WO2023091747A1 - Composés modulateurs, compositions pharmaceutiques et leurs utilisations - Google Patents

Composés modulateurs, compositions pharmaceutiques et leurs utilisations Download PDF

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WO2023091747A1
WO2023091747A1 PCT/US2022/050564 US2022050564W WO2023091747A1 WO 2023091747 A1 WO2023091747 A1 WO 2023091747A1 US 2022050564 W US2022050564 W US 2022050564W WO 2023091747 A1 WO2023091747 A1 WO 2023091747A1
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tlr2
cancer
pharmaceutical composition
modulator
substituted
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Patrick J. STERN
Christopher J. OALMANN
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Axial Therapeutics, Inc.
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/58—Medicinal preparations containing antigens or antibodies raising an immune response against a target which is not the antigen used for immunisation
    • A61K2039/585—Medicinal preparations containing antigens or antibodies raising an immune response against a target which is not the antigen used for immunisation wherein the target is cancer

Definitions

  • the present disclosure relates to modulators of Toll-like receptor (TLR) proteins, and particularly modulators of TLR2, as well as methods of using such compounds to treat cancer and other disorders in combination with immune checkpoint therapy.
  • TLR Toll-like receptor
  • the innate immune system contains several families of germline-encoded pattern recognition receptors (PRRs), including Toll-like receptors (TLRs), Nod-like receptors (NLRs), RIG- 1 -like receptors (RLRs), cytosolic DNA sensors (CDSs) and C-type lectins (CLRs) (Newton and Dixit 2012).
  • PRRs germline-encoded pattern recognition receptors
  • TLRs Toll-like receptors
  • NLRs Nod-like receptors
  • RLRs RIG- 1 -like receptors
  • CDSs cytosolic DNA sensors
  • CLRs C-type lectins
  • PAMPs pathogen-associated molecular patterns
  • PAMPs include lipid-based bacterial cell wall components such as lipoproteins and lipopolysaccharides, microbial protein components such as flagellin, and pathogen nucleic acids in the form of double stranded DNA and single or double stranded RNA.
  • PRRs also recognize self ligands known as damage-associated molecular patterns (DAMPs) released from damaged or dying cells and tissues.
  • DAMPs damage-associated molecular patterns
  • Cells of the innate immune system respond to PAMPs and DAMPs by producing proinflammatory cytokines, chemokines, and co-stimulatory molecules that are involved in clearing the pathogens and damaged-self.
  • innate immune responses essentially shape the downstream adaptive immune responses to generate a more specific and long-lasting immunity (Hoebe et al. 2004; Pasare and Medzhitov 2005). As such, harnessing innate immune signaling pathways is a promising therapeutic strategy to fight infections, immune disorders, and diseases such as cancer.
  • TLRs are a well-studied class of innate immune receptors recognizing a diverse range of lipid-, protein-, nucleic acid-based PAMPs and DAMPs (Kawai and Akira 2011).
  • the engagement of TLRs with their specific ligands leads to the activation of innate immune responses, and evokes adaptive immune responses through the activation of antigen presenting cells (APCs) and by amplifying B- and T-cell effector responses (Pasare and Medzhitov 2005; MacLeod and Wetzler 2007).
  • APCs antigen presenting cells
  • B- and T-cell effector responses Pasare and Medzhitov 2005; MacLeod and Wetzler 2007.
  • Several studies have demonstrated stimulation of TLRs with specific ligands and combinations thereof to fine-tune adaptive immune responses.
  • TLRs the aberrant TLR expression in cancer cells and several TLR polymorphisms identified in tumors indicate a role for TLRs in cancer.
  • the infiltration of TLR-expressing immune cells into the tumor microenvironment further implies the significance of TLRs in relation to cancer (Bennaceur et al. 2009; Sato et al. 2009).
  • the precise contribution of TLRs to cancer remains to be understood. Activation of TLRs can have opposing effects, by either promoting cancer cell apoptosis or promoting tumor progression and survival (Huang et al. 2008).
  • TLRs are promising targets for the development of new and effective therapeutic agents (Kanzler et al. 2007; Wang et al. 2008; So and Ouchi 2010).
  • Several small molecules agonists of TLRs have been identified for use as immune stimulants to boost immunity against cancer (Meyer and Stockfleth 2008).
  • TLR2 requires heterodimerization with either TLR1 or TLR6 for activation and intracellular signal transduction by bacterial acylated lipoproteins.
  • a widely-recognized agonist that activates the heterodimer TLR1/TLR2 is synthetic tri- acylated lipopolypeptide PAM3CSK4, and bacterial diacylated lipopolypeptides such as MALP-2 stimulate the TLR2/TLR6 heterodimer (Muhlradt et al., J. Exp. Med. 1997, 185: 1951).
  • TLR1 and TLR6 evolved from a common precursor gene, and their extracellular domains diversified over time to bind a broad array of pathogen-associated ligands.
  • the intracellular signaling domains of TLR1 and TLR6 are virtually identical, reinforcing the concept that TLR2 may interact with a diversity of extracellular ligands through its binding partners and then elicit a common intracellular signaling response.
  • Reported TLR2 ligands are diverse in physical structure, yet the immune response elicited from these diverse structures appears largely stereotyped (Ozinsky A. et al., 2000.
  • the repertoire for pattern recognition of pathogens by the innate immune system is defined by cooperation between toll-like receptors. PNAS, 97: 13766-13771), eliciting both inflammatory (TNF-alpha, IL6) and immune-suppressive (IL 10) cytokines in vitro and enhancing the efficacy of vaccination in vivo.
  • TLR2/1- and TLR2/6-binding ligands Reported differences in signaling between TLR2/1- and TLR2/6-binding ligands likely arise from differences in expression of the TLR2 and its co-receptors in different cell types, such as dendritic cells or regulatory T cells, that possess different immunoregulatory capacities.
  • TLR agonists are utilized often in vaccine preparations to enhance immune responses to specific administered antigens, such as in tumor vaccines (Duthie et al. Immunol Rev 2011). Local administration, as with a vaccine, can result in greatly enhanced immune activation. Systemic administration of TLR agonists, such as intravenous administration, can result in disseminated inflammation and toxicity.
  • Checkpoint inhibitors for cancer therapy often referred to as checkpoint therapies, cover a broad class of targets present on either the tumor itself, immune cells that interact with the tumor, or immune-regulatory cells present in lymphoid organs.
  • the most prominent targets are the PD1/PD-L1 and CTLA4 pathways, and also include TIGIT, TIM3, 0X40, OX40L, ICOS, CD27, BTLA, LAG-3, KIR, GITR, 4-1BB and others.
  • checkpoint therapy provides benefit in only a subset of treated patients (see, Immune checkpoint inhibitors: recent progress and potential biomarkers, Pramod Darvin, Salman M. Toor, Varun Sasidharan Nair, Eyad Elkord Experimental & Molecular Medicine volume 50, pagesl-11 (2018)). Approximately two-thirds of treated patients do not respond to checkpoint therapy and these non-responders present a significant patient population with unmet medical needs (Alyson Haslam, Vinay Prasad (2019) JAMA Netw Open. 2:el92535).
  • TLR2 Toll-like Receptor 2
  • TLR2 Toll-like Receptor 2
  • Compound B shows pro-inflammatory activity in vitro, eliciting a broad cytokine response from human and mouse macrophage-like cell lines with low nanomolar potency.
  • Compound B significantly restored the efficacy of anti-PDl therapy in vivo in a mouse tumor model where antibiotics treatment abrogated anti-PDl therapy.
  • mice bearing MCA205 fibrosarcoma tumors responded well to anti-PDl therapy with significantly delayed tumor growth and prolonged survival.
  • TLR2 modulating compounds have the potential to enhance ICI therapy by modulating the anti- tumor immune response via activation of TLR2 in the gut.
  • a daily oral dosing regimen with a TLR2 agonist that enhances the efficacy of checkpoint therapy to treat cancer. It is demonstrated that daily oral dosing with a TLR2 agonist can convert checkpoint therapy non-responders into responders and significantly enhance the efficacy of cancer treatment.
  • FIG. 1 depicts TLR2 modulators used in the studies described herein.
  • FIG. 2A depicts a dosing scheme for oral administration of TLR2 agonists in combination with anti-PDl checkpoint therapy in ABX-treated, MCA205 tumor model corresponding to the data of FIG. 3.
  • Mice were pre-treated with antibiotics (ABX) and test compounds for 40 days: 10 days before tumor implant and for 30 days post-implant. Mice were administered anti-PDl at the indicated times. Tumor volume was measured beginning on Day 6.
  • FIG. 2B depicts a dosing scheme for oral administration of TLR2 agonists in combination with anti-PDl checkpoint therapy in ABX-treated, MCA205 tumor model corresponding to the data of FIGs. 4-12.
  • Mice were treated with oral TLR2 agonists and ABX for 40 days. After 10 days of pre-treatment, MCA205 tumor cells were implanted subcutaneously in the flank of the mice. Mice were administered anti-PDl on the indicated days. Tumor volume was measured every 2-3 days beginning on Day 6 post tumor implant.
  • FIG. 3 depicts the results of tumor progression in the MCA205 model with oral diprovocim.
  • Antibiotics suppress the efficacy of anti-PDl checkpoint therapy.
  • Daily oral dosing with 160 pg/kg (4 pg/mouse) diprovocim restores anti-PDl efficacy to similar levels as mice without ABX.
  • FIG. 4 shows that Compound A enhances efficacy of anti-PDl checkpoint inhibitor in mouse tumor model.
  • Daily oral treatment with 1 pg Compound A improves tumor growth suppression by anti-PDl treatment in the MCA205 mouse model.
  • FIG. 5 shows that Compound B enhances efficacy of anti-PDl checkpoint inhibitor in mouse tumor model.
  • Daily oral treatment with 1 pg Compound B improves tumor growth suppression by anti-PDl treatment in the MCA205 mouse model.
  • FIG. 6 shows that Compound C enhances efficacy of anti-PDl checkpoint inhibitor in mouse tumor model.
  • Daily oral treatment with 1 pg Compound C improves tumor growth suppression by anti-PDl treatment in the MCA205 mouse model.
  • FIG. 7 shows that Compound D enhances efficacy of anti-PDl checkpoint inhibitor in mouse tumor model.
  • Daily oral treatment with 1 pg Compound D improves tumor growth suppression by anti-PDl treatment in the MCA205 mouse model.
  • FIG. 8 shows that Pam2CSK4 enhances efficacy of anti-PDl checkpoint inhibitor in mouse tumor model.
  • Daily oral treatment with 0.1 pg Pam2CSK4 improves tumor growth suppression by anti-PDl treatment in the MCA205 mouse model.
  • FIG. 9 shows that Pam3CSK4 enhances efficacy of anti-PDl checkpoint inhibitor in mouse tumor model.
  • Daily oral treatment with 1 pg Pam3CSK4 improves tumor growth suppression by anti-PDl treatment in the MCA205 mouse model.
  • FIG. 10 shows that Diprovocim enhances efficacy of anti-PDl checkpoint inhibitor in mouse tumor model.
  • Daily oral treatment with 1 pg diprovocim improves tumor growth suppression by anti-PDl treatment in the MCA205 mouse model.
  • FIG. 11 shows that Compound B prolongs survival in combination with anti-PDl checkpoint inhibitor treatment in a mouse tumor model.
  • Daily oral treatment with 1 pg Compound B in combination with anti-PDl treatment prolongs survival in the MCA205 mouse model compared to anti-PDl treatment alone.
  • FIG. 12 shows that diprovocim prolongs survival in combination with anti-PDl checkpoint inhibitor treatment in a mouse tumor model.
  • Daily oral treatment with 1 pg diprovocim in combination with anti-PDl treatment prolongs survival in the MCA205 mouse model compared to anti-PDl treatment alone.
  • FIG. 13 shows that Compounds A, B, D, and Diprovocim show fecal exposure following oral dosing. Mice were orally dosed with 20 mg/kg Compound A, B, D, or diprovocim. Fecal pellets were collected pre-dose, and at 1, 3, 5, 8, and 24 hours after dosing. Plasma was collected at 2 hours after dosing. Fecal and plasma concentrations of compounds were assessed by liquid chromatography/mass spectroscopy (LC/MS). [0025] FIG. 14 shows that TLR2 agonists elicit TNF-a in a human macrophage cell line.
  • TTP-1 cells Human macrophages (THP-1 cells, purchased from ATCC, catalog number TIB-202) were differentiated in vitro for 5 days, then treated for 24 hours with TLR2 agonists at 1 pM. Supernatants were collected, and cytokine concentrations were assessed by electro- chemiluminescent enzyme-linked immunosorbent assay (ELISA) on a Meso Scale Discovery (MSD) QuickPlex SQ120 according to the manufacturer’s instructions.
  • ELISA electro- chemiluminescent enzyme-linked immunosorbent assay
  • MSD Meso Scale Discovery
  • FIG. 15 shows that TLR2 agonists elicit IL-2 in a human macrophage cell line.
  • TTP-1 cells Human macrophages (THP-1 cells, purchased from ATCC, catalog number TIB-202) were differentiated in vitro for 5 days, then treated for 24 hours with TLR2 agonists at 1 pM. Supernatants were collected and cytokine concentrations were assessed by MSD according to the manufacturer’s instructions.
  • FIG. 16 shows that TLR2 agonists elicit IL-6 in a human macrophage cell line.
  • TTP-1 cells Human macrophages (THP-1 cells, purchased from ATCC, catalog number TIB-202) were differentiated in vitro for 5 days, then treated for 24 hours with TLR2 agonists at 1 pM. Supernatants were collected and cytokine concentrations were assessed by MSD according to the manufacturer’s instructions.
