)(Rb) or -L-4- to 10-membered heterocyclyl optionally substituted with one or more R3a; L is —CH2—; R3a is halo, C1-C6 alkyl, or —C(O)(C1-C6 alkyl); and each Ra and Rb is independently C1-C6 alkyl.
In some embodiments, X is N; R1 is —CN; R2 is C1-C6 alkyl; R3 is -L-4- to 10-membered heterocyclyl optionally substituted with one or more R3a; L is —CH2—; and R3a is C1-C6 alkyl.
In some embodiments, X is N; R1 is —CN; R2 is methyl; R3 is -L-6-membered heterocyclyl optionally substituted with one or more R3a; L is —CH2—; and R3a is methyl.
In some embodiments, X is N; R1 is C1-C6 alkyl; R2 is C1-C6 alkyl; R3 is -L-4- to 10-membered heterocyclyl optionally substituted with one or more R3a; L is —CH2—; and R3a is C1-C6 alkyl.
In some embodiments, X is N; R1 is methyl; R2 is methyl; R3 is -L-6-membered heterocyclyl optionally substituted with one or more R3a; L is —CH2—; and R3a is methyl.
In some embodiments, X is N; R1 is halo; R2 is C1-C6 alkyl; R3 is -L-4- to 10-membered heterocyclyl optionally substituted with one or more R3a; L is —CH2—; and R3a is halo, C1-C6 alkyl, or —C(O)(C1-C6 alkyl).
In some embodiments, X is N; R1 is C1; R2 is methyl; R3 is -L-5-membered heterocyclyl or -L-6-membered heterocyclyl, wherein the heterocyclyl is optionally substituted with one or more R3a; L is —CH2—; and R3a is F, methyl, or —C(O)(methyl).
In some embodiments, X is N; R1 is halo; R2 is C1-C6 alkyl; R3 is —(C1-C3 alkylene)-C(O)N(Ra)(Rb); and each Ra and Rb independently is C1-C6 alkyl.
In some embodiments, X is N; R1 is C1; R2 is methyl; R3 is —(C1 alkylene)-C(O)N(Ra)(Rb); and each Ra and Rb independently is methyl.
In some embodiments, X is CH; R1 is —CN, halo, or C1-C6 haloalkyl; R2 is H or C1-C6 alkyl; R3 is H, C1-C6 alkyl, —(C1-C3 alkylene)-N(Ra)(Rb), —(C1-C3 alkylene)-C(O)N(Ra)(Rb), —(C1-C3 alkylene)-N(Ra)C(O)(Rb), C1-C6 alkoxyl, or -L-4- to 10-membered heterocyclyl optionally substituted with one or more R3a; L is a covalent bond, —CH2—, —(CH2)2—, —CH2—C(═O)—, or —C(═O)—; R3a is —CN, oxo, halo, —OH, —C(═O)H, —C(O)(C1-C6 alkyl), —CO2(C1-C6 alkyl), —S(O)2(C1-C6 alkyl), —C1-C6 alkyl-CN, C1-C6 alkyl, C1-C6 alkoxyl, C1-C6 haloalkyl, or 4- to 10-membered heterocyclyl; and each Ra and Rb independently is H, C1-C6 alkyl, or 4- to 10-membered heterocyclyl optionally substituted with one or more C1-C6 alkyl.
In some embodiments, X is CH; R1 is —CN; R2 is H or C1-C6 alkyl; R3 is -L-4- to 10-membered heterocyclyl optionally substituted with one or more R3a; L is —CH2—; and R3a is C1-C6 alkyl.
In some embodiments, X is CH; R1 is —CN; R2 is H or methyl; R3 is -L-6-membered heterocyclyl optionally substituted with one or more R3a; L is —CH2—; and R3a is methyl.
In some embodiments, X is CH; R1 is C1-C6 haloalkyl; R2 is C1-C6 alkyl; R3 is H, C1-C6 alkyl, or -L-4- to 10-membered heterocyclyl optionally substituted with one or more R3a; L is —CH2—; and R3a is C1-C6 alkyl.
In some embodiments, X is CH; R1 is —CF3; R2 is methyl; R3 is H, methyl, =L=5=membered heterocyclyl, or -L-6-membered heterocyclyl optionally substituted with one or more R3a; L is —CH2—; and R3a is methyl.
In some embodiments, X is CH; R1 is halo; R2 is C1-C6 alkyl; R3 is —(C1-C3 alkylene)-N(Ra)(Rb), —(C1-C3 alkylene)-C(O)N(R)(Rb), —(C1-C3 alkylene)-N(Ra)C(O)(Rb), C1-C6 alkoxyl, or -L-4- to 10-membered heterocyclyl optionally substituted with one or more R3a; L is a covalent bond, —CH2—, —(CH2)2—, —CH2—C(═O)—, or —C(═O)—; R3a is —CN, oxo, halo, —OH, —C(═O)H, —C(O)(C1-C6 alkyl), —CO2(C1-C6 alkyl), —S(O)2(C1-C6 alkyl), —C1-C6 alkyl-CN, C1-C6 alkyl, C1-C6 alkoxyl, C1-C6 haloalkyl, or 4- to 10-membered heterocyclyl; and each Ra and Rb independently is H, C1-C6 alkyl, or 4- to 10-membered heterocyclyl optionally substituted with one or more C1-C6 alkyl.
In some embodiments, X is CH; R1 is C1; R2 is methyl; R3 is —(C1 alkylene)-N(Ra)(Rb), —(C2 alkylene)-N(Ra)(Rb), —(C3 alkylene)-N(Ra)(Rb), —(C1 alkylene)-C(O)N(R)(Rb), —(C2 alkylene)-C(O)N(Ra)(Rb), —(C2 alkylene)-N(Ra)C(O)(Rb), —CH2—CH2—OCH3, —CH2—CH2—CH2—OCH3, -L-4-membered heterocyclyl, -L-5-membered heterocyclyl, -L-6-membered heterocyclyl, or -L-8-membered heterocyclyl, wherein the heterocyclyl is optionally substituted with one or more R3a; L is a covalent bond, —CH2—, —(CH2)2—, —CH2—C(═O)—, or —C(═O)—; R3a is —CN, oxo, F, —OH, —C(═O)H, —C(O)(methyl), —CO2(methyl), —S(O)2(methyl), methyl-CN, methyl, —CH2—CF2H, —CH2—CF3, —CH2—CH2—OCH3, or 4-membered heterocyclyl; and each Ra and Rb independently is H, methyl, 4-membered heterocyclyl, or 5-membered heterocyclyl optionally substituted with one or more methyl.
In some embodiments, the compound of Formula (I) is of Formula (I-a):
or a prodrug, solvate, or pharmaceutically acceptable salt thereof.
In some embodiments, the compound of Formula (I) is of Formula (I-b):
or a prodrug, solvate, or pharmaceutically acceptable salt thereof.
In some embodiments, the compound of Formula (I) is of Formula (I-al):
or a prodrug, solvate, or pharmaceutically acceptable salt thereof.
In some embodiments, the compound of Formula (I) is of Formula (I-al):
or a prodrug, solvate, or pharmaceutically acceptable salt thereof.
In some embodiments, the compound is selected from the compounds described in Table 1 and prodrugs and pharmaceutically acceptable salts thereof.
In some embodiments, the compound is selected from the compounds described in Table 1 and pharmaceutically acceptable salts thereof.
In some embodiments, the compound is selected from the compounds described in Table 1.
In some aspects, the present disclosure provides a compound being an isotopic derivative (e.g., isotopically labeled compound) of any one of the compounds of the Formulae disclosed herein.
In some embodiments, the compound is an isotopic derivative of any one of the compounds described in Table 1 and prodrugs and pharmaceutically acceptable salts thereof.
In some embodiments, the compound is an isotopic derivative of any one of the compounds described in Table 1 and pharmaceutically acceptable salts thereof.
In some embodiments, the compound is an isotopic derivative of any one of the compounds described in Table 1.
It is understood that the isotopic derivative can be prepared using any of a variety of art-recognised techniques. For example, the isotopic derivative can generally be prepared by carrying out the procedures disclosed in the Schemes and/or in the Examples described herein, by substituting an isotopically labeled reagent for a non-isotopically labeled reagent.
In some embodiments, the isotopic derivative is a deuterium labeled compound.
In some embodiments, the isotopic derivative is a deuterium labeled compound of any one of the compounds of the Formulae disclosed herein.
In some embodiments, the compound is a deuterium labeled compound of any one of the compounds described in Table 1 and prodrugs and pharmaceutically acceptable salts thereof.
In some embodiments, the compound is a deuterium labeled compound of any one of the compounds described in Table 1 and pharmaceutically acceptable salts thereof.
In some embodiments, the compound is a deuterium labeled compound of any one of the compounds described in Table 1.
It is understood that the deuterium labeled compound comprises a deuterium atom having an abundance of deuterium that is substantially greater than the natural abundance of deuterium, which is 0.015%.
In some embodiments, the deuterium labeled compound has a deuterium enrichment factor for each deuterium atom of at least 3500 (52.5% deuterium incorporation at each deuterium atom), at least 4000 (60% deuterium incorporation), at least 4500 (67.5% deuterium incorporation), at least 5000 (75% deuterium), at least 5500 (82.5% deuterium incorporation), at least 6000 (90% deuterium incorporation), at least 6333.3 (95% deuterium incorporation), at least 6466.7 (97% deuterium incorporation), at least 6600 (99% deuterium incorporation), or at least 6633.3 (99.5% deuterium incorporation). As used herein, the term “deuterium enrichment factor” means the ratio between the deuterium abundance and the natural abundance of a deuterium.
It is understood that the deuterium labeled compound can be prepared using any of a variety of art-recognised techniques. For example, the deuterium labeled compound can generally be prepared by carrying out the procedures disclosed in the Schemes and/or in the Examples described herein, by substituting a deuterium labeled reagent for a non-deuterium labeled reagent.
A compound of the invention or a pharmaceutically acceptable salt or solvate thereof that contains the aforementioned deuterium atom(s) is within the scope of the invention. Further, substitution with deuterium (i.e., 2H) may afford certain therapeutic advantages resulting from greater metabolic stability, e.g., increased in vivo half-life or reduced dosage requirements.
For the avoidance of doubt it is to be understood that, where in this specification a group is qualified by “described herein”, the said group encompasses the first occurring and broadest definition as well as each and all of the particular definitions for that group.
A suitable pharmaceutically acceptable salt of a compound of the disclosure is, for example, an acid-addition salt of a compound of the disclosure, which is sufficiently basic, for example, an acid-addition salt with, for example, an inorganic or organic acid, for example hydrochloric, hydrobromic, sulphuric, phosphoric, formic, citric methane sulphonate or maleic acid. In addition, a suitable pharmaceutically acceptable salt of a compound of the disclosure which is sufficiently acidic is an alkali metal salt, for example a sodium or potassium salt, an alkaline earth metal salt, for example a calcium or magnesium salt, an ammonium salt or a salt with an organic base which affords a pharmaceutically acceptable cation, for example a salt with methylamine, dimethylamine, diethylamine, trimethylamine, piperidine, morpholine or tris-(2-hydroxyethyl)amine.
It will be understood that the compounds of any one of the Formulae disclosed herein and any pharmaceutically acceptable salts thereof, comprise stereoisomers, mixtures of stereoisomers, polymorphs of all isomeric forms of said compounds.
As used herein, the term “isomerism” means compounds that have identical molecular formulae but differ in the sequence of bonding of their atoms or in the arrangement of their atoms in space. Isomers that differ in the arrangement of their atoms in space are termed “stereoisomers.” Stereoisomers that are not mirror images of one another are termed “diastereoisomers,” and stereoisomers that are non-superimposable mirror images of each other are termed “enantiomers” or sometimes optical isomers. A mixture containing equal amounts of individual enantiomeric forms of opposite chirality is termed a “racemic mixture.”
As used herein, the term “chiral centre” refers to a carbon atom bonded to four nonidentical substituents.
As used herein, the term “chiral isomer” means a compound with at least one chiral centre. Compounds with more than one chiral centre may exist either as an individual diastereomer or as a mixture of diastereomers, termed “diastereomeric mixture.” When one chiral centre is present, a stereoisomer may be characterised by the absolute configuration (R or S) of that chiral centre. Absolute configuration refers to the arrangement in space of the substituents attached to the chiral centre. The substituents attached to the chiral centre under consideration are ranked in accordance with the Sequence Rule of Cahn, Ingold and Prelog. (Cahn et al., Angew. Chem. Inter. Edit. 1966, 5, 385; errata 511; Cahn et al., Angew. Chem. 1966, 78, 413; Cahn and Ingold, J. Chem. Soc. 1951 (London), 612; Cahn et al., Experientia 1956, 12, 81; Cahn, J. Chem. Educ. 1964, 41, 116).
As used herein, the term “geometric isomer” means the diastereomers that owe their existence to hindered rotation about double bonds or a cycloalkyl linker (e.g., 1,3-cyclobutyl). These configurations are differentiated in their names by the prefixes cis and trans, or Z and E, which indicate that the groups are on the same or opposite side of the double bond in the molecule according to the Cahn-Ingold-Prelog rules.
It is to be understood that the compounds of the present disclosure may be depicted as different chiral isomers or geometric isomers. It is also to be understood that when compounds have chiral isomeric or geometric isomeric forms, all isomeric forms are intended to be included in the scope of the present disclosure, and the naming of the compounds does not exclude any isomeric forms, it being understood that not all isomers may have the same level of activity.
It is to be understood that the structures and other compounds discussed in this disclosure include all atropic isomers thereof. It is also to be understood that not all atropic isomers may have the same level of activity.
As used herein, the term “atropic isomers” are a type of stereoisomer in which the atoms of two isomers are arranged differently in space. Atropic isomers owe their existence to a restricted rotation caused by hindrance of rotation of large groups about a central bond. Such atropic isomers typically exist as a mixture, however as a result of recent advances in chromatography techniques, it has been possible to separate mixtures of two atropic isomers in select cases.
As used herein, the term “tautomer” is one of two or more structural isomers that exist in equilibrium and is readily converted from one isomeric form to another. This conversion results in the formal migration of a hydrogen atom accompanied by a switch of adjacent conjugated double bonds. Tautomers exist as a mixture of a tautomeric set in solution. In solutions where tautomerisation is possible, a chemical equilibrium of the tautomers will be reached. The exact ratio of the tautomers depends on several factors, including temperature, solvent and pH. The concept of tautomers that are interconvertible by tautomerisations is called tautomerism. Of the various types of tautomerism that are possible, two are commonly observed. In keto-enol tautomerism a simultaneous shift of electrons and a hydrogen atom occurs. Ring-chain tautomerism arises as a result of the aldehyde group (—CHO) in a sugar chain molecule reacting with one of the hydroxy groups (—OH) in the same molecule to give it a cyclic (ring-shaped) form as exhibited by glucose.
It is to be understood that the compounds of the present disclosure may be depicted as different tautomers. It should also be understood that when compounds have tautomeric forms, all tautomeric forms are intended to be included in the scope of the present disclosure, and the naming of the compounds does not exclude any tautomer form. It will be understood that certain tautomers may have a higher level of activity than others.
Compounds that have the same molecular formula but differ in the nature or sequence of bonding of their atoms or the arrangement of their atoms in space are termed “isomers”. Isomers that differ in the arrangement of their atoms in space are termed “stereoisomers”. Stereoisomers that are not mirror images of one another are termed “diastereomers” and those that are non-superimposable mirror images of each other are termed “enantiomers”. When a compound has an asymmetric centre, for example, it is bonded to four different groups, a pair of enantiomers is possible. An enantiomer can be characterised by the absolute configuration of its asymmetric centre and is described by the R- and S-sequencing rules of Cahn and Prelog, or by the manner in which the molecule rotates the plane of polarised light and designated as dextrorotatory or levorotatory (i.e., as (+) or (−)-isomers respectively). A chiral compound can exist as either individual enantiomer or as a mixture thereof. A mixture containing equal proportions of the enantiomers is called a “racemic mixture”.
The compounds of this disclosure may possess one or more asymmetric centres; such compounds can therefore be produced as individual (R)- or (S)-stereoisomers or as mixtures thereof. Unless indicated otherwise, the description or naming of a particular compound in the specification and claims is intended to include both individual enantiomers and mixtures, racemic or otherwise, thereof. The methods for the determination of stereochemistry and the separation of stereoisomers are well-known in the art (see discussion in Chapter 4 of “Advanced Organic Chemistry”, 4th edition J. March, John Wiley and Sons, New York, 2001), for example by synthesis from optically active starting materials or by resolution of a racemic form. Some of the compounds of the disclosure may have geometric isomeric centres (E- and Z-isomers). It is to be understood that the present disclosure encompasses all optical, diastereoisomers and geometric isomers and mixtures thereof that possess inflammasome inhibitory activity.
The present disclosure also encompasses compounds of the disclosure as defined herein which comprise one or more isotopic substitutions.
It is to be understood that the compounds of any Formula described herein include the compounds themselves, as well as their salts, and their solvates, if applicable. A salt, for example, can be formed between an anion and a positively charged group (e.g., amino) on a substituted compound disclosed herein. Suitable anions include chloride, bromide, iodide, sulphate, bisulphate, sulphamate, nitrate, phosphate, citrate, methanesulphonate, glutamate, glucuronate, glutarate, malate, maleate, succinate, fumarate, tartrate, tosylate, salicylate, lactate, naphthalenesulphonate, and acetate.
As used herein, the term “pharmaceutically acceptable anion” refers to an anion suitable for forming a pharmaceutically acceptable salt. Likewise, a salt can also be formed between a cation and a negatively charged group (e.g., carboxylate) on a substituted compound disclosed herein. Suitable cations include sodium ion, potassium ion, magnesium ion, calcium ion, and an ammonium cation such as tetramethylammonium ion or diethylamine ion. The substituted compounds disclosed herein also include those salts containing quaternary nitrogen atoms.
It is to be understood that the compounds of the present disclosure, for example, the salts of the compounds, can exist in either hydrated or unhydrated (the anhydrous) form or as solvates with other solvent molecules. Nonlimiting examples of hydrates include monohydrates, dihydrates, etc. Nonlimiting examples of solvates include ethanol solvates, acetone solvates, etc.
As used herein, the term “solvate” means solvent addition forms that contain either stoichiometric or non-stoichiometric amounts of solvent. Some compounds have a tendency to trap a fixed molar ratio of solvent molecules in the crystalline solid state, thus forming a solvate. If the solvent is water the solvate formed is a hydrate; and if the solvent is alcohol, the solvate formed is an alcoholate. Hydrates are formed by the combination of one or more molecules of water with one molecule of the substance in which the water retains its molecular state as H2O.
As used herein, the term “analog” refers to a chemical compound that is structurally similar to another but differs slightly in composition (as in the replacement of one atom by an atom of a different element or in the presence of a particular functional group, or the replacement of one functional group by another functional group). Thus, an analog is a compound that is similar or comparable in function and appearance, but not in structure or origin to the reference compound.
As used herein, the term “derivative” refers to compounds that have a common core structure and are substituted with various groups as described herein.
As used herein, the term “bioisostere” refers to a compound resulting from the exchange of an atom or of a group of atoms with another, broadly similar, atom or group of atoms. The objective of a bioisosteric replacement is to create a new compound with similar biological properties to the parent compound. The bioisosteric replacement may be physicochemically or topologically based. Examples of carboxylic acid bioisosteres include, but are not limited to, acyl sulphonamides, tetrazoles, sulphonates and phosphonates. See, e.g., Patani and LaVoie, Chem. Rev. 96, 3147-3176, 1996.
It is also to be understood that certain compounds of any one of the Formulae disclosed herein may exist in solvated as well as unsolvated forms such as, for example, hydrated forms. A suitable pharmaceutically acceptable solvate is, for example, a hydrate such as hemi-hydrate, a mono-hydrate, a di-hydrate or a tri-hydrate. It is to be understood that the disclosure encompasses all such solvated forms that possess inflammasome inhibitory activity.
It is also to be understood that certain compounds of any one of the Formulae disclosed herein may exhibit polymorphism, and that the disclosure encompasses all such forms, or mixtures thereof, which possess inflammasome inhibitory activity. It is generally known that crystalline materials may be analysed using conventional techniques such as X-Ray Powder Diffraction analysis, Differential Scanning Calorimetry, Thermal Gravimetric Analysis, Diffuse Reflectance Infrared Fourier Transform (DRIFT) spectroscopy, Near Infrared (NTR) spectroscopy, solution and/or solid state nuclear magnetic resonance spectroscopy. The water content of such crystalline materials may be determined by Karl Fischer analysis.
Compounds of any one of the Formulae disclosed herein may exist in a number of different tautomeric forms and references to compounds of Formula (I) include all such forms. For the avoidance of doubt, where a compound can exist in one of several tautomeric forms, and only one is specifically described or shown, all others are nevertheless embraced by Formula (I). Examples of tautomeric forms include keto-, enol-, and enolate-forms, as in, for example, the following tautomeric pairs: keto/enol (illustrated below), imine/enamine, amide/imino alcohol, amidine/amidine, nitroso/oxime, thioketone/enethiol, and nitro/aci-nitro.
Compounds of any one of the Formulae disclosed herein containing an amine function may also form N-oxides. A reference herein to a compound of Formula (I) that contains an amine function also includes the N-oxide. Where a compound contains several amine functions, one or more than one nitrogen atom may be oxidised to form an N-oxide. Particular examples of N-oxides are the N-oxides of a tertiary amine or a nitrogen atom of a nitrogen-containing heterocycle. N-oxides can be formed by treatment of the corresponding amine with an oxidising agent such as hydrogen peroxide or a peracid (e.g. a peroxycarboxylic acid), see for example Advanced Organic Chemistry, by Jerry March, 4th Edition, Wiley Interscience, pages. More particularly, N-oxides can be made by the procedure of L. W. Deady (Syn. Comm. 1977, 7, 509-514) in which the amine compound is reacted with meta-chloroperoxybenzoic acid (mCPBA), for example, in an inert solvent such as dichloromethane.
The compounds of any one of the Formulae disclosed herein may be administered in the form of a prodrug which is broken down in the human or animal body to release a compound of the disclosure. A prodrug may be used to alter the physical properties and/or the pharmacokinetic properties of a compound of the disclosure. A prodrug can be formed when the compound of the disclosure contains a suitable group or substituent to which a property-modifying group can be attached. Examples of prodrugs include derivatives containing in vivo cleavable alkyl or acyl substituents in a compound of the any one of the Formulae disclosed herein.
Accordingly, the present disclosure includes those compounds of any one of the Formulae disclosed herein as defined hereinbefore when made available by organic synthesis and when made available within the human or animal body by way of cleavage of a prodrug thereof. Accordingly, the present disclosure includes those compounds of any one of the Formulae disclosed herein that are produced by organic synthetic means and also such compounds that are produced in the human or animal body by way of metabolism of a precursor compound, that is a compound of any one of the Formulae disclosed herein may be a synthetically-produced compound or a metabolically-produced compound.
A suitable pharmaceutically acceptable prodrug of a compound of any one of the Formulae disclosed herein is one that is based on reasonable medical judgment as being suitable for administration to the human or animal body without undesirable pharmacological activities and without undue toxicity. Various forms of prodrug have been described, for example in the following documents: a) Methods in Enzymology, Vol. 42, p. 309-396, edited by K. Widder, et al. (Academic Press, 1985); b) Design of Pro-drugs, edited by H. Bundgaard, (Elsevier, 1985); c) A Textbook of Drug Design and Development, edited by Krogsgaard-Larsen and H. Bundgaard, Chapter 5 “Design and Application of Pro-drugs”, by H. Bundgaard p. 113-191 (1991); d) H. Bundgaard, Advanced Drug Delivery Reviews, 8, 1-38 (1992); e) H. Bundgaard, et al., Journal of Pharmaceutical Sciences, 77, 285 (1988); f) N. Kakeya, et al., Chem. Pharm. Bull., 32, 692 (1984); g) T. Higuchi and V. Stella, “Pro-Drugs as Novel Delivery Systems”, A.C.S. Symposium Series, Volume 14; H. E. Roche (editor), “Bioreversible Carriers in Drug Design”, Pergamon Press, 1987.
A suitable pharmaceutically acceptable prodrug of a compound of any one of the Formulae disclosed herein that possesses a hydroxy group is, for example, an in vivo cleavable ester or ether thereof. An in vivo cleavable ester or ether of a compound of any one of the Formulae disclosed herein containing a hydroxy group is, for example, a pharmaceutically acceptable ester or ether which is cleaved in the human or animal body to produce the parent hydroxy compound. Suitable pharmaceutically acceptable ester forming groups for a hydroxy group include inorganic esters such as phosphate esters (including phosphoramidic cyclic esters). Further suitable pharmaceutically acceptable ester forming groups for a hydroxy group include C1-C10 alkanoyl groups such as acetyl, benzoyl, phenylacetyl and substituted benzoyl and phenylacetyl groups, C1-C10 alkoxycarbonyl groups such as ethoxycarbonyl, N,N-(C1-C6 alkyl)2carbamoyl, 2-dialkylaminoacetyl and 2-carboxyacetyl groups. Examples of ring substituents on the phenylacetyl and benzoyl groups include aminomethyl, N-alkylaminomethyl, N,N-dialkylaminomethyl, morpholinomethyl, piperazin-1-ylmethyl and 4-(C1-C4 alkyl)piperazin-1-ylmethyl. Suitable pharmaceutically acceptable ether forming groups for a hydroxy group include α-acyloxyalkyl groups such as acetoxymethyl and pivaloyloxymethyl groups.
A suitable pharmaceutically acceptable prodrug of a compound of any one of the Formulae disclosed herein that possesses a carboxy group is, for example, an in vivo cleavable amide thereof, for example an amide formed with an amine such as ammonia, a C1-4alkylamine such as methylamine, a (C1-C4 alkyl)2amine such as dimethylamine, N-ethyl-N-methylamine or diethylamine, a C1-C4 alkoxy-C2-C4 alkylamine such as 2-methoxyethylamine, a phenyl-C1-C4 alkylamine such as benzylamine and amino acids such as glycine or an ester thereof.
A suitable pharmaceutically acceptable prodrug of a compound of any one of the Formulae disclosed herein that possesses an amino group is, for example, an in vivo cleavable amide derivative thereof. Suitable pharmaceutically acceptable amides from an amino group include, for example an amide formed with C1-C10 alkanoyl groups such as an acetyl, benzoyl, phenylacetyl and substituted benzoyl and phenylacetyl groups. Examples of ring substituents on the phenylacetyl and benzoyl groups include aminomethyl, N-alkylaminomethyl, N,N-dialkylaminomethyl, morpholinomethyl, piperazin-1-ylmethyl and 4—(C1-C4 alkyl)piperazin-1-ylmethyl.
The in vivo effects of a compound of any one of the Formulae disclosed herein may be exerted in part by one or more metabolites that are formed within the human or animal body after administration of a compound of any one of the Formulae disclosed herein. As stated hereinbefore, the in vivo effects of a compound of any one of the Formulae disclosed herein may also be exerted by way of metabolism of a precursor compound (a prodrug).
Suitably, the present disclosure excludes any individual compounds not possessing the biological activity defined herein.
Selected compounds of Formula (I-a) may be synthesised according to Scheme 1, wherein R1, R2 and R3 are defined as in the claims. Step (a) is a Sonogashira coupling reaction of ethynyl(trimethyl)silane and 4-bromo-6-chloro-pyridazin-3-amine using a palladium catalyst, e.g., Pd(PPh3)2Cl2, a copper reagent, e.g., CuI, and a base, e.g., triethylamine. Step (b) is a cyclisation reaction using e.g., methylamine and potassium tert-butoxide in N,N-dimethylformamide. Step (c) is an alkylation using an appropriate alkylating agent containing group R3 and a base, e.g., K2CO3. Step (d) is a Suzuki-Miyaura coupling reaction using a palladium catalyst, e.g., SPhos Pd G3, a base, e.g., K2CO3, and an appropriate boron-containing reagent such as a pinacol boronate ester or boronic acid.
Selected compounds of Formula (I-b may be synthesised according to Scheme 1, wherein R1, R2 and R3 are defined as in the claims. Step (d) is a Suzuki-Miyaura coupling reaction using a palladium catalyst, e.g., SPhos Pd G3, a base, e.g., K2CO3, and an appropriate boron-containing reagent such as a pinacol boronate ester or boronic acid. Step (e) is a displacement reaction using e.g., sodium benzenesulfinate. Step (f) is a displacement reaction using an appropriate primary amine containing R3 group, and a base, e.g., K2CO3. Step (g) is a displacement reaction using an azide such as sodium azide or trimethylsilylazide. Step (h) is an azide reduction reaction using e.g., zinc in acetic acid. Step (i) is a cyclisation reaction using e.g., triethoxymethane and an acid catalyst, e.g., hydrochloric acid.
In some aspects, the present disclosure provides a method of preparing a compound of the present disclosure.
In some aspects, the present disclosure provides a method of a compound, comprising one or more steps as described herein.
In some aspects, the present disclosure provides a compound obtainable by, or obtained by, or directly obtained by a method for preparing a compound as described herein.
In some aspects, the present disclosure provides an intermediate as described herein, being suitable for use in a method for preparing a compound as described herein.
The compounds of the present disclosure can be prepared by any suitable technique known in the art. Particular processes for the preparation of these compounds are described further in the accompanying examples.
In the description of the synthetic methods described herein and in any referenced synthetic methods that are used to prepare the starting materials, it is to be understood that all proposed reaction conditions, including choice of solvent, reaction atmosphere, reaction temperature, duration of the experiment and workup procedures, can be selected by a person skilled in the art.
It is understood by one skilled in the art of organic synthesis that the functionality present on various portions of the molecule must be compatible with the reagents and reaction conditions utilised.
It will be appreciated that during the synthesis of the compounds of the disclosure in the processes defined herein, or during the synthesis of certain starting materials, it may be desirable to protect certain substituent groups to prevent their undesired reaction. The skilled chemist will appreciate when such protection is required, and how such protecting groups may be put in place, and later removed. For examples of protecting groups see one of the many general texts on the subject, for example, ‘Protective Groups in Organic Synthesis’ by Theodora Green (publisher: John Wiley & Sons). Protecting groups may be removed by any convenient method described in the literature or known to the skilled chemist as appropriate for the removal of the protecting group in question, such methods being chosen so as to effect removal of the protecting group with the minimum disturbance of groups elsewhere in the molecule. Thus, if reactants include, for example, groups such as amino, carboxy or hydroxy it may be desirable to protect the group in some of the reactions mentioned herein.
By way of example, a suitable protecting group for an amino or alkylamino group is, for example, an acyl group, for example an alkanoyl group such as acetyl, an alkoxycarbonyl group, for example a methoxycarbonyl, ethoxycarbonyl or t-butoxycarbonyl group, an arylmethoxycarbonyl group, for example benzyloxycarbonyl, or an aroyl group, for example benzoyl. The deprotection conditions for the above protecting groups necessarily vary with the choice of protecting group. Thus, for example, an acyl group such as an alkanoyl or alkoxycarbonyl group or an aroyl group may be removed by, for example, hydrolysis with a suitable base such as an alkali metal hydroxide, for example lithium or sodium hydroxide. Alternatively an acyl group such as a tert-butoxycarbonyl group may be removed, for example, by treatment with a suitable acid as hydrochloric, sulphuric or phosphoric acid or trifluoroacetic acid and an arylmethoxycarbonyl group such as a benzyloxycarbonyl group may be removed, for example, by hydrogenation over a catalyst such as palladium on carbon, or by treatment with a Lewis acid for example boron tris(trifluoroacetate). A suitable alternative protecting group for a primary amino group is, for example, a phthaloyl group which may be removed by treatment with an alkylamine, for example dimethylaminopropylamine, or with hydrazine.
A suitable protecting group for a hydroxy group is, for example, an acyl group, for example an alkanoyl group such as acetyl, an aroyl group, for example benzoyl, or an arylmethyl group, for example benzyl. The deprotection conditions for the above protecting groups will necessarily vary with the choice of protecting group. Thus, for example, an acyl group such as an alkanoyl or an aroyl group may be removed, for example, by hydrolysis with a suitable base such as an alkali metal hydroxide, for example lithium, sodium hydroxide or ammonia. Alternatively an arylmethyl group such as a benzyl group may be removed, for example, by hydrogenation over a catalyst such as palladium on carbon.
A suitable protecting group for a carboxy group is, for example, an esterifying group, for example a methyl or an ethyl group which may be removed, for example, by hydrolysis with a base such as sodium hydroxide, or for example a tert-butyl group which may be removed, for example, by treatment with an acid, for example an organic acid such as trifluoroacetic acid, or for example a benzyl group which may be removed, for example, by hydrogenation over a catalyst such as palladium on carbon.
Once a compound of Formula (I) has been synthesised by any one of the processes defined herein, the processes may then further comprise the additional steps of: (i) removing any protecting groups present; (ii) converting the compound Formula (I) into another compound of Formula (I); (iii) forming a pharmaceutically acceptable salt, hydrate or solvate thereof; and/or (iv) forming a prodrug thereof.
The resultant compounds of Formula (I) can be isolated and purified using techniques well known in the art.
Conveniently, the reaction of the compounds is carried out in the presence of a suitable solvent, which is preferably inert under the respective reaction conditions. Examples of suitable solvents comprise but are not limited to hydrocarbons, such as hexane, petroleum ether, benzene, toluene or xylene; chlorinated hydrocarbons, such as trichlorethylene, 1,2-dichloroethane, tetrachloromethane, chloroform or dichloromethane; alcohols, such as methanol, ethanol, isopropanol, n-propanol, n-butanol or tert-butanol; ethers, such as diethyl ether, diisopropyl ether, tetrahydrofuran (THF), 2-methyltetrahydrofuran, cyclopentylmethyl ether (CPME), methyl tert-butyl ether (MTBE) or dioxane; glycol ethers, such as ethylene glycol monomethyl or monoethyl ether or ethylene glycol dimethyl ether (diglyme); ketones, such as acetone, methylisobutylketone (MIBK) or butanone; amides, such as acetamide, dimethylacetamide, dimethylformamide (DMF) or N-methylpyrrolidinone (NMP); nitriles, such as acetonitrile; sulphoxides, such as dimethyl sulphoxide (DMSO); nitro compounds, such as nitromethane or nitrobenzene; esters, such as ethyl acetate or methyl acetate, or mixtures of the said solvents or mixtures with water.
The reaction temperature is suitably between about −100° C. and 300° C., depending on the reaction step and the conditions used.
Reaction times are generally in the range between a fraction of a minute and several days, depending on the reactivity of the respective compounds and the respective reaction conditions. Suitable reaction times are readily determinable by methods known in the art, for example reaction monitoring. Based on the reaction temperatures given above, suitable reaction times generally lie in the range between 10 minutes and 48 hours.
Moreover, by utilising the procedures described herein, in conjunction with ordinary skills in the art, additional compounds of the present disclosure can be readily prepared. Those skilled in the art will readily understand that known variations of the conditions and processes of the following preparative procedures can be used to prepare these compounds.
As will be understood by the person skilled in the art of organic synthesis, compounds of the present disclosure are readily accessible by various synthetic routes, some of which are exemplified in the accompanying examples. The skilled person will easily recognise which kind of reagents and reactions conditions are to be used and how they are to be applied and adapted in any particular instance—wherever necessary or useful—in order to obtain the compounds of the present disclosure. Furthermore, some of the compounds of the present disclosure can readily be synthesised by reacting other compounds of the present disclosure under suitable conditions, for instance, by converting one particular functional group being present in a compound of the present disclosure, or a suitable precursor molecule thereof, into another one by applying standard synthetic methods, like reduction, oxidation, addition or substitution reactions; those methods are well known to the skilled person. Likewise, the skilled person will apply—whenever necessary or useful—synthetic protecting (or protective) groups; suitable protecting groups as well as methods for introducing and removing them are well-known to the person skilled in the art of chemical synthesis and are described, in more detail, in, e.g., P. G. M. Wuts, T. W. Greene, “Greene's Protective Groups in Organic Synthesis”, 4th edition (2006) (John Wiley & Sons).
Compounds of Formula (I) can be prepared as described in the Examples.
Compounds designed, selected and/or optimised by methods described above, once produced, can be characterised using a variety of assays known to those skilled in the art to determine whether the compounds have biological activity. For example, the molecules can be characterised by conventional assays, including but not limited to those assays described below, to determine whether they have a predicted activity, binding activity and/or binding specificity.
Furthermore, high-throughput screening can be used to speed up analysis using such assays. As a result, it can be possible to rapidly screen the molecules described herein for activity, using techniques known in the art. General methodologies for performing high-throughput screening are described, for example, in Devlin (1998) High Throughput Screening, Marcel Dekker; and U.S. Pat. No. 5,763,263. High-throughput assays can use one or more different assay techniques including, but not limited to, those described below.
Various in vitro or in vivo biological assays may be suitable for detecting the effect of the compounds of the present disclosure. These in vitro or in vivo biological assays can include, but are not limited to, enzymatic activity assays, electrophoretic mobility shift assays, reporter gene assays, in vitro cell viability assays, and the assays described herein.
In some embodiments, the biological away is a biological away testing inhibitory activity against IL-1β release upon NLRP3 activation in peripheral blood mononuclear cells (PBMC).
In some embodiments, the biological assay is a PBMC IC50 Determination Assay. In some embodiments, the biological assay is a PBMC IC50 Determination Assay described in Example 13.
In some embodiments, the compounds of the present disclosure may be tested for their inhibitory activity against IL-1β release upon NLRP3 activation in blood cells (e.g., peripheral blood mononuclear cells (PBMC)).
In some embodiments, PBMC may be isolated and seeded into the wells of a plate and incubated for a period of time (e.g., for 3 hours with a lipopolysaccharide). Following incubation, the medium may be exchanged and a compound added to the well (e.g., a compound of the present disclosure) and the cells may be incubated. Next, the cells may be stimulated (e.g., with ATP or nigericin) and the cell culture media collected for further analysis.
In some embodiments, the release of IL-1β into the media may be determined by a quantitative detection of IL-1β in the media (e.g., using ELISA).
In some embodiments, PBMC may be isolated (e.g., from buffy coats). Isolated cells may be seeded into wells and incubated (e.g., for 3 hours with lipopolysaccharide). The compounds of the present disclosure may then be added and the cells incubated. Next, the cells may be stimulated and the media from the wells collected for further analysis.
In some embodiments, the release of IL-1β into the media may be determined by quantitative detection (e.g., of IL-1β in media using HTRF®).
In some aspects, the present disclosure provides a pharmaceutical composition comprising a compound of the present disclosure as an active ingredient.
In some embodiments, the present disclosure provides a pharmaceutical composition comprising a compound described herein and one or more pharmaceutically acceptable carriers or excipients. In some embodiments, the present disclosure provides a pharmaceutical composition comprising at least one compound selected from Table 1.
As used herein, the term “composition” is intended to encompass a product comprising the specified ingredients in the specified amounts, as well as any product which results, directly or indirectly, from combination of the specified ingredients in the specified amounts.
The compounds of present disclosure can be formulated for oral administration in forms such as tablets, capsules (each of which includes sustained release or timed-release formulations), pills, powders, granules, elixirs, tinctures, suspensions, syrups and emulsions. The compounds of present disclosure on can also be formulated for intravenous (bolus or in-fusion), intraperitoneal, topical, subcutaneous, intra-muscular or transdermal (e.g., patch) administration, all using forms well known to those of ordinary skill in the pharmaceutical arts.
The formulation of the present disclosure may be in the form of an aqueous solution comprising an aqueous vehicle. The aqueous vehicle component may comprise water and at least one pharmaceutically acceptable excipient. Suitable acceptable excipients include those selected from the group consisting of a solubility enhancing agent, chelating agent, preservative, tonicity agent, viscosity/suspending agent, buffer, and pH modifying agent, and a mixture thereof.
Any suitable solubility enhancing agent can be used. Examples of a solubility enhancing agent include cyclodextrin, such as those selected from the group consisting of hydroxypropyl-β-cyclodextrin, methyl-β-cyclodextrin, randomly methylated-β-cyclodextrin, ethylated-β-cyclodextrin, triacetyl-β-cyclodextrin, peracetylated-β-cyclodextrin, carboxymethyl-β-cyclodextrin, hydroxyethyl-β-cyclodextrin, 2-hydroxy-3-(trimethylammonio)propyl-β-cyclodextrin, glucosyl-β-cyclodextrin, sulphated β-cyclodextrin (S-β-CD), maltosyl-β-cyclodextrin, β-cyclodextrin sulphobutyl ether, branched-β-cyclodextrin, hydroxypropyl-γ-cyclodextrin, randomly methylated-γ-cyclodextrin, and trimethyl-γ-cyclodextrin, and mixtures thereof.
Any suitable chelating agent can be used. Examples of a suitable chelating agent include those selected from the group consisting of ethylenediaminetetraacetic acid and metal salts thereof, disodium edetate, trisodium edetate, and tetrasodium edetate, and mixtures thereof.
Any suitable preservative can be used. Examples of a preservative include those selected from the group consisting of quaternary ammonium salts such as benzalkonium halides (preferably benzalkonium chloride), chlorhexidine gluconate, benzethonium chloride, cetyl pyridinium chloride, benzyl bromide, phenylmercury nitrate, phenylmercury acetate, phenylmercury neodecanoate, merthiolate, methylparaben, propylparaben, sorbic acid, potassium sorbate, sodium benzoate, sodium propionate, ethyl p-hydroxybenzoate, propylaminopropyl biguanide, and butyl-p-hydroxybenzoate, and sorbic acid, and mixtures thereof.
The aqueous vehicle may also include a tonicity agent to adjust the tonicity (osmotic pressure). The tonicity agent can be selected from the group consisting of a glycol (such as propylene glycol, diethylene glycol, triethylene glycol), glycerol, dextrose, glycerin, mannitol, potassium chloride, and sodium chloride, and a mixture thereof.
The aqueous vehicle may also contain a viscosity/suspending agent. Suitable viscosity/suspending agents include those selected from the group consisting of cellulose derivatives, such as methyl cellulose, ethyl cellulose, hydroxyethylcellulose, polyethylene glycols (such as polyethylene glycol 300, polyethylene glycol 400), carboxymethyl cellulose, hydroxypropylmethyl cellulose, and cross-linked acrylic acid polymers (carbomers), such as polymers of acrylic acid cross-linked with polyalkenyl ethers or divinyl glycol (Carbopols—such as Carbopol 934, Carbopol 934P, Carbopol 971, Carbopol 974 and Carbopol 974P), and a mixture thereof.
In order to adjust the formulation to an acceptable pH (typically a pH range of about 5.0 to about 9.0, more preferably about 5.5 to about 8.5, particularly about 6.0 to about 8.5, about 7.0 to about 8.5, about 7.2 to about 7.7, about 7.1 to about 7.9, or about 7.5 to about 8.0), the formulation may contain a pH modifying agent. The pH modifying agent is typically a mineral acid or metal hydroxide base, selected from the group of potassium hydroxide, sodium hydroxide, and hydrochloric acid, and mixtures thereof, and preferably sodium hydroxide and/or hydrochloric acid. These acidic and/or basic pH modifying agents are added to adjust the formulation to the target acceptable pH range. Hence it may not be necessary to use both acid and base—depending on the formulation, the addition of one of the acid or base may be sufficient to bring the mixture to the desired pH range.
The aqueous vehicle may also contain a buffering agent to stabilise the pH. When used, the buffer is selected from the group consisting of a phosphate buffer (such as sodium dihydrogen phosphate and disodium hydrogen phosphate), a borate buffer (such as boric acid, or salts thereof including disodium tetraborate), a citrate buffer (such as citric acid, or salts thereof including sodium citrate), and ε-aminocaproic acid, and mixtures thereof.
The formulation may further comprise a wetting agent. Suitable classes of wetting agents include those selected from the group consisting of polyoxypropylene-polyoxyethylene block copolymers (poloxamers), polyethoxylated ethers of castor oils, polyoxyethylenated sorbitan esters (polysorbates), polymers of oxyethylated octyl phenol (Tyloxapol), polyoxyl 40 stearate, fatty acid glycol esters, fatty acid glyceryl esters, sucrose fatty esters, and polyoxyethylene fatty esters, and mixtures thereof.
Oral compositions generally include an inert diluent or an edible pharmaceutically acceptable carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavouring agent such as peppermint, methyl salicylate, or orange flavoring.
According to a further aspect of the disclosure there is provided a pharmaceutical composition which comprises a compound of the disclosure as defined hereinbefore, or a pharmaceutically acceptable salt, hydrate or solvate thereof, in association with a pharmaceutically acceptable diluent or carrier.
The compositions of the disclosure may be in a form suitable for oral use (for example as tablets, lozenges, hard or soft capsules, aqueous or oily suspensions, emulsions, dispersible powders or granules, syrups or elixirs), for topical use (for example as creams, ointments, gels, or aqueous or oily solutions or suspensions), for administration by inhalation (for example as a finely divided powder or a liquid aerosol), for administration by insufflation (for example as a finely divided powder) or for parenteral administration (for example as a sterile aqueous or oily solution for intravenous, subcutaneous, intramuscular, intraperitoneal or intramuscular dosing or as a suppository for rectal dosing).
The compositions of the disclosure may be obtained by conventional procedures using conventional pharmaceutical excipients, well known in the art. Thus, compositions intended for oral use may contain, for example, one or more colouring, sweetening, flavouring and/or preservative agents.
An effective amount of a compound of the present disclosure for use in therapy is an amount sufficient to treat or prevent an inflammasome related condition referred to herein, slow its progression and/or reduce the symptoms associated with the condition.
The size of the dose for therapeutic or prophylactic purposes of a compound of Formula (I) will naturally vary according to the nature and severity of the conditions, the age and sex of the animal or patient and the route of administration, according to well-known principles of medicine.
In some aspects, the present disclosure provides a method of inhibiting inflammasome (e.g., the NLRP3 inflammasome) activity (e.g., in vitro or in vivo), comprising contacting a cell with an effective amount of a compound of the present disclosure or a pharmaceutically acceptable salt thereof.
In some aspects, the present disclosure provides a method of inhibiting inflammasome (e.g., the NLRP3 inflammasome) activity (e.g., in vitro or in vivo), comprising contacting a cell with a compound of the present disclosure or a pharmaceutically acceptable salt thereof.
In some aspects, the present disclosure provides a method of treating or preventing a disease or disorder disclosed herein in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound of the present disclosure or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of the present disclosure.
In some aspects, the present disclosure provides a method of treating or preventing a disease or disorder disclosed herein in a subject in need thereof, comprising administering to the subject a compound of the present disclosure or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of the present disclosure.
In some embodiments, the disease or disorder is associated with an implicated inflammasome activity. In some embodiments, the disease or disorder is a disease or disorder in which inflammasome activity is implicated.
In some embodiments, the disease or disorder is an inflammatory disorder, autoinflammatory disorder, an autoimmune disorder, a neurodegenerative disease, or cancer.
In some embodiments, the disease or disorder is an inflammatory disorder, autoinflammatory disorder and/or an autoimmune disorder.
In some embodiments, the disease or disorder is cytokine release syndrome (CRS).
In some embodiments, the disease or disorder is selected from cryopyrin-associated autoinflammatory syndrome (CAPS; e.g., familial cold autoinflammatory syndrome (FCAS), Muckle-Wells syndrome (MWS), chronic infantile neurological cutaneous and articular (CINCA) syndrome/neonatal-onset multisystem inflammatory disease (NOMID)), familial Mediterranean fever (FMF), nonalcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), gout, rheumatoid arthritis, osteoarthritis, Irritable Bowel Disease (IBD), Crohn's disease, chronic obstructive pulmonary disease (COPD), chronic kidney disease (CKD), fibrosis, obesity, type 2 diabetes, multiple sclerosis, dermatological disease (e.g. acne) and neuroinflammation occurring in protein misfolding diseases (e.g., Prion diseases).
In some embodiments, the disease or disorder is a neurodegenerative disease.
In some embodiments, the disease or disorder is Parkinson's disease, Alzheimer's disease, multiple sclerosis, amyotrophic lateral sclerosis, or Huntington's disease.
In some embodiments, the disease or disorder is a dermatological disease.
In some embodiments, the dermatological disease is acne.
In some embodiments, the disease or disorder is cancer.
In some embodiments, the cancer is metastasising cancer, gastrointestinal cancer, skin cancer, non-small-cell lung carcinoma, brain cancer (e.g. glioblastoma) or colorectal adenocarcinoma.
In some aspects, the present disclosure provides a method of treating or preventing an autoinflammatory disorder, an autoimmune disorder, a neurodegenerative disease or cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound of the present disclosure or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of the present disclosure.
In some aspects, the present disclosure provides a method of treating or preventing an autoinflammatory disorder, an autoimmune disorder, a neurodegenerative disease or cancer in a subject in need thereof, comprising administering to the subject a compound of the present disclosure or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of the present disclosure.
In some aspects, the present disclosure provides a method of treating an autoinflammatory disorder, an autoimmune disorder, a neurodegenerative disease or cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound of the present disclosure or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of the present disclosure.
In some aspects, the present disclosure provides a method of treating an autoinflammatory disorder, an autoimmune disorder, a neurodegenerative disease or cancer in a subject in need thereof, comprising administering to the subject a compound of the present disclosure or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of the present disclosure.
In some aspects, the present disclosure provides a method of treating or preventing an inflammatory disorder, autoinflammatory disorder and/or an autoimmune disorder selected from cryopyrin-associated auto-inflammatory syndrome (CAPS), familial cold autoinflammatory syndrome (FCAS), Muckle-Wells syndrome (MWS), chronic infantile neurological cutaneous and articular (CINCA) syndrome/neonatal-onset multisystem inflammatory disease (NOMID), familial Mediterranean fever (FMF), nonalcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), gout, calcium pyrophosphate disposition disease (CPPD), rheumatoid arthritis, osteoarthritis, Irritable Bowel Disease (IBD), Crohn's disease, ulcerative colitis, chronic obstructive pulmonary disease (COPD), atherosclerosis, hypertension, asthma, cardiovascular disease, chronic kidney disease (CKD), fibrosis, cystic fibrosis (CF), obesity, type 2 diabetes, multiple sclerosis, dermatological disease (e.g., acne), clonal hematopoiesis of indeterminate potential (CHIP) and neuroinflammation occurring in protein misfolding diseases (e.g., Prion diseases) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound of the present disclosure or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of the present disclosure.
In some aspects, the present disclosure provides a method of treating or preventing an inflammatory disorder, autoinflammatory disorder and/or an autoimmune disorder selected from cryopyrin-associated auto-inflammatory syndrome (CAPS), familial cold autoinflammatory syndrome (FCAS), Muckle-Wells syndrome (MWS), chronic infantile neurological cutaneous and articular (CINCA) syndrome/neonatal-onset multisystem inflammatory disease (NOMID), familial Mediterranean fever (FMF), nonalcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), gout, calcium pyrophosphate disposition disease (CPPD), rheumatoid arthritis, osteoarthritis, Irritable Bowel Disease (IBD), Crohn's disease, ulcerative colitis, chronic obstructive pulmonary disease (COPD), atherosclerosis, hypertension, asthma, cardiovascular disease, chronic kidney disease (CKD), fibrosis, cystic fibrosis (CF), obesity, type 2 diabetes, multiple sclerosis, dermatological disease (e.g., acne), clonal hematopoiesis of indeterminate potential (CHIP) and neuroinflammation occurring in protein misfolding diseases (e.g., Prion diseases) in a subject in need thereof, comprising administering to the subject a compound of the present disclosure or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of the present disclosure.
In some aspects, the present disclosure provides a method of treating an inflammatory disorder, autoinflammatory disorder and/or an autoimmune disorder selected from cryopyrin-associated auto-inflammatory syndrome (CAPS), familial cold autoinflammatory syndrome (FCAS), Muckle-Wells syndrome (MWS), chronic infantile neurological cutaneous and articular (CINCA) syndrome/neonatal-onset multisystem inflammatory disease (NOMID), familial Mediterranean fever (FMF), nonalcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), gout, calcium pyrophosphate disposition disease (CPPD), rheumatoid arthritis, osteoarthritis, Irritable Bowel Disease (IBD), Crohn's disease, ulcerative colitis, chronic obstructive pulmonary disease (COPD), atherosclerosis, hypertension, asthma, cardiovascular disease, chronic kidney disease (CKD), fibrosis, cystic fibrosis (CF), obesity, type 2 diabetes, multiple sclerosis, dermatological disease (e.g., acne), clonal hematopoiesis of indeterminate potential (CHIP) and neuroinflammation occurring in protein misfolding diseases (e.g., Prion diseases) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound of the present disclosure or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of the present disclosure.
In some aspects, the present disclosure provides a method of treating an inflammatory disorder, autoinflammatory disorder and/or an autoimmune disorder selected from cryopyrin-associated auto-inflammatory syndrome (CAPS), familial cold autoinflammatory syndrome (FCAS), Muckle-Wells syndrome (MWS), chronic infantile neurological cutaneous and articular (CINCA) syndrome/neonatal-onset multisystem inflammatory disease (NOMID), familial Mediterranean fever (FMF), nonalcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), gout, calcium pyrophosphate disposition disease (CPPD), rheumatoid arthritis, osteoarthritis, Irritable Bowel Disease (IBD), Crohn's disease, ulcerative colitis, chronic obstructive pulmonary disease (COPD), atherosclerosis, hypertension, asthma, cardiovascular disease, chronic kidney disease (CKD), fibrosis, cystic fibrosis (CF), obesity, type 2 diabetes, multiple sclerosis, dermatological disease (e.g., acne), clonal hematopoiesis of indeterminate potential (CHIP) and neuroinflammation occurring in protein misfolding diseases (e.g., Prion diseases) in a subject in need thereof, comprising administering to the subject a compound of the present disclosure or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of the present disclosure.
In some aspects, the present disclosure provides a method of treating or preventing cytokine release syndrome (CRS) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound of the present disclosure or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of the present disclosure. In some embodiments, the CRS is associated with COVID-19. In some embodiments, the CRS is associated with adoptive cell therapy.
In some aspects, the present disclosure provides a method of treating or preventing cytokine release syndrome (CRS) in a subject in need thereof, comprising administering to the subject a compound of the present disclosure or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of the present disclosure. In some embodiments, the CRS is associated with COVID-19. In some embodiments, the CRS is associated with adoptive cell therapy.
In some aspects, the present disclosure provides a method of treating cytokine release syndrome (CRS) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound of the present disclosure or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of the present disclosure. In some embodiments, the CRS is associated with COVID-19. In some embodiments, the CRS is associated with adoptive cell therapy.
In some aspects, the present disclosure provides a method of treating cytokine release syndrome (CRS) in a subject in need thereof, comprising administering to the subject a compound of the present disclosure or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of the present disclosure. In some embodiments, the CRS is associated with COVID-19. In some embodiments, the CRS is associated with adoptive cell therapy.
In some aspects, the present disclosure provides a method of treating or preventing a neuroinflammatory disorder or neurodegenerative disease (e.g., Parkinson's disease, Alzheimer's disease, multiple sclerosis, amyotrophic lateral sclerosis, neuroinflammation occurring in protein misfolding diseases, Huntington's disease, traumatic brain injury or tauopathy) in a subject in need thereof, said method comprising administering to the subject a therapeutically effective amount of a compound of the present disclosure or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of the present disclosure.
In some aspects, the present disclosure provides a method of treating or preventing a neuroinflammatory disorder or neurodegenerative disease (e.g., Parkinson's disease, Alzheimer's disease, multiple sclerosis, amyotrophic lateral sclerosis, neuroinflammation occurring in protein misfolding diseases, Huntington's disease, traumatic brain injury or tauopathy) in a subject in need thereof, said method comprising administering to the subject a compound of the present disclosure or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of the present disclosure.
In some aspects, the present disclosure provides a method of treating a neuroinflammatory disorder or neurodegenerative disease (e.g., Parkinson's disease, Alzheimer's disease, multiple sclerosis, amyotrophic lateral sclerosis, neuroinflammation occurring in protein misfolding diseases, Huntington's disease, traumatic brain injury or tauopathy) in a subject in need thereof, said method comprising administering to the subject a therapeutically effective amount of a compound of the present disclosure or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of the present disclosure.
In some aspects, the present disclosure provides a method of treating a neuroinflammatory disorder or neurodegenerative disease (e.g., Parkinson's disease, Alzheimer's disease, multiple sclerosis, amyotrophic lateral sclerosis, neuroinflammation occurring in protein misfolding diseases, Huntington's disease, traumatic brain injury or tauopathy) in a subject in need thereof, said method comprising administering to the subject a compound of the present disclosure or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of the present disclosure.
In some aspects, the present disclosure provides a method of treating or preventing cancer in a subject in need thereof, said method comprising administering to the subject a therapeutically effective amount of a compound of the present disclosure or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of the present disclosure.
In some aspects, the present disclosure provides a method of treating or preventing cancer in a subject in need thereof, said method comprising administering to the subject a compound of the present disclosure or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of the present disclosure.
In some aspects, the present disclosure provides a method of treating cancer in a subject in need thereof, said method comprising administering to the subject a therapeutically effective amount of a compound of the present disclosure or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of the present disclosure.
In some aspects, the present disclosure provides a method of treating cancer in a subject in need thereof, said method comprising administering to the subject a compound of the present disclosure or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of the present disclosure.
In some aspects, the present disclosure provides a compound of the present disclosure or a pharmaceutically acceptable salt thereof for use in inhibiting inflammasome (e.g., the NLRP3 inflammasome) activity (e.g., in vitro or in vivo).
In some aspects, the present disclosure provides a compound of the present disclosure or a pharmaceutically acceptable salt thereof for use in treating or preventing a disease or disorder disclosed herein.
In some aspects, the present disclosure provides a compound of the present disclosure or a pharmaceutically acceptable salt thereof for use in treating a disease or disorder disclosed herein.
