Isoquinoline Derivatives as Inhibitors of BAX and / or BAK, Compositions and Uses Thereof

Abstract

This application relates to isoquinoline compounds of formula (I), processes for their preparation, and compositions comprising same. More particularly, this application relates to compounds of formula (I) having activity as inhibitors of Bcl2 associated X protein (BAX) and / or Bcl-2 antagonist killer (BAK), and further to the use of compounds of formula (I) in the treatment of diseases, disorders, or conditions treatable by inhibiting BAX and / or BAK (e.g., neurodegenerative diseases, disorders, or conditions). [Formula 1] TIFF2025506245000155.tif34159

Description

[Technical field]

[0001] Related Applications This application claims priority to co-pending U.S. Provisional Patent Application No. 63 / 311,088, filed February 17, 2022, the contents of which are incorporated by reference herein in their entirety.

[0002] This application relates to isoquinoline compounds, processes for the preparation of isoquinoline compounds, compositions comprising isoquinoline compounds, and uses (e.g., therapeutic) of isoquinoline compounds. More particularly, this application relates to isoquinoline compounds useful for the treatment and / or prevention of diseases, disorders, or conditions (e.g., neurodegenerative diseases, disorders, or conditions) treatable and / or preventable by inhibiting or blocking Bcl2-associated X protein (BAX) and / or Bcl-2 antagonist killer (BAK). [Background technology]

[0003] Apoptosis, a morphologically defined form of programmed cell death, removes excess or dysfunctional cells to maintain homeostasis (Green and Kroemer, 2004). However, dysregulation of apoptosis can contribute to the development of neurological diseases, low viability of cell therapy, and death of healthy cells during cancer chemotherapy. Furthermore, excessive cell death is an obstacle to biotechnological applications that require cell preparation (Arden and Betenbaugh, 2004; Mergenthaler, Dirnagl and Meisel, 2004; Leber et al., 2010; Octavia et al., 2012; Pang et al., 2017; Afreen et al., 2018; Pemberton, Pogmore and Andews, 2020).

[0004] Bcl-2 family proteins control cell death through a complex series of protein-protein interactions that regulate mitochondrial outer membrane permeabilization (MOMP), an event that leads most cells to apoptotic death (Kale et al., 2018). Bcl-2 family proteins contain a common sequence known as the Bcl-2 homology (BH) motif, which contains binding sites and ligands that mediate protein-protein interactions (Aouacheria et al., 2015). The minimum requirement for classification as a Bcl-2 member consists of having a BH3 motif sufficient to bind to a multi-BH motif protein. Deletion of the BH3 motif renders most Bcl-2 members inactive (Leveille et al., 2010; Chi et al., 2020). Proapoptotic "BH3-only" proteins such as BID and BIM can bind to and activate multi-BH motif executioner proteins such as Bcl2-associated X protein (BAX), Bcl-2 antagonist killer (BAK), and Bcl-2 ovarian killer (BOK) ( Lovell et al. 2008 , Fernandez-Marrero et al. 2017 , Hockings et al. 2015 , Kuwana et al. 2005 ).

[0005] MOMP results from the Bcl-2 family executioner proteins BAX, BAK, and possibly BOK, which enter the mitochondrial outer membrane and oligomerize therein. Oligomerization of BAX and BAK is initiated by direct binding of BH3-only activators Bcl-2 family members. Anti-apoptotic proteins bind and inhibit both apoptosis executioner proteins and BH3-only activators (Kim et al., 2009; Llambi et al., 2011; Shamas-Din et al., 2013). MOMP releases cytochrome c and other pro-apoptotic factors from the mitochondrial intermembrane space, triggering the apoptosis signaling cascade and ultimately the activation of caspases that degrade the cell (Tait and Green, 2010; Westphal et al., 2011; Hill, Mackenzie and Harwig, 2015).

[0006] Inhibition of BAX and BAK oligomerization by compounds such as DAN004 has been shown to block MOMP (Niu et al., 2017). Recently, various other small molecules have been reported that activate or inhibit BAX function (Pogmore, Uehling and Andews, 2021). In addition, BAI1 has been reported to inhibit BAX-mediated cell death associated with doxorubicin-induced cardiomyopathy in mice (Amgalan et al., 2020). Despite significant efforts in developing drugs that directly regulate BAX, no BAX inhibitors have been successfully used in the clinic. Thus, there remains a need for BAX and / or BAK inhibitors with clinical potential. Summary of the Invention

[0007] Thus, the present invention includes compounds of formula I, or pharma- ceutically acceptable salts, solvates, and / or prodrugs thereof. [ka] During the ceremony, Each R1 are independently halo, CN, C 1~4 Alkyl, C 1~4 Haloalkyl, OC 1~4 Alkyl and OC 1~4 haloalkyl; R 2 is absent or R 2 Halo, C 1~4 Alkyl, C 1~4 Haloalkyl, OC 1~4 Alkyl and OC 1~4 haloalkyl; R 3 H and C 1~4 alkyl, L is for C 1~4 Alkylene, C 2~4 Alkenylene, and C 2~4 alkynylene, A is N, NH, and N(C 1~4 alkyl), and containing at least one ring hetero moiety selected from OH, halo, C 1~2 Alkyl, C 1~2 Haloalkyl, OC 1~2 Alkyl and OC 1~2 C optionally substituted with one or two substituents selected from haloalkyl 3~8 is heterocycloalkyl, n is selected from 0, 1, 2, and 3.

[0008] The present application also includes pharmaceutical compositions comprising one or more compounds of the present application and a pharma- ceutically acceptable carrier.

[0009] The present application also includes a method of inhibiting Bcl2 Binding X Protein (BAX) and / or Bcl-2 Antagonist Killer (BAK) in a biological sample or in a cell of a patient, the method comprising administering to the cell an effective amount of one or more compounds of the present application.

[0010] The present application also includes methods of treating or preventing BAX-mediated cell death and / or BAK-mediated cell death in a cell, either in a biological sample or in a patient, comprising administering to the cell an effective amount of one or more compounds of the present application.

[0011] The present application includes a method of inhibiting MOMP in a cell, either in a biological sample or in a patient, comprising administering to the cell an effective amount of one or more compounds according to any one of the present application.

[0012] The present application also includes a method of inhibiting oligomerization of BAX and BAK in a cell, either in a biological sample or in a patient, comprising administering to the cell an effective amount of one or more compounds of the present application.

[0013] The present application also includes a method of treating a disease, disorder, or condition treatable by inhibiting Bcl2-associated X protein (BAX) and / or Bcl-2 antagonist killer (BAK), comprising administering to a subject in need thereof a therapeutically effective amount of one or more compounds of the present application.

[0014] In some embodiments, the disease, disorder, or condition treatable by inhibiting BAX and / or BAK is a neurodegenerative disease, disorder, or condition, neuronal damage associated with ischemia, cardiomyopathy induced by chemotherapeutic agents and / or cell death of donor hematopoietic stem and progenitor cells (HSPCs).

[0015] Other features and advantages of the present application will become apparent from the following detailed description. It should be understood that the description and specific examples show embodiments of the present application, but are merely illustrative, and the claims should not be limited by these embodiments, but should be interpreted in the broadest possible manner consistent with the entire description.

[0016] The present application will now be described in more detail with reference to the accompanying drawings and tables. [Brief description of the drawings]

[0017] [Figure 1] Chemical shift perturbations and computational modeling reveal the putative binding site of dacomitinib on BAX. A. 1H, 15N HSQC of 200uM BAX + 1.5% DMSO (dark bars) vs. 200uM BAX + 400uM dacomitinib (light bars). B. Chemical shift perturbations quantified using CCPNMRv3 software reveal local changes in the chemical environment of residues in the presence of dacomitinib. Residues with chemical shifts above the significance threshold or 1.5x the significance threshold are those that cross the lower and upper dotted lines, respectively. C. Model of BAX (PDB:1F16) mapping residues that give rise to significant CSPs in the ribbon structure (structure on the left) and surface image (structure on the right) using the same shading of the CSP histogram in B. [Diagram 2] Graphs showing A and B below. A. Chemical shift perturbations quantified using CCPNMRv3 software identify local changes in the chemical environment of residues in the presence of exemplary compound I-1. Residues with chemical shifts above the significance threshold or 1.5 times the significance threshold are above the lower and upper dotted lines, respectively. B. BAX mutants V83W, L120W reduced the inhibition of BAX by dacomitinib. 100 nM BAX or V83W L120W BAX and 20 nM cBID were incubated with ANTS / DPX liposomes and 20 uM dacomitinib as indicated. Average values ​​of three independent experiments in duplicate are shown. [Diagram 3]FIG. 1 shows that dacomitinib and exemplary compound I-1 inhibit BAX- and BAK-mediated mitochondrial membrane permeabilization independent of activation signals. Mitochondria isolated from mouse liver were used in these assays because in this tissue BAX is cytoplasmic and therefore absent from heavy membrane isolates. Bcl-XL serves as a positive control for complete inhibition of BAX. Mitochondria-only control for non-specific leakage of isolated mitochondria. A. Dacomitinib and exemplary compound I-1 attenuate cBID-activated BAX-mediated permeabilization of isolated mitochondria. Mitochondria isolated from BAK- / - mouse liver were incubated with 10 nM BAX and 2 nM cBID, as well as the indicated concentrations of each inhibitor. B. Dacomitinib and exemplary compound I-1 attenuate BIM-activated BAX-mediated permeabilization of isolated mitochondria. BIM (2.5 nM) was incubated with mitochondria isolated from WT mouse liver and the indicated concentrations of each inhibitor. Mitochondria were incubated at 37°C for 30 min, then pelleted and SMAC-mCherry fluorescence was measured in the supernatant (released from mitochondria) and resuspended pellet fractions. [Figure 4] Figure 1 shows that kinase and BAX inhibitory activity can be differentiated by chemically modifying the kinase-binding hinge region of dacomitinib. A. Kinase panel evaluating exemplary compound I-1 activity at 1 uM performed by Eurofins. Percent kinase inhibition is calculated by subtracting the activity remaining in the presence of compound from the activity of the kinase with DMSO instead of exemplary compound I-1. Negative values ​​reflect the difference for uninhibited activity of some kinases and are interpreted as no inhibition. B. Liposome membrane permeabilization assay using 20 nM cBID and 100 nM BAX for a titration of dacomitinib and exemplary compound I-1. Each point represents one of two replicates. [Diagram 5]Figure 1 shows that actinomycin D-induced cell death depends on the pro-apoptotic activity of BAX and BAK. A. Actinomycin D-induced BMK cell death is mediated by both BAX and BAK. BMK cells of the indicated genotypes were exposed to the indicated concentrations of actinomycin D for 20-24 hours, after which cell death was assessed by confocal microscopy measurements of TMRE, Annexin V, and Hoechst staining. Data from three replicates are shown fitted to one phase binding curve. B. Baseline cell death in the presence of dacomitinib and exemplary compound I-1 is comparable in BMK cells, independent of Bax and Bak expression. Actinomycin D-induced BAX- and BAK-mediated cell death is highest in cells expressing both BAX and BAK and least in cells expressing neither of the pro-apoptotic proteins (BAX- / -BAK- / -). [Figure 6] Figure 1 shows concentration-dependent inhibition of spontaneous and actinomycin D-induced death of wild-type BMK (BMK-wt) cells by dacomitinib. 0 nM actinomycin D shows that dacomitinib reduced spontaneous cell death in cultures from approximately 30% to less than 10%. Other panels show that cell death caused by 3.125 nM, 6.25 nM, and 12.5 nM concentrations of actinomycin D (shown above the panels) was reduced by the addition of the indicated concentrations of dacomitinib. Bar graphs show the average of four replicates, indicated by individual symbols. A linear classifier was used to analyze micrographs of individual cells stained with the mitochondrial potential-sensitive dye TMRE, the nuclear stain Hoescht, and fluorescent annexin V to classify cells as dead. [Figure 7]Figure 1 shows concentration-dependent inhibition of spontaneous and actinomycin D-induced death of wild-type BMK (BMK-wt) cells by exemplary compound I-1. 0 nM actinomycin D shows the effect of I-1 on spontaneous cell death in culture. Other panels show that cell death caused by 3.125 nM, 6.25 nM, and 12.5 nM concentrations of actinomycin D (shown above the panels) was reduced by addition of the indicated concentrations of I-1. Bar graphs show the average of four replicates, indicated by individual symbols. A linear classifier was used to analyze micrographs of individual cells stained with the mitochondrial potential-sensitive dye TMRE, the nuclear stain Hoescht, and fluorescent annexin V to classify cells as dead. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] I. Definition Unless otherwise indicated, the definitions and embodiments described in this section and other sections are intended to be applicable to all embodiments and aspects of the application described herein as appropriate as would be apparent to one of ordinary skill in the art.

[0019] All features disclosed in this specification (including the claims, abstract and drawings), and all steps in any disclosed method or process, may be combined in any combination, except where at least some of such features and / or steps are mutually exclusive. Each feature disclosed in this specification (including the claims, abstract and drawings) may be replaced by an alternative feature serving the same, equivalent or similar purpose, unless expressly stated otherwise.

[0020] As used herein, "compound of the application" or "compound of the application" and like terms refer to compounds of Formula I and IA, including pharma- ceutically acceptable salts, solvates, and / or prodrugs thereof.

[0021] As used herein, "composition of the application" or "composition of the present application" and like terms refer to a composition that includes one or more compounds of the present application and at least one additional component.

[0022] The term "and / or" as used herein means that the listed items are present or used individually or in combination. In practice, the term means that "at least one" or "one or more" of the listed items are used or present. The term "and / or" with respect to pharma-ceutically acceptable salts and / or solvates thereof means that the compounds of the present application are present as individual salts and hydrates as well as combinations of, for example, salts and solvates of the compounds of the present application.

[0023] As used in this application, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, an embodiment including "a compound" should be understood to present a particular embodiment with one compound, or with two or more additional compounds.

[0024] In embodiments that include an "additional" or "second" component (e.g., an additional compound or second compound), the second component, as used herein, is chemically distinct from the other component or the first component. A "third" component is distinct from the other component, the first component, and the second component, and further recited or "additional" components are similarly distinct.

[0025] As used herein, the terms "comprising" (and any form of "comprising", e.g., "comprise" and "comprises"), "having" (and any form of "having", e.g., "have" and "has"), "including" (and any form of "including", e.g., "include" and "includes"), or "containing" (and any form of "containing", e.g., "contain" and "contains") are inclusive or open-ended and do not exclude additional unrecited elements or process / method steps.

[0026] As used herein, the term "consisting of" and its derivatives are intended to be limiting term specifying the presence of stated features, components, ingredients, groups, elements, and / or steps, and excluding the presence of other unrecited features, components, ingredients, groups, elements, and / or steps.

[0027] As used herein, the term "consisting essentially of" is intended to specify the presence of stated features, components, ingredients, groups, elements, and / or steps, as well as the presence of things that do not materially affect the basic and novel property(s) of those features, components, ingredients, groups, elements, and / or steps.

[0028] As used herein, terms of degree such as "substantially," "about," "approximately," and the like, refer to a reasonable amount of deviation from the term modified thereby that does not significantly alter the end result. These terms of degree should be construed to include a deviation of at least ±5% of the modified term, if that deviation does not negate the meaning of the modified term.

[0029] The term "suitable" as used herein means that the selection of a particular compound or condition will depend on the particular synthetic operation to be performed, the identity of the molecule(s) to be converted, and / or the particular application of the compound, but is well within the skill of one of ordinary skill in the art. All process / method steps described herein should be performed under conditions sufficient to result in the indicated product. As will be apparent to one of ordinary skill in the art, all reaction conditions (e.g., reaction solvent, reaction time, reaction temperature, reaction pressure, ratio of reactants, and whether the reaction should be performed under anhydrous or inert atmosphere, etc.) can be varied to optimize the yield of the desired product, and doing so is within the skill of one of ordinary skill in the art.

[0030] This application refers to numerous chemical terms and abbreviations used by those of ordinary skill in the art. Nonetheless, definitions of selected terms are provided for clarity and consistency.

[0031] The products of the processes of the present application can be isolated according to known methods, for example the compounds can be isolated by evaporation of the solvent, filtration, centrifugation, chromatography, or other suitable methods.

[0032] As will be apparent to one skilled in the art, when certain reaction steps of the present application are carried out in various solvents or solvent systems, the reaction steps can also be carried out in mixtures of suitable solvents or solvent systems.

[0033] As used herein, terms such as "protecting group" or "PG" refer to chemical moieties that protect or mask a reactive portion of a molecule to prevent side reactions at the reactive portion of the molecule while a different portion of the molecule is being manipulated or reacted. After the manipulation or reaction is complete, the protecting group is removed under conditions that do not cause degradation or decomposition of the remaining portion of the molecule. The selection of a suitable protecting group can be made by one skilled in the art. Many standard protecting groups are known in the art and are described, for example, in "Protective Groups in Organic Chemistry" McOmie, JFW Ed., Plenum Press, 1973; Greene, TW and Wuts, PGM, "Protective Groups in Organic Synthesis", John Wiley & Sons, 3rd Edition, 1999; and Kocienski, P. Protecting Groups, 3rd Edition, 2003, Georg Thieme Verlag (The Americas).

[0034] As used herein, the term "cell" refers to a single cell or multiple cells, including cells either in cell culture or within a subject.

[0035] As used herein, the term "subject" includes all members of the animal kingdom, including mammals, and thus the methods and uses of the present application are applicable to both human therapy and veterinary use.

[0036] The term "pharmaceutical acceptable" means compatible with the treatment of a subject.

[0037] The term "pharmaceutically acceptable carrier" means a non-toxic solvent, non-toxic dispersant, non-toxic additive, non-toxic adjuvant, or other non-toxic material that can be mixed with an active ingredient (e.g., one or more compounds of the present application) to form a pharmaceutical composition (i.e., a dosage form that can be administered to a subject).

[0038] The term "pharmaceutically acceptable salt" means either an acid addition salt or a base addition salt that is suitable or compatible with the treatment of a subject.

[0039] Acid addition salts suitable or compatible with the treatment of a subject are any non-toxic organic acid addition salts or non-toxic inorganic acid addition salts of any basic compounds.

[0040] A base addition salt suitable for or compatible with the treatment of a subject is any non-toxic organic or inorganic base addition salt of any acidic compound.

[0041] As used herein, the term "prodrug" means a compound or a salt and / or solvate of a compound that is converted into an active drug after administration.

[0042] The term "solvate" as used herein means a compound or a salt or prodrug of a compound, wherein molecules of a suitable solvent are incorporated into the crystal lattice.

[0043] The term "inert organic solvent" as used herein refers to a solvent that is generally considered to be non-reactive with the functional groups present in the compounds being combined together in any given reaction, and therefore will not interfere with or inhibit the necessary synthetic transformations. Organic solvents are typically non-polar and will dissolve compounds that are insoluble in aqueous solutions.

[0044] As used herein, the term "alkyl," whether used alone or as part of another group, refers to a straight or branched chain saturated alkyl group. The number of carbon atoms possible in the alkyl group referred to is indicated by the "C n1~n2 " prefix. For example, C 1~10 The term alkyl refers to alkyl groups having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms.

[0045] The term "alkylene," whether used alone or as part of another group, refers to a straight or branched saturated alkylene group, i.e., a saturated carbon chain containing substituents at two of its termini. The number of possible carbon atoms in a referenced alkylene group is indicated by the term "C n1~n2 " prefix. For example, C 1~10 The term alkylene refers to alkylene groups having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms. All alkyl groups, unless otherwise indicated, may be fluoro-substituted.

[0046] As used herein, the term "alkenyl," whether used alone or as part of another group, refers to a straight or branched chain unsaturated alkyl group containing at least one double bond. The number of carbon atoms possible in the recited alkylene group is indicated by the "C n1~n2 " prefix. For example, C 2~6 The term alkenyl refers to an alkenyl group having 2, 3, 4, 5, or 6 carbon atoms and at least one double bond.

[0047] The term "alkynyl" as used herein, whether used alone or as part of another group, refers to a straight or branched chain unsaturated alkynyl group containing at least one triple bond. The number of carbon atoms possible in the alkyl group referred to is indicated by the "C n1~n2 " prefix. For example, C 2~6 The term alkynyl refers to alkynyl groups having 2, 3, 4, 5 or 6 carbon atoms.

[0048] As used herein, the term "heterocycloalkyl," whether used alone or as part of another group, refers to a cyclic group containing at least one non-aromatic ring containing 3 to 10 atoms, where one or more of the atoms are O, S, S(O), SO, N, NH, and N(C 1~6Heterocycloalkyl groups are either saturated or unsaturated (i.e., contain one or more double bonds). Heterocycloalkyl groups are represented by the prefix C. n1~n2 , this prefix indicates the number of carbon atoms in the corresponding carbocyclic group, where one or more, preferably one to five, of the ring atoms are replaced with a heteroatom as defined above. The heterocycloalkyl group may be optionally benzo-fused.

[0049] All cyclic groups, including aryl, heteroaryl, heterocycloalkyl, and cycloalkyl groups, contain one ring (i.e., are monocyclic) or multiple rings (i.e., are polycyclic). When a cyclic group contains multiple rings, the rings may be fused, bridged, or spiro-fused.

[0050] The term "benzofused," as used herein, refers to a polycyclic group in which the benzene ring is fused to another ring.

[0051] A first ring is "fused" to a second ring means that the first ring and the second ring share two adjacent atoms between them.

[0052] A first ring is "bridged" with a second ring means that the first ring and the second ring share two non-adjacent atoms between them.

[0053] A first ring is "spirofused" to a second ring means that the first ring and the second ring share one atom between them.

[0054] The term "haloalkyl" as used herein refers to an alkyl group as defined above in which one or more of the available hydrogen atoms has been replaced with a halogen. Thus, for example, "C 1~6"Haloalkyl" (or "C1-C6 haloalkyl") refers to a C1-C6 straight or branched chain alkyl group, as defined above, having one or more halogen substituents.

[0055] The terms "halo" or "halogen," as used herein, whether used alone or as part of another group, refer to a halogen atom and include fluoro, chloro, bromo, and iodo.

[0056] The term "chloroalkyl" as used herein refers to a haloalkyl group, as defined above, in which the halogen atom is chloro.

[0057] The term "fluoroalkyl" as used herein refers to a haloalkyl group, as defined above, in which the halogen atom is fluoro.

[0058] The term "available" in "available hydrogen atom" or "available atom" refers to the fact that one of ordinary skill in the art knows that the atom can be replaced by another atom or group.

[0059] As used herein, the term "optionally substituted" means that the referenced group is unsubstituted or substituted.

[0060] The term "treat" or "treatment" as used herein and as well understood in the art, refers to an approach to obtain beneficial or desired results, such as clinical results. Beneficial or desired clinical results may include, but are not limited to, alleviation or amelioration of one or more signs or symptoms, whether detectable or undetectable, reduction in the severity of a disease, disorder, or condition, stabilization (i.e., not worsening) of the condition of a disease, disorder, or condition, prevention of the spread of a disease, disorder, or condition, delay or slowing of the progression of a disease, disorder, or condition, improvement or alleviation of the condition of a disease, disorder, or condition, reduction in the recurrence of a disease, disorder, or condition, inhibition or reduction of a disease, disorder, or condition, and remission (partial or complete). "Treat" and "treatment" may also mean to prolong survival compared to the expected survival in the absence of treatment. As used herein, the terms "treat" and "treatment" also include prophylactic treatment.

[0061] "Alleviating" a disease, disorder, or condition means that the severity and / or undesirable clinical symptoms of the disease, disorder, or condition are reduced and / or the time course of progression is slowed or delayed compared to when the disease, disorder, or condition is not treated.

[0062] As used herein, the terms "prevention" or "prophylaxis," or synonyms thereof, refer to a reduction in the risk or probability of a subject suffering from a disease, disorder, or condition treatable by inhibition of BAX, or a reduction in the risk or probability of a subject developing symptoms associated with a disease, disorder, or condition treatable by inhibition of BAX.

[0063] As used herein, the term "effective amount" or "therapeutically effective amount" means an amount of a compound or compounds of the present application that is effective, at dosages and for periods of time necessary, to achieve the desired result.

[0064] The term "disease, disorder, or condition treatable by inhibiting Bcl2-associated X protein (BAX) and / or Bcl-2 antagonist killer (BAK)" means that the disease, disorder, or condition to be treated is affected or modulated by and / or has some biological basis (either direct or indirect) that involves BAX activity and / or BAK activity (particularly increased BAX activity). Such diseases respond favorably when BAX activity and / or BAK activity associated with the disease, disorder, or condition is inhibited by one or more of the compounds or compositions of the present application.

[0065] As used herein, the phrase "inhibiting Bcl2 associated X protein (BAX) and / or Bcl-2 antagonist killer (BAK)" refers to inhibiting, blocking, and / or interfering with the activity or function of BAX and / or BAK in a cell, which inhibition, blocking, and / or interfering provides a therapeutic effect to the cell.

[0066] By "inhibition, blocking, and / or interference" is meant any inhibition, blocking, and / or interference that is detectable in the presence of a compound relative to otherwise identical conditions in the absence of the compound.

[0067] The expression "Bcl2 associated X protein (BAX)-mediated cell death and / or Bcl-2 antagonist killer (BAK)-mediated cell death" means that the cell death is influenced or regulated by and / or has some biological basis (either direct or indirect) that involves BAX activity and / or BAK activity (in particular increased BAX activity).

[0068] As used herein, the term "BAX" refers to the Bcl2-associated X protein or any functional variant or analogous form thereof.

[0069] As used herein, the term "BAK" refers to Bcl-2 antagonist killer protein or any functional variant or analogous form thereof.

[0070] As used herein, the term "apoptosis" refers to programmed cell death.

[0071] As used herein, the term "MOMP" refers to mitochondrial outer membrane permeabilization.

[0072] The term "chemotherapeutic agent-induced cardiomyopathy" refers to any detectable increase in cardiomyopathy after chemotherapy treatment compared to cardiomyopathy before chemotherapy treatment.

[0073] The phrase "increasing donor hematopoietic stem and progenitor cell (HSPC) survival during transplant" refers to the presence of a compound of the present application resulting in a detectable increase in HSPC survival during transplant relative to otherwise identical conditions in the absence of the compound of the present application.

[0074] As used herein, the term "dacomitinib" refers to a compound of the following formula having the chemical name (E)-N-[4-(3-chloro-4-fluoroanilino)-7-methoxyquinazolin-6-yl]-4-piperidin-1-ylbut-2-enamide. [ka]

[0075] As used herein, the term "administering" means administering a therapeutically effective amount of a compound, or one or more compounds or compositions of the present application to a cell or subject.

[0076] II. Compounds of the Present Application The present application has prepared isoquinoline compounds and found that the isoquinoline compounds of the present application inhibit Bcl2 associated X protein (BAX) and / or Bcl-2 antagonist killer (BAK). These compounds were obtained from chemical modification of dacomitinib, a known therapeutic agent and known kinase inhibitor. The present applicant has surprisingly found that the resulting compounds have both BAX inhibitory activity and BAK inhibitory activity. Through systematic modification of the quinazoline core structure of dacomitinib, the present applicant has developed the isoquinoline compounds of the present application. The isoquinoline compounds of the present application have been found to be effective inhibitors of actinomycin D-mediated cell death in cells expressing either BAX or BAK, although they do not exhibit kinase inhibitory activity.

[0077] Thus, the present application includes compounds of Formula I, or pharma- ceutically acceptable salts, solvates, and / or prodrugs thereof. [ka] During the ceremony, Each R 1 are independently halo, CN, C 1~4 Alkyl, C 1~4 Haloalkyl, OC 1~4 Alkyl and OC 1~4 haloalkyl; R 2 is absent or R 2 Halo, C 1~4 Alkyl, C 1~4 Haloalkyl, OC 1~4 Alkyl and OC 1~4 haloalkyl; R 3 H and C 1~4 alkyl, L is for C 1~4 Alkylene, C 2~4 Alkenylene, and C 2~4 alkynylene, A is N, NH, and N(C 1~4 alkyl), and containing at least one ring hetero moiety selected from OH, halo, C1~2 Alkyl, C 1~2 Haloalkyl, OC 1~2 Alkyl and OC 1~2 C optionally substituted with one or two substituents selected from haloalkyl 3~8 is heterocycloalkyl, n is selected from 0, 1, 2, and 3.

[0078] The present application includes compounds of Formula I, or pharma- ceutically acceptable salts, solvates, and / or prodrugs thereof. [ka] During the ceremony, Each R 1 Independently, Halo, C 1~4 Alkyl, and C 1~4 haloalkyl; R 2 is absent or R 2 Halo, C 1~4 Alkyl, C 1~4 Haloalkyl, OC 1~4 Alkyl and OC 1~4 haloalkyl; L is for C 1~4 Alkylene, C 2~4 Alkenylene, and C 2~4 alkynylene, A is N, NH, and N(C 1~4 alkyl), and one or two C 1~2 C optionally substituted with alkyl 3~8 is heterocycloalkyl, n is an integer selected from 1 to 3.

[0079] In some embodiments, each R 1 are independently F, Cl, Br, CN, and C 1~4 Alkyl, C 1~4 Haloalkyl, OC 1~4 Alkyl and OC 1~4In some embodiments, each R is selected from haloalkyl. 1 are independently F, Cl, Br, CN, and C 1~4 Alkyl, C 1~4 Fluoroalkyl, C 1~4 Chloroalkyl, OC 1~4 Alkyl, OC 1~4 Fluoroalkyl and OC 1~4 In some embodiments, each R 1 is independently selected from F, Cl, Br, CN, CH3, CH2CH3, CH2CH2CH3, CH(CH3)2, CH(CH3)CH2CH3, CH(CH3)3, CF3, CHF2, CFH2, CH2CHF2, CH2CF3, CH2CFH2, CCl3, CH2CClH2, CCl2H, CH2CCl2H, CH2CCl3, CH3, OCH2CH3, OCH2CH2CH3, OCH(CH3)2, OCH(CH3)CH2CH3, OCH(CH3)3, OCF3, OCHF2, OCH2CHF2, OCH2CF3, OCH2CFH2, OCCl3, OCH2CClH2, OCCl2H, OCH2CCl2H, and CH2CCl3. 1 is independently selected from F, Cl, Br, CN, CH3, CH2CH3, CH2CH2CH3, CH(CH3)2, CF3, CHF2, CFH2, CH2CHF2, CH2CF3, CH2CFH2, CCl3, CH2CClH2, CCl2H, CH2CCl2H, CH2CCl3, OCH3, OCH2CH3, OCH2CH2CH3, OCH(CH3)2, OCF3, OCHF2, OCH2CHF2, OCH2CF3, OCH2CFH2, OCCl3, OCH2CClH2, OCCl2H, OCH2CCl2H, and OCH2CCl3. 1is independently selected from F, Cl, Br, CN, CH3, CH2CH3, CH2CH2CH3, CH(CH3)2, CF3, CHF2, CFH2, CH2CHF2, CH2CF3, CH2CFH2, CCl3, CH2CClH2, CCl2H, CH2CCl2H, CH2CCl3, OCH3, OCF3, OCHF2, OCH2CHF2, OCH2CF3, OCH2CFH2, OCCl3, OCH2CClH2, OCCl2H, OCH2CCl2H, and OCH2CCl3. 1 is independently selected from F, Cl, CN, CH3, CH2CH3, CH2CH2CH3, CH(CH3)2, CF3, CHF2, CFH2, CCl3, CCl2H, OCH3, OCF3, OCHF2, OCCl3, and OCCl2H. In some embodiments, each R 1 is independently selected from F, Cl, CN, CH3, CH2CH3, CH2CH2CH3, CH(CH3)2, CF3, CHF2, CCl3, CCl2H, OCH3, OCF3, OCHF2, OCCl3, and OCCl2H. In some embodiments, each R 1 is independently selected from F, Cl, CN, CH3, CH2CH3, CH2CH2CH3, CH(CH3)2, CF3, CHF2, CCl3, OCH3, OCF3, and OCHF2. 1 is independently selected from F, Cl, CN, CH3, CH2CH3, CH2CH2CH3, CH(CH3)2, CF3, OCH3, and OCF3. 1 is CN. In some embodiments, each R 1 is independently selected from F, Cl, and CF. In some embodiments, each R 1 are independently F, Cl, OCH3, and OC F In some embodiments, each R 1 is independently selected from F, Cl, CH, CF, CHCH, CHCHCH, and CH(CH). 1is independently selected from F, Cl, CH, CHCH, CHCHCH, and CH(CH). 1 is independently selected from F and Cl.