  • FIG. 17 shows that TLR2 agonists elicit TNF-a in a mouse macrophage cell line.
  • Mouse macrophages (RAW264.7 cells, Purchased from ATCC, catalog number TIB-71) were treated in vitro for 24 hours with toll-like receptor 2 (TLR2) agonists at 1 pM. Supernatants were collected and cytokine concentrations were assessed by MSD according to the manufacturer’s instructions.
  • TLR2 toll-like receptor 2
  • FIG. 18 shows that TLR2 agonists elicit IL-2 in a mouse macrophage cell line.
  • Mouse macrophages (RAW264.7 cells, Purchased from ATCC, catalog number TIB-71) were treated in vitro for 24 hours with toll-like receptor 2 (TLR2) agonists at 1 pM.
  • TLR2 toll-like receptor 2
  • FIG. 19 shows that TLR2 agonists elicit IL-6 in a mouse macrophage cell line.
  • Mouse macrophages (RAW264.7 cells, Purchased from ATCC, catalog number TIB-71) were treated in vitro for 24 hours with toll-like receptor 2 (TLR2) agonists at 1 pM.
  • TLR2 toll-like receptor 2
  • FIG. 20 shows that TLR2 agonists elicit IL-ip in a mouse macrophage cell line.
  • Mouse macrophages (RAW264.7 cells, Purchased from ATCC, catalog number TIB-71) were treated in vitro for 24 hours with toll-like receptor 2 (TLR2) agonists at 1 pM. Supernatants were collected and cytokine concentrations were assessed by MSD according to the manufacturer’s instructions.
  • TLR2 toll-like receptor 2
  • FIG. 21 shows that TLR2 agonists elicit IL- 10 in a mouse macrophage cell line.
  • Mouse macrophages (RAW264.7 cells, Purchased from ATCC, catalog number TIB-71) were treated in vitro for 24 hours with toll-like receptor 2 (TLR2) agonists at 1 pM. Supernatants were collected and cytokine concentrations were assessed by MSD according to the manufacturer’s instructions.
  • FIG. 22 shows that TLR2 agonists elicit IFN-y in a mouse macrophage cell line.
  • Mouse macrophages (RAW264.7 cells, Purchased from ATCC, catalog number TIB-71) were treated in vitro for 24 hours with toll-like receptor 2 (TLR2) agonists at 1 pM. Supernatants were collected and cytokine concentrations were assessed by MSD according to the manufacturer’s instructions.
  • FIG. 23 shows that oral diprovocim potentiates systemic inflammatory response.
  • Mice were orally dosed daily with vehicle or diprovocim for 28 days.
  • Primary splenocytes were isolated and treated in vitro for 245 hours with 50 ng/mL phorbol 12- myristate 13-acetate (PMA) and 1 pg/mL ionomycin to non-specifically activate immune cells.
  • Supernatants were collected and cytokine concentrations were assessed by electro- chemiluminescent enzyme-linked immunosorbent assay (ELISA) on a Meso Scale Discovery (MSD) QuickPlex SQ120 according to the manufacturer’s instructions.
  • ELISA electro- chemiluminescent enzyme-linked immunosorbent assay
  • FIG. 24 shows structures of TLR2 agonists representing different chemical scaffolds (Kaur et al., 2021; Chen et al., 2022).
  • FIG. 25 depicts a dosing scheme for oral administration of TLR2 agonists in combination with anti-PDl checkpoint therapy in MC38 tumor model corresponding to the data of FIGs. 26-27.
  • MC38 tumor cells were implanted subcutaneously on Day -10, and mice were randomized on Day 0 when tumors reached ⁇ 50 mm3.
  • Mice were treated daily with oral TLR2 agonists for 35 days, beginning on Day 7 after randomization. Mice were treated once every four days for four doses with anti-PDl intraperitoneally, beginning on Day 7 after randomization. Tumor volume was measured every 2-3 days beginning on Day 1 after randomization.
  • FIG. 26A-C shows that Compound F enhances efficacy of anti-PDl checkpoint therapy in the MC38 mouse tumor model.
  • Each line depicts the tumor volume of an individual mouse in the treatment group for groups treated with anti-PDl alone (FIG. 26A), anti-PDl and 50 pg/kg Compound F (FIG. 26B), and anti-PDl and 500 pg/kg Compound F (FIG. 26C).
  • Compound F improves tumor growth suppression by anti-PDl treatment in the MC38 model.
  • FIG. 27 shows that Compound F prolongs survival in combination with anti-PDl treatment in the MC38 mouse tumor model.
  • the present disclosure relates to methods of treating cancer using TLR2 modulators (e.g., agonists or partial agonists) in combination with anti-cancer immune checkpoint therapy.
  • TLR2 modulators e.g., agonists or partial agonists
  • TLR2 agonists signal through TLR2 receptors on immune cells in the gut, including macrophages and dendritic cells, both of which are antigen presenting cells (Hug et al. (2016) Nutrients 10(2):203; Hou et al. (2008) Immunity 29(2):272-282). TLR2 signals through Myd88 and activates canonical inflammatory pathways inducing a cytokine response within the antigen presenting cells (Koch et al. (2016) Nat Commun 9, 4099). It has been demonstrated that human and mouse macrophage cell lines respond to TLR2 agonists in vitro by producing a broad array of cytokines, consistent with activation of inflammatory signaling pathways ( Figures 14-22).
  • FIG. 23 demonstrates that immune cells in the spleens of mice treated with a TLR2 agonist that is not orally bioavailable produce stronger cytokine responses than immune cells from the spleens of untreated mice, thus demonstrating that agonism of TLR2 in the gut can prime immune cells in systemic circulation for enhanced inflammatory activity.
  • Anti -tumor immunity is largely driven by the activation of tumor-antigen-specific CD8+ T cells and their subsequent cytotoxic activity on tumor cells.
  • the number of tumor-infiltrating CD8+ T cells is one of the strongest correlates of immunotherapy efficacy (Paijens et al. (2021) Cell Mol Immunol 18, 842-859).
  • Immune checkpoint molecules including Programmed Cell Death Protein 1 (PD-1), T-Lymphocyte Associated Protein 4 (CTLA-4), Lymphocyte-Activation Protein 3 (LAG-3), T-Cell Immunoglobulin and Mucin-Domain Containing-3 (TIM-3), and T-Cell Immunoreceptor with Ig and ITIM Domains (TIGIT), are expressed by T cells and through various mechanisms function to suppress T cell activation. Tumor cells exploit these mechanisms to evade detection and destruction by the immune system. Immune checkpoint inhibitors, including neutralizing antibodies targeting PD-1, its ligand PDL-1, CTLA-4, and LAG-3, disrupt these suppressive mechanisms leading to increased immune activity targeting tumor cells (De Giglio et al.
  • T cells are recognized as the primary effector cell type through which these immune checkpoint inhibitors act. While the immune checkpoint inhibitors function by removing suppression of the T cells, these cells must still be activated by antigen presenting cells to exhibit anti-tumor activity. Thus, the activity of TLR2 agonists in the gut, leading to enhanced inflammatory capacity by antigen presenting cells in systemic circulation, should drive stronger activation of T cells in a tumor when paired with any of the immune checkpoint inhibitors acting on T cells or their targets, including anti-PDl, anti-PDL-1, anti-CTLA-4, and anti-LAG-3 therapies.
  • a method of treating cancer in a subject in need thereof comprising administering to the subject an immune checkpoint inhibitor and a TLR2 modulator.
  • the immune checkpoint inhibitor comprises one or more of an inhibitor of PD1 protein, an inhibitor of PD-L1 protein, an inhibitor of CTLA4 protein, and an inhibitor of LAG3 protein.
  • the immune checkpoint inhibitor comprises an inhibitor of PD1 protein.
  • the immune checkpoint inhibitor comprises an inhibitor of PD-L1 protein.
  • the immune checkpoint inhibitor comprises an inhibitor of CTLA4 protein.
  • the immune checkpoint inhibitor comprises one or more antibody selected from anti-PDl, anti-PD-Ll, anti-CTLA4, and anti-LAG3.
  • the immune checkpoint inhibitor comprises an anti-PDl antibody.
  • the immune checkpoint inhibitor comprises an anti-PD-Ll antibody.
  • the immune checkpoint inhibitor comprises an anti- CTLA4 antibody.
  • the immune checkpoint inhibitor is administered in the form of a pharmaceutical composition further comprising a pharmaceutically acceptable carrier.
  • the pharmaceutical composition further comprises an additional agent.
  • the additional agent is an oncolytic agent. Representative oncolytic agents are provided herein.
  • the additional agent is an antibiotic.
  • the antibiotic is one or more of ampicillin, streptomycin, vancomycin, and metronidazole.
  • the pharmaceutical composition is formulated for intravenous administration.
  • the TLR2 modulator is an agonist or a partial agonist of TLR2. In certain embodiments, TLR2 modulator modulates heterodimeric TLR2/TLR1 proteins. In certain embodiments, TLR2 modulator modulates heterodimeric TLR2/TLR6 proteins.
  • TLR2 Various chemical scaffolds are reported to activate TLR2, which include naturally occurring lipoproteins, synthetic lipopeptides, and small heterocyclic molecules. Reports on structure-activity relationship in TLR2 agonist small molecules have been published. See, Kaur et al., TLR2 Agonistic Small Molecules: Detailed Structure-Activity Relationship, Applications, and Future Prospects, Journal of Medicinal Chemistry 2021 64 (1), 233-278.
  • the TLR2 modulator is a lipid.
  • the lipid is a phospholipid.
  • the phospholipid has the structure: wherein each R c independently is a substituted or unsubstituted aliphatic moiety, or a substituted or unsubstituted heteroaliphatic moiety.
  • each R c is independently substituted or unsubstituted alkyl. In some embodiments, each R c is independently branched C10-20 alkyl.
  • each R c is independently substituted or unsubstituted heteroalkyl. In some embodiments, each R c is independently branched C10-20 heteroalkyl.
  • the phospholipid is selected from:
  • the TLR2 modulator is a peptide.
  • the TLR2 modulator may be Pam2CSK, Pam3CSK, or a pharmaceutically acceptable salt thereof.
  • the TLR2 modulator is a non-peptide small molecule.
  • the TLR2 modulator is a compound disclosed in International Patent Publication No. WO2021242923 Al, which compounds are incorporated herein by reference, and are shown in Table 3.
  • the TLR2 modulator is a compound of Formula (I): or a pharmaceutically acceptable salt thereof, wherein:
  • R is substituted arylene, substituted heteroarylene, substituted carbocyclylene, or substituted heterocyclylene.
  • R is substituted with -L 1 -G, wherein L 1 is a bond, optionally substituted arylene, optionally substituted heteroarylene, optionally substituted carbocyclylene, or optionally substituted heterocyclylene, optionally substituted alkylene, optionally substituted alkenylene, optionally substituted alkynylene, optionally substituted heteroalkylene, optionally substituted heteroalkenylene, optionally substituted heteroalkynylene,or a heteroatom selected from O, S, and N, optionally substituted as permitted by valence; and G is optionally substituted alkyl, optionally substituted heteroalkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted carbocyclyl, or optionally substituted heterocyclyl; and
  • G is optionally substituted heteroaliphatic or optionally substituted heterocyclyl.
  • L 1 is triazolyl.
  • L 1 is thiadiazolyl.
  • L 1 is imidazolyl.
  • L 1 is oxadiazolyl.
  • G is selected from:
  • Ci-i6 alkyl optionally substituted Ci-i6 alkyl, Ci-i6 alkyl-(optionally substituted aryl), -NH(CH2) m CH3, -N((CH2) m CH3)2, -N(CH3)(Y), or optionally substituted C3-10 cycloalkyl;
  • X is -NH(CH 2 ) m CH3 , -NH(CH2)m-(optionally substituted aryl), or
  • Y is -H, optionally substituted Ci-i6 alkyl, Ci-i6 alkyl-(optionally substituted aryl), - NH(CH 2 )mCH 3 , -SO2(CH2)i3CH3, or optionally substituted C3-10 cycloalkyl; each Z is independently CH or N; each m is independently 1-16; each n is independently 1-3; and each p is independently 3-8.
  • the compound of Formula (I) has the structure of Formula (II), Formula (III), Formula (IV), or Formula (V): wherein m, L 1 , L 2 , W, X, and Y are as defined above.
  • L 1 is five-membered heteroarylene, e.g., triazolyl, imidazolyl, and thiadiazolyl.
  • the compound of Formula (I) has the structure of Formula (VI): wherein L 1 and m are as defined above.
  • L 1 is five-membered heteroarylene.
  • L 1 is triazolyl.
  • L 1 is thiadiazolyl.
  • L 1 is imidazolyl.
  • L 1 is oxadi azolyl.
  • the TLR2 modulator is selected from (3S,4S)-l-(4-(l-((S)-2- (3 -heptylureido)-3-(hexylamino)-3 -oxopropyl)- lH-imidazol-4-yl)benzoyl)-N3,N4- bis((lS,2R)-2-phenylcyclopropyl)pyrrolidine-3,4-dicarboxamide; (3S,4S)-l-(4-(l-((S)-2- (3-heptylureido)-3-(hexylamino)-3-oxopropyl)-lH-l,2,3-triazol-4-yl)benzoyl)-N3,N4- bis((lS,2R)-2-phenylcyclopropyl)pyrrolidine-3,4-dicarboxamide; (3S,4S)-l-(4-(4-(5-(
  • the TLR2 modulator is Compound A: (3S,4S)-l-(4-(l- ((S)-2-(3-heptylureido)-3-(hexylamino)-3-oxopropyl)-lH-imidazol-4-yl)benzoyl)-N3,N4- bis((lS,2R)-2-phenylcyclopropyl)pyrrolidine-3,4-dicarboxamide, or a pharmaceutically acceptable salt thereof.
