BCL-XL inhibitors

Abstract

The present invention relates to BCL-XL inhibitors, comprising a pyrazolyl ring attached to a heterocycloalkyl, such as piperidinyl, wherein the heterocycloalkyl with the pyrazole ring to which it is attached may form a fused heterocyclic ring system, as well as a method for treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an inventive BCL-XL inhibitor compound. The present invention further provides a method for identifying such BCL-XL inhibitors.

Description

P-656906-PCBCL-XL INHIBITORSSEQUENCE LISTING STATEMENT

[0001] A Sequence Listing conforming to the rules of WIPO Standard ST.26 is hereby incorporated by reference. Said Sequence Listing has been filed as an electronic document via PatentCenter encoded as XML in UTF-8 text. The electronic document, created on April 7, 2026, is entitled “P-656906-PC-ST26.xml”, and is 21,715 bytes in size.FIELD OF THE INVENTION

[0002] The present invention relates to BCL-XL inhibitors, methods for identifying the same, and uses thereof for treatment of cancer.BACKGROUND OF THE INVENTION

[0003] BCL-XL is a key regulator of apoptosis and a challenging cancer target. Apoptosis is a tightly regulated process of programmed cell death essential for cellular development, immune homeostasis, and the elimination of damaged or malignant cells. The intrinsic apoptotic pathway is primarily controlled by the BCL-2 family of proteins, which regulate mitochondrial outer membrane permeabilization (MOMP) — a critical event leading to cell death. This pathway is activated in response to various cytotoxic stress stimuli, including DNA damage, ER-stress, oncogene inactivation, and targeted therapies.

[0004] The BCL-2 family consists of three functionally distinct subgroups. (1) Anti-apoptotic proteins (Bcl-2, Bcl-xL, Bcl-w, Mcl-1, Bfl-1) that prevent apoptosis by sequestering pro-apoptotic family members. (2) Pro-apoptotic effectors (Bax, Bak, Bok) that mediate MOMP and trigger cytochrome c release. (3) BH3-only pro-apoptotic proteins (Bad, Bid, Bim, Bik, Noxa, PUMA, Bmf, Hrk) that regulate apoptosis by inhibiting anti-apoptotic BCL-2 family members or directly activating BAK and BAK. Dysregulation of this tightly balanced network leads to tumor progression and therapy resistance, with BCL-XL emerging as a critical survival factor in many malignancies.

[0005] BCL-XL is a driver of tumor survival and resistance. BCL-XL (encoded by BCL2L1) is one of the most frequently upregulated anti-apoptotic proteins in cancer, enabling tumor cells to evade apoptosis. Its overexpression is associated with poor prognosis, therapy resistance, and disease progression across a range of malignancies, including hematologic cancers (e.g., acuteP-656906-PCmyeloid leukemia (AML) and chronic lymphocytic leukemia (CLL)), solid tumors (e g., colorectal adenocarcinoma, lung cancer, pancreatic cancer, and breast cancer), and other malignancies such as Kaposi’s sarcoma, malignant endothelial cells in skin disorders. The high copy number amplification of BCL2L1 in multiple cancer types makes BCL-XL an essential target for therapeutic intervention. BCL-XL inhibition has been shown to re-sensitize resistant tumors to apoptosis, offering a powerful strategy to enhance the efficacy of chemotherapy, radiation, and targeted therapies.

[0006] Despite its significance as a cancer target, there are no FDA-approved drugs that directly inhibit BCL-XL. The primary challenge lies in dose-limiting platelet toxicity. BCL-XL is crucial for platelet survival, and direct inhibition of its function leads to severe thrombocytopenia. Examples of previous BCL-XL inhibitors that were advanced to clinical stage include Navitoclax (ABT-263), Pelcitoclax (APG-1252), S55746, BM-1197, UBX1325 and BCL-XL degrader DT2216 that is based on ABT-263. Currently, all clinically tested BCL-XL inhibitors and degraders have relied on targeting the canonical BH3 -binding groove, a conserved pocket shared among BCL-2 family proteins. These BCL-XL inhibitors developed to date have targeted the canonical BH3-binding pocket, which is highly conserved among BCL-2 family members. The approach has several limitations. Large molecular weight compounds are required for sufficient binding affinity and inhibition imposing limitations in drug-likeness and pharmacokinetics (PK). Achieving selectivity due to structural similarity with other BCL-2 family proteins (e.g., BCL-2, MCL-1) is challenging and requires extensive medicinal chemistry refinement. Further, doselimiting thrombocytopenia from killing platelets, reducing the therapeutic index of these compounds.

[0007] Given these challenges, there is an unmet need for alternative strategies to selectively inhibit BCL-XL while minimizing platelet toxicity.P-656906-PCSUMMARY OF THE INVENTION

[0008] In one aspect, the present invention provides a BCL-XL inhibitor reprsented by a compound of Formula (I),or a pharmaceutically acceptable salt thereof or a stereoisomer thereof,whereinX is a bond or CH2;A is H, alkyl, cycloalkyl, or aryl;B is heterocycloalkyl, wherein the heterocycloalkyl with the pyrazole ring to which it is attached may form a fused heterocyclic ring system;G is alkyl, alkenyl, alkynlyl, C(=O)RP, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; n is 0, 1, 2, or 3;wherein the aryl and heteroaryl are each optionally substituted with one or more groups independently selected from C1-C5 alkyl, halo, haloalkyl, aryl, CN, ORP, NRpRq, RP0H, CHO, COORP, and CONRpRq;wherein the alkyl, cycloalkyl, heterocycloalkyl, and the fused heterocyclic ring are each optionally substituted with one or more groups independently selected from =0, halo, haloalkyl, CN, RqORPORP, NRPRq, NRpCORq, RP0H, CHO, cyclopropyl, heteroaryl, COORP, and CONRpRq; andRpand Rq are independently H or C1-C5 alkyl;wherein Rpand Rqcan optionally be taken together to form a 3-6 membered ring that can be optionally substituted by one or more groups independently selected independently from OH, =0, or C1-C5 alkoxy.P-656906-PC

[0009] In some embodiments, the compound of Formula (T) is represented by Formula (IT)wherein Ai, A2, A3, A4, and A5 are each independently H, C1-C5 alkyl, halo, CN, ORP, NRpRq, COORp, or CONRp.

[0010] In another aspect, the present invention provides a BCL-XL inhibitor represented by a compound of Formula (IV)or a pharmaceutically acceptable salt thereof or a stereoisomer thereof,whereinRH, R12, R13, R14, and R15 are each independently H, alkyl, halo, haloalkyl, CN, ORP, NRpRq, COORp, or CONRpRq;Re is COORp or CONRpRq;Rm and Rn are each independently H, alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, wherein each of alkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl is optionally substituted with one or more groups independently selected from halo, ORP, C1-C5 alkyl, haloalkyl, CN, NRPRq, RPOH, CHO, cyclopropyl, COORP, and CONRpRq;Rmand Rncan optionally be taken together with the nitrogen atom to which they are attached to form a 3-15 membered ring that is optionally substituted by one or more groups independently selected from =0, C1-C5 alkyl, CN, ORP, NRpRq, RP0H, CHO, cycloalkyl,P-656906-PCheterocyclalkyl, aryl, arylalkyl, heteroaryl, and heteroarylalkyl; andRp and Rq are independently H or C1-C5 alkyl.

[0011] In another aspect, the present invention provides a BCL-XL inhibitor represented by a compound of Formula (V)or a pharmaceutically acceptable salt thereof or a stereoisomer thereof,whereinRa and Rb are each independently H, alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, wherein each of alkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl is optionally substituted with one or more groups independently selected from halo, haloalkyl, CN, ORP, NRpRq, RP0H, CHO, cyclopropyl, COORp, and CONRpRq;Ra and Rb can optionally be taken together with the nitrogen atom to which they are attached to form a 3-15 membered ring that is optionally substituted by one or more groups independently selected independently from =0, C1-C5 alkyl, CN, ORP, NRpRq, RPOH, CHO, cycloalkyl, heterocyclalkyl, aryl, arylalkyl, heteroaryl, and heteroarylalkyl; andRpand Rq are independently H or C1-C5 alkyl.

[0012] In another aspect, the BCL-XL inhibitor of the present invention is represented by any one of the following compounds:P-656906-PC

[0013] In another aspect, the present invention provides a method for treating cancer in a subject in need thereof, comprising administering a therapeutically effective amount of a BCL-XL inhbitor compound of the invention as described herein, or a pharmaceutically acceptable salt thereof or a stereoisomer thereof. In some embodiments, the cancer is acute myeloid leukemia, chronic lymphocytic leukemia, acute lymphoblastic leukemia, lymphoma, multiple myeloma, non-small cell lung cancer, small cell lung cancer, triple-negative breast cancer, colorectal cancer, head and neck squamous cell carcinoma, mesothelioma, glioblastoma, and pancreatic neuroedocrine tumors.

[0014] In a further aspect, the present invention provides a method for identifying BCL-XL inhibitors of the invention, comprising the steps of (i) determining an allosteric site on full length BCL-XL, (ii) selecting compounds that bind to the allosteric site, and (iii) evaluating the selected compounds by experimental screening and validation.

[0015] In some embodiments, the present invention provides a method for the identification of an allosteric site on full length BCL-XL, comprisingP-656906-PC(a) calculating a 3D structural model of the human full-length BCL-XL using a truncated crystal structure of BCL-XL and a full length BAX structure as a template for the conformation of the c-terminal helix 9 of BCL-XL;(b) performing molecular dynamics simulations on the BCL-XL structural model to explore conformational dynamics and using conformations to feed automated binding site identification and analysis tools;(c) analyzing predicted binding sites for druggability based on site properties;(d) validating the predicted binding sites with NMR paramagnetic relaxation enhancement (PRE) experiments using hy-TEMPO probe to identify solvent accessible sites on the surface of BCL-XL accessed by TEMPO probe and confirming the predicted binding sites;(e) selecting a binding Site 2 that is adjacent to the N-terminal intrinsically disordered region (al-a2 loop) of BCL-XL and investigating changes of the binding site in the MD simulation trajectory;(f) maintaining the binding site with minor changes over the course of the simulation despite significant changes in al-a2 loop conformation adjacent to the Site 2; and(g) using mutagenesis of the binding site residues and identifying residues R6 and Al 68 in helices al and a6 as the hot spot residues largely determining the identification of the Site 2 by Sitemap.

[0016] These and other aspects of the invention will be appreciated from the ensuing descriptions of the figures and detailed description of the invention.BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Some embodiments of the invention are herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the invention. In this regard, the description taken with the drawings makes apparent to those skilled in the art how embodiments of the invention may be practiced.

[0018] Figure 1A depicts a homology model of full length BCL-XL protein structure with the flexible loop a.l-a2 (green) and C-terminal helix ct9 (orange).

[0019] Figure IB depicts a BH3 binding hydrophobic canonical pocket (blue) of BCL-XL with the binding of BIM BH3 peptide (magenta), helix a9 (orange) is located on the hydrophobic groove.P-656906-PC

[0020] Figure 1C depicts potential small molecule binding sites identified on the structure of inactive BAX (PDB ID: 1F16) using SiteMap (Schrodinger); similar identified sites were also identified from DoGSiteScorer and FTMap, respectively, and mapped onto the ribbon structure of BAX.

[0021] Figure ID depicts a distribution of four computationally predicted binding sites of BCL-XL FL via the application of SiteMap, DoGSiteScorer and FTMap. All four binding sites are represented in mesh format from C-terminal and N-terminal views.

[0022] Figure IE depicts a ratio of15N-BCL-XL AC in the presence or absence of the soluble paramagnetic agent hy-TEMPO (oxlred), plotted as a function of BCL-XL AC residue number. BCL-XL AC residue intensities classified into four categories: < 20%, 20-40%, 40-60% and 60-100%. Data are representative of two independent experiments.

[0023] Figure IF depicts quantitative and graphical characterisation of druggable binding pockets.

[0024] Figure 1G depicts a size variation of allosteric binding sites (Site (2)) of nine trajectory clusters, which were captured at different frames of 1000-ns MD simulation.

[0025] Figure 1H depicts amino acid residues (yellow) which are estimated to be part of the formation of the predicted binding site, i.e. site (2) showed in green, are mutated into Tryptophan and went through SiteMap analysis.

[0026] Figure II depicts a consistency of identification of Site (2) as a favorable druggable pocket. Transition of allosteric binding site (Site (2)) of each trajectory cluster during the course of 1000-ns molecular dynamics simulation of BCL-XL FL.

[0027] Figure 1 J depicts a distribution of amino acid residues within Site (2).

[0028] Figure IK depicts BCL-XL FL binding site predictions via SiteMap, DoGSiteScorer and FTMap.

[0029] Figure IL depicts a progression of Site (2) of BCL-XL from each trajectory cluster, which is a representative frame based on the calculated RMSD matrix at 1000-ns interval. Site (2) is localized on helices al, a6, and the N-terminal of loop al-a2. Site (2) is colored according to the time interval of simulation time.

[0030] Figure IM depicts an evaluation of four top-ranked binding sites in BCL-XL with mutation in the hot spot residues.

[0031] Figure 2A depicts a hierarchical large-scale molecular docking of BCL-XL using a largeP-656906-PClibrary of 14-million compounds, in which the protein structure was equipped with an enclosing grid box (magenta) and a midpoint (green) for a docked ligand confinement in the allosteric binding site.

[0032] Figure 2B depicts gel filtration chromatograms of wt BCL-XL FL (MW = 26.048 kDa) and BCL-XL R6W A168W (MW= 26.194 kDa). Both protein purifications obtain the elution of proteins of interest at the elution volume of 10.4 mL-12.0 mL.

[0033] Figure 2C depicts a validation of FITC-labeled Bid BH3 (above) and Bax BH3 (bottom) peptides as a competitive binder by comparing their EC50 values between the wild-type and mutated BCL-XL FL proteins in FP direct binding assays.

[0034] Figure 2D depicts a screening of the sixty-seven compounds from the large library screening via fluorescence polarization assay which competes against FITC-BID BH3 (above) and FITC-BAX BH3 (bottom) peptides.

[0035] Figure 2E depicts liposome binding of BCL-XL, tBid and Bax.

[0036] Figure 2F depicts a measured chemical shift perturbations (CSP) of 15N-BCL-XL AC upon each compound titration up to a ratio of 1:4 BCL-XL AC:compound, are plotted as a function of BCL-XL AC residue number. Residues with chemical shift ranges over the significance threshold or 1.5 times the threshold are colored orange and red, respectively.

[0037] Figure 2G depicts a single-dose screening of compounds 17, 31 and 57 with their respective analogs via fluorescence polarization assay which competes against FITC-BID BH3 and FITC-BAX BH3 peptides.

[0038] Figure 2H depicts designed scaffolds. (Panel a) The main core with eight R-groups after R-group analysis from the parental compound 17 and its 33 analogs (left); the importance ranking of each R-group position as predicted by R-group analysis. (Panel b) R-groups of compounds 17j and 17w that indicate the most important R-group position, i.e., R6, are colored in yellow, while a common moiety that shared by compounds 17j and 17w are colored in blue. (Panel c) The first designed scaffold for analogs search to increase their potencies and other pharmacokinetic properties.

[0039] Figure 21 depicts IC50 / EC50values of the three most potent compounds 17, 31 and 57, from fluorescence polarization assays, liposome permeabilization assays, and microscale thermophoresis assays (second, third and fourth column to the left, respectively).

[0040] Figure 2J depicts IC50 / EC50 values of compounds 17j, 17w, 57m and 57j, fromP-656906-PCfluorescence polarization (second and third columns to the left), and liposome permeabilization assays (the rightmost column).

[0041] Figure 2K depicts IC50 / EC50values of compounds ABX-2, ABX-27, ABX-28, ABX-36 and ABX-46, as measured from fluorescence polarization assay, liposome release and microscale thermophoresis assays (second, third and fourth column to the left, respectively). The compounds were assayed on BCL-XL FL WT and BCL-XL FL R6W A168W proteins.

[0042] Figure 3A depicts Caspase 3 / 7 activity of HEL wild-type cell line upon treatment with ABX-27, ABX-28, ABX-2, ABX-36, ABX-46, together with their parent compounds 17j, 17w, 57m and 57j for 24 hrs. All data represent mean ± SEM from n = 3 independent experiments.

[0043] Figure 3B depicts a cell viability (Cell-Titer-Glo) of THP-1 wild-type cell line treated with ABX-27, ABX-28, ABX-2, ABX-36, ABX-46 for 72 hrs. All data represent mean ± SEM from n = 3 independent experiments.

[0044] Figure 3C depicts designed scaffolds. (Panel a) The main core with eight R-groups R-groups after R-group analysis from the parental compound 57 and its 28 analogs (left); the importance ranking of each R-group position as predicted by R-group analysis, based on their chemical and property variability. (Panel b) R-group of compounds 57m and 57j at R2 and A3 that indicate the most important R-group positions, are colored in purple. A common moiety that can be found in both compounds 57m and 57j, are colored in blue. A common R-group moiety of R2 and 3 from compounds 57m and 57j is indicated in a box. (Panel c) The second designed scaffold containing the key functionalities that may improve potency for analogs search.

[0045] Figures 4A and Figure 4B depict an HDX-MS analysis of the effect of ABX-27 (17jw27) binding to BCL-XL in solution, in the ratio of (Figure 4A) 1: 1 and (Figure 4B) 1:10.

[0046] Figure 4C and Figure 4D depict an HDX-MS analysis of the effect of ABT-263 binding to BCL-XL in solution, in the ratio of (Figure 4C) 1:1 and (Figure 4D) 1:10.