In some aspects, the present disclosure provides a compound of the present disclosure or a pharmaceutically acceptable salt thereof for use in treating or preventing an inflammatory disorder, an autoinflammatory disorder, an autoimmune disorder, a neurodegenerative disease or cancer in a subject in need thereof.
In some aspects, the present disclosure provides a compound of the present disclosure or a pharmaceutically acceptable salt thereof for use in treating an inflammatory disorder, an autoinflammatory disorder, an autoimmune disorder, a neurodegenerative disease or cancer in a subject in need thereof.
In some aspects, the present disclosure provides a compound of the present disclosure or a pharmaceutically acceptable salt thereof for use in treating or preventing an inflammatory disorder, an autoinflammatory disorder and/or an autoimmune disorder selected from cryopyrin-associated autoinflammatory syndrome (CAPS; e.g., familial cold autoinflammatory syndrome (FCAS), Muckle-Wells syndrome (MWS), chronic infantile neurological cutaneous and articular (CINCA) syndrome/neonatal-onset multisystem inflammatory disease (NOMID)), familial Mediterranean fever (FMF), nonalcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), gout, rheumatoid arthritis, osteoarthritis, Irritable Bowel Disease (IBD), Crohn's disease, chronic obstructive pulmonary disease (COPD), chronic kidney disease (CKD), fibrosis, obesity, type 2 diabetes, multiple sclerosis and neuroinflammation occurring in protein misfolding diseases (e.g., Prion diseases) in a subject in need thereof.
In some aspects, the present disclosure provides a compound of the present disclosure or a pharmaceutically acceptable salt thereof for use in treating an inflammatory disorder, an autoinflammatory disorder and/or an autoimmune disorder selected from cryopyrin-associated autoinflammatory syndrome (CAPS; e.g., familial cold autoinflammatory syndrome (FCAS), Muckle-Wells syndrome (MWS), chronic infantile neurological cutaneous and articular (CINCA) syndrome/neonatal-onset multisystem inflammatory disease (NOMID)), familial Mediterranean fever (FMF), nonalcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), gout, rheumatoid arthritis, osteoarthritis, Irritable Bowel Disease (IBD), Crohn's disease, chronic obstructive pulmonary disease (COPD), chronic kidney disease (CKD), fibrosis, obesity, type 2 diabetes, multiple sclerosis and neuroinflammation occurring in protein misfolding diseases (e.g., Prion diseases) in a subject in need thereof.
In some aspects, the present disclosure provides a compound of the present disclosure or a pharmaceutically acceptable salt thereof for use in treating or preventing CRS in a subject in need thereof.
In some aspects, the present disclosure provides a compound of the present disclosure or a pharmaceutically acceptable salt thereof for use in treating CRS in a subject in need thereof.
In some aspects, the present disclosure provides a compound of the present disclosure or a pharmaceutically acceptable salt thereof for use in treating or preventing a neuroinflammatory disorder or neurodegenerative disease (e.g., Parkinson's disease, Alzheimer's disease, multiple sclerosis, amyotrophic lateral sclerosis, neuroinflammation occurring in protein misfolding diseases, Huntington's disease, traumatic brain injury or tauopathy) in a subject in need thereof.
In some aspects, the present disclosure provides a compound of the present disclosure or a pharmaceutically acceptable salt thereof for use in treating a neuroinflammatory disorder or neurodegenerative disease (e.g., Parkinson's disease, Alzheimer's disease, multiple sclerosis, amyotrophic lateral sclerosis, neuroinflammation occurring in protein misfolding diseases, Huntington's disease, traumatic brain injury or tauopathy) in a subject in need thereof.
In some aspects, the present disclosure provides a compound of the present disclosure or a pharmaceutically acceptable salt thereof for use in treating or preventing cancer in a subject in need thereof.
In some aspects, the present disclosure provides a compound of the present disclosure or a pharmaceutically acceptable salt thereof for use in treating cancer in a subject in need thereof.
In some aspects, the present disclosure provides use of a compound of the present disclosure or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for inhibiting inflammasome (e.g., the NLRP3 inflammasome) activity (e.g., in vitro or in vivo).
In some aspects, the present disclosure provides use of a compound of the present disclosure or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for treating or preventing a disease or disorder disclosed herein.
In some aspects, the present disclosure provides use of a compound of the present disclosure or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for treating a disease or disorder disclosed herein.
In some aspects, the present disclosure provides use of a compound of the present disclosure or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for treating or preventing an inflammatory disorder, an autoinflammatory disorder, an autoimmune disorder, a neurodegenerative disease or cancer in a subject in need thereof.
In some aspects, the present disclosure provides use of a compound of the present disclosure or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for treating an inflammatory disorder, an autoinflammatory disorder, an autoimmune disorder, a neurodegenerative disease or cancer in a subject in need thereof.
In some aspects, the present disclosure provides use of a compound of the present disclosure or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for treating or preventing an inflammatory disorder, an autoinflammatory disorder and/or an autoimmune disorder selected from cryopyrin-associated auto-inflammatory syndrome (CAPS), familial cold autoinflammatory syndrome (FCAS), Muckle-Wells syndrome (MWS), chronic infantile neurological cutaneous and articular (CINCA) syndrome/neonatal-onset multisystem inflammatory disease (NOMID), familial Mediterranean fever (FMF), nonalcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), gout, calcium pyrophosphate disposition disease (CPPD), rheumatoid arthritis, osteoarthritis, Irritable Bowel Disease (IBD), Crohn's disease, ulcerative colitis, chronic obstructive pulmonary disease (COPD), atherosclerosis, hypertension, asthma, cardiovascular disease, chronic kidney disease (CKD), fibrosis, cystic fibrosis (CF), obesity, type 2 diabetes, multiple sclerosis, dermatological disease (e.g., acne), clonal hematopoiesis of indeterminate potential (CHIP) and neuroinflammation occurring in protein misfolding diseases (e.g., Prion diseases) in a subject in need thereof.
In some aspects, the present disclosure provides use of a compound of the present disclosure or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for treating an inflammatory disorder, an autoinflammatory disorder and/or an autoimmune disorder selected from cryopyrin-associated auto-inflammatory syndrome (CAPS), familial cold autoinflammatory syndrome (FCAS), Muckle-Wells syndrome (MWS), chronic infantile neurological cutaneous and articular (CINCA) syndrome/neonatal-onset multisystem inflammatory disease (NOMID), familial Mediterranean fever (FMF), nonalcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), gout, calcium pyrophosphate disposition disease (CPPD), rheumatoid arthritis, osteoarthritis, Irritable Bowel Disease (IBD), Crohn's disease, ulcerative colitis, chronic obstructive pulmonary disease (COPD), atherosclerosis, hypertension, asthma, cardiovascular disease, chronic kidney disease (CKD), fibrosis, cystic fibrosis (CF), obesity, type 2 diabetes, multiple sclerosis, dermatological disease (e.g., acne), clonal hematopoiesis of indeterminate potential (CHIP) and neuroinflammation occurring in protein misfolding diseases (e.g., Prion diseases) in a subject in need thereof.
In some aspects, the present disclosure provides use of a compound of the present disclosure or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for treating or preventing CRS in a subject in need thereof.
In some aspects, the present disclosure provides use of a compound of the present disclosure or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for treating CRS in a subject in need thereof.
In some aspects, the present disclosure provides use of a compound of the present disclosure or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for treating or preventing a neuroinflammatory disorder or neurodegenerative disease (e.g., Parkinson's disease, Alzheimer's disease, multiple sclerosis, amyotrophic lateral sclerosis, neuroinflammation occurring in protein misfolding diseases, Huntington's disease, traumatic brain injury or tauopathy) in a subject in need thereof.
In some aspects, the present disclosure provides use of a compound of the present disclosure or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for treating a neuroinflammatory disorder or neurodegenerative disease (e.g., Parkinson's disease, Alzheimer's disease, multiple sclerosis, amyotrophic lateral sclerosis, neuroinflammation occurring in protein misfolding diseases, Huntington's disease, traumatic brain injury or tauopathy) in a subject in need thereof.
In some aspects, the present disclosure provides use of a compound of the present disclosure or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for treating or preventing cancer in a subject in need thereof.
In some aspects, the present disclosure provides use of a compound of the present disclosure or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for treating cancer in a subject in need thereof.
In some embodiments, the cancer is metastasising cancer, brain cancer, gastrointestinal cancer, skin cancer, non-small-cell lung carcinoma, head and neck squamous cell carcinoma, myelodysplastic syndromes, chronic myelomonocytic leukemia or colorectal adenocarcinoma.
The present disclosure provides compounds that function as inhibitors of inflammasome activity. The present disclosure therefore provides a method of inhibiting inflammasome activity in vitro or in vivo, said method comprising contacting a cell with an effective amount of a compound, or a pharmaceutically acceptable salt thereof, as defined herein.
Effectiveness of compounds of the disclosure can be determined by industry-accepted assays/disease models according to standard practices of elucidating the same as described in the art and are found in the current general knowledge.
The present disclosure also provides a method of treating a disease or disorder in which inflammasome activity is implicated in a patient in need of such treatment, said method comprising administering to said patient a therapeutically effective amount of a compound, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as defined herein.
On a general level, the compounds of the present disclosure, which inhibit the maturation of cytokines of the IL-1 family, are effective in all therapeutic indications that are mediated or associated with elevated levels of active forms of cytokines belonging to IL-1 family of cytokines (Sims J. et al. Nature Reviews Immunology 10, 89-102 (February 2010).
Exemplary diseases and the corresponding references will be given in the following: inflammatory, autoinflammatory and autoimmune diseases like CAPS (Dinarello, C. A. Immunity. 2004 Mar; 20(3):243-4; Hoffman, H. M. et al. Reumatologia 2005; 21(3)), gout, rheumatoid arthritis (Gabay, C. et al. Arthritis Research & Therapy 2009, 11:230; Schett, G. et al. Nat Rev Rheumatol. 2016 January; 12(1):14-24.), Crohn's disease (Jung Mogg Kim Korean J. Gastroenterol. Vol. 58 No. 6, 300-310), COPD (Mortaz, E. et al. Tanaffos. 2011; 10(2): 9-14.), fibrosis (Gasse, P. et al. Am. J Respir. Crit. Care Med. 2009 May 15; 179(10):903-13), obesity, type 2 diabetes ((Dinarello, C. A. et al. Curr. Opin. Endocrinol. Diabetes Obes. 2010 August; 17(4):314-21)) multiple sclerosis (see EAE-model in Coll, R. C. et al. Nat. Med. 2015 March; 21(3):248-55) and many others (Martinon, F. et al. Immunol. 2009. 27:229-65) like Parkinson's disease or Alzheimer's disease (Michael, T. et al. Nature 493, 674-678 (31 Jan. 2013); Halle, A. et al., Nat. Immunol. 2008 Aug; 9(8):857-65; Saresella, M. et al. Mol. Neurodegener. 2016 Mar. 3; 11:23) and some oncological disorders.
Suitably, the compounds according to the present disclosure can be used for the treatment of a disease selected from the group consisting of cytokine release syndrome (CRS), an inflammatory disease, an autoinflammatory disease, an autoimmune disease, a neurodegenerative disease and cancer. Said inflammatory, autoinflammatory and autoimmune disease is suitably selected from the group consisting of a cryopyrin-associated autoinflammatory syndrome (CAPS, such as for example familial cold autoinflammatory syndrome (FCAS), Muckle-Wells syndrome (MWS), chronic infantile neurological cutaneous and articular (CINCA) syndrome/neonatal-onset multisystem inflammatory disease (NOMID)), familial Mediterranean fever (FMF), nonalcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), chronic kidney disease (CKD), gout, rheumatoid arthritis, osteoarthritis, Irritable Bowel Disease (IBD), Crohn's disease, COPD, fibrosis, obesity, type 2 diabetes, multiple sclerosis, dermatological diseases (e.g., acne) and neuroinflammation occurring in protein misfolding diseases, such as Prion diseases. Said neurodegenerative disease includes, but is not limited, to Parkinson's disease and Alzheimer's disease.
Accordingly, the compounds of the present disclosure can be used for the treatment of a disease selected from the group consisting of cryopyrin-associated autoinflammatory syndrome (CAPS, such as for example familial cold autoinflammatory syndrome (FCAS), Muckle-Wells syndrome (MWS), chronic infantile neurological cutaneous and articular (CINCA) syndrome/neonatal-onset multisystem inflammatory disease (NOMID)), familial Mediterranean fever (FMF), nonalcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), chronic kidney disease (CKD), gout, rheumatoid arthritis, osteoarthritis, Irritable Bowel Disease (IBD), Crohn's disease, COPD, fibrosis, obesity, type 2 diabetes, multiple sclerosis, dermatological diseases (e.g., acne) neuroinflammation occurring in protein misfolding diseases, such as Prion diseases, neurogenerative diseases (e.g., Parkinson's disease, Alzheimer's disease, multiple sclerosis, amyotrophic lateral sclerosis, or Huntington's disease) and oncological disorders.
Inflammatory Disease Associated with Infection
In some embodiments, the disease or disorder is an inflammatory disease.
In some embodiments, the inflammatory disease is associated with an infection.
In some embodiments, the inflammatory disease is associated with an infection by a virus.
In some embodiments, the inflammatory disease is associated with an infection by an RNA virus. In some embodiments, the RNA virus is a single stranded RNA virus. Single stranded RNA viruses include group IV (positive strand) and group V (negative strand) single stranded RNA viruses. Group IV viruses include coronaviruses.
In some embodiments, the inflammatory disease is associated with an infection by a coronavirus. In some embodiments, the coronavirus is Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV 2), SARS coronavirus (SARS CoV) or Middle East respiratory syndrome-related coronavirus (MERS).
In some embodiments, the inflammatory disease is associated with an infection by SARS-CoV 2. In some embodiments, SARS-CoV 2 infection leads to 2019 novel coronavirus disease (COVID-19).
In some embodiments, the inflammatory disease is an inflammatory disease of lung.
In some embodiments, the inflammatory disease of lung is associated with an infection by SARS-CoV 2.
In some embodiments, the inflammatory disease comprises cytokine release syndrome (CRS).
In some embodiments, the cytokine release syndrome (CRS) is associated with an infection by SARS-CoV 2.
In some embodiments, the disease or disorder is an inflammatory disease.
In some embodiments, the inflammatory disease is associated with an immunotherapy.
In some embodiments, the immunotherapy causes cytokine release syndrome (CRS).
The effectiveness of immunotherapies, such as CAR-T, are hampered by the frequency with which such therapies induce cytokine release syndrome. Without wishing to be bound by theory, it is thought that the severity of CRS induced by immunotherapy is mediated by IL-6, IL-1 and NO production (Giavridis et al. Nature Medicine; doi.org/10.1038/s41591-018-0041-7). Alternatively, or in addition, CRS may occur when cells targeted by the adoptive cell therapy undergo pyroptosis, a highly inflammatory form of programmed cell death. Pyroptosis leads to release of factors that stimulate macrophages to produce pro-inflammatory cytokines, leading to CRS (Liu et al. Science Immunology (2020) V: eeax7969).
In some embodiments, the immunotherapy comprises an antibody or an adoptive cell therapy.
In some embodiments, the adoptive cell therapy comprises a CAR-T or TCR-T cell therapy.
In some embodiments, the adoptive cell therapy comprises a cancer therapy. For example, the cancer therapy can be to treat B cell lymphoma or B cell acute lymphoblastic leukemia. For example, the adoptive cells may express a CAR targeting CD19+B cell acute lymphoblastic leukemia cells.
In some embodiments, the adoptive cell therapy comprises administration of T cells, B cells or NK cells.
In some embodiments, the adoptive cell therapy is autologous.
In some embodiments, the adoptive therapy is allogeneic.
Treatment in Cancer; Links with Inflammasome
Chronic inflammation responses have long been observed to be associated with various types of cancer. During malignant transformation or cancer therapy inflammasomes may become activated in response to danger signals and this activation may be both beneficial and detrimental in cancer.
IL-1β expression is elevated in a variety of cancers (including breast, prostate, colon, lung, head and neck cancers and melanomas) and patients with IL-1β producing tumours generally have a worse prognosis (Lewis, Anne M., et al. “Interleukin-1 and cancer progression: the emerging role of interleukin-1 receptor antagonist as a novel therapeutic agent in cancer treatment.” Journal of translational medicine 4.1 (2006): 48).
Cancers derived from epithelial cells (carcinoma) or epithelium in glands (adenocarcinoma) are heterogeneous; consisting of many different cell types. This may include fibroblasts, immune cells, adipocytes, endothelial cells and pericytes amongst others, all of which may be cytokine/chemokine secreting (Grivennikov, Sergei I., Florian R. Greten, and Michael Karin. “Immunity, inflammation, and cancer.” Cell 140.6 (2010): 883-899). This can lead to cancer-associated inflammation through the immune cell infiltration. The presence of leukocytes in tumours is known but it has only recently become evident that an inflammatory microenvironment is an essential component of all tumours. Most tumours (>90%) are the result of somatic mutations or environmental factors rather than germline mutations and many environmental causes of cancer are associated with chronic inflammation (20% of cancers are related to chronic infection, 30% to smoking/inhaled pollutants and 35% to dietary factors (20% of all cancers are linked to obesity) (Aggarwal, Bharat B., R. V. Vijayalekshmi, and Bokyung Sung. “Targeting inflammatory pathways for prevention and therapy of cancer: short-term friend, long-term foe.” Clinical Cancer Research 15.2 (2009): 425-430).
Cancers of the gastrointestinal (GI) tract are frequently associated with chronic inflammation. For example, H. pylori infection is associated with gastric cancer (Amieva, Manuel, and Richard M. Peek. “Pathobiology of Helicobacter pylori-Induced Gastric Cancer.” Gastroenterology 150.1 (2016): 64-78). Colorectal cancer is associated with inflammatory bowel disease (Bernstein, Charles N., et al. “Cancer risk in patients with inflammatory bowel disease.” Cancer 91.4 (2001): 854-862). Chronic inflammation in stomach leads to the upregulation of IL-1 and other cytokines (Basso, D. et al., (1996) Helicobacter pylori infection enhances mucosal interleukin-1 beta, interleukin-6, and the soluble receptor of interleukin-2. Int J Clin Lab Res 26:207-210) and polymorphisms in IL-1β gene can increase risk of gastric cancer (Wang, P. et al., (2007) Association of interleukin-1 gene polymorphisms with gastric cancer: a meta-analysis. Int J Cancer 120:552-562).
In 19% of gastric cancer cases, caspase-1 expression is decreased which correlates with stage, lymph node metastasis and survival (Jee et al., 2005). Mycoplasma hyorhinis is associated with the development of gastric cancer its activation of the NLRP3 inflammasome may be associated with its promotion of gastric cancer metastasis (Xu et al., 2013).
Ultraviolet radiation is the greatest environmental risk for skin cancer which is promoted by causing DNA damage, immunosuppression and inflammation. The most malignant skin cancer, melanoma, is characterised by the upregulation of inflammatory cytokines, all of which can be regulated by IL-1β (Lizar-Molnar, Eszter, et al. “Autocrine and paracrine regulation by cytokines and growth factors in melanoma.” Cytokine 12.6 (2000): 547-554). Systemic inflammation induces an enhancement of melanoma cell metastasis and growth by IL-1-dependent mechanisms in vivo. Using thymoquinone inhibition of metastasis in a B16F10 mouse melanoma model was shown to be dependent on inhibition of the NLRP3 inflammasome (Ahmad, Israr, et al. “Thymoquinone suppresses metastasis of melanoma cells by inhibition of NLRP3 inflammasome.” Toxicology and applied pharmacology 270.1 (2013): 70-76).
NLRP3 contributes to radiotherapy resistance in glioma. Ionising radiation can induce NLRP3 expression whereas NLRP3 inhibition reduced tumour growth and prolonged mouse survival following radiation therapy. NLRP3 inflammasome inhibition can therefore provide a therapeutic strategy for radiation-resistant glioma (Li, Lianling, and Yuguang Liu. “Aging-related gene signature regulated by Nlrp3 predicts glioma progression.” American journal of cancer research 5.1 (2015): 442).
More widely, NLRP3 is considered by the applicants to be involved in the promotion of metastasis and consequently modulation of NLRP3 should plausibly block this. IL-1 is involved in tumour genesis, tumour invasiveness, metastasis, tumour host interactions (Apte, Ron N., et al. “The involvement of IL-1 in tumorigenesis, tumour invasiveness, metastasis and tumour-host interactions.” Cancer and Metastasis Reviews 25.3 (2006): 387-408) and angiogenesis (Voronov, Elena, et al. “IL-1 is required for tumor invasiveness and angiogenesis.” Proceedings of the National Academy of Sciences 100.5 (2003): 2645-2650).
The IL-1 gene is frequently expressed in metastases from patients with several types of human cancers. For example, IL-1mRNA was highly expressed in more than half of all tested metastatic human tumour specimens including specifically non-small-cell lung carcinoma, colorectal adenocarcinoma, and melanoma tumour samples (Elaraj, Dina M., et al. “The role of interleukin 1 in growth and metastasis of human cancer xenografts.” Clinical Cancer Research 12.4 (2006): 1088-1096) and IL-1RA inhibits xenograft growth in IL-1 producing tumours but without anti-proliferative effects in vitro.
Further, IL-1 signalling is a biomarker for predicting breast cancer patients at increased risk for developing bone metastasis. In mouse models IL-1β and its receptor are upregulated in breast cancer cells that metastasise to bone compared with cells that do not. In a mouse model the IL-1 receptor antagonist anakinra reduced proliferation and angiogenesis in addition to exerting significant effects on the tumour environment reducing bone turnover markers, IL-1β and TNF alpha (Holen, Ingunn, et al. “IL-1 drives breast cancer growth and bone metastasis in vivo.” Oncotarget (2016).
IL-18 induced the production of MMP-9 in the human leukemia cell line HL-60, thus favouring degradation of the extracellular matrix and the migration and invasiveness of cancer cells (Zhang, Bin, et al. “IL-18 increases invasiveness of HL-60 myeloid leukemia cells: up-regulation of matrix metalloproteinases-9 (MMP-9) expression.” Leukemia research 28.1 (2004): 91-95). Additionally IL-18 can support the development of tumour metastasis in the liver by inducing expression of VCAM-1 on hepatic sinusoidal endothelium (Carrascal, Maria Teresa, et al. “Interleukin-18 binding protein reduces b16 melanoma hepatic metastasis by neutralizing adhesiveness and growth factors of sinusoidal endothelium.” Cancer Research 63.2 (2003): 491-497).
The fatty acid scavenger receptor CD36 serves a dual role in priming gene transcription of pro-IL-1β and inducing assembly of the NLRP3 inflammasome complex. CD36 and the TLR4-TLR6 heterodimer recognise oxLDL, which initiates a signalling pathway leading to transcriptional upregulation of NLRP3 and pro-IL-1β (signal 1). CD36 also mediates the internalisation of oxLDL into the lysosomal compartment, where crystals are formed that induce lysosomal rupture and activation of the NLRP3 inflammasome (signal 2) (Kagan, J. and Horng T., “NLRP3 inflammasome activation: CD36 serves double duty.” Nature immunology 14.8 (2013): 772-774).
A subpopulation of human oral carcinoma cells express high levels of the fatty acid scavenger receptor CD36 and are unique in their ability to initiate metastasis. Palmitic acid or a high fat diet boosted the metastatic potential of the CD36+ cells. Neutralising anti-CD36 antibodies blocked metastasis in orthotopic mouse models of human oral cancer. The presence of CD36+ metastasis-initiating cells correlates with a poor prognosis for numerous types of carcinomas. It is suggested that dietary lipids may promote metastasis (Pasqual, G, Avgustinova, A., Mejetta, S, Martin, M, Castellanos, A, Attolini, CS-O, Berenguer, A., Prats, N, Toll, A, Hueto, JA, Bescos, C, Di Croce, L, and Benitah, S A. 2017 “Targeting metastasis-initiating cells through the fatty acid receptor CD36” Nature 541:41-45).
In hepatocellular carcinoma exogenous palmitic acid activated an epithelial-mesenchymal transition (EMT)-like program and induced migration that was decreased by the CD36 inhibitor, sulpho-N-succinimidyl oleate (Nath, Aritro, et al. “Elevated free fatty acid uptake via CD36 promotes epithelial-mesenchymal transition in hepatocellular carcinoma.” Scientific reports 5 (2015). Body mass index was not associated with the degree of EMT highlighting that it is actually CD36 and free fatty acids that are important.
Cancer stems cells (CSCs) use CD36 to promote their maintenance. Oxidised phospholipids, ligands of CD36, were present in glioblastoma and the proliferation of CSCs but not non-CSCs increased with exposure to oxidised LDL. CD36 also correlated with patient prognosis.
In addition to direct cytotoxic effects, chemotherapeutic agents harness the host immune system which contributes to anti-tumour activity. However, gemcitabine and 5-FU were shown to activate NLRP3 in myeloid-derived suppressor cells leading to production of IL-1β which curtails anti-tumour efficacy. Mechanistically these agents destabilised the lysosome to release cathepsin B to activate NLRP3. IL-13 drove the production of IL-17 from CD4+ T cells, which in turn blunted the efficacy of the chemotherapy. Higher anti-tumoral effects for both gemcitabine and 5-FU were observed when tumours were established in NLRP3−/− or Caps1−/− mice, or WT mice treated with IL-1RA. Myeloid-derived suppressor cell NLRP3 activation therefore limits the anti-tumour efficacy of gemcitabine and 5-FU (Bruchard, Melanie, et al. “Chemotherapy-triggered cathepsin B release in myeloid-derived suppressor cells activates the Nlrp3 inflammasome and promotes tumour growth.” Nature medicine 19.1 (2013): 57-64.). Compounds of the present disclosure may therefore be useful in chemotherapy to treat a range of cancers.
Compounds of the present disclosure, or pharmaceutically acceptable salts thereof, may be administered alone as a sole therapy or can be administered in addition with one or more other substances and/or treatments. Such conjoint treatment may be achieved by way of the simultaneous, sequential or separate administration of the individual components of the treatment.
For example, therapeutic effectiveness may be enhanced by administration of an adjuvant (i.e. by itself the adjuvant may only have minimal therapeutic benefit, but in combination with another therapeutic agent, the overall therapeutic benefit to the individual is enhanced). Alternatively, by way of example only, the benefit experienced by an individual may be increased by administering the compound of Formula (I) with another therapeutic agent (which also includes a therapeutic regimen) that also has therapeutic benefit.
In the instances where the compound of the present disclosure is administered in combination with other therapeutic agents, the compound of the disclosure need not be administered via the same route as other therapeutic agents, and may, because of different physical and chemical characteristics, be administered by a different route. For example, the compound of the disclosure may be administered orally to generate and maintain good blood levels thereof, while the other therapeutic agent may be administered intravenously. The initial administration may be made according to established protocols known in the art, and then, based upon the observed effects, the dosage, modes of administration and times of administration can be modified by the skilled clinician.
The particular choice of other therapeutic agent will depend upon the diagnosis of the attending physicians and their judgment of the condition of the individual and the appropriate treatment protocol. According to this aspect of the disclosure there is provided a combination for use in the treatment of a disease in which inflammasome activity is implicated comprising a compound of the disclosure as defined hereinbefore, or a pharmaceutically acceptable salt thereof, and another suitable agent.
According to a further aspect of the disclosure there is provided a pharmaceutical composition which comprises a compound of the disclosure, or a pharmaceutically acceptable salt thereof, in combination with a suitable, in association with a pharmaceutically acceptable diluent or carrier.
In addition to its use in therapeutic medicine, compounds of Formula (I) and pharmaceutically acceptable salts thereof are also useful as pharmacological tools in the development and standardisation of in vitro and in vivo test systems for the evaluation of the effects of inhibitors of inflammasome in laboratory animals such as dogs, rabbits, monkeys, rats and mice, as part of the search for new therapeutic agents.
In any of the above-mentioned pharmaceutical composition, process, method, use, medicament, and manufacturing features of the instant disclosure, any of the alternate embodiments of macromolecules of the present disclosure described herein also apply.
The compounds of the disclosure or pharmaceutical compositions comprising these compounds may be administered to a subject by any convenient route of administration, whether systemically/peripherally or topically (i.e., at the site of desired action).
Routes of administration include, but are not limited to, oral (e.g. by ingestion); buccal; sublingual; transdermal (including, e.g., by a patch, plaster, etc.); transmucosal (including, e.g., by a patch, plaster, etc.); intranasal (e.g., by nasal spray); ocular (e.g., by eye drops); pulmonary (e.g., by inhalation or insufflation therapy using, e.g., via an aerosol, e.g., through the mouth or nose); rectal (e.g., by suppository or enema); vaginal (e.g., by pessary); parenteral, for example, by injection, including subcutaneous, intradermal, intramuscular, intravenous, intra-arterial, intracardiac, intrathecal, intraspinal, intracapsular, subcapsular, intraorbital, intraperitoneal, intratracheal, subcuticular, intraarticular, subarachnoid, and intrasternal; by implant of a depot or reservoir, for example, subcutaneously or intramuscularly.
Unless otherwise stated, the following terms used in the specification and claims have the following meanings set out below.
Without wishing to be limited by this statement, it is understood that, while various options for variables are described herein, the disclosure intends to encompass operable embodiments having combinations of the options. The disclosure may be interpreted as excluding the non-operable embodiments caused by certain combinations of the options.
It is to be understood that a compound of the present disclosure may be depicted in a neutral form, a cationic form (e.g., carrying one or more positive charges), or an anionic form (e.g., carrying one or more negative charges), all of which are intended to be included in the scope of the present disclosure. For example, when a compound of the present disclosure is depicted in an anionic form, such depiction also refers to the various neutral forms, cationic forms, and anionic forms of the compound. For another example, when a compound the present disclosure is depicted in an anionic form, such depiction also refers to various salts (e.g., sodium salt) of the anionic form of the compound.
A “therapeutically effective amount” means the amount of a compound that, when administered to a mammal for treating a disease, is sufficient to effect such treatment for the disease. The “therapeutically effective amount” will vary depending on the compound, the disease and its severity and the age, weight, etc., of the mammal to be treated.
As used herein, “alkyl”, “C1, C2, C3, C4, C5 or C6 alkyl” or “C1-C6 alkyl” is intended to include C1, C2, C3, C4, C5 or C6 straight chain (linear) saturated aliphatic hydrocarbon groups and C3, C4, C5 or C6 branched saturated aliphatic hydrocarbon groups. For example, C1-C6 alkyl is intends to include C1, C2, C3, C4, C5 and C6 alkyl groups. Examples of alkyl include, moieties having from one to six carbon atoms, such as, but not limited to, methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, t-butyl, n-pentyl, i-pentyl or n-hexyl. In some embodiments, a straight chain or branched alkyl has six or fewer carbon atoms (e.g., C1-C6 for straight chain, C3-C6 for branched chain), and in another embodiment, a straight chain or branched alkyl has four or fewer carbon atoms.
As used herein, the term “optionally substituted alkyl” refers to unsubstituted alkyl or alkyl having designated substituents replacing one or more hydrogen atoms on one or more carbons of the hydrocarbon backbone. Such substituents can include, for example, alkyl, alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, amino (including alkylamino, dialkylamino, arylamino, diarylamino and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulphhydryl, alkylthio, arylthio, thiocarboxylate, sulphates, alkylsulphinyl, sulphonato, sulphamoyl, sulphonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety.
As used herein, the term “alkenyl” includes unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but that contain at least one double bond. For example, the term “alkenyl” includes straight chain alkenyl groups (e.g., ethenyl, propenyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl, decenyl), and branched alkenyl groups. In certain embodiments, a straight chain or branched alkenyl group has six or fewer carbon atoms in its backbone (e.g., C2-C6 for straight chain, C3-C6 for branched chain). The term “C2-C6” includes alkenyl groups containing two to six carbon atoms. The term “C3-C6” includes alkenyl groups containing three to six carbon atoms.
As used herein, the term “optionally substituted alkenyl” refers to unsubstituted alkenyl or alkenyl having designated substituents replacing one or more hydrogen atoms on one or more hydrocarbon backbone carbon atoms. Such substituents can include, for example, alkyl, alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, amino (including alkylamino, dialkylamino, arylamino, diarylamino and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulphhydryl, alkylthio, arylthio, thiocarboxylate, sulphates, alkylsulphinyl, sulphonato, sulphamoyl, sulphonamido, nitro, trifluoromethyl, cyano, heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety.
As used herein, the term “alkynyl” includes unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but which contain at least one triple bond. For example, “alkynyl” includes straight chain alkynyl groups (e.g., ethynyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, octynyl, nonynyl, decynyl), and branched alkynyl groups. In certain embodiments, a straight chain or branched alkynyl group has six or fewer carbon atoms in its backbone (e.g., C2-C6 for straight chain, C3-C6 for branched chain). The term “C2-C6” includes alkynyl groups containing two to six carbon atoms. The term “C3-C6” includes alkynyl groups containing three to six carbon atoms. As used herein, “C2-C6 alkenylene linker” or “C2-C6 alkynylene linker” is intended to include C2, C3, C4, C5 or C6 chain (linear or branched) divalent unsaturated aliphatic hydrocarbon groups. For example, C2-C6 alkenylene linker is intended to include C2, C3, C4, C5 and C6 alkenylene linker groups.
As used herein, the term “optionally substituted alkynyl” refers to unsubstituted alkynyl or alkynyl having designated substituents replacing one or more hydrogen atoms on one or more hydrocarbon backbone carbon atoms. Such substituents can include, for example, alkyl, alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, amino (including alkylamino, dialkylamino, arylamino, diarylamino and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulphhydryl, alkylthio, arylthio, thiocarboxylate, alkylsulphinyl, sulphonato, sulphamoyl, sulphonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety.
Other optionally substituted moieties (such as optionally substituted cycloalkyl, heterocycloalkyl, aryl, or heteroaryl) include both the unsubstituted moieties and the moieties having one or more of the designated substituents.
As used herein, the term “cycloalkyl” refers to a saturated or partially unsaturated hydrocarbon monocyclic or polycyclic (e.g., fused, bridged, or spiro rings) system having 3 to 30 carbon atoms (e.g., C3-C12, C3-C10, or C3-C8). Examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, 1,2,3,4-tetrahydronaphthalenyl, and adamantyl. In the case of polycyclic cycloalkyl, only one of the rings in the cycloalkyl needs to be non-aromatic.
As used herein, the term “heterocycloalkyl” refers to a saturated or partially unsaturated 3-8 membered monocyclic, 6-12 membered bicyclic (fused, bridged, or spiro rings), or 11-14 membered tricyclic ring system (fused, bridged, or spiro rings) having one or more heteroatoms (such as O, N, S, P, or Se), e.g., 1 or 1-2 or 1-3 or 1-4 or 1-5 or 1-6 heteroatoms, or e.g., 1, 2, 3, 4, 5, or 6 heteroatoms, independently selected from the group consisting of nitrogen, oxygen and sulphur, unless specified otherwise. Examples of heterocycloalkyl groups include, but are not limited to, piperidinyl, piperazinyl, pyrrolidinyl, dioxanyl, tetrahydrofuranyl, isoindolinyl, indolinyl, imidazolidinyl, pyrazolidinyl, oxazolidinyl, isoxazolidinyl, triazolidinyl, oxiranyl, azetidinyl, oxetanyl, thietanyl, 1,2,3,6-tetrahydropyridinyl, tetrahydropyranyl, dihydropyranyl, pyranyl, morpholinyl, tetrahydrothiopyranyl, 1,4-diazepanyl, 1,4-oxazepanyl, 2-oxa-5-azabicyclo[2.2.1]heptanyl, 2,5-diazabicyclo[2.2.1]heptanyl, 2-oxa-6-azaspiro[3.3]heptanyl, 2,6-diazaspiro[3.3]heptanyl, 1,4-dioxa-8-azaspiro[4.5]decanyl, 1,4-dioxaspiro[4.5]decanyl, 1-oxaspiro[4.5]decanyl, 1-azaspiro[4.5]decanyl, 3′H-spiro[cyclohexane-1,1′-isobenzofuran]-yl, 7′H-spiro[cyclohexane-1,5′-furo[3,4-b]pyridin]-yl, 3′H-spiro[cyclohexane-1,1′-furo[3,4-c]pyridin]-yl, 3-azabicyclo[3.1.0]hexanyl, 3-azabicyclo[3.1.0]hexan-3-yl, 1,4,5,6-tetrahydropyrrolo[3,4-c]pyrazolyl, 3,4,5,6,7,8-hexahydropyrido[4,3-d]pyrimidinyl, 4,5,6,7-tetrahydro-1H-pyrazolo[3,4-c]pyridinyl, 5,6,7,8-tetrahydropyrido[4,3-d]pyrimidinyl, 2-azaspiro[3.3]heptanyl, 2-methyl-2-azaspiro[3.3]heptanyl, 2-azaspiro[3.5]nonanyl, 2-methyl-2-azaspiro[3.5]nonanyl, 2-azaspiro[4.5]decanyl, 2-methyl-2-azaspiro[4.5]decanyl, 2-oxa-azaspiro[3.4]octanyl, 2-oxa-azaspiro[3.4]octan-6-yl, and the like. In the case of multicyclic heterocycloalkyl, only one of the rings in the heterocycloalkyl needs to be non-aromatic.
As used herein, the term “aryl” includes groups with aromaticity, including “conjugated,” or multicyclic systems with one or more aromatic rings and do not contain any heteroatom in the ring structure. The term aryl includes both monovalent species and divalent species. Examples of aryl groups include, but are not limited to, phenyl, biphenyl, naphthyl and the like.
As used herein, the term “heteroaryl” is intended to include a stable 5-, 6-, or 7-membered monocyclic or 7-, 8-, 9-, 10-, 11- or 12-membered bicyclic aromatic heterocyclic ring which consists of carbon atoms and one or more heteroatoms, e.g., 1 or 1-2 or 1-3 or 1-4 or 1-5 or 1-6 heteroatoms, or e.g., 1, 2, 3, 4, 5, or 6 heteroatoms, independently selected from the group consisting of nitrogen, oxygen and sulphur. The nitrogen atom may be substituted or unsubstituted (i.e., N or NR wherein R is H or other substituents, as defined). The nitrogen and sulphur heteroatoms may optionally be oxidised (i.e., N→O and S(O)p, where p=1 or 2). It is to be noted that total number of S and O atoms in the aromatic heterocycle is not more than 1. Examples of heteroaryl groups include pyrrole, furan, thiophene, thiazole, isothiazole, imidazole, triazole, tetrazole, pyrazole, oxazole, isoxazole, pyridine, pyrazine, pyridazine, pyrimidine, and the like. Heteroaryl groups can also be fused or bridged with alicyclic or heterocyclic rings, which are not aromatic so as to form a multicyclic system (e.g., 4,5,6,7-tetrahydrobenzo[c]isoxazolyl).
Furthermore, the terms “aryl” and “heteroaryl” include multicyclic aryl and heteroaryl groups, e.g., tricyclic, bicyclic, e.g., naphthalene, benzoxazole, benzodioxazole, benzothiazole, benzoimidazole, benzothiophene, quinoline, isoquinoline, naphthyridine, indole, benzofuran, purine, deazapurine, indolizine.
The cycloalkyl, heterocycloalkyl, aryl, or heteroaryl ring can be substituted at one or more ring positions (e.g., the ring-forming carbon or heteroatom such as N) with such substituents as described above, for example, alkyl, alkenyl, alkynyl, halogen, hydroxyl, alkoxy, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkylaminocarbonyl, aralkylaminocarbonyl, alkenylaminocarbonyl, alkylcarbonyl, arylcarbonyl, aralkylcarbonyl, alkenylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, phosphate, phosphonato, phosphinato, amino (including alkylamino, dialkylamino, arylamino, diarylamino and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulphhydryl, alkylthio, arylthio, thiocarboxylate, sulphates, alkylsulphinyl, sulphonato, sulphamoyl, sulphonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety. Aryl and heteroaryl groups can also be fused or bridged with alicyclic or heterocyclic rings, which are not aromatic so as to form a multicyclic system.
As used herein, the term “substituted,” means that any one or more hydrogen atoms on the designated atom is replaced with a selection from the indicated groups, provided that the designated atom's normal valency is not exceeded, and that the substitution results in a stable compound. When a substituent is oxo or keto (i.e., ═O), then 2 hydrogen atoms on the atom are replaced. Keto substituents are not present on aromatic moieties. Ring double bonds, as used herein, are double bonds that are formed between two adjacent ring atoms (e.g., C=C, C=N or N=N). “Stable compound” and “stable structure” are meant to indicate a compound that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture, and formulation into an efficacious therapeutic agent.
When a bond to a substituent is shown to cross a bond connecting two atoms in a ring, then such substituent may be bonded to any atom in the ring. When a substituent is listed without indicating the atom via which such substituent is bonded to the rest of the compound of a given formula, then such substituent may be bonded via any atom in such formula. Combinations of substituents and/or variables are permissible, but only if such combinations result in stable compounds.
When any variable (e.g., R) occurs more than one time in any constituent or formula for a compound, its definition at each occurrence is independent of its definition at every other occurrence. Thus, for example, if a group is shown to be substituted with 0-2 R moieties, then the group may optionally be substituted with up to two R moieties and R at each occurrence is selected independently from the definition of R. Also, combinations of substituents and/or variables are permissible, but only if such combinations result in stable compounds.
As used herein, the term “hydroxy” or “hydroxyl” includes groups with an —OH or —O—.
As used herein, the term “halo” or “halogen” refers to fluoro, chloro, bromo and iodo.
The term “haloalkyl” or “haloalkoxyl” refers to an alkyl or alkoxyl substituted with one or more halogen atoms.
As used herein, the term “optionally substituted haloalkyl” refers to unsubstituted haloalkyl having designated substituents replacing one or more hydrogen atoms on one or more hydrocarbon backbone carbon atoms. Such substituents can include, for example, alkyl, alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, amino (including alkylamino, dialkylamino, arylamino, diarylamino and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulphhydryl, alkylthio, arylthio, thiocarboxylate, sulphates, alkylsulphinyl, sulphonato, sulphamoyl, sulphonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety.
As used herein, the term “alkoxy” or “alkoxyl” includes substituted and unsubstituted alkyl, alkenyl and alkynyl groups covalently linked to an oxygen atom. Examples of alkoxy groups or alkoxyl radicals include, but are not limited to, methoxy, ethoxy, isopropyloxy, propoxy, butoxy and pentoxy groups. Examples of substituted alkoxy groups include halogenated alkoxy groups. The alkoxy groups can be substituted with groups such as alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, amino (including alkylamino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulphhydryl, alkylthio, arylthio, thiocarboxylate, sulphates, alkylsulphinyl, sulphonato, sulphamoyl, sulphonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moieties. Examples of halogen substituted alkoxy groups include, but are not limited to, fluoromethoxy, difluoromethoxy, trifluoromethoxy, chloromethoxy, dichloromethoxy and trichloromethoxy.
As used herein, the expressions “one or more of A, B, or C,” “one or more A, B, or C,” “one or more of A, B, and C,” “one or more A, B, and C,” “selected from the group consisting of A, B, and C”, “selected from A, B, and C”, and the like are used interchangeably and all refer to a selection from a group consisting of A, B, and/or C, i.e., one or more As, one or more Bs, one or more Cs, or any combination thereof, unless indicated otherwise.
As used herein “Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV 2)” refers to the coronavirus that causes 2019 novel coronavirus disease (COVID-19). COVID-19 was first identified in 2019 in Wuhan, China, and has resulted in an ongoing global pandemic. As of August 2020, more than 25 million cases have been reported globally, resulting in an estimated 848,000 deaths. Common symptoms of COVID-19 include fever, cough, fatigue, shortness of breath, and loss of smell and taste. While many people have mild symptoms, some people develop acute respiratory distress syndrome, possibly caused by cytokine release syndrome (CRS), multi-organ failure, septic shock, and blood clots. Time from exposure to the virus to symptom onset is typically around 5 days but may range from 2 to 14 days.
As used herein “cytokine release syndrome (CRS)” refers to a systemic inflammatory response that can be triggered by a variety of factors, including but not limited to drugs, infections such as SARS-CoV 2, and immunotherapies such as chimeric antigen receptor T cell (CAR-T) therapies. In CRS, large numbers of immune cells (e.g. T cells) are activated and release inflammatory cytokines, which in turn activate additional immune cells. Symptoms include fever, fatigue, loss of appetite, muscle and joint pain, nausea, vomiting, diarrhea, rashes, respiratory insufficiency, low blood pressure, seizures, headache and confusion. CRS may respond to IL-6 receptor inhibition, and high doses of steroids.
As used herein, “adoptive cell therapy” refers to a form of treatment that uses immune cells to treat diseases such as cancer. Immune cells, for example T cells are collected from the subject or another source, grown in large numbers, and implanted into the subject to help the immune system fight the disease. Types of adoptive cell therapy include chimerica antigen receptor T cell (CAR-T) therapy, tumor infiltrating lymphocyte (TIL) therapy, and T cell receptor T cell (TCR-T) therapy.
The term “chimeric antigen receptors (CARs),” as used herein, may refer to artificial T-cell receptors, chimeric T-cell receptors, or chimeric immunoreceptors, for example, and encompass engineered receptors that graft an artificial specificity onto a particular immune effector cell. CARs may be employed to impart the specificity of a monoclonal antibody onto a T cell, thereby allowing a large number of specific T cells to be generated, for example, for use in adoptive cell therapy. For example, CARs may direct specificity of the cell expressing the CAR to a tumor associated antigen. In some embodiments, CARs comprise an intracellular activation domain, a transmembrane domain, and an extracellular domain comprising an antigen binding domain, and optionally an extracellular hinge. The antigen binding domain can be any antigen binding domain known in the art, including antigen binding domains derived from antibodies, Fab, F(ab′)2, nanobodies, single domain antigen binding domains, scFv, VHH, and the like. In particular aspects, CARs comprise fusions of single-chain variable fragments (scFv) derived from monoclonal antibodies, fused to a CD3 transmembrane domain and endodomain. In certain cases, CARs comprise domains for additional co-stimulatory signaling, such as CD3, FcR, CD27, CD28, CD137, DAP10, and/or 0X40.
A “T cell receptor (TCR)” is a protein complex found on the surface of T cells, or T lymphocytes, that is responsible for recognizing fragments of antigen as peptides bound to major histocompatibility complex (MHC) molecules. T cell receptors can be engineered to express antigen binding domains specific to particular antigens and used in the adoptive cell therapies described herein.
It is to be understood that the present disclosure provides methods for the synthesis of the compounds of any of the Formulae described herein. The present disclosure also provides detailed methods for the synthesis of various disclosed compounds of the present disclosure according to the following schemes as well as those shown in the Examples.
It is to be understood that, throughout the description, where compositions are described as having, including, or comprising specific components, it is contemplated those compositions also consist essentially of, or consist of, the recited components. Similarly, where methods or processes are described as having, including, or comprising specific process steps, the processes also consist essentially of, or consist of, the recited processing steps. Further, it should be understood that the order of steps or order for performing certain actions is immaterial so long as the invention remains operable. Moreover, two or more steps or actions can be conducted simultaneously.
It is to be understood that the synthetic processes of the disclosure can tolerate a wide variety of functional groups, therefore various substituted starting materials can be used. The processes generally provide the desired final compound at or near the end of the overall process, although it may be desirable in certain instances to further convert the compound to a pharmaceutically acceptable salt thereof.
It is to be understood that compounds of the present disclosure can be prepared in a variety of ways using commercially available starting materials, compounds known in the literature, or from readily prepared intermediates, by employing standard synthetic methods and procedures either known to those skilled in the art, or which will be apparent to the skilled artisan in light of the teachings herein. Standard synthetic methods and procedures for the preparation of organic molecules and functional group transformations and manipulations can be obtained from the relevant scientific literature or from standard textbooks in the field. Although not limited to any one or several sources, classic texts such as Smith, M. B., March, J., March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 5th edition, John Wiley & Sons: New York, 2001; Greene, T. W., Wuts, P. G. M., Protective Groups in Organic Synthesis, 3rd edition, John Wiley & Sons: New York, 1999; R. Larock, Comprehensive Organic Transformations, VCH Publishers (1989); L. Fieser and M. Fieser, Fieser and Fieser's Reagentsfor Organic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995), incorporated by reference herein, are useful and recognised reference textbooks of organic synthesis known to those in the art
One of ordinary skill in the art will note that, during the reaction sequences and synthetic schemes described herein, the order of certain steps may be changed, such as the introduction and removal of protecting groups. One of ordinary skill in the art will recognise that certain groups may require protection from the reaction conditions via the use of protecting groups. Protecting groups may also be used to differentiate similar functional groups in molecules. A list of protecting groups and how to introduce and remove these groups can be found in Greene, T. W., Wuts, P. G. M., Protective Groups in Organic Synthesis, 3rd edition, John Wiley & Sons: New York, 1999.
It is to be understood that, unless otherwise stated, any description of a method of treatment includes use of the compounds to provide such treatment or prophylaxis as is described herein, as well as use of the compounds to prepare a medicament to treat or prevent such condition. The treatment includes treatment of human or non-human animals including rodents and other disease models.
As used herein, the term “subject” includes human and non-human animals, as well as cell lines, cell cultures, tissues, and organs. In some embodiments, the subject is a mammal. The mammal can be e.g., a human or appropriate non-human mammal, such as primate, mouse, rat, dog, cat, cow, horse, goat, camel, sheep or a pig. The subject can also be a bird or fowl. In some embodiments, the subject is a human.
As used herein, the term “subject in need thereof” refers to a subject having a disease or having an increased risk of developing the disease. A subject in need thereof can be one who has been previously diagnosed or identified as having a disease or disorder disclosed herein. A subject in need thereof can also be one who is suffering from a disease or disorder disclosed herein. Alternatively, a subject in need thereof can be one who has an increased risk of developing such disease or disorder relative to the population at large (i.e., a subject who is predisposed to developing such disorder relative to the population at large). A subject in need thereof can have a refractory or resistant a disease or disorder disclosed herein (i.e., a disease or disorder disclosed herein that does not respond or has not yet responded to treatment). The subject may be resistant at start of treatment or may become resistant during treatment. In some embodiments, the subject in need thereof received and failed all known effective therapies for a disease or disorder disclosed herein. In some embodiments, the subject in need thereof received at least one prior therapy.
As used herein, the term “treating” or “treat” describes the management and care of a patient for the purpose of combating a disease, condition, or disorder and includes the administration of a compound of the present disclosure, or a pharmaceutically acceptable salt, polymorph or solvate thereof, to alleviate the symptoms or complications of a disease, condition or disorder, or to eliminate the disease, condition or disorder. The term “treat” can also include treatment of a cell in vitro or an animal model. It is to be appreciated that references to “treating” or “treatment” include the alleviation of established symptoms of a condition. “Treating” or “treatment” of a state, disorder or condition therefore includes: (1) preventing or delaying the appearance of clinical symptoms of the state, disorder or condition developing in a human that may be afflicted with or predisposed to the state, disorder or condition but does not yet experience or display clinical or subclinical symptoms of the state, disorder or condition, (2) inhibiting the state, disorder or condition, i.e., arresting, reducing or delaying the development of the disease or a relapse thereof (in case of maintenance treatment) or at least one clinical or subclinical symptom thereof, or (3) relieving or attenuating the disease, i.e., causing regression of the state, disorder or condition or at least one of its clinical or subclinical symptoms.
It is to be understood that a compound of the present disclosure, or a pharmaceutically acceptable salt, polymorph or solvate thereof, can or may also be used to prevent a relevant disease, condition or disorder, or used to identify suitable candidates for such purposes.
As used herein, the term “preventing,” “prevent,” or “protecting against” describes reducing or eliminating the onset of the symptoms or complications of such disease, condition or disorder.
It is to be understood that one skilled in the art may refer to general reference texts for detailed descriptions of known techniques discussed herein or equivalent techniques. These texts include Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Inc. (2005); Sambrook et al., Molecular Cloning, A Laboratory Manual (3rd edition), Cold Spring Harbor Press, Cold Spring Harbor, New York (2000); Coligan et al., Current Protocols in Immunology, John Wiley & Sons, N.Y.; Enna et al., Current Protocols in Pharmacology, John Wiley & Sons, N.Y.; Fingl et al., The Pharmacological Basis of Therapeutics (1975), Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA, 18th edition (1990). These texts can, of course, also be referred to in making or using an aspect of the disclosure.
It is to be understood that the present disclosure also provides pharmaceutical compositions comprising any compound described herein in combination with at least one pharmaceutically acceptable excipient or carrier.
As used herein, the term “pharmaceutical composition” is a formulation containing the compounds of the present disclosure in a form suitable for administration to a subject. In one embodiment, the pharmaceutical composition is in bulk or in unit dosage form. The unit dosage form is any of a variety of forms, including, for example, a capsule, an IV bag, a tablet, a single pump on an aerosol inhaler or a vial. The quantity of active ingredient (e.g., a formulation of the disclosed compound or salt, hydrate, solvate or isomer thereof) in a unit dose of composition is an effective amount and is varied according to the particular treatment involved. One skilled in the art will appreciate that it is sometimes necessary to make routine variations to the dosage depending on the age and condition of the patient. The dosage will also depend on the route of administration. A variety of routes are contemplated, including oral, pulmonary, rectal, parenteral, transdermal, subcutaneous, intravenous, intramuscular, intraperitoneal, inhalational, buccal, sublingual, intrapleural, intrathecal, intranasal, and the like. Dosage forms for the topical or transdermal administration of a compound of this disclosure include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, and inhalants. In one embodiment, the active compound is mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants that are required.
As used herein, the term “pharmaceutically acceptable” refers to those compounds, anions, cations, materials, compositions, carriers, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
As used herein, the term “pharmaceutically acceptable excipient” means an excipient that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic and neither biologically nor otherwise undesirable, and includes excipient that is acceptable for veterinary use as well as human pharmaceutical use. A “pharmaceutically acceptable excipient” as used in the specification and claims includes both one and more than one such excipient.
It is to be understood that a pharmaceutical composition of the disclosure is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., ingestion), inhalation, transdermal (topical), and transmucosal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulphite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
It is to be understood that a compound or pharmaceutical composition of the disclosure can be administered to a subject in many of the well-known methods currently used for chemotherapeutic treatment. For example, a compound of the disclosure may be injected into the blood stream or body cavities or taken orally or applied through the skin with patches. The dose chosen should be sufficient to constitute effective treatment but not so high as to cause unacceptable side effects. The state of the disease condition (e.g., a disease or disorder disclosed herein) and the health of the patient should preferably be closely monitored during and for a reasonable period after treatment.
As used herein, the term “therapeutically effective amount,” refers to an amount of a pharmaceutical agent to treat, ameliorate, or prevent an identified disease or condition, or to exhibit a detectable therapeutic or inhibitory effect. The effect can be detected by any assay method known in the art. The precise effective amount for a subject will depend upon the subject's body weight, size, and health; the nature and extent of the condition; and the therapeutic or combination of therapeutics selected for administration. Therapeutically effective amounts for a given situation can be determined by routine experimentation that is within the skill and judgment of the clinician.
It is to be understood that, for any compound, the therapeutically effective amount can be estimated initially either in cell culture assays, e.g., of neoplastic cells, or in animal models, usually rats, mice, rabbits, dogs, or pigs. The animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans. Therapeutic/prophylactic efficacy and toxicity may be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED50 (the dose therapeutically effective in 50% of the population) and LD50 (the dose lethal to 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, and it can be expressed as the ratio, LD50/ED50. Pharmaceutical compositions that exhibit large therapeutic indices are preferred. The dosage may vary within this range depending upon the dosage form employed, sensitivity of the patient, and the route of administration.
Dosage and administration are adjusted to provide sufficient levels of the active agent(s) or to maintain the desired effect. Factors which may be taken into account include the severity of the disease state, general health of the subject, age, weight, and gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy. Long-acting pharmaceutical compositions may be administered every 3 to 4 days, every week, or once every two weeks depending on half-life and clearance rate of the particular formulation.
The pharmaceutical compositions containing active compounds of the present disclosure may be manufactured in a manner that is generally known, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, or lyophilising processes. Pharmaceutical compositions may be formulated in a conventional manner using one or more pharmaceutically acceptable carriers comprising excipients and/or auxiliaries that facilitate processing of the active compounds into preparations that can be used pharmaceutically. Of course, the appropriate formulation is dependent upon the route of administration chosen.
Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringeability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol and sorbitol, and sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilisation. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
Oral compositions generally include an inert diluent or an edible pharmaceutically acceptable carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser, which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebuliser.
Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.
The active compounds can be prepared with pharmaceutically acceptable carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.
It is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the disclosure are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved.
In therapeutic applications, the dosages of the pharmaceutical compositions used in accordance with the disclosure vary depending on the agent, the age, weight, and clinical condition of the recipient patient, and the experience and judgment of the clinician or practitioner administering the therapy, among other factors affecting the selected dosage. Generally, the dose should be sufficient to result in slowing, and preferably regressing, the symptoms of the disease or disorder disclosed herein and also preferably causing complete regression of the disease or disorder. An effective amount of a pharmaceutical agent is that which provides an objectively identifiable improvement as noted by the clinician or other qualified observer. Improvement in survival and growth indicates regression. As used herein, the term “dosage effective manner” refers to amount of an active compound to produce the desired biological effect in a subject or cell.
It is to be understood that the pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.
It is to be understood that, for the compounds of the present disclosure being capable of further forming salts, all of these forms are also contemplated within the scope of the claimed disclosure.