  • the TLR2 modulator is Compound B: (3S,4S)-l-(4-(l- ((S)-2-(3-heptylureido)-3-(hexylamino)-3-oxopropyl)-lH-l,2,3-triazol-4-yl)benzoyl)- N3,N4-bis((l S,2R)-2-phenylcyclopropyl)pyrrolidine-3,4-dicarboxamide, or a pharmaceutically acceptable salt thereof.
  • the TLR2 modulator is Compound C: (3S,4S)-l-(4-(5- ((S)-2-decanamido-3-(hexylamino)-3-oxopropyl)-l,3,4-thiadiazol-2-yl)benzoyl)-N3,N4- bis((lS,2R)-2-phenylcyclopropyl)pyrrolidine-3,4-dicarboxamide, or a pharmaceutically acceptable salt thereof.
  • the TLR2 modulator is Compound D: (3S,4S)-l-(4- ((3S,4S)-3-methoxy-4-(3-tridecylureido)pyrrolidine-l-carbonyl)benzoyl)-N3,N4- bis((lS,2R)-2-phenylcyclopropyl)pyrrolidine-3,4-dicarboxamide, or a pharmaceutically acceptable salt thereof.
  • the TLR2 modulator is Compound F: (3S,4S)-l-(4-(l- ((S)-3-(hexylamino)-2-(3-octylureido)-3-oxopropyl)-lH-l,2,3-triazol-4-yl)benzoyl)- N3,N4-bis((l S,2R)-2-phenylcyclopropyl)pyrrolidine-3,4-dicarboxamide, or a pharmaceutically acceptable salt thereof.
  • the TLR2 modulator is diprovocim, or a pharmaceutically acceptable salt thereof.
  • the TLR2 modulator is Pam2CSK4, or a pharmaceutically acceptable salt thereof.
  • the TLR2 modulator is Pam3CSK4, or a pharmaceutically acceptable salt thereof.
  • the TLR2 modulator is gut-restricted. In certain embodiments, the TLR2 modulator exhibits limited entry into systemic circulation. In certain embodiments, the TLR2 modulator does not enter systemic circulation. In certain embodiments, the TLR2 modulator exerts its effect through receptors in the gut.
  • the TLR2 modulator is a compound shown in FIG. 24.
  • the TLR2 modulator is administered in the form of a pharmaceutical composition further comprising a pharmaceutically acceptable carrier.
  • the pharmaceutical composition further comprises an additional agent.
  • the additional agent is an oncolytic agent. Representative oncolytic agents are provided herein.
  • the additional agent is an antibiotic.
  • the antibiotic is one or more of ampicillin, streptomycin, vancomycin, and metronidazole.
  • the pharmaceutical composition is formulated for IP administration. In certain embodiments, the pharmaceutical composition is formulated for oral administration. In certain embodiments, the pharmaceutical composition is formulated for controlled-release, e.g., sustained-release, in the gastrointestinal tract, lower intestine, or colon of a subject.
  • the cancer is a solid cancer, bladder cancer, breast cancer, cervical cancer, colon and rectal cancer, endometrial cancer, kidney cancer, lip and oral cancer, liver cancer, melanoma, mesothelioma, non-small cell lung cancer, non- melanoma skin cancer, oral cancer, ovarian cancer, pancreatic cancer, prostate cancer, sarcoma, small cell lung cancer or thyroid cancer.
  • the TLR2 modulator and the immune checkpoint inhibitor are administered concurrently. In certain embodiments, the TLR2 modulator and the immune checkpoint inhibitor are administered at different times. In certain embodiments, the TLR2 modulator and the immune checkpoint inhibitor are administered on different dosing schedules. In certain embodiments, the TLR2 modulator is administered to the subject one or more times per day.
  • a pharmaceutical composition comprising a TLR2 modulator, an immune checkpoint inhibitor, and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition further comprises an additional agent.
  • the additional agent is an oncolytic agent. Representative oncolytic agents are provided herein.
  • the additional agent is an antibiotic.
  • the antibiotic is one or more of ampicillin, streptomycin, vancomycin, and metronidazole.
  • the pharmaceutical composition is formulated for IP administration.
  • the pharmaceutical composition is formulated for IV administration.
  • the pharmaceutical composition is formulated for oral administration.
  • the pharmaceutical composition is formulated for controlled-release in the gastrointestinal tract, lower intestine, or colon of a subject.
  • a TLR2 agonist targeting the heterodimer TLR2/TLR1 results in enhanced anti-cancer responses elicited by anti-PDl checkpoint therapy.
  • daily oral administration of TLR2 agonists targeting TLR2/TLR1 heterodimer results in enhanced anti-cancer responses elicited by anti-PDl checkpoint therapy.
  • daily oral administration of TLR2 agonists targeting TLR2/TLR6 heterodimer results in enhanced anti-cancer responses elicited by anti-PDl checkpoint therapy.
  • Checkpoint inhibitors for cancer therapy often referred to as checkpoint therapies, cover a broad class of targets present on either the tumor itself, immune cells that interact with the tumor, or immune-regulatory cells present in lymphoid organs.
  • the most prominent targets are the PD1/PD-L1 and CTLA4 pathways, and also include TIGIT, TIM3, 0X40, OX40L, ICOS, CD27, BTLA, LAG-3, KIR, GITR, 4-1BB and others.
  • One common therapeutic incarnation of checkpoint therapies are antibodies that bind to the target checkpoint protein and block the functional interaction of the target checkpoint protein and its cognate binding partner and thereby disrupt effective immune suppressive mechanisms.
  • checkpoint therapies may include small molecules that limit the functional interaction or activity of an immuno-regulatory protein.
  • Another incarnation for checkpoint therapy may include peptide derived therapeutics that inhibit the functional immune-regulatory mechanisms of target immune molecules.
  • Immune checkpoint therapies are generally useful for cancer therapy and utilized to enhance the immune system in recognizing, suppressing, and eliminating the cancer in the patient.
  • compositions comprising one or more compounds, or pharmaceutically acceptable salts thereof, as described herein, and a pharmaceutically acceptable carrier.
  • compositions described herein can be prepared by any method known in the art of pharmacology.
  • preparatory methods include bringing the compounds described herein (i.e., the one or more “active ingredients”) into association with a carrier or excipient, and/or one or more other accessory ingredients, and then, if necessary and/or desirable, shaping, and/or packaging the product into a desired single- or multi-dose unit.
  • compositions can be prepared, packaged, and/or sold in bulk, as a single unit dose, and/or as a plurality of single unit doses.
  • a “unit dose” is a discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient.
  • the amount of the active ingredient(s) is generally equal to the dosage of the active ingredient which would be administered to a subject and/or a convenient fraction of such a dosage, such as one-half or one-third of such a dosage.
  • compositions described herein will vary, depending upon the identity, size, and/or condition of the subject treated and further depending upon the route by which the composition is to be administered.
  • the composition may comprise between 0.1% and 100% (w/w) active ingredient.
  • the compounds and compositions provided herein can be administered by any route, including enteral (e.g., oral), parenteral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, subcutaneous, intraventricular, transdermal, interdermal, rectal, intravaginal, intraperitoneal, topical (as by powders, ointments, creams, and/or drops), mucosal, nasal, bucal, sublingual; by intratracheal instillation, bronchial instillation, and/or inhalation; and/or as an oral spray, nasal spray, and/or aerosol.
  • enteral e.g., oral
  • parenteral intravenous, intramuscular, intra-arterial, intramedullary
  • intrathecal subcutaneous, intraventricular, transdermal, interdermal, rectal, intravaginal, intraperitoneal
  • topical as by powders, ointments, creams, and/or drops
  • mucosal nasal,
  • Specifically contemplated routes are oral administration, intravenous administration (e.g., systemic intravenous injection), regional administration via blood and/or lymph supply, and/or direct administration to an affected site.
  • intravenous administration e.g., systemic intravenous injection
  • regional administration via blood and/or lymph supply e.g., via blood and/or lymph supply
  • direct administration e.g., direct administration to an affected site.
  • the most appropriate route of administration will depend upon a variety of factors including the nature of the compound (e.g., its stability in the environment of the gastrointestinal tract), and/or the condition of the subject (e.g., whether the subject is able to tolerate oral administration).
  • the pharmaceutical composition is formulated for oral administration.
  • Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules.
  • the pharmaceutical composition is formulated for enteric delivery.
  • the pharmaceutical composition is formulated for controlled release within the lower intestine or colon of a subject.
  • Such a pharmaceutical composition may be further formulated for enteric delivery.
  • the exact amount of a compound required to achieve an effective amount will vary from subject to subject, depending, for example, on species, age, and general condition of a subject, severity of the side effects or disorder, identity of the particular compound(s), mode of administration, and the like.
  • the desired dosage can be delivered three times a day, two times a day, once a day, every other day, every third day, every week, every two weeks, every three weeks, or every four weeks.
  • the desired dosage can be delivered using multiple administrations (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or more administrations).
  • an effective amount of a compound for administration one or more times a day to a 70 kg adult human may comprise about 0.0001 ug to about 1000 mg, about 0.0001 ug to about 100 mg, about 0.0001 ug to about 10 mg, about 0.0001 ug to about 5000 ug, about 0.0001 ug to about 2000 ug, about 0.0001 ug to about 1000 ug, about 0.001 ug to about 1000 ug, about 0.01 ug to about 1000 ug, about 0.1 ug to about 1000 ug, about 1 ug to about 1000 ug, about 1 ug to about 100 ug, about 10 ug to about 1000 ug, or about 100 ug to about 1000 ug, of a compound per unit dosage form.
  • the compounds of the invention may be administered orally at dosage levels sufficient to deliver from about 0.001 ug/kg to about 10 mg/kg, 0.001 ug/kg to about 5000 ug/kg, 0.001 ug/kg to about 1000 ug/kg, 0.001 ug/kg to about 100 ug/kg, from about 0.01 ug/kg to about 50 ug/kg, preferably from about 0.1 ug/kg to about 40 ug/kg, preferably from about 0.5 ug/kg to about 30 ug/kg, from about 0.01 ug/kg to about 10 ug/kg, from about 0.1 ug/kg to about 10 ug/kg, and more preferably from about 1 ug/kg to about 25 ug/kg, of subject body weight per day, one or more times a day, to obtain the desired therapeutic effect.
  • the oral dosing frequency (i.e., dosing schedule) of a TLR2 modulator e.g., a TLR2 agonist or partial agonist
  • a TLR2 modulator e.g., a TLR2 agonist or partial agonist
  • daily dosing promotes an immunizing, tumor-suppressive response and enhances the tumor growth-inhibiting effects of checkpoint therapy.
  • dose ranges as described herein provide guidance for the administration of provided pharmaceutical compositions to an adult.
  • the amount to be administered to, for example, a child or an adolescent can be determined by a medical practitioner or person skilled in the art and can be lower or the same as that administered to an adult.
  • TLR2 is a potent regulator of innate immunity and, when orally administered, may enhance T cell activation through various immune signaling networks. Yet TLR signaling is a blunt stimulus, recruiting diverse arms of the innate and adaptive immune response and enhancing both immunizing and tolerizing effector functions. To bias activation toward cytotoxic immunity and away from tolerizing responses, TLR2 agonists may be combined with other treatments that provide additional context and signaling support for eliciting cytotoxic immunity.
  • a compound, compounds, or composition, as described herein can be administered in combination with one or more additional therapeutically active agents.
  • the compounds or compositions can be administered in combination with additional therapeutically active agents that improve their bioavailability, reduce and/or modify their metabolism, inhibit their excretion, and/or modify their distribution within the body.
  • additional therapeutically active agents that improve their bioavailability, reduce and/or modify their metabolism, inhibit their excretion, and/or modify their distribution within the body.
  • the therapy employed may achieve a desired effect for the same disorder, and/or it may achieve different effects.
  • the compound, compounds, or composition can be administered concurrently with, prior to, or subsequent to, one or more additional therapeutically active agents.
  • each agent will be administered at a dose and/or on a time schedule determined for that agent.
  • the additional therapeutically active agent utilized in this combination can be administered together in a single composition or administered separately in different compositions.
  • the particular combination to employ in a regimen will take into account compatibility of the inventive compound with the additional therapeutically active agent and/or the desired therapeutic effect to be achieved.
  • additional therapeutically active agents utilized in combination be utilized at levels that do not exceed the levels at which they are utilized individually. In some embodiments, the levels utilized in combination will be lower than those utilized individually.
  • TLR2 Toll-like receptor 2
  • CD282 cluster of differentiation 282
  • TLR2 is a membrane protein, a receptor, which is expressed on the surface of certain cells and recognizes foreign substances and passes on appropriate signals to cells of immune and non-immune function, and plays a role in the immune system and is associated with tumorigenesis.
  • the compounds described herein are modulators of TLR2, and promote immune activation to generate antigen-specific T cells and suppress tumor growth in combination with checkpoint inhibition such as anti-PDl.
  • the compounds are also useful in raising the immunologic “set point” of the immune system, thereby enhancing anti- cancer immunity elicited by anti-PDl therapy.
  • the concept of the immune set point postulates that a systemic “level” of inflammation can dictate the intensity and direction of immune responses (Concepts Collide: Genomic, Immune, and Microbial Influences on the Tumor Microenvironment and Response to Cancer Therapy. Andrews MC, Reuben A, Gopalakrishnan V, Wargo JA. Front Immunol . 2018 May 4;9:946.).
  • the immune setpoint is defined functionally and generally assayed by determining a relative difference for a defined immune parameter for a given treatment.
