ENPP1 inhibitors and methods of modulating immune responses

Abstract

The present application provides compounds, compositions and methods for inhibiting ENPP1. Aspects of the subject method include contacting a sample with an ENPP1 inhibitor compound to inhibit the cGAMP hydrolysis activity of ENPP1. In some cases, the ENPP1 inhibitor compound is cell-impermeable. The ENPP1 inhibitor compound can act extracellularly to block the degradation of cGAMP. Pharmaceutical compositions and methods for treating cancer are also provided. Aspects of the method include administering a therapeutically effective amount of an ENPP1 inhibitor to a subject to treat the subject's cancer. In some cases, the cancer is a solid tumor cancer. A method of administering radiation therapy to a subject in combination with administering an ENPP1 inhibitor to a subject is also provided. The radiation therapy can be administered in the subject method in a dose and / or frequency that effectively reduces the radiation damage caused to the subject, but still stimulates an immune response.

Description

[0001] Related Applications

[0002] This application claims priority to and the benefit of U.S. Provisional Patent Application No. 62 / 800,283, filed on February 1, 2019, and U.S. Provisional Patent Application No. 62 / 814,745, filed on March 6, 2019, each of which is incorporated herein by reference in its entirety.

[0003] Government Rights

[0004] This invention was made with government support under Contracts CA190896 and CA228044 awarded by the National Institutes of Health and Contract W81XWH-18-1-0041 awarded by the Department of Defense. The government has certain rights in this invention. Background Art

[0005] Cyclic GMP-AMP (cGAMP) activates the stimulator of interferon genes (STING) pathway, an important anticancer innate immune pathway. The cGAS-cGAMP-STING pathway is activated in the presence of cytoplasmic DNA, which may be due to microbial infection or pathophysiological conditions including cancer and autoimmune diseases. Cyclic GMP-AMP synthase (cGAS) belongs to the nucleotidyltransferase family and is a universal DNA sensor that is activated upon binding to cytoplasmic dsDNA to produce the signaling molecule (2'-5',3'-5') cyclic GMP-AMP (or 2',3'-cGAMP or cyclic GMP-AMP, cGAMP). 2',3'-cGAMP acts as a second messenger during microbial infection, binding and activating STING, leading to the production of type I interferons (IFNs) and other co-stimulatory molecules that trigger immune responses. In addition to its role in infectious diseases, the STING pathway has emerged as a target for cancer immunotherapy and autoimmune diseases.

[0006] Ectonucleotide pyrophosphatase / phosphodiesterase 1 (ENPP1) is the major cGAMP hydrolase that can degrade cGAMP. ENPP1 is a member of the ectonucleotide pyrophosphatase / phosphodiesterase (ENPP) family and is a type II transmembrane glycoprotein comprising two identical disulfide-bonded subunits. ENPP1 has a broad specificity to cleave a variety of substrates including phosphodiester bonds of nucleotides and nucleotide sugars and pyrophosphate bonds of nucleotides and nucleotide sugars. ENPP1 can play a role in hydrolyzing nucleoside 5' triphosphates to their corresponding monophosphates and can also hydrolyze diadenosine polyphosphates. Summary of the invention

[0007] Compounds, compositions and methods for inhibiting ENPP1 are provided. Aspects of the subject method include contacting a sample with an ENPP1 inhibitor compound to inhibit the cGAMP hydrolysis activity of ENPP1. In some cases, the ENPP1 inhibitor compound is cell-impermeable. The ENPP1 inhibitor compound can block the degradation of cGAMP outside the cell. Pharmaceutical compositions and methods for treating cancer are also provided. Aspects of the method include administering a therapeutically effective amount of an ENPP1 inhibitor to a subject to treat the subject's cancer. In some cases, the cancer is a solid tumor cancer. A method of administering radiation therapy to a subject in combination with administering an ENPP1 inhibitor to a subject is also provided. The radiation therapy can be administered in the subject method in a dose and / or frequency that effectively reduces the radiation damage caused to the subject, but still stimulates an immune response.

[0008] These and other advantages and features of the present disclosure will become apparent to those skilled in the art after reading the details of the compositions and methods of use more fully described below. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] The present invention may be best understood from the following detailed description when read in conjunction with the accompanying drawings. The patent or application document contains at least one color drawing. It is emphasized that, in accordance with conventional practice, the various features of the drawings are not drawn to scale. Instead, the dimensions of the various features are arbitrarily expanded or reduced for clarity. The following figures are included in the accompanying drawings. It should be understood that the figures described below are for illustrative purposes only. The figures are not intended to limit the scope of the present teachings in any way.

[0010] Figure 1 , Panels A to J, show evidence that cGAMP is expressed as a soluble factor from 293T cGAS ENPP1 - / - Experimental results of cell output.

[0011] Figure 2 , Panels A to C , show experimental results demonstrating that ENPP1 can regulate extracellular cGAMP.

[0012] Figure 3 , Panels A to F, illustrate the structure and activity of an exemplary ENPP1 inhibitor (Compound 1) in various cellular assays.

[0013] Figure 4 ,Panels A to E , show the results of experiments demonstrating that cancer cells express cGAS in culture and continuously export cGAMP.

[0014] Figure 5, Panels A to I , show experimental results demonstrating that sequestration of extracellular cGAMP reduces tumor-associated dendritic cells in a tumor cGAS- and host STING-dependent manner.

[0015] Figure 6 , Panels A to D, show the expression of ENPP1 - / - Experimental results show that tumors that recruit innate immune infiltrates are less aggressive and more susceptible to IR and anti-CTLA-4 (cytotoxic T lymphocyte-associated antigen 4) therapy.

[0016] Figure 7 , Panels A to C , show experimental results demonstrating that ENPP1 inhibition synergizes with IR treatment and anti-CTLA-4 to exert antitumor effects.

[0017] Figure 8 , Panels A to D, illustrate the use of LC-MS / MS methods and 293T cGAS ENPP1 低 and 293T cGASENPP1 - / - Cell lines were used to assess ENPP1 hydrolysis activity and cGAMP levels.

[0018] Fig. 9 ,Panels A to B, show the explanation CD14 + Schematic diagram and results of an experiment in which primary human peripheral blood mononuclear cells (PBMCs) responded to extracellular cGAMP.

[0019] Fig.10 , Panels A to B, show the results of experiments comparing the ENPP1 inhibitory activity of compound 1 and compound QS1, and show the activity of QS1 in a cell-based assay.

[0020] Fig.11 , Panels A to F, show experimental results demonstrating that exemplary ENPP1 inhibitor Compound 1 (STF-1084) is cell-impermeable, specific for ENPP1, and non-toxic.

[0021] Fig.12 ,Panels A to E ,show the results of experiments demonstrating that cancer cells continuously export cGAMP in culture.