[0080] In some embodiments, each R 1 are independently F, Cl, Br, C 1~4 Alkyl, and C 1~4 In some embodiments, each R is selected from haloalkyl. 1 is independently selected from F, Cl, Br, CH3, CH2CH3, CH2CH2CH3, CH(CH3)2, CH(CH3)CH2CH3, CH(CH3)3, CF3, CHF2, CH2CHF2, CH2CF3, CH2CFH2, CCl3, CH2CClH2, CCl2H, CH2CCl2H, and CH2CCl3. 1 is independently selected from F, Cl, Br, CH3, CH2CH3, CH2CH2CH3, CH(CH3)2, CF3, CHF2, CH2CHF2, CH2CF3, CH2CFH2, CCl3, CH2CClH2, CCl2H, CH2CCl2H, and CH2CCl3. 1 is independently selected from F, Cl, Br, CH, CF, CHF, CHCHF, CHCF, CHCFH, CCl, CHCClH, CClH, CHCClH, and CHCCl. 1 is independently selected from F, Cl, Br, CH, CF, CHF, CCl, and CClH. In some embodiments, each R 1 is independently selected from F, Cl, Br, CHF2, and CF3. In some embodiments, each R 1 is independently selected from F, Cl, Br, CF, and CHF. In some embodiments, each R 1 is independently selected from F, Cl, CF, and CHF. In some embodiments, each R 1 is independently selected from F, Cl, and CF. In some embodiments, each R1 is independently selected from F and Cl.

[0081] In some embodiments, n is 0. In some embodiments, n is 1 or 2. In some embodiments, n is 1, 2, or 3. In some embodiments, n is 2 or 3. In some embodiments, n is 2.

[0082] In some embodiments, R 2 are F, Br, Cl, and C. 1~4 Alkyl, C 1~4 Haloalkyl, OC 1~4 Alkyl and OC 1~4 In some embodiments, R is selected from haloalkyl. 2 are F, Cl, Br, and C 1~4 Alkyl, C 1~4 Fluoroalkyl, C 1~4 Chloroalkyl, OC 1~4 Alkyl, OC 1~4 Fluoroalkyl and OC 1~4 In some embodiments, R 2 are F, Br, Cl, and C. 1~4 Alkyl, C 1~4 Haloalkyl, OC 1~3 Alkyl and OC 1~3 In some embodiments, R is selected from haloalkyl. 2 are F, Br, Cl, and C. 1~3 Alkyl, C 1~3 Fluoroalkyl, C 1~3 Chloroalkyl, OC 1~3 Alkyl, OC 1~3 Fluoroalkyl and OC 1~3 chloroalkyl.

[0083] In some embodiments, R 2is selected from F, Cl, Br, CH3, CH2CH3, CH2CH2CH3, CH(CH3)2, CF3, CHF2, CH2CHF2, CH2CF3, CH2CFH2, CCl3, CH2CClH2, CCl2H, CH2CCl2H, CH2CCl3, OCH3, OCH2CH3, OCH2CH2CH3, OCH(CH3)2, OCF3, OCHF2, OCH2CHF2, OCH2CF3, OCH2CFH2, OCCl3, OCH2CClH2, OCCl2H, OCH2CCl2H, and OCH2CCl3. 2 is selected from F, Cl, Br, CH3, CH2CH3, CF3, CHF2, CCl3, CCl2H, OCH3, OCH2CH3, OCH2CH2CH3, OCH(CH3)2, OCF3, OCHF2, OCH2CHF2, OCH2CF3, OCH2CFH2, OCCl3, OCH2CClH2, OCCl2H, OCH2CCl2H, and OCH2CCl3. 2 is selected from F, Cl, Br, CH3, CH2CH3, CF3, CHF2, CCl3, CCl2H, CH2CCl2H, CH2CCl3, OCH3, OCH2CH3, OCF3, OCHF2, OCH2CHF2, OCH2CF3, OCH2CFH2, OCCl3, OCH2CClH2, OCCl2H, OCH2CCl2H, and OCH2CCl3. 2 is selected from F, Cl, Br, CH3, CH2CH3, OCH3, CF3, CHF2, CCl3, CCl2H, OCF3, OCHF2, OCCl3, and OCCl2H. In some embodiments, R 2 is selected from F, Cl, CH3, CF3, CHF2, CCl3, CCl2H, OCH3, OCH2CH3, OCH2CH2CH3, OCH(CH3)2, OCF3, OCHF2, OCCl3, and OCCl2H. 2 is selected from CF, CHF, CCl, CClH, OCH, OCF, OCHF, OCCl, and OCClH. 2is selected from F, Cl, CH3, CF3, OCH3, OCH2CH3, OCH2CH2CH3, OCH(CH3)2, OCF3, and OCHF2. 2 is CH3. In some embodiments, R 2 is selected from OCH, OCF, OCHF, OCCl, and OCClH. In some embodiments, R 2 is selected from OCH, OCHCH, OCHCHCH, OCH(CH), OCF, and OCHF. 2 is selected from F and Cl. In some embodiments, R 2 is selected from OCH, OCF, and OCCl. In some embodiments, R 2 is OCH3.

[0084] In some embodiments, R 2 is absent.

[0085] In some embodiments, R 3 H and C 1~3 In some embodiments, R 3 is selected from H, CH, CHCH, CHCHCH, and CH(CH). 3 is selected from H and CH. In some embodiments, R 3 is H.

[0086] In some embodiments, L is C 1~4 Alkylene and C 2-4 In some embodiments, L is selected from C 1~4In some embodiments, L is selected from CH2CH2CH2CH2, CH2CH2CH2, CH2CH2, and CH2. In some embodiments, L is selected from CH2CH2CH2CH2, CH2CH2CH2, and CH2CH2. In some embodiments, L is selected from CH2CH2CH2CH2(C3 alkylene) and CH2CH2(C2 alkylene). In some embodiments, L is CH2CH2. In some embodiments, L is CH2CH2CH2.

[0087] In some embodiments, L is C 2~4 In some embodiments, L is selected from CHCHCH2CH2, CHCHCH2, CHCH, CH2CHCHCH2, and CH2CHCH. In some embodiments, L is selected from CHCHCH2CH2, CHCHCH2, CH2CHCHCH2, and CH2CHCH. In some embodiments, L is selected from CHCHCH2 and CH2CHCH.

[0088] In some embodiments, A is selected from N, NH, and N(C 1~4 alkyl), and OH, F, Cl, C 1~2 Alkyl, C 1~2 Fluoroalkyl, C 1~2 Chloroalkyl, OC 1~2 Alkyl, OC 1~2 Fluoroalkyl and OC 1~2 C optionally substituted with one or two substituents selected from chloroalkyl 3~8 In some embodiments, A is selected from the group consisting of N, NH, and N(C 1~4 alkyl), and OH, F, Cl, C 1~2 Alkyl, C 1~2 Fluoroalkyl, C 1~2 Chloroalkyl, OC 1~2 Alkyl, OC 1~2 Fluoroalkyl and OC 1~2C optionally substituted with one or two substituents selected from chloroalkyl 3~6 It is a heterocycloalkyl.

[0089] In some embodiments, A is selected from N, NH, and N(C 1~4 alkyl), and OH, F, Cl, C 1~2 C optionally substituted with one or two substituents selected from alkyl, CF3, CHF2, CCl3, CCl2H, OCH3, OCH2CH3, OCF3, OCHF2, OCH2CHF2, OCH2CF3, OCH2CFH2, OCCl3, OCH2CClH2, OCCl2H, OCH2CCl2H, and OCH2CCl3; 3~6 It is a heterocycloalkyl.

[0090] In some embodiments, C includes at least one ring heteromoiety selected from N, NH, and N. 3~6 Heterocycloalkyl is selected from aziridinyl, azetidinyl, pyrrolidinyl, morpholinyl, piperazinyl, and piperidinyl, and A is OH, F, Cl, C 1~2 A is selected from aziridinyl, azetidinyl, pyrrolidinyl, morpholinyl, piperazinyl, and piperidinyl, optionally substituted with one or two of alkyl, CF3, CHF2, CCl3, CCl2H, OCH3, OCH2CH3, OCF3, OCHF2, OCH2CHF2, OCH2CF3, OCH2CFH2, OCCl3, OCH2CClH2, OCCl2H, OCH2CCl2H, and OCH2CCl3. In some embodiments, A is selected from aziridinyl, azetidinyl, pyrrolidinyl, morpholinyl, piperazinyl, and piperidinyl, and is substituted with one or two of C 1~2 In some embodiments, A is selected from pyrrolidinyl, morpholinyl, piperazinyl, and piperidinyl, and is substituted with OH, F, Cl, C 1~2and optionally substituted with one or two substituents selected from alkyl, CF, CHF, OCH, OCHCH, OCF, OCHF, OCHCHF, OCHCF, and OCHCFH. In some embodiments, A is selected from pyrrolidinyl, morpholinyl, piperazinyl, and piperidinyl, and is substituted with one or two C 1~2 In some embodiments, A is selected from pyrrolidinyl, morpholinyl, piperazinyl, and piperidinyl, and may be substituted with one or two substituents selected from OH, F, Cl, CH3, CH2CH3, CF3, CHF2, OCH3, OCH2CH3, OCF3, OCHF2, OCH2CHF2, OCH2CF3, and OCH2CFH2. In some embodiments, A is selected from pyrrolidinyl, morpholinyl, piperazinyl, and piperidinyl, and may be substituted with one or two substituents selected from OH, F, Cl, CH3, CF3, CHF2, OCH3, OCF3, and OCHF2. In some embodiments, A is selected from pyrrolidinyl, morpholinyl, piperazinyl, and piperidinyl, and may be substituted with one or two substituents selected from F, Cl, CH3, CF3, OCH3, and OCF3. In some embodiments, A is selected from pyrrolidinyl, morpholinyl, piperazinyl, and piperidinyl, and is optionally substituted with one or two CH3. In some embodiments, A is selected from pyrrolidinyl, morpholinyl, piperazinyl, and piperidinyl, and is optionally substituted with one or two CH3.

[0091] In some embodiments, A is selected from pyrrolidinyl, morpholinyl, piperazinyl, and piperidinyl, and is optionally substituted with one or two F. In some embodiments, A is selected from pyrrolidinyl, morpholinyl, piperazinyl, and piperidinyl, and is optionally substituted with one or two F.

[0092] In some embodiments, A is selected from pyrrolidinyl, morpholinyl, piperazinyl, and piperidinyl, and is optionally substituted with one or two substituents selected from OCH3 and OCH3. In some embodiments, A is selected from pyrrolidinyl, morpholinyl, piperazinyl, and piperidinyl, and is optionally substituted with one or two substituents selected from OCH3 and OCH3.

[0093] In some embodiments, A is selected from pyrrolidinyl, piperazinyl, and piperidinyl, and one or two C 1~2 In some embodiments, A is selected from pyrrolidinyl, piperazinyl, and piperidinyl, and is substituted with one or two C 1~2 In some embodiments, A is piperidinyl and is substituted with one or two C 1~2 It may be substituted with alkyl.

[0094] In some embodiments, A is unsubstituted. In some embodiments, A is selected from pyrrolidinyl, morpholinyl, piperazinyl, and piperidinyl. In some embodiments, A is selected from pyrrolidinyl, piperazinyl, and piperidinyl. In some embodiments, A is piperidinyl.

[0095] In some embodiments, n is 2 and each R 1 is independently selected from F, Cl, CN, CH3, CH2CH3, CH2CH2CH3, CH(CH3)2, CF3, OCH3, and OCF3. In some embodiments, n is 2 and each R 1 is independently selected from F, Cl, Br, CH, CF, CHF, CCl, and CClH. In some embodiments, n is 2 and each R 1 is independently selected from F, Cl, Br, CHF2, and CF3. In some embodiments, n is 2 and each R 1is independently selected from F, Cl, CF, and CHF. In some embodiments, n is 2 and each R 1 is independently selected from F, Cl, and CF. In some embodiments, n is 2 and each R 1 is independently selected from F and Cl.

[0096] In some embodiments, n is 2 or 3, and each R 1 is independently selected from F and Cl; R 2 is selected from F, Cl, CH3, CF3, OCH3, OCH2CH3, OCH2CH2CH3, OCH(CH3)2, OCF3, and OCHF2. In some embodiments, n is 2 or 3, and each R 1 is independently selected from F and Cl; R 2 , R 2 In some embodiments, n is 2 or 3, and each R 1 is independently selected from F and Cl; R 2 is absent.

[0097] In some embodiments, the compound of formula I is a compound of formula IA or a pharma- ceutically acceptable salt, solvate, and / or prodrug thereof. [ka] During the ceremony, R 1 , R 2 , L, and n are as defined in formula I; m is an integer selected from 1 to 3.

[0098] In some embodiments, the compound of formula I is selected from the compounds listed below. [Table 1] TIFF2025506245000008.tif240159TIFF2025506245000009.tif242159TIFF2025506245000010.tif242156TIFF2025506245000011.tif242157TIFF2025506245000012.tif242157TIFF2025506245000013.tif231159TIFF2025506245000014.tif47159 or a pharma- ceutically acceptable salt, solvate, and / or prodrug thereof.

[0099] In some embodiments, the compound of formula I is [ka] or a pharma- ceutically acceptable salt, solvate, and / or prodrug thereof.

[0100] In one embodiment, the pharma- ceutically acceptable salt is an acid or base addition salt. The selection of a suitable salt can be made by those skilled in the art (see, for example, S. M. Berge, et al., "Pharmaceutical Salts," J. Pharm. Sci. 1977, 66, 1-19).

[0101] The acid addition salt suitable for or compatible with the treatment of the subject is any non-toxic organic acid addition salt or non-toxic inorganic acid addition salt of any basic compound.Basic compounds that form acid addition salts include, for example, compounds that contain amine groups.Exemplary inorganic acids that form suitable salts include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, and phosphoric acid, as well as acid metal salts, for example, sodium monohydrogen orthophosphate and potassium hydrogen sulfate.Exemplary organic acids that form suitable salts include monocarboxylic acids, dicarboxylic acids, and tricarboxylic acids. Examples of such organic acids include, for example, acetic acid, trifluoroacetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, malonic acid, succinic acid, glutaric acid, fumaric acid, malic acid, tartaric acid, citric acid, ascorbic acid, maleic acid, hydroxymaleic acid, benzoic acid, hydroxybenzoic acid, phenylacetic acid, cinnamic acid, mandelic acid, salicylic acid, 2-phenoxybenzoic acid, p-toluenesulfonic acid, and other sulfonic acids, such as methanesulfonic acid, ethanesulfonic acid, and 2-hydroxyethanesulfonic acid. In one embodiment, mono- or di-acid salts are formed, and such salts exist either in hydrate, solvate, or substantially anhydrous form. Acid addition salts are usually more soluble in water and various hydrophilic organic solvents than their free base forms, and usually exhibit higher melting points. The criteria for selecting a suitable salt are known to those skilled in the art. Other non-pharmacologically acceptable salts (such as, but not limited to, oxalates) may be used, for example, for isolation of the compounds of the present application for experimental purposes or for subsequent conversion to a pharma-ceutically acceptable acid addition salt.

[0102] The base addition salt suitable for or compatible with the treatment of the subject is any non-toxic organic base addition salt or non-toxic inorganic base addition salt of any acidic compound.The acidic compounds that form base addition salts include, for example, compounds that contain a carboxylic acid group.The exemplary inorganic bases that form suitable salts include lithium hydroxide, sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide, or barium hydroxide, and ammonia.The exemplary organic bases that form suitable salts include aliphatic, alicyclic, or aromatic amines, such as isopropylamine, methylamine, trimethylamine, picoline, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purine, piperazine, piperidine, N-ethylpiperidine, polyamine resins, etc. Exemplary organic bases include isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine. The selection of a suitable salt may be useful, for example, if there is an ester function elsewhere in the compound, so that it is not hydrolyzed. The criteria for selecting a suitable salt are known to those skilled in the art.

[0103] Solvates of the compounds of the present application include, for example, those prepared using pharma- ceutically acceptable solvents. Examples of such solvents include water (the resulting solvates are called hydrates) and ethanol. Suitable solvents are those that are physiologically acceptable at the doses administered.

[0104] In an embodiment of the present application, the compounds described herein may have at least one asymmetric center. When a compound has multiple asymmetric centers, they may exist as diastereomers. It is understood that all such isomers and mixtures of isomers in any proportion are encompassed within the scope of the present application. It is further understood that while the stereochemistry of a compound may be as shown for any given compound listed herein, such a compound may also contain a certain amount (e.g., less than 20%, preferably less than 10%, more preferably less than 5%) of a compound of the present application having a different stereochemistry. It is intended that any optical isomer as an isolated, pure or partially purified optical isomer or a racemic mixture thereof is encompassed within the scope of the present application.

[0105] The compounds of the present application may also exist in different tautomeric forms and it is intended that any tautomeric forms that the compounds form, as well as mixtures thereof, are included within the scope of the present application.

[0106] The compounds of the present application may furthermore exist in different polymorphic forms and any polymorphs or mixtures thereof which form are considered to be included within the scope of the present application.

[0107] The compounds of the present application may further be radiolabeled, and therefore all radiolabeled versions of the compounds of the present application are included within the scope of the present application. The compounds of the present application also include compounds that have one or more radioactive atoms incorporated into their structure.

[0108] III. Compositions of the Present Application The compounds of the present application are suitably formulated into compositions using one or more carriers by conventional methods. Thus, the present application also includes compositions comprising one or more compounds of the present application and a carrier. The compounds of the present application are suitably formulated into pharmaceutical compositions for administration to a subject in a biologically compatible form suitable for administration in vivo. Thus, the present application further includes pharmaceutical compositions comprising one or more compounds of the present application and a pharma- ceutically acceptable carrier. In an embodiment of the present application, the pharmaceutical composition is used to treat any of the diseases, disorders, or conditions described herein.

[0109] The compounds of the present application may be administered to a subject in various forms depending on the route of administration selected, as will be apparent to those skilled in the art. For example, the compounds of the present application may be administered orally, by inhalation, parenterally, buccal, sublingual, nasal, rectal, vaginal, patch, pump, minipump, topical, or transdermal administration, and the pharmaceutical compositions may be formulated accordingly. In some embodiments, administration is by pump for periodic or continuous delivery. The selection and preparation of suitable compositions and typical procedures and ingredients are described, for example, in Remington's Pharmaceutical Sciences (2000-20th edition) and The United States Pharmacopeia: The National Formulary (USP 24 NF19) (published in 1999).

[0110] Parenteral administration includes systemic delivery routes other than the gastrointestinal (GI) tract, and includes, for example, intravenous, intraarterial, intraperitoneal, subcutaneous, intramuscular, transepithelial, intranasal, intrapulmonary (e.g., by use of an aerosol), intrathecal, rectal, and topical (including use of a patch or other transdermal delivery device) modes of administration. Parenteral administration can be by continuous infusion over a selected period of time.

[0111] In some embodiments, the compounds of the present application are administered orally, for example, with an inert diluent or with an assimilable edible carrier, or are enclosed in hard or soft shell gelatin capsules, or are compressed into tablets, or are taken directly with dietary food. In some embodiments, the compounds are incorporated with additives and used in the form of ingestible tablets, buccal tablets, lozenges, capsules, caplets, pellets, granules, lozenges, chewing gum, powders, syrups, elixirs, wafers, aqueous solutions, and suspensions. In the case of tablets, the carriers used include lactose, cornstarch, sodium citrate, and salts of phosphoric acid. Pharmaceutically acceptable additives include binders (e.g., pregelatinized maize starch, polyvinylpyrrolidone, or hydroxypropylmethylcellulose), fillers (e.g., lactose, microcrystalline cellulose, or calcium phosphate), lubricants (e.g., magnesium stearate, talc, or silica), disintegrants (e.g., potato starch or sodium starch glycolate), or wetting agents (e.g., sodium lauryl sulfate). In embodiments, tablets are coated by methods well known in the art. For tablets, capsules, caplets, pellets, or granules for oral administration, pH-sensitive enteric coatings such as Eudragits™, which are designed to control the release of active ingredients, are optionally used. Oral dosage forms also include modified release formulations, such as immediate release formulations and sustained release formulations. Examples of modified release formulations include, for example, sustained release (SR), extended release (ER, XR, or XL), time-release or timed-release, controlled release (CR), or continuous release (CR or Contin) formulations used, for example, in the form of coated tablets, osmotic delivery devices, coated capsules, microencapsulated microspheres, agglomerated particles (e.g., molecular sieve-type particles), or hollow permeable fine fiber bundles or hollow permeable staple fibers aggregated or held in fiber packets.The sustained release composition is formulated, for example, as liposomes, or the active compound is protected with differently degradable coatings, such as by microencapsulation, multiple coatings, etc. Liposomal delivery systems include, for example, small unilamellar vesicles, large unilamellar vesicles, and multilamellar vesicles. In some embodiments, liposomes are formed from various phospholipids, such as cholesterol, stearylamine, or phosphatidylcholine. For oral administration in capsule form, useful carriers or diluents include lactose and dried cornstarch.

[0112] In some embodiments, liquid preparations for oral administration may take the form of, for example, solutions, syrups, or suspensions, or may be suitably provided as a dry product for constitution with water or other suitable vehicle before use. When aqueous suspensions and / or emulsions are administered orally, the compounds of the present application are suitably suspended or dissolved in an oily phase in combination with an emulsifying and / or suspending agent. If necessary, certain sweetening and / or flavoring and / or coloring agents are added. Such liquid preparations for oral administration are prepared by conventional means using pharma- ceutically acceptable additives (e.g., suspending agents (e.g., sorbitol syrup, methylcellulose, or edible hydrogenated fats and oils), emulsifying agents (e.g., lecithin or acacia), non-aqueous vehicles (e.g., almond oil, oily esters, or ethyl alcohol), and preservatives (e.g., methyl or propyl p-hydroxybenzoate, or sorbic acid). Useful diluents include lactose and high molecular weight polyethylene glycols.

[0113] The compounds of the present application can also be lyophilized and the resulting lyophilizates used, for example, for the preparation of injectable products.

[0114] In some embodiments, the compounds of the present application are administered parenterally. For example, solutions of the compounds of the present application are prepared in water, suitably mixed with a surfactant such as hydroxypropylcellulose. In some embodiments, dispersions are prepared in glycerol, liquid polyethylene glycols, DMSO, and mixtures thereof, with or without alcohol, or in oils. These preparations contain preservatives to prevent the growth of microorganisms under normal storage and use conditions. Methods for preparing suitable preparations are known to those skilled in the art. For parenteral administration, sterile solutions of the compounds of the present application are usually prepared, and the pH of the solution is appropriately adjusted and buffered. For intravenous use, it is desirable to control the total concentration of solutes so that the formulation is isotonic. For ocular administration, ointments or droppable liquids are delivered by ocular delivery systems known in the art, such as applicators or eye droppers. In some embodiments, such compositions contain a mucus mimetic agent such as hyaluronic acid, chondroitin sulfate, hydroxypropylmethylcellulose, or polyvinyl alcohol, a preservative such as sorbic acid, EDTA, or benzyl chromium chloride, and a conventional amount of diluent or carrier. For pulmonary administration, the diluent or carrier is selected to be appropriate to allow the formation of an aerosol.

[0115] In some embodiments, the compounds of the present application are formulated for parenteral administration by injection (including conventional catheterization or infusion). Preparations for injection are provided, for example, in unit dosage form (e.g., ampoules) or in multi-dose containers with added preservatives. In some embodiments, the compositions take the form of a sterile suspension, solution, or emulsion in an oily or aqueous medium, and further contain formulatory agents such as suspending, stabilizing, and / or dispersing agents. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. Alternatively, the compounds of the present application are preferably in the form of a sterile powder for reconstitution with a suitable medium (e.g., sterile pyrogen-free water) before use.

[0116] In some embodiments, compositions for nasal administration are conveniently formulated as aerosols, nasal drops, gels, and powders. For intranasal administration or administration by inhalation, the compounds of the present application are conveniently delivered in the form of solutions, dry powder formulations, or suspensions from pump spray containers that are pushed or pumped by the patient, or as aerosol sprays from pressurized containers or nebulizers. Aerosol formulations typically include solutions or fine suspensions of active substances in physiologically acceptable aqueous or non-aqueous solvents, and are usually provided in single or multiple doses in sterile form in sealed containers, which may take the form of, for example, cartridges or refills for use with nebulizers. Alternatively, the sealed container is an integrated dispenser, such as a single-dose nasal inhaler, or an aerosol dispenser with a metering valve intended for disposal after use. When the dosage form is an aerosol dispenser, the dosage form contains a propellant. The propellant is, for example, a compressed gas, such as compressed air, or an organic propellant, such as fluorochlorohydrocarbons. Suitable propellants include, but are not limited to, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, heptafluoroalkanes, carbon dioxide, or other suitable gases. In the case of a pressurized aerosol, the dosage unit is suitably determined by providing a valve to deliver a metered amount. In some embodiments, the pressurized container or nebulizer contains a solution or suspension of the active compound. Capsules and cartridges (e.g., made of gelatin) for use in an inhaler or insufflator are formulated, for example, containing a powder mix of the compound of the present application and a suitable powder base, such as lactose or starch. The aerosol dosage form can also take the form of a pump-atomiser.

[0117] Compositions suitable for buccal or sublingual administration include tablets, lozenges, and pastilles, in which the compounds of the present application are formulated with carriers such as sugar, acacia, tragacanth, or gelatin and glycerin. Compositions for rectal administration are conveniently in the form of suppositories containing a conventional suppository base such as cocoa butter.

[0118] Suppository forms of the compounds of the present application are useful for vaginal, urethral and rectal administration. Such suppositories are generally composed of a mixture of substances that are solid at room temperature but melt at body temperature. Materials commonly used to compose such vehicles include, but are not limited to, theobroma oil (also known as cocoa butter), glycerinated gelatin, other glycerides, hydrogenated vegetable oils, mixtures of polyethylene glycols of various molecular weights, and fatty acid esters of polyethylene glycol. For further description of suppository forms, see, for example, Remington's Pharmaceutical Sciences, 16th Ed., Mack Publishing, Easton, PA, 1980, pp. 1530-1533.

[0119] In some embodiments, the compounds of the present application are coupled to soluble polymers as targetable drug carriers. Such polymers include, for example, polyvinylpyrrolidone substituted with palmitoyl residues, pyran copolymers, polyhydroxypropylmethacrylamide-phenol, polyhydroxy-ethylaspartamide-phenol, or polyethylene oxide-polylysine. Furthermore, in some embodiments, the compounds of the present application are coupled to a class of biodegradable polymers useful for achieving controlled release of drugs, such as polylactic acid, polyglycolic acid, copolymers of polylactic acid and polyglycolic acid, polyepsiloncaprolactone, polyhydroxybutyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates, and crosslinked or amphiphilic block copolymers of hydrogels.

[0120] In some embodiments, the compounds of the present application may be combined with viral, non-viral, or other vectors. Viral vectors can include retroviruses, lentiviruses, adenoviruses, herpes viruses, poxviruses, alphaviruses, vaccinia viruses, or adeno-associated viruses. Non-viral vectors can include nanoparticles, cationic lipids, cationic polymers, metal nanoparticles, nanorods, liposomes, micelles, microbubbles, cell-penetrating peptides, or lipospheres. Nanoparticles can include silica, lipids, carbohydrates, or other pharma- ceutically acceptable polymers.

[0121] The compounds of the present application, including their pharma- ceutically acceptable salts and / or solvates, are preferably used per se, but are usually administered in the form of a pharmaceutical composition. In the pharmaceutical composition, one or more compounds of the present application (active ingredients) are combined with a pharma- ceutically acceptable carrier. Depending on the mode of administration, the pharmaceutical composition contains about 0.05 wt% to about 99 wt% or about 0.10 wt% to about 70 wt% of the active ingredient, and about 1 wt% to about 99.95 wt% or about 30 wt% to about 99.90 wt% of the pharma- ceutical acceptable carrier, all percentages by weight being based on the weight of the entire composition.

[0122] IV. Methods and Uses of the Application The compounds of the present application have been shown to inhibit or block Bcl2 associated X protein (BAX) and / or Bcl-2 antagonist killer (BAK), and are therefore useful for inhibiting BAX and / or BAK.

[0123] Accordingly, the present application includes a method of inhibiting Bcl2 Binding X Protein (BAX) and / or Bcl-2 Antagonist Killer (BAK) in a cell, either in a biological sample or in a patient, comprising administering to the cell an effective amount of one or more compounds of the present application.

[0124] The present application also includes the use of one or more compounds of the present application for inhibiting BAX and / or BAK in a cell, as well as the use of one or more compounds of the present application for preparing a medicament for inhibiting BAX and / or BAK in a cell. The present application further includes one or more compounds of the present application for use in inhibiting BAX and / or BAK in a cell.

[0125] In some embodiments, the methods and uses are for inhibiting Bcl2-associated X protein (BAX) in either a biological sample or a patient's cells. In some embodiments, the methods and uses are for inhibiting Bcl-2 antagonist killer (BAK) in either a biological sample or a patient's cells. In some embodiments, the methods and uses are for inhibiting both Bcl2-associated X protein (BAX) and Bcl-2 antagonist killer (BAK) in either a biological sample or a patient's cells.

[0126] In some embodiments, Bcl2-associated X protein (BAX) and / or Bcl-2 antagonist killer (BAK)-mediated cell death is inhibited or prevented by inhibiting Bcl2-associated X protein (BAX) and / or Bcl-2 antagonist killer (BAK). Thus, in some embodiments, the methods and uses are for treating or preventing BAX-mediated cell death and / or BAK-mediated cell death in cells of either a biological sample or a patient. In some embodiments, the BAX-mediated cell death and / or BAK-mediated cell death is apoptosis.