As used herein, the term “pharmaceutically acceptable salts” refer to derivatives of the compounds of the present disclosure wherein the parent compound is modified by making acid or base salts thereof. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines, alkali or organic salts of acidic residues such as carboxylic acids, and the like. The pharmaceutically acceptable salts include the conventional non-toxic salts or the quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. For example, such conventional non-toxic salts include, but are not limited to, those derived from inorganic and organic acids selected from 2-acetoxybenzoic, 2-hydroxyethane sulphonic, acetic, ascorbic, benzene sulphonic, benzoic, bicarbonic, carbonic, citric, edetic, ethane disulphonic, 1,2-ethane sulphonic, fumaric, glucoheptonic, gluconic, glutamic, glycolic, glycollyarsanilic, hexylresorcinic, hydrabamic, hydrobromic, hydrochloric, hydroiodic, hydroxymaleic, hydroxynaphthoic, isethionic, lactic, lactobionic, lauryl sulphonic, maleic, malic, mandelic, methane sulphonic, napsylic, nitric, oxalic, pamoic, pantothenic, phenylacetic, phosphoric, polygalacturonic, propionic, salicylic, stearic, subacetic, succinic, sulphamic, sulphanilic, sulphuric, tannic, tartaric, toluene sulphonic, and the commonly occurring amine acids, e.g., glycine, alanine, phenylalanine, arginine, etc.
In some embodiments, the pharmaceutically acceptable salt is a sodium salt, a potassium salt, a calcium salt, a magnesium salt, a diethylamine salt, a choline salt, a meglumine salt, a benzathine salt, a tromethamine salt, an ammonia salt, an arginine salt, or a lysine salt.
Other examples of pharmaceutically acceptable salts include hexanoic acid, cyclopentane propionic acid, pyruvic acid, malonic acid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, 4-chlorobenzenesulphonic acid, 2-naphthalenesulphonic acid, 4-toluenesulphonic acid, camphorsulphonic acid, 4-methylbicyclo-[2.2.2]-oct-2-ene-1-carboxylic acid, 3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid, muconic acid, and the like. The present disclosure also encompasses salts formed when an acidic proton present in the parent compound either is replaced by a metal ion, e.g., an alkali metal ion, an alkaline earth ion, or an aluminum ion; or coordinates with an organic base such as ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, and the like. In the salt form, it is understood that the ratio of the compound to the cation or anion of the salt can be 1:1, or any ratio other than 1:1, e.g., 3:1, 2:1, 1:2, or 1:3.
It is to be understood that all references to pharmaceutically acceptable salts include solvent addition forms (solvates) or crystal forms (polymorphs) as defined herein, of the same salt.
The compounds, or pharmaceutically acceptable salts thereof, are administered orally, nasally, transdermally, pulmonary, inhalationally, buccally, sublingually, intraperitoneally, subcutaneously, intramuscularly, intravenously, rectally, intrapleurally, intrathecally, and parenterally. In one embodiment, the compound is administered orally. One skilled in the art will recognise the advantages of certain routes of administration.
The dosage regimen utilising the compounds is selected in accordance with a variety of factors including type, species, age, weight, sex and medical condition of the patient; the severity of the condition to be treated; the route of administration; the renal and hepatic function of the patient; and the particular compound or salt thereof employed.
Techniques for formulation and administration of the disclosed compounds of the disclosure can be found in Remington: the Science and Practice of Pharmacy, 19th edition, Mack Publishing Co., Easton, PA (1995). In an embodiment, the compounds described herein, and the pharmaceutically acceptable salts thereof, are used in pharmaceutical preparations in combination with a pharmaceutically acceptable carrier, excipient, adjuvant, or diluent. Suitable pharmaceutically acceptable carriers include inert solid fillers or diluents and sterile aqueous or organic solutions. The compounds will be present in such pharmaceutical compositions in amounts sufficient to provide the desired dosage amount in the range described herein.
All percentages and ratios used herein, unless otherwise indicated, are by weight. Other features and advantages of the present disclosure are apparent from the different examples. The provided examples illustrate different components and methodology useful in practicing the present disclosure. The examples do not limit the claimed disclosure. Based on the present disclosure the skilled artisan can identify and employ other components and methodology useful for practicing the present disclosure.
In the synthetic schemes described herein, compounds may be drawn with one particular configuration for simplicity. Such particular configurations are not to be construed as limiting the disclosure to one or another isomer, tautomer, regioisomer or stereoisomer, nor does it exclude mixtures of isomers, tautomers, regioisomers or stereoisomers; however, it will be understood that a given isomer, tautomer, regioisomer or stereoisomer may have a higher level of activity than another isomer, tautomer, regioisomer or stereoisomer.
All publications and patent documents cited herein are incorporated herein by reference as if each such publication or document was specifically and individually indicated to be incorporated herein by reference. Citation of publications and patent documents is not intended as an admission that any is pertinent prior art, nor does it constitute any admission as to the contents or date of the same. The invention having now been described by way of written description, those of skill in the art will recognise that the invention can be practiced in a variety of embodiments and that the foregoing description and examples below are for purposes of illustration and not limitation of the claims that follow.
As use herein, the phrase “compound of the disclosure” refers to those compounds which are disclosed herein, both generically and specifically.
For exemplary purpose, salts of the compounds of Formula (I) are synthesised and tested in the examples. It is understood that neutral compounds of Formula (I) may be similarly synthesised and tested using the exemplary procedures described in the examples. Further, it is understood that the salts (e.g., hydrochloride salt) of the compounds of Formula (I) may be converted to the corresponding neutral compounds using routine techniques in the art (e.g., pH adjustment and, optionally, extraction (e.g., into an organic phase)).
Compounds of Formula (I) can be prepared using the methods detailed herein. Those skilled in the art may be able to envisage alternative synthetic routes, using a variety of starting materials and reagents to prepare the disclosed compounds of Formula (I) and to make further modifications. For exemplary purpose, salts of some of the compounds of Formula (I) are synthesised and tested in the examples. It is understood that neutral compounds of Formula (I) may be similarly synthesised and tested using the exemplary procedures described in the examples. Further, it is understood that the salts (e.g., hydrochloride salt) of the compounds of Formula (I) may be converted to the corresponding neutral compounds using routine techniques in the art (e.g., pH adjustment and, optionally, extraction (e.g., into an organic phase)).
1H Nuclear Magnetic Resonance (NMR) spectra were recorded at 400 MHz and at 298 K unless otherwise stated; the chemical shifts (6) are reported in parts per million (ppm), relative to the residual solvent peak and the multiplicity reported together with the associated coupling constant (J), where applicable. Spectra were recorded using a Bruker or Varian instrument with 16, 32 or 64 scans.
LC-MS chromatograms and spectra were recorded using an Agilent 1200 or Shimadzu LC-20 AD&MS 2020 instrument using a C-18 column such as a Luna-C18 2.0×30 mm or Xbridge Shield RPC18 2.1×50 mm. Injection volumes were 0.7-8.0 μL and the flow rates were typically 0.8 or 1.2 mL/min. Detection methods were diode array (DAD) or evaporative light scattering (ELSD) as well as positive ion electrospray ionisation. MS range was 100-1000 Da. Solvents were gradients of water and acetonitrile both containing a modifier (typically 0.01-0.04%) such as trifluoroacetic acid or ammonium carbonate.
UPLC-MS analysis was carried out on a Waters Acquity UPLC system consisting of an Acquity I-Class Sample Manager-FL, Acquity I-Class Binary Solvent Manager and an Acquity UPLC Column Manager. UV detection was afforded using an Acquity UPLC PDA detector (scanning from 210 to 400 nm), whilst mass detection was achieved using an Acquity QDa detector (mass scanning from 100-1250 Da; positive and negative modes simultaneously), and ELS detection was achieved using an Acquity UPLC ELS Detector. A Waters Acquity UPLC BEH C18 column (2.1×50 mm, 1.7 mm) was used to separate the analytes.
Samples were typically prepared by dissolution (with or without sonication) into 1 mL of 50% (v/v) MeCN in water. The resulting solutions were then filtered through a 0.2 mm syringe filter before submitting for analysis. All of the solvents, including formic acid and 36% ammonia solution, were purchased as HPLC grade. Solvents were gradients of water and acetonitrile both containing a modifier (typically 0.01-0.04%) such as formic acid or ammonia.
Step 1. 6-Chloro-4-[2-(trimethylsilyl)ethynyl]pyridazin-3-amine. To solution of 4-bromo-6-chloro-pyridazin-3-amine (30 g, 144 mmol) in toluene (300 mL) at 25° C. under N2 were added CuI (0.82 g, 4.32 mmol), TEA (40.0 mL, 288 mmol) Pd(PPh3)2Cl2 (5.05 g, 7.20 mmol) and ethynyl(trimethyl)silane (19.9 mL, 144 mmol). The mixture was stirred at 100° C. for 5 h under N2. The mixture was filtered through a pad of Celite, the filtrate was diluted with H2O (300 mL) and extracted with EtOAc (3×300 mL). The combined organic layers were washed with brine (900 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether/ethyl acetate=10/1 to 3/1) to give the title compound as a yellow solid. Y=52%. 1H NMR (400 MHz, DMSO-d6) δ 7.57 (s, 1H), 6.81 (s, 2H), 0.26 (s, 9H).
Step 2. 3-Chloro-7H-pyrrolo[2,3-c]pyridazine. To a solution of 6-chloro-4-[2-(trimethylsilyl)ethynyl]pyridazin-3-amine (17 g, 75.3 mmol) in DMF (470 mL) at 25° C. under N2 were added potassium tert-butoxide (12.7 g, 113 mmol) and methylamine hydrochloride (1.02 g, 15.1 mmol). The mixture was stirred at 120° C. for 1 h under N2. The reaction mixture was diluted with H2O (500 mL) and extracted with EtOAc (3×500 mL). The combined organic layers were washed with brine (3×1500 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether/ethyl acetate=0/1 to 1/1) to give the title compound as a yellow solid. Y=30%. 1H NMR (400 MHz, DMSO-d6) δ 12.62 (s, 1H), 8.03-7.99 (m, 2H), 6.54 (d, J=3 Hz, 1H).
Step 1. 3-Methyl-5-(trifluoromethyl)phenol. To a solution of 1-bromo-3-methyl-5-(trifluoromethyl)benzene (9.67 g, 40.5 mmol) in dioxane (100 mL) and H2O (10 mL) at 25° C. were added Pd2(dba)3 (741 mg, 809 μmol), ditert-butyl-[2-(1,3,5-triphenylpyrazol-4-yl)pyrazo]-3-yl]phosphane “BippyPhos” (820 mg, 1.62 mmol) and LiOH·H2O (5.09 g, 121 mmol). The mixture was stirred at 100° C. under N2 for 16 h. The reaction mixture was filtered through a pad of Celite. The filtrate was acidified with 2 M HCl aqueous solution (50 mL), and the resulting mixture was extracted with EtOAc (3×100 mL). The combined organic layers were washed with brine (100 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give the title compound as a yellow oil. Y=84%. 1H NMR (400 MHz, CDCl3) δ 7.02 (s, 1H), 6.89 (s, 1H), 6.83 (s, 1H), 2.37 (s, 3H).
Step 2. 2-Iodo-3-methyl-5-(trifluoromethyl)phenol. To a solution of 3-methyl-5-(trifluoromethyl)phenol (10 g, 56.8 mmol) in toluene (250 mL) at 0° C. was added NaH (60% in mineral oil, 3.63 g, 90.8 mmol). The mixture was stirred at 0° C. for 30 min, then treated with a solution of I2 (9.15 mL, 45.4 mmol) in toluene (50 mL). The resulting mixture was stirred at 25° C. for 2 h. The reaction mixture was cooled to 0° C., quenched with H2O (100 mL), and adjusted to pH ˜4 with 2 M HCl. The resulting mixture was extracted with EtOAc (3×300 mL). The combined organic layers were washed with brine (300 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether/ethyl acetate=20/1 to 10/1) to give the title compound as a yellow oil. Y=47%. 1H NMR (400 MHz, DMSO-d6) δ 10.96 (s, 1H), 7.13 (s, 1H), 6.93 (s, 1H), 2.46-2.39 (s, 3H).
Step 3. 1-(Ethoxymethoxy)-2-iodo-3-methyl-5-(trifluoromethyl)benzene. To a solution of 2-iodo-3-methyl-5-(trifluoromethyl)phenol (6.48 g, 21.5 mmol) in DMF (65 mL) at 25° C. were added Cs2CO3 (6.99 g, 21.5 mmol) and chloromethoxyethane (3.58 mL, 38.6 mmol). The mixture was stirred at 25° C. for 16 h. The reaction mixture was diluted with H2O (20 mL) and the resulting mixture was extracted with EtOAc (3×20 mL). The combined organic layers were washed with brine (20 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether/ethyl acetate=20/1 to 10/1) to give the title compound as a yellow oil. Y=78%. 1H NMR (400 MHz, DMSO-d6) δ 7.36 (s, 1H), 7.18 (s, 1H), 5.41 (s, 2H), 3.74 (q, J=7 Hz, 2H), 2.48 (s, 3H), 1.13 (t, J=7 Hz, 3H).
Step 4. 2-[2-(Ethoxymethoxy)-6-methyl-4-(trifluoromethyl)phenyl]-4,4,5,5-tetramethyl-1,3,2-dioxaborolane. To a solution of 1-(ethoxymethoxy)-2-iodo-3-methyl-5-(trifluoromethyl)benzene (6.0 g, 16.7 mmol) in dioxane (60 mL) at 25° C. were added TEA (17.2 mL, 123 mmol), 4,4,5,5-tetramethyl-1,3,2-dioxaborolane (12.1 mL, 83.3 mmol), Pd(OAc)2 (486 mg, 2.17 mmol) and dicyclohexyl-(2-phenylphenyl)phosphane (1.40 g, 4.00 mmol). The mixture was stirred at 80° C. under N2 for 18 h. The reaction mixture was diluted with H2O (60 mL) and the resulting mixture extracted with EtOAc (3×60 mL). The combined organic layers were washed with brine (60 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether/ethyl acetate=20/1 to 10/1) to give the title compound as a yellow oil. Y=83%. 1H NMR (400 MHz, DMSO-d6) δ 7.15 (s, 1H), 7.12 (s, 1H), 5.26 (s, 2H), 3.66 (q, J=7 Hz, 2H), 2.32 (s, 3H), 1.32 (s, 12H), 1.12 (t, J=7 Hz, 3H).
Step 5. 3-Methyl-2-(4,4,5,5-tetramethyl-1, 3,2-dioxaborolan-2-yl)-5-(trifluoromethyl)phenol. To a solution of 2-[2-(ethoxymethoxy)-6-methyl-4-(trifluoromethyl)phenyl]-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (5.0 g, 13.9 mmol) in DCM (150 mL) at 0° C. was added TFA (30.8 mL, 416 mmol). The reaction mixture was stirred at 0° C. for 1 h. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether/ethyl acetate=20/1 to 10/1) to give the title compound as a yellow oil. Y=62%. 1H NMR (400 MHz, DMSO-d6) δ 9.79 (s, 1H), 6.92 (s, 1H), 6.83 (s, 1H), 2.30 (s, 3H), 1.31 (s, 12H).
Step 1. 4-Chloro-2-methoxy-6-methylaniline. To a solution of 2-bromo-4-chloro-6-methylaniline (125 g, 567 mmol) in MeOH (125 mL) at 25° C. were added 30% NaOMe in MeOH (666 mL, 567 mmol) and CuI (124 g, 652 mmol). The mixture was stirred at reflux for 2 h. The reaction mixture was filtered, the filtrate was diluted with saturated aqueous NH4Cl solution (400 mL) and the resulting mixture was extracted with EtOAc (3×400 mL). The combined organic layers were washed with brine (400 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether/ethyl acetate=I/O to 10/1) to give part of the desired product. The impure fractions were combined, evaporated and further purified by prep-HPLC (column: Phenomenex Luna C18 250×150 mm×15 μm; mobile phase: [H2O (10 mM NH4HCO3)—ACN]; gradient: 35-65% B over 22 min). The two clean batches were combined and lyophilised to give the title compound as a yellow solid. Y=73%. 1H NMR (400 MHz, DMSO-d6) δ 6.72 (d, J=2 Hz, 1H), 6.65 (d, J=2 Hz, 1H), 4.54 (s, 2H), 3.76 (s, 3H), 2.05 (s, 3H).
Step 2. 5-Chloro-2-iodo-1-methoxy-3-methylbenzene. To a solution of 4-chloro-2-methoxy-6-methylaniline (37 g, 216 mmol) in ACN (370 mL) at 25° C. was added 12 (52.1 mL, 259 mmol). The mixture was then treated dropwise with tert-butyl nitrite (38.5 mL, 323 mmol). The mixture was stirred at 25° C. for 12 h. The reaction was quenched with saturated aqueous Na2S2O2 solution (740 mL) and extracted with EtOAc (3×740 mL). The combined organic phase was washed with brine (370 mL), dried with anhydrous Na2SO4, filtered and concentrated under vacuum to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether/ethyl acetate=1/0 to 10/1) to give the title compound as a yellow solid. Y=48%. 1H NMR (400 MHz, DMSO-d6) δ 7.05 (d, J=2 Hz, 1H), 6.89 (d, J=2 Hz, 1H), 3.84 (s, 3H), 2.37 (s, 3H).
Step 3. 5-Chloro-2-iodo-3-methylphenol. To a solution of 5-chloro-2-iodo-1-methoxy-3-methylbenzene (58 g, 205 mmol) in DCM (770 mL) at 0° C. was added BBr3 (39.6 mL, 411 mmol). The mixture was stirred at 25° C. for 8 h. The reaction mixture was poured into water (700 mL) and extracted with DCM (3×700 mL). The combined organic phase was washed with brine (2×700 mL), dried with anhydrous Na2SO4, filtered and concentrated under reduced pressure to give the title compound as a yellow solid. Y=quantitative. 1H NMR (400 MHz, DMSO-d6) δ 10.73 (s, 1H), 6.88 (d, J=2 Hz, 1H), 6.72 (d, J=2 Hz, 1H), 2.34 (s, 3H).
Step 4. 5-Chloro-1-(ethoxymethoxy)-2-iodo-3-methylbenzene. To a solution of 5-chloro-2-iodo-3-methylphenol (55 g, 205 mmol) in ACN (550 mL) was added Cs2CO3 (133 g, 410 mmol) and chloromethoxyethane (19.0 mL, 205 mmol). The mixture was stirred at 25° C. for 6 h. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether/ethyl acetate=I/O to 10/1) to give the title compound as a colourless oil. Y=91%. 1H NMR (400 MHz, DMSO-d6) δ 7.10 (d, J=2 Hz, 1H), 6.99 (d, J=2 Hz, 1H), 5.34 (s, 2H), 3.69 (q, J=7 Hz, 2H), 2.39 (s, 3H), 1.13 (t, J=7 Hz, 3H).
Step 5. 2-[4-Chloro-2-(ethoxymethoxy)-6-methylphenyl]-4,4,5,5-tetramethyl-1, 3,2-dioxaborolane. To a solution of 5-chloro-1-(ethoxymethoxy)-2-iodo-3-methylbenzene (25 g, 76.6 mmol) in dioxane (500 mL) at 25° C. under N2 were added TEA (32.0 mL, 230 mmol), 4,4,5,5-tetramethyl-1,3,2-dioxaborolane (22.2 mL, 153 mmol), Pd(OAc)2 (1.72 g, 7.66 mmol) and dicyclohexyl-(2-phenylphenyl)phosphane (5.37 g, 15.3 mmol). The mixture was stirred at 80° C. under N2 for 5 h. The mixture was diluted with H2O (500 mL) and extracted with EtOAc (3×500 mL). The combined organic layers were washed with brine (500 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Welch Xtimate C18 250×100 mm×10 μm; mobile phase: [H2O (10 mM NH4HCO3)—ACN]; gradient: 50-85% B over 20.0 min) and lyophilised to give the title compound as a white solid. Y=75%. 1H NMR (400 MHz, CDCl3) δ 6.88 (s, 1H), 6.81 (s, 1H), 5.17 (s, 2H), 3.72 (q, J=7 Hz, 2H), 2.33 (s, 3H), 1.38 (s, 12H), 1.23 (t, J=7 Hz, 3H).
Step 6. 5-Chloro-3-methyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenol. A mixture of 2-[4-chloro-2-(ethoxymethoxy)-6-methyl-phenyl]-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (6.0 g, 18.4 mmol) and 4 M HCl in EtOAc (30 mL) was stirred at 25° C. for 2 h. The reaction mixture was concentrated under reduced pressure to give the title compound 5-chloro-3-methyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenol as a yellow oil. Y=99%. 1H NMR (400 MHz, DMSO-d6) δ 9.48 (s, 1H), 6.66 (s, 1H), 6.60 (s, 1H), 2.24 (s, 3H), 1.29 (s, 12H).
Step 1. 4-Cyano-2-methoxy-6-methylphenyl trifluoromethanesulfonate. To a solution of 4-hydroxy-3-methoxy-5-methyl-benzonitrile (2.0 g, 11.9 mmol) in DCM (20 mL) at 0° C. were added pyridine (1.93 mL, 23.9 mmol) and Tf2O (1.97 mL, 11.9 mmol). The mixture was stirred at 0° C. under N2 for 1 h. The reaction mixture was diluted with H2O (20 mL) and extracted with DCM (3×20 mL). The combined organic layers were washed with brine (30 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether/ethyl acetate=10/1 to 3/1) to give the title compound as a white solid. Y=85%. 1H NMR (400 MHz, DMSO-d6) δ 7.73 (d, J=1 Hz, 1H), 7.57 (d, J=1 Hz, 1H), 3.94 (s, 3H), 2.34 (s, 3H).
Step 2. 3-Methoxy-5-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzonitrile. To a solution of 4-cyano-2-methoxy-6-methylphenyl trifluoromethanesulfonate (0.75 g, 2.54 mmol) in DMF (7.5 mL) at 25° C. under N2 were added 4,4,5,5-tetramethyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1,3,2-dioxaborolane (968 mg, 3.81 mmol), KOAc (1.12 g, 11.4 mmol) and Pd(dppf)Cl2.CH2Cl2 (415 mg, 508 μmol). The mixture was stirred at 100° C. under N2 for 12 h. The reaction mixture was diluted with H2O (10 mL) and extracted with EtOAc (3×10 mL). The combined organic layers were washed with brine (30 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether/ethyl acetate=10/1 to 5/1) to give the title compound as a white solid. Y=86%. 1H NMR (400 MHz, DMSO-d6) δ 7.28-7.16 (m, 2H), 3.76 (s, 3H), 2.27 (s, 3H), 1.30 (s, 12H).
Step 3. (4-Cyano-2-hydroxy-6-methylphenyl)boronic acid. To a solution of 3-methoxy-5-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzonitrile (1.0 g, 3.66 mmol) in DCM (10 mL) at 0° C. was added BBr3 (882 μL, 9.15 mmol). The mixture was stirred at 0° C. under N2 for 0.5 h. The reaction mixture was quenched with H2O (10 mL), filtered and the filter cake dried under vacuum to give a residue. The residue was triturated with EtOAc (3 ml) for 10 min, filtered and the filter cake was dried to give the title compound as a grey solid.
Step 1. 3-Chloro-7-[(oxan-4-yl)methyl]-7H-pyrrolo[2,3-c]pyridazine. To a solution of 3-chloro-7H-pyrrolo[2,3-c]pyridazine (Intermediate A1, 500 mg, 3.26 mmol) in DMF (5 mL) at 25° C. were added 4-(bromomethyl)tetrahydropyran (700 mg, 3.91 mmol) and K2CO3 (900 mg, 6.51 mmol). The reaction mixture was stirred at 80° C. for 2 h under N2. The reaction mixture was purified by prep-HPLC (column: Phenomenex Luna C18 75×30 mm×3 μm; mobile phase: [H2O (0.1% TFA)—ACN]; gradient: 5-35% B over 8.0 min) and lyophilised to give the title compound as a yellow solid. Y=61%. 1H NMR (400 MHz, DMSO-d6) δ 8.06 (s, 1H), 8.05 (s, 1H), 6.57 (d, J=3 Hz, 1H), 4.33 (d, 2H), 3.84-3.78 (m, 2H), 2.19-2.09 (m, 2H), 2.23-2.15 (m, 1H), 1.38-1.20 (m, 4H).
Step 2. 3-Methyl-2-{7-[(oxan-4-yl)methyl]-7H-pyrrolo[2,3-c]pyridazin-3-yl}-5-(trifluoromethyl)phenol. To a solution of 3-chloro-7-[(oxan-4-yl)methyl]-7H-pyrrolo[2,3-c]pyridazine (150 mg, 596 μmol) in dioxane (7.5 mL) and H2O (2.5 mL) at 25° C. under N2 were added 3-methyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-5-(trifluoromethyl)phenol (Intermediate B1, 180 mg, 596 μmol), Pd(PPh3)4(34 mg, 30 μmol) and Na2CO3 (189 mg, 1.79 mmol). The mixture was stirred at 120° C. for 4 h under N2. The reaction mixture was diluted with H2O (10 mL) and extracted with EtOAc (3×10 mL). The combined organic layers were washed with brine (10 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Phenomenex Luna C18 100×40 mm×5 μm; mobile phase: [H2O (0.04% HCl)—ACN]; gradient: 15-45% B over 8.0 min) and lyophilised to give the title compound as a yellow solid. Y=23%. 1H NMR (400 MHz, DMSO-d6) δ 10.93 (br. s, 1H), 8.74-8.43 (m, 2H), 7.30-7.20 (m, 2H), 7.02 (s, 1H), 4.41 (d, J=7 Hz, 2H), 3.90-3.80 (m, 2H), 3.33-3.20 (m, 2H), 2.34-2.20 (m, 1H), 2.16 (s, 3H), 1.52-1.29 (m, 4H). 1H NMR (400 MHz, DMSO-d6+D2O) δ 8.51-8.37 (m, 2H), 7.24 (s, 1H), 7.13 (s, 1H), 7.00 (d, J=3 Hz, 1H), 4.34 (d, J=7 Hz, 2H), 3.85-3.72 (m, 2H), 3.30-3.20 (m, 2H), 2.25-2.14 (m, 1H), 2.09 (s, 3H), 1.47-1.23 (m, 4H). LCMS (ESI): m/z: [M+H]+=392.1.
Step 1. 3-Chloro-7-[(oxolan-2-yl)methyl]-7H-pyrrolo[2,3-c]pyridazine. To a solution of 3-chloro-7H-pyrrolo[2,3-c]pyridazine (Intermediate A1, 500 mg, 3.26 mmol) in DMF (5 mL) at 25° C. were added 2-(bromomethyl)tetrahydrofuran (645 mg, 3.91 mmol) and K2CO3 (900 mg, 6.51 mmol). The reaction mixture was stirred at 80° C. for 2 h. The reaction mixture was purified by prep-HPLC (column: Phenomenex Luna C18 75×30 mm×3 μm; mobile phase: [H2O (0.1% TFA)—ACN]; gradient: 5-35% B over 8.0 min) and lyophilised to give the title compound as a yellow solid. Y=65%.
Step 2. 3-Methyl-2-{7-[(oxolan-2-yl)methyl]-7H-pyrrolo[2,3-c]pyridazin-3-yl}-5-(trifluoromethyl)phenol. To a solution of 3-chloro-7-[(oxolan-2-yl)methyl]-7H-pyrrolo[2,3-c]pyridazine (150 mg, 631 μmol) in dioxane (7.5 mL) and H2O (2.5 mL) at 25° C. under N2 were added 3-methyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-5-(trifluoromethyl)phenol (Intermediate B1, 191 mg, 631 μmol), Pd(PPh3)4(36 mg, 32 μmol) and Na2CO3 (201 mg, 1.89 mmol). The mixture was stirred at 120° C. for 4 h under N2. The reaction mixture was diluted with H2O (10 mL) and extracted with EtOAc (3×10 mL). The combined organic layers were washed with brine (10 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Waters Xbridge BEH C18 100×30 mm×10 μm; mobile phase: [H2O (10 mM NH4HCO3)—ACN]; gradient: 40-70% B over 8.0 min) and lyophilised to give the title compound as a yellow solid. Y=7%. 1H NMR (400 MHz, DMSO-d6) δ 10.04 (br. s, 1H), 7.93 (d, J=3 Hz, 1H), 7.80 (s, 1H), 7.14 (s, 1H), 7.10 (s, 1H), 6.58 (d, J=3 Hz, 1H), 4.59-4.52 (m, 1H), 4.49-4.42 (m, 1H), 4.39-4.28 (m, 1H), 3.87-3.74 (m, 1H), 3.70-3.61 (m, 1H), 2.08-1.94 (m, 4H), 1.90-1.75 (m, 2H), 1.71-1.59 (m, 1H). 1H NMR (400 MHz, DMSO-d6+D2O) δ 7.91 (d, J=3 Hz, 1H), 7.79 (s, 1H), 7.14 (s, 1H), 7.09 (s, 1H), 6.59 (d, J=3 Hz, 1H), 4.58-4.50 (m, 1H), 4.49-4.40 (m, 1H), 4.37-4.27 (m, 1H), 3.84-3.75 (m, 1H), 3.69-3.59 (m, 1H), 2.08-1.93 (m, 4H), 1.85-1.70 (m, 2H), 1.70-1.57 (m, 1H). LCMS (ESI): m/z: [M+H]+=378.1.
Step 1. 3-Chloro-7-[(oxolan-3-yl)methyl]-7H-pyrrolo[2,3-c]pyridazine. To a solution of 3-chloro-7H-pyrrolo[2,3-c]pyridazine (Intermediate A1, 500 mg, 3.26 mmol) in DMF (5 mL) at 25° C. under N2 were added K2CO3 (900 mg, 6.51 mmol) and 3-(bromomethyl)tetrahydrofuran (537 mg, 3.26 mmol). The mixture was stirred at 80° C. for 3 h. The reaction mixture was diluted with H2O (5 mL) and extracted with EtOAc (3×5 mL). The combined organic layers were washed with brine (3×5 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Waters Xbridge BEH C18 250×50 mm×10 μm; mobile phase: [H2O (10 mM NH4HCO3)—ACN]; gradient: 20-40% B over 10.0 min) and lyophilised to give the title compound as a yellow solid. Y=52%.
1H NMR (400 MHz, DMSO-d6) δ 8.12 (d, 1H), 8.04 (s, 1H), 6.58 (d, 1H), 4.39 (d, 2H), 3.83-3.76 (m, 1H), 3.67-3.58 (m, 2H), 3.53-3.48 (m, 1H), 2.95-2.84 (m, 1H), 1.92-1.83 (m, 1H), 1.66-1.55 (m, 1H).
Step 2. 3-Methyl-2-{7-[(oxolan-3-yl)methyl]-7H-pyrrolo[2,3-c]pyridazin-3-yl}-5-(trifluoromethyl)phenol. To a solution of 3-chloro-7-[(oxolan-3-yl)methyl]-7H-pyrrolo[2,3-c]pyridazine (150 mg, 631 μmol) in dioxane (2 mL) and H2O (0.5 mL) at 25° C. under N2 were added Na2CO3 (201 mg, 1.89 mmol), Pd(dppf)Cl2 (23 mg, 31.6 μmol) and 3-methyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-5-(trifluoromethyl)phenol (Intermediate B1, 381 mg, 1.26 mmol). The mixture was stirred at 80° C. for 12 h. The reaction mixture was diluted with H2O (2 mL) and extracted with EtOAc (3×2 mL). The combined organic layers were washed with brine (3×2 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Waters Xbridge C18 150×50 mm×10 μm; mobile phase: [H2O (10 mM NH4HCO3)—ACN]; gradient 30-60% B over 8.0 min) and lyophilised to give the title compound as a white solid. Y=21%. 1H NMR (400 MHz, DMSO-d6) δ 10.03 (br. s, 1H), 8.03 (d, J=3 Hz, 1H), 7.81 (s, 1H), 7.22-7.05 (m, 2H), 6.60 (d, J=3 Hz, 1H), 4.46 (d, J=8 Hz, 2H), 3.90-3.81 (m, 1H), 3.72-3.63 (m, 2H), 3.61-3.54 (m, 1H), 3.01-2.93 (m, 1H), 2.06 (s, 3H), 2.00-1.88 (m, 1H), 1.76-1.63 (m, 1H). 1H NMR (400 MHz, DMSO-d6+D2O) δ 8.02 (d, J=3 Hz, 1H), 7.81 (s, 1H), 7.15 (s, 1H), 7.11 (s, 1H), 6.60 (d, J=3 Hz, 1H), 4.45 (d, J=8 Hz, 2H), 3.87-3.78 (m, 1H), 3.74-3.60 (m, 2H), 3.58-3.53 (m, 1H), 3.03-2.89 (m, 1H), 2.05 (s, 3H), 1.98-1.88 (m, 1H), 1.78-1.61 (m, 1H). LCMS (ESI): m/z: [M+H]+=378.0.
Step 1. Tert-butyl 4-({3-chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}methyl)piperidine-1-carboxylate. To a solution of tert-butyl 4-(bromomethyl)piperidine-1-carboxylate (4.35 g, 15.6 mmol) in DMF (20 mL) at 25° C. were added 3-chloro-7H-pyrrolo[2,3-c]pyridazine (Intermediate A1, 2.0 g, 13.0 mmol) and K2CO3 (3.60 g, 26.1 mmol). The mixture was stirred at 80° C. for 2 h under N2. The reaction mixture was diluted with H2O (20 mL) and extracted with EtAOc (3×20 mL). The combined organic layers were washed with brine (50 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Phenomenex Luna C18 (250×70 mm, 15 μm); mobile phase: [H2O (0.1% TFA)—ACN]; gradient: 40-70% B over 20 min) and lyophilised to give the title compound as a yellow oil. Y=44%. 1H NMR (400 MHz, DMSO-d6) δ 8.06-8.01 (m, 2H), 6.57 (d, J=3 Hz, 1H), 4.30 (d, J=7 Hz, 2H), 3.95-3.84 (m, 2H), 2.68-2.56 (m, 2H), 2.20-2.06 (m, 1H), 1.43-1.40 (m, 2H), 1.37 (s, 9H), 1.18-1.03 (m, 2H).
Step 2. 4-({3-Chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}methyl)piperidine. A mixture of tert-butyl 4-({3-chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}methyl)piperidine-1-carboxylate (2.0 g, 5.70 mmol) and 4 M HCl in EtOAc (20 mL) was stirred at 25° C. for 2 h. The reaction mixture was filtered and the resulting solid isolated and basified to pH=8˜9 by Na2CO3 aqueous solution. The resulting solution was lyophilised to give a residue (a mixture of product and salts). The residue was triturated with 1:1 DCM/EtOAc, stirred for 30 min, filtered and the filtrate concentrated under reduced pressure to give the title compound as a white solid. Y=98%.
Step 3. 4-({3-Chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}methyl)-1-methylpiperidine hydrochloride. To a solution of 4-({3-chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}methyl)piperidine (0.30 g, 1.20 mmol) in MeOH (5 mL) at 25° C. were added 37% formaldehyde (223 μL, 2.99 mmol) and sodium cyanoborohydride (188 mg, 2.99 mmol). The mixture was stirred at 25° C. for 2 h. The mixture was purified by prep-HPLC (column: Phenomenex Luna C18 100×40 mm×5 μm; mobile phase: [H2O (0.04% HCl)—ACN]; gradient:1-25% B over 8.0 min) and lyophilised to give the title compound as a yellow oil. Y=83%.
Step 4. 3-Methyl-2-{7-[(I-methylpiperidin-4-yl)methyl]-7H-pyrrolo[2,3-c]pyridazin-3-yl}-5-(trifluoromethyl)phenol. To a solution of 4-({3-chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}methyl)-1-methylpiperidine (120 mg, 398 μmol) in H2O (0.4 mL) and dioxane (1.2 mL) at 25° C. under N2 were added Na2CO3 (127 mg, 1.20 mmol), Pd(dppf)Cl2 (15 mg, 20 μmol) and 3-methyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-5-(trifluoromethyl)phenol (Intermediate B1, 120 mg, 398 μmol). The mixture was stirred at reflux under N2 for 12 h. The mixture was filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Waters Xbridge Prep OBD C18 150×40 mm×10 μm; mobile phase: [H2O (10 mM NH4HCO3)—ACN]; gradient: 25-55% B over 8.0 min) and lyophilised to give the title compound as a white solid. Y=25%. 1H NMR (400 MHz, DMSO-d6) δ 7.96 (d, J=3 Hz, 1H), 7.80 (s, 1H), 7.15 (s, 1H), 7.10 (s, 1H), 6.58 (d, J=3 Hz, 1H), 4.35 (d, J=7 Hz, 2H), 2.81-2.66 (m, 2H), 2.12 (s, 3H), 2.05 (s, 3H), 1.99-1.91 (m, 1H), 1.85-1.68 (m, 2H), 1.53-1.41 (m, 2H), 1.37-1.29 (m, 2H). 1H NMR (400 MHz, DMSO-d6+D2O) δ 7.90 (d, J=3 Hz, 1H), 7.78 (s, 1H), 7.14 (s, 1H), 7.08 (s, 1H), 6.59 (d, J=3 Hz, 1H), 4.32 (d, J=7 Hz, 2H), 2.81-2.68 (m, 2H), 2.10 (s, 3H), 2.02 (s, 3H), 1.97-1.87 (m, 1H), 1.86-1.71 (m, 2H), 1.51-1.39 (m, 2H), 1.38-1.24 (m, 2H). LCMS (ESI): m/z: [M+H]+=405.1.
To a solution of 3-chloro-7H-pyrrolo[2,3-c]pyridazine (Intermediate A1, 150 mg, 977 μmol) and 3-methyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-5-(trifluoromethyl)phenol (Intermediate B1, 443 mg, 1.47 mmol) in dioxane (7.5 mL) and H2O (2.5 mL) at 25° C. under N2 were added Pd(PPh3)4(56 mg, 49 μmol) and Na2CO3 (311 mg, 2.93 mmol). The mixture was stirred at reflux under N2 for 3 h. The reaction mixture was diluted with H2O (2 mL) and extracted with EtOAc (3×2 mL). The combined organic layers were washed with brine (2 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Waters Xbridge BEH C18 100×30 mm×10 μm; mobile phase: [H2O (10 mM NH4HCO3)—ACN]; gradient: 20-70% B over 8.0 min) and lyophilised to give the title compound as a white solid. Y=10%. 1H NMR (400 MHz, DMSO-d6) δ 12.41 (s, 1H), 10.01 (s, 1H), 7.91 (d, J=3 Hz, 1H), 7.79 (s, 1H), 7.15 (s, 1H), 7.10 (s, 1H), 6.56 (d, J=3 Hz, 1H), 2.06 (s, 3H). 1H NMR (400 MHz, DMSO-d6+D2O) δ 7.88 (d, J=3 Hz, 1H), 7.78 (s, 1H), 7.14 (s, 1H), 7.08 (s, 1H), 6.58 (d, J=3 Hz, 1H), 2.03 (s, 3H). LCMS (ESI): m/z: [M+H]+=294.2.
Step 1. 3-Chloro-7-methyl-7H-pyrrolo[2,3-c]pyridazine. To a solution of 3-chloro-7H-pyrrolo [2,3-c]pyridazine (Intermediate A1, 500 mg, 3.26 mmol) in DMF (5 mL) at 25° C. under N2 were added CH3I (203 μL, 3.26 mmol) and K2CO3 (675 mg, 4.88 mmol). The mixture was stirred at 25° C. under N2 for 4 h. The reaction mixture was diluted with H2O (5 mL) and extracted with EtOAc (3×5 mL). The combined organic layers were washed with brine (5 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Phenomenex Luna C18 75×30 mm×3 μm; mobile phase: [H2O (0.1% TFA)—ACN]; gradient: 1-30% B over 8.0 min) and lyophilised to give the title compound as a yellow oil. Y=55%. 1H NMR (400 MHz, DMSO-d6) δ 8.25-7.91 (m, 2H), 6.56 (d, J=3 Hz, 1H), 3.96 (s, 3H).
Step 2. 3-Methyl-2-{7-methyl-7H-pyrrolo[2,3-c]pyridazin-3-yl}-5-(trifluoromethyl)phenol. To a solution of 3-chloro-7-methyl-7H-pyrrolo[2,3-c]pyridazine (200 mg, 1.19 mmol) and 3-methyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-5-(trifluoromethyl)phenol (Intermediate B1, 541 mg, 1.79 mmol) in dioxane (10 mL) and H2O (3.3 mL) at 25° C. under N2 were added Pd(PPh3)4(69 mg, 60 μmol) and Na2CO3 (379 mg, 3.58 mmol). The mixture was stirred at reflux under N2 for 12 h. The reaction mixture was diluted with H2O (10 mL) and extracted with EtOAc (3×10 mL). The combined organic layers were washed with brine (10 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Phenomenex Luna C18 100×40 mm×5 μm; mobile phase: [H2O (0.04% HCl)—ACN]; gradient: 20-40% B over 8.0 min) and lyophilised to give the title compound as a yellow solid. Y=23%. 1H NMR (400 MHz, DMSO-d6) δ 11.93 (br. s, 1H), 8.57 (s, 1H), 8.50 (s, 1H), 7.27 (s, 1H), 7.26 (s, 1H), 6.99 (d, J=3 Hz, 1H), 4.08 (s, 3H), 2.15 (s, 3H). 1H NMR (400 MHz, DMSO-d6+D2O) δ 8.54 (d, J=3 Hz, 1H), 8.50 (s, 1H), 7.28 (s, 1H), 7.19 (s, 1H), 7.01 (d, J=3 Hz, 1H), 4.06 (s, 3H), 2.13 (s, 3H). LCMS (ESI): m/z: [M+H]+=308.2.
Step 1. 4-({3-Chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}methyl)-1-(2,2,2-trifluoroethyl)piperidine. To a solution of 4-({3-chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}methyl)piperidine (for synthesis see Compound 4)(300 mg, 1.04 mmol) in DMF (3 mL) at 25° C. under N2 was added TEA (582 μL, 4.18 mmol) and 2,2,2-trifluoroethyl trifluoromethanesulfonate (291 mg, 1.25 mmol). The resulting mixture was stirred at 25° C. under N2 for 8 h. The reaction mixture was diluted with H2O (2 mL) and extracted with EtOAc (3×3 mL). The combined organic layers were washed with brine (3 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether/ethyl acetate=10/1 to 1/1, Rf=0.4) to give the title compound as a white solid. Y=43%. 1H NMR (400 MHz, DMSO-d6) δ 8.05-8.03 (m, 2H), 6.57 (d, J=3 Hz, 1H), 4.30 (d, J=7 Hz, 2H), 3.17-3.04 (m, 2H), 2.88-2.86 (m, 2H), 2.28-2.20 (m, 2H), 1.97-1.89 (m, 1H), 1.45-1.37 (m, 2H), 1.33-1.21 (m, 2H).
Step 2. 5-Chloro-3-methyl-2-(7-{[1-(2,2,2-trifluoroethyl)piperidin-4-yl]methyl}-7H-pyrrolo[2,3-c]pyridazin-3-yl)phenol. To a solution of 4-({3-chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}methyl)-1-(2,2,2-trifluoroethyl)piperidine (100 mg, 301 μmol) in dioxane (0.3 mL) and H2O (0.1 mL) at 25° C. under N2 were added 5-chloro-3-methyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenol (Intermediate B2, 97 mg, 361 μmol), Pd(dppf)Cl2 (22 mg, 30 μmol) and Na2CO3 (96 mg, 0.90 mmol). The mixture was stirred at reflux under N2 for 8 h. The reaction mixture was diluted with H2O (1 mL) and extracted with EtOAc (3×1 mL). The combined organic layers were washed with brine (1 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Waters Xbridge BEH C18 100×30 mm×10 μm; mobile phase: [H2O (10 mM NH4HCO3)—ACN]; gradient: 45-75% B over 8.0 min) and lyophilised to give the title compound as a white solid. Y=26%. 1H NMR (400 MHz, DMSO-d6) δ 9.85 (s, 1H), 7.92 (d, J=3 Hz, 1H), 7.75 (s, 1H), 6.88-6.85 (m, 2H), 6.56 (d, J=3 Hz, 1H), 4.34 (d, J=7 Hz, 2H), 3.19-3.05 (m, 2H), 2.92-2.89 (m, 2H), 2.34-2.19 (m, 2H), 2.07-1.93 (m, 4H), 1.51-1.48 (m, 2H), 1.40-1.26 (m, 2H). 1H NMR (400 MHz, DMSO-d6+D2O) δ 7.90 (d, J=3 Hz, 1H), 7.74 (s, 1H), 6.87-6.85 (m, 2H), 6.56 (d, J=3 Hz, 1H), 4.33 (d, J=7 Hz, 2H), 3.16-3.05 (m, 2H), 2.90-2.88 (m, 2H), 2.29-2.34 (m, 2H), 2.10-1.90 (m, 4H), 1.50-1.47 (m, 2H), 1.40-1.25 (m, 2H). LCMS (ESI): m/z: [M+H]+=439.3.
Step 1. Tert-butyl 3-({3-chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}methyl)piperidine-1-carboxylate. To a solution of 3-chloro-7H-pyrrolo[2,3-c]pyridazine (Intermediate A1, 0.33 g, 2.15 mmol) in DMF (3.3 mL) at 25° C. under N2 were added tert-butyl 3-(bromomethyl)piperidine-1-carboxylate (598 mg, 2.15 mmol) and K2CO3 (594 mg, 4.30 mmol). The mixture was stirred at 25° C. for 12 h under N2. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Waters Xbridge Prep OBD C18 150×40 mm×10 μm; mobile phase: [H2O (10 mM NH4HCO3)—ACN]; gradient: 25-55% B over 8.0 min) and lyophilised to give the title compound as a yellow gum. Y=42%. 1H NMR (400 MHz, DMSO-d6) δ 8.08 (d, J=3 Hz, 1H), 8.06-8.04 (m, 1H), 6.60 (d, J=3 Hz, 1H), 4.31 (d, J=7 Hz, 2H), 3.74-3.61 (m, 2H), 2.89-2.78 (m, 1H), 2.75-2.62 (m, 1H), 2.19-2.03 (m, 1H), 1.71-15.3 (m, 2H), 1.38-1.17 (m, 11H).
Step 2. 3-({3-Chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}methyl)piperidine hydrochloride. A mixture of tert-butyl 3-({3-chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}methyl)piperidine-1-carboxylate (0.30 g, 855 μmol) and 4 M HCl in EtOAc (5 mL) were stirred at 25° C. for 2 h. The reaction mixture was concentrated under reduced pressure to give the title compound as a white solid. Y=98%.
Step 3. 3-({3-Chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}methyl)-1-methylpiperidine. To a solution of 3-({3-chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}methyl)piperidine hydrochloride (290 mg, 1.01 mmol) in MeOH (6 mL) at 25° C. under N2 were added 37% formaldehyde (aq.) (376 μL, 5.05 mmol) and NaOAc (414 mg, 5.05 mmol). The mixture was stirred at 25° C. under N2 for 1 h, then treated with NaBH3CN (317 mg, 5.05 mmol). The mixture was stirred at 25° C. under N2 for 1 h. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Waters Xbridge BEH C18 100×30 mm×10 μm; mobile phase: [H2O (10 mM NH4HCO3)—ACN]; gradient: 1-50% B over 8.0 min) and lyophilised to give the title compound as a white solid. Y=26%. 1H NMR (400 MHz, DMSO-d6) δ 8.05 (d, J=3 Hz, 1H), 8.03 (s, 1H), 6.58 (d, J=3 Hz, 1H), 4.38-4.24 (m, 2H), 2.62-2.52 (m, 1H), 2.47-2.36 (m, 1H), 2.29-2.16 (m, 1H), 2.08 (s, 3H), 1.93-1.83 (m, 1H), 1.81-1.69 (m, 1H), 1.67-1.57 (m, 1H), 1.52-1.33 (m, 2H), 1.03-0.90 (m, 1H).
Step 4. 5-Chloro-3-methyl-2-{7-[(I-methylpiperidin-3-yl)methyl]-7H-pyrrolo[2,3-c]pyridazin-3-yl}phenol. To a solution of 3-({3-chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}methyl)-1-methylpiperidine (40 mg, 118 μmol) in dioxane (0.8 mL) and H2O (0.16 mL) at 25° C. under N2 were added 5-chloro-3-methyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenol (Intermediate B2, 32 mg, 118 μmol), Pd(dppf)Cl2 (9 mg, 12 μmol) and Na2CO3 (37 mg, 353 μmol). The mixture was stirred at 100° C. under N2 for 2 h. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Waters Xbridge BEH C18 100×30 mm×10 μm; mobile phase: [H2O (10 mM NH4HCO3)—ACN]; gradient: 25-55% B over 8.0 min) and lyophilised to give the title compound as a white solid. Y=34%. 1H NMR (400 MHz, DMSO-d6) δ 9.85 (s, 1H), 7.92 (d, J 3 Hz, 1H), 7.75 (s, 1H), 6.92-6.81 (m, 2H), 6.57 (d, J=3 Hz, 1H), 4.42-4.29 (m, 2H), 2.63-2.55 (m, 1H), 2.53-2.52 (m, 1H), 2.31-2.21 (m, 1H), 2.11 (s, 3H), 1.98 (s, 3H), 1.95-1.89 (m, 1H), 1.89-1.77 (m, 1H), 1.71-1.62 (m, 1H), 1.60-1.52 (m, 1H), 1.50-1.36 (m, 1H), 1.09-0.96 (m, 1H). 1H NMR (400 MHz, DMSO-d6+D2O) δ 7.89 (d, J=3 Hz, 1H), 7.74 (s, 1H), 6.89-6.81 (m, 2H), 6.57 (d, J=3 Hz, 1H), 4.43-4.21 (m, 2H), 2.64-2.56 (m, 1H), 2.55-2.52 (m, 1H), 2.31-2.17 (m, 1H), 2.09 (s, 3H), 1.96 (s, 3H), 1.93-1.84 (m, 1H), 1.87-1.76 (m, 1H), 1.68-1.60 (m, 1H), 1.58-1.49 (m, 1H), 1.48-1.35 (m, 1H), 1.07-0.94 (m, 1H). 1H NMR (400 MHz, methanol-d4) δ 7.84-7.80 (m, 2H), 6.84 (s, 1H), 6.81 (d, J=3 Hz, 1H), 6.64 (d, J=3 Hz, 1H), 4.48-4.32 (m, 3H), 2.80 (d, J=11 Hz, 1H), 2.69 (d, J=11 Hz, 1H), 2.44-2.30 (m, 1H), 2.22 (s, 3H), 2.03 (s, 3H), 2.01-1.95 (m, 1H), 1.95-1.85 (m, 1H), 1.80-1.67 (m, 2H), 1.66-1.51 (m, 1H), 1.20-1.06 (m, 1H). LC-MS (ESI): m/z: [M+H]+=371.3.
Step 1. 1-[4-({3-Chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}methyl)piperidin-1-yl]ethan-1-one. To a solution of 3-chloro-7-(4-piperidylmethyl)pyrrolo[2,3-c]pyridazine (for synthesis see Compound 4)(250 mg, 1.0 mmol) in DCM (3 mL) at 0° C. were added TEA (555 μL, 3.99 mmol) and acetyl chloride (142 μL, 1.99 mmol). The mixture was stirred at 25° C. for 2 h. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Waters Xbridge BEH C18 100×30 mm×10 μm; mobile phase: [H2O (10 mM NH4HCO3)—ACN]; gradient: 5-50% B over 8.0 min) and lyophilised to give the title compound as a white solid. Y=82%. 1H NMR (400 MHz, DMSO-d6) δ 8.22-7.76 (m, 2H), 6.58 (d, J=4 Hz, 1H), 4.46-4.17 (m, 3H), 3.80-3.70 (m, 1H), 3.02-2.86 (m, 1H), 2.47-2.36 (m, 1H), 2.25-2.10 (m, 1H), 1.96 (s, 3H), 1.63-1.38 (m, 2H), 1.29-0.89 (m, 2H).
Step 2. 1-(4-{[3-(4-Chloro-2-hydroxy-6-methylphenyl)-7H-pyrrolo[2,3-c]pyridazin-7-yl]methyl}piperidin-1-yl)ethan-1-one. To a solution of 1-[4-({3-chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}methyl)piperidin-1-yl]ethan-1-one (220 mg, 751 μmol) in dioxane (10 mL) and H2O (3 mL) at 25° C. under N2 were added 5-chloro-3-methyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenol (Intermediate B2, 303 mg, 1.13 mmol), Pd(PPh3)4(43 mg, 38 μmol) and Na2CO3 (187 mg, 2.25 mmol). The mixture was stirred at 80° C. under N2 for 5 h. The reaction mixture was diluted with H2O (3 mL) and extracted with EtOAc (3×3 mL). The combined organic layers were washed with brine (3 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Phenomenex Luna C18 100×40 mm×5 μm; mobile phase: [H2O (0.04% HCl)—ACN]; gradient: 10-40% B over 8.0 min) and lyophilised to give the title compound as a white solid. Y=17%. 1H NMR (400 MHz, DMSO-d6) δ 10.90 (br. s, 1H), 8.71 (d, J=4 Hz, 1H), 8.63 (s, 1H), 7.09 (d, J=4 Hz, 1H), 7.06 (d, J=2 Hz, 1H), 7.03 (d, J=2 Hz, 1H), 4.45-4.32 (m, 3H), 3.90-3.70 (m, 1H), 3.05-2.90 (m, 1H), 2.50-2.40 (m, 1H), 2.30-2.20 (m, 1H), 2.11 (s, 3H), 1.98 (s, 3H), 1.69-1.46 (m, 2H), 1.37-1.04 (m, 2H). 1H NMR (400 MHz, DMSO-d6+D2O) δ 8.66 (d, J=4 Hz, 1H), 8.60 (s, 1H), 7.09 (d, J=4 Hz, 1H), 7.02 (d, J=2 Hz, 1H), 6.97 (d, J=2 Hz, 1H), 4.59-4.19 (m, 3H), 3.85-3.70 (m, 1H), 3.00-2.90 (m, 1H), 2.48-2.42 (m, 1H), 2.30-2.10 (m, 1H), 2.08 (s, 3H), 1.97 (s, 3H), 1.71-1.46 (m, 2H), 1.38-1.01 (m, 2H). 1H NMR (400 MHz, MeOD) δ 8.60 (d, J=4 Hz, 1H), 8.56 (s, 1H), 7.12 (d, J=4 Hz, 1H), 7.01 (d, J=2 Hz, 1H), 6.93 (d, J=2 Hz, 1H), 4.64-4.39 (m, 3H), 4.00-3.90 (m, 1H), 3.26-3.03 (m, 1H), 2.71-2.58 (m, 1H), 2.45-3.30 (m, 1H), 2.17 (s, 3H), 2.13 (s, 3H), 1.86-1.61 (m, 2H), 1.53-1.19 (m, 2H). LCMS (ESI): m/z: [M+H]+=399.3.
Step 1. 4-({3-Chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}methyl)-1-(2,2-difluoroethyl)piperidine. To a solution of 3-chloro-7-(4-piperidylmethyl)pyrrolo[2,3-c]pyridazine (for synthesis see Compound 4)(200 mg, 0.80 mmol) in DMF (3 mL) at 25° C. were added K2CO3 (220 mg, 1.60 mmol) and 1,1-difluoro-2-iodo-ethane (153 mg, 0.80 mmol). The resulting mixture was stirred at 80° C. for 12 h. The solution was diluted with H2O (3 mL) and extracted with ethyl acetate (3×3 mL). The combined organic layers were washed with brine (3 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (0-50% petroleum ether in ethyl acetate) to give the title compound as a yellow oil. Y=72%. 1H NMR (400 MHz, DMSO-d6) δ 8.05-8.02 (m, 2H), 6.57 (d, J=3 Hz, 1H), 6.24-5.91 (m, 1H), 4.32-4.26 (m, 2H), 2.87-2.79 (m, 2H), 2.73-2.60 (m, 2H), 2.10-2.02 (m, 2H), 2.01-1.84 (m, 1H), 1.46-1.34 (m, 2H), 1.32-1.21 (m, 2H).
Step 2. 5-Chloro-2-(7-{[1-(2,2-difluoroethyl)piperidin-4-yl]methyl}-7H-pyrrolo[2,3-c]pyridazin-3-yl)-3-methylphenol hydrochloride. To a solution of 5-chloro-3-methyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenol (Intermediate B2, 137 mg, 0.51 mmol) in dioxane (3 mL) and H2O (1 mL) at 25° C. was added 4-({3-chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}methyl)-1-(2,2-difluoroethyl)piperidine (80 mg, 0.25 mmol), Pd(dppf)Cl2 (19 mg, 25 μmol) and Na2CO3 (81 mg, 0.76 mmol). The resulting mixture was stirred at 80° C. under N2 for 2 h. The solution was diluted with H2O (3 mL) and extracted with ethyl acetate (3×3 mL). The combined organic layers were washed with brine (3 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Phenomenex Luna C18 100×40 mm×5 μm; mobile phase: [H2O (0.04% HCl)—ACN]; gradient: 1-30% B over 8.0 min) and lyophilised to give the title compound as a yellow solid. Y=17%. 1H NMR (400 MHz, DMSO-d6) δ 11.47 (br. s, 1H), 10.87 (br. s, 1H), 8.767 (s, 1H), 8.54 (s, 1H), 7.07 (s, 1H), 7.06-6.99 (m, 2H), 6.85-6.50 (m, 1H), 4.43 (d, J=7 Hz, 2H), 3.72-3.55 (m, 4H), 3.11-3.00 (m, 2H), 2.31-2.21 (m, 1H), 2.08 (s, 3H), 1.84-1.72 (m, 4H). 1H NMR (400 MHz, DMSO-d6+D2O) δ 8.64 (d, J=3 Hz, 1H), 8.55 (s, 1H), 7.07 (d, J=3 Hz, 1H), 7.03-6.99 (m, 2H), 6.76-6.43 (m, 1H), 4.49-4.32 (m, 2H), 3.74-3.57 (m, 4H), 3.14-3.00 (m, 2H), 2.31-2.22 (m, 1H), 2.08 (s, 3H), 1.86-1.64 (m, 4H). 1H NMR (400 MHz, MeOD) δ 8.62 (d, J=3 Hz, 1H), 8.58 (s, 1H), 7.14 (d, J=3 Hz, 1H), 7.02 (d, J=2 Hz, 1H), 6.93 (d, J=2 Hz, 1H), 6.62-6.29 (m, 1H), 4.56 (d, J=7 Hz, 2H), 3.80-3.62 (m, 4H), 3.29-3.15 (m, 2H), 2.54-2.43 (m, 1H), 2.17 (s, 3H), 2.03-1.93 (m, 2H), 1.90-1.76 (m, 2H). LCMS (ESI): m/z: [M+H]+=421.1.