[0047] Figure 5A depicts chemical shift perturbations (CSPs) of15N-labeled BCL-XL measured in the presence of 1: 1 and 1:2 BCL-XL: ABX-27 compared (BCL-XL: ABX-27) to unbound BCL-XL are plotted as a function of BCL-XL residue number.

[0048] Figure 5B depicts a mapping of residues undergoing significant CSPs onto the ribbon and surface representation of BCL-XL, as in (Figure 5a) in blue gradient colors. SiteMap-predicted Site (1) and Site (2) are mapped as black and white dots, respectively on the protein structure to illustrate the changes in the canonical pocket and allosteric binding site during the NMRP-656906-PCexperiments.

[0049] Figure 5C depicts ABX-27-induced differences in peak intensity ratio (PRE) of peaks in the presence and absence of the soluble paramagnetic probe hydroxyl-TEMPO.

[0050] Figure 5D depicts a ribbon representation of BCL-XL AC with mapping of residues undergoing significant differences in PRE as shown in (Figure 5c). FTMap-predicted Site (2) is mapped as black dots on the protein structure to validate the localization of the novel allosteric binding site.

[0051] Figure 6 depicts a predicted binding pose of 17jw27 (cyan) in the allosteric site consisting of residues R6 and Al 68 (green) via XP Glide docking.

[0052] Figure 7A provides a close up view of the computationally-predicted lowest energy docked pose of ABX-27 (represented in green stick) that is consistent with biophysical and biochemical studies (left), displaying the graphical representations of hydrogen bond (yellow), green (Pi-cation) and van der Waals interactions (orange) with adjacent residues R6, W24, S28, D29 and Al 68 (represented in ball-and-stick) within Site (2).

[0053] Figure 7B depicts changes in structure and dynamics of the helices al and a6 interface. Transparent surface with ribbon representation of helices al and a6 interface with main residues highlighted in orange (left), and graphical representation of distances between residues at the interface (right). Distances for BCL-XL and BCL-XL-ABX-27 complex MD simulations are represented in black and orange respectively.

[0054] Figure 7C depicts changes in structure and dynamics of the canonical site opening formed by a5, a8, and a9. Transparent surface with ribbon representation of the canonical site opening with main residues highlighted in orange (left), and graphical representation of distances between residues at the canonical site opening (right). Distances for BCL-XL and BCL-XL-ABX-27 complex MD simulations are represented in black and orange respectively.

[0055] Figure 7D depicts an overlay of structures of BCL-XL-ABX-27 complex from every 100 ns intervals from 0 to 1000 ns of MD simulations. ABX-27 color spectrum corresponds to time as described, BCL-XL ribbon structure is colored gray with residues of interest represented as sticks for clarity.

[0056] Figure 8A depicts Ca atoms of ABX-27-bound BCL-XL average RMSD plotted against molecular dynamics (MD) time (blue), compared to the Ca atom average RMSD of apo BCL-XL (orange), over three independent 1000 ns simulations, respectively.P-656906-PC

[0057] Figure 8B depicts Ca atoms of ABX-27-bound BCL-XL average RMSF plotted against BCL-XL residues 1-233 (blue), compared to the Ca atom average RMSF of apo BCL-XL (brown), over three independent 1000 ns simulations, respectively.

[0058] Figures 9A-9L depict ABX-27 inhibits the anti-apoptotic activity of BCL-XL in human cell lines. Figure 9A shows chemical structure of ABX-27. Figures 9B-9D show binding and functional potency of ABX-27 assessed using fluorescence polarization (Figure 9B), liposome permeabilization (Figure 9C), and microscale thermophoresis (Figure 9D) assays against full-length BCL-XL wild-type and R6W / A168W allosteric site mutant. IC50 and ECso values reflect allosteric site engagement. Data are mean ± SEM from n = 3 independent experiments.

[0059] Figures 9E-9F depict caspase 3 / 7 activity of (Figure 9E) wild-type THP-1 less and (Figure 9F) THP-1 with BCL-XL knockdown cell line after a titration of ABX-27, A-1331852 and navitoclax for 24 hrs.

[0060] Figures 9G-9H depict cell viability of (Figure 9G) THP-1 wild-type cell line and (Figure 9H) THP-1 with BCL-XL knockdown cell line treated with atitration of ABX-27, A-1331852 and navitoclax for 72 hrs.

[0061] Figures 9I-9J depict effects of ABX-27, A-1331852 and navitoclax on (Figure 91) caspase-3 / 7 activity (24 hrs) and (Figure 9J) viability activity (72 hrs) in Kasumi cells.

[0062] Figures 9K-9L depict effects of ABX-27, A-1331852 and navitoclax on (Figure 9K) caspase-3 / 7 activity and (Figure 9L) viability activity in platelets from a healthy human subject after 24 h treatment. All data represent mean ± SEM from n = 3 independent experiments.

[0063] Figures 10A-10E depict selectivity and cellular activity profile of ABX-27 compared to BH3 mimetics A-1331852 and navitoclax. Figure 10A depicts competitive fluorescence polarization assays assessing displacement of FITC-labeled BID BH3 and BAX BH3 peptides from recombinant BCL-2 AC by ABX-27, A-1331852, and navitoclax. ABX-27 exhibited markedly reduced affinity for BCL-2 AC, supporting its selectivity for BCL-XL.

[0064] Figure 10B depicts competitive binding of navitoclax and A-1331852 to full-length BCL-XL using the same FITC-labelled BH3 peptides.

[0065] Figure 10C depicts Western blot analysis of cytochrome c release in cytosol fractions of treated Kasumi and THP-1 cells upon treatment with ABX-27 and A-1331852 at the indicated concentrations, followed by digitonin fractionation.

[0066] Figure 10D depicts PARP and caspase-3 cleavage in wild-type Kasumi cells treated withP-656906-PCDMSO, ABX-27 and A-1331852 for 24 hrs. Whole cell lysates were analyzed by western blot with anti -cleaved PARP and anti -cleaved caspase-3.

[0067] Figure 10E depicts effects of ABX-27, A-1331852 and navitoclax on caspase-3 / 7 activity in platelets from healthy human after 24 h treatment. Data in Figures 10A-10B represent mean ± SEM from n = 3 independent experiments, and data in Figures 10C-10D are representative from n = 3 independent experiments.

[0068] Figure 11 depicts in silica ADME and pharmacokinetic profiling of ABX-27.DETAILED DESCRIPTION OF THE INVENTION

[0069] The clinical development of BCL-XL inhibitors has been severely hindered by their low therapeutic index, primarily due to dose-limiting on-target platelet toxicity. To date, BCL-XL inhibitors have relied on targeting the canonical BH3-binding pocket, which requires large molecules to achieve high-affinity binding. This pocket shares high structural similarity with other anti-apoptotic Bcl-2 family members, making selective BCL-XL inhibition particularly challenging and necessitating extensive medicinal chemistry efforts.

[0070] The present invention provides a novel class of small-molecule inhibitors that target BCL-XL through an allosteric binding site, other than the canonical BH3 -binding pocket used by existing BCL-XL inhibitors. Binding to a distinct allosteric site enables more selective and tunable inhibition of BCL-XL. Further, the allosteric inhibitors such as the BCL-XL inhibitors of the invention are significantly smaller than existing BCL-XL inhibitors, allowing for improved druglike properties, solubility, and bioavailability.

[0071] In one aspect, the present invention provides a BCL-XL inhibitor, reprsented by a compound of Formula (I),or a pharmaceutically acceptable salt thereof or a stereoisomer thereof,whereinX is a bond or CFE;P-656906-PCA is H, alkyl, cycloalkyl, or aryl;B is heterocycloalkyl, wherein the heterocycloalkyl with the pyrazole ring to which it is attached may form a fused heterocyclic ring system;G is alkyl, alkenyl, alkynlyl, C(=O)RP, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; n is 0, 1, 2, or 3;wherein the aryl and heteroaryl are each optionally substituted with one or more groups independently selected from C1-C5 alkyl, halo, haloalkyl, aryl, CN, ORP, NRpRq, RPOH, CHO, COORP, and CONRpRq;wherein the alkyl, cycloalkyl, heterocycloalkyl, and the fused heterocyclic ring are each optionally substituted with one or more groups independently selected from =0, halo, haloalkyl, CN, RqORPORP, NRpRq, NRpCORq, RP0H, CHO, cyclopropyl, heteroaryl, COORp, and CONRpRq; andRpand Rq are independently H or C1-C5 alkyl;wherein Rpand Rqcan optionally be taken together to form a 3-6 membered ring that can be optionally substituted by one or more groups independently selected independently from OH, =0, or C1-C5 alkoxy.

[0072] In some embodiments of the compound of the invention, A is aryl. In other embodiments, A is phenyl or methylphenyl.

[0073] In some embodiments, B is heterocycloalkyl. In other embodiments, B is a 5- or 6-membered heterocycloalkyl. In certain embodiments, B is a piperidinyl.

[0074] In some embodiments, n is 0 or 1.

[0075] In some embodiments, Gis heteroaryl.

[0076] In some embodiments, G is pyrrolyl or indazolyl.

[0077] In some embodiments, the compound of Formula (I) is represented by Formula (II)in which Ai, A2, A3, A4, and As are each independently H, C1-C5 alkyl, halo, CN, ORP, NRpRq,P-656906-PCCOORp, or CONRp.

[0078] In some embodiments of the compound of Formula (II), B is heterocycloalkyl. In some embodiments, B is a 5- or 6-membered heterocycloalkyl. In some embodiments, B is a piperidinyl. In some embodiments, n is 0.

[0079] In some embodiments, the compound of Formula (I) is represented by Formula (III)wherein Ai, A2, A3, A4, and As are each independently H, C1-C5 alkyl, halo, haloalkyl, CN, ORP, NRPRq, COORp, or CONRpRq.

[0080] In some embodiments of the compound of Formula (II) or Formula (III), Ai, A2, A3, A4, and As are each independently H or C1-C5 alkyl. In some embodiments, Ai, A2, A3, A4, and As are H. In some embodiments, A3 is C1-C5 alkyl and Ai, A2, A4, and As are H. In some embodiments, G is heteroaryl. In some embodiments, G is pyrrolyl or indazolyl.

[0081] In another aspect, the present invention provides a compound represented by Formula (IV)or a pharmaceutically acceptable salt thereof or a stereoisomer thereof,whereinRn, R12, R13, R14, and R15 are each independently H, alkyl, halo, haloalkyl, CN, ORP, NRpRq, COORp, or CONRpRq;K is COORp or CONRpRq;P-656906-PCRm and Rnare each independently H, alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, wherein each of alkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl is optionally substituted with one or more groups independently selected from halo, ORP, C1-C5 alkyl, haloalkyl, CN, NRpRq, RpOH, CHO, cyclopropyl, COORP, and CONRpRq;Rmand Rncan optionally be taken together with the nitrogen atom to which they are attached to form a 3-15 membered ring that is optionally substituted by one or more groups independently selected independently from =0, C1-C5 alkyl, CN, ORP, NRpRq, RpOH, CHO, cycloalkyl, heterocyclalkyl, aryl, arylalkyl, heteroaryl, and heteroarylalkyl; andRPand R are independently H or C1-C5 alkyl.

[0082] In some embodiments of the compound of Formula (IV), R11, R12, R13, R14, and R15 are each H, C1-C5 alkyl, or halo. In some embodiments, R13 is halo and Rn, R12, R14, and R15 are H. In other embodiments, R13 is chloro and Rn, R12, R14, and R15 are H. In some embodiments, Rn, R12, Rn, R14, and R15 are H or C1-C5 alkyl. In some embodiments, Rn, R12, R13, R14, and R15 are H or methyl.

[0083] In some embodiments, Reis COORp. In some embodiments, Re is COOCH3 or COOC2H5.

[0084] In some embodiments, Rmand Rn are H or aryl, wherein aryl is optionally substituted with halo, haloalkyl, CN, ORP, COORP, orCONRp.

[0085] In some embodiments, Rm and Rnare H or aryl, wherein aryl is substituted with haloalkyl.

[0086] In some embodiments, Rmand Rnare taken together with the nitrogen atom to which they are attached to form a 5- or 6-membered ring that is optionally substituted by =0, C1-C5 alkyl, cycloalkyl, heterocyclalkyl, aryl, or heteroaryl.

[0087] In some embodiments, Rmand Rnare taken together with the nitrogen atom to which they are attached to form a 6-membered ring that is optionally substituted by heteroaryl.P-656906-PC

[0088] In another aspect, the present invention provides a BCL-XL inhibitor represented by a compound of Formula (V)or a pharmaceutically acceptable salt thereof or a stereoisomer thereof,whereinRa and Rb are each independently H, alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, wherein each of alkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl is optionally substituted with one or more groups independently selected from halo, haloalkyl, CN, ORP, NRpRq, RPOH, CHO, cyclopropyl, COORp, and CONRpRq;Ra and Rb can optionally be taken together with the nitrogen atom to which they are attached to form a 3-15 membered ring that is optionally substituted by one or more groups independently selected independently from O, C1-C5 alkyl, CN, ORP, NRPRq, RP0H, CHO, cycloalkyl, heterocyclalkyl, aryl, arylalkyl, heteroaryl, and heteroarylalkyl; andRpand Re, are independently H or C1-C5 alkyl.

[0089] In some embodiments of the compound of Formula (V), Ra and Rb are H, alkyl, cycloalkyl, or heterocycloalkyl, wherein alkyl, cycloalkyl, and heterocycloalkyl are each optionally substituted with halo, haloalkyl, CN, or ORP. In some embodiments, Raand Rb are taken together with the nitrogen atom they are attached to form a 3- 15 membered ring that is optionally substituted by =0 or ORP.

[0090] In a further aspect, the present invention provides a BCL-XL inhibitor represented by any compounds as described herein, e.g., any one of the compounds in Tables 1-3.P-656906-PC

[0091] In a further aspect, the present invention provides a BCL-XL inhibitor represented by any one of the following compounds:

[0092] In other embodiments, the present invention provides a BCL-XL inhibitor represented by any one of the following compounds:P-656906-PCP-656906-PCP-656906-PC

[0093] In other embodiments, the present invention provides a BCL-XL inhibitor represented by any one of the following compounds:P-656906-PCP-656906-PC

[0094] In other embodiments, the present invention provides a BCL-XL inhibitor represented by any one of the following compounds:P-656906-PCP-656906-PC

[0095] In other embodiments, the present invention provides a BCL-XL inhibitor represented by any one of the following compounds:P-656906-PCP-656906-PCP-656906-PCP-656906-PC

[0096] In other embodiments, the present invention provides a BCL-XL inhibitor represented by any one of the following compounds:P-656906-PCP-656906-PCP-656906-PCP-656906-PC

[0097] In some embodiments, one or more of the carbon (C) atoms and nitrogen (N) atoms in the embodiments of the compound of the invention as described herein can be13C isotopes, and N atoms can be15N isotopes, wherein it is possible to have compounds with13C isotopes and15N isotopes. Furthermore, changing the isotopic composition of H / D, C and N can be applied in the assessment of drug pharmacology to determine the pharmacokinetic profile or mode of action of a drug substance. Additionally, stable isotopes may be used as internal standards for the assessment of drug concentration or its metabolites in blood or urine by analytical quantification LC-MS, HPLC-MS, LC-MS-MS or HPLC-MS- MS methods.

[0098] In some embodiments, the compound of the invention as described herein, e.g., any one of formulas (I)-(V), any compounds in Tables 1-3, can be prepared by the methods known in the art.

[0099] In some embodiments, the compound of the invention can be in the form of a pharmaceutically acceptable salt.

[0100] In some embodiments, the phrase “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0101] As used herein, the “pharmaceutically acceptable salt” or “pharmaceutically acceptable salts” refers to derivatives of the disclosed compounds wherein the parent compounds are modified by converting an existing acid or base moiety to its salt form. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like. The pharmaceutically acceptable salts of the compound of the invention as described herein include the conventional non-toxic salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. The pharmaceutically acceptable salts of the compound of the invention can be synthesized from the parent compound which contains a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two, for example, nonaqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile. The chemical modification of a pharmaceutical compound (e.g., a compound of the invention) into a salt is a technique well knownP-656906-PCto pharmaceutical chemists to obtain improved physical and chemical stability, hygroscopicity, flowability and solubility of compounds (e.g. Bastin, etal. Organic Proc. Res. Dev. 4 (2000) 427-435).

[0102] In some embodiments, the pharmaceutically acceptable salt of the compound of the invention is formed from suitable non-toxic organic or inorganic acids, including, but not limited to, acetic acid, fumaric acid, benzenesulfonic acid, tartaric acid, citric acid, gluconic acid, hydroiodic acid, p-toluenesulfonic acid, methanesulfonic acid, phosphoric acid, sulfuric acid, propionic acid, nitric acid, or maleic acid. In other embodiments, suitable acids for preparation of the pharmaceutically acceptable salts include, but are not limited to, inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid, or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid.

[0103] In some embodiments, the pharmaceutically acceptable salt of a compound of the invention can include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hernisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3 -phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and the like.

[0104] In some embodiments, the compound of the invention as described herein can be prepared in the form of its derivatives. In some embodiments, the term “derivatives” includes, but is not limited to, ether derivatives, acid derivatives, amide derivatives, ester derivatives and the like.

[0105] In some embodiments, the compound of the invention as described herein can be prepared in the form of its pharmaceutical products. The term “pharmaceutical product” means a composition suitable for pharmaceutical use (pharmaceutical composition), as defined herein.

[0106] As used herein, “pharmaceutical composition” refers to a therapeutically effective amount of a compound of the invention, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, together with suitable diluents, preservatives, solubilizers, emulsifiers, adjuvant and / or carriers.P-656906-PC

[0107] Accordingly, in another aspect, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of a compound of the invention, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, together with suitable diluents, preservatives, solubilizers, emulsifiers, adjuvant and / or carriers.