  • the immune setpoint offers an explanation for how a non-specific immunoregulatory agent may enhance or diminish a specific immune parameter. For example, if an antibody response or an anti-tumor response is modulated by a treatment that is considered to impact systemic inflammation, then the “immune setpoint” may be considered to be altered by the treatment.
  • the host intestinal microbiome can have significant impact on the efficacy of cancer immunotherapy, especially immune checkpoint therapy.
  • Some components of the microbiome are associated with more effective anti-cancer responses with checkpoint therapy.
  • depletion of the microbiome with antibiotics is generally associated with reduced efficacy of checkpoint therapy in both pre-clinical and clinical settings.
  • the prevailing theory is that depletion of the intestinal microbiome with antibiotics eliminates some essential factor(s) that potentiate the immune setpoint and thereby decrease the efficacy of checkpoint therapy.
  • a mouse tumor model was developed that included daily dosing with broad-spectrum oral antibiotics to render checkpoint therapy less effective. The model was used to determine how orally-dosed TLR2 modulators may influence the efficacy of anti-PDl therapy against established tumors in this preclinical model for checkpoint non-responder patients.
  • the model utilized herein involves the MCA205 mouse fibrosarcoma that is implanted subcutaneously in the flank of C57/Black6 mice. In some treatment groups, the mice are treated daily with an oral antibiotic cocktail. Once tumors are established after 5-10 days, mice are dosed with varying combinations and frequencies of TLR2 modulators and anti-PDl antibodies. Tumor growth and mouse survival are measured through the course of treatment and through longer post-treatment time points.
  • An alternative mouse tumor model uses the MC38 adenocarcinoma cell line.
  • a dosing scheme was derived from the standard checkpoint therapy dosing scheme that results in less effective tumor control by the administered checkpoint therapy.
  • This altered scheme to produce less effective tumor control by checkpoint therapy in MC38 varied both the timing and dose of the standard checkpoint therapy dosing protocol. Through this protocol, enhancement of tumor control with daily oral dosing of a TLR2 agonist or partial agonist by the immune system can be assayed.
  • the in vitro TLR2 assay consists of a cell line that responds to TLR2 engagement by producing a factor that elicits a color change in the culture media and can be readily measured by various colorimetric detection methods. This assay enables the identification of existing and novel compounds that engage TLR2 and, by virtue of compound dilution, to approximate the binding affinity of these compounds for eliciting a TLR2-dependent signal.
  • TLR2 modulators Provided herein is an assay to identify TLR2 modulators and/or characterize the binding affinities of TLR2 modulators.
  • the method comprises the steps:
  • step (3) comprises contacting the cultured cells with candidate TLR2 modulator compounds for a period of 8-16 hours, or for a period of 16- 24 hours.
  • the method comprises comparing the absorbance measured in step (4) to absorbances measured under the same conditions using TLR2 modulator compounds of known binding affinities. For example, for determining compound affinities, control or test compounds are assessed in a 7 point dilution scheme, comprising a top concentration of 10 pM and diluted 10-fold to 10 pM.
  • a method for treating cancer in a subject in need thereof comprising administering to the subject a TLR2 modulator and an immune checkpoint inhibitor, or a pharmaceutical composition thereof, as described herein.
  • TLR2 Toll-like receptor 2
  • the disorder is cancer.
  • the cancer is a solid cancer, bladder cancer, breast cancer, cervical cancer, colon and rectal cancer, endometrial cancer, kidney cancer, lip and oral cancer, liver cancer, melanoma, mesothelioma, non-small cell lung cancer, non- melanoma skin cancer, oral cancer, ovarian cancer, pancreatic cancer, prostate cancer, sarcoma, small cell lung cancer or thyroid cancer.
  • the TLR2 modulator and/or immune checkpoint inhibitor, or a pharmaceutical composition thereof is co-administered with one or more oncolytic agents.
  • the oncolytic agents are selected from checkpoint inhibitors (e.g., pembrolizumab, nivolumab, ipilmumab, atezolizumab, durvalumab, avelumab, and tremelimumab), immuno-oncology (IO) agents such as STING agonists and IDO inhibitors, targeted therapies such as protein kinase inhibitors, PARP inhibitors, nuclear receptor antagonists/degraders/hormone therapies (e.g., imatinib, erlotinib, olaparib, tamoxifen, and fulvestrant), cytotoxic agents (e.g., cyclophosphamide, carboplatin, paclitaxel, doxorubicin, e
  • checkpoint inhibitors e.g., pembroli
  • a method of modulating immune function by orally administering a TLR2 modulator (e.g., an agonist or partial agonist) on a dosing schedule to modulate an immune response.
  • a TLR2 modulator e.g., an agonist or partial agonist
  • the dosing schedule promotes an immunizing response.
  • the dosing schedule promotes enhancement of cytotoxic immunity.
  • the TLR2 modulator is a compound, pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, as described herein.
  • the TLR2 modulator is diprovocim.
  • the oral administration of a TLR2 modulating compound enhances the efficacy of immune checkpoint therapy for cancer.
  • Compounds described herein can comprise one or more asymmetric centers, and thus can exist in various stereoisomeric forms, e.g., enantiomers and/or diastereomers.
  • the compounds described herein can be in the form of an individual enantiomer, diastereomer or geometric isomer, or can be in the form of a mixture of stereoisomers, including racemic mixtures and mixtures enriched in one or more stereoisomer.
  • Isomers can be isolated from mixtures by methods known to those skilled in the art, including chiral high pressure liquid chromatography (HPLC) and the formation and crystallization of chiral salts; or preferred isomers can be prepared by asymmetric syntheses.
  • HPLC high pressure liquid chromatography
  • formulae and structures depicted herein include compounds that do not include isotopically enriched atoms, and also include compounds that include isotopically enriched atoms.
  • compounds having the present structures except for the replacement of hydrogen by deuterium or tritium, replacement of 1 9 F with 18 F, or the replacement of a carbon by a 13 C- or 14 C-enriched carbon are within the scope of the disclosure. Such compounds are useful, for example, as analytical tools or probes in biological assays.
  • range When a range of values (“range”) is listed, it encompasses each value and sub- range within the range.
  • a range is inclusive of the values at the two ends of the range unless otherwise provided.
  • “Ci-6 alkyl” encompasses, C 1 , C 2 , C 3 , C 4 , C 5 , C 6 , C 1-6 , C 1-5 , C 1-4 , C 1-3 , C 1-2 , C 2-6 , C 2-5 , C 2-4 , C 2-3 , C 3-6 , C 3-5 , C 3-4 , C 4-6 , C 4-5 , and C 5-6 alkyl.
  • aliphatic refers to alkyl, alkenyl, alkynyl, and carbocyclic groups.
  • heteroaliphatic refers to heteroalkyl, heteroalkenyl, heteroalkynyl, and heterocyclic groups.
  • alkyl refers to a radical of a straight-chain or branched saturated hydrocarbon group having from 1 to 20 carbon atoms (“C 1-20 alkyl”). In some embodiments, an alkyl group has 1 to 12 carbon atoms (“C 1-12 alkyl”). In some embodiments, an alkyl group has 1 to 10 carbon atoms (“C 1-10 alkyl”). In some embodiments, an alkyl group has 1 to 9 carbon atoms (“C 1-9 alkyl”). In some embodiments, an alkyl group has 1 to 8 carbon atoms (“C 1-8 alkyl”). In some embodiments, an alkyl group has 1 to 7 carbon atoms (“C 1-7 alkyl”).
  • an alkyl group has 1 to 6 carbon atoms (“C 1-6 alkyl”). In some embodiments, an alkyl group has 1 to 5 carbon atoms (“C 1-5 alkyl”). In some embodiments, an alkyl group has 1 to 4 carbon atoms (“C 1-4 alkyl”). In some embodiments, an alkyl group has 1 to 3 carbon atoms (“C 1-3 alkyl”). In some embodiments, an alkyl group has 1 to 2 carbon atoms (“C 1-2 alkyl”). In some embodiments, an alkyl group has 1 carbon atom (“Ci alkyl”). In some embodiments, an alkyl group has 2 to 6 carbon atoms (“C2-6 alkyl”).
  • C1-6 alkyl groups include methyl (Ci), ethyl (C2), propyl (C3) (e.g., n-propyl, isopropyl), butyl (C4) (e.g., n-butyl, tert-butyl, sec-butyl, isobutyl), pentyl (C 5 ) (e.g., n-pentyl, 3-pentanyl, amyl, neopentyl, 3- methyl-2-butanyl, tert-amyl), and hexyl (C 6 ) (e.g., n-hexyl).
  • alkyl groups include n-heptyl (C7), zz-octyl (C 8 ), n-dodecyl (C12), and the like. Unless otherwise specified, each instance of an alkyl group is independently unsubstituted (an “unsubstituted alkyl”) or substituted (a “substituted alkyl”) with one or more substituents (e.g., halogen, such as F).
  • substituents e.g., halogen, such as F
  • the alkyl group is an unsubstituted Ci- 12 alkyl (such as unsubstituted C1-6 alkyl, e.g., -CH3 (Me), unsubstituted ethyl (Et), unsubstituted propyl (Pr, e.g., unsubstituted n-propyl (n-Pr), unsubstituted isopropyl (z- Pr)), unsubstituted butyl (Bu, e.g., unsubstituted zz-butyl (n-Bu), unsubstituted tert-butyl (tert-Bu or /-Bu), unsubstituted sec-butyl (sec-Bu or s-Bu), unsubstituted isobutyl (z-Bu)).
  • unsubstituted Ci- 12 alkyl such as unsubstituted C1-6 alkyl, e.g., -CH3 (M
  • the alkyl group is a substituted C1-12 alkyl (such as substituted C1-6 alkyl, e.g., -CH 2 F, -CHF 2 , -CF 3 , -CH2CH2F, -CH2CHF2, -CH2CF3, or benzyl (Bn)).
  • substituted C1-6 alkyl e.g., -CH 2 F, -CHF 2 , -CF 3 , -CH2CH2F, -CH2CHF2, -CH2CF3, or benzyl (Bn)
  • heteroalkyl refers to an alkyl group, which further includes at least one heteroatom (e.g., 1, 2, 3, or 4 heteroatoms) selected from oxygen, nitrogen, or sulfur within (e.g., inserted between adjacent carbon atoms of) and/or placed at one or more terminal position(s) of the parent chain.
  • a heteroalkyl group refers to a saturated group having from 1 to 20 carbon atoms and 1 or more heteroatoms within the parent chain (“heteroCi-20 alkyl”).
  • a heteroalkyl group refers to a saturated group having from 1 to 12 carbon atoms and 1 or more heteroatoms within the parent chain (“heteroCi-12 alkyl”).
  • a heteroalkyl group is a saturated group having 1 to 11 carbon atoms and 1 or more heteroatoms within the parent chain (“heteroCi-n alkyl”). In some embodiments, a heteroalkyl group is a saturated group having 1 to 10 carbon atoms and 1 or more heteroatoms within the parent chain (“heteroCi-10 alkyl”). In some embodiments, a heteroalkyl group is a saturated group having 1 to 9 carbon atoms and 1 or more heteroatoms within the parent chain (“heteroCi-9 alkyl”). In some embodiments, a heteroalkyl group is a saturated group having 1 to 8 carbon atoms and 1 or more heteroatoms within the parent chain (“heteroCi-8 alkyl”).
  • a heteroalkyl group is a saturated group having 1 to 7 carbon atoms and 1 or more heteroatoms within the parent chain (“heteroCi-7 alkyl”). In some embodiments, a heteroalkyl group is a saturated group having 1 to 6 carbon atoms and 1 or more heteroatoms within the parent chain (“heteroCi-6 alkyl”). In some embodiments, a heteroalkyl group is a saturated group having 1 to 5 carbon atoms and 1 or 2 heteroatoms within the parent chain (“heteroCi-5 alkyl”). In some embodiments, a heteroalkyl group is a saturated group having 1 to 4 carbon atoms and lor 2 heteroatoms within the parent chain (“heteroCi-4 alkyl”).
  • a heteroalkyl group is a saturated group having 1 to 3 carbon atoms and 1 heteroatom within the parent chain (“heteroCi-3 alkyl”). In some embodiments, a heteroalkyl group is a saturated group having 1 to 2 carbon atoms and 1 heteroatom within the parent chain (“heteroCi-2 alkyl”). In some embodiments, a heteroalkyl group is a saturated group having 1 carbon atom and 1 heteroatom (“heteroCi alkyl”). In some embodiments, a heteroalkyl group is a saturated group having 2 to 6 carbon atoms and 1 or 2 heteroatoms within the parent chain (“heteroC2-6 alkyl”).
  • each instance of a heteroalkyl group is independently unsubstituted (an “unsubstituted heteroalkyl”) or substituted (a “substituted heteroalkyl”) with one or more substituents.
  • the heteroalkyl group is an unsubstituted heteroCi-12 alkyl.
  • the heteroalkyl group is a substituted heteroCi-12 alkyl.
  • a group is optionally substituted unless expressly provided otherwise.
  • the term “optionally substituted” refers to being substituted or un substituted.
  • aliphatic, heteroaliphatic, alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl groups are optionally substituted.
  • Optionally substituted refers to a group which is substituted or unsubstituted (e.g., “substituted” or “unsubstituted” alkyl, “substituted” or “unsubstituted” alkenyl, “substituted” or “unsubstituted” alkynyl, “substituted” or “unsubstituted” heteroalkyl, “substituted” or “unsubstituted” heteroalkenyl, “substituted” or “unsubstituted” heteroalkynyl, “substituted” or “unsubstituted” carbocyclyl, “substituted” or “unsubstituted” heterocyclyl, “substituted” or “unsubstituted” aryl or “substituted” or “unsubstituted” heteroaryl group).