Step 1. (1-Methyl-2-oxopiperidin-4-yl)methyl methanesulfonate. To a solution of 4-(hydroxymethyl)-1-methyl-piperidin-2-one (0.40 g, 2.79 mmol) in DCM (5 mL) at 0° C. were added MsCl (238 μL, 3.07 mmol) and DIPEA (730 μL, 4.19 mmol). The mixture was stirred at 0° C. for 2 h. The reaction mixture was quenched by addition of H2O (5 mL) at 0° C., then extracted with DCM (3×5 mL). The combined organic layers were washed with brine (5 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give the title compound as a yellow oil. Y=97%. 1H NMR (400 MHz, DMSO-d6) δ 4.16-4.07 (m, 2H), 3.33-3.24 (m, 2H), 3.19 (s, 3H), 2.80 (s, 3H), 2.36-2.17 (m, 2H), 2.08-1.96 (m, 1H), 1.93-1.81 (m, 1H), 1.62-1.44 (m, 1H).
Step 2. 4-({3-Chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}methyl)-1-methylpiperidin-2-one. To a solution of (1-methyl-2-oxopiperidin-4-yl)methyl methanesulfonate (0.60 g, 2.71 mmol) in DMF (10 mL) at 25° C. were added K2CO3 (750 mg, 5.42 mmol) and 3-chloro-7H-pyrrolo[2,3-c]pyridazine (Intermediate A1, 416 mg, 2.71 mmol). The mixture was stirred at 80° C. for 2 h. The reaction mixture was diluted with H2O (7 mL) and extracted with EtOAc (3×7 mL). The combined organic layers were washed with brine (7 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Waters Xbridge BEH C18 250×50 mm×10 μm; mobile phase: [H2O (10 mM NH4HCO3)—ACN]; gradient: 15-40% B over 10.0 min) and lyophilised to give the title compound as a white solid. Y=40%. 1H NMR (400 MHz, DMSO-d6) δ 8.07 (d, J=3 Hz, 1H), 8.04 (s, 1H), 6.60 (d, J=3 Hz, 1H), 4.46-4.27 (m, 2H), 3.30-3.19 (m, 2H), 3.17-3.14 (m, 1H), 2.77 (s, 3H), 2.16-2.00 (m, 2H), 1.78-1.65 (m, 1H), 1.58-1.43 (m, 1H).
Step 3. 4-{[3-(4-Chloro-2-hydroxy-6-methylphenyl)-7H-pyrrolo[2,3-c]pyridazin-7-yl]methyl}-1-methylpiperidin-2-one. To a solution of 4-({3-chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}methyl)-1-methylpiperidin-2-one (120 mg, 431 μmol) in H2O (1 mL) and dioxane (3 mL) at 25° C. under N2 were added 5-chloro-3-methyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenol (Intermediate B2, 116 mg, 431 μmol), Pd(dppf)Cl2 (16 mg, 22 μmol) and Na2CO3 (137 mg, 1.29 mmol). The mixture was stirred at reflux under N2 for 2 h. The reaction mixture was diluted with H2O (1 mL) and extracted with EtOAc (3×2 mL). The combined organic layers were washed with brine (1 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Waters Xbridge Prep OBD C18 150×40 mm×10 μm; mobile phase: [H2O (10 mM NH4HCO3)—ACN]; gradient: 25-55% B over 8.0 min) and lyophilised to give the title compound as a white solid. Y=17%. 1H NMR (400 MHz, DMSO-d6) δ 9.84 (s, 1H), 7.95 (d, J=3 Hz, 1H), 7.76 (s, 1H), 6.94-6.78 (m, 2H), 6.59 (d, J=3 Hz, 1H), 4.47-4.34 (m, 2H), 3.28-3.20 (m, 2H), 2.79 (s, 3H), 2.60-2.55 (m, 1H), 2.16-2.06 (m, 2H), 1.98 (s, 3H), 1.85-1.75 (m, 1H), 1.62-1.50 (m, 1H). 1H NMR (400 MHz, DMSO-d6+D2O) δ 7.95-7.84 (m, 1H), 7.75 (s, 1H), 6.92-6.78 (m, 2H), 6.60 (d, J=3 Hz, 1H), 4.48-4.28 (m, 2H), 3.31-3.16 (m, 2H), 2.77 (s, 3H), 2.53-2.52 (m, 1H), 2.19-2.04 (m, 2H), 1.94 (s, 3H), 1.85-1.71 (m, 1H), 1.60-1.45 (m, 1H). 1H NMR (400 MHz, methanol-d4) δ 7.86 (d, J=3 Hz, 1H), 7.83 (s, 1H), 6.89-6.78 (m, 2H), 6.65 (d, J=3 Hz, 1H), 4.56-4.36 (m, 2H), 3.52-3.34 (m, 2H), 2.92 (s, 3H), 2.74-2.59 (m, 1H), 2.40-2.14 (m, 2H), 2.03 (s, 3H), 1.97-1.79 (m, 1H), 1.77-1.57 (m, 1H). LCMS (ESI): m/z: [M+H]+=385.3.
Step 1. 4-(2-{3-Chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}ethyl)morpholine. To a solution of 3-chloro-7H-pyrrolo[2,3-c]pyridazine (Intermediate A1, 500 mg, 3.26 mmol) in DMF (5 mL) at 25° C. were added K2CO3 (900 mg, 6.51 mmol) and 4-(2-chloroethyl)morpholine (585 mg, 3.91 mmol). The mixture was stirred at 80° C. for 12 h. The reaction mixture was purified by prep-HPLC (column: Phenomenex Luna C18 75×30 mm×3 μm; mobile phase: [H2O (0.1% TFA)—ACN]; gradient: 1-20% B over 8.0 min) and lyophilised to give the title compound as a yellow solid. Y=58%. 1H NMR (400 MHz, chloroform-d) δ 7.74 (s, 1H), 7.70 (d, J=4 Hz, 1H), 6.52 (d, J=4 Hz, 1H), 4.91 (t, J=7 Hz, 2H), 3.96 (m, 4H), 3.73 (t, J=7 Hz, 2H), 3.65-2.77 (m, 4H).
Step 2. 5-Chloro-3-methyl-2-{7-[2-(morpholin-4-yl)ethyl]-7H-pyrrolo[2,3-c]pyridazin-3-yl}phenol hydrochloride. To a solution of 4-(2-{3-chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}ethyl)morpholine (200 mg, 0.75 mmol) and 5-chloro-3-methyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenol (Intermediate B2, 302 mg, 1.12 mmol) in dioxane (10 mL) and H2O (3 mL) at 25° C. under N2 were added Na2CO3 (238 mg, 2.25 mmol) and Pd(PPh3)4(87 mg, 75 μmol). The mixture was stirred at 80° C. for 12 h. The reaction mixture was diluted with H2O (15 mL) and extracted with EtOAc (3×15 mL). The combined organic layers were washed with brine (15 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Phenomenex Luna C18 100×40 mm×5 μm; mobile phase: [H2O (0.04% HCl)—ACN]; gradient: 5-25% B over 8.0 min) and lyophilised to give the title compound as a yellow solid. Y=8%. 1H NMR (400 MHz, DMSO-d6) δ 11.04 (br. s, 1H), 10.49 (br. s, 1H), 8.61-8.09 (m, 2H), 7.05-6.82 (m, 3H), 5.02-4.83 (s, 2H), 4.00-3.73 (m, 6H), 3.60-3.55 (m, 2H), 3.23-3.14 (m, 2H), 2.06 (s, 3H). 1H NMR (400 MHz, DMSO-d6+D2O) δ 8.26 (d, J=4 Hz, 1H), 8.22 (s, 1H), 6.94 (d, J=2 Hz, 1H), 6.92 (d, J=4 Hz, 1H), 6.89 (d, J=2 Hz, 1H), 4.83 (t, J=7 Hz, 2H), 3.87-3.64 (m, 6H), 3.48-3.22 (m, 4H), 2.00 (s, 3H). 1H NMR (400 MHz, methanol-d4) δ 8.66 (d, J=4 Hz, 1H), 8.59 (s, 1H), 7.17 (d, J=4 Hz, 1H), 7.02 (d, J=2 Hz, 1H), 6.93 (d, J=2 Hz, 1H), 5.06 (t, J=7 Hz, 2H), 4.12 -3.84 (m, 6H), 3.80-3.57 (m, 2H), 3.45-3.32 (m, 2H), 2.19 (s, 3H). LCMS (ESI): m/z: [M+H]+=373.3.
Step 1. 1-(2-{3-Chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}ethyl)-4-methylpiperazine. To a solution of 3-chloro-7H-pyrrolo[2,3-c]pyridazine (Intermediate A1, 500 mg, 3.26 mmol) in DMF (5 mL) at 25° C. were added K2CO3 (1.57 g, 11.40 mmol) and 1-(2-chloroethyl)-4-methyl-piperazine hydrochloride (778 mg, 3.91 mmol). The mixture was stirred at 80° C. for 12 h. The reaction mixture was purified by prep-HPLC (column: Phenomenex Luna C18 75×30 mm×3 μm; mobile phase: [H2O (0.1% TFA)—ACN]; gradient: 1-30% B over 8.0 min) and lyophilised to give the title compound as a yellow solid. Y=19%. 1H NMR (400 MHz, chloroform-d) δ 7.74 (s, 1H), 7.56 (d, J=4 Hz, 1H), 6.53 (d, J=4 Hz, 1H), 4.67 (t, J=6 Hz, 2H), 3.57-2.88 (m, 10H), 2.78 (s, 3H).
Step 2. 5-Chloro-3-methyl-2-{7-[2-(4-methylpiperazin-1-yl)ethyl]-7H-pyrrolo[2,3-c]pyridazin-3-yl}phenol dihydrochloride. To a solution of 1-(2-{3-chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}ethyl)-4-methylpiperazine (150 mg, 536 μmol) and 5-chloro-3-methyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenol (Intermediate B2, 216 mg, 804 μmol) in dioxane (7.5 mL) and H2O (2.5 mL) at 25° C. under N2 were added Na2CO3 (170 mg, 1.61 mmol) and Pd(PPh3)4(62 mg, 54 μmol). The mixture was stirred at 80° C. under N2 for 12 h. The reaction mixture was diluted with H2O (15 mL) and extracted with EtOAc (3×15 mL). The combined organic layers were washed with brine (15 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Phenomenex Luna C18 100×40 mm×5 μm; mobile phase: [H2O (0.04% HCl)—ACN]; gradient: 5-25% B over 8.0 min) and lyophilised to give the title compound as a yellow solid. Y=8%. 1H NMR (400 MHz, DMSO-d6) δ 10.78 (br. s, 2H), 8.72-8.65 (m, 1H), 8.59-8.43 (m, 1H), 7.11-6.97 (m, 3H), 4.79-4.53 (m, 2H), 3.42-3.04 (m, 10H), 2.75 (s, 3H), 2.09 (s, 3H). 1H NMR (400 MHz, DMSO-d6+D2O) δ 8.55 (d, J=3 Hz, 1H), 8.46 (s, 1H), 7.01 (d, J=3 Hz, 1H), 6.99 (s, 1H), 6.93 (s, 1H), 4.57 (t, J=6 Hz, 2H), 3.48-2.79 (m, 10H), 2.71 (s, 3H), 2.05 (s, 3H). 1H NMR (400 MHz, methanol-d4) δ 8.67 (d, J=3 Hz, 1H), 8.57 (s, 1H), 7.13 (d, J=3 Hz, 1H), 7.03 (d, J=2 Hz, 1H), 6.94 (d, J=2 Hz, 1H), 4.98-4.90 (m, 2H), 3.88-3.35 (m, 8H), 3.18-2.96 (m, 2H), 2.94 (s, 3H), 2.20 (s, 3H). LCMS (ESI): m/z: [M+H]+=386.3.
Step 1. (1-Methylpiperidin-2-yl)methyl methanesulfonate. To a solution of (1-methyl-2-piperidyl)methanol (1.0 g, 7.74 mmol) in DCM (15 mL) at 0° C. under N2 were added TEA (2.15 mL, 15.5 mmol) and MsCl (719 μL, 9.29 mmol). The mixture was stirred at 0° C. for 2 h. The reaction mixture was quenched by addition of H2O (15 mL) and extracted with EtOAc (3×15 mL). The combined organic layers were washed with brine (3×15 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give the title compound as a yellow oil. Y=500%.
Step 2. 2-({3-Chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}methyl)-1-methylpiperidine. To a solution of (1-methyl-2-piperidyl)methyl methanesulfonate (709 mg, 3.42 mmol) in DMF (7 mL) at 25° C. under N2 were added K2CO3 (945 mg, 6.84 mmol) and 3-chloro-7H-pyrrolo[2,3-c]pyridazine (Intermediate A1, 350 mg, 2.28 mmol). The mixture was stirred at 50° C. for 12 h. The reaction mixture was purified by prep-HPLC (column: Waters Xbridge C18 150×50 mm×10 μm; mobile phase: [H2O (10 mM NH4HCO3)—ACN]; gradient: 20-60% B over 8.0 min) and lyophilised to give the title compound as a yellow solid. Y=33%. 1H NMR (400 MHz, DMSO-d6) δ 8.08-7.97 (m, 2H), 6.57 (d, J=3 Hz, 1H), 4.73 (dd, J=14, 5 Hz, 1H), 4.31 (dd, J=14, 7 Hz, 1H), 2.86-2.73 (m, 1H), 2.48-2.42 (m, 1H), 2.38 (s, 3H), 2.11-1.96 (m, 1H), 1.61-1.51 (m, 1H), 1.51-1.43 (m, 1H), 1.41-1.28 (m, 1H), 1.26-1.18 (m, 1H), 1.17-1.05 (m, 1H), 1.04-0.93 (m, 1H).
Step 3. 5-Chloro-3-methyl-2-{7-[(I-methylpiperidin-2-yl)methyl]-7H-pyrrolo[2,3-c]pyridazin-3-yl}phenol. To a solution of 2-({3-chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}methyl)-1-methylpiperidine (120 mg, 453 μmol) in dioxane (3 mL) and H2O (1 mL) at 25° C. under N2 were added Na2CO3 (144 mg, 1.36 mmol), Pd(PPh3)4(26 mg, 23 μmol) and 5-chloro-3-methyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenol (Intermediate B2, 183 mg, 680 μmol). The mixture was stirred at 90° C. for 12 h. The reaction mixture was diluted with H2O (2 mL) and extracted with EtOAc (3×2 mL). The combined organic layers were washed with brine (3×2 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Phenomenex Luna C18 100×40 mm×5 μm; mobile phase: [H2O (0.04% HCl)—ACN]; gradient: 1-30% B over 8.0 min) and lyophilised to give the title compound as a yellow solid. Y=24%. 1H NMR (400 MHz, DMSO-d6) δ 11.38-11.21 (m, 1H), 11.02-10.83 (m, 1H), 8.79-8.68 (m, 1H), 8.60 (s, 1H), 7.13-7.05 (m, 2H), 7.01 (s, 1H), 5.10-4.65 (m, 2H), 4.11-3.98 (m, 1H), 3.48-3.39 (m, 1H), 3.10-2.88 (m, 4H), 2.54 (m, 0.2H), 2.11 (s, 2.8H), 1.95-1.31 (m, 6H). 1H NMR (400 MHz, DMSO+D2O-d6) δ 8.76-8.69 (m, 1H), 8.65-8.60 (m, 1H), 7.14 (d, J=3 Hz, 1H), 7.02 (s, 2H), 5.08-4.61 (m, 2H), 4.09-3.69 (m, 1H), 3.51-3.39 (m, 1H), 3.27-2.90 (m, 4H), 2.54 (s, 0.2H), 2.09 (s, 2.8H), 1.89-1.35 (m, 6H). 1H NMR (400 MHz, MeOD) δ 8.69-8.63 (m, 1H), 8.61-8.58 (m, 1H), 7.19 (d, J=3 Hz, 1H), 7.02 (s, 1H), 6.94 (s, 1H), 5.12-4.94 (m, 1.7H), 4.82-4.72 (m, 0.3H), 4.29-3.72 (m, 1H), 3.68-3.35 (m, 1H), 3.27-3.02 (m, 4H), 2.71-2.12 (m, 3H), 2.11-1.49 (m, 6H).
LCMS (ESI): m/z: [M+H]+=371.3.
Step 1. 4-({3-Chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}methyl)-1-(oxetan-3-yl)piperidine. To a solution of 3-chloro-7-(4-piperidylmethyl)pyrrolo[2,3-c]pyridazine (for synthesis see Compound 4)(280 mg, 1.12 mmol) in DCE (3 mL) at 25° C. were added oxetan-3-one (97 mg, 1.34 mmol) and AcOH (64 μL, 1.12 mmol). The mixture was stirred at 25° C. for 1 h. The mixture was cooled to 0° C. and treated with NaBH(OAc)3 (437 mg, 2.23 mmol). The mixture was stirred at 25° C. under N2 for 2 h. The reaction mixture was quenched by addition of H2O (1 mL) and extracted with DCM (3×3 mL). The combined organic layers were washed with brine (3 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Waters Xbridge Prep OBD C18 150×40 mm×10 μm; mobile phase: [H2O (10 mM NH4HCO3)—ACN]; gradient: 15-45% B over 8.0 min) and lyophilised to give the title compound as a white solid. Y=50%. 1H NMR (400 MHz, DMSO-d6) δ 8.05 (d, J=3 Hz, 1H), 8.03 (s, 1H), 6.57 (d, J=3 Hz, 1H), 4.52-4.45 (m, 2H), 4.37 (t, J=7 Hz, 2H), 4.31 (d, J=7 Hz, 2H), 3.35-3.32 (m, 1H), 2.70-2.64 (m, 2H), 1.98-1.88 (m, 1H), 1.71-1.60 (m, 2H), 1.45-1.43 (m, 2H), 1.31-1.22 (m, 2H).
Step 2. 5-Chloro-3-methyl-2-(7-{[1-(oxetan-3-yl)piperidin-4-yl]methyl}-7H-pyrrolo[2,3-c]pyridazin-3-yl)phenol. To a solution of 4-({3-chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}methyl)-1-(oxetan-3-yl)piperidine (170 mg, 554 μmol) in dioxane (1.5 mL) and H2O (0.5 mL) at 25° C. under N2 were added 5-chloro-3-methyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenol (Intermediate B2, 179 mg, 665 μmol), Pd(dppf)Cl2 (41 mg, 55 μmol) and Na2CO3 (176 mg, 1.66 mmol). The mixture was stirred at reflux under N2 for 8 h. The reaction mixture was diluted with H2O (1 mL) and extracted with EtOAc (3×1 mL). The combined organic layers were washed with brine (1 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Waters Xbridge BEH C18 100×30 mm×10 μm; mobile phase: [H2O (10 mM NH4HCO3)—ACN]; gradient: 25-60% B over 8.0 min) and lyophilised to give the title compound as a white solid. Y=13%. 1H NMR (400 MHz, DMSO-d6) δ 9.84 (s, 1H), 7.93 (d, J=3 Hz, 1H), 7.74 (s, 1H), 6.91-6.81 (m, 2H), 6.56 (d, J=3 Hz, 1H), 4.53-4.46 (m, 2H), 4.42-4.33 (m, 4H), 3.38-3.35 (m, 1H), 2.71-2.66 (m, 2H), 2.01-1.97 (m, 4H), 1.70-1.67 (m, 2H), 1.54-1.51 (m, 2H), 1.41-1.24 (m, 2H). 1H NMR (400 MHz, DMSO-d6+D2O) δ 7.90 (d, J=3 Hz, 1H), 7.74 (s, 1H), 6.90-6.81 (m, 2H), 6.57 (d, J=3 Hz, 1H), 4.51-4.45 (m, 2H), 4.41-4.31 (m, 4H), 3.39-3.29 (m, 1H), 2.67-2.61 (m, 2H), 2.06-1.92 (m, 4H), 1.70-1.67 (m, 2H), 1.52-1.49 (m, 2H), 1.40-1.25 (m, 2H). 1H NMR (400 MHz, methanol-d4) δ 7.88-7.79 (m, 2H), 6.92-6.79 (m, 2H), 6.62 (d, J=3 Hz, 1H), 4.70-4.63 (m, 2H), 4.60-4.55 (m, 2H), 4.41 (d, J=7 Hz, 2H), 3.51-3.42 (m, 1H), 2.81-2.79 (m, 2H), 2.19-2.10 (m, 1H), 2.04 (s, 3H), 1.84-1.81 (m, 2H), 1.68-1.65 (m, 2H), 1.52-1.38 (m, 2H).
LCMS (ESI): m/z: [M+H]+=413.3.
Step 1. 3-Chloro-7-[(oxolan-3-yl)methyl]-7H-pyrrolo[2,3-c]pyridazine. To a solution of 3-(bromomethyl)tetrahydrofuran (215 mg, 1.30 mmol) in DMF (2 mL) at 25° C. were added 3-chloro-7H-pyrrolo[2,3-c]pyridazine (Intermediate A1, 200 mg, 1.30 mmol) and K2CO3 (360 mg, 2.60 mmol). The reaction mixture was stirred at 80° C. under N2 for 2 h. The reaction mixture was purified by prep-HPLC (column: Waters Xbridge BEH C18 100×30 mm×10 μm; mobile phase: [H2O (10 mM NH4HCO3)—ACN]; gradient: 20-50% B over 8.0 min) and lyophilised to give the title compound 3 as a yellow gum. Y=42%. 1H NMR (400 MHz, DMSO-d6) δ 8.12 (d, J=3 Hz, 1H), 8.04 (s, 1H), 6.59 (d, J=3 Hz, 1H), 4.44-4.36 (m, 2H), 3.82-3.77 (m, 1H), 3.67-3.59 (m, 2H), 3.55-3.50 (m, 1H), 2.95-2.83 (m, 1H), 1.94-1.83 (m, 1H), 1.68-1.56 (m, 1H).
Step 2. 5-Chloro-3-methyl-2-{7-[(oxolan-3-yl)methyl]-7H-pyrrolo[2,3-c]pyridazin-3-yl}phenol. To a solution of 3-chloro-7-[(oxolan-3-yl)methyl]-7H-pyrrolo[2,3-c]pyridazine (130 mg, 427 μmol) in dioxane (2.6 mL) and H2O (0.52 mL) at 25° C. under N2 were added 5-chloro-3-methyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenol (Intermediate B2, 137 mg, 512 μmol), Pd(dppf)Cl2 (31 mg, 43 μmol) and Na2CO3 (136 mg, 1.28 mmol). The reaction mixture was stirred at 80° C. under N2 for 2 h. The reaction mixture was diluted with H2O (1 mL) and extracted with EtOAc (3×1 mL). The combined organic layers were washed with brine (3 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Waters Xbridge BEH C18 100×30 mm×10 μm; mobile phase: [H2O (10 mM NH4HCO3)—ACN]; gradient: 40-50% B over 8.0 min) and lyophilised to give the title compound as a white solid. Y=10%. 1H NMR (400 MHz, DMSO-d6) δ 9.80 (br. s, 1H), 8.01 (d, J=3 Hz, 1H), 7.75 (s, 1H), 6.86 (d, J=6 Hz, 2H), 6.58 (d, J=3 Hz, 1H), 4.44 (d, J=8 Hz, 2H), 3.89-3.79 (m, 1H), 3.71-3.62 (m, 2H), 3.59-3.52 (m, 1H), 3.02-2.89 (m, 1H), 1.98 (s, 3H), 1.96-1.88 (m, 1H), 1.69 (m, 1H). 1H NMR (400 MHz, DMSO-d6+D2O) δ 7.98 (d, J=3 Hz, 1H), 7.75 (s, 1H), 6.86 (d, J=6 Hz, 2H), 6.58 (d, J=3 Hz, 1H), 4.43 (d, J=8 Hz, 2H), 3.88-3.79 (m, 1H), 3.70-3.61 (m, 2H), 3.59-3.53 (m, 1H), 3.01-2.87 (m, 1H), 1.97 (s, 3H), 1.95-1.87 (m, 1H), 1.72-1.58 (m, 1H). LC-MS (ESI): m/z: [M+H]+=344.2.
Step 1. Methyl 4-({3-chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}methyl)piperidine-1-carboxylate. To a solution of 3-chloro-7-(4-piperidylmethyl)pyrrolo[2,3-c]pyridazine (for synthesis see Compound 4)(200 mg, 798 μmol) in DCM (4 mL) was added TEA (444 μL, 3.19 mmol) and methyl chloroformate (247 μL, 3.19 mmol). The mixture was stirred at 25° C. for 2 h. The reaction mixture cooled to 0° C. and quenched by addition of H2O (4 mL), then extracted with DCM (3×4 mL). The combined organic layers were washed with brine (4 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Phenomenex Luna C18 75×30 mm 3 μm; mobile phase: [H2O (0.1% TFA)—ACN]; gradient: 15-45% B over 8.0 min) and lyophilised to give the title compound as a yellow oil. Y=81%. 1H NMR (400 MHz, DMSO-d6) δ 8.10-7.95 (m, 2H), 6.58 (d, J=3 Hz, 1H), 4.31 (d, J=7 Hz, 2H), 4.03-3.81 (m, 2H), 3.56 (s, 3H), 2.80-2.61 (m, 2H), 2.27-2.09 (m, 1H), 1.51-1.36 (m, 2H), 1.26-1.03 (m, 2H).
Step 2. Methyl 4-{[3-(4-chloro-2-hydroxy-6-methylphenyl)-7H-pyrrolo[2,3-c]pyridazin-7-yl]methyl}piperidine-1-carboxylate. To a solution of methyl 4-({3-chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}methyl)piperidine-1-carboxylate (180 mg, 583 μmol) and 5-chloro-3-methyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenol (Intermediate B2, 250 mg, 933 μmol) in dioxane (9 mL) and H2O (3 mL) at 25° C. under N2 were added Na2CO3 (185 mg, 1.75 mmol) and Pd(PPh3)4(67 mg, 58 μmol). The mixture was stirred at reflux under N2 for 8 h. The reaction mixture was diluted with H2O (9 mL) and extracted with EtOAc (3×9 mL). The combined organic layers were washed with brine (9 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Phenomenex Luna C18 100×40 mm×5 μm; mobile phase: [H2O (0.04% HCl)—ACN]; gradient: 15-45% B over 8.0 min) and lyophilised to give the title compound as a yellow solid. Y=16%. 1H NMR (400 MHz, DMSO-d6) δ 10.48 (br. s, 1H), 8.78-8.04 (m, 2H), 7.05-6.90 (m, 3H), 4.39 (d, J=7 Hz, 2H), 3.97 (d, J=10 Hz, 2H), 3.58 (s, 3H), 2.87-2.71 (m, 2H), 2.31-2.12 (m, 1H), 2.06 (s, 3H), 1.55 (m, 2H), 1.25-1.15 (m, 2H). 1H NMR (400 MHz, DMSO-d6+D2O) δ 8.52-8.31 (m, 2H), 7.09-6.80 (m, 3H), 4.37 (d, J=7 Hz, 2H), 4.03-3.89 (m, 2H), 3.56 (s, 3H), 2.83-2.69 (m, 2H), 2.25-2.13 (m, 1H), 2.05 (s, 3H), 1.61-1.49 (m, 2H), 1.22-1.14 (m, 2H). LCMS (ESI): m/z: [M+H]+=415.3.
Step 1. 4-({3-Chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}methyl)-1-(2-methoxyethyl)piperidin-1-ium trifluoroacetate. To a solution of 3-chloro-7-(4-piperidylmethyl)pyrrolo[2,3-c]pyridazine (for synthesis see Compound 4)(250 mg, 997 μmol) in DMF (5 mL) at 25° C. were added K2CO3 (276 mg, 1.99 mmol) and 1-chloro-2-methoxy-ethane (182 μL, 1.99 mmol). The resulting mixture was stirred at 80° C. for 2 h. The reaction mixture was purified by prep-HPLC (column: Phenomenex Luna C18 75×30 mm×3 μm; mobile phase: [H2O (0.1% TFA)—ACN]; gradient: 1-30% B over 8.0 min) and lyophilised to give the title compound as a colourless oil. Y=47%.
Step 2. 5-Chloro-2-(7-{[1-(2-methoxyethyl)piperidin-4-yl]methyl}-7H-pyrrolo[2,3-c]pyridazin-3-yl)-3-methylphenol hydrochloride. To a solution of 4-({3-chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}methyl)-1-(2-methoxyethyl)piperidin-1-ium trifluoroacetate (180 mg, 426 μmol) in dioxane (9 mL) and H2O (3 mL) at 25° C. under N2 were added 5-chloro-3-methyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenol (Intermediate B2, 235 mg, 874 μmol), Pd(PPh3)4(34 mg, 29 μmol) and Na2CO3 (185 mg, 1.75 mmol). The mixture was stirred at 80° C. for 12 h. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Phenomenex Luna C18 100×40 mm×5 μm; mobile phase: [H2O (0.04% HCl)—ACN]; gradient: 5-30% B over 8.0 min) and lyophilised to give the title compound as a white solid. Y=17%. 1H NMR (400 MHz, DMSO-d6) δ 10.62 (br. s, 1H), 9.98 (br. s, 1H), 8.66-8.22 (m, 2H), 7.21-6.81 (m, 3H), 4.72-4.30 (m, 2H), 3.72-3.65 (m, 2H), 3.55-3.45 (m, 2H), 3.28 (s, 3H), 3.23-3.18 (m, 2H), 2.98-2.84 (m, 2H), 2.30-2.20 (m, 1H), 2.06 (s, 3H), 1.80-1.62 (m, 4H). 1H NMR (400 MHz, DMSO-d6+D2O) δ 8.57-8.30 (m, 2H), 7.03-6.96 (m, 2H), 6.94 (s, 1H), 4.53-4.36 (m, 2H), 3.63-3.60 (m, 2H), 3.50-3.37 (m, 2H), 3.32-3.25 (m, 3H), 3.25-3.15 (m, 2H), 2.95-2.80 (m, 2H), 2.29-2.17 (m, 1H), 2.04 (s, 3H), 1.85-1.75 (m, 2H), 1.69-1.46 (m, 2H). LCMS (ESI): m/z: [M+H]+=415.3.
Step 1. 2-{3-Chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}-N-methylacetamide. To a solution of 3-chloro-7H-pyrrolo [2,3-c]pyridazine (Intermediate A1, 50 mg, 326 μmol) in DMF (0.5 mL) at 25° C. under N2 were added K2CO3 (90 mg, 651 μmol) and 2-chloro-N-methyl-acetamide (35 mg, 326 μmol). The mixture was stirred at 90° C. for 5 h. The reaction mixture was purified directly by prep-HPLC (column: Waters Xbridge BEH C18 100×30 mm×10 μm; mobile phase: [H2O (10 mM NH4HCO3)—ACN]; gradient: 1-35% B over 8.0 min) and lyophilized to give the title compound as a white solid. Y=27%. 1H NMR (400 MHz, DMSO-d6) δ 8.27-8.12 (m, 1H), 8.03 (s, 1H), 7.98 (d, J=3 Hz, 1H), 6.58 (d, J=3 Hz, 1H), 5.06 (s, 2H), 2.62 (d, J=5 Hz, 3H).
Step 2. 2-[3-(4-Chloro-2-hydroxy-6-methylphenyl)-7H-pyrrolo[2,3-c]pyridazin-7-yl]-N-methylacetamide. To a solution of 2-{3-chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}-N-methylacetamide (110 mg, 490 μmol) in dioxane (0.9 mL) and H2O (0.3 mL) at 25° C. under N2 were added Na2CO3 (156 mg, 1.47 mmol), Pd(PPh3)4(28 mg, 24 μmol) and 5-chloro-3-methyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenol (Intermediate B2, 197 mg, 734 μmol). The mixture was stirred at 120° C. for 5 h. The reaction mixture was diluted with H2O (2 mL) and extracted with EtOAc (3×2 mL). The combined organic layers were washed with brine (3×2 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Waters Xbridge BEH C18 100×30 mm×10 μm; mobile phase: [H2O (10 mM NH4HCO3)—ACN]; gradient: 20-50% B over 8.0 min) and lyophilized to give the title compound as a white solid. Y=12%. 1H NMR (400 MHz, DMSO-d6) δ 9.86 (s, 1H), 8.26 (q, J=4 Hz, 1H), 7.87 (d, J=3 Hz, 1H), 7.75 (s, 1H), 6.93-6.80 (m, 2H), 6.57 (d, J=3 Hz, 1H), 5.10 (s, 2H), 2.65 (d, J=5 Hz, 3H), 1.98 (s, 3H). 1H NMR (400 MHz, DMSO-d6+D2O) δ 7.84 (d, J=3 Hz, 1H), 7.74 (s, 1H), 6.90-6.81 (m, 2H), 6.58 (d, J=3 Hz, 1H), 5.09 (s, 2H), 2.64 (s, 3H), 1.96 (s, 3H). LCMS (ESI): m/z: [M+H]+=331.1.
Step 1. 3-{3-Chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}-N-methylpropanamide. To a solution of 3-chloro-7H-pyrrolo[2,3-c]pyridazine (Intermediate A1, 300 mg, 1.95 mmol) in DMF (3 mL) at 25° C. were added K2CO3 (540 mg, 3.91 mmol) and 3-chloro-N-methyl-propanamide (356 mg, 2.93 mmol). The mixture was stirred at 25° C. for 3 h. The reaction mixture was purified by prep-HPLC (column: Waters Xbridge BEH C18 100×30 mm×10 μm; mobile phase: [H2O (10 mM NH4HCO3)—ACN]; gradient: 1-45% B over 8.0 min) and lyophilised to give the title compound as a white solid. Y=64%. 1H NMR (400 MHz, DMSO-d6) δ 8.02 (s, 1H), 7.94 (d, J=3 Hz, 1H), 7.87-7.72 (m, 1H), 6.53 (d, J=3 Hz, 1H), 4.60 (t, J=7 Hz, 2H), 2.71 (t, J=7 Hz, 2H), 2.52 (s, 3H).
Step 2. 3-[3-(4-Chloro-2-hydroxy-6-methylphenyl)-7H-pyrrolo[2,3-c]pyridazin-7-yl]-N-methylpropanamide. To a solution of 3-{3-chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}-N-methylpropanamide (90 mg, 377 μmol) in dioxane (3 mL) and H2O (0.6 mL) at 25° C. under N2 were added 5-chloro-3-methyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenol (Intermediate B2, 101 mg, 377 μmol), Na2CO3 (94 mg, 1.13 mmol) and Pd(dppf)Cl2 (14 mg, 19 μmol). The mixture was stirred at 85° C. under N2 for 12 h. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was diluted with H2O (2 mL) and extracted with EtOAc (2×2 mL). The combined organic layers were washed with brine (2×1 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Waters Xbridge Prep OBD C18 150×40 mm×10 μm; mobile phase: [H2O (10 mM NH4HCO3)—ACN]; gradient: 5-45% B over 8.0 min) and lyophilised to give the title compound as a white solid. Y=23%. 1H NMR (400 MHz, DMSO-d6) δ 9.83 (br. s, 1H), 7.89 (d, J=5 Hz, 1H), 7.83 (d, J=3 Hz, 1H), 7.73 (s, 1H), 6.86 (d, J=6 Hz, 2H), 6.53 (d, J=3 Hz, 1H), 4.66 (t, J=7 Hz, 2H), 2.76 (t, J=7 Hz, 2H), 2.55 (d, J=5 Hz, 3H), 1.98 (s, 3H). 1H NMR (400 MHz, DMSO-d6+D2O) δ 7.89-7.60 (m, 2H), 6.90-6.80 (m, 2H), 6.55 (d, J=3 Hz, 1H), 4.62 (t, J=7 Hz, 2H), 2.73 (t, J=7 Hz, 2H), 2.52-2.51 (m, 3H), 1.91 (s, 3H). LCMS (ESI): m/z: [M+H]+=345.2.
Step 1. 3-Chloro-7-(2-methoxyethyl)-7H-pyrrolo[2,3-c]pyridazine. To a solution of 3-chloro-7H-pyrrolo[2,3-c]pyridazine (Intermediate A1, 300 mg, 1.95 mmol) in DMF (3 mL) at 25° C. were added K2CO3 (540 mg, 3.91 mmol) and 1-chloro-2-methoxy-ethane (267 μL, 2.93 mmol). The mixture was stirred at 25° C. for 3 h. The reaction mixture was purified by prep-HPLC (column: Waters Xbridge BEH C18 100×30 mm×10 μm; mobile phase: [H2O (10 mM NH4HCO3)—ACN]; gradient: 5-50% B over 8.0 min) and lyophilised to give the title compound as a white solid. Y=70%. 1H NMR (400 MHz, DMSO-d6) δ 8.10-7.97 (m, 2H), 6.56 (d, J=3 Hz, 1H), 4.57 (t, J=5 Hz, 2H), 3.76 (t, J=5 Hz, 2H), 3.23 (s, 3H).
Step 2. 5-Chloro-2-[7-(2-methoxyethyl)-7H-pyrrolo[2,3-c]pyridazin-3-yl]-3-methylphenol. To a solution of 3-chloro-7-(2-methoxyethyl)-7H-pyrrolo[2,3-c]pyridazine (90 mg, 425 μmol) in dioxane (4.5 mL) and H2O (0.9 mL) at 25° C. under N2 were added 5-chloro-3-methyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenol (Intermediate B2, 114 mg, 425 μmol), dicyclohexyl-[2-(2,6-dimethoxyphenyl)phenyl]phosphane; methanesulfonate; (2-phenylanilino)palladium(1+) “SPhos Pd G3” (50 mg, 64 μmol) and K2CO3 (235 mg, 1.70 mmol). The mixture was stirred at 120° C. for 12 h under N2. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was diluted with H2O (1 mL) and extracted with EtOAc (3×1 mL). The combined organic layers were washed with brine (2×1 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. This was purified by prep-HPLC (column: Phenomenex Luna C18 75×30 mm×3 μm; mobile phase: [water (0.04% HCl)—ACN]; gradient:10-43% B over 8.0 min) and lyophilised to give the title compound as a white solid. Y=17%. 1H NMR (400 MHz, DMSO-d6) δ 10.75 (br. s, 1H), 8.65-8.45 (m, 2H), 7.09-6.92 (m, 3H), 4.67 (t, J=5 Hz, 2H), 3.83 (t, J=5 Hz, 2H), 3.28 (s, 3H), 2.08 (s, 3H). 1H NMR (400 MHz, DMSO-d6+D2O) δ 8.56 (d, J=3 Hz, 1H), 8.50 (s, 1H), 7.05-6.99 (m, 2H), 6.95 (s, 1H), 4.64 (t, J=5 Hz, 2H), 3.81 (t, J=5 Hz, 2H), 3.25 (s, 3H), 2.06 (s, 3H). LCMS (ESI): m/z: [M+H]+=318.1.
Step 1. (2-{3-Chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}ethyl)dimethylazanium trifluoroacetate. To a solution of 3-chloro-7H-pyrrolo[2,3-c]pyridazine (Intermediate A1, 400 mg, 2.60 mmol) in DMF (6 mL) at 0° C. under N2 was added NaH (60% in mineral oil, 208 mg, 5.21 mmol). The mixture was stirred at 0° C. for 30 min, then treated with a solution of 2-bromo-N,N-dimethyl-ethanamine (475 mg, 3.13 mmol) in DMF (3 mL). The mixture was stirred at 25° C. for 1 h. The reaction mixture was cooled to 0° C. and treated with saturated aqueous NH4Cl (9 mL). The resulting mixture was extracted with EtOAc (3×9 mL). The combined organic layers were washed with brine (9 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Phenomenex Luna C18 75×30 mm×3 μm; mobile phase: [H2O (0.1% TFA)—ACN]; gradient:1-30% B over 8.0 min) and lyophilised to give the title compound as a yellow oil. Y=45%. 1H NMR (400 MHz, DMSO-d6) δ 9.81 (br. s, 1H), 8.27-7.93 (m, 2H), 6.65 (d, J=3 Hz, 1H), 4.79 (t, J=6 Hz, 2H), 3.68 (t, J=6 Hz, 2H), 2.87 (s, 6H).
Step 2. 5-Chloro-2-{7-[2-(dimethylamino)ethyl]-7H-pyrrolo[2,3-c]pyridazin-3-yl}-3-methylphenol hydrochloride. To a solution of (2-{3-chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}ethyl)dimethylazanium trifluoroacetate (100 mg, 295 μmol) and 5-chloro-3-methyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenol (Intermediate B2, 87 mg, 325 μmol) in dioxane (5 mL) and H2O (1 mL) at 25° C. under N2 were added K2CO3 (122 mg, 886 μmol) and dicyclohexyl-[2-(2,6-dimethoxyphenyl)phenyl]phosphane; methanesulfonate;(2-phenylanilino)palladium(1+) “SPhos Pd G3” (23 mg, 30 μmol). The mixture was stirred at reflux under N2 for 4 h. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Phenomenex Luna C18 100×40 mm×5 μm; mobile phase: [H2O (0.04% HCl)—ACN]; gradient: 10-30% B over 8.0 min) and lyophilised to give the title compound as a yellow solid. Y=24%. 1H NMR (400 MHz, DMSO-d6) δ 10.95-10.58 (m, 2H), 8.60 (s, 1H), 8.43 (s, 1H), 7.12-6.92 (m, 3H), 4.92 (t, J=6 Hz, 2H), 3.80-3.70 (m, 2H), 2.86 (d, J=4 Hz, 6H), 2.07 (s, 3H). 1H NMR (400 MHz, DMSO-d6+D2O) δ 8.54 (d, J=3 Hz, 1H), 8.44 (s, 1H), 7.08-6.91 (m, 3H), 4.87 (t, J=6 Hz, 2H), 3.72 (t, J=6 Hz, 2H), 2.89 (s, 6H), 2.05 (s, 3H). LCMS (ESI): m/z: [M+H]+=331.1.
Step 1. 3-Chloro-7-(3-methoxypropyl)-7H-pyrrolo[2,3-c]pyridazine. To a solution of 3-chloro-7H-pyrrolo[2,3-c]pyridazine (Intermediate A1, 300 mg, 1.95 mmol) in DMF (5 mL) at 25° C. were added K2CO3 (540 mg, 3.91 mmol) and 1-chloro-3-methoxy-propane (255 mg, 2.34 mmol). The mixture was stirred at 80° C. for 12 h. The reaction mixture was purified by prep-HPLC (column: Phenomenex Luna C18 75×30 mm×3 μm; mobile phase: [H2O (0.1% TFA)—ACN]; gradient: 10-40% B over 8.0 min) and lyophilised to give the title compound as a yellow oil. Y=68%. 1H NMR (400 MHz, DMSO-d6) δ 8.20-7.82 (m, 2H), 6.57 (d, J=3 Hz, 1H), 4.45 (t, J=7 Hz, 2H), 3.30 (t, J=7 Hz, 2H), 3.20 (s, 3H), 2.30-2.10 (m, 2H).
Step 2. 5-Chloro-2-[7-(3-methoxypropyl)-7H-pyrrolo[2,3-c]pyridazin-3-yl]-3-methylphenol. To a solution of 3-chloro-7-(3-methoxypropyl)-7H-pyrrolo[2,3-c]pyridazine (150 mg, 665 μmol) and 5-chloro-3-methyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenol (Intermediate B2, 178 mg, 665 μmol) in dioxane (3 mL) and H2O (0.6 mL) at 25° C. under N2 were added dicyclohexyl-[2-(2,6-dimethoxyphenyl)phenyl]phosphane; methanesulfonate;(2-phenylanilino)palladium(1+) “SPhos Pd G3” (52 mg, 66 μmol) and K2CO3 (276 mg, 1.99 mmol). The mixture was stirred at reflux under N2 for 4 h. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Phenomenex Luna C18 100×40 mm×5 μm; mobile phase: [H2O (0.04% HCl)—ACN]; gradient: 10-40% B over 8.0 min) and lyophilised to give the title compound as a yellow solid. Y=32%. 1H NMR (400 MHz, DMSO-d6) δ 10.75 (br. s, 1H), 8.63 (s, 1H), 8.51 (s, 1H), 7.10-6.95 (m, 3H), 4.55 (t, J=7 Hz, 2H), 3.38 (t, J=7 Hz, 2H), 3.20 (s, 3H), 2.25-2.10 (m, 2H), 2.08 (s, 3H). 1H NMR (400 MHz, DMSO-d6+D2O) δ 8.55 (s, 1H), 8.47 (s, 1H), 7.03-6.97 (m, 2H), 6.94 (s, 1H), 4.52 (t, J=7 Hz, 2H), 3.35 (t, J=7 Hz, 2H), 3.15 (s, 3H), 2.20-2.08 (m, 2H), 2.05 (s, 3H). LCMS (ESI): m/z: [M+H]+=332.2.
Step 1. Tert-butyl cis-3-fluoro-4-[(methanesulfonyloxy)methyl]piperidine-1-carboxylate. To a solution of tert-butyl cis-3-fluoro-4-(hydroxymethyl)piperidine-1-carboxylate (500 mg, 2.14 mmol) in DCM (5 mL) at 0° C. under N2 were added DIPEA (560 μL, 3.22 mmol) and MsCl (166 μL, 2.14 mmol). The reaction was stirred at 0° C. under N2 for 3 h. The reaction mixture was quenched by addition of H2O (6 mL) and extracted with DCM (2×10 mL). The combined organic layers were washed with brine (2×8 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give the title compound as a yellow solid.
Step 2. Tert-butyl cis-4-({3-chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}methyl)-3-fluoropiperidine-1-carboxylate. To a solution of 3-chloro-7H-pyrrolo[2,3-c]pyridazine (Intermediate A1, 280 mg, 1.82 mmol) in DMF (5 mL) at 25° C. were added K2CO3 (504 mg, 3.65 mmol) and tert-butyl cis-3-fluoro-4-[(methanesulfonyloxy)methyl]piperidine-1-carboxylate (852 mg, 2.73 mmol). The reaction was stirred at 25° C. under N2 for 3 h. The reaction mixture was purified directly by prep-HPLC (column: Phenomenex Luna C18 100×40 mm×5 μm; mobile phase: [H2O (0.04% HCl)—ACN]; gradient: 35-55% B over 8.0 min) and lyophilised to give the title compound as a yellow solid. Y=51%. 1H NMR (400 MHz, DMSO-d6) δ 8.11-7.94 (m, 2H), 6.59 (d, J=3 Hz, 1H), 4.70-4.51 (m, 1H), 4.50-4.33 (m, 2H), 4.28-4.06 (m, 1H), 4.04-3.85 (m, 1H), 3.07-2.67 (m, 2H), 2.46-2.34 (m, 1H), 1.62-1.20 (m, 11H).
Step 3. Cis-4-({3-chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}methyl)-3-fluoropiperidine hydrochloride. A mixture of tert-butyl cis-4-({3-chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}methyl)-3-fluoropiperidine-1-carboxylate (260 mg, 705 μmol) and 4 M HCl in EtOAc (4 mL) was stirred at 25° C. for 2 h. The reaction mixture was concentrated under reduced pressure to give the title compound as a yellow solid. Y=quantitative.
Step 4. Cis-4-({3-chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}methyl)-3-fluoro-1-methylpiperidine. To a solution of cis-4-({3-chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}methyl)-3-fluoropiperidine hydrochloride (200 mg, 655 μmol) in MeOH (6 mL) at 0° C. were added 37% formaldehyde (1.95 mL, 26.2 mmol) and NaBH3CN (82 mg, 1.31 mmol). The reaction was stirred at 0° C. under N2 for 2 h. The reaction mixture was purified directly by prep-HPLC (column: Waters Xbridge BEH C18 100×30 mm×10 μm; mobile phase: [H2O (10 mM NH4HCO3)—ACN]; gradient: 1-55% B over 8.0 min) and lyophilised to give the title compound as a yellow solid. Y=86%. 1H NMR (400 MHz, DMSO-d6) δ 8.14-7.93 (m, 2H), 6.58 (d, J=3 Hz, 1H), 4.58-4.35 (m, 3H), 2.99 (t, J=12 Hz, 1H), 2.74 (d, J=12 Hz, 1H), 2.35-2.16 (m, 1H), 2.13 (s, 3H), 2.06-1.77 (m, 2H), 1.53-1.68 (m, 1H), 1.26 (m, 1H).
Step 5. Cis-5-chloro-2-{7-[(3-fluoro-1-methylpiperidin-4-yl)methyl]-7H-pyrrolo[2,3-c]pyridazin-3-yl}-3-methylphenol. To a solution of 5-chloro-3-methyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenol (Intermediate B2, 91 mg, 340 μmol) in dioxane (4 mL) and H2O (0.8 mL) at 25° C. under N2 were added cis-4-({3-chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}methyl)-3-fluoro-1-methylpiperidine (80 mg, 283 μmol), K2CO3 (156 mg, 1.13 mmol) and dicyclohexyl-[2-(2,6-dimethoxyphenyl)phenyl]phosphane; methanesulfonate;(2-phenylanilino)palladium(1+) “SPhos Pd G3” (33 mg, 42 μmol). The solution was stirred at reflux under N2 for 4 h. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was diluted with H2O (1 mL) and extracted with EtOAc (3×1 mL). The combined organic layers were washed with brine (2×1 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Waters Xbridge BEH C18 100×30 mm×10 μm; mobile phase: [H2O (10 mM NH4HCO3)—ACN]; gradient: 15-60% B over 8.0 min) and lyophilised to give the title compound as a yellow solid. Y=31%. 1H NMR (400 MHz, DMSO-d6) δ 9.84 (br. s, 1H), 7.92 (d, J=3 Hz, 1H), 7.76 (s, 1H), 6.91-6.82 (m, 2H), 6.58 (d, J=3 Hz, 1H), 4.65-4.39 (m, 3H), 3.01 (t, J=12 Hz, 1H), 2.77 (d, J=12 Hz, 1H), 2.30-2.18 (m, 1H), 2.14 (s, 3H), 2.06 (d, J=12 Hz, 1H), 1.98 (s, 3H), 1.87 (t, J=12 Hz, 1H), 1.75-1.55 (m, 1H), 1.43-1.26 (m, 1H). 1H NMR (400 MHz, DMSO-d6+D2O) δ 7.82 (d, J=3 Hz, 1H), 7.74 (s, 1H), 6.83 (m, 2H), 6.60 (d, J=3 Hz, 1H), 4.63-4.35 (m, 3H), 3.09-2.89 (m, 1H), 2.80-2.65 (m, 1H), 2.29-2.13 (m, 1H), 2.10 (s, 3H), 2.06-1.95 (m, 1H), 1.92 (s, 3H), 1.90-1.82 (m, 1H), 1.72-1.52 (m, 1H), 1.39-1.21 (m, 1H). LCMS (ESI): m/z: [M+H]+=389.3.
Step 1. 3-Chloro-7-[(oxan-2-yl)methyl]-7H-pyrrolo[2,3-c]pyridazine. To a solution of 3-chloro-7H-pyrrolo[2,3-c]pyridazine (Intermediate A1, 350 mg, 2.28 mmol) in DMF (4 mL) at 25° C. were added 2-(bromomethyl)tetrahydropyran (351 μL, 2.73 mmol) and t-BuOK (511 mg, 4.56 mmol). The mixture was stirred at 80° C. for 2 h. The reaction mixture was diluted with H2O (4 mL) and extracted with EtAOc (3×4 mL). The combined organic layers were washed with brine (4 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Phenomenex Luna C18 100×40 mm×5 μm; mobile phase: [H2O (0.04% HCl)—ACN]; gradient: 25-50% B over 8.0 min) and lyophilised to give the title compound as a yellow oil. Y=30%. 1H NMR (400 MHz, DMSO-d6) δ 8.02 (s, 1H), 7.97 (d, J=3 Hz, 1H), 6.55 (d, J=3 Hz, 1H), 4.48-4.33 (m, 2H), 3.86-3.79 (m, 1H), 3.77-3.75 (m, 1H), 3.29-3.20 (m, 1H), 1.83-1.72 (m, 1H), 1.65-1.53 (m, 1H), 1.50-1.36 (m, 3H), 1.30-1.12 (m, 1H).
Step 2. 5-Chloro-3-methyl-2-{7-[(oxan-2-yl)methyl]-7H-pyrrolo[2,3-c]pyridazin-3-yl}phenol. To a solution of 3-chloro-7-[(oxan-2-yl)methyl]-7H-pyrrolo[2,3-c]pyridazine (100 mg, 397 μmol) in dioxane (1.5 mL) and H2O (0.3 mL) at 25° C. under N2 were added 5-chloro-3-methyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenol (Intermediate B2, 128 mg, 477 μmol), K2CO3 (165 mg, 1.19 mmol) and SPhos Pd G3 (31 mg, 40 μmol). The mixture was stirred at 80° C. under N2 for 1 h. The reaction mixture was diluted with H2O (1 mL) and extracted with EtOAc (3×1 mL). The combined organic layers were washed with brine (1 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Phenomenex Luna C18 100×40 mm×5 μm; mobile phase: [H2O (0.04% HCl)—ACN]; gradient: 15-45% B over 8.0 min) and lyophilised to give the title compound as a white solid. Y=27%. 1H NMR (400 MHz, DMSO-d6) δ 10.79 (br. s, 1H), 8.59-8.37 (m 2H), 7.10-6.91 (m, 3H), 4.60-4.40 (m, 2H), 3.90-3.75 (m, 2H), 3.31-3.27 (m, 1H), 2.08 (s, 3H), 1.87-1.77 (m, 1H), 1.78-1.64 (m, 1H), 1.56-1.40 (m, 3H), 1.35-1.19 (m, 1H). 1H NMR (400 MHz, DMSO-d6+D2O) δ 8.57-8.46 (m, 2H), 7.04-6.93 (m, 3H), 4.56-4.41 (m, 2H), 3.86-3.74 (m, 2H), 3.31-3.23 (m, 1H), 2.07 (s, 3H), 1.86-1.75 (m, 1H), 1.73-1.64 (m, 1H), 1.54-1.37 (m, 3H), 1.32-1.19 (m, 1H). LCMS (ESI): m/z: [M+H]+=358.2.
Step 1. 2-[4-({3-Chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}methyl)piperidin-1-yl]acetonitrile. To a solution of 3-chloro-7-(4-piperidylmethyl)pyrrolo[2,3-c]pyridazine (for synthesis see Compound 4)(200 mg, 696 μmol) in DMF (2 mL) at 25° C. under N2 were added 2-chloroacetonitrile (53 μL, 836 μmol) and K2CO3 (193 mg, 1.39 mmol). The mixture was stirred at 25° C. under N2 for 2 h. The reaction mixture was purified by prep-HPLC (column: Waters Xbridge Prep OBD C18 150×40 mm×10 μm; mobile phase: [H2O (10 mM NH4HCO3)—ACN]; gradient: 20-50% B over 8.0 min) and lyophilised to give the title compound as a white solid. Y=59%. 1H NMR (400 MHz, DMSO-d6) δ 8.05 (d, J=3 Hz, 1H), 8.03 (s, 1H), 6.58 (d, J=3 Hz, 1H), 4.31 (d, J=7 Hz, 2H), 3.67 (s, 2H), 2.87-2.69 (m, 2H), 2.12-2.02 (m, 2H), 2.00-1.87 (m, 1H), 1.54-1.42 (m, 2H), 1.37-1.17 (m, 2H).
Step 2. 2-(4-{[3-(4-Chloro-2-hydroxy-6-methylphenyl)-7H-pyrrolo[2,3-c]pyridazin-7-yl]methyl}piperidin-1-yl)acetonitrile. To a solution of 2-[4-({3-chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}methyl)piperidin-1-yl]acetonitrile (70 mg, 242 μmol) in dioxane (3.5 mL) and H2O (0.7 mL) at 25° C. under N2 were added 5-chloro-3-methyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenol (Intermediate B2, 65 mg, 242 μmol), dicyclohexyl-[2-(2,6-dimethoxyphenyl)phenyl]phosphane; methanesulfonate;(2-phenylanilino)palladium (1+) “SPhos Pd G3” (28 mg, 36 μmol) and K2CO3 (134 mg, 966 μmol). The mixture was stirred at reflux under N2 for 2 h. The reaction mixture was diluted with H2O (3 mL) and extracted with EtOAc (3×3 mL). The combined organic layers were washed with brine (9 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Phenomenex Luna C18 100×40 mm×5 μm; mobile phase: [H2O (0.04% HCl)—ACN]; gradient: 1-40% B over 8.0 min) and lyophilised to give the title compound as a yellow solid. Y=31%. 1H NMR (400 MHz, DMSO-d6) δ 10.86 (br. s, 1H), 8.70 (s, 1H), 8.58 (s, 1H), 7.07-7.03 (m, 2H), 7.02 (s, 1H), 4.43 (d, J=7 Hz, 2H), 4.33-4.12 (m, 2H), 3.34-3.19 (m, 2H), 2.85-2.71 (m, 2H), 2.26-2.16 (m, 1H), 2.09 (s, 3H), 1.80-1.71 (m, 2H), 1.70-1.57 (m, 2H). 1H NMR (400 MHz, DMSO-d6+D2O) δ 8.62 (d, J=3 Hz, 1H), 8.56 (s, 1H), 7.08 (d, J 3 Hz, 1H), 7.01 (s, 1H), 6.96 (s, 1H), 4.41 (d, J=7 Hz, 2H), 4.19 (s, 2H), 3.25 (d, J=12 Hz, 2H), 2.71 (t, J=12 Hz, 2H), 2.24-2.13 (m, 1H), 2.07 (s, 3H), 1.83-1.67 (m, 2H), 1.59-1.45 (m, 2H). LC-MS (ESI): m/z: [M+H]+=396.2.