[0108] In some embodiments, the compound of the invention as described herein can be prepared in the form of its prodrugs. The term “prodrug” means a substance which can be converted in vivo into a biologically active agent by such reactions as hydrolysis, esterification, de-esterification, activation, salt formation and the like.

[0109] In each of the foregoing and each of the following embodiments, it is to be understood that a compound of the invention also includes any and all hydrates and / or solvates of the compound formulas. It is appreciated that certain functional groups, such as the hydroxy, amino, and like groups form complexes and / or coordination compounds with water and / or various solvents, in the various physical forms of the compounds. Accordingly, the above formulas are to be understood to include and represent those various hydrates and / or solvates.

[0110] As used herein, the term “solvate” refers to compounds that further include a stoichiometric or non-stoichiometric amount of a solvent bound by non-covalent intermolecular forces. If the solvent is water, the solvate is referred to as “hydrate.” Pharmaceutically acceptable solvates and hydrates are complexes that, for example, may include from 1 to about 100, or from 1 to about 10, or from one to about 2.3 or 4 molecules of water or a solvent. It should be understood that the term “compound” in this application covers the compound and solvates of the compound, as well as mixtures thereof.

[0111] In some embodiments, the term “hydrate” includes, but is not limited to, hemihydrate, monohydrate, dihydrate, trihydrate and the like.

[0112] The present invention further provides a method for identifying BCL-XL inhibitors, which includes computational screening and allosteric targeting strategies. The method of the invention can further be used in developing inhibitors against other challenging protein targets.

[0113] In another aspect, the present invention provides a method for identifying BCL-XL inhibitors of the invention, comprising the steps of (i) determining an allosteric site on full length BCL-XL, (ii) selecting compounds that bind to the allosteric site, and (iii) evaluating the selected compounds by experimental screening and validation.

[0114] In some embodiments, determining an allosteric site on full length BCL-XL comprises theP-656906-PCsteps of:(a) calculating a 3D structural model of the human full-length BCL-XL using a truncated crystal structure of BCL-XL and a full length BAX structure as a template for the conformation of the c-terminal helix 9 of BCL-XL;(b) performing molecular dynamics (MD) simulations on the BCL-XL structural model to explore conformational dynamics and using conformations to feed automated binding site identification and analysis tools;(c) analyzing predicted binding sites for druggability based on site properties;(d) validating the predicted binding sites with NMR paramagnetic relaxation enhancement (PRE) experiments using hy-TEMPO probe to identify solvent accessible sites on the surface of BCL-XL accessed by TEMPO probe and confirming the predicted binding sites;(e) selecting a binding Site 2 that is adjacent to the N-terminal intrinsically disordered region (al-a2 loop) of BCL-XL and investigating changes of the binding site in the MD simulation trajectory;(f) maintaining the binding site with minor changes over the course of the simulation despite significant changes in al-a2 loop conformation adjacent to the Site 2; and(g) using mutagenesis of the binding site residues and identifying residues R6 and A168 in helices al and a6 as the hot spot residues largely determining the identification of the Site 2 by Sitemap.

[0115] In some embodiments, selecting compounds that bind to the allosteric site comprises:(h) identifying compounds binding to Site 2 by performing in silico screening using a compound library via a three-tier docking methodology which starts with high throughput virtual screening (HTVS) followed by standard precision (SP) and then by extra-precision mode (XP) screening mode; and(j) selecting compounds for favorable ADMET properties.

[0116] In some embodiments, evaluating the selected compounds by experimental screening and validation comprises:(i) employing various biochemical, biophysical and cell-based assays for BCL-XL binding, BCL-XL inhibition activity and binding to the allosteric site as well as apoptosis related assays to establish production of full length BCL-XL wt and mutant R6W / A168W to interrogate binding to and allosteric effects mediated by Site 2;P-656906-PC(ii) screening the selected compounds using a Fluorescence Polarization (FP) assay for competing BCL-XL-BID and BCL-XL-BAX binding and prioritizing hits with > 70% inhibition for either complex;(iii) establishing a functional assay for full length BCL-XL activity in promoting inhibition of B AX-mediated membrane permeabilization using liposomal membranes;(iv) using FP, liposomal assay, MST direct binding to BCL-XL and HSCQ-NMR analysis using full length BCL-XL wt and R6W / A168W mutant to identify and prioritize lead compounds that show the best binding to BCL-XL and reduce binding / activity to the mutant;(v) preparing and designing new analogs of the lead compounds and screening for binding and activity using the same assays;(vi) screening the new analogs and identifying more potent inhibitors that are also inhibited by R6W / A168W mutant but not by a mutant in the canonical site that typically disrupts established BCL-XL inhibitors G138W to obtain compounds that have potent cellular activity and selectivity for binding to BCL-XL compared to BCL-2;(vii) performing additional experiments with the obtained potent compounds using structural and biophysical HSQC-NMR, NMR-PRE, and HDX-MS analysis that demonstrate specificity for binding to Site 2 and allosteric effects at the C-terminal canonical site of BCL-XL to explain the mechanism of site 2 binding molecules to compete BH3 domains from the BCL-XL canonical site; and(viii) recapitulating the allosteric effects from compound-binding to Site 2 in molecular dynamics simulations that show allosteric conformational changes in the canonical pocket in the the presence of the potent compound by increased interatomic distances that include the C-terminal helix 9.

[0117] The present invention provides an integrated computational and experimental approach to identify, validate and target cryptic pockets of BCL-XL and other conformationally dynamic proteins including other BCL-2 family proteins. Unlike existing BCL-XL inhibitors that bind to the canonical BH3-binding pocket, the present invention identifies and targets a novel allosteric site on full-length BCL-XL. Binding to a distinct allosteric site allows more selective and tunable inhibition of BCL-XL.

[0118] A BCL-XL inhibitor has the potential to address multiple cancer types where BCL-XL is frequently upregulated, contributing to resistance to apoptosis and treatment failure. Based onP-656906-PCprevalence data, previous clinical trials and preclinical data, a number of patients could benefit from a selective BCL-XL inhibitor for several cancer types including acute myeloid leukemia, chronic lymphocytic leukemia, acute lymphoblastic leukemia, lymphoma, multiple myeloma, non-small cell lung cancer, small cell lung cancer, triple-negative breast cancer, colorectal cancer, head and neck squamous cell carcinoma, melanoma, mesothelioma, glioblastoma, and pancreatic neuroedocrine tumors.

[0119] In some embodiments, the compounds of the invention as described herein can be used for the treatment of cancer. Examples of cancer include, but are not limited to, acute myeloid leukemia, chronic lymphocytic leukemia, acute lymphoblastic leukemia, lymphoma, multiple myeloma, non-small cell lung cancer, small cell lung cancer, triple-negative breast cancer, colorectal cancer, head and neck squamous cell carcinoma, melanoma, mesothelioma, glioblastoma, and pancreatic neuroedocrine tumors.

[0120] In a further aspect, the present invention provides a method for treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a BCL-XL inhibitor compound of the invention as described herein, or a pharmaceutically acceptable salt thereof or a stereoisomer thereof.

[0121] In some embodiments of the method of the invention, the cancer is acute myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma, non-small cell lung cancer, small cell lung cancer, triple-negative breast cancer, colorectal cancer, or head and neck squamous cell carcinoma. In some embodiments, the compound of the invention is any one of the BCL-XL inhibitors of formulas (I)-(V). In some embodiments, the compound of the invention is any one of the BCL-XL inhibitors in Tables 1-3. In other embodiments, the compound is a BCL-XL inhibitor represented by any one of the following compounds:P-656906-PC

[0122] It is another aspect of the invention that the BCL-XL inhibitors of the invention as described herein can potentially overcome platelet toxicity with improved therapeutic index. The BCL-XL inhibitors of the invention avoid binding to the BH3 pocket. Platelet toxicity is associated with the disruption of BCL-XL-BAK interaction in platelets. The allosteric targeting approach of the BCL-XL inhibitors described herein has used the disruption of BCL-XL-BID and BCL-XL-BAX interactions in inhibitor optimization, to avoid disruption of BCL-XL-BAX interaction and preserve platelets viability.

[0123] The selective, small-molecule BCL-XL inhibitors of the present invention are designed to induce apoptosis in BCL-XL-dependent tumors while minimizing platelet toxicity. These inhibitors can be used as standalone cancer therapeutics or in combination with existing chemotherapy or targeted therapies.

[0124] In a further aspect, the present invention provides a combination therapy that includes a compound of the invention, or a pharmaceutically acceptable salt thereof or a stereoisomer thereof, and an existing chemotherapy or targeted therapy. For example, BCL-XL inhibitors have been also tested in the clinic for myelofibrosis (Ph3) and diabetic macular edema based (Ph2) on their senolytic activity.

[0125] In some embodiments, a combination of a BCL-XL inhibitor of the invention, or a pharmaceutically acceptable salt thereof or stereoisomer thereof, and an existing agent for cancer treatment. The combination of the invention is effective for treating or preventing cancer in aP-656906-PCsubject in need thereof. The combination of the invention may exhibit a therapeutic synergistic or complementary effect and allow for an improved safety profde of the existing agent in the combination and may minimize toxicity during treatments and result in a decrease in the onset of resistance development.

[0126] As used herein, the term “a therapeutically effective amount” of a compound means an amount of compound that is effective to prevent, alleviate or ameliorate symptoms of disease or prolong the survival of the subject being treated. Determination of a therapeutically effective amount is within the skill in the art. The therapeutically effective amount or dosage of a compound according to this invention can vary within wide limits and may be determined in a manner known in the art. Such dosage will be adjusted to the individual requirements in each particular case including the specific compound(s) being administered, the route of administration, the condition being treated, as well as the patient being treated. In cases of administration of a pharmaceutically acceptable salt, dosages may be calculated as the free form. In general, in the case of oral or parenteral administration to adult humans weighing approximately 70 kg, a daily dosage of about 5 mg to about 500 mg, or from about 10 mg to about 200 mg, can be considered as a therapeutically effective amount, although the upper limit may be exceeded when indicated. The daily dosage can be administered as a single dose or in divided doses, or for parenteral administration, it may be given as continuous infusion.

[0127] Further, a skilled artisan would appreciate that the term “therapeutically effective amount” may encompass the total amount of each active component of the combination therapy or method that is sufficient to show a meaningful patient benefit, i.e., treatment, healing, prevention or amelioration of the relevant medical condition, or an increase in rate of treatment, healing, prevention or amelioration of such conditions. When applied to an individual active ingredient, administered alone, the term refers to that ingredient alone. When applied to a combination, the term refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, serially or simultaneously.

[0128] In some embodiments of the method of the invention, the BCL-XL inhibitor compound of the invention may be administered by any appropriate route. In some embodiments, the BCL-XL inhibitor compound of the invention can be administered by an oral, rectal, nasal, topical (including buccal and sublingual), vaginal, or parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intrathecal, and epidural) route. In other embodiments, the BCL-XL inhibitorP-656906-PCcompound of the invention is administered by an intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion, as well as in vivo electroporation. In some embodiments, the BCL-XL inhibitor compound of the invention is administered orally or a topical, epidermal or mucosal route of administration, for example, intranasally, vaginally, rectally, sublingually or topically.

[0129] In preparing the pharmaceutical compositions in oral dosage form, any of the usual pharmaceutical media may be employed. Thus, for liquid oral preparations, such as, suspensions, elixirs, and solutions, suitable carriers and additives include water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents, and the like. For solid oral preparations such as, chewable, soft-chew, gummy, powders, capsules, and tablets, suitable carriers and additives include starches, sugars, diluents, granulating agents, lubricants, binders, disintegrating agents, and the like. Due to their ease in administration, tablets and capsules represent the most advantageous oral dosage unit form. If desired, tablets may be sugar coated or enteric coated by standard techniques.

[0130] Formulations suitable for parenteral administration may comprise a sterile aqueous preparation of the active compound, which in some embodiments is isotonic with the blood of the recipient (e.g., physiological saline solution). Such formulations may include suspending agents and thickening agents and liposomes or other microparticulate systems which are designed to target the compound to blood components or one or more organs. The formulations may be presented in unit-dose or multi-dose form. Parenteral administration may comprise any suitable form of systemic delivery. Administration may for example be intravenous, intra-arterial, intrathecal, intramuscular, subcutaneous, intramuscular, intra-abdominal (e.g., intraperitoneal), etc., and may be affected by infusion pumps (external or implantable) or any other suitable means appropriate to the desired administration modality.

[0131] In addition to the aforementioned ingredients, the compositions may further include one or more ingredients selected from diluents, buffers, flavoring agents, binders, disintegrants, surface active agents, thickeners, lubricants, preservatives (including antioxidants), and the like. The formulations may be of immediate release, sustained release, delayed-onset release or any other release profile known to one skilled in the art.

[0132] For administration to mammals, it is expected that the physician will determine the actualP-656906-PCdosage and duration of treatment, which will be most suitable for an individual and can vary with the age, weight, genetics and / or response of the particular individual.

[0133] The exact dose and regimen of administration of the composition will necessarily be dependent upon the therapeutic or nutritional effect to be achieved and may vary with the particular formula, the route of administration, and the age and condition of the individual subject to whom the composition is to be administered.

[0134] In some embodiments, the amount of the BCL-XL inhibitor compound of the invention administered in the method of the invention can be an amount selected from about 0.1 mg to 50 mg; from about 0.1 mg to about 40 mg; from about 0.1 mg to about 30 mg; from about 0.1 mg to about 20 m; from about 0.1 mg to about 10 mg; from about 0.25 mg to about 10 mg; from about 0.25 mg to about 8 mg; from about 0.5 mg to about 8 mg; from about 0.5 mg to about 7 mg; and from about 1 mg to about 7 mg. In other embodiments, the amount of the compound of the invention administered can be 0.1 mg, 0.25 mg, 0.3 mg, 0.5 mg, 0.75 mg, 1 mg, 1.5 mg, 2 mg, 2.5 mg, 3 mg, 3.5 mg, 4 mg, 4.5 mg, 5 mg, 5.5 mg, 6 mg, 6.5 mg, 7 mg, 7.5 mg, 8 mg, 8.5 mg, 9 mg, 9.5 mg, 10 mg, or 18 mg.

[0135] Throughout this application, various embodiments of the present disclosure may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0136] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and / or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting. Each literature reference or other citation referred to herein isP-656906-PCincorporated herein by reference in its entirety.

[0137] In the description presented herein, each of the steps of the invention and variations thereof are described. This description is not intended to be limiting and changes in the components, sequence of steps, and other variations would be understood to be within the scope of the present invention.

[0138] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

[0139] At various places in the present specification, substituents of compounds of the invention are disclosed in groups or in ranges. It is specifically intended that the invention includes each and every individual subcombination of the members of such groups and ranges. For example, the term “C1-5 alkyl” is specifically intended to individually disclose methyl, ethyl, C3 alkyl, C4 alkyl, and C5 alkyl.

[0140] In some embodiments, the term “alkyl” is meant to refer to a saturated hydrocarbon group which is straight-chained or branched. Example alkyl groups include methyl (Me), ethyl (Et), propyl (e.g., n-propyl and isopropyl), butyl (e.g., n-butyl, isobutyl, t-butyl), pentyl (e g., n-pentyl, isopentyl, neopentyl), and the like. An alkyl group can contain from 1 to about 20, from 2 to about 20, from 1 to about 10, from 1 to about 8, from 1 to about 6, from 1 to about 4, or from 1 to about 3 carbon atoms.

[0141] In some embodiments, “cycloalkyl” refers to non-aromatic carbocycles including cyclized alkyl, alkenyl, and alkynyl groups. Cycloalkyl groups can include mono- or polycyclic (e.g., having 2, 3 or 4 fused rings) ring systems, including spirocycles. In some embodiments, cycloalkyl groups can have from 3 to about 20 carbon atoms, 3 to about 14 carbon atoms, 3 to about 10 carbon atoms, 3 to 8 carbon atoms, or 3 to 7 carbon atoms. Cycloalkyl groups can further have 0, 1, 2, or 3 double bonds and / or 0, 1, or 2 triple bonds. Also included in the definition of cycloalkyl are moieties that have one or more aromatic rings fused (i.e., having a bond in common with) to the cycloalkyl ring, for example, benzo derivatives of cyclopentane, cyclopentene, cyclohexane, andP-656906-PCthe like. A cycloalkyl group having one or more fused aromatic rings can be attached through the aromatic or non-aromatic portion. One or more ring-forming carbon atoms of a cycloalkyl group can be oxidized, for example, having an oxo or sulfido substituent. Example cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclopentenyl, cyclohexenyl, cyclohexadienyl, cycloheptatrienyl, norbornyl, norpinyl, norcarnyl, adamantyl, and the like.