[0127] In some embodiments, inhibiting BAX and / or BAK inhibits or prevents mitochondrial outer membrane permeabilization (MOMP).Thus, in some embodiments, the methods and uses are for inhibiting MOMP in cells, either in a biological sample or in a patient.

[0128] In some embodiments, inhibiting BAX and / or BAK inhibits oligomerization of BAX and BAK. Thus, in some embodiments, the methods and uses are for inhibiting oligomerization of BAX and / or BAK in cells of either a biological sample or a patient.

[0129] It has been found that the compounds of the present application can inhibit BAX protein activity and / or BAK protein activity, and therefore the compounds of the present application are useful for treating diseases, disorders, or conditions by inhibiting BAX and / or BAK. Thus, the compounds of the present application are useful as pharmaceuticals. Thus, the present application includes pharmaceutical uses of the compounds of the present application.

[0130] Thus, the present application also includes methods of treating a disease, disorder, or condition treatable by inhibiting Bcl2-associated X protein (BAX) and / or Bcl-2 antagonist killer (BAK), comprising administering a therapeutically effective amount of one or more compounds of the present application to a subject in need thereof. In some embodiments, the subject has the disease, disorder, or condition.

[0131] The present application also includes the use of one or more compounds of the present application for treating a disease, disorder, or condition treatable by inhibiting BAX and / or BAK, and the use of one or more compounds of the present application for the preparation of a medicament for treating a disease, disorder, or condition treatable by inhibiting BAX and / or BAK. The application further includes one or more compounds of the present application for use in treating a disease, disorder, or condition treatable by inhibiting BAX and / or BAK.

[0132] In some embodiments, the disease, disorder, or condition treatable by inhibiting BAX and / or BAK is a neurodegenerative disease, disorder, or condition. Thus, in some embodiments, the methods and uses are for treating a neurodegenerative disease, disorder, or condition. In some embodiments, the neurodegenerative disease, disorder, or condition is selected from Alzheimer's disease, Huntington's disease, Parkinson's disease, Friedreich's ataxia, amyotrophic lateral sclerosis (ALS), multiple sclerosis, ischemic brain injury, glaucoma, encephalitis, meningitis, and inflammatory neuronal damage due to trauma (such as, for example, malarial encephalitis or cerebral malaria).

[0133] In some embodiments, the disease, disorder, or condition treatable by inhibiting BAX and / or BAK is nerve damage associated with ischemia.Thus, in some embodiments, the method and use are for treating nerve damage associated with ischemia.In some embodiments, the nerve damage associated with ischemia is due to stroke.Thus, the method and use are for treating nerve damage associated with stroke.

[0134] In some embodiments, the disease, disorder, or condition treatable by inhibiting BAX and / or BAK is chemotherapeutic drug-induced cardiomyopathy. Thus, in some embodiments, the method and use are for treating chemotherapeutic drug-induced cardiomyopathy. In some embodiments, the chemotherapeutic drug is doxorubicin. Thus, in some embodiments, the method and use are for treating doxorubicin-induced cardiomyopathy.

[0135] In some embodiments, the disease, disorder, or condition treatable by inhibiting BAX and / or BAK is cell death of donor hematopoietic stem or progenitor cells (HSPCs). Thus, in some embodiments, the methods and uses are for treating cell death of donor hematopoietic stem or progenitor cells (HSPCs). In some embodiments, cell death of the treated donor hematopoietic stem or progenitor cells (HSPCs) occurs during transplantation, including all stages of the transplantation process, including, for example, removal of HSPCs from their original microenvironment, transportation, cryopreservation, and transplantation. In some embodiments, treating cell death of donor HSPCs is by inhibiting or reducing cell death of donor HSPCs.

[0136] Accordingly, the present application includes a method of increasing the survival rate of donor HSPCs during transplantation, the method comprising administering to a subject in need thereof a therapeutically effective amount of one or more compounds of the present application.

[0137] The present application also includes the use of one or more compounds of the present application for increasing the survival rate of donor HSPCs during transplantation, and the use of one or more compounds of the present application for preparing a medicament for increasing the survival rate of donor HSPCs during transplantation. The present application further includes one or more compounds of the present application for use in increasing the survival rate of donor HSPCs during transplantation.

[0138] In some embodiments, the method and use are for treating or preventing BAX-mediated cell death and / or BAK-mediated cell death in cells, thus increasing the viability of any cell (e.g., cells used in cell-based therapy). Thus, the present application includes a method for increasing the viability of a cell, comprising administering an effective amount of one or more compounds of the present application to a cell in need thereof. Also included are the use of one or more compounds of the present application for increasing the viability of a cell, the use of one or more compounds of the present application for preparing a composition for increasing the viability of a cell, and one or more compounds of the present application for increasing the viability of a cell. In some embodiments, the cell is any cell used in cell-based therapy. In some embodiments, the cell used in cell-based therapy is, for example, but not limited to, transplanted skin cells from burn patients, hair follicles for alopecia, corneal endothelial cells in Fuchs' corneal dystrophy, etc.

[0139] In some embodiments, treating a disease, disorder, or condition treatable by inhibiting BAX and / or BAK is by preventing a disease, disorder, or condition treatable by inhibiting BAX and / or BAK. Thus, in some embodiments, the methods and uses are for preventing a neurodegenerative disease, disorder, or condition, for preventing neuronal damage associated with ischemia, for preventing cardiomyopathy induced by chemotherapeutic agents, and / or for preventing cell death of donor HSPCs.

[0140] The present application also includes a method of treating a disease, disorder, or condition treatable by inhibiting BAX and / or BAK. The method includes administering to a subject in need thereof a therapeutically effective amount of one or more compounds of the present application in combination with another known agent useful in treating a disease, disorder, or condition treatable by inhibiting BAX and / or BAK. In some embodiments, the subject has the disease, disorder, or condition.

[0141] The present application also includes the use of one or more compounds of the present application in combination with another known agent useful for treating a disease, disorder, or condition treatable by inhibiting BAX and / or BAK, and the use of one or more compounds of the present application in combination with another known agent useful for treating a disease, disorder, or condition treatable by inhibiting BAX and / or BAK to prepare a medicament for the treatment of a disease, disorder, or condition treatable by inhibiting BAX and / or BAK. The application further includes one or more compounds of the present application in combination with another known agent useful for treating a disease, disorder, or condition treatable by inhibiting BAX and / or BAK.

[0142] In one embodiment, the disease, disorder, or condition treatable by inhibiting BAX and / or BAK is a neurodegenerative disease, disorder, or condition, neuronal damage associated with ischemia, chemotherapeutic drug-induced cardiomyopathy, and / or donor hematopoietic stem and progenitor cell (HSPC) cell death.

[0143] In one embodiment, the subject is a mammal, hi another embodiment, the subject is a human.

[0144] The compounds of the present application are used alone or in combination with other known agents useful for treating diseases, disorders, or conditions treatable by inhibiting BAX and / or BAK. When used in combination with other agents useful for treating diseases, disorders, or conditions treatable by inhibiting BAX and / or BAK, in one embodiment, the compounds of the present application are administered simultaneously with such agents. As used herein, the term "co-administration" of two substances to a subject means providing each of the two substances such that they are both biologically active in the individual at the same time. The exact details of administration depend on the pharmacokinetics of the two substances in the presence of each other. The exact details of administration depend on the pharmacokinetics of the two substances in the presence of each other, and may include administering the two substances within a few hours of each other, or administering one substance within 24 hours of administering the other, if the pharmacokinetics are favorable. Designing a suitable dosing regimen is a routine task for those skilled in the art. In certain embodiments, the two substances are administered substantially simultaneously, i.e., within minutes of each other, or in a single composition containing both substances. In a further embodiment of the present application, the combination of drugs is administered to the subject in a non-simultaneous manner.In some embodiments, the compound of the present application is administered simultaneously or sequentially with another therapeutic agent in separate unit dosage forms, or administered together in a single unit dosage form.Thus, the present application provides a single unit dosage form that includes one or more compounds of the present application (e.g., compound of formula I), an additional therapeutic agent, and a pharmaceutically acceptable carrier.

[0145] The treatment method includes administering to a subject a therapeutically effective amount of one or more of the compounds of the present application, optionally consisting of a single administration or alternatively including a series of administrations, and optionally including the co-administration or use of one or more other therapeutic agents. For example, in some embodiments, the compounds of the present application can be administered at least once a week. In some embodiments, the compounds can be administered to a subject about once every two or three weeks or once a week to about once a day for a given treatment. In another embodiment, the compounds are administered two, three, four, five, or six times a day. The length of the treatment period will vary depending on a variety of factors (e.g., the severity of the disease, disorder, or condition, the age of the subject, the concentration and / or activity of the compounds of the present application, and / or a combination thereof). It will also be apparent that the effective dosage of the compounds used for treatment may increase or decrease over the course of a particular treatment regimen. Changes in dosage may be effected and revealed by standard diagnostic assays known in the art. In some cases, prolonged administration may be required. For example, the compounds are administered to a subject in an amount and for a period sufficient to treat the subject. In some embodiments, treatment includes prophylactic treatment, for example, a subject with early stage cancer can be treated to prevent its progression, or a subject in remission can be treated with a compound or composition of the present application to prevent recurrence.

[0146] The dosage of the compounds of the present application will vary depending on many factors (e.g., the pharmacodynamic properties of the compounds, the mode of administration, the age, health and weight of the recipient, the nature and extent of the symptoms, the frequency of treatment, and the type of concurrent treatment, if any, as well as the clearance rate of the compounds in the treated subject). Those skilled in the art will be able to determine suitable dosages based on the above factors. The compounds of the present application can be initially administered at suitable dosages, which can be adjusted as necessary depending on the clinical response. The dosage is typically selected to maintain a serum concentration of the compounds of the present application at about 0.01 μg / cc to about 1000 μg / cc, or about 0.1 μg / cc to about 100 μg / cc. As a representative example, the oral dosage of one or more compounds of the present application ranges from about 0.05 mg per day to about 1000 mg per day for an adult, preferably from about 0.1 mg per day to about 500 mg per day, more preferably from about 1 mg per day to about 200 mg per day. For parenteral administration, a representative amount can be administered from about 0.001 mg / kg to about 10 mg / kg, from about 0.01 mg / kg to about 10 mg / kg, from about 0.01 mg / kg to about 1 mg / kg, or from about 0.1 mg / kg to about 1 mg / kg. For oral administration, a representative amount is from about 0.001 mg / kg to about 10 mg / kg, from about 0.1 mg / kg to about 10 mg / kg, from about 0.01 mg / kg to about 1 mg / kg, or from about 0.1 mg / kg to about 1 mg / kg. For administration in suppository form, a representative amount is from about 0.1 mg / kg to about 10 mg / kg or from about 0.1 mg / kg to about 1 mg / kg. The compounds of the present application may be administered in a single daily, weekly, or monthly dosage, or the total daily dosage may be divided into two, three, or four daily dosages.

[0147] In one embodiment, the effective amount varies with factors such as the disease state, age, sex, and / or weight of the subject. In a further embodiment, the amount of a given compound or compounds that corresponds to an effective amount will vary depending on factors such as the given drug(s) or compound(s), pharmaceutical formulation, route of administration, type of condition, disease, or disorder, what subject is being treated, and the like, but can nevertheless be routinely determined by one of skill in the art.

[0148] It is noted above that the term "a compound" includes embodiments in which one or more compounds are referenced, and similarly the term "compounds of the application" includes embodiments in which only one compound is referenced.

[0149] V. Methods for Preparing the Compounds of the Present Application The compounds of the present application can be prepared by various synthetic processes. The selection of certain structural features and / or substituents may influence the selection of one process over another. It is within the skill of the art to select a particular process for preparing a given compound of formula I. Some of the starting materials for preparing the compounds of the present application are available as commercial chemicals. Other starting materials are readily prepared from available precursors using simple transformations well known in the art, for example as described below.

[0150] Compounds of formula I can generally be prepared according to the process shown in the following scheme. In the structural formulas shown below, the symbols are as defined in formula I unless otherwise indicated. As will be appreciated by those skilled in the art, many of the reactions shown in the following schemes are oxygen and water sensitive, and reactions should be carried out under anhydrous, inert atmospheres, if necessary. Reaction temperatures and reaction times are provided for illustrative purposes only, and can be modified to optimize yields, as will be appreciated by those skilled in the art.

[0151] Thus, in some embodiments, compounds of formula I are prepared as shown in Scheme 1. [ka]

[0152] Thus, in some embodiments, a compound of formula A is coupled with an amino compound of formula B to provide an intermediate compound of formula C. The compound of formula C is reduced under suitable conditions to provide an amino compound of formula D. The compound of formula D is then coupled with a compound of formula E (wherein X is halo, such as Br, and LG is a leaving group, such as Cl) under suitable coupling conditions (e.g., in the presence of an organic base such as N,N-diisopropylethylamine) to provide an intermediate compound of formula C containing at least one ring hetero moiety selected from N and NH of formula F. 3~8 Coupling with a heterocyclic compound gives the compound of formula I.

[0153] In an embodiment, compounds of formula A are prepared as shown in Scheme 2. [ka]

[0154] Thus, in some embodiments, a compound of formula G is esterified to form an ester, e.g., a methyl ester compound of formula H, which is then reacted under suitable conditions (e.g., at about 105 o C~120 o The compound of formula K is then combined with a tert-butoxybis(dimethylamino)methane compound of formula J under suitable conditions (e.g., at about 105° C.) to give a compound of formula K. o C~120 o The 2,4-dimethoxybenzylamine compound of formula L is cyclized in a suitable solvent (e.g., toluene) at a temperature of 30° C. to give a compound of formula M. The 2,4-dimethoxybenzyl group is then removed from the compound of formula M in the presence of an acid (e.g., trifluoroacetic acid) to give a compound of formula N, which is reacted with a chlorinating agent (e.g., phosphorus(V) oxychloride) to give a compound of formula A.

[0155] The reaction is typically carried out in a suitable inert organic solvent at a temperature and for a time period that optimizes the yield of the desired compound. Examples of suitable inert organic solvents include, but are not limited to, 2-propanol, dimethylformamide (DMF), 1,4-dioxane, methylene chloride, chloroform, tetrahydrofuran (THF), toluene, and the like.

[0156] The salts of the compounds of the present application are typically formed by dissolving the neutral compound in an inert organic solvent, adding either the desired acid or base, and isolating the resulting salt by filtration or any other known means.

[0157] The formation of solvates of the compounds of the present application varies depending on the compound and the solvate. Usually, solvates are formed by dissolving the compound in a suitable solvent and isolating the solvate by cooling or using an anti-solvent. The solvate is typically dried or azeotroped under ambient conditions. The selection of suitable conditions for forming a particular solvate can be performed by one skilled in the art.

[0158] Prodrugs of the compounds of the present application can be, for example, conventional esters formed with available hydroxy, thiol, amino, or carboxyl groups. For example, available hydroxy or amino groups can be acylated using an activated acid in the presence of a base, optionally in an inert solvent (e.g., an acid chloride in pyridine).

[0159] The formation of a desired compound salt is accomplished using standard techniques, for example, treating a neutral compound with an acid or base in a suitable solvent and isolating the formed salt by filtration, extraction, or any other suitable method.

[0160] The formation of solvates varies depending on the compound and the solvate. Usually, solvates are formed by dissolving the compound in a suitable solvent and isolating the solvate by cooling or using an anti-solvent. The solvate is typically dried or azeotroped under ambient conditions. The selection of suitable conditions for forming a particular solvate can be made by one of ordinary skill in the art. Examples of suitable solvents include ethanol, water, etc. When water is the solvent, the molecule is called a "hydrate". The formation of solvates of the compounds of the present application varies depending on the compound and the solvate. Usually, solvates are formed by dissolving the compound in a suitable solvent and isolating the solvate by cooling or using an anti-solvent. The solvate is typically dried or azeotroped under ambient conditions. The selection of suitable conditions for forming a particular solvate can be made by one of ordinary skill in the art.

[0161] Throughout the processes described herein, it will be understood that suitable protecting groups will be added to and subsequently removed from the various reactants and intermediates, where appropriate, in a manner that will be readily understood by those skilled in the art. Conventional procedures for the use of such protecting groups, and examples of suitable protecting groups, are described, for example, in "Protective Groups in Organic Synthesis", TW Green, PG M Huts, Wiley-Interscience, New York, (1999). It will also be understood that the conversion of a group or substituent to another group or substituent by chemical manipulation can be performed on any intermediate or final product on the synthetic route to the final product, where the types of transformations possible are limited only by the inherent incompatibility of other functional groups possessed by the molecule at that stage with the conditions or reagents used in the transformation. Such inherent incompatibilities, and how to avoid such incompatibilities by carrying out appropriate transformations and synthetic steps in a suitable order, will be readily understood by those skilled in the art. Examples of transformations are given herein, and it will be understood that the transformations described are not limited to only the general groups or substituents for which the transformations are exemplified. References and descriptions of other suitable transformations can be found in "Comprehensive Organic Transformations-A Guide to Functional Group Preparations" RC Lalock, VHC Publishers, Inc. (1989). References and descriptions of other suitable reactions can be found in organic chemistry textbooks, such as "Advanced Organic Chemistry", March, 4th ed. McGraw Hill (1992) or "Organic Synthesis", Smith, McGraw Hill (1994). Techniques for purification of intermediates and final products include, for example, normal and reverse phase chromatography on columns or spinning plates, recrystallization, distillation, and liquid-liquid or solid-liquid extraction, and are readily understood by those skilled in the art. EXAMPLES

[0162] The following non-limiting examples are illustrative of the present application. A. Synthesis and Characterization of Exemplary Compounds of the Present Application Example 1: Preparation of N-(1-((3-chloro-4-fluorophenyl)amino)-6-methoxyisoquinolin-7-yl)-4-(piperidin-1-yl)butanamide (I-1) [ka]

[0163] Step 1: 2-Methyl-4,5-dinitrobenzoic acid [ka] 2-Methyl-4-nitrobenzoic acid (9 g, 49.7 mmol) was added portionwise to a premixed solution of nitric acid (31.3 g, 497 mmol) and sulfuric acid (97 g, 994 mmol) under ice-water bath. The reaction mixture was stirred at room temperature overnight and then poured into 300 g of ice, the resulting solid was filtered, rinsed with water and hexane, and dried under vacuum to give crude 2-methyl-4,5-dinitrobenzoic acid (9.361 g) as an off-white solid. Yield=83%, purity=72%. LCMS: [MH] - =225.27. 1 H NMR (500 MHz, methanol-d4) δ = 8.55 (s, 1H), 7.98 (s, 1H), 2.76 (s, 3H).

[0164] Step 2: 4-Methoxy-2-methyl-5-nitrobenzoic acid [ka] 2-Methyl-4,5-dinitrobenzoic acid (9.361 g, 41.4 mmol) was mixed with potassium hydroxide (11.61 g, 207 mmol) in MeOH (120 mL) and the reaction mixture was stirred for 70 oThe mixture was stirred in a 300° C. oil bath for 90 min. The reaction mixture was acidified with 2N HCl to pH<1 and the resulting orange-red solid was filtered, washed with water and air-dried overnight to give crude 4-methoxy-2-methyl-5-nitrobenzoic acid (8.5 g). Yield=97%, purity=76%. [MH] - =210.16. 1 H NMR(500MHz,DMSO-d6)δ=13.45-12.99(m,1H),8.43-8.29(m,1H),7.32(s,1H),3.99(s,3H),2.64(s,3H).

[0165] Step 3: Methyl 4-methoxy-2-methyl-5-nitrobenzoate [ka] 4-Methoxy-2-methyl-5-nitrobenzoic acid (8.5 g, 40.3 mmol) was mixed with MeOH (150 mL) under ice-water bath, and then thionyl chloride (8.38 g, 70.4 mmol) was added dropwise. The reaction mixture was stirred for 70 o The mixture was stirred in a 0.05 C oil bath for 4.5 h and then at room temperature overnight. The reaction mixture was concentrated by rotary evaporation and the residue was dissolved in EtOAc and DCM, concentrated onto silica gel and purified by Biotage (50 g) column, eluted with 0-15% EtOAc in hexanes. The product was triturated with ether to give methyl 4-methoxy-2-methyl-5-nitrobenzoate (2.56 g) as a pale yellow solid. Yield=28.2%. [M+H] + =226.34. 1 H NMR(500MHz,DMSO-d6)δ=8.38(s,1H),7.36(s,1H),4.00(s,3H),3.83(s,3H),2.63(s,3H)

[0166] Step 4: (E)-2-(2-(dimethylamino)vinyl)-4-methoxy-5-nitrobenzoate methyl [ka] Methyl 4-methoxy-2-methyl-5-nitrobenzoate (2.56 g, 11.37 mmol) was mixed with tert-butoxybis(dimethylamino)methane (2.58 g, 14.78 mmol) to give 115 o The mixture was heated in a 30°C oil bath for 1 hour and 20 minutes. The reaction mixture was diluted with hexanes and the resulting solid was filtered and rinsed with hexanes to give (E)-methyl 2-(2-(dimethylamino)vinyl)-4-methoxy-5-nitrobenzoate (2.946 g) as a dark red solid. Yield=92%. 1 H NMR(500MHz,DMSO-d6)δ=8.40(s,1H),7.73(d,J=13.3Hz,1H),7.17(s,1H),6.28(d,J=13.3Hz,1H),3.98(s,3H),3.79(s,3H),2.98(s,6H).

[0167] Step 5: 2-(2,4-dimethoxybenzyl)-6-methoxy-7-nitroisoquinolin-1(2H)-one [ka] (E)-Methyl 2-(2-(dimethylamino)vinyl)-4-methoxy-5-nitrobenzoate (2.96 g, 10.56 mmol) was mixed with 2,4-dimethoxybenzylamine (2.472 g, 14.79 mmol) in toluene (20 mL), and the reaction mixture was cooled to 125° C. o C oil bath for 1 h. The reaction mixture was cooled, diluted with hexanes and filtered. The solid was triturated to give 2-(2,4-dimethoxybenzyl)-6-methoxy-7-nitroisoquinolin-1(2H)-one (3.31 g) as a yellow solid. Yield=85%. [M+H] + =371.35. 1 H NMR(500MHz,DMSO-d6)δ=8.60(s,1H),7.57(d,J=7.5Hz,1H),7.49(s,1H),7.04(d,J=8.3Hz,1H),6.64(d,J=7.3 Hz,1H),6.59(d,J=2.3Hz,1H),6.47(dd,J=2.3,8.4Hz,1H),5.01(s,2H),4.01(s,3H),3.81(s,3H),3.74(s,3H).

[0168] Step 6: 6-Methoxy-7-nitroisoquinolin-1(2H)-one [ka] 2-(2,4-Dimethoxybenzyl)-6-methoxy-7-nitroisoquinolin-1(2H)-one (3.31 g, 8.94 mmol) was mixed with trifluoroacetic acid (50 mL) and the reaction mixture was diluted with 85 mL of trifluoroacetic acid. o C oil bath for 3 h. The reaction mixture was concentrated using a rotary evaporator, and the residue was mixed with MeOH, concentrated again, and then triturated with EtOAc to give 6-methoxy-7-nitroisoquinolin-1(2H)-one (1.784 g) as a pale green solid. Yield=90.9%. [M+H] + =221.20. 1 H NMR(500MHz,DMSO-d6)δ=11.47(br s,1H),8.57(s,1H),7.51(s,1H),7.40-7.30(m,1H),6.58(d,J=7.1Hz,1H),4.01(s,3H)

[0169] Step 7: 1-Chloro-6-methoxy-7-nitroisoquinoline [ka] 6-Methoxy-7-nitroisoquinolin-1(2H)-one (1.784 g, 8.10 mmol) was mixed with phosphorus(V) oxychloride (37.3 g, 243 mmol) and the reaction mixture was diluted with 100 o C oil bath for 1 h 20 min, then concentrated by rotary evaporation to remove excess POCl3. The residue was diluted with DCM and ice water, neutralized with solid NaHCO3. The DCM layer was concentrated onto silica gel and purified by Biotage column (10 g) eluting with 30-50% EtOAc in hexanes. The resulting product was triturated with ether to give 1-chloro-6-methoxy-7-nitroisoquinoline (1.016 g) as a pale yellow solid. Yield=53%. [M+H] + =239.26.1 H NMR(500MHz,DMSO-d6)δ=8.75(s,1H),8.38(d,J=5.7Hz,1H),7.91-7.86(m,2H),4.07(s,3H)

[0170] Step 8: N-(3-chloro-4-fluorophenyl)-6-methoxy-7-nitroisoquinolin-1-amine hydrochloride [ka] 1-Chloro-6-methoxy-7-nitroisoquinoline (400 mg, 1.676 mmol) was mixed with 3-chloro-4-fluoroaniline (244 mg, 1.68 mmol) in 1,4-dioxane (5 mL), followed by the addition of 4.0 M hydrochloric acid in dioxane (0.84 mL, 3.35 mmol). The reaction suspension was cooled to 100 o C oil bath for 2 h, then diluted with ether and filtered. The solid was rinsed with ether to give N-(3-chloro-4-fluorophenyl)-6-methoxy-7-nitroisoquinolin-1-amine hydrochloride (676 mg). Yield = quantitative. [M-HCl+H] + =348.34. 1 H NMR(500MHz,DMSO-d6)δ=9.36(s,1H),8.04(br d,J=5.0Hz,1H),7.90(br d,J=5.7Hz,1H),7.77(s,1H),7.73-7.66(m,1H),7.54(br t,J=9.0Hz,1H),7.30(d,J=6.4Hz,1H),4.08(s,3H)

[0171] Step 9: N 1 -(3-chloro-4-fluorophenyl)-6-methoxyisoquinoline-1,7-diamine [ka] N-(3-chloro-4-fluorophenyl)-6-methoxy-7-nitroisoquinolin-1-amine hydrochloride (676 mg, 1.76 mmol) was dissolved in MeOH (25 mL), water (18 mL), and THF (10 mL) and 60 oThe mixture was stirred in a 30° C. oil bath, and then ammonium chloride (376 mg, 7.04 mmol) and iron powder 99% (393 mg, 7.04 mmol) were added. The reaction mixture was stirred for 60 o The mixture was stirred in a 35°C oil bath for 90 min. It was then neutralized with solid NaHCO3 and filtered. The filtrate was concentrated using a rotary evaporator and extracted with EtOAc. The organic layer was dried over MgSO4 and filtered. The filtrate was concentrated and the residue was triturated with ether and purified with N 1 -(3-chloro-4-fluorophenyl)-6-methoxyisoquinoline-1,7-diamine (400 mg) was obtained as a light brown solid. [M+H] + =318.38.

[0172] Step 10: N-(1-((3-chloro-4-fluorophenyl)amino)-6-methoxyisoquinolin-7-yl)-4-(piperidin-1-yl)butanamide [ka]

[0173] N 1To a solution of -(3-chloro-4-fluorophenyl)-6-methoxyisoquinoline-1,7-diamine (200 mg, 0.629 mmol) and N,N-diisopropylethylamine (163 mg, 1.259 mmol) in THF (10 mL) was added 4-bromobutyryl chloride (140 mg, 0.755 mmol) in DCM (1 mL) dropwise in an ice-water bath. The reaction mixture was stirred at room temperature for 1 h. Piperidine (536 mg, 6.29 mmol) and sodium iodide (94 mg, 0.629 mmol) were added and the reaction mixture was stirred at room temperature overnight. The reaction mixture was quenched with brine and extracted with EtOAc. The organic layer was concentrated onto silica gel by using a rotary evaporator. The product was purified by Biotage (12 g) and eluted with 50-100% EtOAc in hexanes, followed by 5% MeOH in EtOAc. Pure fractions were combined and concentrated. The residue was triturated with EtOAc-ether to give N-(1-((3-chloro-4-fluorophenyl)amino)-6-methoxyisoquinolin-7-yl)-4-(piperidin-1-yl)butanamide (112.5 mg) as an off-white solid. Yield=37.9%. [M+H] + =471.51. 1 H NMR(500MHz,DMSO-d6)δ=9.37(br s,1H),9.16(s,1H),8.80(br s,1H),8.08(dd,J=2.3,6.7Hz,1H),7.91(d,J=5.6 Hz,1H),7.73(td,J=3.6,8.3Hz,1H),7.39-7.28(m,2H),7.15(d,J=5.7Hz,1H),3.97(s,3H),2.45(br t,J=6.8Hz,2H),2.37-2.23(m,6H),1.76(quin,J=7.1Hz,2H),1.54-1.43(m,4H),1.37(br d,J=4.5Hz,2H).

[0174] Example 2: Preparation of N-(1-((3-chloro-4-fluorophenyl)amino)-6-methoxyisoquinolin-7-yl)-5-(piperidin-1-yl)pentanamide (I-2) [ka] To a solution of N1-(3-chloro-4-fluorophenyl)-6-methoxyisoquinoline-1,7-diamine (0.1 g, 0.314 mmol) in THF (5 mL) was added 5-bromopentanoyl chloride (0.075 g, 0.376 mmol), DIPEA (0.085 g, 0.628 mmol) and the mixture was stirred at 0° C. for 2 h. After 2 h, NaI (0.05 g, 0.314 mmol), piperidine (0.27 g, 3.147 mmol) were added and the reaction was stirred at room temperature for 16 h. After completion of the reaction, the mixture was diluted with brine (20 mL) and extracted with EtOAc (3×10 mL). The combined organic layers were dried over anhydrous Na2SO4 and concentrated under vacuum to give the crude product. The crude product was purified using basic alumina eluting with 0.8% MeOH in DCM to give the title compound (0.072 g, 45.9%) as a light brown solid. 1 H NMR(400MHz,DMSO-d6)δ9.37(s,1H),9.17(s,1H),8.79(s,1H),8.08(s,1H),7.91(d,J=4.4Hz,1H),7.73(s,1H),7.33(s,2 H),7.15(d,J=4.8Hz,1H),3.97(s,3H),2.25-2.29(bs,J=17.6Hz,8H),1.61(s,2H),1.47(s,4H),1.36(s,2H).LCMS:[M+2H] + =487.3.

[0175] Example 3: Preparation of N-(1-((3-chloro-4-fluorophenyl)amino)-6-methoxyisoquinolin-7-yl)-3-(piperidin-1-yl)propenamide (I-3) [ka] N 1To a solution of -(3-chloro-4-fluorophenyl)-6-methoxyisoquinoline-1,7-diamine (0.1 g, 0.314 mmol) in THF (5 mL), 3-bromopropionyl chloride (0.07 g, 0.376 mmol), DIPEA (0.085 g, 0.628 mmol) were added and the mixture was stirred at 0° C. for 2 h. After 2 h, NaI (0.05 g, 0.314 mmol) and piperidine (0.27 g, 3.14 mmol) were added and the mixture was stirred at room temperature for 16 h. After completion of the reaction, the mixture was diluted with brine (20 mL) and extracted with EtOAc (3×10 mL). The combined organic layers were dried over anhydrous Na2SO4 and concentrated in vacuo to give the crude product. The crude product was purified using basic alumina eluting with 0.5% MeOH in DCM to give the title compound (0.05 g, 34.8%) as a light brown solid. 1 H NMR(400MHz,DMSO-d6)δ10.7(s,1H),9.17(s,1H),9.00(s,1H),8.05(s,1H),7.91(s,1H),7.71(s,1H),7.35( s,2H),7.16(s,1H),4.00(s,3H),2.59(t,J=17.6Hz,8H),1.61(s,4H),1.48(s,2H),1.24(s,3H).LCMS:(M+H) + =457.2.