Step 1. Tert-butyl 4-(bromomethyl)-4-fluoropiperidine-1-carboxylate. To a solution of tert-butyl 4-methylenepiperidine-1-carboxylate (1.0 g, 5.07 mmol) in DCM (10 mL) at 0° C. under N2 were added triethylamine trihydrofluoride (2.11 mL, 12.7 mmol) and NBS (1.35 g, 7.60 mmol). The mixture was stirred at 25° C. under N2 for 12 h. The reaction mixture was diluted with H2O (10 mL) and extracted with DCM (3×10 mL). The combined organic layers were washed with brine (30 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, 0-50% ethyl acetate in petroleum ether) to give the title compound as a colourless oil. Y=87%. 1H NMR (400 MHz, chloroform-d) δ 4.12-3.91 (m, 2H), 3.75-3.65 (m, 2H), 3.15-3.00 (m, 2H), 2.02-1.90 (m, 2H), 1.77-1.58 (m, 2H), 1.47 (s, 9H).
Step 2. Tert-butyl 4-({3-chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}methyl)-4-fluoropiperidine-1-carboxylate. To a solution of tert-butyl 4-(bromomethyl)-4-fluoropiperidine-1-carboxylate (500 mg, 1.69 mmol) in DMF (5 mL) at 25° C. under N2 were added 3-chloro-7H-pyrrolo[2,3-c]pyridazine (Intermediate A1, 259 mg, 1.69 mmol) and K2CO3 (467 mg, 3.38 mmol). The mixture was stirred at 100° C. for 12 h under N2. The reaction mixture was diluted with H2O (5 mL) and extracted with EtOAc (3×5 mL). The combined organic layers were washed with brine (15 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, 0-50% ethyl acetate in petroleum ether) to give the title compound as a yellow oil. Y=77%. 1H NMR (400 MHz, DMSO-d6) δ 8.07 (s, 1H), 7.98-7.89 (m, 1H), 6.63 (d, J=3 Hz, 1H), 4.77-4.61 (m, 2H), 3.86-3.72 (m, 2H), 3.00-2.90 (m, 2H), 1.80-1.62 (m, 2H), 1.57-1.47 (m, 2H), 1.39 (s, 9H).
Step 3. 4-({3-Chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}methyl)-4-fluoropiperidin-1-ium trifluoroacetate. To a solution of tert-butyl 4-({3-chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}methyl)-4-fluoropiperidine-1-carboxylate (480 mg, 1.30 mmol) in DCM (5 mL) at 25° C. was added TFA (1.0 mL, 13.5 mmol). The mixture was stirred at 25° C. for 2 h. The reaction mixture was concentrated under reduced pressure to give the title compound as an oil. Y=quantitative.
Step 4. 4-({3-Chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}methyl)-4-fluoro-1-methylpiperidine. To solution of 4-({3-chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}methyl)-4-fluoropiperidin-1-ium trifluoroacetate (0.49 g, 1.82 mmol) in MeOH (5 mL) at 25° C. under N2 were added NaBH3CN (229 mg, 3.65 mmol) and 37% formaldehyde (477 μL, 6.40 mmol). The mixture was stirred at 25° C. under N2 for 1 h. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Waters Xbridge Prep OBD C18 150×40 mm×10 μm; mobile phase: [H2O (10 mM NH4HCO3)—ACN]; gradient: 10-40% B over 8.0 min) and lyophilised to give the title compound as a yellow oil. Y=29%. 1H NMR (400 MHz, DMSO-d6) δ 8.06 (s, 1H), 7.97-7.90 (m, 1H), 6.63 (d, J=3 Hz, 1H), 4.72-4.62 (m, 2H), 2.66-2.54 (m, 2H), 2.21-2.16 (m, 3H), 2.10 (t, J=11 Hz, 2H), 1.90-1.65 (m, 2H), 1.63-1.45 (m, 2H).
Step 5. 5-Chloro-2-{7-[(4-fluoro-1-methylpiperidin-4-yl)methyl]-7H-pyrrolo[2,3-c]pyridazin-3-yl}-3-methylphenol hydrochloride. To a solution of 4-({3-Chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}methyl)-4-fluoro-1-methylpiperidine (140 mg, 495 μmol) in dioxane (5 mL) and H2O (1 mL) at 25° C. under N2 was added 5-chloro-3-methyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenol (Intermediate B2, 133 mg, 495 μmol), Na2CO3 (157 mg, 1.49 mmol) and Pd(dppf)Cl2 (36 mg, 50 μmol). The mixture was stirred at 80° C. under N2 for 2 h. The reaction mixture was diluted with H2O (5 mL) and extracted with EtOAc (3×5 mL). The combined organic layers were washed with brine (15 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Phenomenex Luna C18 100×40 mm×5 μm; mobile phase: [H2O (0.04% HCl)—ACN]; gradient: 1-40% B over 8.0 min) to give the title compound as a yellow solid. Y=67%. 1H NMR (400 MHz, DMSO-d6) δ 10.91-10.42 (m, 2H), 8.52-8.19 (m, 2H), 7.10-6.90 (m, 3H), 5.02-4.76 (m, 2H), 3.40-3.37 (m, 2H), 3.10-2.97 (m, 2H), 2.88-2.74 (m, 3H), 2.36-2.16 (m, 2H), 2.06 (s, 3H), 2.03-1.87 (m, 2H). 1H NMR (400 MHz, DMSO-d6+D2O) δ 8.51-8.29 (m, 2H), 7.06-6.94 (m, 3H), 5.00-4.74 (m, 2H), 3.43-3.36 (m, 2H), 3.10-2.99 (m, 2H), 2.78 (s, 3H), 2.28-2.09 (m, 2H), 2.06 (s, 3H), 2.02-1.89 (m, 2H). LC-MS (ESI): m/z: [M+H]+=389.3.
Step 1. Tert-butyl 3-({3-chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}methyl)azetidine-1-carboxylate. To a solution of tert-butyl 3-(bromomethyl)azetidine-1-carboxylate (1.63 g, 6.51 mmol) in DMF (10 mL) at 25° C. were added 3-chloro-7H-pyrrolo[2,3-c]pyridazine (Intermediate A1, 1.0 g, 6.51 mmol) and K2CO3 (1.80 g, 13.0 mmol). The reaction mixture was stirred at 25° C. under N2 for 8 h. The reaction mixture was diluted with H2O (20 mL) and extracted with EtOAc (3×20 mL). The combined organic layers were washed with brine (50 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Phenomenex Luna C18 (250×70 mm, 15 μm); mobile phase: [H2O (0.04% HCl)—ACN]; gradient: 20-65% B over 20.0 min) and lyophilised to give the title compound as a yellow oil. Y=81%. 1H NMR (400 MHz, DMSO-d6) δ 8.14 (d, J=3 Hz, 1H), 8.04 (s, 1H), 6.58 (d, J=3 Hz, 1H), 4.62 (d, J=7 Hz, 2H), 3.93-3.82 (m, 2H), 3.73-3.72 (m, 2H), 3.14-3.04 (m, 1H), 1.36 (s, 9H).
Step 2. 3-({3-Chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}methyl)azetidine hydrochloride. A mixture of tert-butyl 3-({3-chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}methyl)azetidine-1-carboxylate (1.7 g, 5.27 mmol) and 4 M HCl in EtOAc (7 mL) was stirred at 25° C. for 2 h. The reaction mixture was filtered and dried under reduced pressure to give the title compound as a white solid. Y=quantitative.
Step 3. 3-({3-Chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}methyl)-1-methylazetidine. To a solution of 3-({3-chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}methyl)azetidine hydrochloride (500 mg, 1.93 mmol) in DCE (5 mL) at 25° C. were added 37% formaldehyde (359 μL, 4.82 mmol) and AcOH (552 μL, 9.65 mmol). The reaction mixture was stirred at 25° C. for 1 h, cooled to 0° C. and treated with NaBH(OAc)3 (1.23 g, 5.79 mmol). The resulting mixture was stirred at 25° C. under N2 for 3 h. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Waters Xbridge Prep OBD C18 150×40 mm×10 μm; mobile phase: [H2O (10 mM NH4HCO3)—ACN]; gradient: 5-35% B over 8.0 min) and lyophilised to give the title compound as an oil. Y=57%. 1H NMR (400 MHz, DMSO-d6) δ 8.08-8.04 (m, 2H), 6.59 (d, J=3 Hz, 1H), 4.64 (d, J=7 Hz, 2H), 3.72-3.64 (m, 2H), 3.62-3.53 (m, 2H), 3.16-3.10 (m, 1H), 2.52 (s, 3H).
Step 4. 5-Chloro-3-methyl-2-{7-[(I-methylazetidin-3-yl)methyl]-7H-pyrrolo[2,3-c]pyridazin-3-yl}phenol hydrochloride. To a solution of 3-({3-chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}methyl)-1-methylazetidine (100 mg, 422 μmol) in dioxane (1 mL) and H2O (0.2 mL) at 25° C. under N2 were added 5-chloro-3-methyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenol (Intermediate B2, 136 mg, 507 μmol), K2CO3 (175 mg, 1.27 mmol) and SPhos Pd G3 (33 mg, 42 μmol). The reaction mixture was stirred at 80° C. under N2 for 1 h. The reaction mixture was diluted with H2O (1 mL) and extracted with EtOAc (3×1 mL). The combined organic layers were washed with brine (1 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Phenomenex Luna C18 100×40 mm×5 μm; mobile phase: [H2O (0.04% HCl)—ACN];
Step 1. (3-{3-Chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}propyl)dimethylamine. To a solution of 3-chloro-7H-pyrrolo[2,3-c]pyridazine (Intermediate A1, 300 mg, 1.95 mmol) in DMF (3 mL) at 25° C. were added K2CO3 (540 mg, 3.91 mmol) and 3-chloro-N,N-dimethyl-propan-1-amine (475 mg, 3.91 mmol). The mixture was stirred at 80° C. for 12 h. The mixture was purified by prep-HPLC (column: Phenomenex Luna C18 75×30 mm×3 μm; mobile phase: [H2O (0.1% TFA)—ACN]; gradient: 1-25% B over 8.0 min) to give a residue. The residue was adjusted to pH ˜8 with saturated aqueous NaOH and the resulting mixture was concentrated under reduced pressure to give a residue. The residue was triturated with 10:1 DCM in MeOH, filtered and the filtrate concentrated to give the title compound as an oil. Y=75%.
Step 2. 5-Chloro-2-{7-[3-(dimethylamino)propyl]-7H-pyrrolo[2,3-c]pyridazin-3-yl}-3-methylphenol hydrochloride. To a solution of (3-{3-chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}propyl)dimethylamine (100 mg, 419 μmol) in dioxane (2 mL) and H2O (0.4 mL) at 25° C. under N2 were added 5-chloro-3-methyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenol (Intermediate B2, 112 mg, 419 μmol), K2CO3 (116 mg, 838 μmol) and SPhos Pd G3 (35 mg, 42 μmol). The mixture was stirred at reflux under N2 for 2 h. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Phenomenex Luna C18 100×40 mm×5 μm; mobile phase: [H2O (0.04% HCl)—ACN];
LC-MS (ESI): m/z: [M+H]+=345.2.
Step 1. 2-{3-Chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}-1-(pyrrolidin-1-yl)ethan-1-one. To a solution of 3-chloro-7H-pyrrolo[2,3-c]pyridazine (Intermediate A1, 300 mg, 1.95 mmol) in DMF (3 mL) at 25° C. were added K2CO3 (810 mg, 5.86 mmol) and 2-chloro-1-pyrrolidin-1-yl-ethanone (288 mg, 1.95 mmol). The resulting mixture was stirred at 80° C. for 2 h. The solution was diluted with H2O (5 mL) and extracted with ethyl acetate (3×5 mL). The combined organic layers were washed with brine (5 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Phenomenex Luna C18 100×40 mm×5 μm; mobile phase: [H2O (0.04% HCl)—ACN]; gradient: 5-45% B over 8.0 min) and lyophilised to give the title compound as a yellow solid. Y=52%. 1H NMR (400 MHz, MeOD) δ 8.25 (s, 1H), 8.14 (d, J=3 Hz, 1H), 6.83 (d, J=3 Hz, 1H), 5.38 (s, 2H), 3.71 (t, J=7 Hz, 2H), 3.46 (t, J=7 Hz, 2H), 2.15-2.06 (m, 2H), 1.98-1.87 (m, 2H).
Step 2. 2-[3-(4-Chloro-2-hydroxy-6-methylphenyl)-7H-pyrrolo[2,3-c]pyridazin-7-yl]-1-(pyrrolidin-1-yl)ethan-1-one. To a solution of 2-{3-chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}-1-(pyrrolidin-1-yl)ethan-1-one (90 mg, 340 μmol) in dioxane (2 mL) and H2O (0.4 mL) at 25° C. under N2 were added 5-chloro-3-methyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenol (Intermediate B2, 91 mg, 340 μmol), K2CO3 (188 mg, 1.36 mmol) and dicyclohexyl-[2-(2,6-dimethoxyphenyl)phenyl]phosphane; methanesulfonate;(2-phenylanilino)palladium(1+) “SPhos Pd G3” (40 mg, 51 μmol). The resulting mixture was stirred at 120° C. under N2 for 1 h. The solution was diluted with H2O (2 mL) and extracted with ethyl acetate (3×2 mL). The combined organic layers were washed with brine (2 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Waters Xbridge BEH C18 100×30 mm×10 μm; mobile phase: [H2O (10 mM NH4HCO3)—ACN]; gradient: 25-55% B over 8.0 min) and lyophilised to give the title compound as a white solid. Y=27%. 1H NMR (400 MHz, DMSO-d6) δ 9.88 (s, 1H), 7.83 (d, J=4 Hz, 1H), 7.75 (s, 1H), 6.88-6.84 (m, 2H), 6.58 (d, J=4 Hz, 1H), 5.33 (s, 2H), 3.65 (t, J=7 Hz, 2H), 3.37-3.35 (m, 2H), 2.05-1.92 (m, 5H), 1.88-1.78 (m, 2H). 1H NMR (400 MHz, DMSO-d6+D2O) δ 7.80 (d, J=4 Hz, 1H), 7.74 (s, 1H), 6.88-6.83 (m, 2H), 6.58 (d, J=4 Hz, 1H), 5.31 (s, 2H), 3.64 (t, J=7 Hz, 2H), 3.33 (t, J=7 Hz, 2H), 2.02-1.94 (m, 5H), 1.86-1.77 (m, 2H). LCMS (ESI): m/z: [M+H]+=371.2.
Step 1. 4-({3-Chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}methyl)-1-methanesulfonylpiperidine. To a solution of 3-chloro-7-(4-piperidylmethyl)pyrrolo[2,3-c]pyridazine (for synthesis see Compound 4)(0.20 g, 798 μmol) in DCM (2 mL) at 0° C. under N2 were added MsCl (74 μL, 957 μmol) and TEA (222 μL, 1.60 mmol). The mixture was stirred at 0° C. under N2 for 1 h. The reaction mixture was quenched by addition of H2O (2 mL) and extracted with DCM (3×2 mL). The combined organic layers were washed with brine (6 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give the title compound as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 8.06 (d, J=3 Hz, 1H), 8.05-8.04 (m, 1H), 6.59 (d, J=3 Hz, 1H), 4.35 (d, J=7 Hz, 2H), 3.60-3.45 (m, 2H), 2.70-2.55 (m, 2H), 2.44 (s, 3H), 2.15-2.05 (m, 1H), 1.57-1.54 (m, 2H), 1.40-1.20 (m, 2H).
Step 2. 5-Chloro-2-{7-[(I-methanesulfonylpiperidin-4-yl)methyl]-7H-pyrrolo[2,3-c]pyridazin-3-yl}-3-methylphenol. To a solution of 4-({3-chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}methyl)-1-methanesulfonylpiperidine (100 mg, 304 μmol) in dioxane (5 mL) and H2O (1 mL) at 25° C. under N2 were added 5-chloro-3-methyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenol (Intermediate B2, 98 mg, 365 μmol), dicyclohexyl-[2-(2,6-dimethoxyphenyl)phenyl]phosphane; methanesulfonate;(2-phenylanilino)palladium(1+) “SPhos Pd G3” (36 mg, 46 μmol) and K2CO3 (168 mg, 1.22 mmol). The mixture was stirred at 120° C. under N2 for 0.5 h. The reaction mixture was cooled to 0° C., quenched by addition of H2O (4 mL) and extracted with EtOAc (3×5 mL). The combined organic layers were washed with brine (15 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Phenomenex Luna C18 100×40 mm×5 μm; mobile phase: [H2O (0.04% HCl)—ACN]; gradient: 20-40% B over 8.0 min) and lyophilised to give the title compound as a yellow solid. Y=24%. 1H NMR (400 MHz, DMSO-d6) δ 10.59 (br. s, 1H), 8.73-8.21 (m, 2H), 7.10-6.88 (m, 3H), 4.43 (d, J=7 Hz, 2H), 3.57 (d, J=12 Hz, 2H), 2.84 (s, 3H), 2.70-2.64 (m, 2H), 2.21-2.11 (m, 1H), 2.07 (s, 3H), 1.72-1.62 (m, 2H), 1.45-1.30 (m, 2H). 1H NMR (400 MHz, DMSO-d6+D2O) δ 8.58-8.37 (m, 2H), 7.04-6.90 (m, 3H), 4.41 (d, J=7 Hz, 2H), 2.81 (s, 3H), 2.68-2.62 (m, 2H), 2.20-2.09 (m, 1H), 2.06 (s, 3H), 1.70-1.61 (m, 2H), 1.41-1.29 (m, 2H). 2H obscured by water peak at −3.5 ppm. 1H NMR (400 MHz, MeOD) δ 8.51 (d, J=3 Hz, 1H), 8.47 (s, 1H), 7.06 (d, J=3 Hz, 1H), 6.99 (d, J=2 Hz, 1H), 6.92 (d, J=2 Hz, 1H), 4.50 (d, J=7 Hz, 2H), 3.75 (d, J=12 Hz, 2H), 2.81 (s, 3H), 2.77-2.69 (m, 2H), 2.33-2.18 (m, 1H), 2.15 (s, 3H), 1.80-1.72 (m, 2H), 1.54-1.42 (m, 2H).
LC-MS (ESI): m/z: [M+H]+=435.2.
Step 1. 2-{3-Chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}-N,N-dimethylacetamide. To a solution of 3-chloro-7H-pyrrolo[2,3-c]pyridazine (Intermediate A1, 300 mg, 1.95 mmol) in DMF (3 mL) at 25° C. under N2 were added K2CO3 (540 mg, 3.91 mmol) and 2-chloro-N,N-dimethyl-acetamide (201 μL, 1.95 mmol). The mixture was stirred at 90° C. for 12 h. The reaction mixture was purified by prep-HPLC (column: Waters Xbridge BEH C18 100×30 mm×10 μm; mobile phase: [H2O (10 mM NH4HCO3)—ACN]; gradient: 1-45% B over 8.0 min) and lyophilised to give the title compound as a solid. Y=66%. 1H NMR (400 MHz, DMSO-d6) δ 8.03 (s, 1H), 7.91 (d, J=4 Hz, 1H), 6.58 (d, J=4 Hz, 1H), 5.38 (s, 2H), 3.14 (s, 3H), 2.86 (s, 3H).
Step 2. 2-[3-(4-Chloro-2-hydroxy-6-methylphenyl)-7H-pyrrolo[2,3-c]pyridazin-7yl]-N,N-dimethylacetamide. To a solution of 2-{3-chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}-N,N-dimethylacetamide (100 mg, 419 μmol) in dioxane (5 mL) and H2O (1 mL) at 25° C. under N2 were added 5-chloro-3-methyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenol (Intermediate B2, 146 mg, 545 μmol), K2CO3 (232 mg, 1.68 mmol) and dicyclohexyl-[2-(2,6-dimethoxyphenyl)phenyl]phosphane; methanesulfonate;(2-phenylanilino)palladium(1+) “SPhos Pd G3” (49 mg, 63 μmol). The resulting mixture was stirred at 90° C. under N2 for 3 h. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was diluted with H2O (1 mL) and extracted with EtOAc (3×1 mL). The combined organic layers were washed with brine (2×1 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Waters Xbridge BEH C18 100×30 mm×10 μm; mobile phase: [H2O (10 mM NH4HCO3)—ACN]; gradient: 20-55% B over 8.0 min) and lyophilised to give the title compound as a yellow solid. Y=20%.
1H NMR (400 MHz, DMSO-d6) δ 9.88 (s, 1H), 7.81 (d, J=4 Hz, 1H), 7.75 (s, 1H), 6.91-6.81 (m, 2H), 6.57 (d, J=4 Hz, 1H), 5.42 (s, 2H), 3.18 (s, 3H), 2.88 (s, 3H), 1.98 (s, 3H). 1H NMR (400 MHz, DMSO-d6+D2O) δ 7.75 (d, J=4 Hz, 1H), 7.73 (s, 1H), 6.85-6.83 (m, 2H), 6.59 (d, J=4 Hz, 1H), 5.36 (s, 2H), 3.14 (s, 3H), 2.85 (s, 3H), 1.94 (s, 3H). LCMS (ESI): m/z: [M+H]+=345.1.
Step 1. 3-Chloro-7-(2-chloroethyl)-7H-pyrrolo[2,3-c]pyridazine. To a solution of 3-chloro-7H-pyrrolo[2,3-c]pyridazine (Intermediate A1, 600 mg, 3.91 mmol) in DMF (10 mL) at 25° C. was added K2CO3 (2.16 g, 15.6 mmol). The mixture was stirred at 25° C. for 30 min, then treated with 1-bromo-2-chloro-ethane (486 μL, 5.86 mmol) at 25° C. The mixture was stirred at 25° C. for 4 h. The reaction mixture was diluted with H2O (8 mL) and extracted with EtOAc (3×8 mL). The combined organic layers were washed with brine (8 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Phenomenex Luna C18 100×40 mm×5 μm; mobile phase: [H2O (0.04% HCl)—ACN]; gradient: 20-50% B over 8.0 min) and lyophilised to give the title compound as a white solid. Y=45%. 1H NMR (400 MHz, DMSO-d6) δ 8.11 (d, J=3 Hz, 1H), 8.06 (s, 1H), 6.61 (d, J=3 Hz, 1H), 4.76 (t, J=6 Hz, 2H), 4.13 (t, J=6 Hz, 2H).
Step 2. 1-(2-{3-chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}ethyl)-3-fluoropiperidine. To a solution of 3-chloro-7-(2-chloroethyl)-7H-pyrrolo[2,3-c]pyridazine (150 mg, 694 μmol) in ACN (4 mL) at 25° C. under N2 were added K2CO3 (576 mg, 4.17 mmol) and 3-fluoropiperidine hydrochloride (194 mg, 1.39 mmol). The mixture was stirred at reflux for 2 h. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Waters Xbridge BEH C18 100×30 mm×10 μm; mobile phase: [H2O (10 mM NH4HCO3)—ACN]; gradient: 25-60% B over 8.0 min) and lyophilised to give the title compound as a white solid. Y=51%. 1H NMR (400 MHz, DMSO-d6) δ 8.14-7.94 (m, 2H), 6.55 (d, J=3 Hz, 1H), 4.60-4.42 (m, 3H), 2.88-2.71 (m, 3H), 2.47-2.36 (m, 2H), 2.34-2.25 (m, 1H), 1.86-1.71 (m, 1H), 1.69-1.57 (m, 1H), 1.55-1.40 (m, 1H), 1.39-1.25 (m, 1H).
Step 3. 5-Chloro-2-{7-[2-(3-fluoropiperidin-1-yl)ethyl]-7H-pyrrolo[2,3-c]pyridazin-3-yl}-3-methylphenol hydrochloride. To a solution of 1-(2-{3-chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}ethyl)-3-fluoropiperidine (80 mg, 283 μmol) in dioxane (2 mL) and H2O (0.4 mL) at 25° C. under N2 were added 5-chloro-3-methyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenol (Intermediate B2, 91 mg, 340 μmol), XPhos Pd G3 (24 mg, 29 μmol) and K2CO3 (156 mg, 1.13 mmol). The mixture was stirred at 80° C. under N2 for 3 h. The reaction mixture was diluted with H2O (1 mL) and extracted with EtOAc (3×1 mL). The combined organic layers were washed with brine (1 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Phenomenex Luna C18 100×40 mm×5 μm; mobile phase: [H2O (0.04% HCl)—ACN]; gradient: 10-30% B over 8.0 min) and lyophilised to give the title compound as a white solid. Y=30%. 1H NMR (400 MHz, DMSO-d6) δ 12.16-10.02 (m, 2H), 8.85-8.17 (m, 2H), 7.43-6.66 (m, 3H), 5.25-4.92 (m, 3H), 4.18-3.74 (m, 5H), 3.18-3.04 (m, 1H), 2.07 (s, 3H), 2.03-1.85 (m, 2H), 1.85-1.59 (m, 2H). 1H NMR (400 MHz, DMSO-d6) δ 8.49 (d, J=3 Hz, 1H), 8.43 (s, 1H), 7.05 (d, J=3 Hz, 1H), 6.99 (d, J=2 Hz, 1H), 6.94 (d, J=2 Hz, 1H), 5.21-5.01 (m, 1H), 4.96-4.78 (m, 2H), 3.86-3.79 (m, 1H), 3.70 (t, J=6 Hz, 2H), 3.56-3.27 (m, 2H), 3.13 (s, 1H), 2.03 (s, 3H), 1.98-1.61 (m, 4H). 1H NMR (400 MHz, MeOD) δ 8.66 (d, J=3 Hz, 1H), 8.62 (s, 1H), 7.18 (d, J=3 Hz, 1H), 7.02 (s, 1H), 6.93 (s, 1H), 5.26-5.13 (m, 1H), 5.11-5.00 (m, 2H), 4.28-4.08 (m, 1H), 3.95-3.79 (m, 2H), 3.74-3.62 (m, 1H), 3.55-3.41 (m, 1H), 3.30-3.18 (m, 1H), 2.29-2.05 (m, 5H), 2.00-1.74 (m, 2H). LC-MS (ESI): m/z: [M+H]+=389.2.
Step 1. Tert-butyl 2-({3-chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}methyl)morpholine-4-carboxylate. To a solution of 3-chloro-7H-pyrrolo[2,3-c]pyridazine (Intermediate A1, 300 mg, 1.95 mmol) in DMF (5 mL) at 25° C. were added K2CO3 (540 mg, 3.91 mmol) and tert-butyl 2-(chloromethyl)morpholine-4-carboxylate (460 mg, 1.95 mmol). The mixture was stirred at 80° C. under N2 for 12 h. The mixture was purified by prep-HPLC (column: Phenomenex Luna C18 75×30 mm×3 μm; mobile phase: [H2O (0.1% TFA)—ACN]; gradient: 30-55% B over 8.0 min) to give the title compound as a yellow oil. Y=87%. 1H NMR (400 MHz, DMSO-d6) δ 8.04 (s, 1H), 8.02 (d, J=4 Hz, 1H), 6.59 (d, J=4 Hz, 1H), 4.60-4.40 (m, 2H), 3.87-3.77 (m, 3H), 3.72-3.61 (m, 2H), 3.46-3.39 (m, 1H), 3.35-3.25 (m, 1H), 1.41-1.38 (m, 9H).
Step 2. 2-({3-Chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}methyl)morpholine hydrochloride. A mixture of tert-butyl 2-({3-chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}methyl)morpholine-4-carboxylate (500 mg, 1.42 mmol) and 4 M HCl in EtOAc (10 mL) was stirred at 25° C. for 4 h. The reaction mixture was concentrated under reduced pressure to give the title compound as a yellow solid. Y=quantitative. 1H NMR (400 MHz, DMSO-d6) δ 9.62 (s, 2H), 8.06 (s, 1H), 7.98 (d, J=4 Hz, 1H), 6.60 (d, J=4 Hz, 1H), 4.67-4.57 (m, 1H), 4.54-4.42 (m, 1H), 4.30-4.20 (m, 1H), 3.98-3.85 (m, 1H), 3.75-3.65 (m, 1H), 3.40-3.25 (m, 1H), 3.20-3.08 (m, 1H), 3.00-2.85 (m, 1H), 2.85-2.71 (m, 1H).
Step 3. 2-({3-Chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}methyl)-4-methylmorpholin-4-ium trifluoroacetate. To a solution of 2-({3-chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}methyl)morpholine hydrochloride (300 mg, 1.04 mmol) in MeOH (3 mL) was added aqueous 37% formaldehyde (386 μL, 5.19 mmol) and NaBH3CN (130 mg, 2.07 mmol). The mixture was stirred at 25° C. for 2 h. The mixture was purified by prep-HPLC (column: Phenomenex Luna C18 75×30 mm×3 μm; mobile phase: [H2O (0.1% TFA)—ACN]; gradient: 1-25% B over 8.0 min) and lyophilised to give the title compound as a white solid. Y=76%. 1H NMR (400 MHz, DMSO-d6) δ 10.10 (br. s, 1H), 8.07 (s, 1H), 7.98 (d, J=4 Hz, 1H), 6.61 (d, J=4 Hz, 1H), 4.65-4.48 (m, 2H), 4.20-4.13 (m, 1H), 4.05-3.95 (m, 1H), 3.67-3.50 (m, 2H), 3.40-3.30 (m, 1H), 3.04-2.92 (m, 1H), 2.90-2.75 (m, 4H).
Step 4. 5-Chloro-3-methyl-2-{7-[(4-methylmorpholin-2-yl)methyl]-7H-pyrrolo[2,3-c]pyridazin-3-yl}phenol hydrochloride. To a solution of 2-({3-chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}methyl)-4-methylmorpholin-4-ium trifluoroacetate (100 mg, 263 μmol) and 5-chloro-3-methyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenol (Intermediate B2, 71 mg, 263 μmol) in dioxane (5 mL) and H2O (1 mL) at 25° C. under N2 were added K2CO3 (109 mg, 788 μmol) and dicyclohexyl-[2-(2,6-dimethoxyphenyl)phenyl]phosphane; methanesulfonate;(2-phenylanilino)palladium(1+) “SPhos Pd G3” (20 mg, 26 μmol). The mixture was stirred at 100° C. under N2 for 4 h. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Phenomenex Luna C18 100×40 mm×5 μm; mobile phase: [H2O (0.0 4% HCl)—ACN]; gradient: 10-30% B over 8.0 min) and lyophilised to give the title compound as a yellow solid. Y=36%. 1H NMR (400 MHz, DMSO-d6) δ 11.32 (br. s, 1H), 10.70 (br. s, 1H), 8.45 (s, 2H), 7.05-6.96 (m, 3H), 4.80-4.65 (m, 1H), 4.61-4.50 (m, 1H), 4.41-4.29 (m, 1H), 4.05-3.98 (m, 1H), 3.85-3.75 (m, 1H), 3.65-3.55 (m, 1H), 3.36-3.35 (m, 1H), 3.06-2.90 (m, 2H), 2.78 (d, J=3 Hz, 3H), 2.07 (s, 3H). 1H NMR (400 MHz, DMSO-d6+D2O) δ 8.45 (s, 2H), 7.12-6.86 (m, 3H), 4.73-4.65 (m, 1H), 4.61-4.50 (m, 1H), 4.31-4.22 (m, 1H), 4.05-3.98 (m, 1H), 3.70-3.65 (m, 1H), 3.65-3.60 (m, 1H), 3.40-3.30 (m, 1H), 3.09-2.91 (m, 2H), 2.81 (s, 3H), 2.06 (s, 3H). 1H NMR (400 MHz, MeOD) δ 8.60-8.53 (m, 2H), 7.13 (d, J=3 Hz, 1H), 7.02 (d, J=2 Hz, 1H), 6.94 (d, J=2 Hz, 1H), 4.85-4.80 (m, 1H), 4.75-4.65 (m, 1H), 4.40-4.30 (m, 1H), 4.22-4.12 (m, 1H), 3.90-3.81 (m, 1H), 3.78-3.72 (m, 1H), 3.50-4.45 (m, 1H), 3.19-3.03 (m, 2H), 2.96 (s, 3H), 2.18 (s, 3H). LCMS (ESI): m/z: [M+H]+=373.2.
Step 1. Tert-butyl 3,3-difluoro-4-[(methanesulfonyloxy)methyl]piperidine-1-carboxylate. To a solution of tert-butyl 3,3-difluoro-4-(hydroxymethyl)piperidine-1-carboxylate (500 mg, 1.99 mmol) in DCM (5 mL) at 0° C. under N2 were added DIPEA (693 μL, 3.98 mmol) and MsCl (185 μL, 2.39 mmol). The resulting mixture was stirred at 0° C. under N2 for 1 h. The mixture was quenched with H2O (3 mL) and extracted with DCM (3×3 mL). The combined organic layers were washed with brine (3×3 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give the title compound as a yellow oil. Y=92%.
Step 2. Tert-butyl 4-({3-chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}methyl)-3,3-difluoropiperidine-1-carboxylate. To a solution of tert-butyl 3,3-difluoro-4-(methylsulfonyloxymethyl)piperidine-1-carboxylate (600 mg, 1.82 mmol) in DMF (8 mL) at 25° C. were added K2CO3 (755 mg, 5.47 mmol) and 3-chloro-7H-pyrrolo[2,3-c]pyridazine (Intermediate A1, 280 mg, 1.82 mmol). The resulting mixture was stirred at 80° C. for 12 h. The solution was diluted with H2O (2 mL) and extracted with ethyl acetate (3×2 mL). The combined organic layers were washed with brine (2 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Phenomenex Luna C18 100×40 mm×5 μm; mobile phase: [H2O (0.04% HCl)—ACN]; gradient: 35-75% B over 8.0 min) and lyophilised to give the title compound as a yellow solid. Y=43%. 1H NMR (400 MHz, CDCl3) δ 7.72 (s, 1H), 7.53 (d, J=3 Hz, 1H), 6.47 (d, J=3 Hz, 1H), 5.47-5.08 (m, 1H), 4.84-4.24 (m, 3H), 3.71 (t, J=11 Hz, 1H), 3.50 (t, J=6 Hz, 1H), 2.44 (t, J=6 Hz, 1H), 1.64-1.56 (m, 2H), 1.47 (s, 9H).
Step 3. 4-({3-Chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}methyl)-3,3-difluoropiperidine hydrochloride. A mixture of tert-butyl 4-({3-chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}methyl)-3,3-difluoropiperidine-1-carboxylate (240 mg, 620 μmol) and 4 M HCl in EtOAc (5 mL) was stirred at 25° C. for 1 h. The reaction mixture was concentrated under vacuum to give the title compound as a white solid. Y=quantitative.
Step 4. 4-({3-Chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}methyl)-3,3-difluoro-1-methylpiperidine. To a solution of 4-({3-chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}methyl)-3,3-difluoropiperidine hydrochloride (200 mg, 619 μmol) in DCE (3 mL) and AcOH (0.36 mL) at 0° C. was added aqueous 37% formaldehyde (138 μL, 1.86 mmol). The resulting mixture was stirred at 0° C. for 0.5 h, then treated with NaBH(OAc)3 (393 mg, 1.86 mmol). The resulting mixture was stirred at 0° C. for 0.5 h. The solution was diluted with H2O (3 mL) and extracted with DCM (3×3 mL). The combined organic layers were washed with brine (3 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give the title compound as a yellow oil. Y=97%.
Step 5. 5-Chloro-2-{7-[(3,3-difluoro-1-methylpiperidin-4-yl)methyl]-7H-pyrrolo[2,3-c]pyridazin-3-yl}-3-methylphenol hydrochloride. To a solution of 4-({3-chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}methyl)-3,3-difluoro-1-methylpiperidine (90 mg, 299 μmol) in dioxane (2 mL) and H2O (0.4 mL) at 25° C. under N2 were added 5-chloro-3-methyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenol (Intermediate B2, 80 mg, 299 μmol), dicyclohexyl-[2-(2,6-dimethoxyphenyl)phenyl]phosphane; methanesulfonate;(2-phenylanilino)palladium(1+) “SPhos Pd G3” (35 mg, 45 μmol) and K2CO3 (165 mg, 1.20 mmol). The resulting mixture was stirred at reflux under N2 for 1 h. The solution was diluted with H2O (2 mL) and extracted with ethyl acetate (3×2 mL). The combined organic layers were washed with brine (2 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Phenomenex Luna C18 100×40 mm×5 μm; mobile phase: [H2O (0.04% HCl)—ACN]; gradient: 10-30% B over 8.0 min) and lyophilised to give the title compound as a yellow solid. Y=21%. 1H NMR (400 MHz, DMSO-d6) δ 10.98 (br. s, 1H), 10.62 (br. s, 1H), 8.72-8.26 (m, 2H), 7.06-6.88 (m, 3H), 4.95-4.85 (m, 1H), 4.61-4.47 (m, 1H), 4.00 (s, 1H), 3.39-3.35 (m, 2H), 2.99 (s, 2H), 2.79 (s, 3H), 2.06 (s, 3H), 1.89 (s, 2H). 1H NMR (400 MHz, DMSO-d6+D2O) δ 8.49 (s, 1H), 8.39 (s, 1H), 7.03-6.91 (m, 3H), 4.95-4.80 (m, 1H), 4.60-4.49 (m, 1H), 4.02-3.92 (m, 1H), 3.53-3.40 (m, 2H), 3.08-2.89 (m, 2H), 2.83 (s, 3H), 2.04 (s, 3H), 1.98-1.79 (m, 2H). 1H NMR (400 MHz, MeOD) δ 8.63 (d, J=3 Hz, 1H), 8.59 (s, 1H), 7.15 (d, J=3 Hz, 1H), 7.02 (d, J=2 Hz, 1H), 6.93 (d, J=2 Hz, 1H), 5.10 -5.00 (m, 1H), 4.74-4.63 (m, 1H), 4.08-3.94 (m, 1H), 3.65-3.46 (m, 2H), 3.22-3.05 (m, 2H), 2.99 (s, 3H), 2.19-2.13 (m, 4H), 2.08-1.96 (m, 1H). LCMS (ESI): m/z: [M+H]+=407.2.
Step 1. 2-{3-Chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}-1piperidin-1-yl)ethan-1-one. To a solution of 3-chloro-7H-pyrrolo[2,3-c]pyridazine (Intermediate A1, 150 mg, 977 μmol) in DMF (3 mL) at 25° C. were added K2CO3 (405 mg, 2.93 mmol) and 2-chloro-1-(1-piperidyl)ethanone (158 mg, 977 μmol). The resulting mixture was stirred at 80° C. for 2 h. The solution was diluted with H2O (5 mL) and extracted with ethyl acetate (3×5 mL). The combined organic layers were washed with brine (8 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Phenomenex Luna C18 100×40 mm×5 μm; mobile phase: [H2O (0.04% HCl)—ACN]; gradient: 15-45% B over 8.0 min) to give the title compound as a yellow solid. Y=51%. 1H NMR (400 MHz, DMSO-d6) δ 8.03 (s, 1H), 7.94 (d, J=3 Hz, 1H), 6.58 (d, J=3 Hz, 1H), 5.38 (s, 2H), 3.57-3.53 (m, 2H), 3.44-3.39 (m, 2H), 1.67-1.59 (m, 4H), 1.50-1.42 (m, 2H).
Step 2. 2-[3-(4-Chloro-2-hydroxy-6-methylphenyl)-7H-pyrrolo[2,3-c]pyridazin-7-yl]-1-(piperidin-1-yl)ethan-1-one. To a solution of 2-{3-chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}-1-(piperidin-1-yl)ethan-1-one (90 mg, 323 μmol) in dioxane (3 mL) and H2O (0.6 mL) at 25° C. were added 5-chloro-3-methyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenol (Intermediate B2, 87 mg, 323 μmol), K2CO3 (179 mg, 1.29 mmol) and dicyclohexyl-[2-(2,6-dimethoxyphenyl)phenyl]phosphane; methanesulfonate;(2-phenylanilino)palladium(1+) “SPhos Pd G3” (38 mg, 48 μmol), the resulting mixture was stirred at 120° C. under N2 for 1 h. The solution was diluted with H2O (2 mL) and extracted with ethyl acetate (3×2 mL). The combined organic layers were washed with brine (2 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Phenomenex Luna C18 100×40 mm×5 μm; mobile phase: [H2O (0.04% HCl)—ACN]; gradient: 15-30% B over 8.0 min) and lyophilised to give the title compound as an off-white solid. Y=44%. 1H NMR (400 MHz, DMSO-d6) δ 10.52 (br. s, 1H), 8.39 (br. s, 2H), 6.99 (s, 1H), 6.95 (s, 2H), 5.51 (s, 2H), 3.60-3.55 (m, 2H), 3.48-3.41 (m, 2H), 2.06 (s, 3H), 1.66-1.63 (m, 4H), 1.51-1.46 (m, 2H). 1H NMR (400 MHz, DMSO-d6+D2O) δ 8.39 (s, 2H), 6.99 (s, 1H), 6.99-6.92 (m, 2H), 5.50 (s, 2H), 3.57-2.52 (m, 2H), 3.44-3.41 (m, 2H), 2.05 (s, 3H), 1.67-1.62 (m, 4H), 1.51-1.44 (m, 2H). 1H NMR (400 MHz, MeOD) δ 8.46 (s, 1H), 8.39 (d, J=3 Hz, 1H), 7.06 (d, J=3 Hz, 1H), 6.99 (d, J=2 Hz, 1H), 6.91 (d, J=2 Hz, 1H), 5.57 (s, 2H), 3.69-3.64 (m, 2H), 3.61-3.55 (m, 2H), 2.14 (s, 3H), 1.80-1.72 (m, 4H), 1.65-1.57 (m, 2H).
LCMS (ESI): m/z: [M+H]+=385.2.
Step 1. (Oxan-4-yl)methyl methanesulfonate. To a solution of tetrahydropyran-4-ylmethanol (500 mg, 4.30 mmol) in DCM (6 mL) at 0° C. were added DIPEA (1.50 mL, 8.61 mmol) and MsCl (400 μL, 5.17 mmol). The resulting mixture was stirred at 0° C. for 1 h. The mixture was quenched by H2O (5 mL) and extracted with DCM (3×5 mL). The combined organic layers were washed with brine (3×5 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give the title compound as a colourless oil. Y=96%. 1H NMR (400 MHz, DMSO-d6) δ 4.06 (d, J=6 Hz, 2H), 3.90-3.80 (m, 2H), 3.33-3.27 (m, 2H), 3.17 (s, 3H), 1.98-1.88 (m, 1H), 1.62-1.54 (m, 2H), 1.26-1.18 (m, 2H).
Step 2. 3-Chloro-7-[(oxan-4-yl)methyl]-7H-pyrrolo[2,3-c]pyridazine. To a solution of 3-chloro-7H-pyrrolo[2,3-c]pyridazine (Intermediate A1, 300 mg, 1.95 mmol) in DMF (8 mL) at 25° C. were added K2CO3 (540 mg, 3.91 mmol) and (oxan-4-yl)methyl methanesulfonate (379 mg, 1.95 mmol). The resulting mixture was stirred at 80° C. for 12 h. The mixture was diluted with H2O (8 mL) and extracted with ethyl acetate (3×8 mL). The combined organic layers were washed with brine (8 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Phenomenex Luna C18 100×40 mm×5 μm; mobile phase: [H2O (0.04% HCl)—ACN]; gradient: 10-50% B over 8.0 min) and lyophilised to give the title compound as a yellow solid. Y=51%. 1H NMR (400 MHz, DMSO-d6) δ 8.06 (d, J=3 Hz, 1H), 8.03 (s, 1H), 6.58 (d, J=3 Hz, 1H), 4.31 (d, J=7 Hz, 2H), 3.81 (d, J=11 Hz, 2H), 3.26-3.17 (m, 2H), 2.24-2.14 (m, 1H), 1.37-1.21 (m, 4H).
Step 3. 5-Chloro-3-methyl-2-{7-[(oxan-4-yl)methyl]-7H-pyrrolo[2,3-c]pyridazin-3-yl}phenol. To a solution of 3-chloro-7-[(oxan-4-yl)methyl]-7H-pyrrolo[2,3-c]pyridazine (100 mg, 397 μmol) in dioxane (2 mL) and H2O (0.4 mL) at 25° C. under N2 were added 5-chloro-3-methyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenol (Intermediate B2, 107 mg, 397 μmol), K2CO3 (220 mg, 1.59 mmol) and dicyclohexyl-[2-(2,6-dimethoxyphenyl)phenyl]phosphane; methanesulfonate;(2-phenylanilino)palladium(1+) “SPhos Pd G3” (47 mg, 60 μmol). The resulting mixture was stirred at reflux under N2 for 2 h. The mixture was diluted with H2O (2 mL) and extracted with ethyl acetate (3×2 mL). The combined organic layers were washed with brine (2 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Waters Xbridge Prep OBD C18 150×40 mm×10 μm; mobile phase: [H2O (10 mM NH4HCO3)—ACN]; gradient: 30-60% B over 8.0 min) and lyophilised to give the title compound as a white solid. Y=22%. 1H NMR (400 MHz, DMSO-d6) δ 9.83 (br. s, 1H), 7.93 (d, J=3 Hz, 1H), 7.75 (s, 1H), 6.86 (d, J=5 Hz, 2H), 6.56 (d, J=3 Hz, 1H), 4.35 (d, J=7 Hz, 2H), 3.87-3.80 (m, 2H), 3.28-3.22 (m, 2H), 2.31-2.22 (m, 1H), 1.98 (s, 3H), 1.48-1.40 (m, 2H), 1.39-1.28 (m, 2H). 1H NMR (400 MHz, DMSO-d6+D2O) δ 7.91 (d, J=3 Hz, 1H), 7.74 (s, 1H), 6.86 (d, J=5 Hz, 2H), 6.57 (d, J=3 Hz, 1H), 4.34 (d, J=7 Hz, 2H), 3.86-3.79 (m, 2H), 3.29-3.20 (m, 2H), 2.30-2.19 (m, 1H), 1.96 (s, 3H), 1.47-1.39 (m, 2H), 1.39-1.27 (m, 2H). LCMS (ESI): m/z: [M+H]+=358.2.
Step 1. 1-(2-Chloroethyl)piperidine hydrochloride. To a solution of 2-(1-piperidyl)ethanol (1.03 mL, 7.74 mmol) in DCM (10 mL) at 0° C. were added DMF (60 μL, 774 mol) and SOCl2 (3.09 mL, 42.6 mmol). The mixture was stirred at 40° C. under N2 for 4 h. The reaction mixture was concentrated under reduced pressure to give the title compound as a white solid. Y=70%.
Step 2. 1-(2-{3-Chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}ethyl)piperidine hydrochloride. To a solution of 3-chloro-7H-pyrrolo[2,3-c]pyridazine (Intermediate A1, 300 mg, 1.95 mmol) in DMF (3 mL) at 25° C. were added 1-(2-chloroethyl)piperidine hydrochloride (432 mg, 2.34 mmol) and K2CO3 (810 mg, 5.86 mmol). The mixture was stirred at 80° C. under N2 for 2 h. The reaction mixture was diluted with H2O (4 mL) and extracted with EtOAc (3×4 mL). The combined organic layers were washed with brine (4 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Phenomenex Luna C18 100×40 mm×5 μm; mobile phase: [H2O (0.04% HCl)—ACN]; gradient: 1-15% B over 8.0 min) and lyophilised to give the title as a yellow oil. Y=34%. 1H NMR (400 MHz, DMSO-d6) δ 10.80 (s, 1H), 8.16 (d, J=3 Hz, 1H), 8.08 (s, 1H), 6.64 (d, J=3 Hz, 1H), 4.89-4.87 (m, 2H), 3.66-3.58 (m, 2H), 3.50-3.47 (m, 2H), 3.01-2.86 (m, 2H), 1.84-1.73 (m, 4H), 1.70-1.66 (m, 1H), 1.45-1.29 (m, 1H).
Step 3. 5-Chloro-3-methyl-2-{7-[2-(piperidin-1-yl)ethyl]-7H-pyrrolo[2,3-c]pyridazin-3-yl}phenol hydrochloride. To a solution of 1-(2-{3-chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}ethyl)piperidine hydrochloride (80 mg, 266 μmol) in dioxane (1 mL) and H2O (0.2 mL) at 25° C. under N2 were added 5-chloro-3-methyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenol (Intermediate B2, 86 mg, 319 μmol), K2CO3 (110 mg, 797 μmol) and SPhos Pd G3 (21 mg, 27 μmol). The mixture was stirred at reflux under N2 for 2 h. The reaction mixture was diluted with H2O (1 mL) and extracted with EtOAc (3×1 mL). The combined organic layers were washed with brine (1 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Phenomenex Luna C18 100×40 mm×5 μm; mobile phase: [H2O (0.04% HCl)—ACN]; gradient: 10-30% B over 8.0 min) and lyophilised to give the title compound as a white solid. Y=29%. 1H NMR (400 MHz, DMSO-d6) δ 10.60 (br. s, 2H), 8.70-8.19 (m, 2H), 7.00-6.98 (m, 3H), 4.96 (t, J=7 Hz, 2H), 3.67-3.64 (m, 2H), 3.59-3.56 (m, 2H), 3.04-2.93 (m, 2H), 2.06 (s, 3H), 1.86-1.66 (m, 5H), 1.47-1.32 (m, 1H). 1H NMR (400 MHz, DMSO-d6+D2O) δ 8.59-8.38 (m, 2H), 7.05-6.91 (m, 3H), 4.92 (t, J=7 Hz, 2H), 3.68 (t, J=7 Hz, 2H), 3.61-3.58 (m, 2H), 3.00 (t, J=11 Hz, 2H), 2.06 (s, 3H), 1.89-1.78 (m, 2H), 1.71-1.68 (m, 3H), 1.48-1.33 (m, 1H). LCMS (ESI): m/z: [M+H]+=371.2.
Step 1. Tert-butyl 2-({3-chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}methyl)pyrrolidine-1-carboxylate. To a solution of 3-chloro-7H-pyrrolo[2,3-c]pyridazine (Intermediate A1, 350 mg, 2.28 mmol) in DMF (6 mL) at 25° C. were added K2CO3 (945 mg, 6.84 mmol) and tert-butyl 2-(bromomethyl)pyrrolidine-1-carboxylate (602 mg, 2.28 mmol). The resulting mixture was stirred at 80° C. under N2 for 12 h. The solution was diluted with H2O (6 mL) and extracted with ethyl acetate (3×6 mL). The combined organic layers were washed with brine (6 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Phenomenex Luna C18 100×40 mm×5 μm; mobile phase: [H2O (0.04% HCl)—ACN]; gradient: 30-70% B over 8.0 min) and lyophilised to give the title compound as a yellow gum. Y=33%. 1H NMR (400 MHz, MeOD) δ 8.01 (s, 1H), 7.95-7.78 (m, 1H), 6.65 (d, J=3 Hz, 1H), 4.59-4.28 (m, 3H), 3.47-3.32 (m, 2H), 2.04-1.92 (m, 1H), 1.91-1.77 (m, 3H), 1.37-1.13 (m, 9H).
Step 2. 2-({3-Chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}methyl)pyrrolidine hydrochloride. A mixture of tert-butyl 2-({3-chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}methyl)pyrrolidine-1-carboxylate (200 mg, 594 μmol) and 4 M HCl in EtOAc (3 mL) was stirred at 25° C. for 1 h. The solution was concentrated under vacuum to give the title compound as a white solid. Y=quantitative. 1H NMR (400 MHz, MeOD) δ 8.33-8.26 (m, 2H), 6.86 (d, J=3 Hz, 1H), 4.88-4.84 (m, 2H), 4.25-4.14 (m, 1H), 3.51-3.42 (m, 1H), 3.36-3.32 (m, 1H), 2.38-2.28 (m, 1H), 2.24-2.07 (m, 2H), 1.99-1.85 (m, 1H).
Step 3. 2-({3-Chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}methyl)-1-methylpyrrolidine. To a solution of 2-({3-chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}methyl)pyrrolidine hydrochloride (160 mg, 586 μmol) in DCE (3 mL) and AcOH (0.34 mL) at 0° C. was added aqueous 37% formaldehyde (131 μL, 1.76 mmol). The resulting mixture was stirred at 0° C. for 0.5 h, then treated with NaBH(OAc)3 (372 mg, 1.76 mmol). The resulting mixture was stirred at 0° C. for 0.5 h. The solution was diluted with H2O (3 mL) and extracted with DCM (3×3 mL). The combined organic layers were washed with brine (3 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give the title compound as a yellow oil. Y=82%.
Step 4. 5-Chloro-3-methyl-2-{7-[(I-methylpyrrolidin-2-yl)methyl]-7H-pyrrolo[2,3-c]pyridazin-3-yl}phenol hydrochloride. To a solution of 2-({3-chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}methyl)-1-methylpyrrolidine (70 mg, 279 μmol) in dioxane (2 mL) and H2O (0.4 mL) at 25° C. under N2 were added K2CO3 (154 mg, 1.12 mmol), dicyclohexyl-[2-(2,6-dimethoxyphenyl)phenyl]phosphane; methanesulfonate;(2-phenylanilino)palladium(1+) “SPhos Pd G3” (33 mg, 42 μmol) and 5-chloro-3-methyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenol (Intermediate B2, 75 mg, 279 μmol). The resulting mixture was stirred at reflux under N2 for 1 h. The solution was diluted with H2O (2 mL) and extracted with ethyl acetate (3×2 mL). The combined organic layers were washed with brine (2 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Phenomenex Luna C18 100×40 mm×5 μm; mobile phase: [H2O (0.04% HCl)—ACN]; gradient: 1-35% B over 8.0 min) and lyophilised to give the title compound as a yellow solid. Y=32%. 1H NMR (400 MHz, DMSO-d6) δ 11.25 (br. s, 1H), 10.59 (br. s, 1H), 8.63 (s, 1H), 8.39 (s, 1H), 7.07-6.92 (m, 3H), 5.09-4.99 (m, 1H), 4.96-4.87 (m, 1H), 4.07-3.92 (m, 1H), 3.67-3.65 (m, 1H), 3.17-3.07 (m, 1H), 2.90-2.82 (m, 3H), 2.21-2.11 (m, 1H), 2.07 (s, 3H), 2.03-1.84 (m, 3H). 1H NMR (400 MHz, DMSO-d6+D2O) δ 8.54 (d, J=3 Hz, 1H), 8.40 (s, 1H), 7.02-6.99 (m, 1H), 6.99 (s, 1H), 6.95 (s, 1H), 5.02-4.92 (m, 1H), 4.90-4.82 (m, 1H), 4.05-3.92 (m, 1H), 3.71-3.62 (m, 1H), 3.20-3.10 (m, 1H), 2.90 (s, 3H), 2.22-2.11 (m, 1H), 2.05 (s, 3H), 2.03-1.83 (m, 3H). 1H NMR (400 MHz, MeOD) δ 8.73 (d, J=3 Hz, 1H), 8.62 (s, 1H), 7.18 (d, J=3 Hz, 1H), 7.02 (d, J=2 Hz, 1H), 6.94 (d, J=2 Hz, 1H), 5.17-4.97 (m, 2H), 4.17-4.07 (m, 1H), 3.87-3.79 (m, 1H), 3.30-3.23 (m, 1H), 3.09 (s, 3H), 2.42-2.32 (m, 1H), 2.20 (s, 3H), 2.19-2.02 (m, 3H). LCMS (ESI): m/z: [M+H]+=357.1.
Step 1. 1-(2-{3-Chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}ethyl)pyrrolidin-2-one. To a solution of 3-chloro-7H-pyrrolo[2,3-c]pyridazine (Intermediate A1, 200 mg, 1.30 mmol) in DMF (3 mL) at 25° C. under N2 was added K2CO3 (360 mg, 2.60 mmol) and 1-(2-chloroethyl)pyrrolidin-2-one (192 mg, 1.30 mmol). The mixture was stirred at 80° C. under N2 for 2 h. The reaction mixture was diluted with H2O (3 mL) and extracted with EtOAc (3×3 mL). The combined organic layers were washed with brine (9 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Phenomenex Luna C18 100×40 mm×5 μm; mobile phase: [H2O (0.04% HCl)—ACN]; gradient: 10-25% B over 8.0 min) and lyophilised to give the title compound as a yellow solid. Y=55%. 1H NMR (400 MHz, DMSO-d6) δ 8.04-7.99 (m, 2H), 6.54 (d, J=3 Hz, 1H), 4.59-4.47 (m, 2H), 3.68-3.59 (m, 2H), 3.28 (t, J=7 Hz, 2H), 2.00-1.91 (m, 2H), 1.81-1.75 (m, 2H).