[0022] Fig.13 , Panels A to D , show experimental results demonstrating that sequestration of extracellular cGAMP reduces tumor-associated dendritic cells in a tumor cGAS- and host STING-dependent manner.

[0023] Fig.14 , Panels A to F, show the identified ENPP1 - / -The tumors resulted in an increase in tumor-associated dendritic cells that were less aggressive and more susceptible to IR and anti-CTLA-4 therapy.

[0024] Fig.15 Shown are graphs of data demonstrating that ENPP1 inhibition (eg, using Compound 1; STF-1084) synergizes with IR treatment to increase tumor-associated dendritic cells.

[0025] Fig.16 Schematic diagrams illustrating different modes of cGAMP delivery from synthesizing cells to target cells are shown.

[0026] Fig.17 Shown is a schematic diagram illustrating that cGAMP is a cancer danger signal secreted by cancer cells in the body.

[0027] 18A to 18C Shown are data illustrating that an exemplary ENPP1 inhibitor (Compound 1) can increase the amount of extracellular cGAMP present in a cellular system.

[0028] FIG. 19A to FIG. 19B Shown are experimental schematics and results illustrating that an exemplary ENPP1 inhibitor (Compound 1) can increase cGAMP-stimulated interferon transcription.

[0029] FIG. 20A to FIG. 20B Shown are data illustrating that an exemplary ENPP1 inhibitor (Compound 1) can increase the number of tumor-associated dendritic cells in a mouse tumor model.

[0030] FIG. 21A to FIG. 21C Shown are experimental results illustrating that ENPP1 inhibition synergizes with IR treatment and anti-CTLA-4 to exert anti-tumor effects.

[0031] Fig. 22 Shown is a schematic diagram illustrating that ENPP1 is an innate immune checkpoint that regulates the immune transmitter cGAMP.

[0032] definition

[0033] Before further describing the embodiments of the present disclosure, it should be understood that the present disclosure is not limited to the particular embodiments described, and therefore may of course vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting, as the scope of the present disclosure will be limited only by the appended claims.

[0034] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure belongs. Any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of embodiments of the present disclosure.

[0035] It must be noted that, as used herein and in the appended claims, the singular forms "a / an", "and / and", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a compound" includes not only a single compound but also a combination of two or more compounds, reference to a "substituent" includes a single substituent as well as two or more substituents, and so on.

[0036] In describing and claiming the present invention, certain terms will be used according to the definitions set forth below. It should be understood that the definitions provided herein are not intended to be mutually exclusive. Therefore, some chemical moieties may fall within the definitions of more than one term.

[0037] The phrases "for example," "for instance," "such as," or "including" are intended to introduce examples that further illustrate a more general subject matter. These examples are provided merely to aid in understanding the present disclosure and are not meant to be limiting in any way.

[0038] The publications discussed herein are provided only for their disclosure prior to the filing date of the present application. Nothing herein shall be construed as an admission that the present invention is not entitled to such publications prior to the invention. In addition, the date of publication provided may differ from the actual publication date, which may require independent confirmation.

[0039] The terms "active agent," "antagonist," "inhibitor," "drug," and "pharmacologically active agent" are used interchangeably herein to refer to a chemical material or compound that, when administered to an organism (human or animal), induces a desired pharmacological and / or physiological effect through local and / or systemic action.

[0040] The terms "treatment", "treating" and the like refer to obtaining a desired pharmacological and / or physiological effect, such as reducing tumor burden. The effect may be preventive in terms of completely or partially preventing a disease or its symptoms; and / or the effect may be therapeutic in terms of partially or completely curing a disease and / or side effects attributable to the disease. As used herein, "treatment" is intended to cover any treatment of a disease in a mammal, particularly a human, and includes: (a) preventing a disease or symptoms of a disease from occurring in a subject who may be susceptible to the disease but has not yet been diagnosed with the disease (e.g., including a disease that may be associated with or caused by a primary disease (such as in the case of liver fibrosis that may lead to chronic HCV infection); (b) inhibiting a disease, i.e., curbing its development; and (c) alleviating a disease, i.e., regressing the disease (e.g., reducing tumor burden).

[0041] The term "pharmaceutically acceptable salt" means a salt that is acceptable for administration to a patient such as a mammal (a salt with a counterion that has acceptable mammalian safety for a given dosage regimen). Such salts may be derived from a pharmaceutically acceptable inorganic or organic base and may be derived from a pharmaceutically acceptable inorganic or organic acid. "Pharmaceutically acceptable salt" refers to a pharmaceutically acceptable salt of a compound, which is derived from a variety of organic and inorganic counterions well known in the art, and includes, by way of example only, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, and the like; and when the molecule contains a basic functional group, salts of organic or inorganic acids, such as hydrochloride, hydrobromide, formates, tartrates, benzenesulfonates, methanesulfonates, acetates, maleates, oxalates, and the like.

[0042] The terms "individual", "host", "subject" and "patient" are used interchangeably herein and refer to animals, including but not limited to humans and non-human primates, including monkeys and humans; rodents, including rats and mice; bovines; equines; ovines; felines; canines; etc. "Mammal" means one or more members of any mammalian species, and includes, for example, canines; felines; equines; bovines; ovines; rodents, etc., as well as primates, such as non-human primates and humans. Non-human animal models, such as mammals, such as non-human primates, murines, lagomorphs, etc., can be used for experimental studies.

[0043] The terms "determine," "measure," "evaluate," and "measure" are used interchangeably and include both quantitative and qualitative determination.

[0044] The terms "polypeptide" and "protein" used interchangeably herein refer to a polymeric form of amino acids of any length, which may include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides with modified peptide backbones. The term includes fusion proteins, including but not limited to fusion proteins with heterologous amino acid sequences, fusions containing heterologous and native leader sequences with or without an N-terminal methionine residue; immunolabeled proteins; fusion proteins with detectable fusion partners, such as fusion proteins containing fluorescent proteins, β-galactosidase, luciferase, etc. as fusion partners; etc.

[0045] The terms "nucleic acid molecule" and "polynucleotide" are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides or their analogs. A polynucleotide may have any three-dimensional structure and may perform any known or unknown function. Non-limiting examples of polynucleotides include genes, gene fragments, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, control regions, isolated RNA of any sequence, nucleic acid probes, and primers. Nucleic acid molecules may be linear or circular.

[0046] "Therapeutically effective amount" or "effective amount" means the amount of a compound that, when administered to a mammal or other subject to treat a disease, disorder or condition, is sufficient to effect such treatment for the disease, disorder or condition. The "therapeutically effective amount" will vary depending on the compound, the disease and its severity, and the age, weight, etc., of the subject to be treated.