[0176] Example 4: Preparation of N-(1-((3-chloro-4-fluorophenyl)amino)-6-methoxyisoquinolin-7-yl)-4-(pyrrolidin-1-yl)butanamide (I-4) [ka] N 1To a solution of -(3-chloro-4-fluorophenyl)-6-methoxyisoquinoline-1,7-diamine (0.1 g, 0.314 mmol) in THF (5 mL) was added 4-bromobutanoyl chloride (0.075 g, 0.376 mmol) and DIPEA (0.085 g, 0.628 mmol) and the mixture was stirred at 0° C. for 2 h. After 2 h, NaI (0.05 g, 0.314 mmol) and pyrrolidine (0.23 g, 3.147 mmol) were added and the mixture was stirred at room temperature for 16 h. After completion of the reaction, the mixture was diluted with brine (20 mL) and extracted with EtOAc (3×10 mL). The combined organic layers were dried over anhydrous Na2SO4 and concentrated in vacuum. The crude product obtained was purified using basic alumina eluting with 1.2% MeOH in DCM to give the title compound (0.055 g, 38.3%) as a pale yellow solid. 1 H NMR(400MHz,DMSO-d6)δ9.45(s,1H),9.19(s,1H),8.83(s,1H),8.09(d,J=6.4Hz,1H),7.93(d,J=4.8Hz,1H),7.73(s,1H),7 .33-7.37(t,J=8.8Hz,2H),7.15(d,J=5.6Hz,1H),3.98(s,3H),2.45(s,8H),1.79(t,J=8.6Hz,2H),1.69(s,4H).LCMS:(M+H) + =457.2.

[0177] Example 5: Preparation of N-(1-((3-chloro-4-fluorophenyl)amino)-6-methoxyisoquinolin-7-yl)-4-((2S,6R)-2,6-dimethylmorpholino)butanamide (I-5) [ka] N 1To a solution of -(3-chloro-4-fluorophenyl)-6-methoxyisoquinoline-1,7-diamine (0.15 g, 0.471 mmol) in THF (9 mL) was added 4-bromobutanoyl chloride (0.105 g, 0.566 mmol), DIPEA (0.122 g, 0.944 mmol) and the mixture was stirred at 0° C. for 2 h. After 2 h, NaI (0.071 g, 0.472 mmol) and (2S,6R)-2,6-dimethylmorpholine (0.361 g, 4.712 mmol) were added and the reaction was stirred at room temperature for 16 h. The mixture was diluted with brine (30 mL) and extracted with EtOAc (3×15 mL). The combined organic layers were dried over anhydrous Na2SO4 and concentrated in vacuo. The crude product was purified using basic alumina to give the title compound (0.07 g, 29.6%) as an off-white solid. 1 H NMR(400MHz,DMSO-d6)δ9.4(s,1H),9.19(s,1H),8.82(s,1H),8.09(d,J=4.4Hz,1H ),7.91(d,J=6Hz,1H),7.76(bs,J=8.4Hz,1H),7.35-7.50(t,J=9.6Hz,2H),7.15(d ,J=5.6Hz,1H),3.98(s,3H),3.54(s,2H),3.76(d,J=10.8Hz,2H),2.34(d,J=6.4Hz ,2H),1.8(d,J=6.8Hz,2H),1.60-1.55(t,J=10.4Hz,2H),1.04(s,6H).LCMS:[M+2H] + =503.3.

[0178] Example 6: Preparation of N-(1-((3-chloro-4-fluorophenyl)amino)-6-methoxyisoquinolin-7-yl)-4-morpholinobutanamide (I-6) [ka] N 1To a solution of -(3-chloro-4-fluorophenyl)-6-methoxyisoquinoline-1,7-diamine (0.12 g, 0.377 mmol) in THF (6 mL) was added 4-bromobutanoyl chloride (0.085 g, 0.453 mmol), DIPEA (0.1 g, 0.755 mmol) and the mixture was stirred at 0° C. for 2 h. After 2 h, NaI (0.057 g, 0.377 mmol) and morpholine (0.33 g, 3.776 mmol) were added and the mixture was stirred at room temperature for 16 h. After completion of the reaction, the mixture was diluted with brine (20 mL) and extracted with EtOAc (3×10 mL). The combined organic layers were dried over anhydrous Na2SO4 and concentrated under vacuum to give the crude product. The crude product was purified by silica flash chromatography to give the title compound (0.045 g, 25.2%) as an off-white solid. 1 H NMR(400MHz,DMSO-d6)δ9.39(s,1H),9.17(s,1H),8.81(s,1H),8.09(d,J=4.4Hz,1H),7.91(d,J=5.6Hz,1H),7.72(bs,J=4.8Hz,1H),7.35-7.50 (t,J=9.2Hz,2H),7.15(d,J=5.6Hz,1H),3.96(s,3H),3.56(s,4H),3.16(d,J=4.8Hz,2H),2.34(s,6H),1.79-1.76(t,J=6.8Hz,2H).LCMS:(M+H) + =473.3.

[0179] Example 7: Preparation of N-(1-((3-chloro-4-fluorophenyl)amino)-6-methoxyisoquinolin-7-yl)-4-(methylpiperazin-1-yl)butanamide (I-7) [ka] N 1To a solution of -(3-chloro-4-fluorophenyl)-6-methoxyisoquinoline-1,7-diamine (0.1 g, 0.314 mmol) in THF (5 mL), 4-bromobutanoyl chloride (0.075 g, 0.376 mmol), DIPEA (0.085 g, 0.628 mmol) were added and the mixture was stirred at 0° C. for 2 h. After 2 h, NaI (0.05 g, 0.314 mmol), 1-methylpiperidine (0.32 g, 3.147 mmol) were added and the mixture was stirred at room temperature for 16 h. After completion of the reaction, the mixture was diluted with brine (20 mL) and extracted with EtOAc (3×10 mL). The combined organic layers were dried over anhydrous Na2SO4 and concentrated in vacuo to give the crude product. The crude product was purified using basic alumina eluting with 1% MeOH in DCM to give the title compound (0.047 g, 30.7%) as an off-white solid. 1 H NMR(400MHz,DMSO-d6)δ9.38(s,1H),9.17(s,1H),8.81(s,1H),8.09(d,J=5.2Hz,1H),7.91(d,J=5.2Hz,1H),7.72(s,1H),7.35 -7.31(t,J=10Hz,2H),7.15(d,J=5.6Hz,1H),3.97(s,3H),2.31(bs,12H),2.12(s,3H),1.77-1.75(d,J=7.2Hz,2H).LCMS:(M+H) + =486.2.

[0180] Example 8: Preparation of N-(1-((3-chloro-4-fluorophenyl)amino)-6-methoxyisoquinolin-7-yl)-4-(4,4-difluoropiperidin-1-yl)butanamide (I-8) [ka] To a solution of N1-(3-chloro-4-fluorophenyl)-6-methoxyisoquinoline-1,7-diamine (0.1 g, 0.314 mmol) in THF (5 mL), 4-bromobutanoyl chloride (0.075 g, 0.376 mmol), DIPEA (0.085 g, 0.628 mmol) were added and the mixture was stirred at 0° C. for 2 h. After 2 h, NaI (0.05 g, 0.314 mmol) and 4,4-difluoropiperidine (0.9 g, 6.294 mmol) were added and the mixture was stirred at room temperature for 16 h. After completion of the reaction, the mixture was diluted with brine (20 mL) and extracted with EtOAc (3×10 mL). The combined organic layers were dried over anhydrous Na2SO4 and concentrated under vacuum to give the crude product. The crude product was purified by preparative HPLC purification to give the title compound (0.035 g, 11%) as a white solid. 1 H NMR(400MHz,DMSO-d6)δ9.38(s,1H),9.16(s,1H),8.81(s,1H),8.09(d,J=4.8Hz,1H),7.97(d,J=5.6Hz,1H),7.72(s,1H),7.35-7.50(t,J=8 .8Hz,2H),7.15(d,J=5.6Hz,1H),3.96(s,3H),2.39(m,J=6.8Hz,8H),1.96-1.89(m,J=13.6Hz,4H),1.79-1.76(t,J=6.8Hz,2H).LCMS:[M+H] + =507.4.

[0181] Example 9: Preparation of N-(1-((3-fluorophenyl)amino)-6-methoxyisoquinolin-7-yl)-4-(piperidin-1-yl)butanamide (I-9) [ka] To a solution of N-(1-chloro-6-methoxyisoquinolin-7-yl)-4-(piperidin-1-yl)butanamide (0.15 g, 0.41 mmol), 3-fluoroaniline (50 mg, 0.45 mmol), and t-BuOK (0.093 g, 0.82 mmol) in degassed dioxane (20 V), Xantphos (24 mg, 0.041 mmol) and Pd2(dba)3 (38 mg, 0.041 mmol) were added under nitrogen atmosphere and the mixture was stirred at 100° C. for 2 h. The mixture was quenched with water (10 mL) and extracted with EtOAc (2×20 mL). The combined organic layers were dried over anhydrous Na2SO4 and concentrated under vacuum to give the crude product. The crude product was purified using column chromatography eluting with 12% MeOH in DCM to give the title compound (52 mg, 28.7%) as a light brown solid. 1 H NMR(400MHz,DMSO-d6)δ9.42(s,1H),9.19(s,1H),8.82(s,1H),7.95-7.93(d,J=5.6Hz 1H),7.81-7.78(d,J=11.6Hz,1H),7.57-7.54(d,J=8Hz,1H),7.34-7.26(m,2H),7.17-7.16(d,J=5.2Hz,1H),6.74-6.71(d, LCMS: (M+H) + =436.53.

[0182] Example 10: Preparation of N-(6-methoxy-1-((3-methoxyphenyl)amino)isoquinolin-7-yl)-4-(piperidin-1-yl)butanamide (I-10) [ka] Step 1: tert-Butyl 4-(piperidin-1-yl)butanoate [ka] To a solution of tert-butyl 4-bromobutanoate (10 g, 44.8 mmol) in ACN (10 V) was added piperidine (8 mL, 89.6 mmol) at room temperature and the mixture was stirred at 90° C. for 2 h. After completion of the reaction, the mixture was quenched with water (100 mL), extracted with EtOAc (2×100 mL), the organic layer was dried over anhydrous Na2SO4 and concentrated under vacuum to give the title compound (10 g, 98.1%) as an off-white solid. This crude product was used in the next step without further purification.

[0183] Step 2: 4-(Piperidin-1-yl)butanoic acid [ka] To a stirred solution of tert-butyl 4-(piperidin-1-yl)butanoate (10 g, 43.8 mmol) in dioxane (100 mL) was added concentrated HCl (1V) at room temperature and the mixture was stirred at 90° C. for 16 h. After completion of the reaction, the mixture was concentrated under vacuum to give the crude product. The crude product was triturated with DCM and the solid was filtered and dried to give the title compound (7.5 g, 99.6%) as an off-white solid. 1 H NMR(400MHz,DMSO-d6)δ12.32(s,1H),2.99(s,2H),2.84(s,2H),2.35-2.31(t,J=13.6Hz,2H),1.93-1.92(d,J=7.2Hz,2H),1.77(s,6H),1.38(s,1H).

[0184] Step 3: 1-Chloro-6-methoxyisoquinolin-7-amine [ka] To a stirred solution of 1-chloro-6-methoxy-7-nitroisoquinoline (1 g, 41.8 mmol), MeOH (37 V), water (27 V), and THF (14 V) was added NH4Cl (0.81 g, 15.08 mmol) and Fe (0.84 g, 15.08 mmol) at 70° C. The mixture was stirred at 70° C. for 5 h, quenched with solid NaHCO3 (1 g), the mixture was passed through a bed of celite, and the filtrate was concentrated under vacuum to give the crude product. The crude product was stirred in DCM and filtered. The filtrate was concentrated under vacuum to give the title compound (0.6 g, 68.6%) as a yellow solid. LCMS: (M+H) + =208.65.

[0185] Step 4: N-(1-chloro-6-methoxyisoquinolin-7-yl)-4-(piperidin-1-yl)butanamide [ka] To a stirred solution of 1-chloro-6-methoxyisoquinolin-7-amine (0.6 g, 2.88 mmol) and 4-(piperidin-1-yl)butanoic acid (0.64 g, 3.74 mmol) in DCM (6 mL) was added DIPEA (1.11 g, 8.64 mmol) and T3P (50% solution in EtOAc, 1.8 g, 5.76 mmol) at room temperature. The mixture was stirred at 70° C. for 16 h, quenched with water (20 mL) and extracted with EtOAc (3×20 mL). The combined organic layers were dried over anhydrous Na2SO4 and concentrated under vacuum to give the crude product. The crude product was purified using column chromatography eluting with 10% MeOH in DCM to give the title compound (0.5 g, 48.05%) as a yellow solid. LCMS: (M+H)+=361.87.

[0186] Step 5: N-(6-Methoxy-1-((3-methoxyphenyl)amino)isoquinolin-7-yl)-4-(piperidin-1-yl)butanamide [ka] To a solution of N-(1-chloro-6-methoxyisoquinolin-7-yl)-4-(piperidin-1-yl)butanamide (0.15 g, 0.41 mmol), 3-methoxyaniline (60 mg, 0.49 mmol), and Cs2CO3 (0.27 g, 0.82 mmol) in degassed dioxane (10 V), Xantphos (24 mg, 0.041 mmol) and Pd2(dba)3 (38 mg, 0.041 mmol) were added under nitrogen atmosphere and the mixture was stirred at 100° C. for 2 h. The mixture was quenched with water (10 mL) and extracted with EtOAc (2×20 ml). The combined organic layers were dried over anhydrous Na2SO4 and concentrated under vacuum to give the crude product. The crude product was purified using column chromatography eluting with 12% MeOH in DCM to give the title compound (40 mg, 21.5%) as a light brown solid. 1 H NMR(400MHz,DMSO-d6)δ9.47(s,1H),8.95(s,1H),8.79(s,1H),7.91(s,1H),7.45(s,1H),7.38-7.37(d,J=6.8Hz,2H),7.17-7 .12(d,J=23.2Hz,2H),6.54(s,1H),3.97(s,3H),3.74(s,2H),2.50(s,8H),1.91(s,2H),1.62(s,4H),1.44(s,2H).LCMS:(M+H) + =448.57.

[0187] Example 11: Preparation of N-(6-methoxy-1-(m-tolylamino)isoquinolin-7-yl)-4-(piperidin-1-yl)butanamide (I-11) [ka] To a solution of N-(1-chloro-6-methoxyisoquinolin-7-yl)-4-(piperidin-1-yl)butanamide (0.15 g, 0.41 mmol), m-toluidine (52 mg, 0.49 mmol), and Cs2CO3 (0.27 g, 0.82 mmol) in degassed dioxane (20 V), Xantphos (24 mg, 0.041 mmol) and Pd2(dba)3 (38 mg, 0.041 mmol) were added under nitrogen atmosphere and the mixture was stirred at 100° C. for 2 h. The mixture was quenched with water (10 mL) and extracted with EtOAc (2×20 ml). The combined organic layers were dried over anhydrous Na2SO4 and concentrated under vacuum to give the crude product. The crude product was purified using column chromatography eluting with 12% MeOH in DCM to give the title compound (50 mg, 27.9%) as a yellow solid. 1 H NMR(400MHz,DMSO-d6)δ9.38(s,1H),8.88(s,1H),8.79(s,1H),7.88-7.871(d,J=4.8Hz,1H),7.58-7.56(d,J=8.8Hz,2H),7.30(s,1H),7 .18-7.08(m,2H),6.77-6.75(d,J=6.8Hz,1H),3.96(s,3H),2.46(s,8H),2.29(s,3H),1.79(s,2H),1.52(s,4H),1.39(s,2H).LCMS:(M+H) + =432.57.

[0188] Example 12: Preparation of N-(1-((4-fluorophenyl)amino)-6-methoxyisoquinolin-7-yl)-4-(piperidin-1-yl)butanamide (I-12) [ka] To a solution of N-(1-chloro-6-methoxyisoquinolin-7-yl)-4-(piperidin-1-yl)butanamide (0.15 g, 0.41 mmol), 4-fluoroaniline (50 mg, 0.45 mmol), and Cs2CO3 (0.27 g, 0.82 mmol) in degassed dioxane (20 V), Xantphos (24 mg, 0.041 mmol) and Pd2(dba)3 (38 mg, 0.041 mmol) were added under nitrogen atmosphere and the mixture was stirred at 100 °C for 2 h. The mixture was quenched with water (10 mL), extracted with EtOAc (2x20 mL), and the combined organic layers were dried over anhydrous Na2SO4 and concentrated under vacuum to give the crude product. The crude product was purified using column chromatography eluting with 10% MeOH in DCM to give the title compound (51 mg, 28.2%) as a yellow solid. 1 H NMR(400MHz,DMSO-d6)δ9.40(s,1H),9.01(s,1H),8.79(s,1H),7.85(s,1H),7.75(s,2H),7.30(s,1H),7 .12-7.10(d,J=9.2Hz,3H),3.97(s,3H),2.50(s,8H),1.79(s,2H),1.51(s,4H),1.39(s,2H).LCMS:(M+H) + =436.53.

[0189] Example 13: Preparation of N-(1-((3-chlorophenyl)amino)-6-methoxyisoquinolin-7-yl)-4-(piperidin-1-yl)butanamide (I-13) [ka] To a solution of N-(1-chloro-6-methoxyisoquinolin-7-yl)-4-(piperidin-1-yl)butanamide (0.15 g, 0.41 mmol), 3-chloroaniline (63 mg, 0.45 mmol), and Cs2CO3 (0.27 g, 0.82 mmol) in degassed dioxane (20 V), Xantphos (24 mg, 0.041 mmol) and Pd2(dba)3 (38 mg, 0.041 mmol) were added under nitrogen atmosphere and the mixture was stirred at 100 °C for 2 h. The mixture was quenched with water (10 mL), extracted with EtOAc (2x20 mL), the organic layer was dried over anhydrous Na2SO4 and concentrated under vacuum to give the crude product. The crude product was purified using column chromatography eluting with 8% MeOH in DCM to give the title compound (50 mg, 26.6%) as a yellow solid. 1 H NMR(400MHz,MeOD)δ8.80(s,1H),7.88-7.87(d,J=5.6Hz,1H),7.74(s,1H),7.50-7.78(d,J=7.6Hz,1H),7.40(s,1H),7.29-7.26(d,J=12Hz,1H),7 .18-7.16(d,J=5.6Hz,1H),7.00-6.99(d,J=6.4Hz,1H),4.06(s,3H),2.6 1-2.59(d,J=6Hz,8H),2.01(s,2H),1.68(s,4H),1.53(s,2H).LCMS:(M+H) + =452.98.

[0190] Example 14: Preparation of N-(6-methoxy-1-(p-tolylamino)isoquinolin-7-yl)-4-(piperidin-1-yl)butanamide (I-14) [ka] To a solution of N-(1-chloro-6-methoxyisoquinolin-7-yl)-4-(piperidin-1-yl)butanamide (0.15 g, 0.41 mmol), p-toluidine (53 mg, 0.45 mmol), and Cs2CO3 (0.27 g, 0.82 mmol) in degassed dioxane (20 V), Xantphos (24 mg, 0.041 mmol) and Pd2(dba)3 (38 mg, 0.041 mmol) were added under nitrogen atmosphere and the mixture was stirred at 100 °C for 2 h. The mixture was quenched with water (10 mL), extracted with EtOAc (2x20 mL), the organic layer was dried over anhydrous Na2SO4 and concentrated under vacuum to give the crude product. The crude product was purified using column chromatography eluting with 15% MeOH in DCM to give the title compound (45 mg, 25.1%) as a pale yellow solid. 1 H NMR(400MHz,DMSO-d6)δ9.40(s,1H),8.88(s,1H),8.77(s,1H),7.85-7.83(d,J=5.6Hz,1H),7.63-7.61(d,J=8Hz,2H),7.2 8(s,1H),7.10-7.04(m,3H),3.96(s,3H),2.33-2.27(d,J=25.2Hz,8H),1.78(s,2H),1.50(s,4H),1.39(s,2H).LCMS:(M+H) + =432.57.

[0191] Example 15: Preparation of N-(6-methoxy-1-(phenylamino)isoquinolin-7-yl)-4-(piperidin-1-yl)butanamide (I-15) [ka] To a solution of N-(1-chloro-6-methoxyisoquinolin-7-yl)-4-(piperidin-1-yl)butanamide (0.15 g, 0.41 mmol), aniline (46 mg, 0.45 mmol), and Cs2CO3 (0.27 g, 0.82 mmol) in degassed dioxane (20 V), Xantphos (24 mg, 0.041 mmol) and Pd2(dba)3 (38 mg, 0.041 mmol) were added under nitrogen atmosphere and the mixture was stirred at 100° C. for 2 h. The mixture was quenched with water (10 mL), extracted with EtOAc (2×20 ml), the organic layer was dried over anhydrous Na2SO4 and concentrated under vacuum to give the crude product. The crude product was purified using column chromatography eluting with 12% MeOH in DCM to give the title compound (55 mg, 31.7%) as a yellow solid. 1 H NMR(400MHz,MeOD)δ8.79(s,1H),7.80-7.78(d,J=5.6Hz,1H),7.56-7.54(m,2H),7.35-7.31(m,2H),7.27(s,1H),7.11-7.09(d,J=5.6Hz,1H) ,7.06-7.01(m,1H),4.06(s,3H),2.62-2.55(m,8H),2.03-1.96(m,2H) ,1.70-1.64(m,4H),1.53-1.52(d,J=5.2Hz,2H).LCMS:(M+H)+=418.54.

[0192] Example 16: Preparation of N-(1-((4-fluoro-3-(trifluoromethyl)phenyl)amino)-6-methoxyisoquinolin-7-yl)-4-(piperidin-1-yl)butanamide (I-16) [ka] To a solution of N-(1-chloro-6-methoxyisoquinolin-7-yl)-4-(piperidin-1-yl)butanamide (0.15 g, 0.41 mmol), 4-fluoro-3-(trifluoromethyl)aniline (89 mg, 0.45 mmol) and Cs2CO3 (0.27 g, 0.82 mmol) in degassed dioxane (3 mL), Xantphos (24 mg, 0.041 mmol) and Pd2(dba)3 (38 mg, 0.041 mmol) were added under nitrogen atmosphere and the mixture was stirred at 100° C. for 2 h. After completion of the reaction, the mixture was diluted with water (20 mL) and extracted with EtOAc (2×30 ml), the organic layer was dried over anhydrous Na2SO4 and concentrated to give the crude product. The crude product was purified using column chromatography eluting with 12% MeOH in DCM to give the title compound (65 mg, 31.1%) as a yellow solid. 1 H NMR(400MHz, methanol-d4)δ.8.80(s,1H),7.80-7.94(m,1H),7.90-7.84(m,2H),7.28-7.23(m,2H),7.15-7.13(d, J=5.6Hz,1H),4.05(s,3H),2.59-2.49(m,8H),2.02-1.94(m,2H),1.67-1.62(m,4H),1.51(bs,2H).LCMS:(M+H) + =505.2.

[0193] Example 17: Preparation of N-(1-((3-chloro-4-fluorophenyl)amino)-6-methylisoquinolin-7-yl)-4-(piperidin-1-yl)butanamide (I-17) [ka] Step 1: 4-Methyl-3-nitro-N-(pivaloyloxy)benzamide [ka] To a solution of 4-methyl-3-nitrobenzoic acid (5 g, 27.6 mmol) in cold DCM (50 mL) was added oxalyl chloride (2.5 mL, 30.36 mmol) and DMF (1 mL) at 0° C. and the mixture was stirred at room temperature for 3 h. The mixture was concentrated under N2 atmosphere to give 4-methyl-3-nitrobenzoyl chloride. To a solution of O-pivaloylhydroxylammonium trifluoromethanesulfonate (2.5 g, 16.77 mmol) and NaHCO3 (6.95 g, 82.8 mmol) in cold EtOAc:water (2:1, 50 mL) was added 4-methyl-3-nitrobenzoyl chloride in EtOAc (5 mL) at 0° C. and the mixture was stirred at room temperature for 3 h. After completion of the reaction, the mixture was diluted with water (100 ml) and extracted with EtOAc (3×150 mL). The combined organics were dried over anhydrous Na2SO4 and concentrated in vacuo to give the title compound (3 g, 38.8%) as an off-white solid. 1 H NMR (400MHz, DMSO-d6): δ8.83(s,1H),8.44(s,1H),8.07(d,J 6.4Hz,1H),7.60(d,8Hz,1H),4.50(t,J=5.2Hz,1H),3.37-3.22(m,4H),2.48(s,5H).

[0194] Step 2: 6-Methyl-7-nitroisoquinolin-1(2H)-one [ka] To a stirred solution of 4-methyl-3-nitro-N-(pivaloyloxy)benzamide (3 g, 10.7 mmol) in MeOH (30 mL) was added vinyl acetate (1.17 g, 13.6 mmol), cesium acetate (0.52 g, 2.72 mmol), and pentamethylcyclopentadienylrhodium dichloride dimer (50 mg, 0.9 mmol) under nitrogen atmosphere and the mixture was stirred at 70° C. for 4 h. The mixture was concentrated under vacuum to give the crude product. The crude product was triturated with 5% MeOH in diethyl ether, filtered, and dried to give the title compound (1.7 g, 77.8%) as a brown solid. LCMS: (M+H) + =205.15.

[0195] Step 3: 1-Chloro-6-methyl-7-nitroisoquinoline [ka] 6-Methyl-7-nitroisoquinolin-1(2H)-one (0.250 g, 1.22 mmol) was mixed with phosphorus(V) oxychloride (5 mL) and the reaction mixture was stirred at 130° C. for 16 h. The mixture was concentrated under vacuum and excess POCl3 was removed with toluene. The residue was diluted with EtOAc (50 mL) and an ice-cold 10% solution of NaHCO3 (100 mL). The layers were separated and the organic layer was dried over anhydrous Na2SO4 and concentrated under vacuum to give the crude product. The crude product was triturated with diethyl ether to give the title compound (0.25 g, 91.7%) as a pale yellow solid. LCMS: (M+H) + =223.14.

[0196] Step 4: 1-Chloro-6-methylisoquinolin-7-amine [ka] 60% of 1-chloro-6-methyl-7-nitroisoquinoline (250 mg, 1.12 mmol) in MeOH (8 mL), water (6 mL), and THF (3 mL). o To the C solution was added ammonium chloride (180 mg, 3.243 mmol) and iron powder 99% (181 mg, 3.243 mmol). The mixture was stirred at 60° C. for 4 h. The mixture was neutralized with solid NaHCO3 and filtered. The organic layer of the filtrate was evaporated and extracted with EtOAc (2×20 mL). The organic layer was dried over Na2SO4, concentrated and triturated with ether to give the title compound (180 mg, 83.2%) as a brown solid. LCMS: (M+H) + =193.12.

[0197] Step 5: N-(1-chloro-6-methylisoquinolin-7-yl)-4-(piperidin-1-yl)butanamide [ka] To a solution of 1-chloro-6-methylisoquinolin-7-amine (0.15 g, 0.778 mmol) in DCM (1.5 mL) was added 4-(piperidin-1-yl)butanoic acid (0.15 g, 0.875 mmol), N,N-diisopropylethylamine (1.5 mL), and 50% T3P in EtOAc (0.64 g, 2.02 mmol), and the mixture was stirred at 60° C. for 6 h. The mixture was concentrated and purified using silica gel chromatography. The product was eluted with 10% MeOH in MDC, and the resulting product was triturated with ether to give the title compound (200 mg, 74.3%) as a light brown solid. LCMS [M+H] + =346.31.

[0198] Step 6: N-(1-((3-chloro-4-fluorophenyl)amino)-6-methylisoquinolin-7-yl)-4-(piperidin-1-yl)butanamide [ka] To a stirred solution of N-(1-chloro-6-methylisoquinolin-7-yl)-4-(piperidin-1-yl)butanamide (200 mg, 0.578 mmol) and 3-chloro-4-fluoroaniline (100 mg, 0.687 mmol) in 1,4-dioxane (2 mL) was added 4.0 M hydrochloric acid in dioxane (0.041 g, 1.556 mmol). The mixture was stirred at 100° C. for 6 h. The mixture was concentrated in vacuo to give the title compound (80 mg, 30.4%) as a light brown solid. 1 H NMR (400MHz, methanol-d4): δ8.82(s,1H),7.98(s,1H),7.85(d,J=7.6Hz,1H),7.59 (d,J=6.8Hz,2H),7.55-7.53(m,1H),7.39(d,J=6Hz,1H),3.66(d,J=11.6Hz,2H) ,3.39-3.28(m,2H),3.06(t,J=12.8Hz,2H),2.82-2.78(m,2H),2.63(s,3H),2.2 5-2.24(m,2H),2.04(d,J=14.8,2H),1.94-1.84(m,4H),1.37(s,1H).LCMS:(M+H) +=455.2.

[0199] Example 18: Preparation of N-(1-((3-chloro-4-fluorophenyl)amino)isoquinolin-7-yl)-4-(piperidin-1-yl)butanamide (I-18) [ka] Step 1: 3-Nitro-N-(pivaloyloxy)benzamide [ka] To a cooled solution of 3-nitrobenzoic acid (2.2 g, 13.6 mmol) in DCM (22 mL) and DMF (catalytic amount) was added oxalyl chloride (1.3 ml, 14.48 mmol) at 0° C. and the mixture was stirred at room temperature for 3 h. The mixture was concentrated under N2 atmosphere to give 3-nitrobenzoyl chloride. To a cold solution of O-pivaloylhydroxylammonium trifluoromethanesulfonate (3.8 g, 14.48 mmol) and NaHCO3 (3.3 g, 39.49 mmol) in EtOAc:H2O (12:1, 50 mL) was added 3-nitrobenzoyl chloride in EtOAc (10 mL) at 0° C. and the mixture was stirred at room temperature for 3 h. The mixture was diluted with water (100 mL) and extracted with EtOAc (3×150 mL). The combined organic layers were dried over anhydrous Na2SO4 and concentrated in vacuo to give the title compound (1.7 g, 48.5%) as an off-white solid. The crude product was carried onto the next step without further purification. LCMS: (M+H)+=266.25.