  • substituted means that at least one hydrogen present on a group is replaced with a permissible substituent, e.g, a substituent which upon substitution results in a stable compound, e.g, a compound which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, or other reaction.
  • a “substituted” group has a substituent at one or more substitutable positions of the group, and when more than one position in any given structure is substituted, the substituent is either the same or different at each position.
  • substituted is contemplated to include substitution with all permissible substituents of organic compounds, and includes any of the substituents described herein that results in the formation of a stable compound.
  • the present invention contemplates any and all such combinations in order to arrive at a stable compound.
  • heteroatoms such as nitrogen may have hydrogen substituents and/or any suitable substituent as described herein which satisfy the valencies of the heteroatoms and results in the formation of a stable moiety.
  • carbocyclyl refers to a radical of a non-aromatic cyclic hydrocarbon group having from 3 to 14 ring carbon atoms (“C 3 -i4 carbocyclyl”) and zero heteroatoms in the non-aromatic ring system.
  • a carbocyclyl group has 3 to 14 ring carbon atoms (“C 3 -i4 carbocyclyl”).
  • a carbocyclyl group has 3 to 13 ring carbon atoms (“C 3 -i 3 carbocyclyl”).
  • a carbocyclyl group has 3 to 12 ring carbon atoms (“C 3 -i2 carbocyclyl”).
  • a carbocyclyl group has 3 to 11 ring carbon atoms (“C 3 -u carbocyclyl”). In some embodiments, a carbocyclyl group has 3 to 10 ring carbon atoms (“C 3 -io carbocyclyl”). In some embodiments, a carbocyclyl group has 3 to 8 ring carbon atoms (“C 3 -s carbocyclyl”). In some embodiments, a carbocyclyl group has 3 to 7 ring carbon atoms (“C 3 -7 carbocyclyl”). In some embodiments, a carbocyclyl group has 3 to 6 ring carbon atoms (“C 3 -6 carbocyclyl”).
  • a carbocyclyl group has 4 to 6 ring carbon atoms (“C4-6 carbocyclyl”). In some embodiments, a carbocyclyl group has 5 to 6 ring carbon atoms (“C5-6 carbocyclyl”). In some embodiments, a carbocyclyl group has 5 to 10 ring carbon atoms (“C5-10 carbocyclyl”).
  • Exemplary C 3 -6 carbocyclyl groups include cyclopropyl (C 3 ), cyclopropenyl (C 3 ), cyclobutyl (C4), cyclobutenyl (C4), cyclopentyl (C5), cyclopentenyl (C5), cyclohexyl (C 6 ), cyclohexenyl (C 6 ), cyclohexadienyl (C 6 ), and the like.
  • Exemplary C 3 -s carbocyclyl groups include the aforementioned C 3 -6 carbocyclyl groups as well as cycloheptyl (C7), cycloheptenyl (C7), cycloheptadienyl (C7), cycloheptatrienyl (C7), cyclooctyl (Cs), cyclooctenyl (Cs), bicyclo[2.2.1]heptanyl (C7), bicyclo[2.2.2]octanyl (Cs), and the like.
  • Exemplary C 3 -io carbocyclyl groups include the aforementioned C 3-8 carbocyclyl groups as well as cyclononyl (C 9 ), cyclononenyl (C 9 ), cyclodecyl (C 10 ), cyclodecenyl (C 10 ), octahydro- 1H- indenyl (C 9 ), decahydronaphthalenyl (C 10 ), spiro[4.5]decanyl (C 10 ), and the like.
  • Exemplary C 3-8 carbocyclyl groups include the aforementioned C 3-10 carbocyclyl groups as well as cycloundecyl (C 11 ), spiro[5.5]undecanyl (C 11 ), cyclododecyl (C 12 ), cyclododecenyl (C 12 ), cyclotridecane (C 13 ), cyclotetradecane (C 14 ), and the like.
  • the carbocyclyl group is either monocyclic (“monocyclic carbocyclyl”) or polycyclic (e.g., containing a fused, bridged or spiro ring system such as a bicyclic system (“bicyclic carbocyclyl”) or tricyclic system (“tricyclic carbocyclyl”)) and can be saturated or can contain one or more carbon-carbon double or triple bonds.
  • Carbocyclyl also includes ring systems wherein the carbocyclyl ring, as defined above, is fused with one or more aryl or heteroaryl groups wherein the point of attachment is on the carbocyclyl ring, and in such instances, the number of carbons continue to designate the number of carbons in the carbocyclic ring system.
  • each instance of a carbocyclyl group is independently unsubstituted (an “unsubstituted carbocyclyl”) or substituted (a “substituted carbocyclyl”) with one or more substituents.
  • the carbocyclyl group is an unsubstituted C3-14 carbocyclyl.
  • the carbocyclyl group is a substituted C3-14 carbocyclyl.
  • “carbocyclyl” is a monocyclic, saturated carbocyclyl group having from 3 to 14 ring carbon atoms (“C3-14 cycloalkyl”). In some embodiments, a cycloalkyl group has 3 to 10 ring carbon atoms (“C3-10 cycloalkyl”). In some embodiments, a cycloalkyl group has 3 to 8 ring carbon atoms (“C3-8 cycloalkyl”). In some embodiments, a cycloalkyl group has 3 to 6 ring carbon atoms (“C3-6 cycloalkyl”). In some embodiments, a cycloalkyl group has 4 to 6 ring carbon atoms (“C4-6 cycloalkyl”).
  • a cycloalkyl group has 5 to 6 ring carbon atoms (“C5- 6 cycloalkyl”). In some embodiments, a cycloalkyl group has 5 to 10 ring carbon atoms (“C5-10 cycloalkyl”). Examples of C5-6 cycloalkyl groups include cyclopentyl (C5) and cyclohexyl (C5). Examples of C3-6 cycloalkyl groups include the aforementioned C5-6 cycloalkyl groups as well as cyclopropyl (C3) and cyclobutyl (C4).
  • C3-8 cycloalkyl groups include the aforementioned C3-6 cycloalkyl groups as well as cycloheptyl (C7) and cyclooctyl (C 8 ).
  • each instance of a cycloalkyl group is independently unsubstituted (an “unsubstituted cycloalkyl”) or substituted (a “substituted cycloalkyl”) with one or more substituents.
  • the cycloalkyl group is an unsubstituted C 3-14 cycloalkyl.
  • the cycloalkyl group is a substituted C 3-14 cycloalkyl.
  • heterocyclyl refers to a radical of a 3- to 14- membered non-aromatic ring system having ring carbon atoms and 1 to 4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“3-14 membered heterocyclyl”).
  • heterocyclyl groups that contain one or more nitrogen atoms, the point of attachment can be a carbon or nitrogen atom, as valency permits.
  • a heterocyclyl group can either be monocyclic (“monocyclic heterocyclyl”) or polycyclic (e.g., a fused, bridged or spiro ring system such as a bicyclic system (“bicyclic heterocyclyl”) or tricyclic system (“tricyclic heterocyclyl”)), and can be saturated or can contain one or more carbon-carbon double or triple bonds.
  • Heterocyclyl polycyclic ring systems can include one or more heteroatoms in one or both rings.
  • Heterocyclyl also includes ring systems wherein the heterocyclyl ring, as defined above, is fused with one or more carbocyclyl groups wherein the point of attachment is either on the carbocyclyl or heterocyclyl ring, or ring systems wherein the heterocyclyl ring, as defined above, is fused with one or more aryl or heteroaryl groups, wherein the point of attachment is on the heterocyclyl ring, and in such instances, the number of ring members continue to designate the number of ring members in the heterocyclyl ring system.
  • each instance of heterocyclyl is independently unsubstituted (an “unsubstituted heterocyclyl”) or substituted (a “substituted heterocyclyl”) with one or more substituents.
  • the heterocyclyl group is an unsubstituted 3-14 membered heterocyclyl.
  • the heterocyclyl group is a substituted 3-14 membered heterocyclyl.
  • the heterocyclyl is substituted or unsubstituted, 3- to 7-membered, monocyclic heterocyclyl, wherein 1, 2, or 3 atoms in the heterocyclic ring system are independently oxygen, nitrogen, or sulfur, as valency permits.
  • a heterocyclyl group is a 5-10 membered non-aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-10 membered heterocyclyl”).
  • a heterocyclyl group is a 5-8 membered non- aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-8 membered heterocyclyl”).
  • a heterocyclyl group is a 5-6 membered non- aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-6 membered heterocyclyl”).
  • the 5-6 membered heterocyclyl has 1-3 ring heteroatoms selected from nitrogen, oxygen, and sulfur.
  • the 5-6 membered heterocyclyl has 1-2 ring heteroatoms selected from nitrogen, oxygen, and sulfur.
  • the 5-6 membered heterocyclyl has 1 ring heteroatom selected from nitrogen, oxygen, and sulfur.
  • Exemplary 3 -membered heterocyclyl groups containing 1 heteroatom include azirdinyl, oxiranyl, and thiiranyl.
  • Exemplary 4-membered heterocyclyl groups containing 1 heteroatom include azetidinyl, oxetanyl, and thietanyl.
  • Exemplary 5-membered heterocyclyl groups containing 1 heteroatom include tetrahydrofuranyl, di hydrofuranyl, tetrahydrothiophenyl, dihydrothiophenyl, pyrrolidinyl, dihydropyrrolyl, and pyrrolyl-2,5- dione.
  • Exemplary 5-membered heterocyclyl groups containing 2 heteroatoms include dioxolanyl, oxathiolanyl and dithiolanyl.
  • Exemplary 5-membered heterocyclyl groups containing 3 heteroatoms include triazolinyl, oxadiazolinyl, and thiadiazolinyl.
  • Exemplary 6-membered heterocyclyl groups containing 1 heteroatom include piperidinyl, tetrahydropyranyl, dihydropyridinyl, and thianyl.
  • Exemplary 6-membered heterocyclyl groups containing 2 heteroatoms include piperazinyl, morpholinyl, dithianyl, and dioxanyl.
  • Exemplary 6-membered heterocyclyl groups containing 3 heteroatoms include triazinyl.
  • Exemplary 7-membered heterocyclyl groups containing 1 heteroatom include azepanyl, oxepanyl and thiepanyl.
  • Exemplary 8-membered heterocyclyl groups containing 1 heteroatom include azocanyl, oxecanyl and thiocanyl.
  • Exemplary bicyclic heterocyclyl groups include indolinyl, isoindolinyl, dihydrobenzofuranyl, dihydrobenzothienyl, tetrahydrobenzothienyl, tetrahydrobenzofuranyl, tetrahydroindolyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, decahydroisoquinolinyl, octahydrochromenyl, octahydroisochromenyl, decahydronaphthyridinyl, decahydro- 1,8-naphthyridinyl, octahydropyrrolo[3,2-b]pyrrole, indolinyl,
  • aryl refers to a radical of a monocyclic or polycyclic (e.g., bicyclic or tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or 14 it electrons shared in a cyclic array) having 6-14 ring carbon atoms and zero heteroatoms provided in the aromatic ring system (“ C 6-14 aryl”).
  • an aryl group has 6 ring carbon atoms (“C 6 aryl”; e.g., phenyl).
  • an aryl group has 10 ring carbon atoms (“C 10 aryl”; e.g., naphthyl such as 1-naphthyl and 2-naphthyl).
  • an aryl group has 14 ring carbon atoms (“C 14 aryl”; e.g., anthracyl).
  • Aryl also includes ring systems wherein the aryl ring, as defined above, is fused with one or more carbocyclyl or heterocyclyl groups wherein the radical or point of attachment is on the aryl ring, and in such instances, the number of carbon atoms continue to designate the number of carbon atoms in the aryl ring system.
  • each instance of an aryl group is independently unsubstituted (an “unsubstituted aryl”) or substituted (a “substituted aryl”) with one or more substituents.
  • the aryl group is an unsubstituted C 6-14 aryl.
  • the aryl group is a substituted C 6-14 aryl.
  • Alkyl is a subset of “alkyl” and refers to an alkyl group substituted by an aryl group, wherein the point of attachment is on the alkyl moiety.
  • heteroaryl refers to a radical of a 5-14 membered monocyclic or polycyclic (e.g., bicyclic, tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or 14 it electrons shared in a cyclic array) having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-14 membered heteroaryl”).
  • the point of attachment can be a carbon or nitrogen atom, as valency permits.
  • Heteroaryl polycyclic ring systems can include one or more heteroatoms in one or both rings.
  • Heteroaryl includes ring systems wherein the heteroaryl ring, as defined above, is fused with one or more carbocyclyl or heterocyclyl groups wherein the point of attachment is on the heteroaryl ring, and in such instances, the number of ring members continue to designate the number of ring members in the heteroaryl ring system. “Heteroaryl” also includes ring systems wherein the heteroaryl ring, as defined above, is fused with one or more aryl groups wherein the point of attachment is either on the aryl or heteroaryl ring, and in such instances, the number of ring members designates the number of ring members in the fused polycyclic (aryl/heteroaryl) ring system.
  • Polycyclic heteroaryl groups wherein one ring does not contain a heteroatom e.g., indolyl, quinolinyl, carbazolyl, and the like
  • the point of attachment can be on either ring, e.g., either the ring bearing a heteroatom (e.g., 2- indolyl) or the ring that does not contain a heteroatom (e.g., 5-indolyl).
  • the heteroaryl is substituted or unsubstituted, 5- or 6-membered, monocyclic heteroaryl, wherein 1, 2, 3, or 4 atoms in the heteroaryl ring system are independently oxygen, nitrogen, or sulfur.
  • the heteroaryl is substituted or unsubstituted, 9- or 10-membered, bicyclic heteroaryl, wherein 1, 2, 3, or 4 atoms in the heteroaryl ring system are independently oxygen, nitrogen, or sulfur.