[0142] In some embodiments, “heterocycloalkyl” refers to a non-aromatic heterocycle where one or more of the ring-forming atoms are a heteroatom such as an O, N, or S atom. Heterocycloalkyl groups can include mono- or polycyclic (e.g., having 2, 3 or 4 fused rings) ring systems as well as spirocycles. Example heterocycloalkyl groups include morpholino, thiomorpholino, piperazinyl, tetrahydrofuranyl, tetrahydrothienyl, 2,3 -dihydrobenzofuryl, 1,3 -benzodi oxole, benzo-1,4-dioxane, piperidinyl, pyrrolidinyl, isoxazolidinyl, isothiazolidinyl, pyrazolidinyl, oxazolidinyl, thiazolidinyl, imidazolidinyl, and the like. Also included in the definition of heterocycloalkyl are moieties that have one or more aromatic rings fused (i.e., having a bond in common with) to the nonaromatic heterocyclic ring, for example phthalimidyl, naphthalimidyl, and benzo derivatives of heterocycles. A heterocycloalkyl group having one or more fused aromatic rings can be attached though either the aromatic or non-aromatic portion. Also included in the definition of heterocycloalkyl are moieties where one or more ring-forming atoms are substituted by 1 or 2 oxo or sulfido groups. In some embodiments, the heterocycloalkyl group has from 1 to about 20 carbon atoms, and in further embodiments from about 3 to about 20 carbon atoms. In some embodiments, the heterocycloalkyl group contains 3 to about 20, 3 to about 14, 3 to about 10, 3 to about 7, or 5 to 6 ring-forming atoms. In some embodiments, the heterocycloalkyl group has 1 to about 4, 1 to about 3, or 1 to 2 heteroatoms. In some embodiments, the heterocycloalkyl group contains 0 to 3 double bonds. In some embodiments, the heterocycloalkyl group contains 0 to 2 triple bonds.

[0143] In some embodiments, “aryl” refers to monocyclic or polycyclic (e.g., having 2, 3 or 4 fused rings) aromatic hydrocarbons such as, for example, phenyl, naphthyl, anthracenyl, phenanthrenyl, and the like. In some embodiments, an aryl group has from 6 to about 20 carbon atoms. In some embodiments, “aryl” may be optionally substituted at any one or more positions.

[0144] In some embodiments, “heteroaryl” refers to an aromatic heterocycle having at least one heteroatom ring member such as sulfur, oxygen, or nitrogen. Heteroaryl groups include monocyclic and polycyclic (e.g., having 2, 3 or 4 fused rings) systems. Any ring-forming N atomP-656906-PCin a heteroaryl group can also be oxidized to form an N-oxo moiety. Examples of heteroaryl groups include without limitation, pyridyl, N-oxopyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, triazinyl, furyl, quinolyl, isoquinolyl, thienyl, imidazolyl, thiazolyl, indolyl, pyrryl, oxazolyl, benzofuryl, benzothienyl, benzthiazolyl, isoxazolyl, pyrazolyl, triazolyl, tetrazolyl, indazolyl, 1,2,4-thiadiazolyl, isothiazolyl, benzothienyl, purinyl, carbazolyl, benzimidazolyl, indolinyl, and the like. In some embodiments, the heteroaryl group has from 1 to about 20 carbon atoms, and in further embodiments from about 3 to about 20 carbon atoms. In some embodiments, the heteroaryl group contains 3 to about 14, 3 to about 7, or 5 to 6 ring-forming atoms. In some embodiments, the heteroaryl group has 1 to about 4, 1 to about 3, or 1 to 2 heteroatoms. In some embodiments, “heteroaryl” may be optionally substituted at any one or more positions capable of bearing a hydrogen atom.

[0145] In some embodiments, an “arylalkyl” group refers to an alkyl bound to an aryl, wherein alkyl and aryl are as defined above. An example of an arylalkyl group is a benzyl group.

[0146] In some embodiments, a “heteroarylalkyl” group refers to an alkyl bound to a heteroaryl, wherein alkyl and heteroaryl are as defined above. An example of a heteroarylalkyl group is a pyridylalkyl.

[0147] In some embodiments, “halo” or “halogen” includes fluoro, chloro, bromo, and iodo. A “halogen-substitution” or “halo” substitution designates replacement of one or more hydrogen atoms with F, CI, Br or I.

[0148] In some embodiments, “haloalkyl” refers to an alkyl group having one or more halogen substituents. Example haloalkyl groups include CF3, C2F5, CHF2, CCI3, CHCI2, C2CI5, and the like.

[0149] As used herein, “alkoxy” refers to an -O-alkyl group. Example alkoxy groups include methoxy, ethoxy, propoxy (e.g., n-propoxy and isopropoxy), t-butoxy, and the like.

[0150] It is understood that each of alkyl, cycloalkyl, heterocycloalkyl, alkenyl, cycloalkenyl, alkylene, aryl, and heteroaryl may be optionally substituted with independently selected groups such as alkyl, haloalkyl, hydroxyalkyl, aminoalkyl, carboxylic acid and derivatives thereof, including esters, amides, and nitrites, hydroxy, alkoxy, acyloxy, amino, alky and dialkylamino, acylamino, thio, and the like, and combinations thereof.

[0151] In some embodiments, the term “substituted” refers to the replacement of a hydrogen moiety with a non-hydrogen moiety in a molecule or group. It can refer to “mono-substituted” orP-656906-PC“poly-substituted.” The term “mono-substituted” or “poly-substituted” means substituted with one or more than one substituent up to the valence of the substituted group. For example, a monosubstituted group can be substituted with 1 substituent, and a poly-substituted group can be substituted with 2, 3, 4, or 5 substituents. When a list of possible substituents is provided, the substituents can be independently selected from that group.

[0152] The term “optionally substituted,” or “optional substituents,” as used herein, means that the groups in question are either unsubstituted or substituted with one or more of the substituents specified. When the groups in question are substituted with more than one substituent, the substituents may be the same or different. Such other functional groups illustratively include, but are not limited to, amino, hydroxyl, CN, halo, thiol, alkyl, haloalkyl, heteroalkyl, aryl, arylalkyl, arylheteroalkyl, heteroaryl, heteroarylalkyl, heteroarylheteroalkyl, nitro, sulfonic acids and derivatives thereof, carboxylic acids and derivatives thereof, and the like. Illustratively, any of amino, hydroxyl, CH, thiol, alkyl, haloalkyl, heteroalkyl, aryl, arylalkyl, arylheteroalkyl, heteroaryl, heteroarylalkyl, heteroarylheteroalkyl, and / or sulfonic acid is optionally substituted. In some embodiments, the functional groups are the substituents described herein for any one of variables. Further, when using the terms “independently,” “independently are,” and “independently selected from” mean that the groups in question may be the same or different. Certain of the herein defined terms may occur more than once in the structure, and upon such occurrence each term shall be defined independently of the other.

[0153] As used herein, the terms "treating" or "treatment" includes preventative as well as disorder remitative treatment. The terms "reducing", "suppressing" and "inhibiting" have their commonly understood meaning of lessening or decreasing, or delaying, or reducing, the incidence, severity or pathogenesis of a disease, disorder or condition. In one embodiment, the term treatment refers to delayed progression of, prolonged remission of, reduced incidence of, or amelioration of symptoms associated with the disease, disorder or condition. In one embodiment, the terms "treating" "reducing", "suppressing" or "inhibiting" refer to a reduction in morbidity, mortality, or a combination thereof, in association with the indicated disease, disorder or condition. In one embodiment, the methods of treatment disclosed herein reduce the severity of the disease, or in another embodiment, symptoms associated with the disease, or in another embodiment, reduces the number of biomarkers expressed during disease.

[0154] In one embodiment, the term "administering" refers to bringing a subject in contact with aP-656906-PCcompound of the present disclosure. Administration can be accomplished in vitro, e.g. in a test tube, or in vivo, e.g. in cells or tissues of living organisms, for example humans. In some embodiments, the methods disclosed herein encompass administering the compounds of the present disclosure to a subject.

[0155] The compounds described herein can be asymmetric (e.g., having one or more stereocenters). All stereoisomers, such as enantiomers and diastereomers, are intended unless otherwise indicated. Compounds of the present invention that contain asymmetrically substituted carbon atoms can be isolated in optically active or racemic forms. Methods on how to prepare optically active forms from optically active starting materials are known in the art, such as by resolution of racemic mixtures or by stereoselective synthesis. Many geometric isomers of olefins, C=N double bonds, and the like can also be present in the compounds described herein, and all such stable isomers are contemplated in the present invention. Cis and trans geometric isomers of the compounds of the present invention are described and may be isolated as a mixture of isomers or as separated isomeric forms.

[0156] In the case of the compounds which contain an asymmetric carbon atom, the invention relates to the D form, the L form, and D, L mixtures and also, where more than one asymmetric carbon atom is present, to the diastereomeric forms. Those compounds of the invention which contain asymmetric carbon atoms, and which as a rule accrue as racemates, can be separated into the optically active isomers in a known manner, for example using an optically active acid. However, it is also possible to use an optically active starting substance from the outset, with a corresponding optically active or diastereomeric compound then being obtained as the end product.

[0157] Compounds of the invention also include tautomeric forms. Tautomeric forms result from the swapping of a single bond with an adjacent double bond together with the concomitant migration of a proton. Tautomeric forms include prototropic tautomers which are isomeric protonation states having the same empirical formula and total charge. Example prototropic tautomers include ketone - enol pairs, amide - imidic acid pairs, lactam - lactim pairs, amide -imidic acid pairs, enamine - imine pairs, and annular forms where a proton can occupy two or more positions of a heterocyclic system, for example, 1H- and 3H-imidazole, 1H-, 2H- and 4H-1,2,4-triazole, 1H- and 2H- isoindole, and 1H- and 2H-pyrazole. Tautomeric forms can be in equilibrium or sterically locked into one form by appropriate substitution.

[0158] Compounds of the invention can also include all isotopes of atoms occurring in theP-656906-PCintermediate or final compounds. Isotopes include those atoms having the same atomic number but different mass numbers. For example, isotopes of hydrogen include tritium and deuterium.

[0159] In some embodiments, the term “compound” as used herein, is meant to include all stereoisomers, geometric isomers, tautomers, and isotopes of the structures depicted.

[0160] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.EXAMPLES METHODSHomology modeling of BCL-XL full-length structure

[0161] The full length structural model including the C-terminal helix a9 of BCL-XL was predicted using the I-TASSER modelling server (zhanggroup.org / I-TASSER / ), with the amino acid sequence of human Bax protein (UniProtKB: Q07812) as the homologous structural template and the amino acid sequence of truncated human BCL-XL protein (UniProtKB: Q07817) as the target. The top-ranked model was selected based on the highest global and local accuracy estimations and was used to reassemble continuous fragments into a complete full-length structure. This model then underwent topology refinement, energy minimization, and side-chain fixation using the Protein Preparation panel in Maestro (Schrodinger, LLC).In silico binding site prediction

[0162] Binding sites were identified using SiteMap (Schrodinger, LLC), DoGSiteScorer, and FTMap, automated tools for binding site detection and analysis. The prepared full-length BCL-XL was uploaded to each respective tool, and binding site prediction analyses were performed. For each tool, the druggability of the predicted sites was assessed based on their structural and physicochemical properties, aiding in the selection of potential binding sites for further investigation. Molecular dynamics (MD) simulation of apo-BCL-XL was performed using the Desmond (v7.4, Schrodinger, 2023-2). A hierarchical clustering was conducted based on the RMSD matrix of all atoms of BCL-XL protein structure using Desmond Trajectory Clustering on Maestro. SiteMap binding sites of each trajectory cluster were determined on Maestro.Molecular dynamics simulations

[0163] A low-energy 3D structure of ABX-27 was docked into the pre-generated receptor grid at residues R6 and A168 via Glide XP ligand docking (Maestro version 13.6.127, Schrodinger, 2023-P-656906-PCT). The best docking pose of ABX-27 in the allosteric binding site was selected based on the Glide GScore, 3D structural interaction fingerprints (SIFt) and 2D ligand fingerprints on Maestro. Molecular dynamics (MD) simulations were performed using the Desmond (v7.4, Schrodinger, 2023-2). The prepared apo BCL-XL full-length structure or ABX-27-bound BCL-XL FL structure was loaded into System Builder, where a predefined TIP3P solvent model was applied within a volume-minimized orthorhombic box. The system was neutralized with Na+ions, and 0.15 M NaCl was added under the OPLS4 force field. For apo BCL-XL, the system consisted of 42236 atoms. For the BCL-XL- ABX-27 complex, the system consisted of 42247 atoms. A 1000-ns all-atom MD simulation was conducted with a 1.0-ps recording interval. The system was first relaxed before the production run, which was performed under isothermal-isobaric (NPT) ensemble conditions. The Nose-Hoover Chain thermostat (1.0 ps of relaxation time) and Martyna-Tobias-Klein barostat (2.0 ps of relaxation time) were used to maintain the temperature and pressure, respectively. MD simulations were performed 3 times using different random seed numbers to ensure statistical reliability. Analysis was carried out using the Simulation Interactions Diagram panel in Maestro, where RMSD and RMSF values of the protein backbone, ligand, and protein-ligand interactions were computed over trajectory. Interatomic distances were analyzed across all trajectory frames using Maestro.In Silico Small Molecule Receptor-Based Screening

[0164] A library of purchasable compounds from eMolecules (www.emolecules.com) was converted into 3D structures using LigPrep (Schrodinger, 2020, LLC), with Epik (Schrodinger, 2020, LLC) applied to generate various ionization states (pH 7.0 ± 2.0), stereochemical variations, and tautomeric forms, resulting in an in silico screening library of approximately 14 million compounds. Conformational analysis of ligands was performed using the OPLS3 force field.

[0165] Virtual screening was conducted using grid-based ligand docking with energetics Glide (Schrodinger, 2020, LLC). A receptor grid was generated by defining the centroid of residues R6 and A 168 in the BCL-XL protein structure, with a grid box size of 10A * 10A * 10 A. The 14-million compound library was docked in a three-tiered docking protocol, starting with high-throughput virtual screening (HTVS), followed by standard precision (SP), and concluding with extra-precision (XP) docking. Ligand sampling was set to flexible mode, and post-docking minimization was performed at each stage. Following XP docking, 10,000 top-scoring docked conformers were retained and subjected to hierarchical clustering using the Similarity andP-656906-PCClustering Panel in Maestro. Virtual hits were prioritized based on their Glide GScore. Additionally, Absorption, Distribution, Metabolism, Excretion, and Toxicology (ADMET) properties were assessed using QikProp (Schrodinger, LLC). Selected ligands met key druglikeness criteria, including molecular weight < 500 Da, hydrogen bond donors < 5, hydrogen bond acceptors < 10, partition coefficient (LogP) < 5, number of rotatable bonds < 10. Using the PubChem database, it was confirmed that ABX-27 has not been reported as a hit in previous screens.Analogs design

[0166] The second batch of compound analogs were identified based on similarity search using Molport (www.molport.com / shop / find-chemicals) chemical compounds source. Compounds 17, 31 and 57 were submitted as the query structures and similarity search was launched with a similarity index of 0.6 on the basis of the molecular descriptors. The third batch of compound analogs were designed via R-group analysis with Maestro (Schrodinger, LLC). Parental compounds 17 and 57 and their respective analogs from the second batch compounds were imported to the R-group analysis panel. Their cores were defined with Canvas maximum common substructure (MCS) scheme. Atomic number, aromaticity and bond order were set as the atom equivalences for the Canvas MCS run. The R-group position importance ranking was analyzed by addressing the sensitivity and fingerprint similarity of the property of interest at the studied position. Two final scaffolds were designed and submitted as the query structures to PubChem (pubchem.ncbi.nlm.nih.gov / ) for fingerprint Tanimoto-based 2-dimensional similarity search. Recombinant BCL-XL protein production

[0167] Human full-length (aal-233) wild-type BCL-XL FL (SEQ ID NO: 16), truncated (aal-196) wild-type BCL-XL AC and truncated (1-44; 85-196) wild-type BCL-XL ALAC were cloned in pTYBl vector (New England BioLabs) between the Ndel and SapI restriction sites. Mutations were generated using the QuikChange Lightning site-directed mutagenesis kit (Agilent). Recombinant proteins were expressed in BL21 (DE3) CodonPlus (DE3)-RIPL, grown in Luria-Bertani (LB) broth media and induced with 1 mM isopropyl [3-d- 1 -thiogalactopyranoside (IPTG) and incubated at 18 °C overnight. The bacterial pellet was resuspended in lysis buffer (25 mM Tris-HCl, pH 8.0, 150 mM NaCl, ImM EDTA), lysed by high pressure homogenization, and clarified by ultracentrifugation at 17,000 rpm for 45 min. The supernatant was applied to 5 mL of pre-equilibrated chitin beads (New England BioLabs) in a gravity-flow column and washed withP-656906-PC3 column volumes of lysis buffer. BCL-XL was cleaved by overnight incubation using 30 mM DTT in lysis buffer. Cleaved BCL-XL was eluted with lysis buffer, concentrated with a Centricon spin concentrator (Millipore) and purified by gel filtration using a using a Superdex 75 Increase 10 / 300 GL column (Cytiva), pre-equilibrated with gel filtration buffer (25 mM sodium phosphate, pH 6.5, 1 mM EDTA, 2mM DTT) at 4 °C. Fractions containing purified BCL-XL were combined and concentrated using a 10-kDa cut-off Centricon spin concentrator (Millipore) for prompt use in biochemical and structural studies.Recombinant BAX protein production

[0168] Human full-length (aal-192) wild-type BAX was cloned in pTYBl vector (New England BioLabs) between the Ndel and SapI restriction sites. Recombinant protein was expressed in BL21 (DE3) CodonPlus (DE3)-RIPL, grown in Luria-Bertani (LB) broth media and induced with 1 mM isopropyl 0-d-l -thiogalactopyranoside (IPTG) and incubated at 18 °C overnight. The bacterial pellet was resuspended in lysis buffer (20 mM Tris-HCl, pH 7.6, 250 mM NaCl, ImM EDTA) with the addition of Pierce Protease Inhibitor Tablets, EDTA-free (Thermo Fisher Scientific), lysed by high pressure homogenization, and clarified by ultracentrifugation at 17,000 rpm for 45 min. The supernatant was applied to 5 mL of pre-equilibrated chitin beads (New England BioLabs) in a gravity -flow column and washed with 3 column volumes of lysis buffer. BAX was cleaved by overnight incubation using 50 mM DTT in lysis buffer. Cleaved BAX was eluted with lysis buffer, concentrated with a Centricon spin concentrator (Millipore) and purified by gel filtration using a using a Superdex 75 Increase 10 / 300 GL column (Cytiva), pre-equilibrated with gel filtration buffer (20 mM HEPES, pH 7.2, 150 mM KC1) at 4 °C. Fractions containing purified BAX were combined and concentrated using a 10-kDa cut-off Centricon spin concentrator (Millipore) for prompt use in biochemical studies.Recombinant Bcl-2 AC protein production