[0200] Step 2: 7-Nitroisoquinolin-1(2H)-one [ka] To a stirred solution of 3-nitro-N-(pivaloyloxy)benzamide (1 g, 3.75 mmol) in MeOH (10 mL) was added vinyl acetate (0.48 g, 4.87 mmol), cesium acetate (0.25 g, 1.323 mmol), and pentamethylcyclopentadienylrhodium dichloride dimer (116 mg, 0.37 mmol) under nitrogen atmosphere. The mixture was then stirred at 70° C. for 16 h and concentrated in vacuo to give the crude product. The crude product was triturated with 1% MeOH in diethyl ether to give the title compound (0.7 g, 98.0%) as a brown solid. This crude product was taken on to the next step without further purification.

[0201] Step 3: 1-Chloro-7-nitroisoquinoline [ka] A suspension of 7-nitroisoquinolin-1(2H)-one (0.7 g, 3.68 mmol) and POCl3 (10 mL) was stirred at 100 °C for 2 h. After completion of the reaction, the mixture was concentrated and the residue was diluted with DCM (50 mL) and ice-cold water (50 mL), neutralized with solid NaHCO3, and extracted with DCM (2 x 50 mL). The combined organic layers were concentrated and triturated with diethyl ether to give 1-chloro-7-nitroisoquinoline (0.4 g, 1.92 mmol, 52.1%) as a pale yellow solid. LCMS: (MH) + =208.60.

[0202] Step 4: 1-Chloroisoquinolin-7-amine [ka] To a stirred solution of 1-chloro-7-nitroisoquinoline (0.4 g, 1.92 mmol) in MeOH (37 V), water (27 V), and THF (14 V) was added NH4Cl (0.37 g, 6.92 mmol) and Fe (0.38 g, 6.92 mmol) at 60 °C, and the mixture was stirred at 60 °C for 5 h. After completion of the reaction, the mixture was passed through a bed of Celite and the filtrate was concentrated under vacuum to give the crude product. The crude product was extracted with DCM (2 x 30 mL), dried over anhydrous Na2SO4, and concentrated to give the title compound (0.24 g, 70.1%) as a yellow solid. LCMS: (M+H) + =178.62.

[0203] Step 5: N-(1-chloroisoquinolin-7-yl)-4-(piperidin-1-yl)butanamide [ka] To a stirred solution of 1-chloroisoquinolin-7-amine (0.15 g, 0.84 mmol) and 4-(piperidin-1-yl)butanoic acid (0.22 g, 1.26 mmol) in DCM (10V) was added DIPEA (0.32 g, 2.52 mmol) and T3P (50% solution, 0.5 g, 1.68 mmol) at room temperature. The mixture was stirred at 70 °C for 16 h. After completion of the reaction, the mixture was diluted with water (20 mL), extracted with EtOAc (3 x 20 mL), the organic layer was dried over anhydrous Na2SO4 and concentrated under vacuum to give the crude product. The crude product was purified using column chromatography eluting with 8-11% MeOH in DCM to give the title compound (0.1 g, 35.9%) as a yellow solid. LCMS: (M+H) + =331.84.

[0204] Step 6: N-(1-((3-chloro-4-fluorophenyl)amino)isoquinolin-7-yl)-4-(piperidin-1-yl)butanamide [ka] To a solution of N-(1-chloroisoquinolin-7-yl)-4-(piperidin-1-yl)butanamide (0.1 g, 0.301 mmol), 3-chloro-4-fluoroaniline (52.64 mg, 0.36 mmol), and Cs2CO3 (0.19 g, 0.6 mmol) in degassed dioxane (10 V), Xantphos (17.45 mg, 0.030 mmol) and Pd2(dba)3 (27.6 mg, 0.030 mmol) were added under nitrogen atmosphere, and the mixture was stirred at 100° C. for 2 h. After completion of the reaction, the mixture was diluted with water (20 mL) and extracted with EtOAc (2×20 mL), and the organic layer was dried over anhydrous Na2SO4 and concentrated to give the crude product. The crude product was purified using column chromatography eluting with 12% MeOH in DCM to give the title compound (65 mg, 48.9%) as an off-white solid. 1 H NMR (400MHz, methanol d4) δ8.59(s,1H),7.90-7.86(m,2H),7.81-7.79(d,J=8.8Hz,1H),7.72-7.69(dd,J=2Hz,J=2Hz,1H),7.57-7.53(m,1H) ,7.23-7.17(m,3H),3.27-3.106(m,6H),2.67-2.64(t,J=13.6,2H),2.18-2.11(m,2H),1.86(s,4H),1.68(s,2H),1.30(s,1H).LCMS:(M+H) + =440.95.

[0205] Example 19: Preparation of N-(1-((2-chloro-4-fluorophenyl)amino)-6-methoxyisoquinolin-7-yl)-4-(piperidin-1-yl)butanamide (I-19) [ka] To a solution of N-(1-chloro-6-methoxyisoquinolin-7-yl)-4-(piperidin-1-yl)butanamide (100 mg, 0.276 mmol) in dioxane (2 mL) was added Cs2CO3 (179 mg, 0.552 mmol), 2-chloro-4-fluoroaniline (44 mg, 0.304 mmol) and the mixture was degassed with N2 for 10 min. To this was added Xantphos (15 mg, 0.027 mmol) and Pd2(dba)3 (25 mg, 0.276 mmol) and the mixture was stirred at 100 °C for 2 h. The mixture was concentrated and the residue was purified by silica gel chromatography eluting with 5-20% MeOH / DCM to give the title compound (25 mg, 26.9%) as a light brown solid. 1 H NMR(400MHz,MeOD-d4):δ9.44(s,1H),8.87(s,1H),8.66(s,1H),7.79-7.7 7(d,J=5.2Hz,1H),7.71-7.67(d,J=6Hz,1H),7.50-7.48(d,J=6.4Hz,1H),7 .39(s,1H),7.33(s,1H),7.26-7.18(m,1H),7.09-7.08(d,J=5.2Hz,1H),4 .21(s,2H),2.51(s,8H),1.87(s,2H)1.57(s,4H),1.24(s,2H);LCMS:(M+1) + =470.97.

[0206] Example 20: Preparation of N-(1-((4-fluoro-3-methylphenyl)amino)-6-methoxyisoquinolin-7-yl)-4-(piperidin-1-yl)butanamide (I-20) [ka] To a solution of N-(1-chloro-6-methoxyisoquinolin-7-yl)-4-(piperidin-1-yl)butanamide (100 mg, 0.276 mmol) in dioxane (2 mL) was added Cs2CO3 (180 mg, 0.552 mmol), 4-fluoro-3-methylaniline (38 mg, 0.304 mmol) and the mixture was degassed with N2 for 10 min. To this was added Xantphos (16 mg, 0.027 mmol) and Pd2(dba)3 (25 mg, 0.276 mmol) and the mixture was stirred at 100 °C for 2 h. The mixture was concentrated and the residue was purified using silica gel chromatography eluting with 5-20% MeOH / DCM to give the title compound (35 mg, 28.1%) as a grey fluffy solid. 1 H NMR(400MHz,MeOD-d4):δ9.364(s,1H),8.92(s,1H),8.79(s,1H),7.87-7.85(d,J=5.6Hz,1H),7.63-7.61(d,J=7.2Hz,2H),7.30( s,1H),7.12-7.03(m,1H),3.97(s,3H)2.32-2.30(d,J=6Hz,6H),2.24(s,3H),1.77(s,2H),1.50(s,4H),1.31(s,1H);LCMS:(M+1) + =470.97.

[0207] Example 21: Preparation of N-(1-((3-chloro-4-methylphenyl)amino)-6-methoxyisoquinolin-7-yl)-4-(piperidin-1-yl)butanamide (I-21) [ka] To a solution of N-(1-chloro-6-methoxyisoquinolin-7-yl)-4-(piperidin-1-yl)butanamide (0.15 g, 0.41 mmol), 3-chloro-4-methylaniline (70 mg, 0.45 mmol), and Cs2CO3 (0.27 g, 0.82 mmol) in degassed dioxane (3 mL), Xantphos (24 mg, 0.041 mmol) and Pd2(dba)3 (38 mg, 0.041 mmol) were added under nitrogen atmosphere and the mixture was stirred at 100 °C for 2 h. The mixture was diluted with water (25 mL) and extracted with EtOAc (2 x 30 mL). The combined organic layers were dried over anhydrous Na2SO4 and concentrated to give the crude product. The crude product was purified using column chromatography eluting with 12% MeOH in DCM to give the title compound (65 mg, 33.6%) as a yellow solid. 1 H NMR (400MHz, methanol-d4) δ8.767(s,1H),7.84-7.82(d,J=6Hz,1H),7.70-7.70 (d,J=2Hz,1H),7.41-7.38(dd,J=2.4Hz,J=2Hz,1H),7.26(s,1H),7.23-7.21( d,J=8Hz,1H),7.120-7.11(d,J=5.6Hz,1H),4.05(s,3H),2.58-2.48(m,8H),2 .02-1.96(m,2H),1.67-1.62(m,4H),1.51-1.50(d,J=4.4Hz,2H);LCMS:(M+H) + =567.01.

[0208] Example 22: Preparation of N-(1-((4-fluoro-3-methoxyphenyl)amino)-6-methoxyisoquinolin-7-yl)-4-(piperidin-1-yl)butanamide (I-22) [ka] To a solution of N-(1-chloro-6-methoxyisoquinolin-7-yl)-4-(piperidin-1-yl)butanamide (100 mg, 0.276 mmol) in dioxane (2 mL), Cs2CO3 (180 mg, 0.552 mmol), 4-fluoro-3-methoxyaniline (42 mg, 0.304 mmol) were added and the mixture was degassed with N2 for 10 min. To this, Xantphos (16 mg, 0.027 mmol) and Pd2(dba)3 (25 mg, 0.276 mmol) were added and the mixture was stirred at 100 °C for 2 h. The mixture was concentrated and the residue was purified by silica gel chromatography and the product was eluted with 12-20% MeOH / DCM to give the title compound (35 mg, 27.2%) as a yellow solid. 1 H NMR(400MHz,MeOD-d4):δ9.38(s,1H),8.96(s,1H),8.78(s,1H),7.88-7.87(d,J=5 .6Hz,1H),7.61-7.59(t,J=6Hz,2H),7.42-7.40(t,J=8.8Hz,1H)7.30(s,1H),7.13- 7.08(m,2H)3.96(s,3H),3.82(s,3H),2.50-2.44(t,J=22Hz,3H),2.31-2.27(m,J= 13.6Hz,6H)1.80-1.74(m,2H)1.48-1.47(d,J=4.8Hz,4H),1.37(s,2H);LCMS:(M+H) + =467.5.

[0209] Example 23: Preparation of N-(1-((3,5-dichloro-4-fluorophenyl)amino)-6-methoxyisoquinolin-7-yl)-4-(piperidin-1-yl)butanamide (I-23) [ka] To a solution of N-(1-chloro-6-methoxyisoquinolin-7-yl)-4-(piperidin-1-yl)butanamide (100 mg, 0.276 mmol) in dioxane (2 mL) was added Cs2CO3 (180 mg, 0.552 mmol), 3,5-dichloro-4-fluoroaniline (54 mg, 0.304 mmol) and the mixture was degassed with N2 for 10 min. To this was added Xantphos (15 mg, 0.027 mmol) and Pd2(dba)3 (25 mg, 0.276 mmol) and the mixture was stirred at 100 °C for 2 h. The mixture was concentrated and the residue was purified by silica gel chromatography eluting with 5-20% MeOH / DCM to give the title compound (60 mg, 43.0%) as a light brown solid. 1 H NMR (400MHz, methanol-d4): δ8.79(s,1H),7.90-7.89(d,J=5.6Hz,1H),7.76-7.75(d,J=6Hz,2H),7.27(s,1H),7.18-7.16(d,J=6Hz ,1H),4.06(s,3H),2.60-2.53(m,8H),2.01-1.98(t,J=15.2Hz,2H),1.69-1.64(m,4H),1.53-1.51(d,J=4.8Hz,2H).LCMS:(M+1) + =505.42.

[0210] Example 24: Preparation of N-(1-((3-chloro-4-methoxyphenyl)amino)-6-methoxyisoquinolin-7-yl)-4-(piperidin-1-yl)butanamide (I-24) [ka] To a solution of N-(1-chloro-6-methoxyisoquinolin-7-yl)-4-(piperidin-1-yl)butanamide (100 mg, 0.276 mmol) in dioxane (2 mL) was added Cs2CO3 (180 mg, 0.552 mmol), 3-chloro-4-methoxyaniline (46 mg, 0.304 mmol) and the mixture was degassed with N2 for 10 min. To this was added Xantphos (15 mg, 0.027 mmol) and Pd2(dba)3 (25 mg, 0.276 mmol) and the mixture was stirred at 100 °C for 2 h. The mixture was evaporated and the residue was purified by silica gel chromatography and the product was eluted with 5-20% MeOH / DCM to give the title compound (35 mg, 27.2%) as a yellow solid. 1 H NMR(400MHz,MeOD-d4):δ8.77(s,1H),7.79-7.77(d,J=5.6Hz 1H),7.65-7.64(d,J=2.4Hz,1H),7.44-7.42(m,1H),7.26(s,1H),7.08-7.05(m,2H),4.09-7.05(d,J=14Hz,3H)3.89(s,3H),2.74 -2.67(m,6H),2.62-2.59(t,J=14.4Hz,2H)2.06-1.99(m,2H)1.73-1.70(t,J=11.6Hz,4H),1.56(s,2H),1.33(s,2H);LCMS:(M+H) + =483.5.

[0211] Example 25: Preparation of N-(1-((3-chloro-5-fluorophenyl)amino)-6-methoxyisoquinolin-7-yl)-4-(piperidin-1-yl)butanamide (I-25) [ka] To a solution of N-(1-chloro-6-methoxyisoquinolin-7-yl)-4-(piperidin-1-yl)butanamide (100 mg, 0.276 mmol) in dioxane (2 mL), Cs2CO3 (180 mg, 0.552 mmol) and 3-chloro-5-fluoroaniline (46 mg, 0.304 mmol) were added and the mixture was degassed with N2 for 10 min. To this mixture, Xantphos (15 mg, 0.027 mmol) and Pd2(dba)3 (25 mg, 0.276 mmol) were added and the mixture was stirred at 100 °C for 2 h. The mixture was concentrated and the residue was purified by silica gel chromatography eluting with 10-20% MeOH / DCM to give the title compound (35 mg, 26.9%) as a white solid. 1 H NMR(400MHz,MeOD-d4):δ8.81(s,1H),7.96-7.94(d,J=5.6Hz,1H),7.52-7.51(d, J=4Hz,1H),7.49(s,1H),7.31(s,1H),7.23-7.23(d,J=5.6Hz,2H),6.77-6.75(d, J=8.4Hz)4.07(s,3H)2.78-2.71(m,3H),2.64-2.61(t,J=14Hz,6H),2.06-2.02(t ,J=16Hz,2H)1.74-1.71(t,J=11.2Hz,4H),1.57(s,2H),1.30(s,1H);LCMS:(M+H) + =471.49.

[0212] Example 26: Preparation of N-(6-chloro-1-((3-chloro-4-fluorophenyl)amino)isoquinolin-7-yl)-4-(piperidin-1-yl)butanamide (I-26) [ka] Step 1: 4-Chloro-3-nitro-N-(pivaloyloxy)benzamide [ka] To a solution of 4-chloro-3-nitrobenzoic acid (1 g, 4.96 mmol) in DCM (10 mL) was added oxalyl chloride (4 mL, 5.449 mmol) and DMF (catalytic amount) at 0° C., and the mixture was stirred at room temperature for 2 h. After 2 h, the mixture was concentrated. To a solution of O-pivaloylhydroxylammonium-trifluoromethanesulfonate (0.8 g, 6.770 mmol) and NaHCO3 (0.83 g, 9.523 mmol) in EtOAc (10 ml) and water (10 mL), the acid chloride prepared above was added dropwise, and the mixture was stirred at room temperature for 4 h. The mixture was quenched with saturated NaHCO3 solution (20 mL) and extracted with EtOAc (200 mL). The organic layer was dried over anhydrous Na2SO4 and concentrated under vacuum to give the title compound (1.3 g, 87.1%) as an off-white solid. The crude product was triturated with pentane and taken onto the next step. LCMS: (M+H) + =301.

[0213] Step 2: 6-Chloro-7-nitroisoquinolin-1(2H)-one [ka] To a stirred solution of 4-chloro-3-nitro-N-(pivaloyloxy)benzamide (1 g, 3.325 mmol) in MeOH (10 mL) was added vinyl acetate (0.42 g, 4.87 mmol), cesium acetate (0.25 g, 1.323 mmol), and pentamethylcyclopentadienylrhodium dichloride dimer (40 mg, 0.06 mmol) under nitrogen and the mixture was stirred at 70° C. for 16 h. The mixture was concentrated under vacuum to give the crude product. The crude product was triturated with 1% MeOH in diethyl ether. The product was filtered and dried over Na2SO4 to give the title compound (0.35 g, 46.9%) as a brown solid. LCMS: (M+H) + =225.2.

[0214] Step 3: 1,6-Dichloro-7-nitroisoquinoline [ka] 6-Chloro-7-nitroisoquinolin-1(2H)-one (0.3 g, 1.335 mmol) was mixed with phosphorus oxychloride (6 mL) and the mixture was diluted with 100 o The mixture was stirred at RT for 1 h. The mixture was concentrated and residual POCl3 was removed by stripping with toluene. The residue was diluted with ice-cold water (50 mL), extracted with DCM (2 x 20 mL) and backwashed with saturated NaHCO3 solution. The organic layer was concentrated and triturated with diethyl ether to give 1-chloro-6-methoxy-7-nitroisoquinoline (0.23 g, 1.05 mmol, 78.24%) as a pale yellow solid which was used directly in the next step.

[0215] Step 4: 6-Chloro-N-(3-chloro-4-fluorophenyl)-7-nitroisoquinolin-1-amine [ka] To a suspension of 1,6-dichloro-7-nitroisoquinoline (300 mg, 1.23 mmol) and 3-chloro-4-fluoroaniline (185 mg, 1.27 mmol) in 1,4-dioxane (5 mL) was added 4 M hydrochloric acid in dioxane (0.6 mL, 2.46 mmol), and the mixture was diluted to 100 o C for 2 h. The mixture was diluted with diethyl ether and the resulting precipitate was filtered. The solid was rinsed with ether to give the title compound (300 mg, 69.0%) as a brown solid. LCMS: (M+H) + =354.2.

[0216] Step 5: 6-chloro-N1-(3-chloro-4-fluorophenyl)isoquinoline-1,7-diamine [ka] To a solution of 6-chloro-N-(3-chloro-4-fluorophenyl)-7-nitroisoquinolin-1-amine (320 mg, 0.908 mmol) in MeOH (12 mL), water (9 mL), and THF (12 mL) was added ammonium chloride (179 mg, 3.346 mmol) and iron powder (182 mg, 2.542 mmol) to form a mixture. The mixture was stirred at 60° C. for 90 min, filtered, concentrated, and extracted with EtOAc (2×20 mL). The organic layer was dried over Na2SO4, concentrated, and the residue was triturated with ether to give the title compound (220 mg, 0.679 mmol, 75.2%) as a brown solid. LCMS [M+H] + =324.2.

[0217] Step 6: N-(6-chloro-1-((3-chloro-4-fluorophenyl)amino)isoquinolin-7-yl)-4-(piperidin-1-yl)butanamide [ka] 6-Chloro-N 1 To a solution of -(3-chloro-4-fluorophenyl)isoquinoline-1,7-diamine (200 mg, 0.621 mmol) in DCM (2 mL) was added 4-(piperidin-1-yl)butanoic acid (240 mg, 1.401 mmol), DIPEA (1 mL, 8.61 mmol), and 50% T3P in EtOAc (0.8 mL, 2.79 mmol) at room temperature, and the mixture was stirred for 60 o The mixture was stirred at C for 16 h. The reaction was quenched by the addition of MeOH, the mixture was concentrated in vacuo and purified on silica eluting with 10% MeOH in CH2Cl2. The final compound was triturated with ether to give the title compound (85 mg, 78.2%) as a light brown solid. 1H NMR (400MHz, methanol-d4) δ8.62(s,1H),7.97-7.91(m,3H),7.59-7.56(m,1H),7.23-7.14(m,3H),4.85(s,2H),3.66(d,J=15 .1Hz,1H),2.61-2.36(m,8H),2.16(s,1H),2.05-2.01(m,2H),1.68(s,4H),1.53(s,1H),1.33(d,J=19.2Hz,2H).LCMS(M+H) + =475.39.

[0218] Example 27: Preparation of N-(1-((3,4-dichlorophenyl)amino)-6-methoxyisoquinolin-7-yl)-4-(piperidin-1-yl)butanamide (I-27) [ka] To a solution of N-(1-chloro-6-methoxyisoquinolin-7-yl)-4-(piperidin-1-yl)butanamide (150 mg, 0.414 mmol) in dioxane (2 mL), Cs2CO3 (269 mg, 0.829 mmol), 3,4-dichloroaniline (73 mg, 0.455 mmol) were added, and the mixture was degassed with N2 for 10 min. To this, Xantphos (23 mg, 0.414 mmol) and Pd2(dba)3 (37 mg, 0.414 mmol) were added, and the mixture was stirred at 100 °C for 2 h. After completion of the reaction, the mixture was concentrated, and the residue was purified by silica gel chromatography eluting with 15-20% MeOH / DCM to give the title compound (35 mg, 17.3%) as a brown solid. 1 H NMR(400MHz,MeOD-d4):δ8.81(s,1H),7.90-7.92(m,2H),7.55-7.53(m,1H),7.43-7.40(d,J=8.8Hz,1H),7.30(s,1H),7.19-7.18( d,J=6Hz,1H),4.08(s,3H),2.93-2.86(m,5H),2.67-2.64(t,J=16.4Hz,2H),1.80-1.74(m,4H),1.51(s,2H).LCMS:(M+1)+=487.00.

[0219] Example 28: Preparation of N-(1-((3-ethyl-4-fluorophenyl)amino)-6-methoxyisoquinolin-7-yl)-4-(piperidin-1-yl)butanamide (I-28) [ka] Step 1: 3-Ethyl-4-fluoroaniline [ka] To a suspension of 10% Pd / C (50% wet, w / 2, 0.125 g) in EtOAc (4 mL) was added 3-ethynyl-4-fluoroaniline (0.25 g, 0.184 mmol) and the mixture was stirred under hydrogen purge for 3 h. The mixture was filtered through a bed of celite and washed with methanol. The organic layer was concentrated under vacuum to give the product (150 mg, 0.01 mmol) as a yellow oil. LCMS: (M+H) + =465.2.

[0220] Step 2: N-(1-((3-ethyl-4-fluorophenyl)amino)-6-methoxyisoquinolin-7-yl)-4-(piperidin-1-yl)butanamide [ka] To a solution of N-(1-chloro-6-methoxyisoquinolin-7-yl)-4-(piperidin-1-yl)butanamide (150 mg, 0.414 mmol) in dioxane (2 mL) was added Cs2CO3 (269 mg, 0.829 mmol), 3-ethyl-4-fluoroaniline (63 mg, 0.455 mmol) and the mixture was degassed with N2 for 10 min. Xantphos (23 mg, 0.414 mmol) and Pd2(dba)3 (37 mg, 0.414 mmol) were added and the mixture was stirred at 100 °C for 2 h. The mixture was concentrated and the residue was purified by silica gel chromatography eluting with 5-20% MeOH / DCM to give the title compound (30 mg, 15.6%) as an off-white solid. 1H NMR (400MHz, methanol-d4): δ8.79(s,1H),7.78-7.76(d,J=6Hz 1H),7.45-7.35(m,2H),7.27(s,1H),7.09-7.07(t,J=6Hz,1H),6.99(s,1H),4.06(s,3H),3.37-3.31(m,1H),2.88-2.83(t,J LCMS: (M+H) + =465.2.

[0221] Example 29: Preparation of N-(1-((3-chloro-4-fluorophenyl)amino)-5-methoxyisoquinolin-7-yl)-4-(piperidin-1-yl)butanamide (I-29) [ka] Step 1: 3-Methoxy-5-nitro-N-(pivaloyloxy)benzamide [ka] To a cold solution of 3-methoxy-5-nitrobenzoic acid (2.1 g, 10.65 mmol) in DCM (20 mL) was added oxalyl chloride (1.2 mL, 8.51 mmol) and DMF (catalytic amount) at 0° C. and the mixture was stirred at room temperature for 3 h. The mixture was concentrated under N2 atmosphere to give 3-methoxy-5-nitrobenzoyl chloride. To a cooled solution of O-pivaloylhydroxylammonium trifluoromethanesulfonate (1.2 g, 10.08 mmol) and NaHCO3 (1.7 g, 20.23 mmol) in EtOAc (10 mL) and water (10 mL) was added a solution of 3-methoxy-5-nitrobenzoyl chloride in EtOAc (5 mL) at 0° C. and the mixture was stirred at room temperature for 4 h. The mixture was diluted with water (100 mL) and extracted with EtOAc (3×150 mL). The combined organic layers were dried over anhydrous Na2SO4 and concentrated in vacuo to give the title compound (1.4 g, 46.6%) as an off-white solid. The crude product was taken on to the next step. LCMS: (M+H) + =296.2.

[0222] Step 2: 5-Methoxy-7-nitroisoquinolin-1(2H)-one [ka] To a stirred solution of 3-methoxy-5-nitro-N-(pivaloyloxy)benzamide (1.0 g, 3.375 mmol) in MeOH (10 ml) was added vinyl acetate (0.47 g, 5.549 mmol), cesium acetate (0.26 g, 1.352 mmol), and pentamethylcyclopentadienylrhodium dichloride dimer (40 mg, 0.060 mmol) under nitrogen. After addition, the mixture was stirred at 70° C. for 16 hours. After completion of the reaction, the mixture was concentrated under vacuum to give the crude product. The crude product was triturated with 1% MeOH in diethyl ether. The solid was filtered and dried over Na2SO4 to give the title compound (0.72 g, 97.0%) as a brown solid. LCMS: (M+H) + =221.4.

[0223] Step 3: 1-Chloro-5-methoxy-7-nitroisoquinoline [ka] A suspension of 5-methoxy-7-nitroisoquinolin-1(2H)-one (0.7 g, 3.179 mmol) in phosphorus oxychloride (14 mL) was o C for 1 h. After completion of the reaction, the mixture was concentrated. The residue was diluted with DCM (100 mL) and ice-cold water (100 mL) and neutralized with solid NaHCO3. The layers were separated and the organic layer was dried over Na2SO4, concentrated and triturated with diethyl ether to give the title compound (0.68 g, 89.6%) as a pale yellow solid. LCMS: (M+H) + =239.2

[0224] Step 4: 1-Chloro-5-methoxyisoquinolin-7-amine [ka] 60% of 1-chloro-5-methoxy-7-nitroisoquinoline (0.9 g, 2.588 mmol) in MeOH (34 mL), water (23 mL), and THF (13 mL). o To solution C, ammonium chloride (0.5 g, 9.301 mmol) and iron powder (0.52 g, 9.325 mmol) were added, and the mixture was heated for 60 o C for 90 min. The mixture was filtered, the organic solvent was evaporated and the product was extracted with EtOAc (2x100 mL). The combined organic layers were dried over Na2SO4, concentrated and triturated with ether to give the title compound (0.73 g, 92.8%) as a brown solid. LCMS: [M+H] + =209.0.

[0225] Step 5: N-(6-chloro-1-((3-chloro-4-fluorophenyl)amino)isoquinolin-7-yl)-4-(piperidin-1-yl)butanamide [ka] To a solution of 1-chloro-5-methoxyisoquinolin-7-amine (0.3 g, 1.435 mmol) in DCM (3 mL) was added 4-(piperidin-1-yl)butanoic acid (0.37 g, 2.152 mmol), N,N-diisopropylethylamine (5 mL), and 50% T3P in EtOAc (2.5 mL, 11.5 mmol), and the mixture was stirred for 60 o C for 16 h. The mixture was concentrated and purified using silica eluting with 10% MeOH in MDC to give the title compound (251 mg, 48.4%) as a light brown solid. LCMS: [M+H] + =362.

[0226] Step 6: N-(1-((3-chloro-4-fluorophenyl)amino)-5-methoxyisoquinolin-7-yl)-4-(piperidin-1-yl)butanamide [ka] To a solution of 1-chloro-5-methoxyisoquinolin-7-amine (0.15 g, 0.41 mmol), 3-chloro-4-fluoroaniline (90 mg, 0.61 mmol), and Cs2CO3 (0.53 g, 1.62 mmol) in degassed 1,4-dioxane (2 mL), tris(dibenzylideneacetone)dipalladium (20 mg, 0.024 mmol), and (9,9-dimethyl-9H-xanthene-4,5-diyl)bis(diphenylphosphane) (20 mg, 0.034 mmol) were added under N2 atmosphere, and the mixture was heated to 110°C. o C for 1 h. The mixture was concentrated and purified by flash chromatography on neutral alumina. The product was eluted with 6% MeOH in DCM and triturated with n-pentane to give the title compound (33 mg, 18%) as a brown solid. 1 H NMR (400MHz, methanol-d4) δ8.08(s,1H),7.86(s,1H),7.47-7.38(m,2H),7.21-7.14(m,2H),4.01(s, 3H),2.56-2.45(m,8H),2.03-1.94(m,2H),1.66-1.64(m,5H),1.32(d,J=19.2Hz,1H);LCMS:[M+H]+ =471.

[0227] Example 30: Preparation of N-(1-((4-fluoro-3-isopropylphenyl)amino)-6-methoxyisoquinolin-7-yl)-4-(piperidin-1-yl)butanamide (I-30) [ka] Step 1: 1-Fluoro-4-nitro-2-(prop-1-en-2-yl)benzene [ka] In a flame-dried flask, KHMDS (21.6 mL, 20% in THF, 21.85 mmol) was added under N2 for 5 min to Ph3P + To a stirred suspension of CH3Br- (7.6 g, 21.85 mmol) was added dropwise at -78 °C. A yellow color developed upon addition. After 5 min at -78 °C, the suspension was stirred at room temperature for 5 min and cooled again to -78 °C. 1-(2-fluoro-5-nitrophenyl)ethan-1-one (2 g, 10.9 mmol) was then added dropwise over 5 min. A deep red color developed upon addition. The mixture was warmed to room temperature, concentrated, and the residue was purified by silica gel chromatography eluting with 7% EtOAc in hexanes to give the product (300 mg, 1.66 mmol). 1 H NMR (400MHz, chloroform-d): δ8.26(d,J=3.2Hz,1H),8.24-8.14(m,1H),7.28-7.1 6(m,2H),5.40-5.38(t,J=8Hz,2H),2.19-2.19(d,J=0.8Hz,3H),1.45(s,1H).