  • a heteroaryl group is a 5-10 membered aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-10 membered heteroaryl”).
  • a heteroaryl group is a 5-8 membered aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-8 membered heteroaryl”).
  • a heteroaryl group is a 5-6 membered aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-6 membered heteroaryl”).
  • the 5-6 membered heteroaryl has 1-3 ring heteroatoms selected from nitrogen, oxygen, and sulfur.
  • the 5-6 membered heteroaryl has 1-2 ring heteroatoms selected from nitrogen, oxygen, and sulfur.
  • the 5-6 membered heteroaryl has 1 ring heteroatom selected from nitrogen, oxygen, and sulfur.
  • each instance of a heteroaryl group is independently unsubstituted (an “unsubstituted heteroaryl”) or substituted (a “substituted heteroaryl”) with one or more substituents.
  • the heteroaryl group is an unsubstituted 5-14 membered heteroaryl.
  • the heteroaryl group is a substituted 5-14 membered heteroaryl.
  • Exemplary 5-membered heteroaryl groups containing 1 heteroatom include pyrrolyl, furanyl, and thiophenyl.
  • Exemplary 5-membered heteroaryl groups containing 2 heteroatoms include imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, and isothiazolyl.
  • Exemplary 5-membered heteroaryl groups containing 3 heteroatoms include triazolyl, oxadiazolyl, and thiadiazolyl.
  • Exemplary 5-membered heteroaryl groups containing 4 heteroatoms include tetrazolyl.
  • Exemplary 6-membered heteroaryl groups containing 1 heteroatom include pyridinyl.
  • Exemplary 6-membered heteroaryl groups containing 2 heteroatoms include pyridazinyl, pyrimidinyl, and pyrazinyl.
  • Exemplary 6- membered heteroaryl groups containing 3 or 4 heteroatoms include triazinyl and tetrazinyl, respectively.
  • Exemplary 7-membered heteroaryl groups containing 1 heteroatom include azepinyl, oxepinyl, and thiepinyl.
  • Exemplary 5,6-bicyclic heteroaryl groups include indolyl, isoindolyl, indazolyl, benzotri azolyl, benzothiophenyl, isobenzothiophenyl, benzofuranyl, benzoisofuranyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzoxadi azolyl, benzthiazolyl, benzisothiazolyl, benzthiadi azolyl, indolizinyl, and purinyl.
  • Exemplary 6,6-bicyclic heteroaryl groups include naphthyridinyl, pteridinyl, quinolinyl, isoquinolinyl, cinnolinyl, quinoxalinyl, phthalazinyl, and quinazolinyl.
  • Exemplary tricyclic heteroaryl groups include phenanthridinyl, dibenzofuranyl, carbazolyl, acridinyl, phenothiazinyl, phenoxazinyl, and phenazinyl.
  • Heteroaralkyl is a subset of “alkyl” and refers to an alkyl group substituted by a heteroaryl group, wherein the point of attachment is on the alkyl moiety.
  • saturated or “fully saturated” refers to a moiety that does not contain a double or triple bond, e.g., the moiety only contains single bonds.
  • alkylene is the divalent moiety of alkyl
  • alkenylene is the divalent moiety of alkenyl
  • alkynylene is the divalent moiety of alkynyl
  • heteroalkylene is the divalent moiety of heteroalkyl
  • heteroalkenylene is the divalent moiety of heteroalkenyl
  • heteroalkynylene is the divalent moiety of heteroalkynyl
  • carbocyclylene is the divalent moiety of carbocyclyl
  • heterocyclylene is the divalent moiety of heterocyclyl
  • arylene is the divalent moiety of aryl
  • heteroarylene is the divalent moiety of heteroaryl.
  • pharmaceutically acceptable salt refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response, and the like, and are commensurate with a reasonable benefit/risk ratio.
  • Pharmaceutically acceptable salts are well known in the art. For example, Berge et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66, 1-19, incorporated herein by reference.
  • Pharmaceutically acceptable salts of the compounds of this invention include those derived from suitable inorganic and organic acids and bases.
  • Examples of pharmaceutically acceptable, nontoxic acid addition salts are salts of an amino group formed with 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 or by using other methods known in the art such as ion exchange.
  • inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid
  • organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid or by using other methods known in the art such as ion exchange.
  • salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemi sulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pec
  • Salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium, and N + (CI-4 alkyl)4 ⁇ salts.
  • Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like.
  • Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, lower alkyl sulfonate, and aryl sulfonate.
  • a formula includes compounds that do not include isotopically enriched atoms (e.g., isotopes of hydrogen, nitrogen, and oxygen) and also compounds that do include isotopically enriched atoms.
  • Compounds that include isotopically enriched atoms may be useful, for example, as analytical tools and/or probes in biological assays.
  • a “subject” to which administration is contemplated refers to a human (i.e., male or female of any age group, e.g., pediatric subject (e.g., infant, child, or adolescent) or adult subject (e.g., young adult, middle-aged adult, or senior adult)) or non-human animal.
  • the non-human animal is a mammal (e.g., primate (e.g., cynomolgus monkey or rhesus monkey), commercially relevant mammal (e.g., cattle, pig, horse, sheep, goat, cat, or dog), or bird (e.g., commercially relevant bird, such as chicken, duck, goose, or turkey)).
  • the non-human animal is a fish, reptile, or amphibian.
  • the non-human animal may be a male or female at any stage of development.
  • the non-human animal may be a transgenic animal or genetically engineered animal.
  • administer refers to implanting, absorbing, ingesting, injecting, inhaling, or otherwise introducing a compound described herein, or a composition thereof, in or on a subject.
  • treatment refers to reversing, alleviating, delaying the onset of, or inhibiting the progress of a disease described herein.
  • treatment may be administered after one or more signs or symptoms of the disease have developed or have been observed.
  • treatment may be administered in the absence of signs or symptoms of the disease.
  • treatment may be administered to a susceptible subject prior to the onset of symptoms (e.g., in light of a history of symptoms and/or in light of exposure to a pathogen). Treatment may also be continued after symptoms have resolved, for example, to delay and/or prevent recurrence.
  • modulator refers to a compound that modulates the activity of the protein.
  • a modulator may be an agonist or a partial-agonist.
  • prevent refers to a prophylactic treatment of a subject who is not and was not with a disease but is at risk of developing the disease or who was with a disease, is not with the disease, but is at risk of regression of the disease.
  • the subject is at a higher risk of developing the disease or at a higher risk of regression of the disease than an average healthy member of a population of subjects.
  • cancer refers to a class of diseases characterized by the development of abnormal cells that proliferate uncontrollably and have the ability to infiltrate and destroy normal body tissues. See, e.g., Stedman ’s Medical Dictionary, 25th ed.; Hensyl ed.; Williams & Wilkins: Philadelphia, 1990.
  • Exemplary cancers include, but are not limited to, acoustic neuroma; adenocarcinoma; adrenal gland cancer; anal cancer; angiosarcoma (e.g., lymphangiosarcoma, lymphangioendotheliosarcoma, hemangiosarcoma); appendix cancer; benign monoclonal gammopathy; biliary cancer (e.g., cholangiocarcinoma); bladder cancer; breast cancer (e.g., adenocarcinoma of the breast, papillary carcinoma of the breast, mammary cancer, medullary carcinoma of the breast); brain cancer (e.g., meningioma, glioblastomas, glioma (e.g., astrocytoma, oligodendroglioma), medulloblastoma); bronchus cancer; carcinoid tumor; cervical cancer (e.g., cervical adenocarcinoma); choriocar
  • hematopoietic cancers e.g., leukemia such as acute lymphocytic leukemia (ALL) (e.g., B-cell ALL, T-cell ALL), acute myelocytic leukemia (AML) (e.g., B-cell AML, T-cell AML), chronic myelocytic leukemia (CML) (e.g., B-cell CML, T-cell CML), and chronic lymphocytic leukemia (CLL) (e.g., B-cell CLL, T-cell CLL)); lymphoma such as Hodgkin lymphoma (HL) (e.g., B-cell HL, T-cell HL) and non-Ho
  • Wilms tumor, renal cell carcinoma); liver cancer (e.g., hepatocellular cancer (HCC), malignant hepatoma); lung cancer (e.g., bronchogenic carcinoma, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), adenocarcinoma of the lung); leiomyosarcoma (LMS); mastocytosis (e.g., systemic mastocytosis); muscle cancer; myelodysplastic syndrome (MDS); mesothelioma; myeloproliferative disorder (MPD) (e.g., polycythemia vera (PV), essential thrombocytosis (ET), agnogenic myeloid metaplasia (AMM) a.k.a.
  • HCC hepatocellular cancer
  • lung cancer e.g., bronchogenic carcinoma, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), adenocarcinoma of the lung
  • myelofibrosis MF
  • chronic idiopathic myelofibrosis chronic myelocytic leukemia (CML), chronic neutrophilic leukemia (CNL), hypereosinophilic syndrome (HES)
  • neuroblastoma e.g., neurofibromatosis (NF) type 1 or type 2, schwannomatosis
  • neuroendocrine cancer e.g., gastroenteropancreatic neuroendoctrine tumor (GEP-NET), carcinoid tumor
  • osteosarcoma e.g., bone cancer
  • ovarian cancer e.g., cystadenocarcinoma, ovarian embryonal carcinoma, ovarian adenocarcinoma
  • papillary adenocarcinoma pancreatic cancer
  • pancreatic cancer e.g., pancreatic andenocarcinoma, intraductal papillary mucinous neoplasm (IPMN), Islet cell tumors
  • Anti-cancer agents also referred to herein as oncolytic agents, encompass biotherapeutic anti-cancer agents as well as chemotherapeutic agents.
  • biotherapeutic anti-cancer agents include, but are not limited to, interferons, cytokines (e.g., tumor necrosis factor, interferon a, interferon y), vaccines, hematopoietic growth factors, monoclonal serotherapy, immunostimulants and/or immunodulatory agents (e.g., IL-1, 2, 4, 6, or 12), immune cell growth factors (e.g., GM- CSF) and antibodies (e.g., Herceptin (trastuzumab), T-DM1, AVASTIN (bevacizumab), ERBITUX (cetuximab), Vectibix (panitumumab), Rituxan (rituximab), Bexxar (tositumomab)).
  • interferons e.g., tumor necrosis factor, interferon a, interferon y
  • vaccines e.g., hematopoietic growth factors, monoclonal sero
  • chemotherapeutic agents include, but are not limited to, anti- estrogens (e.g., tamoxifen, raloxifene, and megestrol), LHRH agonists (e.g., goscrclin and leuprolide), anti-androgens (e.g., flutamide and bicalutamide), photodynamic therapies (e.g., vertoporfin (BPD-MA), phthalocyanine, photosensitizer Pc4, and demethoxy- hypocrellin A (2BA-2-DMHA)), nitrogen mustards (e.g., cyclophosphamide, ifosfamide, trofosfamide, chlorambucil, estramustine, and melphalan), nitrosoureas (e.g., carmustine (BCNU) and lomustine (CCNU)), alkyl sulphonates (e.g., busulfan and treosulfan), triazen
  • a “protein,” “peptide,” or “polypeptide” comprises a polymer of amino acid residues linked together by peptide bonds.
  • the term refers to proteins, polypeptides, and peptides of any size, structure, or function. Typically, a protein will be at least three amino acids long.
  • a protein may refer to an individual protein or a collection of proteins. Inventive proteins preferably contain only natural amino acids, although non-natural amino acids (i.e., compounds that do not occur in nature but that can be incorporated into a polypeptide chain) and/or amino acid analogs as are known in the art may alternatively be employed.
  • amino acids in a protein may be modified, for example, by the addition of a chemical entity such as a carbohydrate group, a hydroxyl group, a phosphate group, a famesyl group, an isofamesyl group, a fatty acid group, a linker for conjugation or functionalization, or other modification.
  • a protein may also be a single molecule or may be a multi-molecular complex.
  • a protein may be a fragment of a naturally occurring protein or peptide.
  • a protein may be naturally occurring, recombinant, synthetic, or any combination of these.
  • the additional agent is an antibiotic.
  • antibiotics include, but are not limited to, penicillins (e.g., penicillin, amoxicillin), cephalosporins (e.g., cephalexin), macrolides (e.g., erythromycin, clarithormycin, azithromycin, troleandomycin), fluoroquinolones (e.g., ciprofloxacin, levofloxacin, ofloxacin, delafloxacin), sulfonamides (e.g., co-trimoxazole, trimethoprim), tetracyclines (e.g., tetracycline, chlortetracycline, oxytetracycline, demeclocycline, methacycline, sancycline, doxycline, aureomycin, terramycin, minocycline, 6-deoxytetracycline, lymecycline, meclocycline, metha
  • penicillins e.
  • inflammatory disease refers to a disease caused by, resulting from, or resulting in inflammation.
  • inflammatory disease may also refer to a dysregulated inflammatory reaction that causes an exaggerated response by macrophages, granulocytes, and/or T-lymphocytes leading to abnormal tissue damage and/or cell death.
  • An inflammatory disease can be either an acute or chronic inflammatory condition and can result from infections or non-infectious causes.
  • Inflammatory diseases include, without limitation, atherosclerosis, arteriosclerosis, autoimmune disorders, multiple sclerosis, systemic lupus erythematosus, polymyalgia rheumatica (PMR), gouty arthritis, degenerative arthritis, tendonitis, bursitis, psoriasis, cystic fibrosis, arthrosteitis, rheumatoid arthritis, inflammatory arthritis, Sjogren’s syndrome, giant cell arteritis, progressive systemic sclerosis (scleroderma), ankylosing spondylitis, polymyositis, dermatomyositis, pemphigus, pemphigoid, diabetes (e.g., Type I), myasthenia gravis, Hashimoto’s thyroiditis, Graves’ disease, Goodpasture’s disease, mixed connective tissue disease, sclerosing cholangitis, inflammatory bowel disease, Crohn’s disease, ulcerative colitis, per
  • compositions of the present invention are typically formulated in dosage unit form for ease of administration and uniformity of dosage. It will be understood, however, that the total daily usage of the compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment.