[0047] The term "unit dosage form" as used herein refers to physically discrete units suitable as unit dosages for human and animal subjects, each unit containing a predetermined amount of a compound (e.g., an aminopyrimidine compound as described herein) calculated to produce the desired effect in combination with a pharmaceutically acceptable diluent, carrier or vehicle. The specifications for the unit dosage form depend on the specific compound used and the effect to be achieved, as well as the pharmacodynamics associated with each compound in the host.

[0048] The terms "pharmaceutically acceptable excipient", "pharmaceutically acceptable diluent", "pharmaceutically acceptable carrier" and "pharmaceutically acceptable adjuvant" refer to excipients, diluents, carriers or adjuvants that can be used to prepare pharmaceutical compositions that are generally safe, non-toxic and have no biological or other adverse effects, and include excipients, diluents, carriers and adjuvants that are acceptable for veterinary use as well as human pharmaceutical use. "Pharmaceutically acceptable excipients, diluents, carriers and adjuvants" as used in the specification and claims include one and more than one such excipients, diluents, carriers and adjuvants.

[0049] The term "pharmaceutical composition" is intended to encompass compositions suitable for administration to a subject such as a mammal, particularly a human. Typically, a "pharmaceutical composition" is sterile and preferably free of contaminants that can cause an undesirable response in a subject (e.g., one or more compounds in the pharmaceutical composition are pharmaceutical grade). A pharmaceutical composition can be designed to be administered to a subject or patient in need via many different routes of administration (including oral, buccal, rectal, parenteral, intraperitoneal, intradermal, intratracheal, intramuscular, subcutaneous, etc.).

[0050] The phrase "having the formula" or "having the structure" is not intended to be limiting and is used in the same manner as the term "comprising" is generally used. The term "independently selected from" is used herein to indicate that the listed elements, for example, R groups, etc., may be the same or different.

[0051] The terms "may," "optionally," "optionally," or "may optionally" mean that the subsequently described event may or may not occur, and thus the description includes instances where the event occurs and instances where the event does not occur. For example, the phrase "optionally substituted / optionally substituted with" means that non-hydrogen substituents may or may not be present on a given atom, and thus the description includes structures where the non-hydrogen substituents are present and structures where the non-hydrogen substituents are not present.

[0052] "Acyl" refers to the radicals HC(O)-, alkyl-C(O)-, substituted alkyl-C(O)-, alkenyl-C(O)-, substituted alkenyl-C(O)-, alkynyl-C(O)-, substituted alkynyl-C(O)-, cycloalkyl-C(O)-, substituted cycloalkyl-C(O)-, cycloalkenyl-C(O)-, substituted cycloalkenyl-C(O)-, aryl-C(O)-, substituted aryl -C(O)-, heteroaryl-C(O)-, substituted heteroaryl-C(O)-, heterocyclyl-C(O)-, and substituted heterocyclyl-C(O)-, wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocycle, and substituted heterocycle are as defined herein. For example, acyl includes the "acetyl" group CH3C(O)-.

[0053] The term "alkyl" generally refers to a branched or unbranched saturated hydrocarbon group (i.e., a monoradical hydrocarbon group), although not necessarily containing 1 to about 24 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, octyl, decyl, etc., and cycloalkyl groups such as cyclopentyl, cyclohexyl, etc. Typically, although not necessarily, the alkyl groups herein may contain 1 to about 18 carbon atoms, and such groups may contain 1 to about 12 carbon atoms. The term "lower alkyl" means an alkyl group of 1 to 6 carbon atoms. "Substituted alkyl" refers to an alkyl group substituted with one or more substituent groups, and this includes examples in which two hydrogen atoms from the same carbon atom in the alkyl substituent are replaced, such as in the case of a carbonyl group (i.e., the substituted alkyl group may include a -C(=O)- moiety). The terms "heteroatom-containing alkyl" and "heteroalkyl" refer to an alkyl substituent in which at least one carbon atom is replaced by a heteroatom, as described in further detail below. If not otherwise stated, the terms "alkyl" and "lower alkyl" include straight chain, branched, cyclic, unsubstituted, substituted and / or heteroatom-containing alkyl or lower alkyl, respectively.

[0054] The term "substituted alkyl" is intended to include alkyl groups as defined herein wherein one or more carbon atoms in the alkyl chain are optionally replaced by heteroatoms such as -O-, -N-, -S-, -S(O)n- (wherein n is 0 to 2), -NR- (wherein R is hydrogen or alkyl), having from 1 to 5 substituents selected from the group consisting of alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxy, oxo, thioketo, , carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclyloxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclyloxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-aryl, -SO2-heteroaryl and -NRaRb, wherein R' and R" may be the same or different and are selected from hydrogen, optionally substituted alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, heteroaryl and heterocycle.

[0055] The term "alkenyl" refers to a straight chain, branched or cyclic hydrocarbon group of 2 to about 24 carbon atoms containing at least one double bond, such as vinyl, n-propenyl, isopropenyl, n-butenyl, isobutenyl, octenyl, decenyl, tetradecenyl, hexadecenyl, eicosene, tetracosene, etc. Usually, although not necessarily, the alkenyl group herein can contain 2 to about 18 carbon atoms, and for example can contain 2 to 12 carbon atoms. The term "lower alkenyl" means an alkenyl group of 2 to 6 carbon atoms. The term "substituted alkenyl" refers to an alkenyl substituted by one or more substituent groups, and the terms "heteroatom-containing alkenyl" and "heteroalkenyl" refer to an alkenyl in which at least one carbon atom is replaced by a heteroatom. If not otherwise specified, the terms "alkenyl" and "lower alkenyl" include straight chain, branched, cyclic, unsubstituted, substituted and / or heteroatom-containing alkenyl and lower alkenyl, respectively.

[0056] The term "alkynyl" refers to a straight or branched hydrocarbon group of 2 to 24 carbon atoms containing at least one triple bond, such as ethynyl, n-propynyl, etc. Typically, although not necessarily, the alkynyl groups herein may contain 2 to about 18 carbon atoms, and such groups may also contain 2 to 12 carbon atoms. The term "lower alkynyl" means an alkynyl group of 2 to 6 carbon atoms. The term "substituted alkynyl" refers to an alkynyl substituted by one or more substituent groups, and the terms "heteroatom-containing alkynyl" and "heteroalkynyl" refer to an alkynyl in which at least one carbon atom is replaced by a heteroatom. If not otherwise specified, the terms "alkynyl" and "lower alkynyl" include straight, branched, unsubstituted, substituted and / or heteroatom-containing alkynyl and lower alkynyl, respectively.

[0057] The term "alkoxy" refers to an alkyl group bound by a single terminal ether linkage; that is, an "alkoxy" group can be represented as -O-alkyl, wherein alkyl is as defined above. A "lower alkoxy" group refers to an alkoxy group containing 1 to 6 carbon atoms, and includes, for example, methoxy, ethoxy, n-propoxy, isopropoxy, tert-butoxy, and the like. Substituents represented herein as "C1-C6 alkoxy" or "lower alkoxy" can, for example, contain 1 to 3 carbon atoms, and as a further example, such substituents can contain 1 or 2 carbon atoms (i.e., methoxy and ethoxy). The names "-OMe" and "MeO-" refer to methoxy.