[0228] Step 2: 3-Ethyl-4-fluoroaniline [ka] To a suspension of 10% Pd / C (50% wet, w / 2, 0.150 g) in EtOAc (40 mL) was added 1-fluoro-4-nitro-2-(prop-1-en-2-yl)benzene (0.3 g, 1.65 mmol) and the mixture was stirred with hydrogen purging for 3 h. After the reaction was complete, the mixture was filtered through a bed of celite and washed with methanol. The organic layer was concentrated under vacuum to give the product (110 mg, 0.718 mmol) as a yellow oil. LCMS: (M+H)+=154.22.

[0229] Step 3: N-(1-((4-fluoro-3-isopropylphenyl)amino)-6-methoxyisoquinolin-7-yl)-4-(piperidin-1-yl)butanamide [ka] To a solution of N-(1-chloro-6-methoxyisoquinolin-7-yl)-4-(piperidin-1-yl)butanamide (100 mg, 0.276 mmol) in dioxane (2 mL) was added Cs2CO3 (270 mg, 0.823 mmol), 4-fluoro-3-isopropylaniline (46 mg, 0.304 mmol) and the mixture was degassed with N2 for 10 min. Xantphos (15 mg, 0.276 mmol) and Pd2(dba)3 (25 mg, 0.276 mmol) were added and the mixture was stirred at 100 °C for 2 h. The mixture was evaporated and the residue was purified by silica gel chromatography eluting with 5-20% MeOH / DCM to give the title compound (28 mg, 27.2%) as a pale yellow solid. 1H NMR(400MHz,MeOD-d4):δ9.21(s,1H),7.55(s,1H),7.48-7.46(t,J=9.2Hz,1H),7.42( s,1H),7.31-7.28(m,4H),4.16(s,3H),3.61-3.49(m,1H)3.25-3.23(m,4H),3.01-2.9 9(t,J=11.6Hz,2H),2.77-2.73(t,J=13.6Hz,2H),2.19-2.15(t,J=16.4Hz,2H)2.00(s ,1H)1.96(s,1H)1.85-1.79(t,J=25.6Hz,4H)1.34-1.30(t,J=16.8Hz,6H).LCMS:(M+H) + =479.00.

[0230] Example 31: Preparation of N-(1-((3-chloro-4-fluorophenyl)amino)-6-methoxyisoquinolin-7-yl)-N-methyl-4-(piperidin-1-yl)butanamide (I-31) [ka] Step 1: 4-((1-chloro-6-methoxyisoquinolin-7-yl)amino)-4-oxobutanoic acid [ka] To a stirred solution of 1-chloro-6-methoxyisoquinolin-7-amine (1 g, 4.79 mmol) and monomethyl succinate (0.94 g, 7.19 mmol) in DCM (10V), DIPEA (1.86 g, 14.42 mmol) and T3P (50% in EtOAc) (3.04 g, 9.58 mmol) were added and the mixture was stirred at 70° C. for 16 h. After completion of the reaction, the mixture was quenched with water (200 mL), extracted with EtOAc (3×200 mL), the organic layer was dried over anhydrous Na2SO4 and concentrated under vacuum to give the crude product. The crude product was purified using column chromatography eluting with 70% EtOAc in hexane to give the title compound (0.8 g, 51.7%) as a brown solid. LCMS: (M+H) + =322.75.

[0231] Step 2: Methyl 4-((1-chloro-6-methoxyisoquinolin-7-yl)(methyl)amino)-4-oxobutanoate [ka] To a stirred solution of methyl 4-((1-chloro-6-methoxyisoquinolin-7-yl)amino)-4-oxobutanoate (0.8 g, 2.48 mmol) and t-BuOK (0.55 g, 4.96 mmol) in DMSO (10 mL) was added MeI (1.05 g, 7.45 mmol) and the reaction was stirred at room temperature for 2 h. The mixture was quenched with water (150 mL) and extracted with DCM (2x200 mL). The combined organic layers were dried over anhydrous Na2SO4 and concentrated under vacuum to give the crude product. The crude product was purified using column chromatography eluting with 60% EtOAc in hexane to give the title compound (0.6 g, 1.78 mmol, 71.9%) as a brown solid. LCMS: (M+H) + =336.77.

[0232] Step 3: N-(1-chloro-6-methoxyisoquinolin-7-yl)-4-hydroxy-N-methylbutanamide [ka] To a stirred solution of methyl 4-((1-chloro-6-methoxyisoquinolin-7-yl)(methyl)amino)-4-oxobutanoate (0.6 g, 1.78 mmol) in MeOH (10 V) was added NaBH4 (0.66 g, 17.8 mmol) at 50° C. The mixture was stirred at 50° C. for 24 h. After completion of the reaction, the mixture was concentrated to give the crude product. The crude product was purified using column chromatography eluting with 5% MeOH in DCM to give the title compound (0.5 g, 71.9%) as a light brown solid. LCMS: (M+H) + =308.76.

[0233] Step 4: N-(1-chloro-6-methoxyisoquinolin-7-yl)-N-methyl-4-oxobutanamide [ka] To a stirred solution of N-(1-chloro-6-methoxyisoquinolin-7-yl)-4-hydroxy-N-methylbutanamide (0.5 g, 1.62 mmol) in DMSO (10V), IBX (0.68 g, 2.43 mmol) was added portionwise and the mixture was stirred at room temperature for 24 h. After completion of the reaction, the mixture was quenched with water (50 mL) and extracted with EtOAc (3×50 mL). The combined organic layers were dried over anhydrous Na2SO4 and concentrated under vacuum to give the crude product. The crude product was purified using column chromatography eluting with 4% MeOH in DCM to give the title compound (0.4 g, 1.30 mmol, 80.5%). LCMS: (M+H) + =306.75.

[0234] Step 5: N-(1-chloro-6-methoxyisoquinolin-7-yl)-N-methyl-4-(piperidin-1-yl)butanamide [ka] To a stirred solution of N-(1-chloro-6-methoxyisoquinolin-7-yl)-N-methyl-4-oxobutanamide (0.4 g, 1.32 mmol), piperidine (0.22 g, 2.6 mmol) in MeOH (10V), STAB (0.41 g, 1.95 mmol) was added after 30 min at room temperature and the mixture was stirred at room temperature for 24 h. The mixture was quenched with water (20 mL), extracted with EtOAc (3x20 mL), the organic layer was dried over anhydrous Na2SO4 and concentrated under vacuum to give the crude product. The crude product was purified using column chromatography eluting with 10% MeOH in DCM to give the title compound (0.25 g, 0.66 mmol, 51%) as a red-brown solid. LCMS: (M+H) + =375.90.

[0235] Step 6: N-(1-((3-chloro-4-fluorophenyl)amino)-6-methoxyisoquinolin-7-yl)-N-methyl-4-(piperidin-1-yl)butanamide [ka] To a solution of N-(1-chloro-6-methoxyisoquinolin-7-yl)-N-methyl-4-(piperidin-1-yl)butanamide (0.15 g, 0.39 mmol), 3-methoxyaniline (63 mg, 0.42 mmol), and Cs2CO3 (0.25 g, 0.78 mmol) in degassed dioxane (10 V), Xantphos (22.5 mg, 0.039 mmol) and Pd2(dba)3 (35.71 mg, 0.039 mmol) were added under nitrogen atmosphere and the mixture was stirred at 100 °C for 1 h. The mixture was quenched with water (10 mL) and extracted with EtOAc (2x20 mL). The combined organic layers were dried over anhydrous Na2SO4 and concentrated under vacuum to give the crude product. The crude product was purified using column chromatography eluting with 10-12% MeOH in DCM to give the title compound (40 mg, 21.5%) as a light brown solid. 1 H NMR (400MHz, methanol-d4) δ8.33(s,1H),7.95-7.90(m,2H),7.58-7.54(m,1H),7.40(s,1H),7.3-7.18(m,2H),4.03(s,3H),3.27(s,3H), 2.44(bs,4H),2.35-2.30(s,2H),2.24-2.16(m,1H),2.10-2.03(m,1H),1.82-1.74(m,2H),1.58-1.52(m,4H),1.43(s,2H);LCMS:(M+H) + =485.00.

[0236] Example 32: Preparation of N-(1-((3-chloro-4-methylphenyl)amino)isoquinolin-7-yl)-4-(piperidin-1-yl)butanamide (I-32) [ka] To a solution of N-(1-chloroisoquinolin-7-yl)-4-(piperidin-1-yl)butanamide (150 mg, 0.452 mmol) in degassed dioxane (3 mL) was added Cs2CO3 (294 mg, 0.904 mmol), 3-chloro-4-methylaniline (70.4 mg, 0.497 mmol). Xantphos (26 mg, 0.0452 mmol) and Pd2(dba)3 (41.38 mg, 0.0452 mmol) were added and the mixture was stirred at 100 °C for 2 h. The mixture was concentrated and the residue was purified by silica gel chromatography eluting with 5-20% MeOH / DCM to give the title compound (40 mg, 20.3%) as a brown solid. 1 H NMR(400MHz,MeOD-d4)δ8.58(s,1H),7.87(s,1H),7.78-7.76(d,J=8.8Hz,2H),7.67-7.65(d,J=8.4Hz,1H),7.45-7.43(d,J=7.2Hz ,1H),7.25-7.23(d,J=8Hz,1H),7.16-7.14(d,J=4.8Hz,1H),2.56-2.51(m,8H),1.99(s,2H),1.66(s,4H),1.51(s,2H).LCMS:(M+H) + =436.98.

[0237] Example 33: Preparation of N-(1-((4-fluoro-3-(trifluoromethyl)phenyl)amino)isoquinolin-7-yl)-4-(piperidin-1-yl)butanamide (I-33) [ka] To a solution of N-(1-chloroisoquinolin-7-yl)-4-(piperidin-1-yl)butanamide (150 mg, 0.452 mmol) in degassed dioxane (3 mL) was added Cs2CO3 (294 mg, 0.904 mmol), 4-fluoro-3-(trifluoromethyl)aniline (89 mg, 0.497 mmol). Xantphos (26 mg, 0.0452 mmol) and Pd2(dba)3 (41.38 mg, 0.0452 mmol) were added and the mixture was stirred at 100 °C for 2 h. The mixture was concentrated and the residue was purified by silica gel chromatography eluting with 12-20% MeOH / DCM to give the title compound (50 mg, 23.3%) as a brown solid. 1 H NMR(400MHz,MeOD-d4)δ8.65(s,1H),8.08-8.07(d,J=3.6Hz,1H),7.97-7.91(m,2H),7.81-7.79(d,J=8.8Hz,1H),7.67-7.65(d,J=8.8Hz,1H),7.3 2-7.27(t,J=19Hz,1H),7.21-7.19(d,J=5.6Hz,1H),2.53-2.48(m,8H),2 .03-1.92(m,2H),1.67-1.64(t,J=10.8Hz,4H),1.51(s,2H);LCMS:(M+H) + =475.

[0238] Example 34: Preparation of N-(1-((3,4-dichloro-2-fluorophenyl)amino)isoquinolin-7-yl)-4-(piperidin-1-yl)butanamide (I-34) [ka] To a solution of N-(1-chloroisoquinolin-7-yl)-4-(piperidin-1-yl)butanamide (150 mg, 0.452 mmol) in degassed dioxane (3 mL) was added Cs2CO3 (294 mg, 0.904 mmol), 3,4-dichloro-2-fluoroaniline (89 mg, 0.497 mmol), Xantphos (26 mg, 0.0452 mmol), and Pd2(dba)3 (41.38 mg, 0.0452 mmol) and the mixture was stirred at 100° C. for 2 h. The mixture was concentrated and the residue was purified by silica gel chromatography eluting with 5-20% MeOH / DCM to give the title compound (50 mg, 23.3%) as a brown solid. 1 H NMR(400MHz,MeOD-d4)δ8.62(s,1H),7.87-7.80(m,2H),7.74-7.68(m,2H),7.38-7.36(d,J=8.8Hz,1H),7.2 3-7.23(d,J=5.2Hz,1H),2.52-2.46(m,8H),2.02-1.94(m,2H),1.66-1.63(m,4H),1.50(s,2H).LCMS:(M+H) + =475.39.

[0239] Example 35: Preparation of N-(1-((3,4-dichlorophenyl)amino)isoquinolin-7-yl)-4-(piperidin-1-yl)butanamide (I-35) [ka] To a solution of N-(1-chloroisoquinolin-7-yl)-4-(piperidin-1-yl)butanamide (100 mg, 0.276 mmol) in dioxane (2 mL) was added Cs2CO3 (179 mg, 0.552 mmol), 3,4-dichloroaniline (44 mg, 0.304 mmol) and the reaction vessel was flushed with N2 for 10 min. To the reaction was added Xantphos (15 mg, 0.027 mmol) and Pd2(dba)3 (25 mg, 0.276 mmol) and the mixture was stirred at 100 °C for 2 h. The mixture was concentrated and the residue was purified by silica gel chromatography eluting with 5-20% MeOH / DCM to give the title compound (60 mg, 43.5%) as an off-white solid. 1H NMR(400MHz,MeOD-d4):δ8.61(s,1H),8.02(s,1H),7.95-7.94(d,J=5.2Hz,1H),7.81-7.79(d,J=9.2Hz,1H),7.67-7.56(m,2H),7.45-7.43(d, LCMS: (M+1) + =457.40.

[0240] Example 36: Preparation of N-(1-((3,5-dichloro-4-fluorophenyl)amino)isoquinolin-7-yl)-4-(piperidin-1-yl)butanamide (I-36) [ka] To a solution of N-(1-chloroisoquinolin-7-yl)-4-(piperidin-1-yl)butanamide (100 mg, 0.276 mmol) in dioxane (2 mL) was added Cs2CO3 (179 mg, 0.552 mmol) and 3,5-dichloro-4-fluoroaniline (44 mg, 0.304 mmol) and the reaction was degassed with N2 for 10 min. To this mixture was added Xantphos (15 mg, 0.027 mmol) and Pd2(dba)3 (25 mg, 0.276 mmol) and the mixture was stirred at 100 °C for 2 h. The reaction mixture was concentrated and the residue was purified by silica gel chromatography eluting with 5-15% MeOH / DCM to give the title compound (25 mg, 49.0%) as an off-white solid. 1H NMR(400MHz,MeOD-d4):δ8.62(s,1H),7.96-7.95(d,J=5.6Hz,1H),7.87-7.86(d,J=6Hz,2H),7.81-7.79(d,J=8.8Hz,1H),7.66-7.64( d,J=8Hz,1H),7.23-7.22(d,J=5.6Hz,1H),2.57-2.50(m,8H),2.04-1.98(m,2H),1.68-1.65(t,J=5.6Hz,4H),1.52(s,2H),LCMS:(M+1) + =475.39.

[0241] Example 37: Preparation of N-(1-((3,4-dichloro-5-fluorophenyl)amino)isoquinolin-7-yl)-4-(piperidin-1-yl)butanamide (I-37) [ka] To a solution of N-(1-chloroisoquinolin-7-yl)-4-(piperidin-1-yl)butanamide (0.1 g, 0.30 mmol) in 1,4-dioxane (1 mL) was added 3,4-dichloro-5-fluoroaniline (0.06 g, 0.33 mmol) and Cs2CO3 (0.196 g, 0.60 mmol) and the reaction was flushed with N2 for 20 min. Tris(dibenzylideneacetone)dipalladium (0.028 g, 0.03 mmol) and (9,9-dimethyl-9H-xanthene-4,5-diyl)bis(diphenylphosphane) (0.018 g, 0.03 mmol) were added and the reaction was stirred at 100 °C for 2 h. The mixture was diluted with water (10 mL) and extracted with EtOAc (3 x 10 mL). The combined organic layers were dried over anhydrous Na2SO4 and concentrated under vacuum. The crude product obtained was purified by column chromatography eluting with 20% MeOH in DCM to give the title compound (0.07 g, 48.9%) as a yellow solid. 1H NMR (400MHz, methanol-d4) δ8.63(s,1H),8.00-8.02(d,J=5.6Hz,1H),7.80-7.82(d,J=9.6Hz,3H),7.65(m,1H),7.25-7.27( d,J=5.6Hz,1H),2.63(m,6H),2.55(t,2H),2.01(t,2H),1.67(m,4H),1.54-1.55(d,J=4.4Hz,2H),0.91(s,1H);LCMS[M+1] + =475.1.

[0242] Example 38: Preparation of N-(1-((4,5-dichloro-2-fluorophenyl)amino)isoquinolin-7-yl)-4-(piperidin-1-yl)butanamide (I-38) [ka] To a solution of N-(1-chloroisoquinolin-7-yl)-4-(piperidin-1-yl)butanamide (100 mg, 0.276 mmol) in dioxane (2 mL) was added Cs2CO3 (179 mg, 0.552 mmol), 4,5-dichloro-2-fluoroaniline (44 mg, 0.304 mmol) and the reaction was degassed with N2 for 10 min. To this mixture was added Xantphos (15 mg, 0.027 mmol) and Pd2(dba)3 (25 mg, 0.276 mmol) and the reaction was stirred at 100 °C for 2 h. The mixture was concentrated and the residue was purified by silica gel chromatography eluting with 10-20% MeOH / DCM to give the title compound (50 mg, 34.9%) as an off-white solid. 1 H NMR(400MHz,MeOD-d4):δ8.61(s,1H),8.23-8.20(d,J=7.2Hz,1H),7.94-7. 93(d,J=1.6Hz,1H),7.84-7.82(d,J=8.8Hz,1H),7.77-7.75(d,J=8.4Hz,1H ),7.47-7.45(d,J=10.4Hz,1H),7.26-7.25(d,J=4Hz,1H),2.81-2.55(m,8H ),2.08-2.03(m,2H),1.74-1.73(d,J=5.2Hz,4H),1.59(s,2H).LCMS:(M+1)+ =475.39.

[0243] Example 39: Preparation of N-(1-((3-chloro-2-fluorophenyl)amino)isoquinolin-7-yl)-4-(piperidin-1-yl)butanamide (I-39) [ka] To a solution of N-(1-chloroisoquinolin-7-yl)-4-(piperidin-1-yl)butanamide (100 mg, 0.276 mmol) in dioxane (2 mL) was added Cs2CO3 (179 mg, 0.552 mmol) and 3-chloro-2-fluoroaniline (44 mg, 0.304 mmol) and the reaction was degassed with N2 for 10 min. To this mixture was added Xantphos (15 mg, 0.027 mmol) and Pd2(dba)3 (25 mg, 0.276 mmol) and the reaction was stirred at 100 °C for 2 h. The mixture was concentrated and the residue was purified by silica gel chromatography eluting with 15-20% MeOH / DCM to give the title compound (45 mg, 33.9%) as an off-white solid. 1 H NMR(400MHz,MeOD-d4):δ8.62(s,1H),7.86-7.80(m,2H),7.75-7.73(d,J=8.8Hz,1H),7.68-7.67(d,J=7.2Hz,2H) ,7.30-7.15(m,3H),2.54-2.49(m,8H),2.03-1.96(m,2H),1.67-1.65(t,J=10.8Hz,4H),1.51(s,2H);LCMS:(M+1) + =441.0.

[0244] Example 40: Preparation of N-(1-((3-chloro-2,4-difluorophenyl)amino)isoquinolin-7-yl)-4-(piperidin-1-yl)butanamide (I-40) [ka] To a solution of N-(1-chloroisoquinolin-7-yl)-4-(piperidin-1-yl)butanamide (100 mg, 0.276 mmol) in dioxane (2 mL) was added Cs2CO3 (179 mg, 0.552 mmol), 3,4-dichloroaniline (44 mg, 0.304 mmol) and the mixture was degassed with N2 for 10 min. To this mixture was added Xantphos (15 mg, 0.027 mmol) and Pd2(dba)3 (25 mg, 0.276 mmol) and the reaction was stirred at 100 °C for 2 h. The mixture was concentrated and the residue was purified by silica gel chromatography eluting with 5-20% MeOH / DCM to give the title compound (70 mg, 50.6%) as an off-white solid. 1 H NMR(400MHz,MeOD-d4):δ8.62(s,1H),7.81-7.79(d,J=7.2Hz,2H),7.72-7.70(d,J=8.4Hz,1H),7.58-7.57(d,J=5.6Hz,1 LCMS: (M+1) + =458.94.

[0245] Example 41: Preparation of N-(1-((3-cyanophenyl)amino)isoquinolin-7-yl)-4-(piperidin-1-yl)butanamide (I-41) [ka] To a solution of N-(1-chloroisoquinolin-7-yl)-4-(piperidin-1-yl)butanamide (100 mg, 0.276 mmol) in dioxane (2 mL) was added Cs2CO3 (179 mg, 0.552 mmol) and 3-aminobenzonitrile (44 mg, 0.304 mmol) and the reaction flask was flushed with N2 for 10 min. Xantphos (15 mg, 0.027 mmol) and Pd2(dba)3 (25 mg, 0.276 mmol) were added and the reaction was stirred at 100 °C for 2 h. The mixture was concentrated and the residue was purified by silica gel chromatography eluting with 5-20% MeOH / DCM to give the title compound (60 mg, 48.2%) as an off-white solid. 1 H NMR(400MHz,MeOD-d4):δ8.65(s,1H),8.20(s,1H),7.98-7.94(t,J=6Hz,2H),7.83-7.81(d,J=8.8Hz,1H),7.70-7.67(d,J=8.8Hz,1H),7 .52-7.48(t,J=7.2Hz,1H)7.36-7.24(m,2H),2.60-2.51(m,8H),2.03-1.92(m,2H),1.70-1.67(t,J=5.2Hz,4H),1.45(s,2H).LCMS:(M+1) + =414.52.

[0246] Example 42: Preparation of N-(1-((3-chloro-4-fluorophenyl)amino)-6-methoxyisoquinolin-7-yl)-3-(piperidin-1-yl)propenamide (I-42) [ka] N 1To a solution of -(3-chloro-4-fluorophenyl)-6-methoxyisoquinoline-1,7-diamine (0.1 g, 0.314 mmol) in THF (5 mL) was added 3-bromopropanoyl chloride (0.064 g, 0.376 mmol) and DIPEA (0.085 g, 0.628 mmol) and the reaction was stirred at 0° C. for 2 h. After 2 h, NaI (0.05 g, 0.314 mmol) and pyrrolidine (0.25 g, 3.147 mmol) were added and the mixture was stirred at room temperature for 16 h. The mixture was diluted with brine (20 mL) and extracted with EtOAc (3×10 mL). The combined organic layers were dried over anhydrous Na2SO4 and concentrated in vacuo. The crude product obtained was purified using basic alumina eluting with 0.8% MeOH in DCM to give the title compound (0.035 g, 25.1%) as a pale brown solid. 1 H NMR (400MHz, methanol-d4) δ8.90(s,1H),7.85-7.81(m,2H),7.52-7.49(m,1H),7.28(s,1H),7.21-7.17(t, LCMS: (M+H) + =442.92.

[0247] Example 43: Preparation of N-(1-((3-chloro-4-fluorophenyl)amino)-6-methoxyisoquinolin-7-yl)-4-(3,3-difluoropyrrolidin-1-yl)butanamide (I-43) [ka] Step 1: Methyl 4-((1-((3-chloro-4-fluorophenyl)amino)-6-methoxyisoquinolin-7-yl)amino)-4-oxobutanoate [ka] N 1To a stirred solution of -(3-chloro-4-fluorophenyl)-6-methoxyisoquinoline-1,7-diamine (0.9 g, 2.83 mmol) and monomethyl succinate (0.56 g, 4.24 mmol) in DCM (5.6 mL) was added DIPEA (5.6 mL) and T3P (50% in EtOAc, 2.7 g, 8.49 mmol) and the reaction was stirred at 70 °C for 16 h. The mixture was quenched with water (100 mL) and extracted with EtOAc (3x100 mL). The combined organic layers were dried over anhydrous Na2SO4 and concentrated under vacuum to give the crude product. The crude product was purified using column chromatography and the product was eluted with 90% EtOAc in hexane to give the title compound (0.7 g, 57.2%) as a brown solid. LCMS: (M+H) + =432.09.

[0248] Step 2: N-(1-((3-chloro-4-fluorophenyl)amino)-6-methoxyisoquinolin-7-yl)-4-hydroxybutanamide [ka] To a stirred solution of methyl 4-((1-((3-chloro-4-fluorophenyl)amino)-6-methoxyisoquinolin-7-yl)amino)-4-oxobutanoate (0.7 g, 1.62 mmol) in MeOH (7 mL) was added NaBH4 (0.61 g, 16.2 mmol) portionwise at 50 °C and the reaction was stirred at 50 °C for 24 h. The mixture was concentrated and the crude product was purified by column chromatography eluting with 5-6% MeOH in DCM to give the title compound (0.6 g, 91.7%) as an off-white solid. LCMS: (M+H) + =404.18.

[0249] Step 3: Synthesis of N-(1-((3-chloro-4-fluorophenyl)amino)-6-methoxyisoquinolin-7-yl)-4-oxobutanamide [ka] To a stirred solution of N-(1-((3-chloro-4-fluorophenyl)amino)-6-methoxyisoquinolin-7-yl)-4-hydroxybutanamide (0.6 g, 1.49 mmol) in DMSO (6 mL) was added IBX (0.42 g, 2.98 mmol) in portions and the reaction was stirred at room temperature for 24 h. The mixture was diluted with water (50 mL) and extracted with EtOAc (3×50 mL). The combined organic layers were dried over anhydrous Na2SO4 and concentrated under vacuum. The resulting crude product was purified using column chromatography eluting with 4-5% MeOH in DCM to give the title compound (0.45 g, 75.4%) as a light brown solid. LCMS: (M+H) + =402.26.

[0250] Step 4: N-(1-((3-chloro-4-fluorophenyl)amino)-6-methoxyisoquinolin-7-yl)-4-(3,3-difluoropyrrolidin-1-yl)butanamide [ka] To a stirred solution of N-(1-((3-chloro-4-fluorophenyl)amino)-6-methoxyisoquinolin-7-yl)-4-oxobutanamide (0.15 g, 0.37 mmol) and 3,3-difluoropyrrolidine hydrochloride (0.26 g, 1.87 mmol) in MeOH (3 mL), NaCNBH3 (0.069 g, 1.12 mmol) was added after 30 min at room temperature and the reaction was stirred at 35 °C for 24 h. The mixture was diluted with water (20 mL) and extracted with EtOAc (3 x 20 mL). The combined organic layers were dried over anhydrous Na2SO4 and concentrated under vacuum. The resulting crude product was purified using column chromatography eluting with 8-10% MeOH in DCM to give the title compound (0.018 g, 9.7%) as an off-white solid. 1H NMR (400MHz, methanol-d4) δ8.93(s,1H),7.92-7.82(m,2H),7.62-7.53(m,1H),7.39(s,1H),7.36-7.13(m,2H),4.15(s,3H), 3.04(t,J=13.3Hz,2H),2.88(t,J=7.0Hz,3H),2.42-2.28(m,3H),2.08-1.97(m,2H),1.48(s,3H),1.37(s,1H).LCMS:(M+H) + =493.

[0251] Example 44: Preparation of N-(1-((3,5-dichloro-2-fluorophenyl)amino)isoquinolin-7-yl)-4-(piperidin-1-yl)butanamide (I-44) [ka] To a solution of N-(1-chloroisoquinolin-7-yl)-4-(piperidin-1-yl)butanamide (0.1 g, 0.30 mmol) in degassed 1,4-dioxane (1 mL) was added 3,5-dichloro-2-fluoroaniline (0.06 g, 0.33 mmol), Cs2CO3 (0.196 g, 0.60 mmol), tris(dibenzylideneacetone)dipalladium (0.028 g, 0.03 mmol), and (9,9-dimethyl-9H-xanthene-4,5-diyl)bis(diphenylphosphane) (0.018 g, 0.03 mmol) and the reaction was stirred at 100° C. for 2 h. The mixture was diluted with water (10 mL) and extracted with EtOAc (3×10 mL). The combined organic layers were dried over anhydrous Na2SO4 and concentrated under vacuum to give the crude product. The crude product was purified by column chromatography, the product was eluted with 20% MeOH in DCM to give the title compound (0.070 g, 48.9%) as an off-white solid. 1 H NMR (400MHz, methanol-d4) δ8.62(s,1H),7.94(s,2H),7.77(dd,2H),7.23(m,2H),2.71(m,5H),2.58( m,2H),2.02(m,2H),1.73(m,4H),1.57-1.58(d,J=4Hz,2H),1.28-1.30(d,J=8Hz,1H).LCMS:[M+1]+ =475.10.

[0252] Example 45. Preparation of N-(1-((4-chloro-2,3-difluorophenyl)amino)isoquinolin-7-yl)-4-(piperidin-1-yl)butanamide (I-45) [ka] To a solution of N-(1-chloroisoquinolin-7-yl)-4-(piperidin-1-yl)butanamide (100 mg, 0.276 mmol) in dioxane (2 mL) was added Cs2CO3 (179 mg, 0.552 mmol) and 4-chloro-2,3-difluoroaniline (44 mg, 0.304 mmol) and the reaction was degassed with N2 for 10 min. To this mixture was added Xantphos (15 mg, 0.027 mmol) and Pd2(dba)3 (25 mg, 0.276 mmol) and the reaction was stirred at 100 °C for 2 h. After completion of the reaction, the mixture was concentrated and the residue was purified by silica gel chromatography eluting with 10-20% MeOH / DCM to give the title compound (60 mg, 50.6%) as an off-white solid. 1 H NMR(400MHz,MeOD-d4):δ8.63(s,1H),7.88-7.82(m,2H),7.75-7.73(d,J=8Hz,1H),7.53(s,1H),7.30-7.23(m,2H),2 .73-2.53(m,8H),2.07-2.01(m,2H),1.73-1.70(t,J=12Hz,4H),1.56(s,2H),1.34-1.31(d,J=12Hz,1H).LCMS:(M+1) + =459.0.

[0253] Example 46. Preparation of N-(1-((3-chloro-4-fluorophenyl)amino)-6-ethoxyisoquinolin-7-yl)-4-(piperidin-1-yl)butanamide (I-46) [ka] Step 1: 6-Fluoro-3,4-dihydroisoquinolin-1(2H)-one [ka] To a 0° C. cooled solution of 5-fluoro-2,3-dihydro-1H-inden-1-one (10 g, 15.02 mmol) in CHCl (100 mL) was added methanesulfonic acid (43.62 mL, 150.2 mmol) and sodium azide (8.66 g, 30.03 mmol) in portions. The reaction was stirred at 0° C. for 2 h. After 2 h, 20% aqueous NaOH (4 V) was added and the reaction was stirred at room temperature for 30 min. The mixture was diluted with water (1000 mL) and extracted with CHCl (3×500 mL). The combined organic layers were dried over NaSO and concentrated. The resulting crude product was purified using column chromatography eluting with 50% EtOAc:Hexane to give the title compound (6.5 g, 59.0%) as an off-white solid. LCMS: (M+H) + =166.0.

[0254] Step 2. 6-Fluoro-7-nitro-3,4-dihydroisoquinolin-1(2H)-one [ka] To a solution of 6-fluoro-3,4-dihydroisoquinolin-1(2H)-one (6.5 g, 39.149 mmol) in cold sulfuric acid (65 mL) was added potassium nitrate portionwise at 0° C. The reaction was stirred at the same temperature for 2 h. After completion of the reaction, the reaction mixture was poured into ice water. The resulting precipitate was collected by suction filtration to give the product (8 g, 96.7%) as a yellow solid. LCMS: (M+H) + =211.1.