[0169] Human truncated (aal-218) wild-type Bcl-2 AC was cloned in pGex-6p-l vector (New England BioLabs). Recombinant proteins were expressed in BL21 (DE3) CodonPlus (DE3)-RIPL, grown in Luria-Bertani (LB) broth media and induced with 1 mM isopropyl [3-d-l-thiogalactopyranoside (IPTG) and incubated at 37 °C for 4 hours. The bacterial pellet was resuspended in lysis buffer (Ixphosphate-buffered saline (PBS), pH 7.2, 1% Triton X-100, 1 mM EDTA) with the addition of lysozyme, PMSF and Pierce Protease Inhibitor Tablets, EDTA-free (Thermo Fisher Scientific), lysed by high pressure homogenization, and clarified byP-656906-PCultracentrifugation at 17,000 rpm for 45 min. The supernatant was applied to 5 mL of preequilibrated Glutathione Sepharose 4B beads (Cytiva) in a gravity-flow column and washed with 5 column volumes of PBS / Triton X-100, followed by another 5 column volumes of PBS. Bcl-2 AC was cleaved by overnight incubation using 50u thrombin per 1 mL PBS. Cleaved Bcl-2 AC was eluted with PBS, concentrated with a Centricon spin concentrator (Millipore) and purified by gel filtration using a using a Superdex 200 Increase 10 / 300 GL column (Cytiva), pre-equilibrated with gel filtration buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl) at 4 °C. Fractions containing purified Bcl-2 DC were combined and concentrated using a 10-kDa cut-off Centricon spin concentrator (Millipore) for prompt use in biochemical studies.Site-directed mutagenesis

[0170] Mutagenic primers were designed on QuikChange Primer Design Program (www.agilent.com / store / primerDesignProgram.jsp) by using the DNA sequence of BCL-XL (www.uniprot.or / uniprotkb / Q07817 / entry). The single-site complementary mutagenic primer sequences are 5’- CTCAGAAAATCAAC AACCAGTTCCC AATTGCTCTGGCTC ATATGTATATC-3 ’ (R6W) (SEQ ID NO:1), 5’-AGGTTGCCATCCACCATGCAATACGGCTAACCAGAACCTGC-3’ (A168W) (SEQ ID NO:2), 5’-ACCTTTACGGCTTTCTGCGCATGCATTATTACCATACAGTTCCACAAA. GG-3 ’ (A200C) (SEQ ID NO:3) and 5’-AGCTAAAAAAGGCAACAATACGCCACCAATTAAC ACCATCACGAAAC-3 ’ (G138W) (SEQ ID NO:4) for pTYBl plasmid. The PCR-based plasmid mutagenesis was performed by using the QuikChange Lightning Multi Site-Directed Mutagenesis Kit (Agilent Technologies). Transformation of the PCR product was performed using XLIO-Gold ultracompetent cells (Agilent Technologies). The plasmid DNA was isolated and purified using QIAprep Spin Miniprep Kit (Qiagen). The nucleotide sequence of the newly-constructed plasmid was validated via Sanger Sequencing service of Genewiz (Azenta Life Sciences).Fluorescence polarization binding assays

[0171] Fluorescence polarization assays (FPA) were performed as previously described. Firstly, direct binding curves were generated by incubating FITC-labelled BH3 peptides (25 nM) with serial dilutions of BCL-XL. Fluorescence polarization was measured every 10 min for 60 minutes on a F200 PRO microplate reader (TECAN). Reported curves are the 10-minute time-point. ForP-656906-PCthe competition assay in the screen, each compound at 10 pM were combined with recombinant BCL-XL at EC75 concentration as determined by the direct binding assay followed by the addition of FITC-Bid BH3 and FITC-Bax BH3 (5 nM). For titration assays of the top hits, a serial dilution of ABX-27 or analogues was combined with recombinant BCL-XL at EC75 concentration as determined by the direct binding assay followed by the addition of FITC-Bid BH3 and FITC-Bax BH3 (5 nM). EC50 values were calculated by nonlinear regression analysis four-parameter [inhibitor] versus response with restraints for 100% top and 0% bottom calculated by the mP of saturated BCL-XL + FITC-Bid BH3 or FITC-Bax BH3 and BH3 peptide alone, respectively. Data were analyzed and graphed using GraphPad Prism 9 software.Microscale thermophoresis

[0172] Recombinant BCL-XL mutants (BCL-XL FL A200C, BCL-XL FL A200C R6W A168W and BCL-XL A200C G138W) were labeled at cysteine using the Monolith Protein Labeling Kit Red Maleimide 2nd generation (NanoTemper Technologies) according to the instructions of the manufacturer. Specifically, 35 uM BCL-XL was incubated with around 2 equivalents of dye in MST buffer (100 mM potassium monophosphate, pH 7.4, 150mM NaCl) in the dark at room temperature (22-25 °C) for 30 min. Unreacted dye was quenched using 10 mM DTT and removed using Column B (NanoTemper Technologies). To determine the KD of the compounds, 50 nM labeled BCL-XL was incubated with increasing concentrations of ABX-27 or analogs in MST buffer supplemented with 0.25% CHAPS. Samples were loaded into standard glass capillaries (Monolith NT.155 Capillaries) and analyzed by MST using a Monolith NT.115 Blue / Red, LED power and IR laser power of 40%. Samples showed no aggregation according to post-run analysis using the Monolith data collection software (Nanotemper). Fraction bound and error were generated by NanoTemper software (MO. Affinity Analysis) and KD values were calculated by nonlinear regression analysis four-parameter [agonist] versus response on GraphPad Prism 9. Liposomal permeabilization assay

[0173] Lipids (Avanti Polar Lipids) at the indicated ratio: phosphatidylcholine 48%, phosphatidylinositol 10%, dioleoyl phosphatidylserine 10%, phosphatidylethanolamine 28%, and tetraoleoyl cardiolipin 4% were mixed in total of 4 mg, dried and resuspended in 10 mM HEPES, pH 7.0, 200 mM KC1, and 5 mM MgCh buffer with 12.5 mM 8-aminonaphthalene- 1,3,6-trisulfonic acid (ANTS) dye and 45 mM p-xylene-bis-pyridinium bromide (DPX) quencher (Molecular Probes) using a water bath sonicator. Liposomes were formed by extrusion of theP-656906-PCsuspension using polycarbonate membranes of 0.2 pm pore size (Avanti Polar Lipids). ANTS / DPX encapsulated liposomes were purified from non-encapsulated ANTS / DPX by gel filtration over a 10 mL Sepharose CL2B resin (GE Healthcare Life Sciences) gravity flow column.200 nM BAX, 160 nM BCL-XL, compounds (in a dose-dependent concentration range), and 30 nM tBid were added into a 96-well microplate (Coming), prior to the addition of liposomes (10 pL from 50 mM total lipid stock) in assay buffer to a final volume of 100 pl. ANTS / DPX release was quantified based on the increase in fluorescence intensity that occurs when the ANTS fluorophore is separated from the DPX quencher upon release from the liposomes into the supernatant. Fluorescence (Lex = 355 nm and Xem = 520 nm) was measured over time at 30.0°C using SpectraMax iD5 microplate reader. The percentage release of ANTS / DPX at any given time point was calculated as percentage release = ((F - F0) / (F100 - F0)) A x 100, where F0 and F100 are baseline and maximal fluorescence, respectively. 1% Triton treatment is used to determine the maximum amount of liposomal release per assay, and this value sets the 100% value.NMR samples and spectroscopy

[0174] For NMR studies, uniformly15N-labeled BCL-XL protein was produced by growing the bacteria in minimal medium and15NH4Cl as previously described. Following similar purification of BCL-XL as with unlabeled protein, each NMR sample was prepared in NMR buffer (25 mM sodium phosphate, pH 6.5, 1 mM EDTA), 10% D2O within a 3mm Bruker Biospin unracked 4” NMR tube (Bruker Biospin).1H-15N-HSQC spectra were recorded on15N-labeled BCL-XL at 50 pM in the presence and absence of ligand in molar excess equivalence. NMR spectra were acquired at 25 °C (298.15 K) on a Bruker 600 MHz spectrometer equipped with a cryoprobe, processed using TopSpin 3.6.2 and analyzed using CcpNmr Analysis 2.5.2. BCL-XL chemical shifts assignments were applied as previously generated for BCL-XL and deposited (BMRB Entry 25466 and BMRB entry 50276) and buffer matching and acquisition temperature were considered to minimize errors. Optimized parameters during data collection are spectral Width= 14.0289ppm (F2) and 29.0001ppm (Fl); acquisition time= 0.1216512 sec (F2) and 0.0283548 sec (Fl); number of scans= 32; number of dummy scans= 16. The weighted average chemical shift perturbation (CSP) was calculated as √(ΔδH)2+ (Δδ15N / 5)2in p.p.m and the significance threshold for backbone amide chemical shift changes was calculated based on the average chemical shift across all residues plus 1.5 s.d., in accordance with standard methods.

[0175] The absence of a bar indicates no chemical shift difference, the presence of a proline, or aP-656906-PCresidue that is overlapped or missing and therefore not used in the analysis. Solvent accessible surface area was probed by the addition of 10 mM hy-TEMPO (Sigma) to 50 pM15N-BCL-XL with and without 100 pM ABX-27 measured using standard1H-15N-HSQC incorporating an increased recycle delay of 2 seconds. PRE were calculated as the ratio of peak intensities of BCL-XL in the presence of hy-TEMPO to BCL-XL without hy-TEMPO (Ipara / Idia). Mapping of chemical shifts and PRE data onto the BCL-XL structure was performed with PYMOL (Schrodinger, LLC). Software was made available through the SBGrid collaborative network. Hydrogen-deuterium exchange mass spectrometry

[0176] BCL-XL stock solutions (50 pM) were prepared in a buffer containing 100 mM sodium phosphate, 150 mM NaCl, pH 7.2 H2O. The solutions were allowed to incubate at room temperature for 30 min. Ligand-bound complexes of BCL-XL were prepared by mixing BCL-XL with ABX-27 and ABT-263, respectively at 1:1 and 1:10 molar ratios. These mixtures were incubated at room temperature for 30 minutes and then stored at 0 °C until further use in HDXMS studies. Functional deuterated exchange reactions were initiated by diluting of 3 pL of stock solution (BCL-XL, BCL-XL-lx ABX-27, BCL-XL-lOx ABX-27, BCL-XL-lx ABT-263 and BCL-XL-lOx ABT-263) into 9 pL of D2O buffer (100 mM sodium phosphate, 150 mM NaCl, pDREAD 7.2). The mixtures were incubated at 0 °C for various time points: 10, 1000 and 10000 sec. The exchange reactions were quenched by adding 18 pL of ice-cold quench buffer (0.6 M Glycine, 1.6 M GuHCl, pH 2.4) to achieve a final pH of 2.5. The quenched samples were incubated at 0 °C for 1 minute, immediately frozen on dry ice and stored at -80 °C before LC / MS analysis. Additionally, un-deuterated and equilibrium-deuterated control samples are also prepared as previously described. The frozen samples were subsequently loaded onto a cryogenic autosampler where they were automatically thawed at 4 °C. The samples were then passed over an immobilized pepsin column (16 pL bed volume) at a flow rate of 25 pL / min for digestion. The resulting proteolytic peptides were collected on a trap column and separated using Acclaim PepMap RSLC C18 reverse phase analytical column (ThermoSci, 0.3 * 50 mm, 2 pm, 100 A) with a linear acetonitrile gradient (5%-45% over 30 min). The eluate was directed into an OrbiTrap Elite Mass Spectrometer (ThermoFisher Scientific, San Jose, CA) for MS analysis. Instruments settings were carefully optimized to minimize back-exchange during the process. Data acquisition was performed in either MS1 profile mode or data-dependent MS / MS mode, depending on the experimental requirements. Peptide identification was facilitated using Proteome DiscovererP-656906-PCsoftware (ThermoFisher). The centroids of the mass envelopes of deuterated peptides were calculated using HDExaminer (Sierra Analytics Inc, Modesto, CA) and subsequently converted to corresponding deuterium incorporation level with appropriate corrections applied for back-exchange.Reagents

[0177] Fluorescein-labelled BH3 peptides corresponding to the BH3 domain of BIM (FITC-Ahx-EIWIAQELRRIGDEFNAYYA (SEQ ID NO:5)), BID (FITC-Ahx-EDIIRNIARHLAQVGDSMDR (SEQ ID NO:6)), BAD (FITC-b-Ala-NLWAAQRYGRELRRMSDEFVDSFKK (SEQ ID NO:7)), BAK (FITC-Ahx-HQQPSSTMGQVGRQLAIIGDDINRRYDSEFQTMLQH (SEQ ID NO:8)) and BAX (FITC-Ahx-QDASTKKLSECLKRIGDELDSNMELQRMIAA (SEQ ID NO:9)), were synthesized, purified at >95% purity, and verified by HPLC and mass spectrometry, by GenScript. All screening compounds were provided by Molport and ChemBridge Corporation at >90% purity and catalog numbers and molecular information are detailed in Supplementary Table.Navitoclax / ABT-263 (Cat. #7680) and A-1331852 (Cat. # 7661) were provided by Tocris Bioscience. The peptides were reconstituted in 100% DMSO at a concentration of 10 mM, then diluted in aqueous buffers for assays. For cellular and in vitro assays, compounds were reconstituted in 100% DMSO to prepare 20 mM and 10 mM stock solutions and diluted in cell culture medium or aqueous buffers for assays. ABX-27 was additionally synthesized to confirm its activity. The synthesis and analytical characterization of ABX-27 (>95% purity) is provided in Example 17.Cell Lines

[0178] HEL WT cell line (Cat. #YC-C090) was purchased from Ubigene. THP-1, HEL and Kasumi were gifted from the Marina Konopleva laboratory. All cells were maintained in a 37°C incubator with 5% CO2. Cells were maintained in RPMI media supplemented with 10% fetal bovine serum with 100 U / mL pen / strep. Cell lines were authenticated by their vendor and the Albert Einstein College of Medicine Genomics Core Facility. Morphology, karyotyping, and STR profiling were performed to confirm the identity of human cell lines and to rule out both intra- and inter-species contamination. Cell lines were checked regularly for mycoplasma contamination using DNA staining protocol with Hoechst (Life Technologies, 33342) dye and any suspected contamination was further tested using Myco-sniff™ mycoplasma PCR detection kit (MPP-656906-PCBiomedical, 093050201).CRISPR-Cas9 mediated knock out of BCL2L1

[0179] CRISPR RNP complex was used to target Bcl2Ll using the following sgRNA sequences. SgRNA 1: CCTTGGATCCAGGAGAACGG (SEQ ID NO: 10), sgRNA 2: TTCCGGGATGGGGTAAACTG (SEQ ID NO: 11). Electroporation was performed with SF Cell Line 4D-Nucleofector X Kit according to the manufacturer’s instructions (Lonza, V4XC-2032) using the program FF120. The cells were then cultured in RPM1 1640 medium supplemented with 10% FBS and 1% PenStrep. Cell growth was monitored every 24 h using CellTiter-Glo assay. Real-Time PCR

[0180] BCL2L1 mRNA expression was measured three days after CRISPR-Cas9 mediated knock-out using PowerUp SYBR Green master mix (Applied Biosystems, A25742) and primers for BCL2L1 forward: GCCACTTACCTGAATGACCACC (SEQ ID NO: 12), BCL2L1 reverse: AACCAGCGGTTGAAGCGTTCCT (SEQ ID NO: 13), GAPDH forward:GTCTCCTCTGACTTCAACAGCG (SEQ ID NO: 14), GAPDH reverse:ACCACCCTGTTGCTGTAGCCAA (SEQ ID NO: 15) Briefly, PCR was performed in a 20 pL reaction volume using the Bio-Rad CFX-96 Touch Real-Time PCR System with the following reaction conditions: enzyme activation for 30 seconds at 95°C, followed by 39 cycles of denaturation for 5 seconds at 95 °C and annealing / extension for 5 seconds at 58 °C, followed by the melt curve from 65 °C for 5 seconds to 95 °C for 5 seconds in 0.5 °C increment. Relative fold change in mRNA levels were calculated by the AACT method.Subcellular fractionation

[0181] To isolate the cytosolic and mitochondrial cellular fractions, cells were centrifuged at 500g for 5 mins and washed in ice-cold PBS. Cells were then resuspended in a digitonin lysis buffer (10 mM KC1, 5 mM MgCh, 1 mM EGTA, 1 mM EDTA, 250 mM sucrose, 20 mM Hepes, 0.025% digitonin, pH 7.2) supplemented with Halt protease inhibitor cocktail (Thermofisher Cat. 1861279) and incubated on ice for 15 mins. Cells were then centrifuged at 15,000 g for 10 mins at 4°c. Supernatant was collected as the cytoplasmic fraction and stored at -20°c before use. The remaining cell pellet was resuspended in 1% Triton-X buffer supplemented with Halt protease inhibitor cocktail (Thermofisher Cat. 1861279), and incubated on ice for 1 hour followed by centrifugation at 15,000 g for 10 mins at 4 °C. Supernatant was collected as the mitochondrialP-656906-PCfraction and stored at -20 °C before use.Western blotting and quantification