[0058] The term "substituted alkoxy" refers to the groups substituted alkyl-O-, substituted alkenyl-O-, substituted cycloalkyl-O-, substituted cycloalkenyl-O-, and substituted alkynyl-O-, wherein substituted alkyl, substituted alkenyl, substituted cycloalkyl, substituted cycloalkenyl, and substituted alkynyl are as defined herein.

[0059] The term "aryl", unless otherwise indicated, refers to an aromatic substituent, which generally, but not necessarily, contains 5 to 30 carbon atoms and contains a single aromatic ring or multiple aromatic rings fused together, directly connected or indirectly connected (so that different aromatic rings are bound to a common group, such as a methylene or ethylene moiety). The aryl group can, for example, contain 5 to 20 carbon atoms, and as a further example, the aryl group can contain 5 to 12 carbon atoms. For example, the aryl group can contain one aromatic ring or two or more aromatic rings that are fused or connected (i.e., biaryl, aryl-substituted aryl, etc.). Examples include phenyl, naphthyl, biphenyl, diphenyl ether, diphenylamine, benzophenone, etc. "Substituted aryl" refers to an aryl moiety substituted by one or more substituent groups, and the terms "heteroatom-containing aryl" and "heteroaryl" refer to such aryl substituents, wherein at least one carbon atom is replaced by a heteroatom, as will be described in further detail below. Aryl is intended to include stable cyclic, heterocyclic, polycyclic and polyheterocyclic unsaturated C3-C 14 moiety (such as but not limited to phenyl, biphenyl, naphthyl, pyridyl, furanyl, thienyl, imidazolyl, pyrimidinyl and oxazolyl), which may be further substituted with 1-5 members selected from the group consisting of hydroxy, C1-C8 alkoxy, C1-C8 branched or straight chain alkyl, acyloxy, carbamoyl, amino, N-acylamino, nitro, halogen, trifluoromethyl, cyano and carboxyl (see, e.g., Katritzky, Handbook of Heterocyclic Chemistry). If not otherwise indicated, the term "aryl" includes unsubstituted, substituted and / or heteroatom-containing aromatic substituents.

[0060] The term "aralkyl" refers to an alkyl group having an aryl substituent, and the term "alkaryl" refers to an aryl group having an alkyl substituent, wherein "alkyl" and "aryl" are as defined above. Typically, the aralkyl and alkaryl groups herein contain 6 to 30 carbon atoms. Aralkyl and alkaryl groups may, for example, contain 6 to 20 carbon atoms, and as a further example, such groups may contain 6 to 12 carbon atoms.

[0061] The term "alkylene" refers to a dialkyl group. Unless otherwise indicated, such groups include saturated hydrocarbon chains containing 1 to 24 carbon atoms, which may be substituted or unsubstituted, may contain one or more alicyclic groups and may contain heteroatoms. "Low alkylene" refers to an alkylene linker containing 1 to 6 carbon atoms. Examples include methylene (--CH2--), ethylene (--CH2CH2--), propylene (--CH2CH2CH2--), 2-methylpropylene (--CH2--CH(CH3)--CH2--), hexylene (--(CH2)6--) and the like.

[0062] Similarly, the terms "alkenylene," "alkynylene," "arylene," "aralkylene," and "alkarylene" refer to alkenyl, alkynyl, aryl, aralkyl, and alkaryl groups, respectively.

[0063] The term "amino" refers to the group -NRR', wherein R and R' are independently hydrogen or non-hydrogen substituents, wherein non-hydrogen substituents include, for example, alkyl, aryl, alkenyl, aralkyl, and substituted and / or heteroatom-containing variations thereof.

[0064] The terms "halo" and "halogen" are used in the conventional sense to refer to chloro, bromo, fluoro, or iodo substituents.

[0065] "Carboxyl", "carboxyl" or "carboxylate" refers to -CO2H or a salt thereof.

[0066] "Cycloalkyl" refers to a cycloalkyl group of 3 to 10 carbon atoms having a single or multiple cyclic rings (including fused rings, bridged rings and spiro ring systems). Examples of suitable cycloalkyl groups include, for example, adamantyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, etc. Such cycloalkyl groups include, for example, monocyclic structures such as cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, etc., or polycyclic structures such as adamantyl, etc.

[0067] The term "substituted cycloalkyl" refers to a cycloalkyl group having 1 to 5 substituents or 1 to 3 substituents selected from alkyl, substituted alkyl, alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxy, oxo, thioketo, carboxy, carboxyalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclyloxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclyloxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl and -SO2-heteroaryl.

[0068] The term "heteroatom-containing" as in "heteroatom-containing alkyl" (also referred to as a "heteroalkyl" group) or "heteroatom-containing aryl" (also referred to as a "heteroaryl" group) refers to a molecule, linkage, or substituent in which one or more carbon atoms are replaced by atoms other than carbon, such as nitrogen, oxygen, sulfur, phosphorus, or silicon, typically nitrogen, oxygen, or sulfur. Similarly, the term "heteroalkyl" refers to an alkyl substituent containing a heteroatom, the term "heterocycloalkyl" refers to a cycloalkyl substituent containing a heteroatom, the term "heterocyclic" or "heterocycle" refers to a cyclic substituent containing a heteroatom, the terms "heteroaryl" and "heteroaromatic" refer to "aryl" and "aromatic" substituents containing a heteroatom, respectively, and the like. Examples of heteroalkyl groups include alkoxyaryl, alkylthio-substituted alkyl, N-alkylated aminoalkyl, and the like. Examples of heteroaryl substituents include pyrrolyl, pyrrolidinyl, pyridinyl, quinolinyl, indolyl, furanyl, pyrimidinyl, imidazolyl, 1,2,4-triazolyl, tetrazolyl, and the like, and examples of heteroatom-containing alicyclic groups are pyrrolidino, morpholino, piperazino, piperidino, tetrahydrofuranyl, and the like.