[0255] Step 3. Synthesis of 6-fluoro-7-nitroisoquinolin-1(2H)-one [ka] 0% of 6-fluoro-7-nitro-3,4-dihydroisoquinolin-1(2H)-one (8 g, 38.06 mmol) oC Add manganese(IV) oxide (65.6 g, 761.32 mmol) in small portions to the cooled 1,2-dichloroethane (160 mL) solution and dilute the reaction to 100%. o C for 48 h. The mixture was concentrated and the resulting crude product was purified by column chromatography eluting with 20% MeOH:CH2Cl2 to give the title compound (6.7 g, 84.6%) as a yellow solid. LCMS: (M+H) + =209.4.

[0256] Step 4: Synthesis of 1-chloro-6-fluoro-7-nitroisoquinoline [ka] A suspension of 6-fluoro-7-nitroisoquinolin-1(2H)-one (6.7 g, 32.18 mmol) in phosphoryl chloride (70 mL) was o C for 16 h. The phosphoryl chloride was removed by distillation and the mixture was dissolved in CH2Cl2 (500 mL) and quenched by addition of saturated sodium bicarbonate solution. The organic layer was separated, dried over Na2SO4 and concentrated to give the product (5.5 g, 75.9%) as a yellow solid. LCMS: (M+H) + =227.06.

[0257] Step 5: N-(3-chloro-4-fluorophenyl)-6-fluoro-7-nitroisoquinolin-1-amine [ka] 0% of 2-chloro,3-fluoroaniline (2.8 g, 19.42 mmol) o C1,4-dioxane (60 mL) solution was slowly added with 4 M hydrochloric acid in dioxane (55 mL) and the mixture was cooled to 0 o C for 20 minutes. To this mixture was added 1-chloro-6-fluoro-7-nitroisoquinoline (5.5 g, 24.27 mmol), and the mixture was stirred for 60 oC for 16 h. The mixture was diluted with water (100 ml) and extracted with EtOAc (2×100 mL). The organic layer was dried over Na2SO4 and concentrated. The crude product obtained was purified using column chromatography eluting with 10% EtOAc:Hexane to give the title compound (4.3 g, 52.7%) as a reddish solid. 1 H NMR(400MHz,DMSO-d6):δ9.87(s,1H),9.54(d,J=8Hz,1H),8.20(d,J=5.6Hz,1H),8.16(d,J=4.4H) z,1H),7.99(d,J=12Hz,1H),7.83(bs,1H),7.42(t,J=8.8Hz,1H),7.28(J=7.6Hz,1H).LCMS:[M+1] + =336.

[0258] Step 6: N-(3-chloro-4-fluorophenyl)-6-ethoxy-7-nitroisoquinolin-1-amine [ka] 0% of ethanol (0.122 g, 2.681 mmol) o To a cooled solution of THF (6 mL) was added potassium tert-butoxide (0.3 g, 2.68 mmol) and the mixture was stirred at 30° C. for 15 min. To this mixture was added N-(3-chloro-4-fluorophenyl)-6-fluoro-7-nitroisoquinolin-1-amine (0.6 g, 1.78 mmol) and the reaction was stirred at 50° C. for 16 h. The mixture was diluted with water (50 mL) and the resulting precipitate was collected by filtration and dried to give the product (0.6 g, 92.9%) as a yellow solid. LCMS: (M+H) + =362.1.

[0259] Step 7: N1-(3-chloro-4-fluorophenyl)-6-ethoxyisoquinoline-1,7-diamine [ka] To a stirred solution of N-(3-chloro-4-fluorophenyl)-6-ethoxy-7-nitroisoquinolin-1-amine (0.6 g, 1.66 mmol) in methanol (20 mL), water (10 mL), and THF (7 mL) was added iron (0.368 g, 6.64 mmol) and ammonium chloride (0.355 g, 6.64 mmol). The reaction was stirred at 100° C. overnight. The mixture was filtered using a bed of celite, and the filtrate was concentrated, diluted with water (50 mL), and extracted with EtOAc (3×30 mL). The organic layer was washed with brine, dried over Na2SO4, and concentrated to give the product (0.17 g, 30.9%) as a brown solid. LCMS: [M+H] + =332.20.

[0260] Step 8: N-(1-((3-chloro-4-fluorophenyl)amino)-6-methoxyisoquinolin-7-yl)-4-(piperidin-1-yl)butanamide [ka] 0% of N1-(3-chloro-4-fluorophenyl)-6-ethoxyisoquinoline-1,7-diamine (0.13 g, 0.392 mmol) o C To a cooled solution of THF (1.2 mL) was added DIPEA (0.13 mL, 0.78 mmol) and 4-bromobutyryl chloride (0.078 g, 0.47 mmol) and the reaction was brought to 0°C. o C for 2 h. To this mixture was added sodium iodide (0.065 g, 0.393 mmol) and piperidine (0.35 mL, 0.392 mmol) and the reaction was stirred at room temperature overnight. The mixture was diluted with water (30 mL) and extracted with EtOAc (3×20 mL). The combined organic layers were dried over Na2SO4 and concentrated. The resulting crude product was purified using column chromatography and the product was eluted with 10% MeOH:DCM to give the title compound (0.093 g, 48.9%) as an off-white solid. 1H NMR(400MHz,MeOD-d4):δ8.77(s,1H),7.85(d,J=8Hz,1H),7.81(d,J=4Hz,1H) ,7.50(d,J=8Hz,1H),7.27(s,1H),7.19(t,J=8Hz,1H),7.13(d,J=8Hz,1H),4. 32(m,J=8Hz,2H),2.99(m,5H),2.68(t,J=8Hz,2H),2.10(s,2H),1.80(s,3H), 1.63(s,2H),1.56(t,J=4Hz,3H),1.33(m,2H),0.92(t,J=8Hz,1H).LCMS:(M+H) + =485.0.

[0261] Example 47: Preparation of (R)-N-(1-((3-chloro-4-fluorophenyl)amino)-6-methoxyisoquinolin-7-yl)-4-(3-methoxypyrrolidin-1-yl)butanamide ((R)I-47) [ka] To a stirred solution of N-(1-((3-chloro-4-fluorophenyl)amino)-6-methoxyisoquinolin-7-yl)-4-oxobutanamide (0.1 g, 0.24 mmol) and (R)-3-methoxypyrrolidine hydrochloride (0.34 g, 2.4 mmol) in MeOH (2 mL), NaCNBH3 (0.046 g, 0.747 mmol) was added after 30 min at room temperature and the reaction was stirred at 35 °C for 24 h. The mixture was diluted with water (20 mL) and extracted with EtOAc (3 x 20 mL). The combined organic layers were dried over anhydrous Na2SO4 and concentrated under vacuum. The resulting crude product was purified using column chromatography eluting with 8-10% MeOH in DCM to give the title compound (0.068 g, 56.1%) as a pale yellow solid. 1H NMR (400MHz, methanol-d4) δ8.80(s,1H),7.82(dd,J=17.6,6.6Hz,2H),7.50(d,J =8.6Hz,1H),7.28(s,1H),7.19(t,J=9.0Hz,2H),4.06(s,5H),3.22(d,J=11.9H z,2H),3.08(dd,J=11.7,5.0Hz,1H),2.99(t,J=8.0Hz,3H),2.66(t,J=7.0Hz, 2H),2.19(dd,J=14.2,6.9Hz,1H),2.10-2.02(m,4H),1.30(s,1H).LCMS:(M+H) + =487.1.

[0262] Example 48: Preparation of (S)-N-(1-((3-chloro-4-fluorophenyl)amino)-6-methoxyisoquinolin-7-yl)-4-(3-methoxypyrrolidin-1-yl)butanamide ((S)I-47) [ka] To a stirred solution of N-(1-((3-chloro-4-fluorophenyl)amino)-6-methoxyisoquinolin-7-yl)-4-oxobutanamide (0.1 g, 0.24 mmol) and (S)-3-methoxypyrrolidine hydrochloride (0.34 g, 2.4 mmol) in MeOH (2 mL), NaCNBH3 (0.046 g, 0.747 mmol) was added after 30 min at room temperature and the reaction was stirred at 35 °C for 24 h. The mixture was diluted with water (20 mL) and extracted with EtOAc (3 x 20 mL). The combined organic layers were dried over Na2SO4 and concentrated. The resulting crude product was purified using column chromatography eluting with 5-6% MeOH in DCM to give the title compound (0.032 g, 26.4%) as a pale yellow solid. 1H NMR (400MHz, methanol-d4) δ8.79(s,1H),7.84(s,2H),7.50(m,1H),7.28(s,1H),7.24-7.11(m,2H),4.06(d,J=3.7 Hz,4H),2.90(s,3H),2.74(s,3H),2.61(s,2H),2.13(s,1H),2.01(s,3H),1.94(s,1H),1.30(s,2H).LCMS:(M+H) + =487.2.

[0263] Example 49: (R)-N-(1-((3-chloro-4-fluorophenyl)amino)-6-methoxyisoquinolin-7-yl)-4-(3-fluoropyrrolidin-1-yl)butanamide ((R)I-48) [ka] To a stirred solution of N-(1-((3-chloro-4-fluorophenyl)amino)-6-methoxyisoquinolin-7-yl)-4-oxobutanamide (0.1 g, 0.24 mmol) and (R)-3-fluoropyrrolidine hydrochloride (0.31 g, 2.4 mmol) in MeOH (2 mL), NaCNBH3 (0.046 g, 0.747 mmol) was added after 30 min at room temperature and the reaction was stirred at 35° C. for 24 h. The mixture was diluted with water (20 mL) and extracted with EtOAc (3×20 mL). The combined organic layers were dried over anhydrous Na2SO4 and concentrated under vacuum. The resulting crude product was purified using column chromatography eluting with 4% MeOH in DCM to give the title compound (0.065 g, 55.0%) as a pale yellow solid. 1 H NMR (400MHz, methanol-d4) δ8.79(s,1H),7.87-7.79(m,2H),7.54-7.46(m,1H),7.28(s,1H),7.20(d,J=9.0Hz,1H),7.16-7.11(m,1H),5.27-5 .11(m,1H),4.06(s,3H),3.00(dt,J=23.6,12.1Hz,2H),2.62(dq,J=14.4,7.7Hz,5H),2.47(q,J=8.0Hz,1H),2.26-1.94(m,4H).LCMS:(M+H) + =475.35.

[0264] Example 50: (S)-N-(1-((3-chloro-4-fluorophenyl)amino)-6-methoxyisoquinolin-7-yl)-4-(3-fluoropyrrolidin-1-yl)butanamide ((S)I-48) [ka] To a stirred solution of N-(1-((3-chloro-4-fluorophenyl)amino)-6-methoxyisoquinolin-7-yl)-4-oxobutanamide (0.1 g, 0.24 mmol) and (S)-3-fluoropyrrolidine hydrochloride (0.31 g, 2.4 mmol) in MeOH (2 mL), NaCNBH3 (0.046 g, 0.747 mmol) was added after 30 min at room temperature and the reaction was stirred at 35 °C for 24 h. The mixture was diluted with water (20 mL) and extracted with EtOAc (3 x 20 mL). The combined organic layers were dried over anhydrous Na2SO4 and concentrated under vacuum. The resulting crude product was purified by column chromatography eluting with 3-4% MeOH in DCM to give the title compound (0.064 g, 54.2%) as a pale yellow solid. 1 H NMR (400MHz, methanol-d4) δ 8.79 (s, 1H), 7.84-7.80 (m, 2H), 7.51-7.47 (m, 1H), 7.27 (s, 1H), 7.20-7.12 (m, 2H), 5.28-5.11 (m, 1H), 4.05 (s, 3H), 2.97 (t, J = 11.8Hz, 2H), 2.74 ( dd,J=11.9,5.0Hz,1H),2.63(dq,J=21.6,6.5Hz,4H),2.47(q,J=8.0Hz,1H),2.22(dd d,J=27.6,14.0,7.6Hz,1H),2.13-2.02(m,1H),1.98(m,2H),1.30(s,1H).LCMS:(M+H) + =475.37.

[0265] Compounds I-49 to I-53 are prepared using methods similar to those described above.

[0266] B. Biology Example 51 i. Method details Protein purification and site-specific fluorophore labeling BAX and BCL-X L were expressed with a C-terminal intein-chitin binding domain that was used for purification according to the manufacturer's recommended procedure (NEB) (Kale et al., 2014; Pogmore et al., 2016). BID and BIM contain N-terminal histidine tags for nickel column purification (Chi et al., 2020), which is used both for routine purification and to remove free dye in solution after site-specific labeling of proteins.

[0267] Fluorescence-based assays using purified proteins were performed using single cysteine ​​mutants that allow site-specific labeling. WT BAX contains two endogenous cysteines (C62 and C126). C62A BAX was used to label C126. Like BAX, BID contains two endogenous cysteines (C30 and C126). Therefore, C30A BID was used to label C126. For compound screening, several batches of BAX were purified and mixed together to obtain more consistent data for multiple assays.

[0268] Expression of recombinant proteins for purification For standard protein purification: BAX, BID, BIM, and cBAK proteins were expressed in Escherichia coli BL21-AI cells and purified using the BCL-X Lwere expressed in DH5α cells. Proteins were purified as previously reported (Moldoveanu et al., 2006; Pogmore et al., 2016; Liu et al., 2019; Bogner et al., 2020). For each purification, bacteria were transformed with the required expression plasmid and a single colony was picked from an agar plate containing 100 μg / mL ampicillin and used to inoculate an overnight starter culture of 100 mL of Luria-Bertani (LB) broth supplemented with 100 μg / mL ampicillin. The next day, 50 mL of the overnight culture was used to inoculate two to four 1.5 L flasks containing LB supplemented with 100 μg / mL ampicillin. The flasks were then incubated in a shaking incubator at 200 rpm until the OD600 reached 0.7–0.8. Protein expression was induced by adding 3 g L-arabinose (for BL21-AI cells) or 1 mM IPTG (final concentration for DH5α cells). The incubator temperature was then reduced to 20°C for 24 h. Bacteria were harvested by centrifugation and the resulting bacterial pellet was stored at -20°C.

[0269] 15 For N protein expression: WT BAX was expressed in Escherichia coli BL21-AI cells with some slight differences. Instead of picking a single colony, multiple colonies were selected from ampicillin-containing LB agar plates, and the clone expressing the highest amount of BAX, as determined by immunoblotting, was stored as a 25% glycerol stock for subsequent high-yield purification. Overnight cultures (100 mL) were used to inoculate four 1.5 L flasks containing minimal medium supplemented with 100 μg / mL ampicillin as reported (Grant, Marshall and Ikura, 2019). Once the cultures reached an OD600 of 0.8, 3 g of arabinose was added to each flask, the incubator temperature was reduced to 20 °C, and the cultures were left to incubate for 24 h. Bacteria were harvested as above, and the resulting bacterial pellets were stored at -20 °C until used for purification.

[0270] BAX and 15 Purification of N-BAX Standard BAX purification: Bacterial pellets containing full-length BAX fused to the chitin-binding domain and modified self-cleaving intein were thawed at room temperature and resuspended in 50 mL of BAX lysis buffer (10 mM HEPES, pH 7, 100 mM NaCl, 0.2% CHAPS, 1 mg DNase, 2 mM PMSF, and 2x protease inhibitor cocktail). The resulting solution was removed via an 18-gauge syringe needle, after which the cells were lysed by French press. The lysed bacterial solution was centrifuged at 20000xg for 30 min at 4°C. The pellet was discarded, and 1 mL of chitin resin (50% slurry in ethanol) was added per large 1.5 L flask to the supernatant fraction (approximately 45-50 mL total). The supernatant was then incubated in a rotor for 2 h at 4°C, and then the solution was passed through a 15 mL BioRad gravity column to separate the chitin beads from the supernatant. The supernatant was collected and passed through the column twice. The column was then washed with 50 mL of BAX wash buffer (10 mM HEPES, pH 7, 500 mM NaCl, 0.5% CHAPS). Once the BAX wash buffer had passed through the column, 5 mL of BAX wash buffer supplemented with 300 mM hydroxylamine (pH was returned to pH 7 after adding hydroxylamine) was added to the column and allowed to run until 1-2 mL of buffer was visible above the chitin bead bed. At that point the column was capped. The column was then incubated at 4°C for 24 hours to allow time for self-cleavage by the intein to release the protein. Six 0.5 mL fractions were taken from the column and analyzed for protein concentration by Bradford assay. Typically the first 3-4 fractions, which contained the most protein, were pooled and passed three times over a DEAE-Sepharose column of 0.25 mL bed volume equilibrated with BAX wash buffer. The flow-through was then dialyzed (12-14 kDa pore size) in 1 L of BAX dialysis buffer (10 mM HEPES, pH 7, 200 mM NaCl, 10% glycerol) at 4° C. The buffer was changed twice with 3-4 hour intervals, then dialysis was continued overnight in fresh 1 L of buffer and collected the next day.The concentration of purified BAX recovered from the dialysis tubing was measured by absorbance at 280 nm and was either used for site-specific labeling with fluorophores (BAX C62A, 126C) or aliquoted, flash frozen in liquid nitrogen, and stored at -80°C for future use.

[0271] 15 NBAX purification: from the bacterial pellet obtained using the same protocol as above, with an additional step and omitting the dialysis. 15 N BAX was purified. The eluted fractions from the DEAE column were further purified by gel filtration using a manually packed G25 fine column equilibrated with BAX phosphate buffer (50 mM potassium phosphate, 50 mM NaCl, pH 6) on a BioRAD NGC liquid chromatography system. Fractions containing BAX monomer were pooled, concentrated using 15 kDa cutoff Centricon spin concentrators (Millipore), stored at 4°C, and subjected to NMR experiments within 24 hours. These additional steps did not make any difference in BAX activity, but did reduce aggregation during NMR.

[0272] Purification of cBAK The purification of calpain-truncated BAK (cBAK), lacking the C-terminal 24 amino acids, has been reported previously (Moldoveanu et al., 2006). Briefly, residues 1-186 of BAK with an N-terminal FLAG tag and a C-terminal calpain recognition site, as well as a six-histidine tag. The bacterial pellet was lysed, centrifuged, and the supernatant containing soluble cBAK was run on a gravity-flow nickel-NTA agarose column, followed by further purification by gel filtration chromatography and anion exchange using HPLC. On a smaller scale to identify optimal cleavage conditions, the purified protein was subjected to limited proteolysis by calpain in vitro. The cleaved protein was confirmed by the appearance of a lower band on an SDS-PAGE gel. Samples for SDS-Page were taken at 10-min intervals to find the optimal time (1 h) for the calpain reaction to cleave cBAK from the calpain recognition site and the histidine tag. The cleaved fraction was then separated by anion exchange. Fractions containing cBAK were pooled, flash frozen using liquid nitrogen, and stored at -80°C.

[0273] Purification of cBID In cells, BID is activated by cleavage by caspase 8. Therefore, after purifying BID from bacterial lysates, it was cleaved by adding His-tagged caspase 8. Cleaved BID (cBID) contains two fragments (p7 and p15) that spontaneously dissociate upon interaction with membranes. The cleaved form of BID (tBID aka p15), which activates BAX and BAK, remains membrane-bound, while the p7 fragment is released into solution. Thus, when cBID participates in a biochemical reaction, tBID accumulates at any membranes added.

[0274] To purify cBID, a bacterial pellet of cells containing full-length BID was thawed at room temperature and resuspended in 30 mL of BID lysis buffer (10 mM HEPES, pH 7, 100 mM NaCl, 10 mM imidazole, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 1 mg of bovine DNase I). Cells were lysed using a French press, and the lysate was cleared by centrifugation at 20,000 x g for 30 min, and the pellet was discarded. The supernatant containing BID was added to nickel-NTA agarose beads (total volume 1 mL) equilibrated in a gravity-flow column with BID lysis buffer. To maximize binding, the cleared lysate was passed through the beads three times. The beads were then washed with 10 mL of BID wash buffer (10 mM HEPES, pH 7, 300 mM NaCl, 10 mM imidazole, 1% CHAPS, and 10% glycerol). 1 mL fractions were collected using BID elution buffer (10 mM HEPES, 100 mM NaCl, 0.1% CHAPS, 200 mM imidazole, and 10% glycerol). Eluted protein was detected by spectroscopic analysis of a small aliquot of each fraction after addition of Bradford reagent, and protein-containing fractions were pooled for activation by site-specific labeling and / or cleavage. Purified BID or fluorophore-labeled BID was incubated in BID cleavage buffer (50 mM HEPES, pH 7, 100 mM NaCl, 0.1% CHAPS, 10% glycerol, 1 mM EDTA, and 10 mM DTT) and 500 units of caspase 8 (1 unit / uL cleavage reaction) on a rotor for approximately 2 days at room temperature. Cleavage efficiency was determined visually after SDS polyacrylamide gel electrophoresis and Coomassie blue staining. The resulting solution of cBID was dialyzed (12-14 kDa pore size) in 1 L of BID dialysis buffer (10 mM HEPES, pH 7, 100 mM NaCl, 0.1 mM EDTA, and 10% glycerol) for 4 h, after which the buffer was exchanged and dialysis continued for another 4 h. The sample was then added to a third liter of dialysis buffer and left overnight. Labeled or unlabeled cBID was then aliquoted, flash frozen in liquid nitrogen, and stored at -80 °C.

[0275] Purification of BIM To purify BIM, we used full-length BIM L The bacterial pellet containing BIM was thawed at room temperature and resuspended in 30 mL of BIM lysis buffer (20 mM Tris-HCl, pH 8, 20 mM NaCl, 1% CHAPS, 20% glycerol, 5 mM imidazole, 0.5 mM DTT, 1 mM PMSF, and 1 mg of bovine DNase I). Cells were lysed using a French press, the lysate was centrifuged at 20,000xg for 30 min, the pellet was discarded, and the supernatant containing BIM was mixed with 2 mL of nickel-NTA agarose beads (50% slurry in ethanol) and allowed to incubate for 2 h at 4°C in a rotor. The solution was then poured into a 10 mL BioRad gravity flow column and the flow-through was passed over the beads twice. 50 mL of BIM wash buffer (20 mM Tris, pH 8, 50 mM NaCl, 0.5% CHAPS, 20% glycerol, and 10 mM imidazole) was then loaded onto the column. 10 mL was eluted from the column using BIM elution buffer (20 mM HEPES, pH 7, 10 mM NaCl, 0.3% CHAPS, 20% glycerol, and 300 mM imidazole). The eluate was then loaded onto a smaller column with 400 uL bed volume of high performance phenyl sepharose (HPPS) beads. The beads were then washed with 5 mL of BIM HPPS wash buffer (20 mM HEPES, pH 7, 100 mM NaCl, 0.3% CHAPS, and 20% glycerol). Three 1 mL elution fractions containing BIM were collected using BIM HPPS elution buffer (20 mM HEPES, pH 7, 0.3% CHAPS, and 20% glycerol). The presence of eluted protein was detected by Bradford reagent, and the BIM-containing fractions were pooled. To remove CHAPS, the pooled fractions were dialyzed (12–14 kDa pore size) against a total of 3 L of BIM dialysis buffer (20 mM HEPES, pH 7, 1 mM NaCl, 20% glycerol) in a volume of 1 L. As with the other proteins, 1 liter was used for each dialysis step, with a change of solution after 4 h, and the third liter was left overnight at 4 °C. The final sample containing BIM was removed from the dialysis tube, aliquoted, flash frozen in liquid nitrogen, and stored at -80 °C.

[0276] BCL-X L Purification of BCL-X L To purify full-length BCL-X L Thaw the bacterial pellet containing BCL-X at room temperature and aliquot it into 50 mL of BCL-X. L The cells were resuspended in lysis buffer (20 mM Tris-HCl, pH 8, 500 mM NaCl, 1% CHAPS, 0.5 mM EDTA, 1 mg bovine DNase I, and 2 mM PMSF). The resulting solution was removed via an 18-gauge syringe needle, after which the cells were lysed using a French press. The lysed bacterial solution was centrifuged at 20,000xg for 30 minutes at 4°C. The pellet was discarded, and 1 mL of chitin resin (50% slurry in ethanol) was added per large 1.5 L flask to the supernatant fraction (approximately 45-50 mL of supernatant total). The supernatant was then incubated in a rotor for 2 hours at 4°C, and the solution was then poured into a 10 mL BioRad gravity column. The flow-through fraction was collected and passed through the column twice. The column was then immersed in 50 mL of BCL- XL The column was washed with wash buffer (20 mM Tris-HCl, pH 8, 200 mM NaCl, 0.2% CHAPS, 20% glycerol, and 1 mM PMSF). After the wash buffer had passed through the column, the column was washed with 5 mL of BCL-X supplemented with 300 mM hydroxylamine (pH was returned to pH 8 with concentrated NaOH after addition of hydroxylamine). L Wash buffer was added to the column and capped when 1-2 mL of buffer appeared above the chitin bead bed. The column was then incubated at 4°C for 24 hours to allow the intein-chitin binding domain to be cleaved from the protein. Six 0.5 mL fractions were taken from the column and analyzed for concentration by Bradford assay. The first 3-4 fractions usually contain the most protein, so they were pooled and passed over a 0.3 mL bed volume of high performance phenyl sepharose (HPPS) beads in a small column. The column was then washed with 5 mL of BCL-X L Wash with washing buffer. BCL-X LElute BCL-X by collecting three 1 mL fractions using elution buffer (Tris-HCl, pH 8, 0.2% CHAPS, and 20% glycerol). L The BCL-X was eluted from the HPPS beads. The most protein-containing fractions (as determined by Bradford Reagent) were pooled for dialysis. The pooled fractions were then eluted in 1 L of BCL-X. L The purified BCL-X was then dialyzed at 4°C in dialysis buffer (Tris-HCl, pH 8, and 20% glycerol) with two fresh changes of buffer every 3-4 hours, and the final 1 liter was left overnight and harvested the next day. L was removed from the dialysis tubing and BCL-X was analyzed by absorbance at 280 nm. L The concentration of was measured and either immediately used for fluorescent site-specific labeling or aliquoted, flash frozen in liquid nitrogen, and stored at -80°C for future use.

[0277] Site-specific labeling of proteins BAX and cBID were labeled using the single cysteine ​​mutants mentioned above and a 15-fold molar excess of Alexa647-C2-maleimide and Alexa568-C5-maleimide dissolved in DMSO, respectively. Proteins were labeled in their respective dialysis buffers supplemented with 0.5% CHAPS for 4-5 hours with rotation at room temperature. It is advisable that the concentration of DMSO does not exceed 10% as this will affect the labeling efficiency. To remove excess dye from the labeled protein, BID can be run again on a nickel-NTA agarose column and undergo the same steps as above, while BAX can be passed through a G-25 fine desalting column and eluted using BAX dialysis buffer. Due to the strong color development of the Alexa dye, the labeled protein can be seen on the beads, which aids in the recovery of the labeled protein-containing fraction. The efficiency of the labeling reaction was determined by dividing the concentration of the fluorophore (measured by absorbance at the dye's absorption maximum) by the concentration of the protein, measured by absorbance at 280 nm. Labeled proteins were only used if the labeling efficiency was at least 80%.

[0278] Preparation of liposomes Liposome preparation and dye release assay were performed as previously reported (Kale et al., 2014). The composition of liposomes (mol%) to make a 1 mg / mL lipid film is as follows: 48% phosphatidylcholine, 28% phosphatidylethanolamine, 10% phosphatidylinositol, 10% dioleoylphosphatidylserine, and 4% tetraoleoylcardiolipin. Each lipid (dissolved in chloroform) was mixed in a glass test tube. A constant stream of nitrogen was directed into the test tube and the tube was rotated to form a lipid film on the glass. The tube was flushed with nitrogen and covered with parafilm. The dried lipid film was then stored at -20°C until required. The lipid film was rehydrated with 1 mL of assay buffer (10 mM HEPES, pH 7.2, 200 mM KCl, and 5 mM MgCl2) and vortexed for 10 seconds. The solution was frozen and thawed 10 times by rotating the tube in liquid nitrogen and then in warm water, and the resuspended lipid solution was then extruded 11 times through a 100 nM pore size membrane to produce a homogenous solution of liposomes.

[0279] For ANTS / DPX encapsulated liposomes: 5.3 mg of ANTS and 18.1 mg of DPX were added to the dried lipid film along with 1 mL of assay buffer and then processed as above.

[0280] For Tb:DPA encapsulated liposomes: 1 mL of assay buffer supplemented with 2 mM Tb(III) chloride and 6 mM DPA was added to the dried lipid film and then processed as above.

[0281] Liposome permeabilization and dye release assay The choice of ANTS / DPX or terbium:DPA liposomes for dye release assays was determined depending on the properties of the compound assayed for BAX inhibitory activity that affected the fluorescence of ANTS or terbium. Compounds with overlapping fluorescence with either of the encapsulated dyes or that altered the fluorescence of dye-encapsulated liposomes in the absence of added protein were assayed with the other fluorophore to ensure that the compound did not simply partition into or permeabilize the liposomes.

[0282] For the ANTS / DPX dye release assay: ANTS (fluorophore, excitation 355 nm, emission 520 nm) and DPX (collision quencher) are encapsulated in reconstituted liposomes. Upon membrane permeabilization, ANTS and DPX diffuse out of the liposomes, decreasing the effective concentration of DPX and thus decreasing quenching by ANTS and increasing ANTS fluorescence. ANTS / DPX liposomes (8 uL of mg / mL liposome preparation per 100 uL reaction) were added to individual wells of a multiwell plate (final volume of 100 μL for 96-well plates or total volume of 30 μL for 384-well plates) in assay buffer (10 mM HEPES, pH 7.2, 200 mM KCl, and 5 mM MgCl2). Background fluorescence of compounds and liposomes dissolved in DMSO was measured for 10 min to ensure stability of the fluorescent signal (F0). Unless stated explicitly, 20 nM cBID and 100 nM BAX were added sequentially to each well and mixed thoroughly. The fluorescence intensity of ANTS was measured every minute for 2-3 hours until the signal stabilized at a plateau (F). Finally, 2 μL of 10% TritonX-100 was added to each well of the 96-well plate (1 μL / well for 384-well plate) and mixed carefully to avoid forming air bubbles. The plate was then read a third time to assess the fluorescence of ANTS at the time when the liposomes were completely dissociated in each well (F). 100 The percentage of dye released from liposomes by the action of cBID and BAX is calculated as follows:

number

[0283] For the Terbium:DPA dye release assay: Association of Tb:DPA results in a fluorescent complex (excitation 280 nm, emission 490 nm) when encapsulated in liposomes. When liposomes are permeabilized, Tb:DPA diffuses into solution where EDTA chelates with Tb and reduces fluorescence. Tb:DPA liposomes (8 uL of 0.5 mg / mL lipid preparation per 100 uL reaction) were added to each well of a 96-well (100 μL final volume) or 384-well (30 μL total volume) containing EDTA assay buffer (10 mM HEPES, pH 7.2, 200 mM KCl, 5 mM MgCl2, and 5 mM EDTA). The background fluorescence of the liposomes in the presence of compound (if used) dissolved in DMSO was measured for 10 min until the signal was stable (F0). Unless stated explicitly, 20 nM cBID and 100 nM BAX were added sequentially to each well and mixed thoroughly. The fluorescence intensity of ANTS was measured kinetically every minute for 2-3 h until the signal stabilized at a plateau (F). Finally, 5 μL of 20% CHAPS was added to each well of the 96-well plate and 2 μL for the 384-well plate and mixed carefully to avoid excessive air bubble formation. The plates were then read a third time to assess the fluorescence of Tb:DPA at the time of complete dissociation of liposomes in each well (F). 100 The percentage of dye released from liposomes by the action of cBID and BAX is calculated as follows:

number

[0284] Measurement of BAX binding to liposomes and mitochondria Compounds were incubated with membranes for 10 min at 37° C. before adding purified proteins. Alexa568, 126C BAX, and in certain cases 20 nM BIDmt1 (M97A, D98A (BCL-X LA BID mutant that cannot be inhibited by DMSO was added to all reactions.