[0182] Cells were lysed with Pierce RIPA buffer (Thermofisher Cat. 89900) supplemented with Halt protease inhibitor cocktail (Thermofisher Cat. 1861279). Whole-cell lysates were electrophoretically separated on 4-12% NuPage gels (Life Technologies), transferred to mobilon-FL PVDF membranes (Millipore) and subjected to immunoblotting. For visualization of proteins with Odyssey Infrared Imaging System (LLCOR Biosciences), membranes were blocked in Intercept Blocking Buffer (LLCOR Biosciences Cat. 927-7000) for 1 h at room temperature. Primary antibodies were incubated overnight at 4 °C. After washing, membranes were incubated with IRDye800- conjugated goat anti-mouse TgG (1:5,000 dilution) / IRDye680RD-conjugated goat anti-Rabbit IgG (1:5,000 dilution) secondary antibodies (LLCOR Biosciences) for the Odyssey Infrared Imaging System. Protein levels were quantified by fluorescence intensity (Odyssey Infrared Imaging System) using the Image Studio 3.1 software. Antibodies were used to detect the following proteins on membrane: BCL-XL (Cell Signaling Cat. 2764), BCL-2 (Cell Signaling Cat.4223), Cleaved Caspase-3 (Cell Signaling Cat. 9661), Cleaved PARP (Cell Signaling Cat. 5625), COX-IV (Cell Signaling Cat. 4850), Cytochrome C (Cell Signaling Cat. 4272S), 0-Actin (Sigma Cat. A1978), P-Tubulin (Cell Signaling Cat. 2146).Cell viability assay

[0183] Cells (2 x 103cells / well) were seeded in Corning® 384-well white plates and incubated with serial dilutions of Navitoclax (ABT263), A-1331852, ABX-27 and select compounds or vehicle (1% DMSO) using a TECAN D300e Digital Dispenser in 25 pl. Cell viability was assessed at 72 h post-treatment, by addition of CellTiter-Glo® Assay reagents according to the manufacturer’s protocol (Promega), and luminescence measured using a F200 PRO microplate reader (TECAN). Viability assays were performed in at least triplicate and the data normalized to 1% vehicle-treated control wells. IC50 values were determined by nonlinear regression analysis using GraphPad Prism 10 software (GraphPad).Caspase 3 / 7 activation assay

[0184] Cells (2 x 103cells / well) were seeded in Corning® 384-well white plates and incubated with serial dilutions of Navitoclax (ABT263), A-1331852, ABX-27 and select compounds or vehicle (1% DMSO) using a TECAN D300e Digital Dispenser in 25 pl. Caspase 3 / 7 activity wasP-656906-PCassessed at 24 h post-treatment by the addition of Caspase 3 / 7 Gio® Assay reagents according to the manufacturer’ s protocol (Promega), and luminescence measured using a F200 PRO microplate reader (TEC AN).Statistical analysis and sample size determination

[0185] All numerical results are reported as mean + SEM and represent data from a minimum of three independent experiments unless otherwise stated. Experiments in cells in culture were performed on different days to confirm reproducibility of the procedures. Statistical significance was performed by: unpaired two-tailed t-test of the means (in instances of single comparisons), one-way analysis of variance (ANOVA) followed by the Bonferroni post-hoc test (in instances of multiple comparisons with one independent variable). Statistical analysis was performed for all assays and significant differences, as determined by significance level p < 0.05, are noted in graphical representations. The following software programs were utilized: Microsoft Excel 365 (Version 2305) for basic data handling, Prism software (v9 - Graph Pad Software Inc) for data analysis, and Odyssey (LICORBio) for acquisition of gel scans, Schrodinger Software Suite (Releases 2017-2023 - Schrodinger, LLC) for structural and small molecule data analysis.EXAMPLE 1IDENTIFICATION OF ALLOSTERIC SITE ON FULL LENGTH BCL-XL AND IN SILICO SCREENING

[0186] A 3D structural model of the human full-length BCL-XL was first calculated using an available truncated crystal structure of BCL-XL and the full-length BAX structure as a template for the conformation of the c-terminal helix 9 of BCL-XL (Figure 1A and Figure IB). Molecular dynamics simulations were performed on the BCL-XL structural model to explore conformational dynamics and several conformations were used to feed automated binding site identification and analysis tools SiteMap, DoGSiteScorer and FTMap. Predicted binding sites were analyzed for druggability based on site properties (Figure ID). Further, the predicted binding sites were validated with NMR paramagnetic relaxation enhancement (PRE) experiments using hy-TEMPO probe to identify solvent accessible sites on the surface of BCL-XL accessed by TEMPO probe and the predicted binding sites were confirmed (Figure IE and Figure IF).

[0187] Binding Site 2 was selected that is adjacent to the N-terminal intrinsically disordered region (al-a2 loop) of BCL-XL and changes of the binding site in the MD simulation trajectory wereP-656906-PCinvestigated (Figure 1G). The binding site was maintained with minor changes over the course of the simulation despite significant changes in al-a2 loop conformation adjacent to the site 2. Mutagenesis of the binding site residues R6 and A 168 in helices al and a6 were performed as these were identified as the hot spot residues largely determining the identification of the Site 2 by Sitemap (Figure 1H). Next, to identify compounds binding to Site 2, in silico screening was performed using a 14 million compounds library via a three-tier docking methodology which starts with high throughput virtual screening (HTVS) followed by standard precision (SP) and then by extra-precision mode (XP) screening mode (Figure 2A).

[0188] Following compound clustering and filtering for favorable ADMET properties, a selection of 67 compounds were purchased from commercial vendors for evaluation.EXAMPLE 2EXPERIMENTAL SCREENING, HIT IDENTIFICATION AND VALIDATION

[0189] To screen the in silico hits, various biochemical, biophysical and cell-based assays were employed for BCL-XL binding, BCLXL inhibition activity and binding to the allosteric site as well as apoptosis related assays. A production of full length BCL-XL wt and mutant R6W / A168W was established to interrogate binding to and allosteric effects mediated by Site 2 (Figure 2B).

[0190] Interestingly, among BH3 domain / peptides binding to the canonical pocket of BCL-XL by Fluorescence Polarization (FP) assay, BCL-XL R6W / A168W mutant reduced only binding of BID BH3 and BAX BH3 (Figure 2C) while BAK BH3 binding to BCL-XL was increased, suggesting differential inhibition activity of BCL-XL-BAX / BID and BCL-BAK complexes, mediated by Site 2 and potentially small molecules binding to Site 2. Next, the 67 compounds were screened using the FP assay for competing BCL-XL-BID and BCL-XL-BAX binding and 17 hits with > 70% inhibition were prioritized for either complex (Figure 2D). A functional assay was also established for full length BCL-XL activity in promoting inhibition of BAX-mediated membrane permeabilization using liposomal membranes (Figure 2E). Using FP, liposomal assay, MST direct binding to BCL-XL and HSCQ-NMR analysis using full length BCL-XL wt and R6W / A168W mutant, 3 different compounds (compound 17, compound 31 and compound 57) were identified and prioritized, which showed the best binding to BCL-XL and reduced binding / activity to the mutant (Figure 21, Figure 2F). Next, the analogs of these 3 compounds were obtained and screened for binding and activity using the same assays (Figure 2J, Figure 2G) resulting in compound 17j, compound 17w, compound 57m, compound 57j as best inhibitors. The R-groupP-656906-PCanalysis was performed to design novel analogs 17jw and 57mj (Figure 2H and Figure 3C). The analogs of 17jw and 57mj were then screened and more potent inhibitors were identified, which are also inhibited by R6W / A168W mutant but not by a mutant in the canonical site that typically disrupts established BCL-XL inhibitors G138W (Figure 2K).

[0191] Several compounds were evaluated in cells and demonstrated activity in inducing caspase-3 / 7 activation and loss of viability in AML cell lines, e.g. HEL (more sensitive to BCL-XL inhibition) and THP-1 (less sensitive to BCL-XL inhibition). Among the hits, 17jw27 -(ABX-27) was focused on, which also had potent cellular activity (Figure 3A and Figure 3B) and selectivity for binding to BCL-XL compared to BCL-2 (Figure 10A). 17jw27 compound also demonstrated induction of apoptosis and loss of viability in THP-1 and Kasumi AML cells while it had no effect on THP-1 cells with BCL-XL knockdown (Figures 9E-9J). Our experiments used ABT-263 (BCL-XL / BCL-2 inhibitor) and A1331852 (BCL-XL inhibitor) for comparison. Additional experiments were performed with 17jw27 using structural and biophysical HSQC-NMR (Figure 5A and Figure 5B), NMR-PRE (Figure 5C and Figure 5D) and HDX-MS (Figures 4A-4D) analysis that demonstrate specificity for binding to Site 2 and allosteric effects at the C-terminal canonical site of BCL-XL to explain the mechanism of site 2 binding molecules to compete BH3 domains from the BCL-XL canonical site. These allosteric effects from 17jw27-binding to Site 2 were also recapitulated in molecular dynamics simulations that showed allosteric conformational changes in the canonical pocket in the presence of 17jw27 by increased interatomic distances that include the C-terminal helix 9 (Figure 6, Figures 7B-7D, and Figures 8D-8E).

[0192] As used herein and throughout the application, the terms “17jw27” and “ABX-27” can be used interchangeably. Compound 17jw27 represents the same compound as Compound ABX-27 as shown in their chemical structures (e.g., Table 3).

[0193] A list of compound structures tested are provided in Tables 1-3.

[0194] Experiments have also shown apoptotic activity through BCL-XL inhibition from 17jw27 in solid tumors cell lines including NSCLC and CRC cell lines (not shown here), and additional readouts such as mitochondrial cytochrome c release.P-656906-PCEXAMPLE 3IDENTIFICATION AND CHARACTERIZATION OF A NOVEL ALLOSTERIC BINDING SITE OF BCL-XL FL

[0195] The identification and characterization of a novel allosteric binding site of BCL-XL FL (SEQ ID NO: 16) are illustrated in the results and figures below.

[0196] Figure 1A depicts a homology model of full length BCL-XL protein structure with the flexible loop ctl -ci2 (green) and Ctenninal helix a.9 (orange).

[0197] Figure IB depicts a BH3 binding hydrophobic canonical pocket (blue) of BCL-XL with the binding of BIM BH3 peptide (magenta), helix a9 (orange) is located on the hydrophobic groove.

[0198] Figure ID depicts a distribution of four computationally predicted binding sites of BCL-XL FL via the application of SiteMap, DoGSiteScorer and FTMap. All four binding sites are represented in mesh format from C-terminal and N-terminal views.

[0199] Figure IE depicts a ratio of15N-BCL-XL AC in the presence or absence of the soluble paramagnetic agent hy-TEMPO (Iox / Ired), plotted as a function of BCL-XL AC residue number. BCL-XL AC residue intensities classified into four categories: < 20%, 20-40%, 40-60% and 60-100%. Data are representative of two independent experiments.

[0200] Figure IF depicts quantitative and graphical characterisation of druggable binding pockets. Mapping of residues undergoing significant PRE effects from (e) onto the surface representation of BCL-XL AC with SiteMap binding site localizations (represented in mesh format). Extensive PREs within site (2) confirm the presence of a relatively deep solvent exposed cavity at the proposed interaction site.

[0201] Figure 1G depicts a size variation of allosteric binding sites (Site (2)) of nine trajectory clusters, which were captured at different frames of 1000-ns MD simulation. Sizes of Site (2) shifts via the localization of the relevant amino acid residues: Ins (Ml, R6, V10, W24, F27, S28, D29, V163, S164, R165, A167, A168, A171); 90ns (R6, V10, W24, S25, F27, S28, D29, V30, E31, E32, N33, R34, T35, V161, V163, S164, R165, A168); 170ns (Ml, Q3, R6, E7, V10, W24, S25, Q26, F27, S28, V30, E31, E32, N33, Q160, V163, S164); 220ns (Ml, Q3, R6, V10, W24, S25, Q26, F27, S28, D29, V30, E31, E32, N33, R34, Q160, V161, V163, S164, A167); 350ns (Q3, R6, E7, V10, W24, S25, F27, S28, D29, V30, E31, E32, N33, S164); 500ns (R6, V10, W24, D29, V30, E31, E32, R34, T35, E36, P38, Pl 16, T118, Al 19, L162, S164, R165, A167, A168, W169); 670nsP-656906-PC(Q3, R6, E7, V9, VI 0, W24, S28, D29, V30, E31, E32, N33, R34, E36, A37, P38, SI 64, R165, A167, A168, A171); 760ns (Q3, R6, E7, V10, L17, S23, W24, S25, Q26, F27, S28, D29, V30, E31, E32, N33, R34, T35, V155, Q160, V161, V163, S164, R165, A167); and 940ns (Q3, R6, E7, V10, W24, F27, S28, D29, V30, E31, N33, R34, T35, E36, A37, P38, Pl 16, G117, T118, Al 19, Y120, Q121, V161, L162, V163, S164, R165, A167, A168,, W169).

[0202] Figure II depicts a consistency of identification of Site (2) as a favorable druggable pocket. Transition of allosteric binding site (Site (2)) of each trajectory cluster during the course of 1000-ns molecular dynamics simulation of BCL-XL FL. Every trajectory cluster is a representative frame based on the calculated RMSD matrix at 1000-ns interval. Each trajectory cluster was submitted to SiteMap (Maestro) for binding site assessment. The RMSD plot was produced via Simulation Interactions Diagram (Maestro).

[0203] Figure 1 J depicts a distribution of amino acid residues within Site (2). A zoomed-in view of the novel allosteric binding site i.e., Site (2) with the colored amino acid residues as reported by the in silico binding site prediction methodologies. The involved amino acid residues are colored based on their nature of basic (blue), acidic (red), hydrophobic (yellow) and polar (green). The flexible loop of ctl-a.2 are removed for a clearer magnification purpose.EXAMPLE 4BCL-XL FL BINDING SITE PREDICTIONS VIA SITEMAP, DOGSITE SCORER AND FTMAP

[0204] Figure IK provides that the evaluation of binding sites on SiteMap is based on SiteScore (based on site points), Dscore (druggability), Volume (estimation of the “shrink-wrap” volume on protein surface) and Balance (ratio between hydrophobic and hydrophilic character). The evaluation of binding sites on DoGSiteScorer is based on Simple Score (a linear combination of pocket volume, enclosure and lipophilic character), Drug Score (druggability), Volume and Surface (ratio between pocket hull and surface grid points). The evaluation of binding sites on FTMap is based on the number of probe clusters bind to consensus sites.P-656906-PCEXAMPLE 5COMPUTATIONAL LOCALIZATION OF DRUGGABLE BINDING POCKETS AND HOT SPOT RESIDUES OF BCL-XL

[0205] Figure 1C provides potential small molecule binding sites identified on the structure of inactive BAX (PDB ID: 1F16) using SiteMap (Schrodinger); similar identified sites were also identified from DoGSiteScorer and FTMap, respectively, and mapped onto the ribbon structure of BAX.

[0206] Figure IL depicts a progression of Site (2) of BCL-XL from each trajectory cluster, which is a representative frame based on the calculated RMSD matrix at 1000-ns interval. Site (2) is localized on helices al, a6, and the N-terminal of loop al-a2. Site (2) is colored according to the time interval of simulation time.

[0207] Figure 1H illustrates amino acid residues (yellow) which are estimated to be part of the formation of the predicted binding site, i.e. site (2) showed in green, are mutated into Tryptophan and went through SiteMap analysis. The single mutations of R6W and A168W respectively and double mutation of R6W and A168W trigger the prediction of other binding sites except the initial site (2), making both residues R6 and Al 68 are the hot spot residues of the functional site in protein-ligand interaction interface.

[0208] Figure IM depicts an evaluation of four top-ranked binding sites in BCL-XL with mutation in the hot spot residues, hi silico mutagenesis of residues R6 and Al 68 into W6 and W168 (represented by sticks in in red and green, respectively). The single mutants (R6W and A168W) and double mutant (R6W A168W) were subjected to SiteMap binding site identification. Four topranked binding sites for each single mutant and double mutant were shown in mesh format. The absence of residues R6 and Al 68 lead to the low propensity of Site (2) in becoming a functional site that favors for protein-ligand interaction.EXAMPLE 6LARGE-SCALE COMPUTATIONAL STRUCTURE-BASED SCREENING OF BCL-XL,AND CHARACTERIZATION OF POTENT BINDING CANDIDATES

[0209] Figure 2C provides a validation of FITC-labeled Bid BH3 (above) and Bax BH3 (bottom) peptides as a competitive binder by comparing their EC50 values between the wild-type and mutated BCL-XL FL proteins in FP direct binding assays. Data are mean ± SEM from n = 3 independent experiments.P-656906-PC

[0210] Figure 2A provides a hierarchical large-scale molecular docking of BCL-XL using a large library of 14-million compounds, in which the protein structure was equipped with an enclosing grid box (magenta) and a midpoint (green) for a docked ligand confinement in the allosteric binding site. The docking process started with high-throughput virtual screening (HTVS), followed by standard precision (SP) and extras precision (XP) modes. 10,000 virtual hits were clustered based on their similarity fingerprints and ranked from their docking scoring functions, followed by Lipinski Rule of 5 (Ro5) evaluation. Finally, 67 hit candidates were selected and proceeded to binding characterization and evaluation.

[0211] Figure 2D provides a screening of the sixty-seven compounds from the large library screening via fluorescence polarization assay which competes against FITC-BID BH3 (above) and FITC-BAX BH3 (bottom) peptides. A threshold value of 30% (blue line) is set to further characterize compounds that show binding potency (< 30%). Data are mean ± SEM from n = 3 independent experiments.