  • the specific therapeutically effective dose level for any particular subject or organism will depend upon a variety of factors including the disease, disorder, or condition being treated and the severity of the disorder; the activity of the specific active ingredient employed; the specific composition employed; the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific active ingredient employed; the duration of the treatment; drugs used in combination or coincidental with the specific active ingredient employed; and like factors well known in the medical arts.
  • an “effective amount” of a compound described herein refers to an amount sufficient to elicit the desired biological response.
  • An effective amount of a compound described herein may vary depending on such factors as the desired biological endpoint, the pharmacokinetics of the compound, the condition being treated, the mode of administration, and the age and health of the subject.
  • an effective amount is a therapeutically effective amount.
  • an effective amount is a prophylactic treatment.
  • an effective amount is the amount of a compound described herein in a single dose.
  • an effective amount is the combined amounts of a compound described herein in multiple doses.
  • a “therapeutically effective amount” of a compound described herein is an amount sufficient to provide a therapeutic benefit in the treatment of a condition or to delay or minimize one or more symptoms associated with the condition.
  • a therapeutically effective amount of a compound means an amount of therapeutic agent, alone or in combination with other therapies, which provides a therapeutic benefit in the treatment of the condition.
  • the term “therapeutically effective amount” can encompass an amount that improves overall therapy, reduces or avoids symptoms, signs, or causes of the condition, and/or enhances the therapeutic efficacy of another therapeutic agent.
  • small molecule refers to molecules, whether naturally- occurring or artificially created (e.g., via chemical synthesis) that have a relatively low molecular weight.
  • a small molecule is an organic compound (e.g., it contains carbon).
  • the small molecule may contain multiple carbon-carbon bonds, stereocenters, and other functional groups (e.g., amines, hydroxyl, carbonyls, and heterocyclic rings, etc.).
  • the molecular weight of a small molecule is not more than about 1,000 g/mol, not more than about 900 g/mol, not more than about 800 g/mol, not more than about 700 g/mol, not more than about 600 g/mol, not more than about 500 g/mol, not more than about 400 g/mol, not more than about 300 g/mol, not more than about 200 g/mol, or not more than about 100 g/mol.
  • the molecular weight of a small molecule is at least about 100 g/mol, at least about 200 g/mol, at least about 300 g/mol, at least about 400 g/mol, at least about 500 g/mol, at least about 600 g/mol, at least about 700 g/mol, at least about 800 g/mol, or at least about 900 g/mol, or at least about 1,000 g/mol. Combinations of the above ranges (e.g., at least about 200 g/mol and not more than about 500 g/mol) are also possible.
  • the small molecule is a therapeutically active agent such as a drug (e.g., a molecule approved by the U.S.
  • the small molecule may also be complexed with one or more metal atoms and/or metal ions.
  • the small molecule is also referred to as a “small organometallic molecule.”
  • Small molecules include, but are not limited to, radionuclides and imaging agents.
  • the TLR2 modulator is one that has already been deemed safe and effective for use in humans or animals by the appropriate governmental agency or regulatory body.
  • drugs approved for human use are listed by the FDA under 21 C.F.R. ⁇ 330.5, 331 through 361, and 440 through 460, incorporated herein by reference; drugs for veterinary use are listed by the FDA under 21 C.F.R. ⁇ 500 through 589, incorporated herein by reference.
  • TLRs are a class of cell-surface receptors expressed on many different cell types that recognize conserved structural motifs present on pathogen-derived molecules.
  • TLR2 recognizes bacteria-derived lipopeptides.
  • One known agonist is a synthetic lipopeptide known as Pam3CSK4, which consists of a peptide-like backbone with 3 lipophilic tails. More recently, Diprovocim was demonstrated to be a potent agonist of TLR2 despite possessing no extended lipid domains.
  • an assay to identify and characterize the binding affinities of novel TLR2 agonists is provided herein.
  • the in vitro TLR2 assay consists of a cell line that responds to TLR2 engagement by producing a factor that elicits a color change in the culture media and can be readily measured by various colorimetric detection methods. This assay enables the identification of existing and novel compounds that engage TLR2 and, by virtue of compound dilution, to approximate the binding affinity of these compounds for eliciting a TLR2 signal.
  • the TLR2 screening assay was developed from the HEK-Blue TLR2 reporter cell line that is commercially available from Invivogen (San Diego, CA).
  • the reporter cell line consists of HEK-293 embryonic kidney cells that have been engineered to express human TLR2 and with an NF-kB promoter driving expression of Secreted Alkaline Phosphatase (SEAP).
  • SEAP Secreted Alkaline Phosphatase
  • downstream signaling results in activation of NF-kB pathway activation and secretion of SEAP.
  • SEAP mediates a color change from clear to indigo that is readily assessed by measuring absorbance at 600 nm.
  • HEK-Blue TLR2 reporter cells are cultured according to manufacturer’s specifications until a confluence of 70-90%, at which point the cells are trypsinized, counted, and plated into 96-well plates at approximately 40,000 cells per well. Cells are cultured overnight and the TLR2 assay is performed 16-24 hours post-plating into 96 well plates.
  • control or test compounds are assessed in a 7-point dilution scheme, comprising a top concentration of 10 micromolar and diluted 10-fold to 10 picomolar.
  • Compounds are diluted from stocks dissolved in DMSO and a 1% DMSO final concentration is maintained in all testing wells.
  • the assay consists of removing the cell culture media after cells have been cultured overnight and replacing the media with HEK-Blue Detection media with TLR2 agonists at indicated dilution schemes.
  • the Detection media contains a chemical substrate that turns from clear to indigo when acted upon by SEAP.
  • the HEK-Blue cells are then cultured overnight in Detection media with diluted compounds. After overnight culture with agonists, each well of the plate is assayed in a plate reader at 600 nanometer absorbance.
  • HEK-Blue TLR2 cells are diluted in complete DME culture medium and plated into 96-well plates at 40,000 cells per well and cultured overnight.
  • TLR2 agonists are diluted 100-fold from DMSO stocks into each well, yielding a final DMSO concentration of 1% in all wells.
  • Treated cells are cultured overnight and the following day are assayed in plate reader for Absorbance at 600 nanometers.
  • TLR2 agonists measure cytokine release from purified immune cells or immortalized cell lines derived from immune cells. Cytokine release is measured by standard ELISA approaches or multiplexed analysis using the Meso Scale Discovery (MSD) platform. Assay
  • Purified RAW 264.7 mouse macrophage cell line cells or human THP-1 monocytic leukemia cell line cells are plated at approximately 40,000 cells per well. Cells are treated with agonist compounds and assays 6-24 hours post-treatment for cytokines released into the cell culture media. Cytokine concentrations are measured using standard ELISA or MSD methods.
  • Immortalized immune cells are particularly well-suited to TLR studies, as they express a repertoire of TLRs and the primary function of their normal progenitors are to respond to infection that are often detected through TLR engagement by pathogen-derived moieties.
  • Immortalized immune cells from human, mouse and rat have been used to demonstrate TLR2 engagement by the compounds described herein.
  • the tumor growth model is a highly sensitive method for determining cytotoxic T cell function. Mice are treated with potentially therapeutic compounds and a bolus of cultured cancer cell lines are implanted orthotopically into the animal, usually on the flank. Treatment may be initiated for some time before cancer cell implantation, after implantation, or both before and after. The rate and extent of tumor growth over time is a good indicator of the immune-potentiating and cancer-suppressive activities of the tested compounds.
  • mice are treated orally with TLR2 modulator (e.g., an agonist or partial agonist) and a combination of these additional compounds for varying amounts of time before and/or after implantation of cancer cells.
  • TLR2 modulator e.g., an agonist or partial agonist
  • These tumor studies may include administration of immune checkpoint therapies at varied times and doses when tumor growth has been established in the model. Tumor growth is monitored for alterations of progression from the control group.
  • Compound A is (3S,4S)-l-(4-(l-((S)-2-(3-heptylureido)-3-(hexylamino)-3- oxopropyl)-lH-imidazol-4-yl)benzoyl)-N3,N4-bis((lS,2R)-2- phenylcyclopropyl)pyrrolidine-3,4-dicarboxamide.
  • Compound B is (3S,4S)-l-(4-(l-((S)-2-(3-heptylureido)-3-(hexylamino)-3- oxopropyl)-lH-l,2,3-triazol-4-yl)benzoyl)-N3,N4-bis((lS,2R)-2- phenylcyclopropyl)pyrrolidine-3,4-dicarboxamide, and its synthesis and characterization are described in International Patent Publication No. WO2021242923 Al.
  • Compound C is (3S,4S)-l-(4-(5-((S)-2-decanamido-3-(hexylamino)-3- oxopropyl)-l,3,4-thiadiazol-2-yl)benzoyl)-N3,N4-bis((lS,2R)-2- phenylcyclopropyl)pyrrolidine-3,4-dicarboxamide, and its synthesis and characterization are described in International Patent Publication No. WO2021242923 Al.
  • Compound D is (3S,4S)-l-(4-((3S,4S)-3-methoxy-4-(3- tridecylureido)pyrrolidine-l-carbonyl)benzoyl)-N3,N4-bis((lS,2R)-2- phenylcyclopropyl)pyrrolidine-3,4-dicarboxamide, and its synthesis and characterization are described in International Patent Publication No. WO2021242923 Al.
  • Compound E is (R)-3-(((R)-2-amino-3-(((S)-3 -hydroxy- 1 -methoxy- 1- oxopropan-2-yl)amino)-3-oxopropyl)thio)propane-l,2-diyl dipalmitate, and its synthesis and characterization are described in “Structure- Activity Relationships in Toll-like Receptor-2 Agonistic Diacylthioglycerol Lipopeptides
  • Compound F is (3S,4S)-l-(4-(l-((S)-3-(hexylamino)-2-(3-octylureido)-3- oxopropyl)-lH-l,2,3-triazol-4-yl)benzoyl)-N3,N4-bis((lS,2R)-2-phenylcyclopropyl)pyrrolidine- 3,4-dicarboxamide, and its synthesis and characterization are described in International Patent Publication No. WO2021242923 Al .
  • Diprovocim-1 (3 S,3 'S,4S,4'S)-1, 1 '-(1,4- phenylenedicarbonyl)bis[N-[(lS,2R)-2-phenylcyclopropyl]-3,4- pyrrolidinedicarboxamide; CAS# 2170867-89-5) is a TLR1/TLR2 agonist, and is commercially available.
  • the compound Pam3CSK4 is S-(2,3-bis(palmitoyloxy)propyl)-N-palmitoyl-L- cysteinyl-L-seryl-L-lysyl-L-lysyl-L-lysyl-L-lysine, and is commercially available.
  • the compound Pam2CSK4 is S-(2,3-bis(palmitoyloxy)propyl)-L-cysteinyl-L- seryl-L-lysyl-L-lysyl-L-lysyl-L-lysine, and is commercially available.
  • mice Dose administered of test compounds in mouse tumor study [00184] After 10 days of treatment, 500,000 MCA205 mouse fibrosarcoma cells in 100 pL sterile phosphate buffered saline (PBS) were implanted subcutaneously into the right flank of each mouse. Mice were orally treated daily with Vehicle or ABX or ABX and TLR2 agonist for 29 days after tumor cell inoculation. Additionally, mice were treated intraperitoneally with 100 pg anti-mouse PD1 clone RMP1-14 (ICHOR BIO #ICH1132) on days 6, 9, 12, and 15 after tumor cell inoculation. Beginning on day 6 after tumor cell inoculation, tumor volume was measured every 2-3 days until the conclusion of the study.
  • PBS sterile phosphate buffered saline
  • Tumor volume data up to Day 25 were entered into Prism (GraphPad) and analyzed with a restricted maximum likelihood (REML) mixed-effects model with time, treatment, and time-treatment interaction effects factored. Time effects were significant, and statistically significant treatment effects were reported as follows: # p ⁇ 0.1, * p ⁇ 0.05, ** p ⁇ 0.01, *** p ⁇ 0.001.
  • Tumor volume data after Day 25 were excluded from analysis as mice began to die and group sizes changed, confounding the results for later time points.
  • Survival data were entered into Prism (GraphPad) and plotted as Kaplan-Meier survival curves. Log- rank test was used to compare survival curves between treatment groups. For both survival and tumor volume analyses, anti-PDl treated mice were compared to vehicle- treated mice and to anti-PDl + ABX-treated mice. All TLR2 modulator-treated mice were compared to the anti-PDl + ABX-treated mice.
  • Fecal samples were homogenized in a mixture of 20:80 acetonitrile:water (ACN: water) to generate a 10X dilution based on the weight of the sample. Homogenates were then futher diluted 5X with blank plasma. Plasma samples were extracted by combining 10 pL of plasma with 60 pL of acetonitrile. The samples were vortexed briefly and centrifuges for 5 minutes at 3000 revolutions per minute. 50 pL of the supernatant was transferred to a clean plate and diluted with 50 pL of water. Samples were analyzed using at Triple Quad 5500 high performance liquid chromatography (HPLC)- tandem mass spectrometer (MS/MS) using electrospray ionization.