[0285] For ANTS liposomes, 200ug / mL lipid in a total reaction volume of 300uL was incubated at 37°C for 1 hour, after which the mixture was subjected to size exclusion chromatography using a 2.5mL CL-2B column. Twelve 200uL fractions were collected for each reaction, and 100uL was transferred to a 96-well plate (corning3881). ANTS (excitation 355nm, emission 520nm) and Alexa568 (excitation 578nm, emission 603nm) fluorescence was measured on a TCAN M1000, and the gain was adjusted to obtain the same fluorescence intensity values ​​between the two dyes.

[0286] Isolated BAK - / - For mitochondria, 1 mg / mL protein (measured by Bradford assay) in 100 uL total reaction volume was incubated at 37°C for 1 hour, then spun down to pellet mitochondria. Supernatants were transferred to different wells and pellets were resuspended in 100 uL assay buffer. Alexa568 BAX fluorescence was measured in the supernatant and pellet fractions to determine the percentage of BAX in the membrane. The nM amount of Alexa568, 126C BAX was then determined by multiplying the amount of BAX used in the assay (100 nM or 200 nM as indicated) by the fraction bound to the membrane. To compare BIDmt1-bound BAX in the presence or absence of BAX inhibitors (dacomitinib or I-1), the ratio of Alexa568-BAX fluorescence intensities was used to determine the nM amount of BAX bound to the membrane. Each drug condition represents the amount of BAX in the membrane compared to the absence of compound.

[0287] Mitochondrial isolation and permeabilization assay Animal breeding and handling were performed in accordance with local regulations after approval by the Animal Care Committee of Sunnybrook Research Institute, Toronto. The isolation of mouse-liver mitochondria and their use with Bcl-2 family proteins to measure activity and protein-protein interactions was previously reported in detail (Pogmore et al., 2016). In mouse liver, BAX is either cytoplasmic or loosely bound to mitochondria. Thus, BAX is absent in isolated mitochondria. Thus, when mitochondria are isolated from livers from BAK- / - mice, they contain neither BAX nor BAK. Briefly, BAK - / -Transgenic mice (The Jackson Laboratory, stock number 004183) were sacrificed and liver tissue was harvested. Four to five mouse livers were washed in AT buffer (300 mM trehalose, 10 mM HEPES-KOH, pH 7.7, 10 mM KCl, 1 mM EGTA, 1 mM EDTA, and 0.1% BSA) until red blood cells were visually absent. The washed tissue was minced using sharp surgical scissors in 5 mL of ice-cold AT buffer for each liver used. The minced tissue was homogenized with four strokes of a motorized Potter-Elvehjem homogenizer. The homogenate was centrifuged at 600 x g for 10 min, the supernatant was collected, and centrifuged again at 3500 x g for 15 min. The supernatant was then collected, taking care to avoid the floating fat layer. The heavy membrane fraction containing mitochondria was obtained by centrifugation at 5500xg for 10 min. The pellet was then resuspended in the desired amount of AT buffer, aliquoted for single use, flash frozen using liquid nitrogen, and stored at -80°C. Prior to use in the assay, mitochondria were quickly thawed, washed once with AT-KCl buffer (300mM trehalose, 10mM HEPES-KOH, pH 7.7, 80mM KCl, 1mM EGTA, 1mM EDTA, and 0.1% BSA), and pelleted by centrifugation of the solution at 10000xg. The supernatant was discarded and the remaining pellet was resuspended in renaturation buffer (300 mM trehalose, 10 mM HEPES-KOH, pH 7.7, 80 mM KCl, 1 mM EGTA, 1 mM EDTA, 0.1% BSA, 5 mM succinate, 2 mM ATP, 10 μM phosphocreatine, and 10 μg / mL creatine kinase) at a protein concentration of 1 mg / mL (determined by Bradford assay) for immediate use as a source of membranes for FRET measurements.

[0288] BAX stably expresses a fusion protein consisting of the SMAC uptake peptide and the fluorescent protein mCherry. - / - / BAK - / - or BAX - / -Mitochondria from baby mouse kidney (BMK) cells were isolated and used as previously reported (Niu et al., 2017). SMAC-mCherry BMK cells were maintained in DMEM containing 10% FBS supplemented with 3 μg / mL blasticidin. Cells were scraped from tissue culture dishes, placed on ice, harvested with PBS, and centrifuged at 500 x g for 4 min to pellet the cells. The supernatant was removed and the cell pellet was resuspended in lysis buffer (20 mM HEPES, pH 7, 250 mM sucrose, 150 mM KCl, 1 mM EDTA, and 2x protease inhibitor cocktail), and the cells were lysed by nitrogen cavitation at 250 psi for 10 min with the containment chamber surrounded by ice. Cell debris was removed by centrifugation at 2000 x g for 4 min at 4 °C. The supernatant was retained and centrifuged at 13,000×g for 10 min to pellet the heavy membranes containing mitochondria. The pellet was then diluted in lysis buffer to a protein concentration of 0.2 mg / ml (measured by Bradford assay) and used immediately for permeabilization experiments.

[0289] BAX - / - / BAK - / - For BMK SMAC-mCherry mitochondria, mitochondria were fully permeabilized using 2 nM cBID and 20 nM BAX. After 30 min incubation at 37°C, 50 μL of the reaction was centrifuged to pellet the mitochondria and the supernatant was separated. The pellet was resuspended in an equal volume of lysis buffer. The percentage of SMAC-mCherry release was determined by fluorimetric analysis of the supernatant and pellet fractions by measuring the fluorescence intensity (F) of mCherry (excitation 580, emission 610 on a Tecan M1000).

[0290] Forster resonance energy transfer Detailed methods for FRET assays using isolated mitochondria have been reported previously (Pogmore et al., 2016). Briefly, compounds were dissolved in DMSO and incubated at 37 °C with 1 mg / mL mitochondria and 5 nM Alexa568-cBID (FRET donor), followed by titration with labeled Alexa647-BAX (FRET acceptor) or unlabeled BAX (to account for changes in donor fluorescence due to changes in the chemical environment upon protein binding). Measurements were taken every minute for 1–2 h until the fluorescence intensity of the donor fluorophore reached a plateau. The fluorescence intensity of the donor-labeled protein in the presence of the labeled acceptor protein (F DA ) is the fluorescence intensity of the donor fluorophore in the presence of unlabeled acceptor protein (F D ) and subtracting the quotient to obtain the FRET efficiency (E). Because the background of Alexa dye in the presence of mitochondria is negligible, the formula for calculating E is:

number

[0291] Kinase assay A peptide containing the kinase domain of EGFR (amino acids 695-1210) was used to evaluate the inhibitory potency of the inhibitors. The ADP-Glo ​​Kinase Assay (Promega V9101) and EGFR kinase enzyme (Promega V3831) were used according to the supplier's instructions. Briefly, 26 μL reactions were set up in wells of a 384-well plate as follows: 5 μL kinase reaction, 5 μL ADP depletion reagent, and 10 uL luciferase reagent. For the kinase reaction, 1 μL of inhibitor (dissolved in DMSO) was mixed with 2 μL EGFR peptide (0.1 mg / μL), 1 μL polyGlu4Tyr1 peptide (0.2 μg / μL), 1 μL reaction buffer, and 1 uL ATP (final concentration 5 μM). The kinase reaction was incubated for 1 h at room temperature. 5 μL of ATP depletion / kinase stop reagent was mixed into each well and incubated for 40 min at room temperature. 10 μL of kinase detection reagent containing luciferase was mixed into each well and incubated at room temperature for 30 min for bioluminescence detection of ADP generated from the kinase reaction in step 1. The bioluminescence signal was detected using a Tecan Safire Fluorescence Imaging Kit equipped with a blank emission filter and a black plug covering the excitation filter. 2 The integration time was increased to 0.5 μs to detect sufficient emission per reading.

[0292] For the kinase panel, % inhibition was measured using standardized assay conditions at the KinaseProfiler service (Eurofins Pharma Discovery Services). Compounds were sent for testing as dry powders.

[0293] Microscale Thermophoresis MST measurements were performed with a Monolith NT.115 at 60% LED power, 25 s IR laser time (5 s final off time), 50% IR laser power, and machine temperature set at 25 °C. Each reaction differed only in the concentration of compound. Each reaction contained 100 nM Alexa647-labeled BAX, 0.5% DMSO, and pH 7.3 phosphate buffer (100 mM KCl and 150 mM NaCl). The reaction tubes were sonicated for approximately 5 s to dissolve the compounds at higher concentrations and then transferred to the capillary tube. In liposomal dye release experiments, no difference in activity was observed with sonicated BAX, similar to other studies (Garner et al., 2019).

[0294] NMR spectroscopy and chemical shift perturbation All experiments were performed using independent samples for each measurement and were referenced against DMSO to account for the solvent-induced environmental changes. 15 N-labeled BAX was prepared as above in 50 mM potassium phosphate, pH 6, 50 mM NaCl, and 10% D2O. Samples were mixed and then 40 μL was placed in a 1.7 mm sample tube. No change in pH was observed upon addition of dacomitinib to samples containing BAX at the concentrations used here. NMR experiments used full-length WT BAX at a concentration of 200 μM. Experiments were performed at 25 °C on a 600 MHz Bruker Avance magnet. Chemical shift perturbations (CSPs) were calculated in CCPNMRv3 using automated analysis, with all peaks corresponding to their extremes, using the following equation:

number

[0295] In the above CSP formula, α is 1 For H nuclei 15 The relative weighting of the chemical shift changes of N is shown. Hx and Δδ Nxare the observed changes in proton and nitrogen chemical shifts for residue x, respectively. Note that all amino acid positions on the HSQC were manually inspected to use the extrema of the nearest identified peak. The absence of a bar in the CSP graph means no chemical shift, the presence of proline, or a missing residue peak that was not used in the analysis. The significance threshold was determined as the average of the chemical shifts for all residues plus the standard deviation. To indicate the significance of a residue, shifts above the significance threshold are colored light gray, while shifts above 1.5 times the significance threshold are colored dark gray.

[0296] cell culture Baby mouse kidney (BMK) cells were cultured as described by (Mathew et al., 2008). BMK double knockout, SMAC-mCherry, and BAX - / - The cell lines were generated in-house as reported (Niu et al., 2017). BMK lines were grown in DMEM supplemented with 10% fetal bovine serum and 1x MEM non-essential amino acids in a humidified incubator at 37°C and 5% CO2.

[0297] For cell death assays using actinomycin D, 3000WT or DKO BMK cells were cultured in 384-well plates and incubated overnight. The next morning, cells were treated with actinomycin D for 2 h, followed by the addition of BAX inhibitor. Actinomycin D and BAX inhibitor (if applicable) were then incubated for 24 h, followed by staining with Hoechst, AnnexinV-Alexa488, and TMRE for 30 min. Cells were imaged by confocal microscopy (Opera Phenix-PerkinElmer) and then analyzed with Harmony (PerkinElmer) software. Cell death was scored by three criteria: loss of TMRE signal, appearance of AnnexinV-Alexa488 signal, and nuclear shrinkage visualized by Hoechst.

[0298] ii.Results Structure of a kinase inhibitor that blocks cBID-BAX-mediated liposome permeabilization Dacomitinib, a 4-anilinoquinazoline-containing compound, was identified in a screen for compounds that inhibit c-BID-activated BAX-mediated liposome permeabilization.

[0299] Compounds bind directly to BAX Identifying the binding sites of dacomitinib and compound I-1 would ideally be achieved by high-resolution 3D structures of BAX and the compounds. However, such measurements are difficult due to technical challenges such as the dynamic structure of BAX and the limited solubility of the compounds. Therefore, to identify where dacomitinib and I-1 bind to BAX, 2D structures were used. 1 H- 15 N heteronuclear single quantum coherence (HSQC) NMR analysis 15 This was performed with N-labeled BAX (Figure 1A). Analysis of the chemical shift perturbations (CSPs) of BAX in the presence of dacomitinib (Figure 1B,C) and I-1 (Figure 2A) reveals significant chemical shifts localized in the pockets of α-helices 4, 5, and 6. Additional CSPs adjacent to α-helices 4, 5, and 6 are less significant and are likely the result of adaptive conformational changes induced upon dacomitinib binding to BAX. To further corroborate the proposed location of the dacomitinib and I-1 binding sites, we used the V83W, L120W mutant of BAX (Garner et al., 2019), which is thought to block the BAI1 binding site between α-helices 4, 5, and 6. These mutations in the recombinant protein were also observed to prevent the inhibition of BAX by dacomitinib (Figure 2B). Taken together, these data suggest that dacomitinib and I-1 bind to the cytosolic conformation of BAX near the previously identified BAI1 binding site and that the binding does not inhibit BH3 peptide binding. The NMR data further suggest that the effect of I-1 binding on the structure of BAX is slightly more pronounced for I-1, consistent with tighter binding.

[0300] Hit compounds reduced BAX / BAK permeabilization of the outer mitochondrial membrane To evaluate the inhibitory activity of dacomitinib and I-1 against BAX- or BAK-mediated mitochondrial permeabilization, we used BAX-mCherry-expressing mitochondrial permeabilization assays (MSA-MSCs) and mitochondrial permeabilization assays (MSCs) for the treatment of BAX- or BAK-mediated mitochondrial permeabilization in mice expressing Smac-mCherry (a fluorescent protein located in the interm - / - BAK - / - Baby Mouse Kidney (BMK) cells or BAX - / - Heavy membranes were isolated from BMK cells. Fractions of heavy membranes from these cells were incubated with sufficient recombinant BAX and cBID proteins to release 60-80% of Smac-mCherry from the intermembrane space of mitochondria. A concentration-dependent decrease in the percentage of Smac-mCherry release was measured in reactions where compounds were added at concentrations up to 60 uM (Figure 3A). In agreement with the results obtained using liposomes, the inhibition curves were similar for both compounds, suggesting that they have similar activity. Unexpectedly, much higher concentrations of compound were required to inhibit MOMP than were required to inhibit liposome permeabilization. This could be due to nonspecific attachment of compounds to the membrane fraction, degradation of compounds by microsomal and mitochondrial enzymes present in the heavy membrane fraction, or poor solubility of compounds in incubations containing heavy membranes. To assay inhibition of full-length BAK, the same concentration range of compounds was incubated with recombinant activator proteins BIM and BAX. - / - As a positive control for both experiments, the anti-apoptotic protein BCL-X was added to the incubation containing the heavy membrane fraction isolated from mitochondria (Figure 3B). L were used to inhibit BH3 protein activators and BAX or BAK. Collectively, the data suggest that dacomitinib and I-1 are equally effective inhibitors of BAX and BAK, independent of the activator BH3 protein.

[0301] Kinase binding is not essential for BAX inhibition Compound I-1, a variant of dacomitinib lacking both the Michael acceptor and the hinge-bound N-1 nitrogen of the quinazoline ring, has no appreciable kinase inhibitory activity at or below 10 uM (Table 1). Indeed, removal of the hinge-bound nitrogen resulted in minimal activity across a diverse panel of 58 kinases (Figure 4A). Nevertheless, unexpectedly, exemplary compound I-1 retained comparable BAX inhibitory activity to the comparator compound dacomitinib when assayed for inhibition of cBID-activated BAX-mediated permeabilization of mitochondrial-like liposomes (Figure 4B). In summary, the data for compound I-1 clearly demonstrate that the quinazoline core structure and kinase inhibition of dacomitinib-type compounds are not required for BAX inhibition. Furthermore, compound I-1 meets the predictive requirements of drug-likeness for molecules with favorable ADME. [Table 2]

[0302] BAX inhibition rescues baby mouse kidney cells from actinomycin D-induced BAX- and BAK-mediated cell death To test the effects of dacomitinib and compound I-1 on cell death, it is necessary to utilize a method that induces cell death in a manner that is solely dependent on BAX and BAK. Actinomycin D is an inducer of BAX and BAK-mediated apoptosis (Figure 5A), and therefore actinomycin D kills approximately 80% of wild-type cells but not BAX. - / - , B.A.K. - / -A concentration that did not kill double knockout BMK cells was used (Figure 5A-B). Single knockouts of BAX or BAK were also used to evaluate the contribution of individual execution proteins to actinomycin D-induced apoptosis. At 100 nM, actinomycin D induced cell death equally in both single knockout cell types, indicating that the drug results in the activation of both execution proteins. Furthermore, cell death in the single knockouts was approximately half that of the wild type, suggesting that at that concentration of actinomycin D, the two execution proteins contributed equally to the observed cell death. At higher concentrations of the drug, cell death was further increased in cells expressing BAX over BAK, an effect that may be related to the total protein concentration(s) of execution protein or BH3 protein in the cells. Based on the data in Figure 5A, concentrations of 25 nM and 100 nM of actinomycin D were chosen for wildtype and knockout cell lines, respectively, to obtain a comparable dynamic range for inhibitor titration. Based on data from preliminary experiments, each cell type was protected against the indicated concentrations of actinomycin D using either 1.25 uM or 2.5 uM concentrations of dacomitinib or I-1 (Figure 5B). Based on these data, compound I-1 was the most effective inhibitor of actinomycin D-mediated cell death in cells expressing either BAX or BAK. However, comparison of titration data for each of the inhibitors across multiple replicates suggests that, overall, the various molecules inhibit BAX and BAK more or less equally.

[0303] Inhibition of BAX and BAK protects BMK cells from apoptosis induced by actinomycin D Wild-type BMK cells (BMKwt) were treated with different concentrations of actinomycin D and dacomitinib (Figure 6) or compound I-1 (Figure 7). Cell death was assessed using a linear classifier based on measurements of mitochondrial membrane potential measured by TMRE (tetramethylrhodamine ester), nuclear shrinkage visualized with Hoechst, and binding of fluorescent annexin V. Cells showed good resistance to dacomitinib and I-1, which reduced the amount of spontaneous cell death in cultures (0 mM actinomycin D). Dacomitinib (Figure 6) and compound I-1 (Figure 7) reduced cell death induced by concentrations of actinomycin D that did not kill BMK cells lacking both BAX and BAK. Taken together, these data indicate that dacomitinib and I-1 inhibit cell death resulting from actinomycin D-mediated activation of BAX and / or BAK.

[0304] iii. Discussion Dacomitinib, an FDA-approved kinase inhibitor, has surprisingly been found to have off-target inhibitory activity against BAX and BAK. To the applicant's knowledge, Dacomitinib is the first FDA-approved compound found to inhibit both BAX and BAK. Dacomitinib is a well-tolerated, orally available small molecule, which has become a scaffold for the study of further pharmacologic useful inhibitors of BAX and BAK. Furthermore, unexpectedly, it has been demonstrated that the kinase inhibitory activity of Dacomitinib is not required for the inhibition of BAX and BAK. In line with this view, compound I-1 has been identified as an example of a new family of compounds with an isoquinoline core structure that lacks kinase inhibitory activity but still inhibits both BAX and BAK. Without wishing to be limited by theory, NMR data suggest that dacomitinib and I-1 bind BAX in a solution-accessible pocket at the junction between α-helices 4, 5, and 6 (Figures 1-2).

[0305] Example 52: SMAC mCherry assay Bax- / -Bak- / - double knockout baby mouse kidney (BMK) cells stably expressing the SMAC-(1-56)-mCherry fusion protein were cultured for 37 h in DMEM complete medium (DMEM supplemented with 10% FBS and 1x penicillin-streptomycin). oCells were cultured at 37°C in 10 cm diameter tissue culture treated dishes in a humidified incubator with 5% CO2 in air. Cells were washed with PBS, incubated with 1x trypsin, and resuspended in DMEM complete medium. Dissociated cells were seeded at 6000 cells per well in 50uL of DMEM complete medium in 384-well Perkin-Elmer Phenoplates and incubated at room temperature for 30 minutes followed by overnight incubation at 37°C in a humidified incubator with 5% CO2 in air. Recombinant Bax and recombinant cBid were thawed quickly between fingers and compound stocks at 25mM in DMSO were thawed at room temperature. Eight-point titration curves for each compound were generated in a 384-well source plate containing Trehalose-Hepes buffer (THB, 135 mM trehalose, 50 mM KCl, 10 mM HEPES-KOH, pH 7.4, 5 mM succinate, 20 uM EDTA, 20 uM EGTA, 0.1% BSA) supplemented with 1 / 1000 DRAQ5, 0.0025% digitonin, 20 nM Bax, and 0.5 nM cBid. The source plate also contained control wells of THB supplemented with 1 / 1000 DRAQ5, 0.0025% digitonin, and 20 nM Bax (negative control), or 20 nM Bax and 0.5 nM cBid (positive control). Using a Vantage automated liquid handler, DMEM complete media was removed from cells growing in a 384-well Phenoplate. Cells were washed twice with 30uL of THB, then 25uL from the 384-well source plate containing THB protein and compounds was transferred to cells growing in the 384-well plate. Cells were incubated for 60-80 minutes at 37C in a humidified incubator with 5% CO2 in air, then imaged on an Opera Phenix microscope. Two image channels were acquired: 1) a DRAQ5 channel to visualize nuclei and aid in cell segmentation, and 2) a SMAC-mCherry channel to visualize mitochondria bearing SMAC-mCherry.Data were processed using PerkinElmer Harmony software, cells were segmented using the DRAQ5 channel, and the mean intensity of SMAC-mCherry for each cell was calculated. Single cell object level data was exported and then analyzed using R code. A cutoff value for SMAC-mCherry intensity per cell was calculated as 1.33 times the mean SMAC-mCherry intensity per cell of the THB+Bax control. Cells with SMAC-mCherry intensity below the cutoff value were recorded as "releasing". Percentage SMAC-mCherry release was calculated as the number of cells recorded as "releasing" divided by the total number of cells multiplied by 100. Dose-response curves were fitted to a 4-parameter log-logistic model to determine IC for each compound. 50 value was determined.

[0306] Exemplary compounds of the present application have IC values ​​in the SMA CmCherry assay in the following ranges: 50 A: 0.1-10 uM, B: 11-50 uM, C: 51-100 uM, D: >100 uM. Specific ranges for the exemplary compounds of formula (I) are shown in Table 2. [Table 3] TIFF2025506245000136.tif242159TIFF2025506245000137.tif242159TIFF20255062450 00138.tif235159TIFF2025506245000139.tif235159TIFF2025506245000140.tif237159 TIFF2025506245000141.tif230159TIFF2025506245000142.tif230159TIFF2025506245000143 .tif230159TIFF2025506245000144.tif205159A:0.1~10uM, B:11~50uM, C:51~100uM, D:>100uM.

[0307] While the present application has been described with reference to what are presently considered to be the preferred embodiments, it is to be understood that the present application is not limited to the disclosed embodiments, but rather, the present application is intended to cover various modifications and equivalents included within the spirit and scope of the appended claims.

[0308] All publications, patents, and patent applications are incorporated herein by reference in their entirety as if each was specifically and individually indicated to be incorporated by reference in its entirety. In the event that a different definition is found in a document incorporated herein by reference for any term in this application, the definition set forth herein shall control for that term.

[0309] Full citations for documents referred to herein Many publications are cited herein, full citations for which are set forth below, and each of which is incorporated by reference in its entirety into this disclosure as if each was specifically and individually indicated to be incorporated by reference.

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Claims

1. A compound of formula I: or a pharmaceutically acceptable salt, solvate, and / or prodrug thereof: 【Chemistry 1】 During the ceremony, Each R １ are independently halo, CN, C １～４ Alkyl, C １～４ Haloalkyl, OC １～４ Alkyl, and OC １～４ haloalkyl; R ２ is absent or R ２ Ha, Halo, C １～４ Alkyl, C １～４ Haloalkyl, OC １～４ Alkyl, and OC １～４ haloalkyl; R ３ is H and C １～４ alkyl, L is C １～４ Alkylene, C ２～４ Alkenylene, and C ２～４ alkynylene, A is N, NH, and N(C １～４ alkyl), and includes at least one ring hetero moiety selected from OH, halo, C １～２ Alkyl, C １～２ Haloalkyl, OC １～２ Alkyl, and OC １～２ C optionally substituted with one or two substituents selected from haloalkyl ３～８ is heterocycloalkyl, The compound of formula I, or a pharmaceutically acceptable salt, solvate, and / or prodrug thereof, wherein n is selected from 0, 1, 2, and 3.

2. Each R １ are independently F, Cl, Br, CN, C １～４ Alkyl, C １～４ Haloalkyl, OC １～４ Alkyl, and OC １～４ The compound of claim 1 selected from haloalkyl.

3. Each R １ are independently F, Cl, Br, CN, CH ３ , C.H. ２ CH ３ , C.H. ２ CH ２ CH ３ , CH(CH ３ ) ２ , C.F. ３ , CHF ２ , C.F.H. ２ , C.H. ２ CHF ２ , C.H. ２ CF ３ , C.H. ２ CFH ２ , CCl ３ , C.H. ２ CClH ２ , CCl ２ H, CH ２ CCl ２ H, CH ２ CCl ３ , OCH ３ , OCH ２ CH ３ , OCH ２ CH ２ CH ３ , OCH(CH ３ ) ２ , OCF ３ , OCHF ２ , OCH ２ CHF ２ , OCH ２ CF ３ , OCH ２ CFH ２ , OCCl ３ , OCH ２ CClH ２ , OCCl ２ H, OCH ２ CCl ２ H, and OCH ２ CCl ３ 3. The compound of claim 2, selected from:

4. 2. The compound of claim 1, wherein n is 2 or 3, or n is 0.

5. R ２ are F, Br, Cl, C １～３ Alkyl, C １～３ Fluoroalkyl, C １～３ Chloroalkyl, OC １～３ Alkyl, OC １～３ Fluoroalkyl, and OC １～３ 2. The compound of claim 1, wherein the compound is selected from chloroalkyl.

6. R ２ are F, Cl, CH ３ , C.F. ３ , OCH ３ , OCH ２ CH ３ , OCH ２ CH ２ CH ３ , OCH(CH ３ ) ２ , OCF ３ , and OCHF ２ selected from, or 6. The compound of claim 5, wherein R2 is absent.

7. R ３ is H and CH ３ 2. The compound of claim 1 selected from:

8. L is C １～４ Alkylene and C ２～４ The compound of claim 1 selected from alkenylene.

9. L is CH ２ CH ２ CH ２ (C ３ alkylene) and CH ２ CH ２ (C ２ 9. The compound of claim 8, wherein the alkylene is selected from the group consisting of aryl, arylalkylene, arylsulfonyl ...

10. L is CHCHCH ２ and CH ２ 9. The compound of claim 8, wherein the compound is selected from CHCH.

11. A is N, NH, and N(C １～４ alkyl), and OH, F, Cl, C １～２ Alkyl, CF ３ , CHF ２ , CCl ３ , CCl ２ H, OCH ３ , OCH ２ CH ３ , OCF ３ , OCHF ２ , OCH ２ CHF ２ , OCH ２ CF ３ , OCH ２ CFH ２ , OCCl ３ , OCH ２ CClH ２ , OCCl ２ H, OCH ２ CCl ２ H, and OCH ２ CCl ３ C optionally substituted with one or two substituents selected from ３～６ The compound of claim 1 which is a heterocycloalkyl.

12. A is selected from aziridinyl, azetidinyl, pyrrolidinyl, morpholinyl, piperazinyl, and piperidinyl, and is selected from OH, F, Cl, C １－２ Alkyl, CF ３ , CHF ２ , CCl ３ , CCl ２ H, OCH ３ , OCH ２ CH ３ , OCF ３ , OCHF ２ , OCH ２ CHF ２ , OCH ２ CF ３ , OCH ２ CFH ２ , OCCl ３ , OCH ２ CClH ２ , OCCl ２ H, OCH ２ CCl ２ H, and OCH ２ CCl ３ The compound of claim 11, optionally substituted with one or two of:

13. The compound of formula I is a compound of formula IA below, or a pharmaceutically acceptable salt, solvate, and / or prodrug thereof: 【Chemistry 2】 During the ceremony, R １ , R ２ , L, and n are as defined for formula I of claim 1; 2. The compound of claim 1, wherein m is an integer selected from 1 to 3. 【Request Item 14】 【Table 1】

15. A pharmaceutical composition comprising one or more compounds according to any one of claims 1 to 14 and a pharmaceutically acceptable carrier.

16. In cells of either a biological sample or a patient, for use in inhibiting Bcl-2 associated X protein (BAX) and / or Bcl-2 antagonist killer (BAK), for use in treating or preventing BAX-mediated cell death and / or BAK-mediated cell death, for use in inhibiting mitochondrial outer membrane permeabilization (MOMP), or For use in inhibiting the oligomerization of BAX and BAK, The pharmaceutical composition of claim 15.

17. 16. The pharmaceutical composition of claim 15 for use in treating a disease, disorder, or condition treatable by inhibiting Bcl-2 associated X protein (BAX) and / or Bcl-2 antagonist killer (BAK).

18. The disease, disorder, or condition treatable by inhibiting BAX and / or BAK is a neurodegenerative disease, disorder, or condition. The disease, disorder, or condition treatable by inhibiting BAX and / or BAK is neuronal damage associated with ischemia, The disease, disorder, or condition treatable by inhibiting BAX and / or BAK is cardiomyopathy induced by a chemotherapy drug, or a pharmaceutical composition, or The disease, disorder, or condition treatable by inhibiting BAX and / or BAK is cell death of donor hematopoietic stem and progenitor cells (HSPCs); 18. The pharmaceutical composition of claim 17.

19. The pharmaceutical composition of claim 15 for use in increasing the survival rate of donor hematopoietic stem and progenitor cells (HSPCs) during transplantation.

20. 16. The pharmaceutical composition of claim 15, for use in treating a disease, disorder, or condition treatable by inhibiting BAX and / or BAK, characterized in that the pharmaceutical composition is used in combination with another known drug useful in treating a disease, disorder, or condition treatable by inhibiting BAX and / or BAK.

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