[0069] "Heteroaryl" refers to an aromatic group having 1 to 15 carbon atoms, such as 1 to 10 carbon atoms and 1 to 10 heteroatoms selected from oxygen, nitrogen and sulfur in the ring. Such heteroaryl groups can have a monocycle (such as pyridyl, imidazolyl or furyl) or multiple condensed rings (for example, as in the case of groups such as indolizinyl, quinolyl, benzofuran, benzimidazolyl or benzothienyl) in the ring system, wherein at least one ring in the ring system is aromatic, provided that the connection point is through the atom of the aromatic ring. In certain embodiments, one or more nitrogen and / or sulfur ring atoms of the heteroaryl group are optionally oxidized to provide N-oxide (N→O), sulfinyl or sulfonyl moieties. The term includes, for example, pyridyl, pyrrolyl, indolyl, thienyl and furyl. Unless otherwise limited by the definition of heteroaryl substituents, such heteroaryl groups may be optionally substituted with 1 to 5 substituents, or 1 to 3 substituents, selected from acyloxy, hydroxy, thiol, acyl, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, substituted alkyl, substituted alkoxy, substituted alkenyl, substituted alkynyl, substituted cycloalkyl, substituted cycloalkenyl, amino, substituted amino, aminoacyl, acylamino, alkaryl, aryl. , aryloxy, azido, carboxyl, carboxylalkyl, cyano, halogen, nitro, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclyloxy, aminoacyloxy, oxyacylamino, thioalkoxy, substituted thioalkoxy, thioaryloxy, thioheteroaryloxy, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl and -SO2-heteroaryl, and trihalomethyl.

[0070] The terms "heterocycle", "heterocyclic" and "heterocyclyl" refer to a saturated or unsaturated group having a single ring or multiple fused rings (including fused bridged ring systems and spirocyclic ring systems) and having 3 to 15 ring atoms (including 1-4 heteroatoms). These ring heteroatoms are selected from nitrogen, sulfur and oxygen, wherein in the fused ring system, one or more rings can be cycloalkyl, heterocycloalkyl, aryl or heteroaryl, provided that the point of attachment is through a non-aromatic ring. In certain embodiments, one or more nitrogen and / or sulfur atoms of the heterocyclic group are optionally oxidized to provide N-oxide, -S(O)- or -SO2- moieties.

[0071] Examples of heterocyclic and heteroaryl groups include, but are not limited to, azetidine, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, indoline, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, phenanthroline, isothiazole, phenazine, isoxazole, phenoxazine, Phenothiazine, imidazolidine, imidazoline, piperidine, piperazine, indoline, phthalimide, 1,2,3,4-tetrahydroisoquinoline, 4,5,6,7-tetrahydrobenzo[b]thiophene, thiazole, thiazolidine, thiophene, benzo[b]thiophene, morpholinyl, thiomorpholinyl (also known as thiomorpholinyl), 1,1-dioxothiomorpholinyl, piperidinyl, pyrrolidine, tetrahydrofuranyl, and the like.

[0072] Unless otherwise limited by the definition of heterocyclic substituents, such heterocyclic groups may be optionally substituted with 1 to 5 or 1 to 3 substituents selected from the group consisting of alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxy, oxo, thioketo, carboxy, carboxyalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclyloxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclyloxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl, -SO2-heteroaryl, and fused heterocycle.

[0073] "Hydrocarbyl" refers to a monovalent hydrocarbon group containing 1 to about 30 carbon atoms (including 1 to about 24 carbon atoms, further including 1 to about 18 carbon atoms and further including about 1 to 12 carbon atoms), including straight chain, branched, cyclic, saturated and unsaturated substances, such as alkyl groups, alkenyl groups, aryl groups, etc. The hydrocarbon group can be substituted with one or more substituent groups. The term "heteroatom-containing hydrocarbon group" refers to a hydrocarbon group in which at least one carbon atom is replaced by a heteroatom. Unless otherwise indicated, the term "hydrocarbyl" should be interpreted as including substituted and / or heteroatom-containing hydrocarbon moieties.

[0074] As mentioned in some of the foregoing definitions, "substituted" as in "substituted hydrocarbyl," "substituted alkyl," "substituted aryl," etc., means that at least one hydrogen atom bonded to a carbon (or other) atom in the hydrocarbyl, alkyl, aryl or other moiety is replaced with one or more non-hydrogen substituents. Examples of such substituents include, but are not limited to, functional groups and hydrocarbon moieties C1-C24 alkyl (including C1-C18 alkyl, further including C1-C12 alkyl, and further including C1-C6 alkyl), C2-C24 alkenyl (including C2-C18 alkenyl, further including C2-C12 alkenyl, and further including C2-C6 alkenyl), C2-C24 alkynyl (including C2-C18 alkynyl, further including C2-C12 alkynyl, and further including C2-C6 alkynyl), C5-C30 aryl (including C5-C20 aryl, and further including C5-C12 aryl), and C6-C30 aralkyl (including C6-C20 aralkyl, and further including C6-C12 aralkyl). The above hydrocarbon moieties may be further substituted by one or more functional groups or additional hydrocarbon moieties, such as those specifically listed. Unless otherwise stated, any group described herein should be interpreted as including substituted and / or heteroatom-containing moieties in addition to unsubstituted groups.

[0075] "Sulfonyl" refers to the groups SO2-alkyl, SO2-substituted alkyl, SO2-alkenyl, SO2-substituted alkenyl, SO2-cycloalkyl, SO2-substituted cycloalkyl, SO2-cycloalkenyl, SO2-substituted cycloalkenyl, SO2-aryl, SO2-substituted aryl, SO2-heteroaryl, SO2-substituted heteroaryl, SO2-heterocycle, and SO2-substituted heterocycle, wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocycle, and substituted heterocycle are as defined herein. Sulfonyl includes, for example, methyl-SO2-, phenyl-SO2-, and 4-methylphenyl-SO2-.

[0076] The term "functional group" means a chemical group such as a halogen, a hydroxyl, a thiol, a C1-C24 alkoxyl, a C2-C24 alkenyloxyl, a C2-C24 alkynyloxyl, a C5-C20 aryloxyl, an acyl group (including a C2-C24 alkylcarbonyl (-CO-alkyl) and a C6-C20 arylcarbonyl (-CO-aryl)), an acyloxy group (-O-acyl), a C2-C24 alkoxycarbonyl (-(CO)-O-alkyl), a C6-C20 aryloxycarbonyl (-(CO)-O-aryl), a halocarbonyl (-CO)-X, wherein X is a halogen), a C2-C24 alkylcarbonate (carbonat o)(-O-(CO)-O-alkyl), C6-C20 aryl carbonate (-O-(CO)-O-aryl), carboxyl (-COOH), carboxylate (-COO-), carbamoyl (-(CO)-NH2), monosubstituted C1-C24 alkylcarbamoyl (-(CO)-NH(C1-C24 alkyl)), disubstituted alkylcarbamoyl (-(CO)-N(C1-C24 alkyl)2), monosubstituted arylcarbamoyl (-(CO)-NH-aryl), thiocarbamoyl (-(CS)-NH2), urea (-N H-(CO)-NH2), cyano (-C≡N), isocyano (-N+≡C-), cyano (-OC≡N), isocyano (-O-N+≡C-), isothiocyano (-SC≡N), azido (-N=N+=N-), formyl (-(CO)-H), thioformyl (-(CS)-H), amino (-NH2), mono- and di-(C1-C24 alkyl) substituted amino, mono- and di-(C5-C20 aryl) substituted amino, C2-C24 alkylamido (-NH-(CO)-alkyl), C5-C20 arylamido (-NH-(CO)- imino (-CR=NH, wherein R=hydrogen, C1-C24 alkyl, C5-C20 aryl, C6-C20 alkaryl, C6-C20 aralkyl, etc.), alkylimino (-CR=N(alkyl), wherein R=hydrogen, alkyl, aryl, alkaryl, etc.), arylimino (-CR=N(aryl), wherein R=hydrogen, alkyl, aryl, alkaryl, etc.), nitro (-NO2), nitroso (-NO), sulfo (-SO2-OH), sulfonate (-SO2-O-), C1-C24 alkylthio (-S-alkyl; also known as "alkylthio"), arylthio (-S-aryl;Also known as "arylthio"), C1-C24 alkylsulfinyl (-(SO)-alkyl), C5-C20 arylsulfinyl (-(SO)-aryl), C1-C24 alkylsulfonyl (-SO2-alkyl), C5-C20 arylsulfonyl (-SO2-aryl), phosphono (-P(O)(OH)2), phosphonato (-P(O)(O-)2), phosphinate (-P(O)(O-)), phosphate (-PO2) and phosphino (-PH2), mono- and di-(C1-C24 alkyl) substituted phosphino, mono- and di-(C5-C20 aryl) substituted phosphine. In addition, if the particular group allows, the aforementioned functional groups can be further substituted with one or more additional functional groups or with one or more hydrocarbyl moieties such as those specifically listed above. ;