Step 2. 1-{2-[3-(4-Chloro-2-hydroxy-6-methylphenyl)-7H-pyrrolo[2,3-c]pyridazin-7-yl]ethyl}pyrrolidin-2-one. To a solution of 1-(2-{3-chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}ethyl)pyrrolidin-2-one (80 mg, 302 μmol) in dioxane (4 mL) and H2O (0.8 mL) at 25° C. under N2 were added K2CO3 (125 mg, 907 μmol), SPhos Pd G3 (24 mg, 30 μmol) and 5-chloro-3-methyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenol (Intermediate B2, 97 mg, 363 μmol). The mixture was stirred at reflux under N2 for 0.5 h. The reaction mixture was diluted with H2O (1 mL) and extracted with EtOAc (3×1 mL). The combined organic layers were washed with brine (1×3 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Phenomenex Luna C18 100×40 mm×5 μm; mobile phase: [H2O (0.04% HCl)—ACN]; gradient: 10-30% B over 8.0 min) and lyophilised to give the title compound as a yellow solid. Y=27%. 1H NMR (400 MHz, DMSO-d6) δ 10.80 (br. s, 1H), 8.66 (s, 1H), 8.56 (s, 1H), 7.06-6.95 (m, 3H), 4.65 (t, J=5 Hz, 2H), 3.69 (t, J=5 Hz, 2H), 3.45-3.40 (m, 2H), 2.07 (s, 3H), 1.99-1.89 (m, 2H), 1.89-1.80 (m, 2H). 1H NMR (400 MHz, DMSO-d6+D2O) δ 8.63 (d, J=3 Hz, 1H), 8.56 (s, 1H), 7.13-6.81 (m, 3H), 4.63 (t, J=5 Hz, 2H), 3.67 (t, J=5 Hz, 2H), 3.41 (t, J=7 Hz, 2H), 2.06 (s, 3H), 1.96-1.88 (m, 2H), 1.88-1.77 (m, 2H). LC-MS (ESI): m/z: [M+H]+=371.2.
Step 1. (4-Methylmorpholin-3-yl)methyl methanesulfonate. To a solution of (4-methylmorpholin-3-yl)methanol (450 mg, 3.43 mmol) in DCM (9 mL) at 0° C. under N2 were added TEA (955 μL, 6.86 mmol) and MsCl (398 μL, 5.15 mmol). The mixture was stirred at 25° C. for 3 h. The reaction mixture was cooled to 0° C. and quenched by addition of H2O (8 mL). The mixture was extracted with DCM (3×5 mL). The combined organic layers were washed with brine (5 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give the title compound as an oil. Y=quantitative.
Step 2. 3-({3-Chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}methyl)-4-methylmorpholine hydrochloride. To a solution of 3-chloro-7H-pyrrolo[2,3-c]pyridazine (Intermediate A1, 250 mg, 1.63 mmol) in DMF (0.3 mL) at 25° C. under N2 were added K2CO3 (675 mg, 4.88 mmol) and (4-methylmorpholin-3-yl)methyl methanesulfonate (511 mg, 2.44 mmol). The mixture was stirred at 25° C. for 2 h. The reaction mixture was purified by prep-HPLC (column: Phenomenex Luna C18 100×40 mm×5 μm; mobile phase: [H2O (0.04% HCl)—ACN]; gradient: 15-40% B over 8.0 min) and lyophilised to give the title compound as a white solid. Y=35%. 1H NMR (400 MHz, DMSO-d6) δ 12.15-11.50 (m, 1H), 8.26-7.99 (m, 2H), 6.66 (d, J=3 Hz, 1H), 5.01-4.68 (m, 2H), 3.95-3.66 (m, 5H), 3.49-3.40 (m, 1H), 3.33-3.15 (m, 1H), 3.07-2.90 (m, 3H).
Step 3. 5-Chloro-3-methyl-2-{7-[(4-methylmorpholin-3-yl)methyl]-7H-pyrrolo[2,3-c]pyridazin-3-yl}phenol hydrochloride. To a solution of 3-({3-chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}methyl)-4-methylmorpholine hydrochloride (100 mg, 375 μmol) in dioxane (1.5 mL) and H2O (0.3 mL) at 25° C. under N2 were added 5-chloro-3-methyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenol (Intermediate B2, 121 mg, 450 μmol), SPhos Pd G3 (29 mg, 37 μmol) and K2CO3 (207 mg, 1.50 mmol). The mixture was stirred at 80° C. under N2 for 3 h. The reaction mixture was quenched by addition of H2O (1 mL) and extracted with EtOAc (3×1 mL). The combined organic layers were washed with brine (1 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Phenomenex Luna C18 100×40 mm×5 μm; mobile phase: [H2O (0.04% HCl)—ACN]; gradient: 10-30% B over 8.0 min) and lyophilised to give the title compound as a white solid. Y=22%. 1H NMR (400 MHz, DMSO-d6) δ 12.32-10.05 (m, 2H), 8.79-8.26 (m, 2H), 7.13-6.86 (m, 3H), 5.15-4.73 (m, 2H), 4.08-3.72 (m, 6H), 3.18-3.08 (m, 1H), 3.04 (s, 3H), 2.08 (s, 3H). 1H NMR (400 MHz, DMSO-d6+D2O) δ 8.65-8.28 (m, 2H), 7.20-6.77 (m, 3H), 5.10-4.69 (m, 2H), 4.14-3.79 (m, 6H), 3.31-3.21 (m, 1H), 3.07 (s, 3H), 2.05 (s, 3H). 1H NMR (400 MHz, MeOD) δ 8.75-8.48 (m, 2H), 7.19 (d, J=3 Hz, 1H), 7.02 (d, J=1 Hz, 1H), 6.94 (d, J=1 Hz, 1H), 5.20-5.01 (m, 2H), 4.26-3.82 (m, 5H), 3.67-3.55 (m, 1H), 3.45-3.35 (m, 1H), 3.25 (s, 3H), 2.19 (s, 3H). LCMS (ESI): m/z: [M+H]+=373.2.
Step 1. (Oxan-3-yl)methyl methanesulfonate. To a solution tetrahydropyran-3-ylmethanol (150 mg, 1.29 mmol) in DCM (3 mL) at 0° C. were added DIPEA (450 μL, 2.58 mmol) and MsCl (120 μL, 1.55 mmol). The resulting mixture was stirred at 0° C. for 1 h. The mixture was quenched with H2O (3 mL) and extracted with DCM (3×3 mL). The combined organic layers were washed with brine (3×3 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give the title compound as a colourless oil. Y=88%.
Step 2. 3-Chloro-7-[(oxan-3-yl)methyl]-7H-pyrrolo[2,3-c]pyridazine. To a solution of 3-chloro-7H-pyrrolo[2,3-c]pyridazine (Intermediate A1, 200 mg, 1.30 mmol) in DMF (5 mL) at 25° C. were added K2CO3 (540 mg, 3.91 mmol) and (oxan-3-yl)methyl methanesulfonate (253 mg, 1.30 mmol). The resulting mixture was stirred at 80° C. under N2 for 12 h. The solution was diluted with H2O (5 mL) and extracted with ethyl acetate (3×5 mL). The combined organic layers were washed with brine (5 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Phenomenex Luna C18 100×40 mm×5 μm; mobile phase: [H2O (0.04% HCl)—ACN]; gradient: 10-55% B over 8.0 min) and lyophilised to give the title compound as a yellow solid. Y=40%. 1H NMR (400 MHz, DMSO-d6) δ 8.05 (d, J=3 Hz, 1H), 8.03 (s, 1H), 6.58 (d, J=3 Hz, 1H), 4.32 (d, J=7 Hz, 2H), 3.73-3.65 (m, 1H), 3.62-3.58 (m, 1H), 3.39-3.31 (m, 1H), 3.25-3.15 (m, 1H), 2.28-2.14 (m, 1H), 1.67-1.56 (m, 2H), 1.48-1.36 (m, 1H), 1.30-1.18 (m, 1H).
Step 3. 5-Chloro-3-methyl-2-{7-[(oxan-3-yl)methyl]-7H-pyrrolo[2,3-c]pyridazin-3-yl}phenol. To a solution of 3-chloro-7-[(oxan-3-yl)methyl]-7H-pyrrolo[2,3-c]pyridazine (90 mg, 358 μmol) in dioxane (2 mL) and H2O (0.4 mL) at 25° C. under N2 were added 5-chloro-3-methyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenol (Intermediate B2, 96 mg, 358 μmol), K2CO3 (198 mg, 1.43 mmol) and dicyclohexyl-[2-(2,6-dimethoxyphenyl)phenyl]phosphane; methanesulfonate;(2-phenylanilino)palladium(1+) “SPhos Pd G3” (42 mg, 54 μmol). The resulting mixture was stirred at reflux under N2 for 1 h. The solution was diluted with H2O (2 mL) and extracted with ethyl acetate (3×2 mL). The combined organic layers were washed with brine (2 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Phenomenex Luna C18 100×40 mm×5 μm; mobile phase: [H2O (0.04% HCl)—ACN]; gradient: 20-30% B over 8.0 min) and lyophilised to give the title compound as a yellow solid. Y=19%. 1H NMR (400 MHz, DMSO-d6) δ 10.78 (br. s, 1H), 8.65 (s, 1H), 8.55 (s, 1H), 7.06-7.02 (m, 2H), 7.01 (s, 1H), 4.47-4.34 (m, 2H), 3.75-3.66 (m, 2H), 3.31-3.27 (m, 2H), 2.35-2.24 (m, 1H), 2.09 (s, 3H), 1.77-1.60 (m, 2H), 1.53-1.41 (m, 1H), 1.40-1.28 (m, 1H). 1H NMR (400 MHz, DMSO-d6+D2O) δ 8.60 (d, J=3 Hz, 1H), 8.53 (s, 1H), 7.05 (d, J=3 Hz, 1H), 7.00 (s, 1H), 6.96 (s, 1H), 4.49-4.31 (m, 2H), 3.71-3.69 (m, 2H), 3.43-3.34 (m, 1H), 3.28-3.21 (m, 1H), 2.31-2.19 (m, 1H), 2.07 (s, 3H), 1.74-1.59 (m, 2H), 1.50-1.38 (m, 1H), 1.37-1.27 (m, 1H). LCMS (ESI): m/z: [M+H]+=358.2.
Step 1. 3-Chloro-7-[(oxolan-2-yl)methyl]-7H-pyrrolo[2,3-c]pyridazine. To a solution of 3-chloro-7H-pyrrolo[2,3-c]pyridazine (Intermediate A1, 200 mg, 1.30 mmol) in DMF (3 mL) at 25° C. were added K2CO3 (540 mg, 3.91 mmol) and 2-(bromomethyl)tetrahydrofuran (215 mg, 1.30 mmol). The resulting mixture was stirred at 80° C. under N2 for 12 h. The solution was diluted with H2O (3 mL) and extracted with ethyl acetate (3×3 mL). The combined organic layers were washed with brine (3 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Phenomenex Luna C18 100×40 mm×5 μm; mobile phase: [H2O (0.04% HCl)—ACN]; gradient: 10-55% B over 8.0 min) and lyophilised to give the title compound as a yellow solid. Y=55%. 1H NMR (400 MHz, DMSO-d6) δ 8.03 (s, 1H), 8.01 (d, J=4 Hz, 1H), 6.57 (d, J=4 Hz, 1H), 4.52-4.38 (m, 2H), 4.33-4.22 (m, 1H), 3.81-3.72 (m, 1H), 3.66-3.63 (m, 1H), 2.02-1.90 (m, 1H), 1.84-1.71 (m, 2H), 1.64-1.53 (m, 1H).
Step 2. 5-Chloro-3-methyl-2-{7-[(oxolan-2-yl)methyl]-7H-pyrrolo[2,3-c]pyridazin-3-yl}phenol. To a solution of 3-chloro-7-[(oxolan-2-yl)methyl]-7H-pyrrolo[2,3-c]pyridazine (90 mg, 379 μmol) in dioxane (2 mL) and H2O (0.4 mL) at 25° C. under N2 were added 5-chloro-3-methyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenol (Intermediate B2, 102 mg, 379 μmol), K2CO3 (209 mg, 1.51 mmol) and dicyclohexyl-[2-(2,6-dimethoxyphenyl)phenyl]phosphane; methanesulfonate;(2-phenylanilino)palladium(1+) “SPhos Pd G3” (44 mg, 57 μmol). The resulting mixture was stirred at reflux under N2 for 1 h. The solution was diluted with H2O (2 mL) and extracted with ethyl acetate (3×2 mL). The combined organic layers were washed with brine (2 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Phenomenex Luna C18 100×40 mm×5 μm; mobile phase: [H2O (0.04% HCl)—ACN]; gradient: 20-30% B over 8.0 min) and lyophilised to give the title compound as a yellow solid. Y=46%. 1H NMR (400 MHz, DMSO-d6) δ 10.77 (br. s, 1H), 8.60 (s, 1H), 8.52 (s, 1H), 7.05-6.99 (m, 3H), 4.63-4.56 (m, 1H), 4.51-4.43 (m, 1H), 4.39-4.27 (m, 1H), 3.88-3.76 (m, 1H), 3.70-3.62 (m, 1H), 2.08 (s, 3H), 2.06-2.00 (m, 1H), 1.91-1.80 (m, 2H), 1.73-1.63 (m, 1H). 1H NMR (400 MHz, DMSO-d6+D2O) δ 8.58 (d, J=3 Hz, 1H), 8.52 (s, 1H), 7.03 (d, J=3 Hz, 1H), 7.00 (s, 1H), 6.96 (s, 1H), 4.61-4.55 (m, 1H), 4.49-4.42 (m, 1H), 4.35-4.26 (m, 1H), 3.82-3.75 (m, 1H), 3.69-3.65 (m, 1H), 2.07 (s, 3H), 2.05-1.98 (m, 1H), 1.88-1.79 (m, 2H), 1.70-1.60 (m, 1H).
LCMS (ESI): m/z: [M+H]+=344.1.
Step 1. Tert-butyl N-(2-{3-chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}ethyl)carbamate. To a solution of 3-chloro-7H-pyrrolo[2,3-c]pyridazine (Intermediate A1, 400 mg, 2.60 mmol) in DMF (5 mL) were added K2CO3 (720 mg, 5.21 mmol) and tert-butyl N-(2-bromoethyl)carbamate (700 mg, 3.13 mmol). The mixture was stirred at 80° C. for 2 h. The reaction mixture was purified by prep-HPLC (column: Phenomenex Luna C18 75×30 mm×3 μm; mobile phase: [TFA-ACN]; gradient: 25-55% B over 8.0 min) and lyophilised to give the title compound as an oil. Y=56%. 1H NMR (400 MHz, DMSO-d6) δ 8.00 (s, 1H), 7.94 (d, J=3 Hz, 1H), 6.94 (t, J=5 Hz, 1H), 6.54 (d, J=3 Hz, 1H), 4.42 (t, J=6 Hz, 2H), 3.39 (d, J=6 Hz, 2H), 1.26 (s, 9H).
Step 2. 2-{3-Chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}ethan-1-amine hydrochloride. A mixture of tert-butyl N-(2-{3-chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}ethyl)carbamate (380 mg, 1.28 mmol) and 4 M HCl in EtOAc (5 mL) was stirred at 25° C. for 1 h. The reaction mixture was concentrated under reduced pressure to give the title compound as a yellow solid. Y=84%. 1H NMR (400 MHz, DMSO-d6) δ 8.31 (br. s, 3H), 8.10 (d, J=4 Hz, 1H), 8.05 (s, 1H), 6.61 (d, J=4 Hz, 1H), 4.68 (t, J=6 Hz, 2H), 3.43-3.35 (m, 2H).
Step 3. N-(2-{3-Chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}ethyl)acetamide. To a solution of 2-{3-chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}ethan-1-amine hydrochloride (220 mg, 944 μmol) in DCM (2.2 mL) were added DIPEA (493 μL, 2.83 mmol) and acetyl chloride (134 μL, 1.89 mmol). The mixture was stirred at 25° C. for 2 h. The reaction mixture was diluted with H2O (2 mL) and extracted with DCM (3×2 mL). The combined organic layers were washed with brine (8 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was dissolved in MeOH (3 mL) and then treated with saturated aqueous NaHCO3 (250 L). The mixture was stirred at 25° C. for 10 h. The reaction mixture was purified by prep-HPLC (column: Phenomenex Luna C18 75×30 mm×3 μm; mobile phase: [H2O (0.1% TFA)—ACN]; gradient: 1-30% B over 8.0 min) and lyophilised to give the title compound as a yellow oil. Y=71%. 1H NMR (400 MHz, DMSO-d6) δ 8.02 (s, 1H), 7.98 (d, J=4 Hz, 1H), 7.94 (m, 1H), 6.55 (d, J=4 Hz, 1H), 4.44 (t, J=6 Hz, 2H), 3.55-3.45 (m, 2H), 1.70 (s, 3H).
Step 4. N-{2-[3-(4-Chloro-2-hydroxy-6-methylphenyl)-7H-pyrrolo[2,3-c]pyridazin-7-yl]ethyl}acetamide. To a solution of N-(2-{3-chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}ethyl)acetamide (130 mg, 545 μmol) and 5-chloro-3-methyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenol (Intermediate B2, 161 mg, 599 μmol) in dioxane (6.5 mL) and H2O (1.3 mL) at 25° C. under N2 were added SPhos Pd G3 (43 mg, 54 μmol) and K2CO3 (226 mg, 1.63 mmol). The mixture was stirred at reflux under N2 for 4 h. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Waters Xbridge BEH C18 100×30 mm×10 μm; mobile phase: [H2O (10 mM NH4HCO3)—ACN]; gradient: 5-50% B over 8.0 min) and lyophilised to give the title compound as a white solid. Y=19%. 1H NMR (400 MHz, DMSO-d6) δ 9.86 (br. s, 1H), 8.03 (t, J=6 Hz, 1H), 7.87 (d, J=4 Hz, 1H), 7.74 (s, 1H), 6.85 (d, J=6 Hz, 2H), 6.55 (d, J=4 Hz, 1H), 4.50 (t, J=6 Hz, 2H), 3.56 (q, J=6 Hz, 2H), 1.98 (s, 3H), 1.74 (s, 3H). 1H NMR (400 MHz, DMSO-d6+D2O) δ 7.82 (d, J=4 Hz, 1H), 7.73 (s, 1H), 6.85-6.80 (m, 2H), 6.56 (d, J=4 Hz, 1H), 4.48 (t, J=6 Hz, 2H), 3.54 (t, J=6 Hz, 2H), 1.95 (s, 3H), 1.72 (s, 3H). LCMS (ESI): m/z: [M+H]+=345.2.
Step 1. 1-(2-{3-Chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}ethyl)pyrrolidin-3-ol. To a solution of 3-chloro-7-(2-chloroethyl)pyrrolo[2,3-c]pyridazine (for synthesis see Compound 33)(180 mg, 833 μmol) in ACN (2 mL) at 25° C. under N2 were added pyrrolidin-3-ol (287 μL, 3.55 mmol) and K2CO3 (576 mg, 4.17 mmol). The mixture was stirred at 85° C. under N2 for 0.5 h. The reaction mixture was quenched by H2O (2 mL) and extracted with EtOAc (3×2 mL). The combined organic layers were washed with brine (3×2 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Waters Xbridge BEH C18 100×30 mm×10 μm; mobile phase: [H2O (10 mM NH4HCO3)—ACN]; gradient: 1-55% B over 8.0 min) and lyophilised to give the title compound as a yellow oil. Y=90%. 1H NMR (400 MHz, DMSO-d6) δ 8.10-7.95 (m, 2H), 6.55 (d, J=3 Hz, 1H), 4.65 (d, J=4 Hz, 1H), 4.49 (t, J=6 Hz, 2H), 4.22-4.03 (m, 1H), 2.87 (t, J=6 Hz, 2H), 2.80-2.70 (m, 1H), 2.65-2.57 (m, 1H), 2.35-2.25 (m, 1H), 1.99-1.82 (m, 1H), 1.60-1.39 (m, 1H).
Step 2. 1-(2-{3-Chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}ethyl)pyrrolidin-3-one. To a solution of oxalyl chloride (98 μL, 1.12 mmol) in DCM (1.5 mL) at −78° C. under N2 was added DMSO (176 μL, 2.25 mmol). The mixture was stirred at −78° C. under N2 for 15 min, then treated with a solution of 1-(2-{3-chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}ethyl)pyrrolidin-3-ol (150 mg, 562 μmol) in DCM (1.5 mL). The mixture was stirred at −78° C. under N2 for 1 h. The mixture was treated with TEA (470 μL, 3.37 mmol) at −78° C., then stirred at 0° C. for 2 h. The reaction mixture was quenched by addition of saturated aqueous NH4Cl (2 mL) and extracted with DCM (3×2 mL). The combined organic layers were washed with brine (3×2 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Waters Xbridge BEH C18 100×30 mm×10 μm; mobile phase: [H2O (10 mM NH4HCO3)—ACN]; gradient: 1-45% B over 8.0 min) and lyophilised to give the title compound as a yellow solid. Y=67%. 1H NMR (400 MHz, DMSO-d6) δ 8.10 (d, J=3 Hz, 1H), 8.01 (s, 1H), 6.56 (d, J=3 Hz, 1H), 4.55 (t, J=7 Hz, 2H), 3.07-2.96 (m, 4H), 2.89 (t, J=7 Hz, 2H), 2.26 (t, J=7 Hz, 2H).
Step 3. 1-{2-[3-(4-Chloro-2-hydroxy-6-methylphenyl)-7H-pyrrolo[2,3-c]pyridazin-7-yl]ethyl}pyrrolidin-3-one. To a solution of 1-(2-{3-chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}ethyl)pyrrolidin-3-one (70 mg, 264 μmol) in dioxane (1 mL) and H2O (0.2 mL) at 25° C. under N2 were added 5-chloro-3-methyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenol (Intermediate B2, 85 mg, 317 μmol), K2CO3 (146 mg, 1.06 mmol) and SPhos Pd G3 (21 mg, 26 μmol). The mixture was stirred at 80° C. under N2 for 3 h. The reaction mixture was cooled to 0° C., quenched with H2O (1 mL) and extracted with EtOAc (3×1 mL). The combined organic layers were washed with brine (1 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Waters Xbridge BEH C18 100×30 mm×10 μm; mobile phase: [H2O (10 mM NH4HCO3)—ACN]; gradient: 30-60% B over 8.0 min) and lyophilised to give the title compound as a white solid. Y=24%. 1H NMR (400 MHz, DMSO-d6) δ 9.86 (br. s, 1H), 7.99 (d, J=3 Hz, 1H), 7.74 (s, 1H), 7.02-6.74 (m, 2H), 6.56 (d, J=3 Hz, 1H), 4.60 (t, J=7 Hz, 2H), 3.11-3.02 (m, 4H), 2.94 (t, J=7 Hz, 2H), 2.29 (t, J=7 Hz, 2H), 1.97 (s, 3H). 1H NMR (400 MHz, DMSO-d6+D2O) δ 9.86 (br. s, 0.1H), 7.97 (d, J=3 Hz, 1H), 7.73 (s, 1H), 6.94-6.80 (m, 2H), 6.56 (d, J=3 Hz, 1H), 4.59 (t, J=7 Hz, 2H), 3.11-2.99 (m, 4H), 2.93 (t, J=7 Hz, 2H), 2.28 (t, J=7 Hz, 2H), 1.96 (s, 3H). LCMS (ESI): m/z: [M+H]+=371.2.
Step 1. [(2S)-1-Methylpiperidin-2-yl]methyl methanesulfonate. To a solution of [(2S)-1-methyl-2-piperidyl]methanol (750 mg, 5.80 mmol) in DCM (7.5 mL) at 0° C. were added DIPEA (2.02 mL, 11.6 mmol) and MsCl (674 μL, 8.71 mmol). The solution was stirred at 0° C. for 2 h. The reaction mixture was quenched with H2O (8 mL) and extracted with DCM (2×7 mL). The combined organic layers were washed with brine (2×6 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give the[(2S)-1-methyl-2-piperidyl]methyl methanesulfonate (900 mg, 4.34 mmol, 74.79% yield) as a yellow oil.
Step 2. (2S)-2-({3-Chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}methyl)-1-methylpiperidine. To a solution of [(2S)-1-methylpiperidin-2-yl]methyl methanesulfonate (810 mg, 3.91 mmol) in DMF (9 mL) at 25° C. was added 3-chloro-7H-pyrrolo[2,3-c]pyridazine (Intermediate A1, 400 mg, 2.60 mmol) and K2CO3 (720 mg, 5.21 mmol). The solution was stirred at 90° C. under N2 for 2 h. The reaction mixture was purified by prep-HPLC (column: Waters Xbridge BEH C18 250×50 mm×10 μm; mobile phase: [H2O (10 mM NH4HCO3)—ACN]; gradient: 20-40% B over 10.0 min) and lyophilised to give the title compound as a yellow solid. Y=61%. 1H NMR (400 MHz, DMSO-d6) δ 8.09-7.95 (m, 2H), 6.57 (d, J=4 Hz, 1H), 4.75-4.65 (m, 1H), 4.35-4.25 (m, 1H), 2.85-2.70 (m, 1H), 2.48-2.42 (m, 1H), 2.38 (s, 3H), 2.15-2.00 (m, 1H), 1.65-1.54 (m, 1H), 1.52-1.44 (m, 1H), 1.39-1.28 (m, 1H), 1.26-1.18 (m, 1H), 1.14-0.93 (m, 2H).
Step 3. 5-Chloro-3-methyl-2-(7-{[(2S)-1-methylpiperidin-2-yl]methyl}-7H-pyrrolo[2,3-c]pyridazin-3-yl)phenol hydrochloride. To a solution of (2S)-2-({3-chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}methyl)-1-methylpiperidine (100 mg, 378 μmol) in dioxane (5 mL) and H2O (1 mL) at 25° C. under N2 were added 5-chloro-3-methyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenol (Intermediate B2, 152 mg, 567 μmol), K2CO3 (209 mg, 1.51 mmol) and dicyclohexyl-[2-(2,6-dimethoxyphenyl)phenyl]phosphane; methanesulfonate;(2-phenylanilino)palladium(1+) “SPhos Pd G3” (44 mg, 57 μmol). The solution was stirred at 90° C. under N2 for 3 h. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was diluted with H2O (2 mL) and extracted with EtOAc (2×1 mL). The combined organic layers were washed with brine (2×1 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Phenomenex Luna C18 100×40 mm×5 μm; mobile phase: [H2O (0.04% HCl)—ACN]; gradient: 10-30% B over 8.0 min) and lyophilised to give the title compound Y=23%.
1H NMR (400 MHz, DMSO-d6) δ 11.35 (br. s, 1H), 10.84 (br. s, 1H), 8.71 (s, 1H), 8.56 (s, 1H), 7.11-7.08 (m, 2H), 6.99 (s, 1H), 5.14-4.67 (m, 2H), 4.03 (s, 0.4H), 3.73 (s, 0.6H), 3.45 (s, 1H), 3.08-2.85 (m, 4H), 2.54 (s, 0.2H), 2.10 (s, 2.8H), 1.80-1.75 (m, 5H), 1.59-1.29 (m, 1H). 1H NMR (400 MHz, DMSO-d6+D2O) δ 8.70-8.42 (m, 2H), 7.10 (s, 1H), 7.00-6.97 (m, 2H), 5.02-4.58 (m, 2H), 4.10-4.01 (s, 0.4H), 3.70 (s, 0.6H), 3.47-3.42 (s, 1H), 3.26-2.88 (m, 4H), 2.54 (s, 0.3H), 2.06 (s, 2.7H), 1.92-1.32 (m, 6H). 1H NMR (400 MHz, MeOD) δ 8.71-8.55 (m, 2H), 7.18 (d, J=3 Hz, 1H), 7.02 (d, J=2 Hz, 1H), 6.93 (d, J=2 Hz, 1H), 5.12-4.96 (m, 1.7H), 4.81-4.75 (m, 0.3H), 4.28-3.75 (m, 1H), 3.66-3.46 (m, 1H), 3.42-3.35 (m, 0.4H), 3.26-3.09 (m, 3.6H), 2.69-2.16 (m, 3H), 2.10-1.52 (m, 6H). LCMS (ESI): m/z: [M+H]+=371.3.
Synthesised in an analogous way to Compound 46, starting from [(2R)-1-methyl-2-piperidyl]methanol. 1H NMR (400 MHz, DMSO-d6) δ 11.08 (s, 1H), 10.73 (s, 1H), 8.72-8.36 (m, 2H), 7.03-6.98 (m, 3H), 5.10-4.72 (m, 2H), 4.11-4.05 (m, 0.3H), 3.47-3.44 (m, 0.7H), 3.44 (s, 1H), 3.38-3.30 (m, 1H), 3.07-2.91 (m, 4H), 2.09 (s, 3H), 1.86-1.71 (m, 5H), 1.59-1.38 (m, 1H). 1H NMR (400 MHz, DMSO-d6+D2O) δ 8.47-8.38 (m, 2H), 7.03 (d, J=3 Hz, 1H), 6.97 (s, 1H), 6.92 (s, 1H), 4.93-4.53 (m, 2H), 4.09-3.98 (m, 0.3H), 3.67-3.65 (m, 0.7H), 3.59-3.37 (m, 1H), 3.05-3.00 (m, 4H), 2.03 (s, 3H), 1.84-1.71 (m, 3H), 1.66-1.36 (m, 3H).
LCMS (ESI): m/z: [M+H]+=371.3.
Step 1. 3-Chloro-7-[(oxetan-3-yl)methyl]-7H-pyrrolo[2,3-c]pyridazine. To a solution of 3-(bromomethyl)oxetane (197 mg, 1.30 mmol) in DMF (3 mL) at 25° C. under N2 were added K2CO3 (360 mg, 2.60 mmol) and 3-chloro-7H-pyrrolo[2,3-c]pyridazine (0.20 g, 1.30 mmol). The mixture was stirred at 25° C. under N2 for 12 h. The reaction mixture was purified by prep-HPLC (column: Phenomenex Gemini-NX 80×40 mm×3 μm; mobile phase: [H2O (10 mM NH4HCO3)—ACN]; gradient: 5-35% B over 8.0 min) and lyophilised to give the title compound as a grey solid. Y=48%. 1H NMR (400 MHz, DMSO-d6) δ 8.11 (d, J=3 Hz, 1H), 8.04 (s, 1H), 6.58 (d, J=3 Hz, 1H), 4.71 (d, J=8 Hz, 2H), 4.63 (dd, J=8, 6 Hz, 2H), 4.45 (t, J=6 Hz, 2H), 3.61-3.47 (m, 1H).
Step 2. 5-Chloro-3-methyl-2-{7-[(oxetan-3-yl)methyl]-7H-pyrrolo[2,3-c]pyridazin-3-yl}phenol. To a solution of 3-chloro-7-[(oxetan-3-yl)methyl]-7H-pyrrolo[2,3-c]pyridazine (0.13 g, 581 μmol) in dioxane (3 mL) and H2O (0.6 mL) at 25° C. under N2 were added K2CO3 (241 mg, 1.74 mmol), SPhos Pd G3 (45 mg, 58 μmol) and 5-chloro-3-methyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenol (Intermediate B2, 156 mg, 581 μmol). The mixture was stirred at 80° C. under N2 for 2 h. The reaction mixture was diluted with H2O (1 mL) and extracted with EtOAc (3×1 mL). The combined organic layers were washed with brine (3 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Waters Xbridge BEH C18 100×30 mm×10 μm; mobile phase: [H2O (10 mM NH4HCO3)—ACN]; gradient: 25-55% B over 8.0 min) and lyophilised to give the title compound as a solid. Y=21%. 1H NMR (400 MHz, DMSO-d6) δ 9.88 (br. s, 1H), 7.99 (d, J=3 Hz, 1H), 7.75 (s, 1H), 6.84 (d, J=5 Hz, 2H), 6.57 (d, J=3 Hz, 1H), 4.76 (d, J=8 Hz, 2H), 4.72-4.64 (m, 2H), 4.50 (t, J=6 Hz, 2H), 3.66-3.53 (m, 1H), 1.97 (s, 3H). 1H NMR (400 MHz, DMSO-d6+D2O) δ 7.97 (d, J=3 Hz, 1H), 7.75 (s, 1H), 6.85 (d, J=5 Hz, 2H), 6.57 (d, J=3 Hz, 1H), 4.75 (d, J=8 Hz, 2H), 4.71-4.63 (m, 2H), 4.49 (t, J=6 Hz, 2H), 3.64-3.52 (m, 1H), 1.96 (s, 3H). LC-MS (ESI): m/z: [M+H]+=330.1.
Step 1. Tert-butyl 7-(methoxymethylidene)-4-azaspiro[2.5]octane-4-carboxylate. To a solution of (methoxymethyl)triphenylphosphanium chloride (5.71 g, 16.7 mmol) in THE (20 mL) at 0° C. was added potassium tert-butoxide (1.87 g, 16.7 mmol). The reaction mixture was stirred at 0° C. for 1 h, then treated with a solution of tert-butyl 7-oxo-4-azaspiro[2.5]octane-4-carboxylate (2.5 g, 11.1 mmol) in THF (20 mL). The resulting mixture was stirred at 25° C. for 12 h. The solution was diluted with H2O (30 mL) and extracted with ethyl acetate (3×30 mL). The combined organic layers were washed with brine (30 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, 3:1 petroleum ether/ethyl acetate) to give the title compound as a colourless oil. Y=57%. 1H NMR (400 MHz, DMSO-d6) δ 6.02-5.84 (m, 1H), 3.55-3.45 (m, 3H), 3.30-3.23 (m, 2H), 2.11 (t, J=5 Hz, 1H), 2.02 (s, 1H), 1.96 (t, J=5 Hz, 1H), 1.83 (s, 1H), 1.39 (s, 9H), 0.77-0.71 (m, 2H), 0.58-0.52 (m, 2H).
Step 2. Tert-butyl 7-formyl-4-azaspiro[2.5]octane-4-carboxylate. To a solution of tert-butyl 7-(methoxymethylene)-4-azaspiro[2.5]octane-4-carboxylate (1.6 g, 6.32 mmol) in ACN (160 mL) at 0° C. was added 1 M HCl in H2O (7.6 mL). The resulting mixture was stirred at 25° C. for 12 h. The reaction mixture was quenched by saturated aqueous NaHCO3 solution (20 mL) and extracted with ethyl acetate (3×20 mL). The combined organic layers were washed with brine (3×20 mL), dried over Na2SO4, filtered and concentrated under vacuum to give the title compound as a colourless oil. Y=66%. 1H NMR (400 MHz, CDCl3) δ 9.67-9.64 (m, 1H), 3.98-3.89 (m, 1H), 3.14-3.04 (m, 1H), 2.71-2.62 (m, 1H), 1.91-1.83 (m, 2H), 1.64-1.59 (m, 1H), 1.46 (s, 9H), 1.43-1.37 (m, 1H), 1.20-1.13 (m, 1H), 0.88-0.79 (m, 1H), 0.71-0.64 (m, 1H), 0.55-0.47 (m, 1H).
Step 3. Tert-butyl 7-(hydroxymethyl)-4-azaspiro[2.5]octane-4-carboxylate. To a solution of tert-butyl 7-formyl-4-azaspiro[2.5]octane-4-carboxylate (1.0 g, 4.18 mmol) in EtOH (20 mL) at 0° C. was added NaBH4 (237 mg, 6.27 mmol). The resulting mixture was stirred at 0° C. for 1 h. The mixture was quenched with H2O (10 mL), concentrated under vacuum and extracted with ethyl acetate (3×10 mL). The combined organic layers were washed with brine (10 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give the title compound as a colourless oil. Y=84%. 1H NMR (400 MHz, DMSO-d6) δ 4.42 (t, J=5 Hz, 1H), 3.88-3.78 (m, 1H), 3.24-3.21 (m, 1H), 2.85-2.74 (m, 1H), 1.81-1.74 (m, 1H), 1.63-1.56 (m, 1H), 1.51-1.43 (m, 1H), 1.41-1.34 (m, 10H), 1.07-0.97 (m, 3H), 0.80-0.73 (m, 1H), 0.48-0.41 (m, 1H), 0.41-0.32 (m, 1H).
Step 4. Tert-butyl 7-(bromomethyl)-4-azaspiro[2.5]octane-4-carboxylate. To a solution of tert-butyl 7-(hydroxymethyl)-4-azaspiro[2.5]octane-4-carboxylate (800 mg, 3.32 mmol) in DCM (10 mL) at 0° C. were added CBr4 (1.32 g, 3.98 mmol) and PPh3 (1.13 g, 4.31 mmol). The resulting mixture was stirred at 25° C. for 1 h. The solution was concentrated in vacuum and the residue purified by column chromatography (SiO2, petroleum ether/ethyl acetate=10:1 to 5:1) to give the title compound as a yellow oil. Y=66%. 1H NMR (400 MHz, DMSO-d6) δ 3.85 (d, J=12 Hz, 1H), 3.45 (d, J=6 Hz, 2H), 2.81 (t, J=12 Hz, 1H), 2.07-1.94 (m, 1H), 1.71 (d, J=12 Hz, 1H), 1.60-1.58 (m, 1H), 1.39 (s, 9H), 1.19-1.03 (m, 3H), 0.86-0.77 (m, 1H), 0.51-0.37 (m, 2H).
Step 5. Tert-butyl 7-({3-chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}methyl)-4-azaspiro[2.5]octane-4-carboxylate. To a solution of tert-butyl 7-(bromomethyl)-4-azaspiro[2.5]octane-4-carboxylate (620 mg, 2.04 mmol) in DMF (6 mL) at 25° C. were added K2CO3 (845 mg, 6.11 mmol) and 3-chloro-7H-pyrrolo[2,3-c]pyridazine (Intermediate A1, 313 mg, 2.04 mmol). The resulting mixture was stirred at 80° C. under N2 for 12 h. The solution was diluted with H2O (10 mL) and extracted with ethyl acetate (3×10 mL). The combined organic layers were washed with brine (10 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Phenomenex Luna C18 100×40 mm×5 μm; mobile phase: [H2O (0.04% HCl)—ACN]; gradient: 20-60% B over 8.0 min) and lyophilised to give the title compound as a yellow gum. Y=56%. 1H NMR (400 MHz, MeOD) δ 8.01 (s, 1H), 7.98 (d, J=3 Hz, 1H), 6.64 (d, J=3 Hz, 1H), 4.34 (d, J=7 Hz, 2H), 4.01-3.91 (m, 1H), 2.94-2.81 (m, 1H), 2.56-2.46 (m, 1H), 1.79-1.70 (m, 1H), 1.56-1.48 (m, 1H), 1.44 (s, 9H), 1.29-1.15 (m, 2H), 0.94-0.84 (m, 1H), 0.86-0.76 (m, 1H), 0.53-0.46 (m, 1H), 0.42-0.35 (m, 1H).
Step 6. 7-({3-Chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}methyl)-4-azaspiro[2.5]octane hydrochloride. A mixture of tert-butyl 7-({3-chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}methyl)-4-azaspiro[2.5]octane-4-carboxylate (250 mg, 663 μmol) and 4 M HCl in EtOAc (3 mL) was stirred at 25° C. for 1 h. The solution was concentrated under vacuum to give the title compound as a yellow oil. Y=quantitative.
Step 7. 7-({3-Chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}methyl)-4-methyl-4-azaspiro[2.5]octane. To a solution of 7-({3-chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}methyl)-4-azaspiro[2.5]octane hydrochloride (200 mg, 639 μmol) in DCE (2 mL) and AcOH (0.24 mL) at 0° C. was added aqueous 37% formaldehyde (143 μL, 1.92 mmol). The resulting mixture was stirred at 0° C. for 0.5 h, then treated with NaBH(OAc)3 (406 mg, 1.92 mmol). The resulting mixture was stirred at 0° C. for 1 h, then concentrated under vacuum. The residue was purified by prep-HPLC (column: Waters Xbridge Prep OBD C18 150×40 mm×10 μm; mobile phase: [H2O (10 mM NH4HCO3)—ACN]; gradient: 25-55% B over 8.0 min) and lyophilised to give the title compound as a white solid. Y=92%.
Step 8. 5-Chloro-3-methyl-2-[7-({4-methyl-4-azaspiro[2.5]octan-7-yl}methyl)-7H-pyrrolo[2,3-c]pyridazin-3-yl]phenol hydrochloride. To a solution of 7-({3-chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}methyl)-4-methyl-4-azaspiro[2.5]octane (140 mg, 481 μmol) in dioxane (2 mL) and H2O (0.4 mL) at 25° C. under N2 were added 5-chloro-3-methyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenol (Intermediate B2, 129 mg, 481 μmol), dicyclohexyl-[2-(2,6-dimethoxyphenyl)phenyl]phosphane; methanesulfonate;(2-phenylanilino)palladium(1+) “SPhos Pd G3” (56 mg, 72 μmol) and K2CO3 (266 mg, 1.93 mmol). The resulting mixture was stirred at 80° C. under N2 for 1 h. The solution was diluted with H2O (3 mL) and extracted with ethyl acetate (3×3 mL). The combined organic layers were washed with brine (3 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Agilent Poroshell EC C18 3×30 mm×2.7 μm; mobile phase: [H2O (0.04% HCl)—ACN]; gradient: 5-30% B over 15 min) and lyophilised to give the title compound as a yellow solid. Y=9%. 1H NMR (400 MHz, DMSO-d6) δ 11.30-10.63 (m, 2H), 8.78-8.33 (m, 2H), 7.10-6.94 (m, 3H), 4.56-4.39 (m, 2H), 3.36-3.10 (m, 2H), 2.85 (d, J=5 Hz, 3H), 2.47-2.40 (m, 1H), 2.30-2.20 (m, 1H), 2.08 (s, 3H), 1.93-1.78 (m, 1H), 1.71-1.57 (m, 1H), 1.52-1.25 (m, 1H), 1.17-0.97 (m, 2H), 0.79-0.52 (m, 2H). 1H NMR (400 MHz, DMSO-d6+D2O) δ 8.63-8.43 (m, 2H), 7.10-6.95 (m, 3H), 4.52-4.41 (m, 2H), 3.30-2.95 (m, 2H), 2.87 (s, 3H), 2.48-2.39 (m, 1H), 2.29-2.19 (m, 1H), 2.07 (s, 3H), 1.91-1.79 (m, 1H), 1.66-1.40 (m, 1H), 1.23-1.16 (m, 1H), 1.14-0.99 (m, 2H), 0.78-0.58 (m, 2H).
1H NMR (400 MHz, MeOD) δ 8.60 (d, J=3 Hz, 1H), 8.57 (s, 1H), 7.13 (d, J=3 Hz, 1H), 7.02 (d, J=2 Hz, 1H), 6.93 (d, J=2 Hz, 1H), 4.63-4.53 (m, 2H), 3.55-3.37 (m, 2H), 3.04 (s, 3H), 2.66-2.59 (m, 1H), 2.42-2.30 (m, 1H), 2.17 (s, 3H), 2.06-1.93 (m, 1H), 1.87-1.66 (m, 1H), 1.31-1.09 (m, 3H), 0.97-0.71 (m, 2H). LCMS (ESI): m/z: [M+H]+=397.3.
Step 1. 1-{3-Chloro-7H-pyrrolo[2,3-c]pyridazine-7-carbonyl}-4-methylpiperazine hydrochloride. To a solution of 3-chloro-7H-pyrrolo[2,3-c]pyridazine (Intermediate A1, 200 mg, 1.30 mmol) in ACN (4 mL) at 25° C. under N2 were added K2CO3 (720 mg, 5.21 mmol) and 4-methylpiperazine-1-carbonyl chloride hydrochloride (324 μL, 1.95 mmol). The mixture was stirred at 25° C. for 12 h. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Phenomenex Luna C18 100×40 mm×5 μm; mobile phase: [H2O (0.04% HCl)—ACN]; gradient: 1-25% B over 8.0 min) and lyophilised to give the title compound as a white solid. Y=58%. 1H NMR (400 MHz, DMSO-d6) δ 11.67-11.36 (m, 1H), 8.18 (s, 1H), 8.12 (d, J=4 Hz, 1H), 6.82 (d, J=4 Hz, 1H), 4.22-4.05 (m, 2H), 3.69-3.56 (m, 2H), 3.54-3.39 (m, 2H), 3.35-3.13 (m, 2H), 2.95-2.74 (m, 3H).
Step 2. 5-Chloro-3-methyl-2-[7-(4-methylpiperazine-1-carbonyl)-7H-pyrrolo[2,3-c]pyridazin-3-yl/phenol hydrochloride. To a solution of 1-{3-chloro-7H-pyrrolo[2,3-c]pyridazine-7-carbonyl}-4-methylpiperazine hydrochloride (100 mg, 357 μmol) in dioxane (0.5 mL) and H2O (0.1 mL) at 25° C. under N2 were added 5-chloro-3-methyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenol (Intermediate B2, 115 mg, 429 μmol), K2CO3 (198 mg, 1.43 mmol) and SPhos Pd G3 (28 mg, 36 μmol). The mixture was stirred at 80° C. for 3 h. The reaction mixture was cooled to 0° C., quenched with H2O (1 mL) and extracted with EtOAc (3×1 mL). The combined organic layers were washed with brine (1 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Phenomenex Luna C18 100×40 mm×5 μm; mobile phase: [H2O (0.04% HCl)—ACN]; gradient: 10-30% B over 8.0 min) and lyophilised to give the title compound as a white solid. Y=43%. 1H NMR (400 MHz, DMSO-d6) δ 11.42 (s, 1H), 10.45 (br. s, 1H), 8.38-8.30 (m, 1H), 8.29-8.21 (m, 1H), 7.02 (d, J=4 Hz, 1H), 6.98 (s, 1H), 6.95 (s, 1H), 4.26-4.17 (m, 2H), 3.71-3.65 (m, 2H), 3.55-3.46 (m, 2H), 3.33-3.18 (m, 2H), 2.94-2.71 (m, 3H), 2.05 (s, 3H). 1H NMR (400 MHz, DMSO-d6+D2O) δ 8.24 (d, J=4 Hz, 1H), 8.20 (s, 1H), 7.01 (d, J=4 Hz, 1H), 6.93 (s, 1H), 6.90 (s, 1H), 4.48-3.99 (m, 2H), 3.72-3.38 (m, 4H), 3.36-3.13 (m, 2H), 2.86 (s, 3H), 2.01 (s, 3H). LCMS (ESI): m/z: [M+H]+=386.3.
Step 1. Trans-tert-butyl 3-fluoro-4-[(methanesulfonyloxy)methyl]piperidine-1-carboxylate. To a solution of trans-tert-butyl 3-fluoro-4-(hydroxymethyl)piperidine-1-carboxylate (500 mg, 2.14 mmol) in DCM (5 mL) at 0° C. under N2 were added DIPEA (597 μL, 3.43 mmol) and MsCl (199 μL, 2.57 mmol). The mixture was stirred at 25° C. for 3 h. The reaction mixture was cooled to 0° C., quenched with H2O (5 mL) and extracted with DCM (3×5 mL). The combined organic layers were washed with brine (5 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give the title compound as a yellow oil.
Step 2. Trans-tert-butyl 4-({3-chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}methyl)-3-fluoropiperidine-1-carboxylate. To a solution of 3-chloro-7H-pyrrolo[2,3-c]pyridazine (Intermediate A1, 210 mg, 1.37 mmol) in DMF (3 mL) at 25° C. under N2 were added K2CO3(567 mg, 4.10 mmol) and trans-tert-butyl 3-fluoro-4-[(methanesulfonyloxy)methyl]piperidine-1-carboxylate (639 mg, 2.05 mmol). The mixture was stirred at 80° C. for 2 h. The reaction mixture was purified by prep-HPLC (column: Phenomenex Luna C18 100×40 mm×5 μm; mobile phase: [H2O (0.04% HCl)—ACN]; gradient: 25-75% B over 8.0 min) and lyophilised to give the title compound as a white solid. Y=quantitative.
Step 3. Trans-4-({3-chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}methyl)-3-fluoropiperidine hydrochloride. A mixture of trans-tert-butyl 4-({3-chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}methyl)-3-fluoropiperidine-1-carboxylate (250 mg, 678 μmol) and 4 M HCl in EtOAc (5 mL) was stirred at 25° C. for 1 h. The reaction mixture was concentrated under reduced pressure to give the title compound as a white solid. Y=quantitative.
Step 4. Trans-4-({3-chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}methyl)-3-fluoro-1-methylpiperidine. To a solution of trans-4-({3-chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}methyl)-3-fluoropiperidine hydrochloride (206 mg, 675 μmol) in MeOH (4 mL) at 0° C. under N2 were added aqueous 37% formaldehyde (2.01 mL, 27.0 mmol) and NaBH3CN (85 mg, 1.35 mmol). The mixture was stirred at 0° C. for 2 h. The reaction mixture was concentrated under reduced pressure to removed MeOH. The residue was diluted with H2O (1 mL), and extracted with EtOAc (3×1 mL). The combined organic layers were washed with brine (1 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Waters Xbridge BEH C18 100×30 mm×10 μm; mobile phase: [H2O (10 mM NH4HCO3)—ACN]; gradient: 20-50% B over 8.0 min) and lyophilised to give the title compound as a white solid. Y=52%. 1H NMR (400 MHz, DMSO-d6) δ 8.06-7.98 (m, 2H), 6.57 (d, J=3 Hz, 1H), 4.70-4.36 (m, 3H), 3.13-3.01 (m, 1H), 2.69-2.58 (m, 1H), 2.19 (s, 4H), 2.03-1.70 (m, 2H), 1.45-1.20 (m, 2H).
Step 5. Trans-5-chloro-2-{7-[(3-fluoro-1-methylpiperidin-4-yl)methyl]-7H-pyrrolo[2,3-c]pyridazin-3-yl}-3-methylphenol hydrochloride. To a solution of trans-4-({3-chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}methyl)-3-fluoro-1-methylpiperidine (90 mg, 318 μmol) in dioxane (1 mL) and H2O (0.2 mL) at 25° C. under N2 were added 5-chloro-3-methyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenol (Intermediate B2, 103 mg, 382 μmol), K2CO3 (176 mg, 1.27 mmol) and SPhos Pd G3 (25 mg, 32 μmol). The mixture was stirred at 80° C. under N2 for 3 h. The reaction mixture was cooled to 0° C., quenched with H2O (1 mL) and extracted with EtOAc (3×1 mL). The combined organic layers were washed with brine (1 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Phenomenex Luna C18 100×40 mm×5 μm; mobile phase: [H2O (0.04% HCl)—ACN]; gradient: 10-30% B over 8.0 min) and lyophilised to give the title compound as a white solid. Y=18%. 1H NMR (400 MHz, DMSO-d6) δ 11.63-10.01 (m, 2H), 8.78-8.34 (m, 2H), 7.08-7.05 (m, 1H), 7.05-7.02 (m, 1H), 7.01-6.96 (m, 1H), 5.20-4.45 (m, 3H), 3.73-3.64 (m, 2H), 3.10-2.86 (m, 3H), 2.77-2.53 (m, 3H), 2.08 (s, 3H), 1.90-1.63 (m, 2H). 1H NMR (400 MHz, DMSO-d6+D2O) δ 8.40 (d, J=3 Hz, 1H), 8.31 (s, 1H), 6.97 (d, J=3 Hz, 1H), 6.94 (s, 2H), 5.04-4.79 (m, 1H), 4.76-4.52 (m, 2H), 3.70-3.65 (m, 1H), 3.40-3.36 (m, 1H), 3.28-2.98 (m, 2H), 2.85-2.53 (m, 4H), 2.12-1.89 (m, 4H), 1.77-1.59 (m, 1H). 1H NMR (400 MHz, MeOD) δ 8.82-8.38 (m, 2H), 7.13 (d, J=3 Hz, 1H), 7.01 (d, J=1 Hz, 1H), 6.93 (d, J=1 Hz, 1H), 5.18-4.99 (m, 2H), 4.72-4.64 (m, 1H), 3.86-3.47 (m, 3H), 3.14-2.65 (m, 5H), 2.39-2.02 (m, 4H), 1.94-1.69 (m, 1H). LCMS (ESI): m/z: [M+H]+=389.3.
Step 1. Tert-butyl 6-[(methanesulfonyloxy)methyl]-2-azaspiro[3.3]heptane-2-carboxylate. To a solution of tert-butyl 6-(hydroxymethyl)-2-azaspiro[3.3]heptane-2-carboxylate (500 mg, 2.20 mmol) in DCM (5 mL) at 0° C. were added DIPEA (766 μL, 4.40 mmol) and MsCl (204 μL, 2.64 mmol). The solution was stirred at 0° C. under N2 for 3 h. The reaction mixture was quenched by addition of H2O (6 mL) and extracted with DCM (2×8 mL). The combined organic layers were washed with brine (2×9 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give the title compound as a yellow oil. Y=98%.
Step 2. Tert-butyl 6-({3-chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}methyl)-2-azaspiro[3.3]heptane-2-carboxylate. To a solution of tert-butyl 6-[(methanesulfonyloxy)methyl]-2-azaspiro[3.3]heptane-2-carboxylate (1.31 g, 4.30 mmol) in DMF (8 mL) at 25° C. were added 3-chloro-7H-pyrrolo[2,3-c]pyridazine (Intermediate A1, 550 mg, 3.58 mmol) and K2CO3 (990 mg, 7.16 mmol). The reaction mixture was stirred at 80° C. under N2 for 5 h. The reaction mixture was purified by prep-HPLC (column: Phenomenex Luna C18 250×50 mm×15 μm; mobile phase: [H2O (0.05% HCl)—ACN]; gradient: 20-70% B over 10 min) and lyophilised to give the title compound as a white solid. Y=44%. 1H NMR (400 MHz, DMSO-d6) δ 8.10-7.98 (m, 2H), 6.56 (d, J=3 Hz, 1H), 4.38 (d, J=8 Hz, 2H), 3.87-3.73 (m, 4H), 2.77-2.66 (m, 1H), 2.22-2.11 (m, 2H), 2.05-1.92 (m, 2H), 1.35 (s, 9H).
Step 3. 6-({3-Chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}methyl)-2-azaspiro[3.3]heptane. A mixture of tert-butyl 6-({3-chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}methyl)-2-azaspiro[3.3]heptane-2-carboxylate (350 mg, 965 μmol) in DCM (8.75 mL) at 25° C. under N2 was added TFA (1.0 mL, 13.5 mmol). The reaction was stirred for 3 h then concentrated under reduced pressure to give a residue. The residue was dissolved in MeOH, the resulting mixture was adjusted to pH=−8 and then concentrated under reduced pressure to give the title compound as a yellow solid. Y=95%.
Step 4. 6-({3-Chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}methyl)-2-methyl-2-azaspiro[3.3]heptane. To a solution of 7-(2-azaspiro[3.3]heptan-6-ylmethyl)-3-chloro-pyrrolo[2,3-c]pyridazine (70 mg, 213 μmol) in MeOH (7 mL) at 0° C. were added 37% formaldehyde solution (15 μL 202 μmol) and NaBH3CN (27 mg, 426 μmol). The solution was stirred at 0° C. under N2 for 3 h. The reaction mixture was purified by prep-HPLC (column: Waters Xbridge BEH C18 100×30 mm×10 μm; mobile phase: [H2O (10 mM NH4HCO3)—ACN]; gradient: 20-65% B over 8.0 min) and lyophilised to give the title compound as a yellow oil. Y=76%. 1H NMR (400 MHz, DMSO-d6) δ 8.12-7.95 (m, 2H), 6.55 (d, J=3 Hz, 1H), 4.37 (d, J=8 Hz, 2H), 3.14-2.96 (m, 4H), 2.74-2.63 (m, 1H), 2.17-2.01 (m, 5H), 1.95-1.84 (m, 2H).
Step 5. 5-Chloro-3-methyl-2-[7-({2-methyl-2-azaspiro[3.3]heptan-6-yl}methyl)-7H-pyrrolo[2,3-c]pyridazin-3-yl]phenol hydrochloride. To a solution of 6-({3-chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}methyl)-2-methyl-2-azaspiro[3.3]heptane (73 mg, 264 μmol) in dioxane (3 mL) and H2O (0.6 mL) at 25° C. under N2 were added 5-chloro-3-methyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenol (Intermediate B2, 106 mg, 396 μmol), K2CO3 (146 mg, 1.06 mmol) and dicyclohexyl-[2-(2,6-dimethoxyphenyl)phenyl]phosphane; methanesulfonate;(2-phenylanilino)palladium(1+) (31 mg, mol). The solution was stirred at 50° C. under N2 for 3 h. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was diluted with H2O (2 mL) and extracted with EtOAc (3×2 mL). The combined organic layers were washed with brine (2×2 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Phenomenex Luna C18 100×40 mm×5 μm; mobile phase: [H2O (0.04% HCl)—ACN]; gradient: 10-25% B over 8.0 min) and lyophilised to give the title compound as a yellow solid. Y=31%. 1H NMR (400 MHz, DMSO-d6) δ 10.97 (br. s, 2H), 8.62 (s, 1H), 8.50 (s, 1H), 7.09 (s, 1H), 6.99 (s, 2H), 4.47 (d, J=8 Hz, 2H), 4.24-4.01 (m, 2H), 3.98-3.80 (m, 2H), 2.86-2.64 (m, 4H), 2.42-2.21 (m, 2H), 2.20-2.00 (m, 5H).
1H NMR (400 MHz, DMSO-d6+D2O) 8.52-8.39 (m, 2H), 7.09 (d, J=3 Hz, 1H), 7.01 (s, 1H), 6.93 (s, 1H), 4.47 (d, J=8 Hz, 2H), 4.26-4.07 (m, 2H), 3.93-3.87 (m, 2H), 2.86-2.69 (m, 4H), 2.43-2.34 (m, 2H), 2.33-2.27 (m, 2H), 2.16 (s, 3H). 1H NMR (400 MHz, methanol-d4) δ 8.59-8.51 (m, 2H), 7.09 (d, J=3 Hz, 1H), 7.01 (s, 1H), 6.93 (s, 1H), 4.57 (d, J=8 Hz, 2H), 4.40-4.25 (m, 2H), 4.10-4.00 (m, 2H), 2.99-2.84 (m, 4H), 2.56-2.47 (m, 1H), 2.46-2.39 (m, 1H), 2.35-2.22 (m, 2H), 2.16 (s, 3H). LCMS (ESI): m/z: [M+H]+=383.3.