[0212] Figure 21 provides IC50 / EC50values of the three most potent compounds 17, 31 and 57, from fluorescence polarization assays, liposome permeabilization assays, and microscale thermophoresis assays (second, third and fourth column to the left, respectively). The compounds were assayed on BCL-XL FL WT and BCL-XL FL R6W A168W proteins. Data are mean ± SEM from n = 3 independent experiments.EXAMPLE 7PRODUCTION OF RECOMBINANT BCL-XL PROTEINSAND VALIDATION OF BIOCHEMICAL BINDING ASSAYS

[0213] Figure 2B provides gel filtration chromatograms of wt BCL-XL FL (MW = 26.048 kDa) and BCL-XL R6W A168W (MW= 26.194 kDa). Both protein purifications obtain the elution of proteins of interest at the elution volume of 10.4 mL-12.0 mL.

[0214] Figure 2C provides a validation of FITC-labeled BIM BH3, BAD BH3 and BAK BH3 peptides as a competitive binder by comparing their EC50 values between wt BCL-XL FL and BCL-XL R6W A168W via FP direct binding assays. Data are mean ± SEM from n = 3 independent experiments.

[0215] Figure 2E describes a liposome binding of BCL-XL, tBid and Bax. Both BCL-XL FL and BCL-XL AC proteins, at the indicated concentrations, were incubated with liposomes LiposomesP-656906-PCencapsulated with ANTS and DPX were incubated with 200 nM Bax, 30 nM tBid, and the indicated concentrations of (i) BCL-XL FL and (ii) BCL-XL AC, respectively. Membrane permeabilization was assayed by an increase of ANTS fluorescence, (iii) The correlation between tBid concentration (30 nM) and the IC50 values of BCL-XL FL and BCL-XL AC for inhibition of liposome permeabilization assays. The full-length protein of BCL-XL has lower activity to inhibit membrane binding by Bax via tBid sequestration, due to the hindrance binding from its helix a.9. Data are mean ± SEM from n = 3 independent experiments.EXAMPLE 8BIOPHYSICAL STUDY OF THE SELECTED FIRST BATCH HIT CANDIDATES AND FURTHER BIO ANALYSIS LIGAND BINDING ASSAYS OF THE SECOND BATCH COMPOUNDS

[0216] Figure 2F provides a measured chemical shift perturbations (CSP) of 15N-BCL-XL AC upon each compound titration up to a ratio of 1:4 BCL-XL AC:compound, are plotted as a function of BCL-XL AC residue number. Residues with chemical shift ranges over the significance threshold or 1.5 times the threshold are colored orange and red, respectively. Data are representative of three independent replicates.

[0217] Figure 2G provides a single-dose screening of compounds 17, 31 and 57 with their respective analogs via fluorescence polarization assay which competes against FITC-BID BH3 and FITC-BAX BH3 peptides. A threshold value of 20% (blue line) is set to further characterize compounds that show binding potency (< 20%).

[0218] Figure 2 J provides IC50 / EC50 values of compounds 17j, 17w, 57m and 57j, from fluorescence polarization (second and third columns to the left), and liposome permeabilization assays (the rightmost column). Data are mean ± SEM from / ? = 3 independent experiments.EXAMPLE 9R-GROUP ENUMERATION AND ANALOG SCAFFOLD DESIGN FOR ADX COMPOUNDS

[0219] Figure 2H describes (a) The main core with eight R-groups after R-group analysis from the parental compound 17 and its 33 analogs (left); the importance ranking of each R-group position as predicted by R-group analysis. The importance value of a position is the range of the property over the R groups at that position, averaged over all structures containing a given R groupP-656906-PCat that position, (b) R-groups of compounds 17j and 17w that indicate the most important R-group position, i.e., R6, are colored in yellow, while a common moiety that shared by compounds 17j and 17w are colored in blue. The colored moieties at the specific sites influence compound binding potency and properties, aiding in the design of more effective and optimized analogs, (c) The first designed scaffold for analogs search to increase their potencies and other pharmacokinetic properties.

[0220] Figure 3C provides (a) The main core with eight R-groups after R-group analysis from the parental compound 57 and its 28 analogs (left); the importance ranking of each R-group position as predicted by R-group analysis, based on their chemical and property variability, (b) R-group of compounds 57m and 57j at R2 and R3 that indicate the most important R-group positions, are colored in purple. A common moiety that can be found in both compounds 57m and 57j, are colored in blue. A common R-group moiety of R2 and R3 from compounds 57m and 57j is indicated in a box. (d) The second designed scaffold containing the key functionalities that may improve potency for analogs search.EXAMPLE 10MEASUREMENT OF THE BINDING AFFINITY AND ACTIVITY OF ABX COMPOUNDS VIA BIOCHEMICAL AND IN VITRO ASSAYS

[0221] Figure 2K provides IC50 / EC50values of compounds ABX-27, ABX-28, ABX-36 and ABX-46, as measured from fluorescence polarization assay, liposome release and microscale thermophoresis assays (second, third and fourth column to the left, respectively). The compounds were assayed on BCL-XL FL WT and BCL-XL FL R6W A168W proteins. Data are mean ± SEM from n = 3 independent experiments. Data are mean ± SEM from n = 3 independent experiments.

[0222] Figure 3A provides Caspase 3 / 7 activity of HEL wild-type cell line upon treatment with ABX-27, ABX-28, ABX-2, ABX-36, ABX-46, together with their parent compounds 17j, 17w, 57m and 57j for 24 hrs. All data represent mean ± SEM from n = 3 independent experiments.

[0223] Figure 3B shows a cell viability (Cell-Titer-Glo) of THP-1 wild-type cell line treated with ABX-27, ABX-28, ABX-2, ABX-36, ABX-46 for 72 hrs. All data represent mean ± SEM from n = 3 independent experiments.EXAMPLE 11HDX-MS ANALYSIS OF THE LIGAND-INDUCED EFFECTS OF ABX-27 ON THEP-656906-PCSTRUCTURE OF BCL-XL

[0224] Figure 4A and Figure 4B provide an HDX-MS analysis of the effect of ABX-27 (17jw27) binding to BCL-XL (SEQ ID NO: 16) in solution, in the ratio of 1:1 (Figure 4A) and 1:10 (Figure 4B) The relative difference of % deuterium incorporation of the ABX-27: BCL-XL complex minus the % deuterium incorporation of BCL-XL monomer conformation over 10 sec, 1000 sec and 10000 sec. Regions with reduced or increased deuterium exchange upon addition of BCL-XL are colored blue and red respectively. Views of allosteric and canonical pockets of BCL-XL ribbon structure showing the residues undergoing protection from deuterium incorporation in the ABX-27-BCL-XL complex (17jw27: BCL-XL complex) using blue and light blue color depending on the reduction in deuterium incorporation, according to their concentration ratios. Residues undergoing higher deuterium incorporation in the ABX-27-BCL-XL complex are mapped with pink. Data are representative of two independent experiments.

[0225] Figure 4C and Figure 4D depict an HDX-MS analysis of the effect of ABT-263 binding to BCL-XL (SEQ ID NO: 16) in solution, in the ratio of (Figure 4C) 1:1 and (Figure 4D) 1:10. The relative difference of % deuterium incorporation of the ABT-263: BCL-XL complex minus the % deuterium incorporation of BCL-XL monomer conformation over 10 sec, 1000 sec and 10000 sec. Regions with reduced or increased deuterium exchange upon addition of BCL-XL are colored blue and red respectively. Views of allosteric and canonical pockets of BCL-XL ribbon structure showing the residues undergoing protection from deuterium incorporation in the ABT-263-BCL-XL complex using blue and light blue color depending on the reduction in deuterium incorporation, according to their concentration ratios. Residues undergoing higher deuterium incorporation in the ABT-263-BCL-XL complex are mapped with pink. Data are representative of two independent experiments.EXAMPLE 12NMR-BASED EVIDENCE OF ABX-27-MEDIATED TO BCL-XL INHIBITION

[0226] Figure 5C depicts ABX-27-induced differences in peak intensity ratio (PRE) of peaks in the presence and absence of the soluble paramagnetic probe hydroxyl-TEMPO. Percentage change in PRE is plotted with respect to BCL-XL AC residue number. Residues that exhibit increased or decreased PRE are labeled in shades of red and blue, respectively, the color gradient corresponding to the figure key. Residues associated with the N-terminal allosteric binding site and other helices are highlighted in green and grey respectively.P-656906-PC

[0227] Figure 5D depicts a ribbon representation of BCL-XL AC with mapping of residues undergoing significant differences in PRE as shown in (c). FTMap-predicted Site (2) is mapped as black dots on the protein structure to validate the localization of the novel allosteric binding site.EXAMPLE 13ABX-27 BINDS TO BCL-XL AT A NOVEL ALLOSTERIC SITE

[0228] Figure 5A depicts chemical shift perturbations (CSPs) of15N-labeled BCL-XL measured in the presence of 1:1 and 1:2 BCL-XL: ABX-27 compared (BCL-XL:17jw27 compound) to unbound BCL-XL are plotted as a function of BCL-XL residue number. Residues with chemical shift perturbations over the significance threshold or two times the significance threshold are labeled light blue or dark blue, respectively. The black dotted line represents the average CSP. Residues that are part of the eight helices of the BCL-XL AC structure are designated with bluecolored shading.

[0229] Figure 5B depicts a mapping of residues undergoing significant CSPs onto the ribbon and surface representation of BCL-XL, as in (Figure 5A) in blue gradient colors. SiteMap-predicted Site (1) and Site (2) are mapped as black and white dots, respectively on the protein structure to illustrate the changes in the canonical pocket and allosteric binding site during the NMR experiments.EXAMPLE 14ALLOSTERIC INHIBITION MECHANISM DUE TO THE BINDING OF ABX-27 IN SITE (2) OF BCL-XL

[0230] Figure 7A provides a close up view of the computationally-predicted lowest energy docked pose of ABX-27 (represented in green stick) that is consistent with biophysical and biochemical studies (left), displaying the graphical representations of hydrogen bond (yellow), green (Pi-cation) and van der Waals interactions (orange) with adjacent residues R6, W24, S28, D29 and Al 68 (represented in ball-and-stick) within Site (2). Ligand interaction diagram of Maestro shows the docking efficiency of ABX-27 was strengthened via hydrogen bonding with residue S28 and Pi-cation interaction with residue R6 (right).

[0231] Figure 7B depicts Changes in structure and dynamics of the helices al and a6 interface. Transparent surface with ribbon representation of helices al and a6 interface with main residues highlighted in orange (left), and graphical representation of distances between residues at theP-656906-PCinterface (right). Distances for BCL-XL and BCL-XL-ABX-27 complex MD simulations are represented in black and orange respectively.

[0232] Figure 7C depicts Changes in structure and dynamics of the canonical site opening formed by a5, a8, and a9. Transparent surface with ribbon representation of the canonical site opening with main residues highlighted in orange (left), and graphical representation of distances between residues at the canonical site opening (right). Distances for BCL-XL and BCL-XL-ABX-27 complex MD simulations are represented in black and orange respectively. In Figure 7B and Figure 7C, distances represent the mean difference of n = 3 BCL-XL and BCL-XL-ABX-27 MD simulations.

[0233] Figure 7D depicts an overlay of structures of BCL-XL-ABX-27 complex from every 100 ns intervals from 0 to 1000 ns of MD simulations. ABX-27 color spectrum corresponds to time as described, BCL-XL ribbon structure is colored gray with residues of interest represented as sticks for clarity.EXAMPLE 15ABX-27 BINDS SELECTIVELY TO BCL-XL AND CREATES SYNERGISTIC STABILITY TO BCL-XL ABX-27 COMPLEX

[0234] Figure 10A describes a selectivity of compounds (i) A-1331852, (ii) ABT-263 and (iii) ABX-27 towards BCL-XL, are examined against Bcl-2 AC protein via dose-dependent FP binding assay. Data are mean ± SEM from n = 3 independent experiments.1H-15N HSQC of 50 μM.

[0235] Figure 8A depicts Ca atoms of ABX-27-bound BCL-XL average RMSD plotted against molecular dynamics (MD) time (blue), compared to the Ca atom average RMSD of apo BCL-XL (orange), over three independent 1000 ns simulations, respectively.

[0236] Figure 8B depicts Ca atoms of ABX-27-bound BCL-XL average RMSF plotted against BCL-XL residues 1-233 (blue), compared to the Ca atom average RMSF of apo BCL-XL (brown), over three independent 1000 ns simulations, respectively.

[0237] ABX-27 bound full-length BCL-XL with nanomolar affinity (IC50 = 163-268 nM in FP and IC50 = 199 in MST assays) and displayed strong selectivity for the allosteric site over the BH3 groove (Figures 9A-9D). Selectivity was further evaluated against BCL-2 AC, the closest BCL-XL homolog. In dose-dependent FP assays, ABX-27 displayed stronger binding to BCL-XL than BCL-2, confirming preferential selectivity to BCL-XL (Figure 10A). This selectivity wasP-656906-PCcomparable to the BCL-XL-selective inhibitor A-1331852 in contrast to the BCL-XL / BCL-2 inhibitor navitoclax (Figures 10A-10B). To confirm cellular on-target selectivity, ABX-27-induced apoptosis was compared in THP-1 leukemia wild-type (BCL-XL WT) cells versus BCL-XL knockdown (KD) cells. Caspase-3 / 7 activation was pronounced in BCL-XL WT cells (>0.5 pM) but minimal in BCL-XL KD cells even at 20 pM (Figures 9E-9F). This selectivity was comparable to A-1331852 and navitoclax. Moreover, the cell viability assay in THP-1 cells supported BCL-XL-dependent activity for ABX-27 consistent with its caspase-3 / 7 activation (Figures 9G-9H)

[0238] Kasumi-1 leukemia cells also showed sensitivity to ABX-27 in caspase-3 / 7 activation and cell viability assays (Figures 9I-9J). Western blot analysis confirmed apoptotic signaling, with increased PARP and caspase-3 cleavage following ABX-27 treatment (Figure 10C). Moreover, separation of cytosolic from mitochondrial fractions revealed the release of cytochrome c into the cytosol upon ABX-27 treatment (Figure 10D). These findings are consistent with ABX-27 acting through BCL-XL to induce mitochondrial outer membrane permeabilization and activate downstream apoptotic signalling. Strikingly, ABX-27 did not induce measurable caspase-3 / 7 activation or cytotoxicity in human platelets, in contrast to A-1331852 and navitoclax, which induced robust apoptotic responses. (Figures 9K-9L, Figure 10E). This differential effect may reflect distinct mechanisms of BCL-XL-BH3 complex disruption by allosteric versus orthosteric inhibition in platelets, although further studies are required to elucidate the underlying molecular basis. Thus, ABX-27 demonstrated a favorable balance of potency, selectivity, and functional activity in BCL-XL-dependent leukemia models.

[0239] Figure 11 shows in silico ADME and pharmacokinetic profiling of ABX-27. Computational assessment of ABX-27 reveals favorable physicochemical, pharmacokinetic, and drug-like properties. The compound exhibits desirable lipophilicity, high gastrointestinal absorption, blood-brain barrier permeability, and oral bioavailability. It meets key drug-likeness and medicinal chemistry filters, supporting its developability as an orally bioavailable, CNS-permeable, allosteric BCL-XL inhibitor.EXAMPLE 16SYNTHESIS OF BCL-XL INHIBITOR ABX-27

[0240] General Methods and Materials

[0241] All chemical reagents and solvents were obtained from commercial sources (Aldrich,P-656906-PCAcros, Fisher, Ambeed) and used without further purification unless otherwise noted. Analytical thin layer chromatography (TLC) was performed on glass or aluminum-backed Silicycle silica gel plates (250 mm film thickness, indicator F254). Compounds were visualized using a dual wavelength (254 and 365 nm) UV lamp, and / or staining with KMnO4 stains. NMR spectra were recorded on Bruker Avance IIIHD 600 MHz or Bruker Avance IIIHD 300 MHz system. 'H chemical shifts (8) are reported relative to tetramethyl silane (TMS, 0.00 / 0.00 ppm) as an internal standard or to residual solvent (CDCI3: 7.26 / 77.16 ppm; DMSO-d6: 2.50 / 39.52 ppm).-

[0242] Synthesis of BCL-XL Inhibitor ABX-27

[0243] tert-butyl 3-(l / / -pyi azol-5-yl)piperidine-l-caiboxylate (2): To a 50 mL round bottom flask with a magnetic stir bar, 3-(l / / -pyrazol-5-yl)piperidine hydrochloride (1, 1 g, 4.46 mmol, 1 equiv.) was added and dissolved in 9 mL DCM. DIPEA (3.09 mL, 17.85 mmol, 4 equiv.) was added to the mixture dropwise and the solution was stirred for an additional 5 min. BocsO (1.13 mL, 4.91 mmol, 1.1 equiv.) dissolved in 3 mL DCM was then added dropwise to the flask over 5 min. The reaction mixture was stirred overnight, and the reaction was monitored using TLC. After completion of the reaction, the reaction mixture was transferred to a separatory funnel, and the organic layer was washed successively with distilled water (2 >< 5 mL) and brine (2 x 5 mL). The organic layer was then dried over sodium sulfate before being filtered and concentrated in vacuo. The crude oily liquid was purified via flash chromatography. Isolated 1.08 g, 96%. ColorlessP-656906-PCviscous liquid.