[0077] "Connection" or "connector" as in "connector group", "connector moiety", etc. means a connecting portion that connects two groups via a covalent bond. The connecting group can be a straight chain, branched, cyclic or single atom. Examples of such connecting groups include alkyl, alkenylene, alkynylene, arylene, alkarylene, aralkylene, and a connecting portion containing functional groups including but not limited to: amido (-NH-CO-), urea (-NH-CO-NH-), imide (-CO-NH-CO-), oxo (-O-), thio (-S-), oxadioxy (-OO-), carbonyldioxy (-O-CO-O-), alkyldioxy (-O-(CH2)nO-), oxadioxyimino (-O-NH-), oxadioxy (-NH-), carbonyl (-CO-), etc. In some cases, one, two, three, four or five or more carbon atoms of the connecting group backbone can be optionally substituted by sulfur, nitrogen or oxygen heteroatoms. The bonds between the backbone atoms may be saturated or unsaturated, and typically no more than one, two or three unsaturated bonds are present in the backbone of the linker. The linker may contain one or more substituents, for example, having alkyl, aryl or alkenyl groups. The linker may include, but is not limited to, one or more poly(ethylene glycol) units (e.g., -(CH2-CH2-O)-); ethers; thioethers; amines; alkyls (e.g., (C1-C 12 ) alkyl), which can be straight or branched, such as methyl, ethyl, n-propyl, 1-methylethyl (isopropyl), n-butyl, n-pentyl, 1,1-dimethylethyl (tert-butyl), etc. The linking group backbone can include cyclic groups, such as aryl, heterocyclic or cycloalkyl groups, wherein 2 or more atoms of the cyclic group, such as 2, 3 or 4 atoms are included in the backbone. The linking group can be cleavable or non-cleavable. Any convenient orientation and / or connection of the linking group to the connected group can be used.

[0078] When the term "substituted" appears before a list of possible substituted groups, it is intended that this term applies to every member of this group. For example, the phrase "substituted alkyl and aryl" should be interpreted as "substituted alkyl and substituted aryl".

[0079] In addition to the disclosure herein, the term "substituted" when used to modify a specified group or radical may also mean that one or more hydrogen atoms of the specified group or radical are independently each replaced with the same or different substituents as defined below.

[0080] In addition to the groups disclosed for each term herein, the following are used to replace one or more hydrogen atoms on a saturated carbon atom in the specified group or radical (any two hydrogen atoms on a single carbon can be replaced by =O, =NR 70 , =N-OR 70 , =N2 or =S) is -R unless otherwise specified. 60 , halogen, =O, -OR 70 、-SR 70 、-NR 80 R 80 , trihalomethyl, -CN, -OCN, -SCN, -NO, -NO2, =N2, -N3, -SO2R 70 、-SO2O – M + 、-SO2OR 70 、-OSO2R 70 、-OSO2O – M + 、-OSO2OR 70 、-P(O)(O – )2(M + )2、-P(O)(OR 70 ) – M + 、-P(O)(OR 70 )2. -C(O)R 70 、-C(S)R 70 、-C(NR 70 )R 70 、-C(O)O – M + 、-C(O)OR 70 、-C(S)OR 70 、-C(O)NR 80 R 80 、-C(NR 70 )NR 80 R 80 、-OC(O)R 70 、-OC(S)R 70 、-OC(O)O- M + 、-OC(O)OR 70 、-OC(S)OR 70 、-NR 70 C(O)R 70 、-NR 70 C(S)R 70 、-NR 70 CO2 – M + 、-NR 70 CO2R 70 、-NR 70 C(S)OR 70 、-NR 70 C(O)NR 80 R 80 、-NR 70 C(NR 70 )R 70 and-NR 70 C(NR 70 )NR 80 R 80 , where R 60 is selected from the group consisting of optionally substituted alkyl, cycloalkyl, heteroalkyl, heterocycloalkylalkyl, cycloalkylalkyl, aryl, aralkyl, heteroaryl and heteroaralkyl, each R 70 are independently hydrogen or R 60 ; Each R 80 Independently R 70 Alternatively, two R 80’ Together with the nitrogen atom to which they are bound, they form a 5-, 6- or 7-membered heterocycloalkyl group which may optionally contain 1 to 4 identical or different further heteroatoms selected from O, N and S, wherein N may be substituted with -H or C1-C3 alkyl; and each M + is a counterion with a net single positive charge. Each M + can independently be, for example, an alkali metal ion such as K + 、Na + , Li + ; ammonium ions, such as + N(R 60 )4; or alkaline earth metal ions, such as [Ca 2+ ] 0.5 , [Mg 2+ ] 0.5 or[Ba 2+ ] 0.5(“Subscript 0.5” means that one counter ion of a divalent alkaline earth metal ion can be the ionized form of the compound of the present invention, and the other is a typical counter ion such as chloride, or both ionized compounds disclosed herein can be used as counter ions for such divalent alkaline earth metal ions, or the secondary ionized compounds of the present invention can be used as counter ions for such divalent alkaline earth ions). As a specific example, -NR 80 R 80 It is intended to include -NH2, -NH-alkyl, N-pyrrolidinyl, N-piperazinyl, 4N-methyl-piperazin-1-yl and N-morpholinyl.