Step 1. (1-Acetylpiperidin-3-yl)methyl methanesulfonate. To a solution of 1-[3-(hydroxymethyl)-1-piperidyl]ethanone (900 mg, 5.72 mmol) in DCM (9 mL) at 0° C. under N2 were added DIPEA (1.99 mL, 11.5 mmol) and MsCl (443 μL, 5.72 mmol). The mixture was stirred at 0° C. for 2 h. The mixture was poured into water (15 mL) and extracted with ethyl acetate (3×15 mL). The combined organic phase was washed with brine (3×15 mL), dried with anhydrous Na2SO4, filtered and concentrated under reduced pressure to give the title compound as a yellow oil. Y=74%.
Step 2. I-[3-({3-Chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}methyl)piperidin-1-yl]ethan-1-one. To a solution of (1-acetylpiperidin-3-yl)methyl methanesulfonate (500 mg, 2.12 mmol) and 3-chloro-7H-pyrrolo[2,3-c]pyridazine (Intermediate A1, 218 mg, 1.42 mmol) in DMF (5 mL) at 20° C. under N2 was added K2CO3 (587 mg, 4.25 mmol). The mixture was stirred at 80° C. for 4 h. The mixture was poured into saturated NH4Cl aqueous solution (8 mL) and the resulting mixture extracted with ethyl acetate (3×8 mL). The combined organic phase was washed with brine (3×8 mL), dried with anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Waters Xbridge BEH C18 250×50 mm×10 μm; mobile phase: [H2O (10 mM NH4HCO3)—ACN]; gradient: 15-40% B over 10 min) and lyophilised to give the title compound as a yellow oil. Y=24%. 1H NMR (400 MHz, DMSO-d6) δ 8.10-8.02 (m, 2H), 6.63-6.56 (m, 1H), 4.46-4.39 (m, 0.4H), 4.36-4.26 (m, 1.6H), 4.06-3.93 (m, 1H), 3.69-3.53 (m, 1H), 3.11-2.91 (m, 1H), 2.79-2.66 (m, 0.4H), 2.62-2.53 (m, 0.6H), 2.28-2.00 (m, 1H), 1.99-1.82 (m, 3H), 1.73-1.15 (m, 4H).
Step 3. 1-(3-{[3-(4-Chloro-2-hydroxy-6-methylphenyl)-7H-pyrrolo[2,3-c]pyridazin-7-yl]methyl}piperidin-1-yl)ethan-1-one. To a solution of 1-[3-({3-chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}methyl)piperidin-1-yl]ethan-1-one (50 mg, 171 μmol) and 5-chloro-3-methyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenol (Intermediate B2, 69 mg, 256 μmol) in dioxane (1 mL) and H2O (0.3 mL) at 25° C. under N2 were added SPhos Pd G3 (13 mg, 17 μmol) and K2CO3 (71 mg, 512 μmol). The mixture was stirred at 90° C. under N2 for 2 h. The mixture was poured into saturated NH4Cl aqueous solution (2 mL) and the resulting mixture was extracted with ethyl acetate (3×2 mL). The combined organic phase was washed with brine (3×2 mL), dried over anhydrous Na2SO4, filtered and concentrated under vacuum. The residue was purified by prep-HPLC (column: Phenomenex Luna C18 100×40 mm×5 μm; mobile phase: [H2O (0.04% HCl)—ACN]; gradient: 20-30% B over 8.0 min) and lyophilised to give the title compound as an off-white solid. Y=44%. 1H NMR (400 MHz, DMSO-d6) δ 10.78 (br. s, 1H), 8.70 (s, 1H), 8.60 (s, 1H), 7.08 (d, J=2 Hz, 1H), 7.02 (s, 2H), 4.56-4.32 (m, 2H), 4.08-4.02 (m, 1H), 3.71-3.59 (m, 1H), 3.20-2.95 (m, 1H), 2.82-2.62 (m, 1H), 2.33-1.87 (m, 7H), 1.78-1.60 (m, 2H), 1.46-1.18 (m, 2H). 1H NMR (400 MHz, DMSO-d6+D2O) δ 8.71-8.56 (m, 2H), 7.12-7.07 (m, 1H), 7.02 (d, J=2 Hz, 1H), 6.96 (d, J=2 Hz, 1H), 4.55-4.27 (m, 2H), 4.13-3.91 (m, 1H), 3.74-3.65 (m, 1H), 3.23-2.92 (m, 1H), 2.79-2.65 (m, 1H), 2.20-1.89 (m, 7H), 1.78-1.55 (m, 2H), 1.42-1.21 (m, 2H). 1H NMR (400 MHz, MeOD) δ 8.66-8.58 (m, 1H), 8.57-8.52 (m, 1H), 7.20-7.10 (m, 1H), 7.01 (s, 1H), 6.93 (s, 1H), 4.56-4.42 (m, 2H), 4.29-4.24 (m, 0.3H), 4.12-4.01 (m, 0.7H), 3.89-3.78 (m, 0.3H), 3.77-3.62 (m, 0.7H), 3.28-2.77 (m, 2H), 2.42-2.22 (m, 1H), 2.21-1.98 (m, 6H), 1.92-1.71 (m, 2H), 1.62-1.31 (m, 2H).
LCMS (ESI): m/z: [M+H]+=399.3.
To a solution of (4-cyano-2-hydroxy-6-methyl-phenyl)boronic acid (Intermediate B3, 100 mg, 567 μmol) in dioxane (3 mL) and H2O (1 mL) at 25° C. were added K2CO3 (157 mg, 1.13 mmol) SPhos Pd G3 (29 mg, 38 μmol) and 3-chloro-7-[(1-methyl-4-piperidyl)methyl]pyrrolo[2,3-c]pyridazine (for synthesis see Compound 4)(0.10 g, 378 μmol). The mixture was stirred at 80° C. under N2 for 1 h. The reaction mixture was diluted with H2O (3 mL) and extracted with EtOAc (3×3 mL). The combined organic layers were washed with brine (9 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Waters Xbridge BEH C18 100×30 mm×10 μm; mobile phase: [H2O (10 mM NH4HCO3)—ACN]; gradient: 15-45% B over 8.0 min) and lyophilised to give the title compound as an off-white solid. Y=22%. 1H NMR (400 MHz, DMSO-d6) δ 9.86 (br. s, 1H), 7.95 (d, J=3 Hz, 1H), 7.80 (s, 1H), 7.27 (s, 1H), 7.15 (s, 1H), 6.58 (d, J=3 Hz, 1H), 4.34 (d, J=7 Hz, 2H), 2.81-2.67 (m, 2H), 2.12 (s, 3H), 2.02 (s, 3H), 2.01-1.88 (m, 1H), 1.86-1.69 (m, 2H), 1.57-1.42 (m, 2H), 1.39-1.24 (m, 2H). 1H NMR (400 MHz, DMSO-d6+D2O) δ 7.92 (d, J=3 Hz, 1H), 7.79 (s, 1H), 7.26 (s, 1H), 7.15 (s, 1H), 6.59 (d, J=3 Hz, 1H), 4.33 (d, J=7 Hz, 2H), 2.79-2.67 (m, 2H), 2.10 (s, 3H), 2.00 (s, 3H), 1.98-1.87 (m, 1H), 1.86-1.71 (m, 2H), 1.53-1.39 (m, 2H), 1.38-1.24 (m, 2H). LC-MS (ESI): m/z: [M+H]+=362.3.
Step 1. Tert-butyl 4-({3-chloro-7H-pyrroio[2,3-c]pyridazin-7-yl}methyl)-4-cyanopiperidine-1-carboxylate. To a solution of tert-butyl 4-cyano-4-(hydroxymethyl)piperidine-1-carboxylate (500 mg, 2.08 mmol) in toluene (6 mL) at 0° C. were added 3-chloro-7H-pyrrolo[2,3-c]pyridazine (Intermediate A1, 320 mg, 2.08 mmol), triphenylphosphine (819 mg, 3.12 mmol) and diisopropyl azodicarboxylate (605, 3.12 mmol). The resulting mixture was stirred at 80° C. for 2 h. The solution was concentrated under vacuum. The residue was purified by prep-HPLC (column: Phenomenex Luna C18 100×40 mm×5 μm; mobile phase: [H2O (0.04% HCl)—ACN]; gradient: 30-70% B over 8.0 min) and lyophilised to give the title compound as a yellow solid. Y=19%. 1H NMR (400 MHz, chloroform-d) δ 7.82 (d, J=4 Hz, 1H), 7.74 (s, 1H), 6.58 (d, J=4 Hz, 1H), 4.82-4.52 (m, 2H), 4.33-4.05 (m, 2H), 3.03-2.93 (m, 2H), 1.83-1.73 (m, 3H), 1.51-1.39 (m, 10H).
Step 2. 4-({3-Chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}methyl)piperidine-4-carbonitrile hydrochloride. A mixture of tert-butyl 4-({3-chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}methyl)-4-cyanopiperidine-1-carboxylate (150 mg, 399 μmol) and 4 M HCl in EtOAc (4 mL) was stirred at 25° C. for 1 h. The solution was concentrated under vacuum to give the title compound as a white solid. Y=96%. 1H NMR (400 MHz, DMSO-d6) δ 9.22-8.82 (m, 2H), 8.11 (s, 1H), 8.09 (d, J=3 Hz, 1H), 6.70 (d, J=3 Hz, 1H), 4.82 (s, 2H), 3.45-3.37 (m, 2H), 2.93-2.82 (m, 2H), 2.09-2.00 (m, 4H).
Step 3. 4-({3-Chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}methyl)-1-methylpiperidine-4-carbonitrile. To a solution of 4-({3-chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}methyl)piperidine-4-carbonitrile hydrochloride (120 mg, 384 μmol) in DCE (3 mL) and AcOH (0.33 mL) at 0° C. was added 37% formaldehyde solution (86 μL, 1.15 mmol). The resulting mixture was stirred at 0° C. for 0.5 h and treated with NaBH(OAc)3 (244 mg, 1.15 mmol). The resulting mixture was stirred at 0° C. for 0.5 h. The solution was diluted with H2O (2 mL) and extracted with DCM (3×2 mL).
The combined organic layers were washed with brine (2 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Waters Xbridge Prep OBD C18 150×40 mm×10 μm; mobile phase: [H2O (10 mM NH4HCO3)—ACN]; gradient: 20-50% B over 8.0 min) and lyophilised to give the title compound as a yellow oil. Y=72%. 1H NMR (400 MHz, DMSO-d6) δ 8.08 (s, 1H), 8.06 (d, J=3 Hz, 1H), 6.67 (d, J=3 Hz, 1H), 4.73 (s, 2H), 2.81-2.75 (m, 2H), 2.17 (s, 3H), 2.05-1.93 (m, 2H), 1.85-1.73 (m, 4H).
Step 4. 4-{[3-(4-Chloro-2-hydroxy-6-methylphenyl)-7H-pyrrolo[2,3-c]pyridazin-7-yl]methyl}-1-methylpiperidine-4-carbonitrile. To a solution of 4-({3-chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}methyl)-1-methylpiperidine-4-carbonitrile (80 mg, 276 μmol) in dioxane (3 mL) and H2O (0.6 mL) at 25° C. under N2 were added 5-chloro-3-methyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenol (Intermediate B2, 74 mg, 276 μmol), dicyclohexyl-[2-(2,6-dimethoxyphenyl)phenyl]phosphane; methanesulfonate;(2-phenylanilino)palladium(1+) “SPhos Pd G3” (32 mg, 41 μmol) and K2CO3 (153 mg, 1.10 mmol). The resulting mixture was stirred at 80° C. under N2 for 1 h. The solution was diluted with H2O (2 mL) and extracted with EtOAc (3×2 mL). The combined organic layers were washed with brine (2 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Phenomenex Luna C18 100×40 mm×5 μm; mobile phase: [H2O (0.04% HCl)—ACN]; gradient: 10-25% B over 8.0 min) and lyophilised to give the title compound as a light yellow solid. Y=50%. 1H NMR (400 MHz, DMSO-d6) δ 11.48-11.11 (m, 1H), 11.01-10.56 (m, 1H), 8.65-8.37 (m, 2H), 7.13-7.02 (m, 2H), 6.98 (s, 1H), 5.11-4.84 (m, 2H), 3.56-3.52 (m, 2H), 3.05-2.95 (m, 2H), 2.89-2.76 (m, 3H), 2.41-2.22 (m, 4H), 2.07 (s, 3H). 1H NMR (400 MHz, DMSO-d6, T=353 K) δ 10.81-10.19 (m, 1H), 8.11 (s, 1H), 7.99 (s, 1H), 6.92 (d, J=1 Hz, 1H), 6.90 (d, J=1 Hz, 1H), 6.81-6.78 (m, 1H), 4.87 (s, 2H), 3.61-3.49 (m, 2H), 3.25-3.23 (m, 2H), 2.82 (s, 3H), 2.30-2.21 (m, 4H), 2.04 (s, 3H). 1H NMR (400 MHz, DMSO-d6+D2O) δ 10.79-10.32 (m, 0.25H), 8.62-8.45 (m, 2H), 7.10 (d, J=2 Hz, 1H), 7.01-6.92 (m, 2H), 5.08-4.83 (m, 2H), 3.55-3.52 (m, 2H), 3.08-2.96 (m, 2H), 2.91-2.78 (m, 3H), 2.31-2.18 (m, 4H), 2.06 (s, 3H). 1H NMR (400 MHz, MeOD) δ 8.62 (d, J=3 Hz, 1H), 8.55 (s, 1H), 7.16 (d, J=3 Hz, 1H), 7.00 (d, J=1 Hz, 1H), 6.92 (d, J=1 Hz, 1H), 4.95 (s, 2H), 3.73-3.65 (m, 2H), 3.29-3.18 (m, 2H), 2.95 (s, 3H), 2.42-2.27 (m, 4H), 2.16 (s, 3H). LCMS (ESI): m/z: [M+H]+=396.3.
Step 1. Tert-butyl 4-({3-choro-7H-pyrrolo[2,3-c]pyridazin-7-yl}methyl)-4-hydroxypiperidine-1-carboxylate. To a solution of 3-chloro-7H-pyrrolo[2,3-c]pyridazine (Intermediate A1, 200 mg, 1.30 mmol) in DMF (4 mL) at 25° C. under N2 were added Cs2CO3 (1.27 g, 3.91 mmol) and tert-butyl 1-oxa-6-azaspiro[2.5]octane-6-carboxylate (333 mg, 1.56 mmol). The mixture was stirred at 80° C. for 12 h. The reaction mixture was purified by prep-HPLC (column: Phenomenex Luna C18 100×40 mm×5 μm; mobile phase: [H2O (0.04% HCl)—ACN]; gradient: 25-45% B over 8.0 min) and lyophilised to give the title compound as a white solid. Y=59%. 1H NMR (400 MHz, DMSO-d6) δ 8.13-7.90 (m, 2H), 6.70-6.48 (m, 1H), 4.53-4.31 (m, 2H), 3.72-3.57 (m, 2H), 3.11-2.89 (m, 2H), 1.54-1.21 (m, 13H).
Step 2. 4-({3-Chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}methyl)piperidin-4-ol hydrochloride. A mixture of tert-butyl 4-({3-chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}methyl)-4-hydroxypiperidine-1-carboxylate (280 mg, 763 μmol) and 4 M HCl in EtOAc (3 mL) was stirred at 25° C. for 6 h. The reaction mixture was concentrated under reduced pressure to give the title compound as a white solid. Y=quantitative.
Step 3. 4-({3-Chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}methyl)-1-methylpiperidin-4-ol hydrochloride. To a solution of 4-({3-chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}methyl)piperidin-4-ol hydrochloride (130 mg, 429 μmol) in MeOH (2 mL) at 0° C. under N2 were added 37% formaldehyde solution (223 μL 3.00 mmol) and NaBH3CN (54 mg, 858 μmol). The mixture was stirred at 0° C. for 2 h. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Phenomenex Luna C18 100×40 mm×5 μm; mobile phase: [H2O (0.04% HCl)—ACN]; gradient: 1-25% B over 8.0 min) and lyophilised to give the title compound as a white solid. Y=66%. 1H NMR (400 MHz, DMSO-d6) δ 10.41-9.62 (m, 1H), 8.06 (s, 1H), 8.02-7.93 (m, 1H), 6.72-6.50 (m, 1H), 4.71-4.34 (m, 2H), 3.32-3.22 (m, 2H), 3.13-2.94 (m, 2H), 2.86-2.71 (m, 3H), 2.03-1.74 (m, 2H), 1.73-1.43 (m, 2H).
Step 4. 4-{[3-(4-Chloro-2-hydroxy-6-methylphenyl)-7H-pyrrolo[2,3-c]pyridazin-7-yl]methyl}-1-methylpiperidin-4-ol hydrochloride. To a solution of 4-({3-chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}methyl)-1-methylpiperidin-4-ol hydrochloride (70 mg, 249 μmol) in dioxane (1 mL) and H2O (0.2 mL) at 25° C. under N2 were added 5-chloro-3-methyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenol (Intermediate B2, 80 mg, 299 μmol), K2CO3 (138 mg, 997 μmol) and SPhos Pd G3 (19 mg, 25 μmol). The mixture was stirred at 80° C. for 3 h. The reaction mixture was diluted with H2O (1 mL) and extracted with EtOAc (3×1 mL). The combined organic layers were washed with brine (1 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Phenomenex Luna C18 100×40 mm×5 μm; mobile phase: [H2O (0.04% HCl)—ACN]; gradient: 10-25% B over 8.0 min) and lyophilised to give the title compound as a white solid. Y=38%. 1H NMR (400 MHz, DMSO-d6) δ 11.06-10.24 (m, 2H), 8.62-8.35 (m, 2H), 7.19-6.81 (m, 3H), 5.83-5.33 (m, 1H), 4.92-4.17 (m, 2H), 3.31-3.19 (m, 2H), 3.13-2.99 (m, 2H), 2.88-2.67 (m, 3H), 2.12-1.90 (m, 5H), 1.80-1.58 (m, 2H). 1H NMR (400 MHz, DMSO-d6+D2O) δ 8.59-8.35 (m, 2H), 7.20-6.63 (m, 3H), 4.73-4.42 (m, 2H), 3.41-3.19 (m, 2H), 3.16-2.97 (m, 2H), 2.87-2.71 (m, 3H), 2.07 (s, 3H), 2.02-1.90 (m, 2H), 1.77-1.56 (m, 2H). 1H NMR (400 MHz, DMSO-d6+D2O, T=353 K) δ 8.54-8.14 (m, 2H), 7.09-6.85 (m, 3H), 4.74-4.40 (m, 2H), 3.50-3.28 (m, 2H), 3.18-3.05 (m, 2H), 2.94-2.69 (m, 3H), 2.11-1.94 (m, 5H), 1.78-1.61 (m, 2H). LC-MS (ESI): m/z: [M+H]+=387.3.
Step 1. Methyl I-acetylpiperidine-2-carboxylate. To a mixture of methyl piperidine-2-carboxylate (1.0 g, 6.98 mmol) and TEA (1.94 mL, 14.0 mmol) in DCM (5 mL) at 0° C. under N2 was added acetyl chloride (596 μL, 8.38 mmol). The reaction mixture was stirred at 0° C. for 1 h. The reaction mixture was poured into water (10 mL) and the resulting mixture was extracted with ethyl acetate (3×10 mL). The combined organic phase was washed with brine (3×10 mL), dried with anhydrous Na2SO4, filtered and concentrated under vacuum. The residue was purified by column chromatography (SiO2, 1/1 petroleum ether/ethyl acetate) to give the title compound as a white oil. Y=93%.
Step 2. I-[2-(Hydroxymethyl)piperidin-1-yl]ethan-1-one. To a solution of methyl 1-acetylpiperidine-2-carboxylate (900 mg, 4.86 mmol) in THE (8 mL) at 0° C. under N2 was added 2 M LiBH4 in THE (4.86 mL, 9.72 mmol). The mixture was stirred at 0° C. under N2 for 2 h. The reaction mixture was quenched with 1 M HCl (2 mL) and the resulting mixture extracted with ethyl acetate (3×15 mL). The combined organic phase was washed with brine (15 mL), dried with anhydrous Na2SO4, filtered and concentrated under vacuum. The residue was purified by column chromatography (SiO2, 1/1 petroleum ether/ethyl acetate) to give the title compound as a white oil. Y=79%. 1H NMR (400 MHz, methanol-d4) δ 4.77-3.52 (m, 4H), 3.24-2.58 (m, 1H), 2.19-2.04 (m, 3H), 1.88-1.29 (m, 6H).
Step 3. 1-[2-({3-Chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}methyl)piperidin-1-yl]ethan-1-one. To a solution of 1-[2-(hydroxymethyl)piperidin-1-yl]ethan-1-one (200 mg, 1.27 mmol) and 3-chloro-7H-pyrrolo[2,3-c]pyridazine (Intermediate A1, 195 mg, 1.27 mmol) in toluene (1 mL) at 25° C. under N2 were added PPh3 (501 mg, 1.91 mmol) and di-tert-butyl azodicarboxylate (439 mg, 1.91 mmol). The mixture was stirred at 80° C. for 3 h. The reaction solution was poured into water (2 mL). The resulting mixture was extracted with ethyl acetate (3×2 mL). The combined organic phase was washed with brine (3×2 mL), dried with anhydrous Na2SO4, filtered and concentrated under vacuum. The residue was purified by prep-HPLC (column: Waters Xbridge BEH C18 100×30 mm×10 μm; mobile phase: [H2O (10 mM NH4HCO3)—ACN]; gradient: 40-75% B over 8.0 min) and lyophilised to give the title compound as a white solid. Y=27%. 1H NMR (400 MHz, DMSO-d6) δ 8.78-7.62 (m, 2H), 6.94-6.39 (m, 1H), 5.51-3.51 (m, 4.6H), 3.11-2.75 (m, 0.4H), 1.81-1.19 (m, 9H).
Step 4. 1-(2-{[3-(4-Chloro-2-hydroxy-6-methylphenyl)-7H-pyrrolo[2,3-c]pyridazin-7-yl]methyl}piperidin-1-yl)ethan-1-one. To a solution of 1-[2-({3-chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}methyl)piperidin-1-yl]ethan-1-one (70 mg, 239 μmol) and 5-chloro-3-methyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenol (Intermediate B2, 96 mg, 359 μmol) in dioxane (1 mL) and H2O (0.3 mL) at 25° C. under N2 were added SPhos Pd G3 (19 mg, 24 μmol) and K2CO3 (149 mg, 1.08 mmol). The mixture was stirred at 90° C. for 2 h. The reaction mixture was poured into saturated ammonium chloride aqueous solution (1 mL). The aqueous phase was extracted with ethyl acetate (3×1 mL). The combined organic phase was washed with brine (1 mL), dried with anhydrous Na2SO4, filtered and concentrated under vacuum. The residue was purified by prep-HPLC (column: Phenomenex Luna C18 100×40 mm×5 μm; mobile phase: [H2O (0.04% HCl)—ACN]; gradient: 15-45% B over 8.0 min) and lyophilised to give the title compound as a yellow solid. Y=11%. 1H NMR (400 MHz, DMSO-d6) δ 11.14-10.40 (m, 1H), 8.77-8.21 (m, 2H), 7.40-6.70 (m, 3H), 5.10-4.33 (m, 3H), 3.64-3.55 (m, 1.5H), 3.05-2.95 (m, 0.5H), 2.12-2.00 (m, 3H), 1.89-1.21 (m, 9H). 1H NMR (400 MHz, DMSO-d6+D2O) δ 8.61-8.39 (m, 2H), 7.41-6.55 (m, 3H), 5.08-4.30 (m, 3H), 3.57-2.90 (m, 2H), 2.12-1.94 (m, 3H), 1.85-1.16 (m, 9H). 1H NMR (400 MHz, DMSO-d6, T=353 K) δ 8.60-8.15 (m, 2H), 7.16-6.79 (m, 3H), 5.18-4.30 (m, 3H), 3.75-3.44 (m, 1.5H), 3.10-2.82 (m, 0.5H), 2.04 (s, 3H), 1.85-1.21 (m, 9H). LCMS (ESI): m/z: [M+H]+=399.3.
Step 1. Tert-butyl 2-{3-chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}acetate. To a solution of 3-chloro-7H-pyrrolo[2,3-c]pyridazine (Intermediate A1, 1.7 g, 11.1 mmol) in DMF (17 mL) were added K2CO3 (3.06 g, 22.1 mmol) and tert-butyl 2-bromoacetate (1.63 mL, 11.1 mmol). The mixture was stirred at 25° C. for 2 h. The reaction mixture was purified by prep-HPLC (column: Waters Xbridge BEH C18 250×50 mm×10 μm; mobile phase: [H2O (10 mM NH4HCO3)—ACN]; gradient: 35-55% B over 10 min) to give the title compound as a yellow solid. Y=74%. 1H NMR (400 MHz, DMSO-d6) δ 8.07 (s, 1H), 8.02 (d, J=3 Hz, 1H), 6.61 (d, J=3 Hz, 1H), 5.20 (s, 2H), 1.41 (s, 9H).
Step 2. 2-[3-(4-Chloro-2-hydroxy-6-methylphenyl)-7H-pyrrolo[2,3-c]pyridazin-7-yl]acetic acid. To a solution of tert-butyl 2-{3-chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}acetate (1.9 g, 7.10 mmol) and 5-chloro-3-methyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenol (Intermediate B2, 2.48 g, 9.23 mmol) in dioxane (95 mL) and H2O (19 mL) at 25° C. under N2 were added SPhos Pd G3 (554 mg, 710 μmol) and K2CO3 (2.94 g, 21.3 mmol). The mixture was stirred at reflux for 8 h under N2. The reaction mixture was poured into water (60 mL) and extracted with EtOAc (60 mL). The combined organic phase was washed with brine (60 mL), dried with anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Phenomenex Luna C18 250×50 mm×15 μm; mobile phase: [H2O (0.1% TFA)—ACN]; gradient: 1-40% B over 10 min) and lyophilised to give the title product as a white solid. Y=40%. 1H NMR (400 MHz, DMSO-d6) δ 10.33 (br. s, 1H), 8.47-8.08 (m, 2H), 6.95 (s, 1H), 6.90 (s, 1H), 6.84 (s, 1H), 5.30 (s, 2H), 2.02 (s, 3H).
Step 3. 2-[3-(4-Chloro-2-hydroxy-6-methylphenyl)-7H-pyrrolo[2,3-c]pyridazin-7-yl]-N-methyl-N-(oxetan-3-yl)acetamide. To a solution of 2-[3-(4-chloro-2-hydroxy-6-methylphenyl)-7H-pyrrolo[2,3-c]pyridazin-7-yl]acetic acid (100 mg, 315 μmol) in ACN (0.5 mL) at 25° C. under N2 were added N-methyloxetan-3-amine (41 mg, 472 μmol), COMU (202 mg, 472 μmol) and NMM (104 μL, 944 μmol). The mixture was stirred at reflux for 1 h. The reaction mixture was diluted with H2O (20 mL) and extracted with EtOAc (3×10 mL). The combined organic layers were washed with brine (2×15 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: CD02-Waters Xbridge BEH C18 150×25×10 μm; mobile phase: [water (NH4HCO3)—ACN]; gradient: 20-50% B over 10 min) and lyophilised to give the title compound as a white solid. Y=28%. 1H NMR (400 MHz, DMSO-d6) δ 9.87 (s, 1H), 7.82-7.72 (m, 2H), 6.86 (d, J=3 Hz, 2H), 6.57 (d, J=3 Hz, 1H), 5.53-5.21 (m, 3H), 4.92-4.58 (m, 4H), 3.29-3.05 (m, 3H), 1.97 (s, 3H). 1H NMR (400 MHz, DMSO-d6+D2O) δ 7.84-7.65 (m, 2H), 6.85 (d, J=3 Hz, 2H), 6.58 (d, J=3 Hz, 1H), 5.50-5.23 (m, 3H), 4.92-4.60 (m, 4H), 3.33-3.02 (m, 3H), 1.96 (s, 3H). 1H NMR (400 MHz, DMSO-d6+D2O, T=353 K) δ 7.75 (d, J=3 Hz, 1H), 7.73 (s, 1H), 6.85 (d, J=3 Hz, 2H), 6.57 (d, J=3 Hz, 1H), 5.40 (s, 2H), 5.35-5.16 (m, 1H), 4.72 (s, 4H), 3.35 (s, 3H), 2.00 (s, 3H). LCMS (ESI): m/z: [M+H]+=387.0.
To a solution of 2-[3-(4-chloro-2-hydroxy-6-methylphenyl)-7H-pyrrolo[2,3-c]pyridazin-7-yl]acetic acid (for synthesis see Compound 58)(100 mg, 315 μmol) and (3S)-N-methyltetrahydrofuran-3-amine hydrochloride (65 mg, 472 μmol) in ACN (1 mL) at 25° C. were added COMU (202 mg, 472 μmol) and NMM (104 μL, 944 μmol). The mixture was stirred at reflux for 5 h. The reaction mixture was added to H2O (5 mL) and the resulting mixture was extracted with ethyl acetate (3×5 mL). The combined organic layers were washed with brine (3×5 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: CD02-Waters Xbridge BEH C18 150×25×10 μm; mobile phase: [water (NH4HCO3)—ACN]; gradient: 21-51% B over 10 min) and lyophilised to give the title compound as an off-white solid. Y=21%. 1H NMR (400 MHz, DMSO-d6) δ 9.88 (br. s, 1H), 7.81 (d, J=3 Hz, 1H), 7.75 (s, 1H), 6.86 (d, J=4 Hz, 2H), 6.57 (d, J=4 Hz, 1H), 5.64-5.32 (m, 2H), 5.20-4.75 (m, 1H), 4.03-3.91 (m, 1H), 3.87-3.76 (m, 1H), 3.73-3.54 (m, 2H), 3.15-2.72 (m, 3H), 2.42-2.09 (m, 1H), 2.08-1.67 (m, 4H). 1H NMR (400 MHz, DMSO-d6+D2O) δ 7.80 (d, J=3 Hz, 1H), 7.75 (s, 1H), 6.86 (d, J=4 Hz, 2H), 6.58 (d, J=4 Hz, 1H), 5.59-5.25 (m, 2H), 5.16-4.79 (m, 1H), 4.03-3.89 (m, 1H), 3.87-3.74 (m, 1H), 3.71-3.56 (m, 2H), 3.15-2.69 (m, 3H), 2.41-2.05 (m, 1H), 2.03-1.79 (m, 4H). 1H NMR (400 MHz, DMSO-d6+D2O, T=353 K) δ 7.77 (d, J=3 Hz, 1H), 7.73 (s, 1H), 6.85 (d, J=4 Hz, 2H), 6.58 (d, J=4 Hz, 1H), 5.51-5.36 (m, 2H), 5.13-4.78 (m, 1H), 4.05-3.88 (m, 1H), 3.78-3.50 (m, 3H), 3.10-2.75 (m, 3H), 2.36-2.09 (m, 1H), 2.04-1.90 (m, 4H). LCMS (ESI): m/z: [M+H]+=401.0.
Synthesised in an analogous way to Compound 59, starting from (3R)-N-methyltetrahydrofuran-3-amine hydrochloride. 1H NMR (400 MHz, DMSO-d6) δ 9.87 (s, 1H), 7.81 (d, J=3 Hz, 1H), 7.75 (s, 1H), 6.91-6.78 (m, 2H), 6.57 (d, J=3 Hz, 1H), 5.59-5.33 (m, 2H), 5.13-4.83 (m, 1H), 3.98-3.96 (m, 1H), 3.90-3.50 (m, 3H), 3.09-2.78 (m, 3H), 2.14-2.11 (m, 1H), 1.98-1.85 (m, 4H). 1H NMR (400 MHz, DMSO-d6, T=353 K) δ 9.63 (br. s, 1H), 7.79 (d, J=3 Hz, 1H), 7.74 (s, 1H), 6.86-6.84 (m, 2H), 6.57 (d, J=3 Hz, 1H), 5.44 (s, 2H), 5.01-4.98 (m, 1H), 4.07-3.91 (m, 1H), 3.75-3.64 (m, 3H), 3.06-2.83 (m, 3H), 2.03-2.01 (m, 1H), 1.96-1.83 (m, 4H). 1H NMR (400 MHz, DMSO-d6+D2O) δ 7.80 (d, J=3 Hz, 1H), 7.75 (s, 1H), 6.90-6.83 (m, 2H), 6.58 (d, J=3 Hz, 1H), 5.55-5.36 (m, 2H), 5.08-4.80 (m, 1H), 4.03-3.89 (m, 1H), 3.86-3.53 (m, 3H), 3.13-2.76 (m, 3H), 2.40-2.09 (m, 1H), 2.02-1.81 (m, 4H). LCMS(ESI): m/z: [M+H]+=401.0.
To a solution of 2-[3-(4-chloro-2-hydroxy-6-methyl-phenyl)pyrrolo[2,3-c]pyridazin-7-yl]acetic acid (for synthesis see Compound 58)(100 mg, 315 μmol) and (3S)-N,1-dimethylpyrrolidin-3-amine (65 mg, 567 μmol) in ACN (1 mL) at 25° C. under N2 were added COMU (202 mg, 472 μmol) and NMM (104 μL, 944 μmol). The mixture was stirred at reflux for 5 h. The reaction mixture was diluted with water (3 mL) and extracted with ethyl acetate (3×3 mL). The combined organic layers were washed with brine (5 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Phenomenex Luna C18 150×25 mm×10 μm; mobile phase: [water (HCl)—ACN]; gradient: 2-32% B over 10 min) and lyophilised to give the title compound as a yellow solid. Y=16%. 1H NMR (400 MHz, DMSO-d6) δ 12.10-10.37 (m, 2H), 8.50-8.48 (m, 2H), 7.44-6.76 (m, 3H), 5.94-5.37 (m, 2H), 5.23-4.81 (m, 1H), 3.43-3.33 (m, 1H), 3.30-2.98 (m, 5H), 2.93-2.67 (m, 4H), 2.38-2.00 (m, 5H). 1H NMR (400 MHz, DMSO-d6+D2O) δ 8.73-8.27 (m, 2H), 7.23-6.73 (m, 3H), 5.79-5.33 (m, 2H), 5.07-4.64 (m, 1H), 3.64-3.52 (m, 2H), 3.29-3.01 (m, 4H), 2.94-2.66 (m, 4H), 2.39-1.95 (m, 5H). 1H NMR (400 MHz, DMSO-d6+D2O, T=353 K) δ 8.47-8.15 (m, 2H), 7.05-6.88 (m, 3H), 5.50 (s, 2H), 5.01-4.65 (m, 1H), 3.73-3.71 (m, 2H), 3.29-2.74 (m, 8H), 2.41-2.10 (m, 2H), 2.05 (s, 3H). LCMS (ESI): m/z: [M+H]+=414.0.
Synthesised in an analogous way to Compound 61, starting from (3R)-N,1-dimethylpyrrolidin-3-amine. 1H NMR (400 MHz, DMSO-d6) δ 11.68-10.55 (m, 2H), 8.70-8.25 (m, 2H), 7.09-6.75 (m, 3H), 5.81-5.43 (m, 2H), 5.15-4.85 (m, 1H), 3.64-3.54 (m, 2H), 3.23-3.01 (m, 4H), 2.91-2.68 (m, 4H), 2.35-2.02 (m, 5H). 1H NMR (400 MHz, DMSO-d6+D2O) δ 8.52-8.29 (m, 2H), 7.10-6.83 (m, 3H), 5.49 (s, 2H), 5.16-4.63 (m, 1H), 3.66-3.50 (m, 2H), 3.29-2.99 (m, 4H), 2.96-2.64 (m, 4H), 2.39-1.92 (m, 5H). 1H NMR (400 MHz, DMSO-d6+D2O, T=353 K) δ 8.36-8.24 (m, 2H), 7.06-6.86 (m, 3H), 5.49 (s, 2H), 5.06-4.66 (m, 1H), 3.79-3.51 (m, 2H), 3.17-3.14 (m, 4H), 2.85-2.82 (s, 4H), 2.42-2.00 (m, 5H). LCMS (ESI): m/z: [M+H]+=414.1.
Step 1. 4-({3-Chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}methyl)piperidine-1-carbaldehyde. To a solution of 3-chloro-7-(4-piperidylmethyl)pyrrolo[2,3-c]pyridazine (for synthesis see Compound 4)(100 mg, 399 μmol) in isopropyl formate (0.7 mL) and MeOH (1.5 mL) at 25° C. was added K2CO3 (83 mg, 598 μmol). The mixture was stirred at 50° C. under N2 for 2 h. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (0-100% ethyl acetate in petroleum ether) and lyophilised to give the title compound as a yellow gum. Y=88%.
Step 2. 4-{[3-(4-Chloro-2-hydroxy-6-methylphenyl)-7H-pyrrolo[2,3-c]pyridazin-7-yl]methyl}piperidine-1-carbaldehyde. To a solution of 4-[(3-chloropyrrolo[2,3-c]pyridazin-7-yl)methyl]piperidine-1-carbaldehyde (88 mg, 316 μmol) in dioxane (0.8 mL) and H2O (0.2 mL) at 25° C. under N2 were added 5-chloro-3-methyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenol (Intermediate B2, 102 mg, 379 μmol), K2CO3 (175 mg, 1.26 mmol) and dicyclohexyl-[2-(2,6-dimethoxyphenyl)phenyl]phosphane; methanesulfonate;(2-phenylanilino)palladium(1+) “SPhos Pd G3” (25 mg, 32 μmol). The mixture was stirred at 80° C. under N2 for 3 h. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was diluted with H2O (5 mL) and extracted with EtOAc (3×5 mL). The combined organic layers were washed with brine (5 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (0-100% ethyl acetate (10% MeOH)/petroleum ether), then further purified by prep-HPLC (column: Phenomenex Luna C18 150×25 mm×10 μm; mobile phase: [water (HCl)—ACN]; gradient: 15-35% B over 10 min), and then lyophilised to give the title compound as a yellow solid. Y=45%. 1H NMR (400 MHz, DMSO-d6) δ 11.03 (br. s, 1H), 8.73 (d, J=3 Hz, 1H), 8.64 (s, 1H), 7.98 (s, 1H), 7.12-7.10 (m, 2H), 7.01 (d, J=1 Hz, 1H), 4.42 (d, J=7 Hz, 2H), 4.19-4.15 (m, 1H), 3.71-3.68 (m, 1H), 3.05-2.95 (m, 1H), 2.65-2.50 (m, 1H), 2.36-2.22 (m, 1H), 2.10 (s, 3H), 1.65-1.58 (m, 2H), 1.29-1.06 (m, 2H). 1H NMR (400 MHz, DMSO-d6+D2O) δ 8.70 (d, J=3 Hz, 1H), 8.63 (s, 1H), 7.95 (s, 1H), 7.11 (d, J=3 Hz, 1H), 7.03-6.99 (m, 2H), 4.40 (d, J=7 Hz, 2H), 4.18-4.16 (m, 1H), 3.64-3.62 (m, 1H), 3.05-2.95 (m, 1H), 2.65-2.50 (m, 1H), 2.36-2.21 (m, 1H), 2.09 (s, 3H), 1.64-1.57 (m, 2H), 1.28-1.04 (m, 2H). 1H NMR (400 MHz, MeOD) δ 8.60 (d, J=3 Hz, 1H), 8.56 (s, 1H), 8.01 (s, 1H), 7.12 (d, J=3 Hz, 1H), 7.01 (d, J=1 Hz, 1H), 6.93 (d, J=1 Hz, 1H), 4.50 (d, J=7 Hz, 2H), 4.41-4.30 (m, 1H), 3.83-3.70 (m, 1H), 3.20-3.05 (m, 1H), 2.75-2.60 (m, 1H), 2.48-2.36 (m, 1H), 2.17 (s, 3H), 1.82-1.66 (m, 2H), 1.43-1.21 (m, 2H). LCMS (ESI): m/z: [M+H]+=385.0.
Step 1. 4-({3-Chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}methyl)-1-methylpyrrolidin-2-one. To a solution of 3-chloro-7H-pyrrolo[2,3-c]pyridazine (Intermediate A1, 500 mg, 3.26 mmol), 4-(hydroxymethyl)-1-methyl-pyrrolidin-2-one (631 mg, 4.88 mmol) and PPh3 (1.71 g, 6.51 mmol) in THE (10 mL) at 0° C. was added diethyl azodicarboxylate (1.18 mL, 6.51 mmol). The mixture was stirred at 25° C. for 1.5 h. The reaction mixture was filtered and the filtrate concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: CD05-Phenomenex Luna C18 150×40×10 μm; mobile phase: [water (HCOOH)—ACN]; gradient: 3-33% B over 11 min) and lyophilised to give the title compound as a yellow solid. Y=30%. 1H NMR (400 MHz, DMSO-d6) δ 8.13 (d, J=3 Hz, 1H), 8.05 (s, 1H), 6.60 (d, J=3 Hz, 1H), 4.48-4.45 (m, 2H), 3.38-3.36 (m, 1H), 3.20-3.17 (m, 1H), 3.15-2.99 (m, 1H), 2.67 (s, 3H), 2.37-2.31 (m, 1H), 2.16-2.10 (m, 1H).
Step 2. 4-{[3-(4-Chloro-2-hydroxy-6-methylphenyl)-7H-pyrrolo[2,3-c]pyridazin-7-yl]methyl}-1-methylpyrrolidin-2-one. To a solution of 4-({3-chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}methyl)-1-methylpyrrolidin-2-one (130 mg, 491 μmol) in dioxane (1.2 mL) and H2O (0.24 mL) at 25° C. under N2 were added 5-chloro-3-methyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenol (Intermediate B2, 198 mg, 737 μmol), K2CO3 (271 mg, 1.96 mmol) and SPhos Pd G3 (38 mg, 49 μmol). The mixture was stirred at 80° C. under N2 for 2 h. The reaction mixture was filtered and the filtrate concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: CD02-Waters Xbridge BEH C18 150×25×10 μm; mobile phase: [water (NH4HCO3)—ACN]; gradient: 22-52% B over 10 min) and lyophilised to give the title compound as an off-white solid. Y=36%. 1H NMR (400 MHz, DMSO-d6) δ 9.82 (br. s, 1H), 8.01 (d, J=3 Hz, 1H), 7.76 (s, 1H), 6.87-6.85 (m, 2H), 6.58 (d, J=3 Hz, 1H), 4.50 (d, J=7 Hz, 2H), 3.43-3.37 (m, 1H), 3.26-3.22 (m, 1H), 3.12-2.99 (m, 1H), 2.68 (s, 3H), 2.37-2.34 (m, 1H), 2.21-2.18 (m, 1H), 1.98 (s, 3H). 1H NMR (400 MHz, DMSO-d6+D2O) δ 8.00 (d, J=3 Hz, 1H), 7.76 (s, 1H), 6.89-6.84 (m, 2H), 6.59 (d, J=3 Hz, 1H), 4.50 (d, J=7 Hz, 2H), 3.38 (s, 1H), 3.26-3.23 (m, 1H), 3.13-3.00 (m, 1H), 2.68 (s, 3H), 2.45-2.30 (m, 1H), 2.25-2.10 (m, 1H), 1.97 (s, 3H). LCMS (ESI): m/z: [M+H]+=371.0.
Synthesised in an analogous way to Compound 64, starting from 5-(hydroxymethyl)-1-methyl-pyrrolidin-2-one. 1H NMR (400 MHz, DMSO-d6) δ 9.85 (s, 1H), 7.89 (d, J=3 Hz, 1H), 7.78 (s, 1H), 6.92-6.76 (m, 2H), 6.61 (d, J=3 Hz, 1H), 4.80-4.75 (m, 1H), 4.55-4.49 (m, 1H), 4.12-4.10 (m, 1H), 2.78 (s, 3H), 2.03-2.00 (m, 2H), 1.97 (s, 3H), 1.93-1.77 (m, 2H). 1H NMR (400 MHz, DMSO-d6+D2O) δ 7.85 (d, J=3 Hz, 1H), 7.77 (s, 1H), 6.92-6.78 (m, 2H), 6.62 (d, J=3 Hz, 1H), 4.78-4.73 (m, 1H), 4.54-4.48 (m, 1H), 4.11-4.09 (m, 1H), 2.77 (s, 3H), 2.07-1.73 (m, 7H). LCMS (ESI): m/z: [M+H]+=371.1.
Synthesised in an analogous way to Compound 64, starting from 3-(hydroxymethyl)-1-methyl-pyrrolidin-2-one. 1H NMR (400 MHz, DMSO-d6) δ 9.83 (br. s, 1H), 7.88 (d, J=3 Hz, 1H), 7.76 (s, 1H), 6.89-6.84 (m, 2H), 6.58 (d, J=3 Hz, 1H), 4.83 (dd, J=14, 5 Hz, 1H), 4.46 (dd, J=14, 8 Hz, 1H), 3.27-3.18 (m, 2H), 3.13-3.02 (m, 1H), 2.76 (s, 3H), 2.12-1.89 (m, 4H), 1.85-1.63 (m, 1H). 1H NMR (400 MHz, DMSO-d6+D2O) δ 7.80 (d, J=3 Hz, 1H), 7.74 (s, 1H), 6.85-6.83 (m, 2H), 6.58 (d, J=3 Hz, 1H), 4.80 (dd, J=14, 5 Hz, 1H), 4.43 (dd, J=14, 8 Hz, 1H), 3.27-3.03 (m, 3H), 2.72 (s, 3H), 2.05-1.92 (m, 4H), 1.78-1.63 (m, 1H). LCMS (ESI): m/z: [M+H]+=371.0.
Step 1. 4-{3-Chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}-1-methylpiperidine hydrochloride. To a solution of 3-chloro-7H-pyrrolo[2,3-c]pyridazine (Intermediate A1, 0.30 g, 1.95 mmol) in toluene (3 mL) at 25° C. were added 1-methylpiperidin-4-ol (503 μL, 4.30 mmol), diisopropyl azodicarboxylate (568 μL, 2.93 mmol) and triphenylphosphine (769 mg, 2.93 mmol). The mixture was stirred at 50° C. under N2 for 12 h. The reaction mixture was filtered and the filtrate concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Phenomenex Luna C18 100×40 mm×5 μm; mobile phase: [H2O (0.04% HCl)—ACN]; gradient: 5-20% B over 8.0 min) and lyophilised to give the title compound as a yellow solid. Y=11%. 1H NMR (400 MHz, DMSO-d6) δ 10.83 (s, 1H), 8.11-7.97 (m, 2H), 6.70-6.58 (m, 1H), 5.15-5.04 (m, 1H), 3.58-3.55 (m, 2H), 3.34-3.20 (m, 2H), 2.79 (d, J=5 Hz, 3H), 2.55-2.51 (m, 2H), 2.29-2.25 (m, 2H).
Step 2. 5-Chloro-3-methyl-2-[7-(I-methylpiperidin-4-yl)-7H-pyrrolo[2,3-c]pyridazin-3-yl]phenol. To a solution of 4-{3-chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}-1-methylpiperidine hydrochloride (50 mg, 174 μmol) in dioxane (0.6 mL) and H2O (0.2 mL) at 25° C. were added 5-chloro-3-methyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenol (Intermediate B2, 70 mg, 261 μmol), K2CO3 (72 mg, 522 μmol) and SPhos Pd G3 (14 mg, 17 μmol). The mixture was stirred at reflux under N2 for 3 h. The reaction mixture was diluted with H2O (1 mL) and the resulting mixture extracted with EtOAc (3×1 mL). The combined organic layers were washed with brine (3 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Waters Xbridge BEH C18 100×30 mm×10 μm; mobile phase: [H2O (10 mM NH4HCO3)—ACN]; gradient: 30-60% B over 8.0 min) and lyophilised to give the title compound as an off-white solid. Y=32%. 1H NMR (400 MHz, DMSO-d6) δ 9.84 (s, 1H), 8.08 (d, J=3 Hz, 1H), 7.74 (s, 1H), 6.89-6.82 (m, 2H), 6.59 (d, J=3 Hz, 1H), 4.98-4.82 (m, 1H), 3.01-2.98 (m, 2H), 2.30 (s, 3H), 2.27-2.19 (m, 4H), 2.06-2.00 (m, 2H), 1.98 (s, 3H). 1H NMR (400 MHz, DMSO-d6+D2O) δ 8.04 (d, J=3 Hz, 1H), 7.74 (s, 1H), 6.89-6.82 (m, 2H), 6.59 (d, J=3 Hz, 1H), 4.99-4.76 (m, 1H), 3.00-2.97 (m, 2H), 2.28 (s, 3H), 2.26-2.12 (m, 4H), 2.05-1.99 (m, 2H), 1.96 (s, 3H). LC-MS (ESI): m/z: [M+H]+=357.2.
Step 1. (4-Cyano-2-hydroxyphenyl)boronic acid. To a solution of (4-cyano-2-hydroxy-phenyl)boronic acid (583 mg, 3.58 mmol) in DCM (4.8 mL) at 0° C. was added 2 M BBr3 in DCM (3.58 mL, 7.16 mmol). The mixture was stirred at 25° C. under N2 for 2 h. The reaction mixture was quenched by addition of H2O (5 mL) and filtered. The filter cake was dried under reduced pressure to give the crude product. The crude product was further triturated with EtOAc (5 mL) for 10 min, filtered, then the filter cake dried under reduced pressure to give the title compound as a white solid.
Step 2. 3-Hydroxy-4-{7-[(I-methylpiperidin-4-yl)methyl]-7H-pyrrolo[2,3-c]pyridazin-3-yl}benzonitrile. To a solution of (4-cyano-2-hydroxy-phenyl)boronic acid (148 mg, 907 μmol) in dioxane (1.2 mL) and H2O (0.4 mL) at 25° C. under N2 were added K2CO3 (188 mg, 1.36 mmol), SPhos Pd G3 (35 mg, 45 μmol) and 3-chloro-7-[(1-methyl-4-piperidyl)methyl]pyrrolo[2,3-c]pyridazine (for synthesis see Compound 4)(0.12 g, 453 μmol). The mixture was stirred at 80° C. under N2 for 12 h. The reaction mixture was diluted with H2O (3 mL) and extracted with EtOAc (3×3 mL). The combined organic layers were washed with brine (9 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Phenomenex Luna C18 100×40 mm×5 μm; mobile phase: [H2O (0.04% HCl)—ACN]; gradient: 10-25% B over 8.0 min) and lyophilised to give the crude product. This was further purified by prep-HPLC (column: Waters Xbridge BEH C18 100×30 mm×10 μm; mobile phase: [H2O (10 mM NH4HCO3)—ACN]; gradient: 25-65% B over 8.0 min) and lyophilised to give the title as a white solid. Y=4%. 1H NMR (400 MHz, DMSO-d6) δ 14.12 (br. s, 1H), 8.80 (s, 1H), 8.24 (d, J=8 Hz, 1H), 8.14 (d, J=3 Hz, 1H), 7.48-7.34 (m, 2H), 6.75 (d, J=3 Hz, 1H), 4.36 (d, J=7 Hz, 2H), 2.79-2.75 (m, 2H), 2.13 (s, 3H), 2.01-1.89 (m, 1H), 1.86-1.75 (m, 2H), 1.50-1.40 (m, 2H), 1.36-1.24 (m, 2H). 1H NMR (400 MHz, DMSO-d6+D2O) δ 8.76 (s, 1H), 8.22 (d, J=8 Hz, 1H), 8.11 (d, J=3 Hz, 1H), 7.47-7.36 (m, 2H), 6.75 (d, J=3 Hz, 1H), 4.36 (d, J=7 Hz, 2H), 2.75-2.72 (m, 2H), 2.12 (s, 3H), 1.99-1.90 (m, 1H), 1.87-1.71 (m, 2H), 1.50-1.40 (m, 2H), 1.36-1.26 (m, 2H). LC-MS (ESI): m/z: [M+H]+=348.3.
Step 1. (3S)-3-[(Tert-butyldimethylsilyl)oxy]pyrrolidin-2-one. To a solution of (3S)-3-hydroxypyrrolidin-2-one (1.0 g, 9.89 mmol) in DMF (5 mL) at 25° C. under N2 were added TBSCl (1.70 mL, 13.9 mmol), imidazole (808 mg, 11.9 mmol) and DMAP (121 mg, 989 μmol). The mixture was stirred at 40° C. for 2 h. The reaction mixture was diluted with water (10 mL) and extracted with ethyl acetate (3×15 mL). The combined organic layers were washed with brine (15 mL), dried over sodium sulfate, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether/ethyl acetate=100/1 to 30/1) to give the title compound as a white solid. Y=94%. 1H NMR (400 MHz, DMSO-d6) δ 7.70 (s, 1H), 4.19 (t, J=8 Hz, 1H), 3.21-3.02 (m, 2H), 2.35-2.22 (m, 1H), 1.85-1.70 (m, 1H), 0.87 (s, 9H), 0.08 (s, 6H).
Step 2. (3S)-3-[(Tert-butyldimethylsilyl)oxy]-1-methylpyrrolidin-2-one. To a solution of (3S)-3-[tert-butyl(dimethyl)silyl]oxypyrrolidin-2-one (1.5 g, 6.96 mmol) in DMF (10 mL) at 0° C. under N2 was added 60% NaH in mineral oil (334 mg, 8.36 mmol). The reaction was stirred at 0° C. for 30 min, then treated with CH3I (650 μL, 10.5 mmol). The mixture was stirred at 25° C. under N2 for 1 h. The reaction mixture was diluted with water (10 mL) and extracted with ethyl acetate (3×15 mL). The combined organic layers were washed with brine (15 mL), dried over sodium sulfate, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether/ethyl acetate=50/1 to 5/1) to give the title compound as a white solid. Y=91%. 1H NMR (400 MHz, DMSO-d6) δ 4.26 (t, J=8 Hz, 1H), 3.29-3.14 (m, 2H), 2.71 (s, 3H), 2.35-2.21 (m, 1H), 1.80-1.65 (m, 1H), 0.87 (s, 9H), 0.09 (d, J=2 Hz, 6H).
Step 3. (3S)-3-Hydroxy-1-methylpyrrolidin-2-one. A solution of (3S)-3-[tert-butyl(dimethyl)silyl]oxy-1-methyl-pyrrolidin-2-one (1.0 g, 4.36 mmol) in 2 M HCl in MeOH (5 mL) was stirred at 25° C. for 1 h. The reaction mixture was concentrated to give the title compound as a white solid. Y=quantitative. 1H NMR (400 MHz, DMSO-d6) δ 4.04 (t, J=8 Hz, 1H), 3.30-3.05 (m, 2H), 2.70 (s, 3H), 2.30-2.15 (m, 1H), 1.75-1.55 (m, 1H).
Step 4. (3R)-3-{3-Chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}-1-methylpyrrolidin-2-one. To a solution of (3S)-3-hydroxy-1-methyl-pyrrolidin-2-one (375 mg, 3.26 mmol) and 3-chloro-7H-pyrrolo[2,3-c]pyridazine (Intermediate A1, 500 mg, 3.26 mmol) in THE (5 mL) under N2 were added diethyl azodicarboxylate (710 μL, 3.91 mmol) and triphenylphosphine (1.28 g, 4.88 mmol). The mixture was stirred at 25° C. for 12 h. The reaction mixture was diluted with water (10 mL) and extracted with ethyl acetate (3×15 mL). The combined organic layers were washed with brine (15 mL), dried over sodium sulfate, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: CD05—Phenomenex Luna C18 150×40×10 μm; mobile phase: [water (HCOOH)—ACN]; gradient: 5-35% B over 11 min) and then further purified by column chromatography (SiO2, 33-100% ethyl acetate in petroleum ether) to give the title compound as a white solid. Y=45%. 1H NMR (400 MHz, CDCl3) δ 7.69 (s, 1H), 7.58 (d, J=3 Hz, 1H), 6.49 (d, J=3 Hz, 1H), 5.58 (t, J=9 Hz, 1H), 3.70-3.63 (m, 1H), 3.61-3.53 (m, 1H), 3.02 (s, 3H), 2.92-2.76 (m, 1H), 2.50-2.30 (m, 1H).
Step 5. (3R)-3-[3-(4-Chloro-2-hydroxy-6-methylphenyl)-7H-pyrrolo[2,3-c]pyridazin-7-yl]-1-methylpyrrolidin-2-one. To a solution of (3R)-3-{3-chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}-1-methylpyrrolidin-2-one (130 mg, 519 μmol) and 5-chloro-3-methyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenol (Intermediate B2, 181 mg, 674 μmol) in dioxane (1 mL) and H2O (0.2 mL) under N2 were added K2CO3 (215 mg, 1.56 mmol) and SPhos Pd G3 (40 mg, 52 μmol). The mixture was stirred at 80° C. under N2 for 2 h. The reaction mixture was diluted with water (5 mL) and extracted with ethyl acetate (3×5 mL). The combined organic layers were washed with brine (5 mL), dried over sodium sulfate, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: CD02—Waters Xbridge BEH C18 150×25×10 μm; mobile phase: [water (NH4HCO3)—ACN]; gradient: 20-50% B over 10 min) and lyophilised to give the title as an off-white solid. Y=24%. 1H NMR (400 MHz, DMSO-d6) δ 9.85 (s, 1H), 7.92 (d, J=3 Hz, 1H), 7.77 (s, 1H), 6.92-6.81 (m, 2H), 6.61 (d, J=3 Hz, 1H), 5.74 (t, J=10 Hz, 1H), 3.66-3.45 (m, 2H), 2.88 (s, 3H), 2.72-2.53 (m, 2H), 1.98 (s, 3H). 1H NMR (400 MHz, DMSO-d6+D2O) δ 7.85 (d, J=3 Hz, 1H), 7.75 (s, 1H), 6.86-6.82 (m, 2H), 6.63 (d, J=3 Hz, 1H), 5.68 (t, J=10 Hz, 1H), 3.63-3.43 (m, 2H), 2.86 (s, 3H), 2.70-2.59 (m, 1H), 2.56-2.51 (m, 0.5H), 2.48-2.43 (m, 0.5H), 1.95 (s, 3H). 1H NMR (400 MHz, MeOD) δ 7.84-7.82 (m, 2H), 6.86-6.80 (m, 2H), 6.67 (d, J=3 Hz, 1H), 5.74 (t, J=9 Hz, 1H), 3.80-3.57 (m, 2H), 3.01 (s, 3H), 2.86-2.72 (m, 1H), 2.68-2.56 (m, 1H), 2.03 (s, 3H). LCMS (ESI): m/z: [M+H]+=357.0.