[0244] rf-butyl 3-(LH-pyrazoI-5-yl)piperidine-l-carboxylate (3): The compound 3 was prepared according to a reported procedure with slight modifications (Molecular Diversity (2017), 21(2), 317-323). 2 (1.08 g, 4.30 mmol, 1 equiv.) was dissolved in DCM (43 mL), and N-bromosuccinimide (841.31 mg, 4.73 mmol, 1.10 equiv.) was added in portions. The mixture was stirred at room temperature for 2 h, and the reaction was monitored using TLC. After completion of the reaction, the reaction mixture was transferred to a separatory funnel, and the organic layer was washed successively with saturated sodium thiosulphate solution (1 x 10 mL), distilled water (2 x 10 mL) and brine (1 x 10 mL). The organic layer was then dried over sodium sulfate before being fdtered and concentrated in vacuo. The crude product was purified via flash chromatography on silica gel (hexanes-ethyl acetate). Isolated 710 mg, 50%, white powder.

[0245] toT-butyl 3-(4-(p-tolyl)-l / / -pyrazol-5-yl)piperidine-l-carboxylate (4): To a 20 mL glass vial with a stir bar, 3 (200 mg, 0.61 mmol, 1 equiv.) was added and dissolved in dioxanewater (4 mL, 4:1 ratio). Pd2(dba)3 (16.64 mg, 0.03 equiv.), X-Phos (17.32 mg, 0.06 equiv.), potassium carbonate (167.4 mg, 2 equiv.) and / i-tolylboronic acid (165 mg, 2 equiv.) was added successively. The vial was sealed, and the reaction mixture was heated at 65 °C for 16 h. The reaction was monitored with TLC. After completion, DCM (5 mL) was added to the reaction mixture. The organic layer was extracted with distilled water (2 x 2 mL), The organic layer was then dried over anh. sodium sulfate, filtered and the solvent removed in vacuo. The crude product was used in the next steps without further purification.

[0246] 3-(4-(p-tolyl)-TH-pyrazol-5-yl)piperidine (5): 100 mg of 4 was dissolved in 2 mL DCM in a 20 mL glass vial loaded with a stir bar. 2 mL of trifluoroacetic acid was added dropwise to the solution using a syringe. The reaction mixture was stirred under an argon balloon for 2 h, after which, TLC showed complete conversion of the starting material. The solvent was removed in vacuo. The crude product was neutralized with a saturated solution of sodium bicarbonate, and extracted with DCM (3 x 3 mL). The organic layer was then dried over anh. sodium sulfate, filtered and the solvent was removed in vacuo. The crude product was purified on preparative TLC (1000 micron). ^NMR (600 MHz, CDC13) 57.59 (s, 1H), 7.21 (q, J= 7.8 Hz, 4H), 3.37 (t, J= 5.5 Hz, 1H), 3.27 (tq, J= 12.3, 5.9, 4.0Hz, 2H), 3.11 - 3.01 (m, 2H), 2.38 (s, 3H), 1.94 (td, J= 10.7, 10.3, 5.7 Hz, 1H), 1.82 (dq, J= 10.2, 5.4, 4.6 Hz, 2H), 1.66 (ddt, J= 14.8, 10.1, 5.5 Hz, 1H).

[0247] ( 1 / / -pyi iol-2-yl)(3-(4-( / i-tolyl)-l / / -pyrazol-5-yl)piperidin- 1 -yl)iiiethanone (6): To a 4P-656906-PCmL glass vial with a stir bar, 5 (20 mg, 0.08 mmol, 1 equiv.) was added and dissolved in DCM (0.4 mL). 177-pyrrole-2-carboxylic acid (11 mg, 1.2 equiv.), EDC. HC1 (19 mg, 1.2 equiv.), and DIPEA (29 pL, 2 equiv.) were added successively. DCM (300 pL) was added down the glass wall to rinse down any reagent. The reaction mixture was stirred for 12 h and monitored with TLC. After completion, DCM (5 mL) was added to the reaction mixture. The organic layer was washed with distilled water (2 x 2 mL) and brine (1 x 1 mL). The organic layer was then dried over anh. sodium sulfate, and the solvent was removed in vacuo. The crude product was purified over preparative TLC using 80% ethyl acetate in hexanes (Rf 0.4). Isolated 16 mg, 57%, white powder.1H NMR (600 MHz, CDC13) 6 10.32 (s, 1H), 7.60 (s, 1H), 7.27 (d, J= 11.8 Hz, 2H), 7.21 (d, J = 7.9 Hz, 2H), 6.92 (d, J= 3.1 Hz, 1H), 6.34 (s, 1H), 6.16 (s, 1H), 4.54 (d, J= 12.8 Hz, 1H), 4.42 (s, 1H), 3.54 (s, 1H), 3.31 (d, J= 11.4 Hz, 1H), 2.39 (s, 3H), 2.10 (s, 1H), 1.95 (dtd, J= 13.8, 10.6, 3.8 Hz, 1H), 1.74 (d, J = 262 Hz, 1H), 1.62 (qd, J = 10.7, 6.8 Hz, 1H). LCMSffl / z: [M + H]+calculated for chemical formula C20H23N4O; Exact Mass: 335.19; found, 335.4.P-656906-PCTABLE 1. List of screened compounds with their molecular structures and percent competition against FITC-BID BH3 and FITC-BAX BH3 peptides bound to BCL-XL in fluorescence polarization assays.P-656906-PCP-656906-PCP-656906-PCP-656906-PCP-656906-PCP-656906-PCP-656906-PCP-656906-PCTABLE 2. List of screened second-generation compounds with their molecular structures and percent competition against FITC-BID BH3 and FITC-BAX BH3 peptides bound to BCL-XL in fluorescence polarization assays.P-656906-PCP-656906-PCP-656906-PCP-656906-PCP-656906-PCP-656906-PCP-656906-PCP-656906-PCP-656906-PCP-656906-PCP-656906-PCTABLE 3. List of screened third-generation compounds with their molecular structures and showing competition against FITC-BID BH3 and FITC-BAX BH3 peptides bound to BCL-XL in fluorescence polarization assays.P-656906-PCP-656906-PCP-656906-PCP-656906-PCP-656906-PCP-656906-PCP-656906-PC

[0248] While certain features of the invention have been illustrated and described herein, many modifications, substitutions, changes, and equivalents will now occur to those of ordinary skill in the art. It is, therefore, to be understood that the appended claims are intended to cover all such modifications and changes as fall within the true spirit of the invention.

Claims

P-656906-PCCLAIMSWhat is claimed:

1. A compound represented by Formula (I)or a pharmaceutically acceptable salt thereof or a stereoisomer thereof,whereinX is a bond or CH2;A is H, alkyl, cycloalkyl, or aryl;B is heterocycloalkyl, wherein the heterocycloalkyl with the pyrazole ring to which it is attached may form a fused heterocyclic ring system;G is alkyl, alkenyl, alkynlyl, C(=O)RP, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; n is 0, 1, 2, or 3;wherein the aryl and heteroaryl are each optionally substituted with one or more groups independently selected from C1-C5 alkyl, halo, haloalkyl, aryl, CN, ORP, NRpRq, RPOH, CHO, COORP, and CONRpRq;wherein the alkyl, cycloalkyl, heterocycloalkyl, and the fused heterocyclic ring are each optionally substituted with one or more groups independently selected from =0, halo, haloalkyl, CN, RqORPORP, NRpRq, NRpCORq, RP0H, CHO, cyclopropyl, heteroaryl, COORp, and CONRpRq; andRpand Re, are independently H or C1-C5 alkyl;wherein Rpand Rqcan optionally be taken together to form a 3-6 membered ring that can be optionally substituted by one or more groups independently selected independently from OH, =0, or C1-C5 alkoxy.

2. The compound according to claim 1, wherein A is aryl.

3. The compound according to claim 1, wherein A is phenyl or methylphenyl.P-656906-PC4. The compound according to claim 1, wherein B is heterocycloalkyl.

5. The compound according to claim 1, wherein B is a 5- or 6-membered heterocycloalkyl.

6. The compound according to claim 1, wherein B is a piperidinyl.

7. The compound according to claim 1, wherein n is 0 or 1.

8. The compound according to claim 1, wherein G is heteroaryl.

9. The compound according to claim 1, wherein G is pyrrolyl or indazolyl.

10. The compound according to claim 1, wherein Formula (I) is represented by Formula (II)wherein Ai, A2, A3, A4, and As are each independently H, C1-C5 alkyl, halo, CN, ORP, NRpRq, COORp, or CONRp.

11. The compound according to claim 10, wherein B is heterocycloalkyl.

12. The compound according to claim 10, wherein B is a 5- or 6-membered heterocycloalkyl.

13. The compound according to claim 10, wherein B is a piperidinyl.

14. The compound according to claim 10, wherein n is 0.P-656906-PC15. The compound according to claim 1, wherein Formula (I) is represented by Formula (III)wherein Ai, A2, A3, A4, and As are each independently H, C1-C5 alkyl, halo, haloalkyl, CN, ORP, NRpRq, COORp, or CONRP.

16. The compound according to any one of claims 10-15, wherein Ai, A2, A3, A4, and As are each independently H or C1-C5 alkyl.

17. The compound according to any one of claims 10-15, wherein Ai, A2, A3, A4, and As are H.

18. The compound according to any one of claims 10-15, wherein A3 is C1-C5 alkyl and Ai, A2, A4, and As are H.

19. The compound according to any one of claims 10-15, wherein G is heteroaryl.

20. The compound according to any one of claims 10-15, wherein G is pyrrolyl or indazolyl.

21. A compound represented by Formula (IV)or a pharmaceutically acceptable salt thereof or a stereoisomer thereof,whereinP-656906-PCRn, R12, R13, Ri4, and Ris are each independently H, alkyl, halo, haloalkyl, CN, ORP, NRpRq, COORp, or CONRpRq;R« is COORp or CONRpRq;Rmand Rnare each independently H, alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, wherein each of alkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl is optionally substituted with one or more groups independently selected from halo, ORP, C1-C5 alkyl, haloalkyl, CN, NRpRq, RPOH, CHO, cyclopropyl, COORP, and CONRpRq;Rmand Rncan optionally be taken together with the nitrogen atom to which they are attached to form a 3-15 membered ring that is optionally substituted by one or more groups independently selected independently from =0, C1-C5 alkyl, CN, 0RP, NRPRq, RP0H, CHO, cycloalkyl, heterocyclalkyl, aryl, arylalkyl, heteroaryl, and heteroarylalkyl; andRpand Rq are independently H or C1-C5 alkyl.

22. The compound according to claim 21, wherein Rn, R12, R13, R14, and R15 are each H, C1-C5 alkyl, or halo.

23. The compound according to claim 21, wherein R1 is halo and Rn, R12, R14, and R15 are H.

24. The compound according to claim 21, wherein R13 is chloro and Rn, R12, R14, and R15 are H.

25. The compound according to claim 21, wherein Rn, R12, R13, R14, and R15 are H or C1-C5 alkyl.

26. The compound according to claim 21, wherein Rn, R12, R13, R14, and R15 are H or methyl.

27. The compound according to claim 21, R, is COORp.

26. The compound according to claim 21, R, is COOCH3 or COOC2H5.

27. The compound according to claim 21, wherein Rmand Rn are H or aryl, wherein aryl is optionally substituted with halo, haloalkyl, CN, ORP, COORp, or CONRp.

28. The compound according to claim 21, wherein Rmand Rnare H or aryl, wherein aryl is substituted with haloalkyl.P-656906-PC29. The compound according to claim 21, wherein Rmand Rnare taken together with the nitrogen atom to which they are attached to form a 5- or 6-membered ring that is optionally substituted by =0, C1-C5 alkyl, cycloalkyl, heterocyclalkyl, aryl, or heteroaryl.

30. The compound according to claim 21, wherein Rmand Rnare taken together with the nitrogen atom to which they are attached to form a 6-membered ring that is optionally substituted by heteroaryl.

31. A compound represented by Formula (V)or a pharmaceutically acceptable salt thereof or a stereoisomer thereof,whereinRa and Rb are each independently H, alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, wherein each of alkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl is optionally substituted with one or more groups independently selected from halo, haloalkyl, CN, ORP, NRpRq, RPOH, CHO, cyclopropyl, COORp, and CONRpRq;Ra and Rb can optionally be taken together with the nitrogen atom to which they are attached to form a 3-15 membered ring that is optionally substituted by one or more groups independently selected independently from =0, C1-C5 alkyl, CN, 0RP, NRPRq, RpOH, CHO, cycloalkyl, heterocyclalkyl, aryl, arylalkyl, heteroaryl, and heteroarylalkyl; andRpand Rq are independently H or C1-C5 alkyl.P-656906-PC32. The compound according to claim 31, wherein Ra and Rb are H, alkyl, cycloalkyl, or heterocycloalkyl, wherein alkyl, cycloalkyl, and heterocycloalkyl are each optionally substituted with halo, haloalkyl, CN, or ORP.

33. The compound according to claim 31, wherein Raand Rb are taken together with the nitrogen atom they are attached to form a 3-15 membered ring that is optionally substituted by =0 or ORP.

34. A compound represented by any one of the following structuresP-656906-PC 35. A compound represented by any one of the following structuresP-656906-PCP-656906-PC36. A compound represented by any one of the following structuresP-656906-PCP-656906-PC37. A compound represented by any one of the following structuresP-656906-PCP-656906-PC38. A compound represented by any one of the following structuresP-656906-PCP-656906-PCP-656906-PCP-656906-PC39. A compound represented by any one of the following structuresP-656906-PCP-656906-PCP-656906-PCP-656906-PC40. A method for treating cancer in a subject in need thereof, comprising administering a therapeutically effective amount of a compound, or a pharmaceutically acceptable salt thereof or a stereoisomer thereof, according to any one of claims 1-39.

41. The method according to claim 40, wherein the cancer is acute myeloid leukemia, chronic lymphocytic leukemia, acute lymphoblastic leukemia, lymphoma, multiple myeloma, non-small cell lung cancer, small cell lung cancer, triple-negative breast cancer, colorectal cancer, head and neck squamous cell carcinoma, melanoma, mesothelioma, glioblastoma, and pancreatic neuroedocrine tumors.

42. A method for identifying BCL-XL inhibitors, comprising the steps of:(i) determining an allosteric site on full length BCL-XL;(ii) selecting compounds that bind to the allosteric site; and(iii) evaluating the selected compounds by experimental screening and validation.

43. The method according to claim 42, wherein determining an allosteric site on full length BCL-XL comprises:(a) calculating a 3D structural model of the human full-length BCL-XL using a truncated crystal structure of BCL-XL and a full length BAX structure as a template for the conformation of the c-terminal helix 9 of BCL-XL;(b) performing molecular dynamics (MD) simulations on the BCL-XL structural model to explore conformational dynamics and using conformations to feed automated binding site identification and analysis tools;(c) analyzing predicted binding sites for druggability based on site properties;(d) validating the predicted binding sites with NMR paramagnetic relaxation enhancement (PRE) experiments using hy-TEMPO probe to identify solvent accessible sites on the surface of BCL-XL accessed by TEMPO probe and confirming the predicted binding sites;(e) selecting a binding Site 2 that is adjacent to the N-terminal intrinsically disordered region (al-a2 loop) of BCL-XL and investigating changes of the binding site in the MD simulation trajectory;(f) maintaining the binding site with minor changes over the course of the simulation despite significant changes in al-a2 loop conformation adjacent to the Site 2; andP-656906-PC(g) using mutagenesis of the binding site residues and identifying residues R6 and A 168 in helices al and a6 as the hot spot residues largely determining the identification of the Site 2 by Sitemap.

44. The method according to claim 42, wherein selecting compounds that bind to the allosteric site comprises:(h) identifying compounds binding to Site 2 according to claim 42 by performing in silico screening using a compound library via a three-tier docking methodology which starts with high throughput virtual screening (HTVS) followed by standard precision (SP) and then by extraprecision mode (XP) screening mode; and(i) selecting compounds for favorable ADMET properties.

45. The method according to claim 42, wherein evaluating the selected compounds by experimental screening and validation, comprises:(i) employing various biochemical, biophysical and cell-based assays for BCL-XL binding, BCL-XL inhibition activity and binding to the allosteric site as well as apoptosis related assays to establish production of full length BCL-XL wt and mutant R6W / A168W to interrogate binding to and allosteric effects mediated by Site 2;(ii) screening the selected compounds using a Fluorescence Polarization (FP) assay for competing BCL-XL-BTD and BCL-XL-BAX binding and prioritizing hits with > 70% inhibition for either complex;(iii) establishing a functional assay for full length BCL-XL activity in promoting inhibition of B AX-mediated membrane permeabilization using liposomal membranes;(iv) using FP, liposomal assay, MST direct binding to BCL-XL and HSCQ-NMR analysis using full length BCL-XL wt and R6W / A168W mutant to identify and prioritize lead compounds that show the best binding to BCL-XL and reduce binding / activity to the mutant;(v) preparing and designing new analogs of lead compounds and screening for binding and activity using the same assays;(vi) screening the new analogs and identifying more potent inhibitors that are also inhibited by R6W / A168W mutant but not by a mutant in the canonical site that typically disrupts established BCL-XL inhibitors G138W to obtain compounds that have potent cellular activity and selectivity for binding to BCL-XL compared to BCL-2;P-656906-PC(vii) performing additional experiments with the obtained potent compounds using structural and biophysical HSQC-NMR, NMR-PRE, and HDX-MS analysis that demonstrate specificity for binding to Site 2 and allosteric effects at the C-terminal canonical site of BCL-XL to explain the mechanism of site 2 binding molecules to compete BH3 domains from the BCL-XL canonical site;(viii) recapitulating the allosteric effects from compound-binding to Site 2 in molecular dynamics simulations that show allosteric conformational changes in the canonical pocket in the presence of the potent compound by increased interatomic distances that include the C-terminal helix 9.

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