  • HPLC high performance liquid chromatography
  • MS/MS tandem mass spectrometer
  • ng/mL nanogram/milliliter
  • g ng/gram
  • feces ng/gram Concentrations in either nanogram/milliliter (ng/mL) (plasma) or ng/gram (g) (feces) were calculated based on a standard curve from 1-5000 ng/mL. The limit of quantitation for plasma and feces was 5 ng/mL and 50 ng/mL, respectively.
  • Mouse RAW cells (RAW264.7 cells, Purchased from ATCC, catalog number TIB-71) were grown at 37°C, 5% CO2 in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin, and 2 mM glutamine.
  • RAW cells were plated in tissue culture (TC)-treated 96-well plates at 40,000 cells/100 pL/well and incubated overnight to adhere. Cells were treated with TLR2 agonists or Dimethyl sulfoxide (DMSO), to a final DMSO concentration of 1%. After 24 hours of treatment, plates were spun at lOOOx g for 3 mins and supernatants were collected.
  • DMEM Modified Eagle’s Medium
  • DMSO Dimethyl sulfoxide
  • Human THP-1 cells (purchased from ATCC, catalog number TIB-202) were grown at 37°C, 5% CO2 in RPMI-1640 supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin, 2 mM glutamine, and 0.05 mM beta-mercaptoethanol.
  • THP-1 cells were plated in TC-treated 96-well plates at 40,000 cells/100 pL/well and treated for 3 days with 10 ng/mL phorbol 12-myristate 13-acetate (PMA) to induce differentiation into mature macrophages. Media was then aspirated and replaced with standard culture media.
  • PMA phorbol 12-myristate 13-acetate
  • mice Five C57/B16 mice per group were dosed daily for 28 days via oral gavage with 0.5% methylcellulose 0.5% Tween-80 (vehicle) or 100 pg diprovocim in 0.5% methylcellulose 0.5% Tween-80. On Day 28, mice were sacrificed and spleens were removed and processed to single cells.
  • spleens were physical dissociated by passing through a 70 pm cell strainer, washed with phosphate buffered saline (PBS), treated for 5 mins with eBioscience RBC lysis buffer (Thermo-Fisher 00-4333-57), washed again with PBS, and resuspended in RPMI-1640 containing 0.5% fetal bovine serum, 1% penicillin/streptomycin, 2 mM glutamine, and 0.05 mM beta-mercaptoethanol.
  • PBS phosphate buffered saline
  • eBioscience RBC lysis buffer Thermo-Fisher 00-4333-57
  • splenocytes were plated at 1 million cells/well in tissue culture-treated 96-well plates and treated with 50 ng/mL phorbol 12-myristate 13-acetate (PMA) and 1 pg/mL ionomycin to non-specifically activate immune cells. Cells were incubated at 37°C, 5% CO2. After 24 hours of treatment, plates were spun at lOOOx g for 3 mins and supernatants were collected. Supernatants were assayed at a 1 :4 dilution in the MSD V- Plex Mouse Pro-inflammatory Panel (Meso Scale Diagnostics catalog number K15048D) according to manufacturer’s instructions. Cytokine concentrations were plotted in Prism (GraphPad) and analyzed by ANOVA with Sidak’s multiple comparisons post hoc test, with statistically significant findings reported as follows: **** p ⁇ 0.0001.
  • Step-1 Preparation of methyl 4-(l-(3-(benzyloxy)-2-((tert-butoxycarbonyl)amino)-3- oxopropyl)-lH-imidazol-4-yl)benzoate.
  • Step-2 Preparation of methyl 4-(l-(2-amino-3-(benzyloxy)-3-oxopropyl)-lH- imidazol-4-yl)benzoate
  • Methyl 4-( 1 -(2-amino-3 -(benzyloxy)-3 -oxopropyl)- lH-imidazol-4- yl)benzoate (1.7 g) was dissolved in dry Dichloromethane (15.0 mL) and cooled to 0°C. Trifluoroacetic acid (9.0 mL) was added drop wise over 10 min and the reaction mixture was warmed to room temperature and stirred 16 h. The solvent was removed under reduced pressure and saturated sodium bicarbonate solution was added.
  • Step-3 Preparation of methyl 4-(l-(3-(benzyloxy)-2-(3-heptylureido)-3-oxopropyl)- lH-imidazol-4-yl)benzoate.
  • Step-4 Preparation of 2-(3-heptylureido)-3-(4-((R)-4-(methoxycarbonyl)cyclohexa- l,5-dien-l-yl)-lH-imidazol-l-yl)propanoic acid.
  • Step-5 Preparation of methyl 4-(l-(2-(3-heptylureido)-3-(hexylamino)-3-oxopropyl)-
  • reaction mixture was stirred for 10 min then triethylamine (0.35 mL, 2.51 mmol) was added drop wise.
  • the reaction mixture was stirred at room temperature for 16 h.
  • the mixture was diluted with ethyl acetate (50mL), washed with saturated aq. sodium bicarbonate (2x50 mL) and brine (2x50mL).
  • the organic layer was dried over sodium sulphate and concentrated under reduced pressure.
  • Step-6 Preparation of 4-(l-(2-(3-heptylureido)-3-(hexylamino)-3-oxopropyl)-lH-
  • Lithium hydroxide monohydrate (0.08 g, 1.92 mmol) was added to a stirred solution of methyl 4-(l-(2-(3-heptylureido)-3-(hexylamino)-3-oxopropyl)-lH-imidazol-4- yl) benzoate (0.24 g, 0.48 mmol) in THF: water (8, 8 mL) at 0°C and stirred for 16 h.
  • Step-7 Preparation (3S,4S)-l-(4-(l-(2-(3-heptylureido)-3-(hexylamino)-3- oxopropyl)-lH-imidazol-4-yl)benzoyl)-N3,N4-bis((lS,2R)-2- phenylcyclopropyl)pyrrolidine-3,4-dicarboxamide.
  • N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (0.13 g, 0.70 mmol), Hydroxybenzotriazole (0.094 g, 0.7 mmol) and ((3S,4S)-N3,N4-bis((lS,2R)-2- phenylcyclopropyl)pyrrolidine-3,4-dicarboxamide TFA salt (0.23g , 0.56 mmol, WO2021242923 Al 2021-12-02) were added sequentially.
  • the reaction mixture was stirred for 10 min then tri ethylamine (0.19 mL, 1.4 mmol) was added drop wise and stirring continued for 16 hrs.
  • mice 7-8 week old female C57BL/6 mice were inoculated subcutaneously on the flank with IxlO 5 MC38 cells in 100 pL 50% Matrigel in Dulbecco’s Modified Eagle Medium. 10 days after inoculation, 10 mice were randomized to each treatment group based on tumor volume, with an average tumor volume of ⁇ 60 mm 3 . Mice were injected intraperitoneally with 10 mg/kg anti-mouse PD1 clone RMP1-14 (ICHOR BIO #ICH1132) on days 7, 11, 15, and 19 after randomization.
  • ICHOR BIO #ICH1132 anti-mouse PD1 clone
  • One treatment group received only anti-PDl treatment, and one group was additionally treated with 50 pg/kg Compound F by oral gavage once daily from day 7 after randomization until the end of the study. Beginning on day 1 after randomization, tumor volume was measured every 2-3 days for 42 days. Tumor volume data were entered into Prism (GraphPad) and analyzed with a restricted maximum likelihood (REML) mixed- effects model with time, treatment, and time-treatment interaction effects factored. Time effects were significant (p ⁇ 0.0001), and both treatment and interaction effects were not statistically significant (p>0.05). Survival data were entered into Prism (GraphPad) and plotted as Kaplan-Meier survival curves. Log-rank test was used to compare survival curves between treatment groups.
  • MERS restricted maximum likelihood
  • TLR2 (HEK-Blue) Assay Average values of all runs. A: 0.1-50 nM; B: 51-350 nM; C: 351-1000 nM; D: >1000 nM. Compounds and data as reported in WO2021242923 Al, Tables 1-4.
  • a pharmaceutical composition comprising a TLR2 modulator, an immune checkpoint inhibitor, and a pharmaceutically acceptable carrier.
  • A2 The pharmaceutical composition of Al, formulated for oral administration.
  • A3 The pharmaceutical composition of any one of A1-A2, formulated for control led-rel ease within the gastrointestinal tract, lower intestine, or colon of a subject.
  • a method of enhancing the efficacy of a cancer therapy comprising orally administering a TLR2 modulator in combination with at least one immune checkpoint inhibitor, or a pharmaceutical composition of any one of A1-A3, for treatment of a subject with a cancer.
  • a method of treating cancer in a subject in need thereof comprising orally administering to the subject a TLR2 modulator and an immune checkpoint inhibitor, or a pharmaceutical composition of any one of Al -A3.
  • A6 The method of A5, wherein the subject responds to the immune checkpoint inhibitor (e.g., the subject’s cancer is treated, for example, tumor volume decreases), and wherein the subject’s response is improved relative to the subject’s response to the immune checkpoint inhibitor in the absence of the TLR2 modulator.
  • the immune checkpoint inhibitor e.g., the subject’s cancer is treated, for example, tumor volume decreases
  • TLR2 Toll-like receptor 2
  • A8 The method of any one of A4-A7, wherein the TLR2 modulator and the immune checkpoint inhibitor are administered concurrently, or at different times.
  • A9 The method of any one of A4-A8, wherein the immune checkpoint inhibitor comprises an inhibitor of PD1 protein.
  • A10. The method of any one of A4-A9, wherein the immune checkpoint inhibitor comprises an inhibitor of PD-L1 protein.
  • A14 The method of A13, wherein the dosing schedule promotes an immunizing response.
  • A15 The method of A13, wherein the dosing schedule promotes enhancement of cytotoxic immunity.
  • A16 The method of any one of A4-A15, wherein the TLR2 modulator is an agonist or partial agonist of TLR2, and further modulates heterodimeric TLR2/TLR1 proteins.
  • A17 The method of any one of A4-A15, wherein the TLR2 modulator is an agonist or partial agonist of TLR2, and further modulates heterodimeric TLR2/TLR6 proteins.
  • A18 The method of any one of A4-15, wherein the TLR2 modulator is diprovocim or a pharmaceutically acceptable salt thereof.
  • A19 The method of any one of A4-A15, wherein the TLR2 modulator is
  • A20 The method of any one of A4-A15, wherein the TLR2 modulator is
  • A21 The method of any one of A4-A15, wherein the TLR2 modulator is selected from compounds: A, B, C, D, E, and pharmaceutically acceptable salts thereof.
  • A22 The method of any one of A4-A15, wherein the TLR2 modulator is compound A or a pharmaceutically acceptable salt thereof.
  • A23 The method of any one of A4-A15, wherein the TLR2 modulator is compound B or a pharmaceutically acceptable salt thereof.
  • A24 The method of any one of A4-A15, wherein the TLR2 modulator is compound C or a pharmaceutically acceptable salt thereof.
  • A25 The method of any one of A4-A15, wherein the TLR2 modulator is compound D or a pharmaceutically acceptable salt thereof.
  • A26 The method of any one of A4-A15, wherein the TLR2 modulator is compound E or a pharmaceutically acceptable salt thereof.
  • A27 The method of any one of A4-A26, wherein the cancer is a solid cancer, bladder cancer, breast cancer, cervical cancer, colon and rectal cancer, endometrial cancer, kidney cancer, lip and oral cancer, liver cancer, melanoma, mesothelioma, non-small cell lung cancer, non-melanoma skin cancer, oral cancer, ovarian cancer, pancreatic cancer, prostate cancer, sarcoma, small cell lung cancer or thyroid cancer.
  • the cancer is a solid cancer, bladder cancer, breast cancer, cervical cancer, colon and rectal cancer, endometrial cancer, kidney cancer, lip and oral cancer, liver cancer, melanoma, mesothelioma, non-small cell lung cancer, non-melanoma skin cancer, oral cancer, ovarian cancer, pancreatic cancer, prostate cancer, sarcoma, small cell lung cancer or thyroid cancer.
  • A28 The method of any one of A4-A27, further comprising administering one or more additional oncolytic agents.
  • A29 The method of A28, wherein the oncolytic agents are selected from checkpoint inhibitors (e.g., pembrolizumab, nivolumab, ipilmumab, atezolizumab, durvalumab, avelumab, and tremelimumab), immuno-oncology (IO) agents (e.g., STING agonists or IDO inhibitors), targeted therapies such as protein kinase inhibitors, PARP inhibitors, nuclear receptor antagonists/degraders/hormone therapies (e.g., imatinib, erlotinib, olaparib, tamoxifen, and fulvestrant), cytotoxic agents (e.g., cyclophosphamide, carboplatin, paclitaxel, doxorubicin, epothilone, irinotecan, etoposide, azacytidine, vinblastine, and bleomycin),
  • A30 The method of any one of A4-A29, wherein the subject is a mammal.
  • A31 The method of A30, wherein the mammal is human.
  • A32 The method of any one of A4-A31, wherein the TLR2 modulator is administered on a once per day dosing schedule.
  • A33 The method of any one of A4-A31, wherein the TLR2 modulator is administered on a twice per day dosing schedule.
  • A34 The method of any one of A4-A31, wherein the TLR2 modulator is administered three times per day dosing schedule.
  • the present disclosure encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the listed claims is introduced into another claim.
  • any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim.
  • elements are presented as lists, e.g., in Markush group format, each subgroup of the elements is also disclosed, and any element(s) can be removed from the group.

Abstract

La présente divulgation concerne des méthodes de modulation des effets d'une thérapie de point de contrôle immunitaire à l'aide de modulateurs de TLR2, et en particulier des méthodes associées de traitement du cancer.
PCT/US2022/050564 2021-11-22 2022-11-21 Composés modulateurs, compositions pharmaceutiques et leurs utilisations WO2023091747A1 (fr)

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