Step 1. 5-({3-Chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}methyl)-1-methylpiperidin-2-one. To a solution of 3-chloro-7H-pyrrolo[2,3-c]pyridazine (Intermediate A1, 30 mg, 195 μmol) in toluene (1 mL) at 0° C. were added 5-(hydroxymethyl)-1-methyl-piperidin-2-one (28 mg, 195 μmol), triphenylphosphine (77 mg, 293 μmol) and diisopropyl azodicarboxylate (57 μL, 293 μmol). The resulting mixture was stirred at 25° C. for 12 h. The solution was concentrated under vacuum to give a residue. The residue was purified by prep-HPLC (column: Phenomenex Luna C18 100×40 mm×5 μm; mobile phase: [H2O (0.04% HCl)—ACN]; gradient: 15-55% B over 8.0 min) and lyophilised to give the title compound as a yellow solid. Y=18%. 1H NMR (400 MHz, DMSO-d6) δ 8.09 (d, J=3 Hz, 1H), 8.05 (s, 1H), 6.61 (d, J=3 Hz, 1H), 4.42 (d, J=7 Hz, 2H), 3.15-3.09 (m, 2H), 2.74 (s, 3H), 2.36-2.15 (m, 2H), 1.75-1.64 (m, 1H), 1.58-1.45 (m, 1H), 1.26-1.10 (m, 1H).
Step 2. 5-{[3-(4-Chloro-2-hydroxy-6-methylphenyl)-7H-pyrrolo[2,3-c]pyridazin-7-yl]methyl}-1-methylpiperidin-2-one. To a solution of 5-({3-chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}methyl)-1-methylpiperidin-2-one (100 mg, 359 μmol) and 5-chloro-3-methyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenol (Intermediate B2, 193 mg, 718 μmol) in dioxane (5 mL) and H2O (1 mL) at 25° C. under N2 were added K2CO3 (149 mg, 1.08 mmol) and dicyclohexyl-[2-(2,6-dimethoxyphenyl)phenyl]phosphane; methanesulfonate;(2-phenylanilino)palladium(1+) “SPhos Pd G3” (42 mg, 54 μmol). The mixture was stirred at 80° C. for 8 h. The reaction mixture was poured into water (5 mL) and the resulting mixture extracted with EtOAc (3×5 mL). The combined organic phase was washed with brine (5 mL), dried with anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Phenomenex Luna C18 100×40 mm×5 μm; mobile phase: [H2O (0.04% HCl)—ACN]; gradient: 20-30% B over 8.0 min) and lyophilised to give the title compound as a yellow solid. Y=21%. 1H NMR (400 MHz, DMSO-d6) δ 10.85 (s, 1H), 8.71 (d, J=3 Hz, 1H), 8.60 (s, 1H), 7.09 (d, J=3 Hz, 1H), 7.04 (s, 1H), 7.02 (s, 1H), 4.51 (d, J=7 Hz, 2H), 3.29-3.14 (m, 2H), 2.78 (s, 3H), 2.65-2.55 (m, 1H), 2.38-2.18 (m, 2H), 2.10 (s, 3H), 1.90-1.75 (m, 1H), 1.69-1.52 (m, 1H). 1H NMR (400 MHz, DMSO-d6+D2O) δ 8.70 (d, J=3 Hz, 1H), 8.62 (s, 1H), 7.11 (d, J=3 Hz, 1H), 7.04 (d, J=2 Hz, 1H), 6.98 (d, J=2 Hz, 1H), 4.50 (d, J=7 Hz, 2H), 3.31-3.12 (m, 2H), 2.77 (s, 3H), 2.64-2.55 (m, 1H), 2.36-2.16 (m, 2H), 2.10 (s, 3H), 1.86-1.73 (m, 1H), 1.66-1.52 (m, 1H). LCMS (ESI): m/z: [M+H]+=385.3.
Step 1. 6-Chloro-N3-[(1-methylpiperidin-4-yl)methyl]pyridazine-3,4-diamine. To a solution of 3,6-dichloropyridazin-4-amine (3.0 g, 18.3 mmol) in decan-1-ol (30 mL) were added (1-methyl-4-piperidyl)methanamine (2.81 g, 22.0 mmol) and DIPEA (6.37 mL, 36.6 mmol). The reaction was stirred at 140° C. under N2 for 48 h. The reaction mixture was purified by prep-HPLC (column: Waters Xbridge BEH C18 250×70 mm×10 μm; mobile phase: [H2O (0.05% NH4OH)—ACN]; gradient: 0-30% B over 20 min) and lyophilised to give the title compound as yellow oil. Y=21%. 1H NMR (400 MHz, DMSO-d6) δ 6.36 (s, 1H), 6.27 (s, 2H), 6.03 (t, J=5 Hz, 1H), 3.24-3.21 (m, 2H), 2.73-2.70 (m, 2H), 2.13 (s, 3H), 1.79-1.76 (m, 2H), 1.70-1.65 (m, 3H), 1.20-1.13 (m, 2H).
Step 2. 4-({3-Chloro-7H-imidazo[4,5-c]pyridazin-7-yl}methyl)-1-methylpiperidine. To a solution of 6-chloro-N3-[(1-methyl-4-piperidyl)methyl]pyridazine-3,4-diamine (0.30 g, 1.17 mmol) in diethoxymethoxyethane (10 mL) at 25° C. under N2 was added aqueous 4 M HCl (147 L, 588 μmol). The mixture was stirred at 100° C. under N2 for 12 h. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Waters Xbridge Prep OBD C18 150×40 mm×10 μm; mobile phase: [H2O (0.05% NH4OH+10 mM NH4HCO3)—ACN]; gradient: 1-30% B over 8.0 min) and lyophilised to give the title compound as a white solid. Y=32%. 1H NMR (400 MHz, DMSO-d6) δ 8.88 (s, 1H), 8.25 (s, 1H), 4.31 (d, J=7 Hz, 2H), 2.72 (d, J=12 Hz, 2H), 2.11 (s, 3H), 2.02-1.88 (m, 1H), 1.81-1.71 (m, 2H), 1.50-1.40 (m, 2H), 1.33-1.20 (m, 2H).
Step 3. 5-Chloro-3-methyl-2-{7-[(I-methylpiperidin-4-yl)methyl]-7H-imidazo[4,5-c]pyridazin-3-yl}phenol. To a solution of 4-({3-chloro-7H-imidazo[4,5-c]pyridazin-7-yl}methyl)-1-methylpiperidine (88 mg, 331 μmol) in dioxane (3 mL) and H2O (0.6 mL) at 25° C. under N2 were added K2CO3 (137 mg, 993 μmol), SPhos Pd G3 (26 mg, 33 μmol) and 5-chloro-3-methyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenol (Intermediate B2, 89 mg, 331 μmol). The mixture was stirred at 80° C. under N2 for 0.5 h. The reaction mixture was diluted with H2O (3 mL) and extracted with EtOAc (3×3 mL). The combined organic layers were washed with brine (9 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Waters Xbridge BEH C18 100×30 mm×10 μm; mobile phase: [H2O (10 mM NH4HCO3)—ACN]; gradient: 5-50% B over 8.0 min) and lyophilised to give the title compound as an off-white solid. Y=24%. 1H NMR (400 MHz, DMSO-d6) δ 9.21 (br. s, 1H), 8.79 (s, 1H), 7.90 (s, 1H), 6.87 (d, J=8 Hz, 2H), 4.35 (d, J=7 Hz, 2H), 2.75 (d, J=12 Hz, 2H), 2.13 (s, 3H), 2.09-2.00 (m, 1H), 1.98 (s, 3H), 1.90-1.75 (m, 2H), 1.54-1.51 (m, 2H)), 1.34-1.30 (m, 2H). 1H NMR (400 MHz, DMSO-d6+D2O) δ 8.78 (s, 1H), 7.89 (s, 1H), 6.87 (d, J=8 Hz, 2H), 4.35 (d, J=7 Hz, 2H), 2.74 (d, J=12 Hz, 2H), 2.12 (s, 3H), 2.07-1.99 (m, 1H), 1.98 (s, 3H), 1.85-1.75 (m, 2H), 1.53-1.50 (m, 2H), 1.34-1.30 (m, 2H). LC-MS (ESI): m/z: [M+H]+=372.3.
Step 1. Tert-butyl 4-{[(4-amino-6-chloropyridazin-3-yl)amino]methyl}piperidine-1-carboxylate. To a solution of 3,6-dichloropyridazin-4-amine (5.0 g, 30.5 mmol) in decan-1-ol (8.73 mL, 45.7 mmol) was added tert-butyl 4-(aminomethyl)piperidine-1-carboxylate (7.19 g, 33.5 mmol) and DIPEA (15.9 mL, 91.5 mmol). The reaction mixture was stirred at 140° C. for 48 h in a sealed tube. The reaction mixture was concentrated under reduced pressure and the residue purified by prep-HPLC (column: Welch Xtimate C18 250×100 mm×10 μm; mobile phase: [H2O (0.05% NH3H2O+10 mM NH4HCO3)—ACN]; gradient: 10-45% B over 20 min) and lyophilised to give the title compound as yellow solid.
Step 2. Tert-butyl 4-({3-chloro-7H-imidazo[4,5-c]pyridazin-7-yl}methyl)piperidine-1-carboxylate. To a solution of tert-butyl 4-{[(4-amino-6-chloropyridazin-3-yl)amino]methyl}piperidine-1-carboxylate (3.4 g, 9.95 mmol) in triethoxymethane (70 mL) was added 4 M HCl in EtOAc (1.24 mL, 5.0 mmol). The solution was stirred at 100° C. under N2 for 12 h. The reaction mixture was diluted with H2O (20 mL) and extracted with EtOAc (3×20 mL). The combined organic layers were washed with brine (50 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Welch Xtimate C18 250×70 mm 10 μm; mobile phase: [H2O (10 mM NH4HCO3)—ACN]; gradient: 25-50% B over 20 min) and lyophilised to give the title compound as a yellow solid. Y=60%. 1H NMR (400 MHz, DMSO-d6) δ 8.88 (s, 1H), 8.29 (s, 1H), 4.34 (d, J=7 Hz, 2H), 3.95-3.92 (m, 2H), 2.72-2.60 (m, 2H), 2.27-2.15 (m, 1H), 1.53-1.49 (m, 2H), 1.40 (s, 9H), 1.20-1.07 (m, 2H).
Step 3. 4-({3-Chloro-7H-imidazo[4,5-c]pyridazin-7-yl}methyl)piperidine hydrochloride. A mixture of tert-butyl 4-({3-chloro-7H-imidazo[4,5-c]pyridazin-7-yl}methyl)piperidine-1-carboxylate (1.5 g, 4.26 mmol) and aqueous 4 M HCl (10 mL) was stirred at 25° C. for 1 h. The reaction mixture was filtered and the filter cake dried under reduced pressure to give the title compound as a white solid. Y=81%. 1H NMR (400 MHz, DMSO-d6) δ 9.07 (br. s, 1H), 8.94 (s, 1H), 8.86 (br., 1H), 8.28 (s, 1H), 4.36-4.30 (m, 2H), 3.55-3.35 (m, 2H), 2.88-2.72 (m, 2H), 2.36-2.24 (m, 1H), 1.70-1.67 (m, 2H), 1.56-1.42 (m, 2H).
Step 4. 1-[4-({3-Chloro-7H-imidazo[4,5-c]pyridazin-7-yl}methyl)piperidin-1-yl]ethan-1-one. To a solution of 4-({3-chloro-7H-imidazo[4,5-c]pyridazin-7-yl}methyl)piperidine hydrochloride (150 mg, 521 μmol) in DCM (3 mL) at 0° C. were added DIPEA (311 μL, 1.79 mmol) and acetyl chloride (55 μL, 775 μmol). The resulting mixture was stirred at 25° C. for 1 h. The solution was diluted with H2O (2 mL) and extracted with DCM (3×2 mL). The combined organic layers were washed with brine (3×2 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give the title compound as a yellow oil. Y=86%.
Step 5. 1-(4-{[3-(4-Chloro-2-hydroxy-6-methylphenyl)-7H-imidazo[4,5-c]pyridazin-7-yl]methyl}piperidin-1-yl)ethan-1-one. To a solution of 1-[4-({3-chloro-7H-imidazo[4,5-c]pyridazin-7-yl}methyl)piperidin-1-yl]ethan-1-one (150 mg, 511 μmol) in dioxane (3 mL) and H2O (0.6 mL) at 25° C. under N2 were added 5-chloro-3-methyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenol (Intermediate B2, 137 mg, 511 μmol), dicyclohexyl-[2-(2,6-dimethoxyphenyl)phenyl]phosphane; methanesulfonate;(2-phenylanilino) palladium(1+) “SPhos Pd G3” (60 mg, 77 μmol) and K2CO3 (282 mg, 2.04 mmol). The resulting mixture was stirred at 80° C. under N2 for 1 h. The solution was diluted with H2O (3 mL) and extracted with EtOAc (3×3 mL). The combined organic layers were washed with brine (3×3 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Phenomenex Luna C18 100×40 mm×5 μm; mobile phase: [H2O (0.04% HCl)—ACN]; gradient: 25-35% B over 8.0 min) and lyophilised to give the title compound as a yellow solid. Y=36%. 1H NMR (400 MHz, DMSO-d6) δ 10.78 (br. s, 1H), 9.26 (s, 1H), 8.57 (s, 1H), 7.05 (s, 1H), 6.98 (s, 1H), 4.41-4.34 (m, 3H), 3.83-3.79 (m, 1H), 3.03-2.95 (m, 1H), 2.49-2.45 (m, 1H), 2.36-2.23 (m, 1H), 2.08 (s, 3H), 1.98 (s, 3H), 1.72-1.58 (m, 2H), 1.33-1.10 (m, 2H). 1H NMR (400 MHz, DMSO-d6+D2O) δ 9.23 (s, 1H), 8.56 (s, 1H), 6.98 (s, 1H), 6.96 (s, 1H), 4.41-4.32 (m, 3H), 3.84-3.82 (m, 1H), 3.03-2.92 (m, 1H), 2.49-2.44 (m, 1H), 2.34-2.21 (m, 1H), 2.07 (s, 3H), 1.97 (s, 3H), 1.70-1.58 (m, 2H), 1.31-1.08 (m, 2H). 1H NMR (400 MHz, MeOD) δ 9.32 (s, 1H), 8.73 (s, 1H), 7.03 (s, 1H), 6.96 (s, 1H), 4.63-4.51 (m, 1H), 4.50 (d, J=7 Hz, 2H), 4.06-3.91 (m, 1H), 3.19-3.07 (m, 1H), 2.69-2.59 (m, 1H), 2.48-2.35 (m, 1H), 2.20 (s, 3H), 2.11 (s, 3H), 1.87-1.73 (m, 2H), 1.48-1.26 (m, 2H).
LCMS (ESI): m/z: [M+H]+=400.3.
To a solution of (4-cyano-2-hydroxy-6-methyl-phenyl)boronic acid (Intermediate B3, 60 mg, 339 μmol) in dioxane (0.9 mL) and H2O (0.3 mL) at 25° C. under N2 were added K2CO3 (94 mg, 677 μmol) SPhos Pd G3 (18 mg, 23 μmol) and 4-({3-chloro-7H-imidazo[4,5-c]pyridazin-7-yl}methyl)-1-methylpiperidine (for synthesis see Compound 71)(0.06 g, 226 μmol). The mixture was stirred at 80° C. under N2 for 12 h. The reaction mixture was diluted with H2O (3 mL) and extracted with EtOAc (3×3 mL). The combined organic layers were washed with brine (9 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Waters Xbridge BEH C18 100×30 mm×10 μm; mobile phase: [H2O (10 mM NH4HCO3)—ACN]; gradient: 5-35% B over 8.0 min) and lyophilised to give the title compound as a white solid. Y=26%. 1H NMR (400 MHz, DMSO-d6) δ 8.81 (s, 1H), 7.95 (s, 1H), 7.24 (s, 1H), 7.13 (s, 1H), 4.48-4.22 (m, 2H), 2.81-2.68 (m, 2H), 2.12 (s, 3H), 2.07-1.89 (m, 4H), 1.85-1.69 (m, 2H), 1.60-1.43 (m, 2H), 1.42-1.25 (m, 2H). 1H NMR (400 MHz, DMSO-d6+D2O) δ 8.78 (s, 1H), 7.93 (s, 1H), 7.23 (s, 1H), 7.12 (s, 1H), 4.49-4.23 (m, 2H), 2.80-2.66 (m, 2H), 2.10 (s, 3H), 2.05-1.90 (m, 4H), 1.85-1.69 (m, 2H), 1.57-1.41 (m, 2H), 1.41-1.25 (m, 2H). 1H NMR (400 MHz, methanol-d4) δ 8.77 (s, 1H), 7.97 (s, 1H), 7.21 (s, 1H), 7.10 (s, 1H), 4.47 (d, J=7 Hz, 2H), 2.99-2.86 (m, 2H), 2.28 (s, 3H), 2.24-2.14 (m, 1H), 2.09 (s, 3H), 2.08-2.01 (m, 2H), 1.78-1.62 (m, 2H), 1.54-1.41 (m, 2H).
LC-MS (ESI): m/z: [M+H]+=363.3.
Step]. Tert-butyl (3R)-3-{[(4-amino-6-chloropyridazin-3-yl)amino]methyl}pyrrolidine-carboxylate. To a solution of 3,6-dichloropyridazin-4-amine (3.0 g, 18.3 mmol) in decan-ol (5.24 mL, 27.4 mmol) at 25° C. under N2 were added DIPEA (9.56 mL, 54.9 mmol) and tert-butyl (3R)-3-(aminomethyl)pyrrolidine-carboxylate (4.40 g, 22.0 mmol). The solution was stirred at 140° C. under N2 for 48 h. The reaction mixture was purified by prep-HPLC (column: Welch Xtimate C18 250×10 mm×10 m; mobile phase: [H2O (0.05 0 NH3H2O+10 M NH4HCO3)—ACN]; gradient: 15-42% B over 20min ) and lyophilised to give the title compound. Y=14%.
Step 2. Tert-butyl (3R)-3-({3-chloro-7H-imidazo[4,5-c-pyridazin-7-yl}methyl)pyrrolidine-1-carboxylate. To a solution of tert-butyl (3R)-3-c{[(4-amino-6-chloropyridazin-3-yl)amino]methyl}pyrrolidine-1-carboxylate (430 mg, 1.31 mmol) in triethoxymethane (9 mL) at 25° C. was added aqueous 4 M HCl (164 μL, 0.656 mmol). The solution was stirred at 100° C. under N2 for 24 h. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Waters Xbridge BEH C18 250×50 mm×10 μm; mobile phase: [H2O (10 mM NH4HCO3)—ACN]; gradient: 15-55 00 B over 10 min) and lyophilised to give the title compound as a white solid. Y=81% 1H NMR (400 MHz, DMSO-d6) δ 8.91 (s, 1H), 8.27 (s, 1H), 4.56-4.31 (m, 2H), 3.41-3.34 (m, 2H), 3.25-3.03 (m, 2H), 2.96-2.75 (m, 1H), 1.96-1.81 (m, 1H), 1.76-1.61 (m, 1H), 1.37 (s, 9H).
Step 3. (3S)-3-({3-Chloro-7H-imidazo[4, 5-c]pyridazin-7-yl}methyl)pyrrolidine hydrochloride. A mixture of tert-butyl (3R)-3-({3-chloro-7H-imidazo[4,5-c]pyridazin-7-yl}methyl)pyrrolidine-1-carboxylate (370 mg, 1.10 mmol) and 4 M HCl in EtOAc (4 mL) was stirred at 25° C. under N2 for 1 h. The reaction mixture was concentrated under reduced pressure to give the title compound as a white solid. Y=quantitative.
Step 4. (3S)-3-({3-Chloro-7H-imidazo[4,5-c]pyridazin-7-yl}methyl)-1-methylpyrrolidine. To a solution of (3S)-3-({3-chloro-7H-imidazo[4,5-c]pyridazin-7-yl}methyl)pyrrolidine hydrochloride (347 mg, 1.27 mmol) in DCE (6 mL) at 0° C. under N2 were added 37% formaldehyde aqueous solution (471 μL, 6.33 mmol) and AcOH (316 L). The mixture was stirred at 0° C. for 15 min, then treated with NaBH(OAc)3 (322 mg, 1.52 mmol). The solution was stirred at 0° C. under N2 for 1 h. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Waters Xbridge BEH C18 100×30 mm×10 μm; mobile phase: [H2O (10 mM NH4HCO3)—ACN]; gradient: 2-20% B over 8.0 min) and lyophilised to give the title compound as a white solid. Y=27%. 1H NMR (400 MHz, DMSO-d6) δ 8.95 (s, 1H), 8.26 (s, 1H), 4.43-4.31 (m, 2H), 2.91-2.81 (m, 1H), 2.57-2.52 (m, 1H), 2.43-2.30 (m, 3H), 2.21 (s, 3H), 1.90-1.79 (m, 1H), 1.57-1.48 (m, 1H).
Step 5. 5-Chloro-3-methyl-2-(7-{[(3S)-1-methylpyrrolidin-3-yl]methyl}-7H-imidazo[4,5-c]pyridazin-3-yl)phenol hydrochloride. To a solution of (3S)-3-({3-Chloro-7H-imidazo[4,5-c]pyridazin-7-yl}methyl)-1-methylpyrrolidine (60 mg, 238 μmol) in dioxane (3 mL) and H2O (0.6 mL) at 25° C. under N2 were added 5-chloro-3-methyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenol (Intermediate B2, 160 mg, 596 μmol), K2CO3 (99 mg, 715 μmol) and dicyclohexyl-[2-(2,6-dimethoxyphenyl)phenyl]phosphane; methanesulfonate; (2-phenylanilino)palladium(1+) “SPhos Pd G3” (28 mg, 36 μmol). The solution was stirred at reflux under N2 for 2 h. The reaction mixture was diluted with H2O (2 mL) and extracted with EtOAc (3×2 mL). The combined organic layers were washed with brine (2×2 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Phenomenex Luna C18 100×40 mm×5 μm; mobile phase: [H2O (0.04% HCl)—ACN]; gradient: 20-25% B over 8.0 min) and lyophilised to give the title compound as a yellow solid. Y=66%. 1H NMR (400 MHz, DMSO-d6) δ 11.27-10.83 (m, 1H), 10.40 (br. s, 1H), 9.12 (d, J=4 Hz, 1H), 8.27 (d, J=2 Hz, 1H), 6.96 (s, 1H), 6.94 (s, 1H), 4.72-4.53 (m, 2H), 3.55-3.46 (m, 2H), 3.24-2.95 (m, 3H), 2.80 (m, 3H), 2.29-2.22 (m, 0.5H), 2.15-1.94 (m, 4H), 1.97-1.81 (m, 0.5H). 1H NMR (400 MHz, DMSO-d6+D2O) δ 8.96 (d, J=4 Hz, 1H), 8.15 (d, J=2 Hz, 1H), 6.92 (s, 1H), 6.89 (s, 1H), 4.64-4.48 (m, 2H), 3.73-3.44 (m, 2H), 3.31-2.92 (m, 3H), 2.90-2.70 (m, 3H), 2.28-2.18 (m, 0.5H), 2.15-1.92 (m, 4H), 1.87-1.76 (m, 0.5H). LCMS (ESI): m/z: [M+H]+=358.2.
Synthesised in an analogous way to Compound 74, starting from tert-butyl (3S)-3-(aminomethyl)pyrrolidine-1-carboxylate. 1H NMR (400 MHz, DMSO-d6) δ 11.34-10.85 (m, 1H), 10.40 (br. s, 1H), 9.13 (d, J=7 Hz, 1H), 8.28 (d, J=5 Hz, 1H), 6.97 (s, 1H), 6.94 (s, 1H), 4.65-4.58 (m, 2H), 3.70-3.59 (m, 1H), 3.57-3.44 (m, 1H), 3.31-2.94 (m, 3H), 2.85-2.75 (m, 3H), 2.30-1.81 (m, 5H). 1H NMR (400 MHz, DMSO-d6+D2O) δ 9.03 (d, J=7 Hz, 1H), 8.23 (d, J=5 Hz, 1H), 6.94 (s, 1H), 6.92 (s, 1H), 4.66-4.48 (m, 2H), 3.69-3.60 (m, 1H), 3.60-3.48 (m, 1H), 3.33-2.95 (m, 3H), 2.83 (d, J=14 Hz, 3H), 2.30-1.75 (m, 5H). LCMS (ESI): m/z: [M+H]+=358.2.
Step 1. 4-{[3-(2-Methoxy-4,6-dimethylphenyl)-7H-imidazo[4,5-c]pyridazin-7-yl]methyl}-1-methylpiperidine hydrochloride. To a solution of 3-chloro-7-[(1-methyl-4-piperidyl)methyl]imidazo[4,5-c]pyridazine (for synthesis see Compound 71)(200 mg, 753 μmol) in dioxane (4 mL) and H2O (0.8 mL) at 25° C. under N2 were added (2-methoxy-4,6-dimethyl-phenyl)boronic acid (163 mg, 903 μmol), K2CO3 (312 mg, 2.26 mmol) and SPhos Pd G3 (59 mg, 75 μmol). The reaction mixture was stirred at 80° C. under N2 for 1 h. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Phenomenex Luna C18 100×40 mm×5 μm; mobile phase: [H2O (0.04% HCl)—ACN]; gradient: 1-30% B over 8.0 min) and lyophilised to give the title compound as a yellow solid. Y=63%. 1H NMR (400 MHz, DMSO-d6) δ 10.34 (s, 1H), 9.00 (s, 1H), 8.10 (s, 1H), 6.87 (s, 1H), 6.81 (s, 1H), 4.40 (d, J=7 Hz, 2H), 3.66 (s, 3H), 3.41-3.38 (m, 2H), 2.97-2.84 (m, 2H), 2.69 (d, J=5 Hz, 3H), 2.37 (s, 3H), 2.31-2.25 (m, 1H), 1.98 (s, 3H), 1.83-1.80 (m, 2H), 1.71-1.65 (m, 2H).
Step 2. 3,5-Dimethyl-2-{7-[(1-methylpiperidin-4-yl)methyl]-7H-imidazo[4,5-c]pyridazin-3-yl}phenol. To a solution of 4-{[3-(2-methoxy-4,6-dimethylphenyl)-7H-imidazo[4,5-c]pyridazin-7-yl]methyl}-1-methylpiperidine hydrochloride (0.19 g, 473 μmol) in DCM (3 mL) at 0° C. was added 2 M BBr3 in DCM (473 μL, 946 μmol). The reaction mixture was stirred at 0° C. under N2 for 3 h. The reaction mixture was quenched by addition of H2O (1 mL) and adjusted to pH ˜7 with saturated Na2CO3 aqueous solution. The resulting mixture was concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Waters Xbridge Prep OBD C18 150×40 mm×10 μm; mobile phase: [H2O (10 mM NH4HCO3)—ACN]; gradient: 15-45% B over 8.0 min) and lyophilised to give the title as an off-white solid. Y=36%. 1H NMR (400 MHz, DMSO-d6) δ 9.29 (br. s, 1H), 8.77 (s, 1H), 7.83 (s, 1H), 6.63 (s, 1H), 6.61 (s, 1H), 4.35 (d, J=7 Hz, 2H), 2.75 (d, J=11 Hz, 2H), 2.25 (s, 3H), 2.12 (s, 3H), 2.02-2.00 (m, 1H), 1.96 (s, 3H), 1.84-1.73 (m, 2H), 1.54-1.51 (m, 2H), 1.34-1.30 (m, 2H). 1H NMR (400 MHz, DMSO-d6+D2O) δ 8.76 (s, 1H), 7.82 (s, 1H), 6.63 (s, 1H), 6.61 (s, 1H), 4.34 (d, J=7 Hz, 2H), 2.74 (d, J=11 Hz, 2H), 2.25 (s, 3H), 2.12 (s, 3H), 2.02-2.00 (m, 1H), 1.95 (s, 3H), 1.84-1.76 (m, 2H), 1.53-1.50 (m, 2H), 1.34-1.31 (m, 2H). LCMS (ESI): m/z: [M+H]+=352.3.
Step 1. 4-(Benzenesulfonyl)-3,6-dichloropyridazine. To a solution of 3,4,6-trichloropyridazine (5.0 g, 27.3 mmol) in THF (40 mL) and DMSO (10 mL) at 25° C. was added sodium benzenesulfinate (5.16 g, 28.6 mmol). The resulting mixture was stirred at 25° C. for 1 h. The solution was diluted with H2O (50 mL) and extracted with EtOAc (3×50 mL). The combined organic layers were washed with brine (3×50 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether/ethyl acetate=10:1 to 3:1) to give the title compound as a white solid. Y=42%. 1H NMR (400 MHz, DMSO-d6) δ 8.61 (s, 1H), 8.11-8.06 (m, 2H), 7.88-7.82 (m, 1H), 7.74-7.68 (m, 2H).
Step 2. 2-{[4-(Benzenesulfonyl)-6-chloropyridazin-3-yl]amino}-N,N-dimethylacetamide. To a solution of 4-(benzenesulfonyl)-3,6-dichloro-pyridazine (0.50 g, 1.73 mmol) in dioxane (8 mL) at 25° C. were added 2-amino-N,N-dimethyl-acetamide (212 mg, 2.08 mmol) and K2CO3 (1.08 g, 7.78 mmol). The mixture was stirred at 100° C. under N2 for 2 h. The reaction mixture was diluted with H2O (10 mL) and extracted with EtOAc (3×10 mL). The combined organic layers were washed with brine (30 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether/ethyl acetate=10/1 to 3/1) to give the title compound as a yellow solid. Y=52%. 1H NMR (400 MHz, DMSO-d6) δ 8.16-8.08 (m, 3H), 7.85-7.75 (m, 1H), 7.73-7.65 (m, 2H), 7.64-7.51 (m, 1H), 4.28 (d, J=4 Hz, 2H), 2.98 (s, 3H), 2.93 (s, 3H).
Step 3. 2-[(4-Azido-6-chloropyridazin-3-yl)amino]-N,N-dimethylacetamide. To a solution of 2-[[4-(benzenesulfonyl)-6-chloro-pyridazin-3-yl]amino]-N,N-dimethyl-acetamide (650 mg, 1.83 mmol) in dioxane (10.4 mL) and DMSO (2.6 mL) at 25° C. was added NaN3 (238 mg, 3.66 mmol). The reaction mixture was stirred at 50° C. under N2 for 48 h. The reaction mixture was quenched by addition of saturated Na2CO3 aqueous solution (10 mL) and extracted with EtOAc (3×10 mL). The combined organic layers were washed with brine (30 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether/ethyl acetate=10/1 to 1/1) to give the title compound as a yellow solid. Y=77%. 1H NMR (400 MHz, DMSO-d6) δ 7.53 (s, 1H), 6.37 (t, J 5 Hz, 1H), 4.17 (d, J=5 Hz, 2H), 3.00 (s, 3H), 2.87 (s, 3H).
Step 4. 2-[(4-Amino-6-chloropyridazin-3-yl)amino]-N,N-dimethylacetamide. To a solution of 2-[(4-azido-6-chloro-pyridazin-3-yl)amino]-N,N-dimethyl-acetamide (0.33 g, 1.29 mmol) in DCM (3 mL) and AcOH (1 mL) at 0° C. was added Zn (236 mg, 3.61 mmol). The reaction mixture was stirred at 25° C. under N2 for 2 h. The reaction mixture was quenched by addition of aqueous 1 M HCl (3 mL), diluted with H2O (3 ml) and extracted with DCM (3×3 mL). The combined organic layers were washed with brine (9 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give the title compound as a solid. 1H NMR (400 MHz, DMSO-d6) δ 6.48-6.34 (m, 3H), 6.26 (t, J=5 Hz, 1H), 4.21 (d, J=5 Hz, 2H), 3.04 (s, 3H), 2.86 (s, 3H).
Step 5. 2-{3-Chloro-7H-imidazo[4,5-c]pyridazin-7-yl}-N,N-dimethylacetamide. To a solution of 2-[(4-amino-6-chloropyridazin-3-yl)amino]-N,N-dimethylacetamide (0.50 g, 2.18 mmol) in triethoxymethane (10 mL) at 25° C. was added 4 M HCl in H2O (272 μL, 1.09 mmol). The mixture was stirred at 100° C. under N2 for 12 h. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Phenomenex Luna C18 100×40 mm×5 μm; mobile phase: [H2O (0.04% HCl)—ACN]; gradient: 5-20% B over 8.0 min) and lyophilised to give the title compound as a gum. Y=13%. 1H NMR (400 MHz, DMSO-d6) δ 8.76 (s, 1H), 8.28 (s, 1H), 5.45 (s, 2H), 3.15 (s, 3H), 2.88 (s, 3H).
Step 6. 2-[3-(4-Chloro-2-hydroxy-6-methylphenyl)-7H-imidazo[4,5-c]pyridazin-7yl]-N,N-dimethylacetamide. To a solution of 5-chloro-3-methyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenol (Intermediate B2, 101 mg, 376 μmol) in dioxane (1.8 mL) and H2O (0.6 mL) at 25° C. were added K2CO3 (104 mg, 751 μmol), SPhos Pd G3 (20 mg, 25 μmol) and 2-{3-chloro-7H-imidazo[4,5-c]pyridazin-7-yl}-N,N-dimethylacetamide (60 mg, 250 μmol). The reaction mixture was stirred at 80° C. under N2 for 2 h. The reaction mixture was diluted with H2O (3 mL) and extracted with EtOAc (3×3 mL). The combined organic layers were washed with brine (9 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Phenomenex Luna C18 100×40 mm×5 μm; mobile phase: [H2O (0.04% HCl)—ACN]; gradient: 20-30% B over 8.0 min) and lyophilised to give the title compound as a yellow solid. Y=18%. 1H NMR (400 MHz, DMSO-d6) δ 10.45 (br. s, 1H), 8.97 (s, 1H), 8.36 (s, 1H), 7.10-6.91 (m, 2H), 5.53 (s, 2H), 3.18 (s, 3H), 2.90 (s, 3H), 2.04 (s, 3H). 1H NMR (400 MHz, DMSO-d6+D2O) δ 8.98 (s, 1H), 8.39 (s, 1H), 7.01-6.95 (m, 2H), 5.52 (s, 2H), 3.16 (s, 3H), 2.89 (s, 3H), 2.04 (s, 3H). LC-MS (ESI): m/z: [M+H]+=346.2.
Step 1. Tert-butyl 4-({[4-(benzenesulfonyl)-6-chloropyridazin-3-yl]amino}methyl)-4-fluoropiperidine-1-carboxylate. To a solution of 4-(benzenesulfonyl)-3,6-dichloro-pyridazine (for synthesis see Compound 77)(3.2 g, 11.1 mmol) in dioxane (50 mL) at 25° C. were added K2CO3 (6.88 g, 49.8 mmol) and tert-butyl 4-(aminomethyl)-4-fluoro-piperidine-1-carboxylate (3.09 g, 13.3 mmol). The resulting mixture was stirred at 100° C. for 2 h. The solution was diluted with H2O (50 mL) and extracted with EtOAc (3×50 mL). The combined organic layers were washed with brine (3×50 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether/ethyl acetate=10:1 to 2:1) to give the title compound as a white solid. Y=89%. 1H NMR (400 MHz, DMSO-d6) δ 8.14-8.08 (m, 3H), 7.84-7.79 (m, 1H), 7.73-7.64 (m, 2H), 6.80 (t, J=6 Hz, 1H), 3.91-3.82 (m, 2H), 3.75-3.65 (m, 2H), 2.98-2.81 (m, 2H), 1.54-1.44 (m, 4H), 1.38 (s, 9H).
Step 2. Tert-butyl 4-{[(4-azido-6-chloropyridazin-3-yl)amino]methyl}-4-fluoropiperidine-1-carboxylate. To a solution of tert-butyl 4-[[[4-(benzenesulfonyl)-6-chloro-pyridazin-3-yl]amino]methyl]-4-fluoro-piperidine-1-carboxylate (1.2 g, 2.47 mmol) in dioxane (12 mL) and DMSO (3 mL) at 25° C. was added NaN3 (483 mg, 7.42 mmol). The resulting mixture was stirred at 50° C. for 48 h. The reaction mixture was cooled to 0° C., quenched by addition of saturated NaHCO3 aqueous solution (10 mL), and extracted with EtOAc (3×10 mL). The combined organic layers were washed with brine (10 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether/ethyl acetate=10:1 to 2:1) to give the title compound as a yellow oil. 1H NMR (400 MHz, DMSO-d6) δ 7.51 (s, 1H), 6.55 (t, J=6 Hz, 1H), 3.83-3.65 (m, 4H), 3.05-2.89 (m, 2H), 1.76-1.49 (m, 4H), 1.39 (s, 9H).
Step 3. Tert-butyl 4-{[(4-amino-6-chloropyridazin-3-yl)amino]methyl}-4-fluoropiperidine-1-carboxylate. To a solution of tert-butyl 4-{[(4-azido-6-chloropyridazin-3-yl)amino]methyl}-4-fluoropiperidine-1-carboxylate (1.1 g, 2.85 mmol) in DCM (15 mL) and AcOH (3 mL) at 0° C. was added Zn (522 mg, 7.98 mmol). The resulting mixture was stirred at 25° C. for 2 h. The reaction mixture was quenched by addition of aqueous 1 M HCl (3 mL), and extracted with DCM (3×10 mL). The combined organic phase was washed with brine (10 mL), dried with anhydrous Na2SO4, filtered and concentrated under vacuum to give the title compound as a yellow gum. Y=quantitative.
Step 4. Tert-butyl 4-({3-chloro-7H-imidazo[4,5-c]pyridazin-7-yl}methyl)-4-fluoropiperidine-1-carboxylate. To a solution of tert-butyl 4-{[(4-amino-6-chloropyridazin-3-yl)amino]methyl}-4-fluoropiperidine-1-carboxylate (1.03 g, 2.86 mmol) in triethoxymethane (20 mL) at 25° C. was added 4 M HCl in H2O (358 μL, 1.43 mmol). The resulting mixture was stirred at 100° C. for 12 h. The solution was diluted with H2O (20 mL) and extracted with EtOAc (3×20 mL). The combined organic layers were washed with brine (3×20 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether/ethyl acetate=10:1 to 0:1) to give the title compound as a yellow solid. Y=40%. 1H NMR (400 MHz, DMSO-d6) δ 8.79 (s, 1H), 8.31 (s, 1H), 4.75 (s, 1H), 4.69 (s, 1H), 3.90-3.76 (m, 2H), 3.05-2.79 (m, 2H), 1.86-1.67 (m, 2H), 1.64-1.55 (m, 2H), 1.40 (s, 9H).
Step 5. 4-({3-Chloro-7H-imidazo[4,5-c]pyridazin-7-yl}methyl)-4-fluoropiperidine. A mixture of tert-butyl 4-({3-chloro-7H-imidazo[4,5-c]pyridazin-7-yl}methyl)-4-fluoropiperidine-1-carboxylate (200 mg, 541 μmol) and 4 M HCl in EtOAc (4 mL) was stirred at 25° C. for 1 h. The mixture was concentrated under vacuum to give a residue. The residue was dissolved in MeOH (2 mL) then adjusted to pH˜9 with MeONa. The resulting mixture was concentrated under vacuum to give the title compound as a white solid. Y=96%.
Step 6. 4-({3-Chloro-7H-imidazo[4,5-c]pyridazin-7-yl}methyl)-4-fluoro-1-methylpiperidine. To a solution of 4-({3-chloro-7H-imidazo[4,5-c]pyridazin-7-yl}methyl)-4-fluoropiperidine (140 mg, 519 μmol) in DCE (3 mL) and AcOH (0.3 mL) at 0° C. was added 37% formaldehyde aqueous solution (116 μL, 1.56 mmol). The resulting mixture was stirred at 0° C. for 0.5 h, treated with NaBH(OAc)3 (330 mg, 1.56 mmol) and the resulting mixture stirred at 0° C. for 0.5 h. The solution was diluted with H2O (2 mL) and extracted with DCM (3×2 mL). The combined organic layers were washed with brine (2 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Waters Xbridge Prep OBD C18 150×40 mm×10 μm; mobile phase: [H2O (10 mM NH4HCO3)—ACN]; gradient: 10-40% B over 8.0 min) and lyophilised to give the title compound as a white solid. Y=81%. 1H NMR (400 MHz, DMSO-d6) δ 8.78 (s, 1H), 8.30 (s, 1H), 4.71 (s, 1H), 4.66 (s, 1H), 2.62-2.54 (m, 2H), 2.16 (s, 3H), 2.05 (t, J=11 Hz, 2H), 1.93-1.73 (m, 2H), 1.58 (t, J=11 Hz, 2H).
Step 7. 5-Chloro-2-{7-[(4-fluoro-1-methylpiperidin-4-yl)methyl]-7H-imidazo[4,5-c]pyridazin-3-yl}-3-methylphenol. To a solution of 4-({3-chloro-7H-imidazo[4,5-c]pyridazin-7-yl}methyl)-4-fluoro-1-methylpiperidine (120 mg, 423 μmol) in dioxane (3 mL) and H2O (0.6 mL) at 25° C. under N2 were added 5-chloro-3-methyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenol (Intermediate B2, 114 mg, 423 μmol), dicyclohexyl-[2-(2,6-dimethoxyphenyl)phenyl]phosphane; methanesulfonate;(2-phenylanilino)palladium(1+) “SPhos Pd G3” (50 mg, 63 μmol) and K2CO3 (234 mg, 1.69 mmol). The resulting mixture was stirred at 80° C. under N2 for 1 h. The solution was diluted with H2O (2 mL) and extracted with EtOAc (3×2 mL). The combined organic layers were washed with brine (3×2 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Phenomenex Luna C18 100×40 mm×5 μm; mobile phase: [H2O (0.04% HCl)—ACN]; gradient: 20-35% B over 8.0 min) and lyophilised to give the title compound as a yellow solid. Y=50%. 1H NMR (400 MHz, DMSO-d6) δ 11.14-10.21 (m, 2H), 9.02-8.87 (m, 1H), 8.31-8.15 (m, 1H), 6.97 (d, J=2 Hz, 1H), 6.94 (d, J=2 Hz, 1H), 5.03-4.80 (m, 2H), 3.42-3.34 (m, 2H), 3.12-2.98 (m, 2H), 2.89-2.73 (m, 3H), 2.40-2.18 (m, 2H), 2.07-1.95 (m, 5H). 1H NMR (400 MHz, DMSO-d6+D2O) δ 9.02-8.88 (m, 1H), 8.31-8.20 (m, 1H), 6.97-6.90 (m, 2H), 5.01-4.77 (m, 2H), 3.45-3.36 (m, 2H), 3.12-3.01 (m, 2H), 2.90-2.74 (m, 3H), 2.31-2.09 (m, 2H), 2.08-1.95 (m, 5H). 1H NMR (400 MHz, DMSO-d6+D2O, T=353 K) δ 8.79 (s, 1H), 8.03 (s, 1H), 6.92 (s, 1H), 6.90 (s, 1H), 4.91-4.75 (m, 2H), 3.45-3.38 (m, 2H), 3.17-3.01 (m, 2H), 2.80 (s, 3H), 2.37-2.18 (m, 2H), 2.07-1.98 (m, 5H). LCMS (ESI): m/z: [M+H]+=390.3.
Step 1. Tert-butyl (3S)-3-{[(4-amino-6-chloropyridazin-3-yl)amino]methyl}piperidine-1-carboxylate. To a solution of 3,6-dichloropyridazin-4-amine (3.0 g, 18.3 mmol) in decan-1-ol (30 mL) at 25° C. in a sealed tube were added DIPEA (6.37 mL, 36.6 mmol) and tert-butyl (3S)-3-(aminomethyl)piperidine-1-carboxylate (4.70 g, 22.0 mmol). The mixture was stirred at 150° C. under N2 for 12 h. The reaction mixture was purified by prep-HPLC (column: Waters Xbridge BEH C18 250×70 mm×10 μm; mobile phase: [H2O (0.05% NH3H2O)—ACN]; gradient: 30-50% B over 20 min) and lyophilised to give the title compound as a white solid. Y=18%.
Step 2. Tert-butyl (3S)-3-({3-chloro-7H-imidazo[4,5-c]pyridazin-7-yl}methyl)piperidine-1-carboxylate. To a solution of tert-butyl (3S)-3-{[(4-amino-6-chloropyridazin-3-yl)amino]methyl}piperidine-1-carboxylate (800 mg, 2.34 mmol) in triethoxymethane (16 mL) was added 4 M HCl in H2O (293 μL, 1.17 mmol). The mixture was stirred at 70° C. for 12 h. The reaction mixture was purified by prep-HPLC (column: Waters Xbridge BEH C18 250×50 mm×10 μm; mobile phase: [H2O (10 mM NH4HCO3)—ACN]; gradient: 20-50% B over 10 min) and lyophilised to give the title compound as a yellow gum. Y=97%.
Step 3. (3R)-3-({3-Chloro-7H-imidazo[4,5-c]pyridazin-7-yl}methyl)piperidine hydrochloride. A mixture of tert-butyl (3S)-3-({3-chloro-7H-imidazo[4,5-c]pyridazin-7-yl}methyl)piperidine-1-carboxylate (500 mg, 1.42 mmol) and 4 M HCl in EtOAc (10 mL) was stirred at 25° C. for 3 h. The reaction mixture was concentrated under reduced pressure to give the title compound as a white solid. Y=98%. 1H NMR (400 MHz, DMSO-d6) δ 9.31-9.07 (m, 1H), 8.91 (s, 1H), 8.76-8.73 (m, 1H), 8.28 (s, 1H), 4.52-4.31 (m, 2H), 3.28-3.05 (m, 3H), 2.82-2.64 (m, 2H), 1.85-1.52 (m, 3H), 1.38-1.24 (m, 1H).
Step 4. (3R)-3-({3-Chloro-7H-imidazo[4,5-c]pyridazin-7-yl}methyl)-1-methylpiperidine. To a solution of (3R)-3-({3-chloro-7H-imidazo[4,5-c]pyridazin-7-yl}methyl)piperidine hydrochloride (300 mg, 1.04 mmol) in MeOH (6 mL) at 0° C. under N2 were added 37% formaldehyde aqueous solution (620 μL, 8.33 mmol) and NaBH3CN (131 mg, 2.08 mmol). The mixture was stirred at 0° C. for 2 h. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Phenomenex Luna C18 100×40 mm×5 μm; mobile phase: [H2O (0.04% HCl)—ACN]; gradient: 1-15% B over 8.0 min) and lyophilised to give the crude product. This was further purified by prep-HPLC (column: Waters Xbridge BEH C18 100×30 mm×10 μm; mobile phase: [H2O (10 mM NH4HCO3)—ACN]; gradient: 1-30% B over 8.0 min) and lyophilised to give the title compound as a yellow oil. Y=25%. 1H NMR (400 MHz, DMSO-d6) δ 8.88 (s, 1H), 8.26 (s, 1H), 4.33 (J=7 Hz, 2H), 2.55-2.51 (m, 1H), 2.27-2.29 (m, 1H), 1.94 (s, 3H), 1.94-1.91 (m, 1H), 1.79-1.75 (m, 1H), 1.66-1.63 (m, 1H), 1.55-1.51 (m, 1H), 1.44-1.41 (m, 1H), 1.24-1.20 (m, 1H), 1.01-0.97 (m, 1H).
Step 5. 5-Chloro-3-methyl-2-(7-{[(3R)-1-methylpiperidin-3-yl]methyl}-7H-imidazo[4,5-c]pyridazin-3-yl)phenol hydrochloride. To a solution of (3R)-3-({3-chloro-7H-imidazo[4,5-c]pyridazin-7-yl}methyl)-1-methylpiperidine (70 mg, 263 μmol) and 5-chloro-3-methyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenol (Intermediate B2, 177 mg, 659 μmol) in dioxane (3.5 mL) and H2O (0.7 mL) at 25° C. under N2 were added K2CO3 (109 mg, 790 μmol) and SPhos Pd G3 (41 mg, 53 μmol). The mixture was stirred at 90° C. under N2 for 4 h. The reaction mixture was poured into water (5 mL) and the resulting mixture extracted with EtOAc (3×5 mL). The combined organic phase was washed with brine (5 mL), dried with anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Phenomenex Luna C18 100×40 mm×5 μm; mobile phase: [H2O (0.04% HCl)—ACN]; gradient: 20-25% B over 8.0 min) and lyophilised to give the title compound as a yellow solid. Y=13%. 1H NMR (400 MHz, DMSO-d6) δ 10.83-9.81 (m, 2H), 9.13-8.94 (m, 1H), 8.14 (s, 1H), 6.94-6.92 (m, 2H), 4.83-4.38 (m, 2H), 3.45-3.18 (m, 2H), 3.02-2.58 (m, 6H), 2.03 (s, 3H), 1.94-1.49 (m, 3H), 1.38-1.17 (m, 1H). 1H NMR (400 MHz, DMSO-d6+D2O) δ 9.05-8.91 (m, 1H), 8.27-7.91 (m, 1H), 7.11-6.73 (m, 2H), 4.42 (d, J=7 Hz, 2H), 3.45-3.15 (m, 2H), 2.89-2.56 (m, 6H), 2.01 (s, 3H), 1.91-1.48 (m, 3H), 1.35-1.20 (m, 1H). 1H NMR (400 MHz, DMSO-d6+D2O, T=393 K) δ 8.73 (s, 1H), 7.91 (s, 1H), 6.93-6.88 (m, 2H), 4.49 (s, 2H), 3.54-3.23 (m, 2H), 2.93-2.57 (m, 6H), 2.05 (s, 3H), 1.96-1.74 (m, 3H), 1.44-1.26 (m, 1H). LCMS (ESI): m/z: [M+H]+=372.3.
Synthesised in an analogous way to Compound 79, starting from tert-butyl (3R)-3-(aminomethyl)piperidine-1-carboxylate. 1H NMR (400 MHz, DMSO-d6) δ 11.02-10.39 (m, 1H), 9.37-9.00 (m, 1H), 8.41-8.19 (m, 1H), 7.00-6.92 (m, 2H), 4.51-4.42 (m, 2H), 3.47-3.30 (m, 2H), 2.91-2.78 (m, 2H), 2.76-2.64 (m, 4H), 2.06 (s, 3H), 1.91-1.70 (m, 3H), 1.36-1.21 (m, 1H). 1H NMR (400 MHz, DMSO-d6+D2O) δ 8.93 (s, 1H), 8.18 (s, 1H), 6.99-6.80 (m, 2H), 4.42 (d, J=7 Hz, 2H), 3.45-3.29 (m, 2H), 2.95-2.64 (m, 6H), 2.00 (s, 3H), 1.89-1.85 (m, 1H), 1.79-1.56 (m, 2H), 1.30-1.23 (m, 1H). 1H NMR (400 MHz, DMSO-d6+D2O, T=353 K) δ 8.78 (s, 1H), 7.99 (s, 1H), 6.89 (s, 2H), 4.52-4.37 (m, 2H), 3.42-3.32 (m, 2H), 2.94-2.68 (m, 5H), 2.65-2.56 (m, 1H), 2.00 (s, 3H), 1.92-1.84 (m, 1H), 1.81-1.59 (m, 2H), 1.42-1.26 (m, 1H). LCMS (ESI): m/z: [M+H]+=372.3.
The biological activity of the compounds of the present disclosure was determined utilising the assay described herein.
The compounds of the present disclosure were tested for their inhibitory activity against IL-1β release upon NLRP3 activation in peripheral blood mononuclear cells (PBMC).
Protocol A. PBMC were isolated from buffy coats by density gradient centrifugation on Histopaque-1077 (Sigma, cat no. 10771). Isolated cells were seeded into the wells of a 96-well plate and incubated for 3 h with lipopolysaccharide (LPS). Following medium exchange, the compounds of the present disclosure were added (a single compound per well) and the cells were incubated for 30 min. Next, the cells were stimulated either with ATP (5 mM) or nigericin (10 μM) for 1 h and the cell culture media from the wells were collected for further analysis.
The release of IL-1β into the media was determined by a quantitative detection of IL-1β in the media using an IL-1β enzyme-linked immunosorbent assay (ELISA) Ready-SET-Go!, eBioscience cat. No. 88-7261-88. Briefly, in a first step, high affinity binding plates (Corning, Costar 9018 or NUNC Maxisorp Cat No. 44-2404) were coated overnight at 4° C. with specific capture antibody included in the kit (anti-human IL-1β ref. 14-7018-68). Subsequently, plates were blocked with blocking buffer for 1 h at room temperature (rt) and after washing with a buffer (PBS with 0.05% Tween-20) incubated with protein standard and culture media. After 2 h of incubation at rt, plates were washed and incubated with biotinylated detection antibody included in the kit (anti-human IL-1β Biotin ref. 33-7110-68) for 1 h at rt. Plates were washed and incubated with HRP-streptavidin for 30 min at rt and washed again. The signal was developed after addition of 3,3′,5,5′-tetramethylbenzidine-peroxidase (TMB) until colour appeared and the reaction was stopped by 2 M H2SO4. A microplate spectrophotometer (BioTek) was used to detect signals with 450 nm. The detection range of IL-1β ELISA was 2-150 ng/mL.
Protocol B. PBMC were isolated from buffy coats by density gradient centrifugation on Histopaque-1077 (Sigma, cat no. 10771). Isolated cells were seeded into the wells (280,000 cells/well) of a 96-well plate and incubated for 3 h with lipopolysaccharide (LPS, 1 μg/mL diluted 1000× from a 1 mg/mL stock solution). The compounds of the present disclosure were added (a single compound per well) and the cells were incubated for 30 min. Next, the cells were stimulated with ATP (5 mM final concentration diluted 20× from a 100 mM stock solution) for 1 h and the cell culture media from the wells were collected for further analysis.
The release of IL-1β into the media was determined by quantitative detection of IL-1β in the media using HTRF®, CisBio cat. No. 62HIL1BPEH. Briefly, cell culture supernatant were dispensed directly into the assay plate containing antibodies labelled with the HTRF® donor and acceptor. A microplate spectrophotometer (BMG) was used to detect signals at 655 nm and 620 nm. The detection range of IL-1β HTRF® was 39-6500 pg/mL.
The compounds of the present disclosure were tested for their inhibitory activity against IL-1β release upon NLRP3 activation in phorbol 12-myristate 13-acetate (PMA)-differentiated THP-1 cells.
THP-1 cells stored in liquid nitrogen were thawed in a 37° C. water bath. The cells were added into complete RPMI 1640 medium (FBS (56 mL), 100× Penicillin-Streptomycin (5.6 mL) and 0.05 mM 2-Mercaptoethanol in RPMI 1640 Medium (500 mL)) and centrifuged for 5 min at 1000 rpm. The supernatant was discarded and the cells resuspended into new cell culture medium for culturing. Cells were cultured in cell culture medium and passaged every 3-4 days to maintain cell density between 4×105 and 1.5×106 cells/mL. The cells were diluted to 0.8×106 cells/mL and grown in a T150 flask with PMA (final concentration 5 ng/mL). The flask was incubated in a 37° C. incubator with a humidified atmosphere of 5% CO2 for −40 hours. After −40 hours of PMA differentiation, the flask was washed with DPBS. Cells were digested with Trypsin LE for about 5 min at room temperature, and digestion was terminated using complete RPMI 1640 medium. Cells were centrifuged and resuspended (1000 rpm, 3 min). The supernatant was discarded and cells washed with DPBS, centrifuged and resuspended cells (1000 rpm, 5 min). Cells were plated at 30,000 cells/well in a 384-well PP plate and treated with 25 ng/mL LPS solution diluted in serum-free medium (45 μL). The plate was incubated in a 37° C. incubator with a humidified atmosphere of 5% CO2 for 2 hours. Compounds (from 10 mM DMSO solution) were dispensed and serially diluted by 1:3 eight times, with final 0.5% DMSO concentration. NLRP3 inflammasome was activated with nigericin (5 μL), the plate was centrifuged (1000 rpm, 30 s) and incubated in a 37° C. incubator with a humidified atmosphere of 50% CO2 for 2 hours. 35 μL/well of supernatant was removed and IL-1β levels determined by ELISA assay (Mabtech 3416-1H-20) according to the manufacturer's instructions.
The determination of the IC50 values was preformed using the Graph Pad Prism software and the measured IC50 values of compounds of the present disclosure are shown in Table A below (“++++++” means <0.03 μM; “+++++” means >0.03 μM and <0.1 μM; “++++” means >0.1 and <0.3 μM; “+++” means >0.3 and <1.0 μM; “++” means >1.0 and <3.0 μM; “+” means≥3 and <10 μM μM). ND means not determined. These results show that the compounds of the present disclosure are capable of inhibiting IL-1β release upon inflammasome activation.
The details of one or more embodiments of the disclosure are set forth in the accompanying description above. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, the preferred methods and materials are now described. Other features, objects, and advantages of the disclosure will be apparent from the description and from the claims. In the specification and the appended claims, the singular forms include plural referents unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. All patents and publications cited in this specification are incorporated by reference.
The foregoing description has been presented only for the purposes of illustration and is not intended to limit the disclosure to the precise form disclosed, but by the claims appended hereto.
This application claims priority to, and the benefit of, U.S. Provisional Application No. 63/614,187, filed on Dec. 22, 2023, the entire contents of which are incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| 63614187 | Dec 2023 | US |