[0081] Except as disclosed herein, a "substituted" alkene, alkyne, aryl, and heteroaryl group having a hydrogen substituent on an unsaturated carbon atom is, unless otherwise specified, -R 60 , halogen, -O - M + 、-OR 70 、-SR 70 , -S – M + 、-NR 80 R 80 , trihalomethyl, -CF3, -CN, -OCN, -SCN, -NO, -NO2, -N3, -SO2R 70 、-SO3 – M + 、-SO3R 70 、-OSO2R 70 、-OSO3 – M + 、-OSO3R 70 、-PO3 -2 (M + )2、-P(O)(OR 70 ) – M + 、-P(O)(OR 70 )2. -C(O)R 70 、-C(S)R 70 、-C(NR 70 )R 70 、-CO2 – M + 、-CO2R 70 、-C(S)OR 70 、-C(O)NR 80 R 80 、-C(NR 70 )NR 80 R 80 、-OC(O)R 70 、-OC(S)R70 、-OCO2 – M + 、-OCO2R 70 、-OC(S)OR 70 、-NR 70 C(O)R 70 、-NR 70 C(S)R 70 、-NR 70 CO2 – M + 、-NR 70 CO2R 70 、-NR 70 C(S)OR 70 、-NR 70 C(O)NR 80 R 80 、-NR 70 C(NR 70 )R 70 and-NR 70 C(NR 70 )NR 80 R 80 , where R 60 , R 70 , R 80 and M + As defined above, provided that in the case of a substituted alkene or alkyne, the substituent is not -O - M + 、-OR 70 、-SR 70 or -S – M + .

[0082] In addition to the groups disclosed for each term herein, "substituted" substituent groups for hydrogen on nitrogen atoms in heteroalkyl and cycloheteroalkyl groups are, unless otherwise specified, -R 60 , -O - M + 、-OR 70 、-SR 70 , -S - M + 、-NR 80 R 80 , trihalomethyl, -CF3, -CN, -NO, -NO2, -S(O)2R 70 、-S(O)2O - M + 、-S(O)2OR 70 、-OS(O)2R 70 、-OS(O)2O - M +、-OS(O)2OR 70 、-P(O)(O - )2(M + )2、-P(O)(OR 70 ) - M + 、-P(O)(OR 70 )(OR 70 )、-C(O)R 70 、-C(S)R 70 、-C(NR 70 )R 70 、-C(O)OR 70 、-C(S)OR 70 、-C(O)NR 80 R 80 、-C(NR 70 )NR 80 R 80 、-OC(O)R 70 、-OC(S)R 70 、-OC(O)OR 70 、-OC(S)OR 70 、-NR 70 C(O)R 70 、-NR 70 C(S)R 70 、-NR 70 C(O)OR 70 、-NR 70 C(S)OR 70 、-NR 70 C(O)NR 80 R 80 、-NR 70 C(NR 70 )R 70 and-NR 70 C(NR 70 )NR 80 R 80 , where R 60 , R 70 , R 80 and M + As defined above.

[0083] In addition to the disclosure herein, in certain embodiments, a substituted group has 1, 2, 3, or 4 substituents, 1, 2, or 3 substituents, 1 or 2 substituents, or 1 substituent.

[0084] Unless otherwise indicated, nomenclature of substituents not explicitly defined herein is derived by naming the terminal portion of the functionality first, followed by naming the adjacent functionality toward the point of attachment. For example, the substituent "aralkyloxycarbonyl" refers to the group (aryl)-(alkyl)-OC(O)-.

[0085] For any group containing one or more substituents disclosed herein, it is of course understood that such groups do not contain any substitution or substitution pattern that is sterically impractical and / or synthetically infeasible. In addition, the subject compounds include all stereochemical isomers resulting from the substitution of these compounds.

[0086] In certain embodiments, the substituents may contribute to the optical isomerism and / or stereoisomerism of the compound. Salts, solvates, hydrates and prodrug forms of the compound are also of interest. The disclosure encompasses all such forms. Therefore, the compounds described herein include salts, solvates, hydrates, prodrugs and isomeric forms thereof, including pharmaceutically acceptable salts, solvates, hydrates, prodrugs and isomers thereof. In certain embodiments, the compound may be metabolized into a pharmaceutically active derivative.

[0087] Unless otherwise specified, a reference to an atom is intended to include all isotopes of that atom. For example, a reference to H is intended to include 1 H. 2 H (i.e. D) and 3 H (i.e. T), and the reference to C is intended to include 12 C and all isotopes of carbon (such as 13 C).

[0088] It will be apparent to those skilled in the art after reading this disclosure that each of the various embodiments described and illustrated herein has discrete components and features that can be easily separated or combined with any of the features in the other several embodiments without departing from the scope or spirit of the invention. Any of the described methods can be performed in the order of events described or in any other order that is logically possible.

[0089] Although the apparatus and method have been or will be described for the sake of grammatical fluency and functional interpretation, it should be expressly understood that unless expressly recited under 35 U.S.C. §112, the claims should not be interpreted as necessarily being limited in any way to the interpretation of "method" or "step", but should be given the full meaning and equivalent scope of the definitions provided in the claims in accordance with the judicial doctrine of equivalents, and in the event that the claims are expressly recited under 35 U.S.C. §112, they should be given full legal equivalents under 35 U.S.C. §112.

[0090] Definitions for other terms and concepts appear throughout the detailed description. DETAILED DESCRIPTION OF THE INVENTION

[0092] As summarized above, aspects of the present disclosure include compounds, compositions and methods for inhibiting ENPP1. Aspects of the methods include contacting a sample with an ENPP1 inhibitor compound to inhibit the cGAMP hydrolysis activity of ENPP1. These compounds, compositions and methods are found to be useful in a variety of applications requiring inhibition of ENPP1.

[0093] Also provided are pharmaceutical compositions and methods for treating cancer using the subject ENPP1 inhibitor compounds.Aspects of the methods include administering to a subject a therapeutically effective amount of an ENPP1 inhibitor compound to inhibit the hydrolysis of cGAMP and treat cancer in the subject.

[0094] ENPP1-Inhibitor Compounds

[0095] The subject ENPP1 inhibitor compound may include a core structure based on an aryl or heteroaryl ring system connected to a hydrophilic head group, such as a quinazoline or quinoline group. The linker between the aryl or heteroaryl ring system and the hydrophilic head group may include a monocyclic aryl, a heteroaryl, a carbocyclic ring or a heterocyclic ring and one or more non-cyclic linking parts. The quinazoline or quinoline core structure may be substituted by a linker at the 4-position. The aryl or heteroaryl ring system is optionally further substituted. The present disclosure includes compounds having a quinoline core structure substituted by a linker at the 4-position and substituted by a cyano group at the 3-position. In some cases, the linker includes a 1,4-disubstituted 6-membered aryl or heteroaryl cyclic group, such as a phenyl or substituted phenyl. In some cases, the linker includes a 1,4-disubstituted 6-membered saturated heterocyclic or carbocyclic ring, such as an N1,4-disubstituted piperidine ring or an N1,N4-disubstituted piperazine ring. Other aspects of the subject ENPP1 inhibitor compounds are described below and in PCT Application No. PCT / US2018 / 050018, filed September 7, 2018, by Li et al., the disclosure of which is incorporated herein by reference in its entirety.

[0096] The term "hydrophilic head group" refers to a group attached to a core aryl or heteroaryl ring system that is hydrophilic and well solvated in an aqueous environment, such as under physiological conditions, and has low permeability to cell membranes. In some cases, low permeability to cell membranes means a permeability coefficient of 10 -4 cm / s or less, such as 10 -5 cm / s or less, 10 - 6 cm / s or less, 10 -7 cm / s or less, 10 -8 cm / s or less, 10 -9cm / s or less, or even less, as measured by passive diffusion through a membrane (e.g., a cell monolayer, such as a colorectal Caco-2 or kidney MDCK cell line) via any convenient isolated hydrophilic head group. See, e.g., Yang and Hinner, Methods Mol Biol. 2015; 1266: 29–53. The hydrophilic head group can impart improved water solubility and reduced cell permeability to the molecule to which it is attached. The hydrophilic head group can be any convenient hydrophilic group that is well solvated in an aqueous environment and has low permeability to the membrane. In some cases, the hydrophilic group is a discrete functional group (e.g., as described herein) or a substituted form thereof. In general, the permeability of a charged group or a larger non-charged polar group is low. In some cases, the hydrophilic head group is charged, such as positively or negatively charged. In some embodiments, the hydrophilic head group itself is not cell permeable and imparts cell impermeability to the subject compound. It should be understood that the hydrophilic head group or its prodrug form can be selected to provide the cell permeability required for the subject compound. In some cases, the hydrophilic head group is a neutral hydrophilic group. In some cases, the hydrophilic head group is included in the prodrug form, thus including a precursor portion that can be removed in vivo. In some cases, the subject compound is cell permeable.

[0097] The hydrophilic head group can be any convenient group capable of binding or chelating zinc ions, or a prodrug form thereof. In some cases, the hydrophilic head group is a phosphorus-containing group. Target phosphorus-containing groups that can be used for the subject ENPP1 inhibitors include, but are not limited to, phosphonic acid or phosphonate, phosphonate, phosphate, phosphate, thiophosphate, thiophosphate, phosphoramidate, and thiophosphoramidate, or a salt thereof, or a prodrug form thereof (e.g., as described herein).

[0098] Exemplary target ENPP1 inhibitor compounds including quinazoline and isoquinoline ring systems are listed in the compound structures of Formulas (I)-(XVb) and Tables 1-2.

[0099] In some cases, the subject ENPP1 inhibitor compound is a compound of formula (I), or a prodrug, a pharmaceutically acceptable salt, or a solvate thereof:

[0100]

[0101] in,

[0102] X 1 is a hydrophilic head group (e.g., as described herein);

[0103] A is a ring system selected from the group consisting of aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocycle, and substituted heterocycle;

[0104] L 1 and L 2 are independently a covalent bond or a linker;

[0105] Z 3 Not present or selected from NR 22 , O and S;

[0106] Z 2 CR 12 or N;

[0107] Z 1 CR 11 or N;

[0108] R 1 is selected from the group consisting of H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkylaryl, substituted alkylaryl, alkylheteroaryl, substituted alkylheteroaryl, alkenylaryl (e.g., vinylaryl), substituted alkenylaryl, alkenylheteroaryl (e.g., vinylheteroaryl), substituted alkenylheteroaryl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocycle, and substituted heterocycle;

[0109] R 11 and R 12 independently selected from H, cyano, trifluoromethyl, halogen, alkyl, and substituted alkyl;

[0110] R 22 is selected from the group consisting of H, alkyl, and substituted alkyl; and

[0111] R 2 To R 5 is independently selected from H, OH, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, -OCF3, halogen, cyano, amine, substituted amine, amide, heterocycle, and substituted heterocycle; or wherein R 2 and R 3 , R 3 and R 4 , or R 4 and R 5 Together with the carbon atoms to which they are attached, they provide a fused ring (eg, a 5- or 6-membered monocyclic ring) selected from heterocycle, substituted heterocycle, cycloalkyl, substituted cycloalkyl, aryl, and substituted aryl.

[0112] In certain embodiments of Formula (I), Z 3 In certain embodiments of Formula (I), Z 3 NR22 , where R 22 Selected from H, C (1-6) Alkyl and substituted C (1-6) In some cases, Z 3 is NH. In some cases, Z 3 NR 22 , and R 22 C (1-6) In some cases, Z 3 NR 22 , and R 22 To replace C (1-6) In certain cases of formula (I), Z 3 is O. In certain cases of formula (I), Z 3 For S.

[0113] In some cases of Formula (I), Z 1 CR 11 , and R 11 is selected from hydrogen, cyano, trifluoromethyl, halogen, alkyl and substituted alkyl hydrogen. In some cases, the alkyl or substituted alkyl is C 1-5 In some cases of Formula (I), Z 1 CR 11 , and R 11 is hydrogen. In some cases, R 11 It is cyano.

[0114] In some cases, R 11 is trifluoromethyl. In some cases, R 11 is a halogen, such as Br, I, Cl or F. In some cases, R 11 is an alkyl group, such as C 1-5 In some cases, R 11 is a substituted alkyl group, such as a substituted C 1-5 alkyl.

[0115] In some cases of Formula (I), Z 2 CR 12 , and R 12 is selected from hydrogen, cyano, trifluoromethyl, halogen, alkyl and substituted alkyl hydrogen. In some cases, the alkyl or substituted alkyl is C 1-5 In some cases of Formula (I), Z 2 CR 12 , and R 12 is hydrogen. In some cases, R 12 In some cases, R 12 is trifluoromethyl. In some cases, R12 is a halogen, such as Br, I, Cl or F. In some cases, R 12 is an alkyl group, such as C 1-5 In some cases, R 12 is a substituted alkyl group, such as a substituted C 1-5 alkyl.

[0116] In certain embodiments of Formula (I), Z 1 and Z 2 At least one of is N. In certain embodiments of Formula (I), Z 1 CR 11 , and Z 2 is N. In certain cases of formula (I), Z 1 is N, and Z 2 CR 12 In certain cases of Formula (I), Z 1 CR 11 , and Z 2 CR 12 In certain cases of Formula (I), Z 1 is N, and Z 2 is N.

[0117] In certain embodiments of Formula (I), L 1 and L 2 Each is a covalent bond. In some cases, L 1 and L 2 Each is a linker. In some cases, L 1 is a covalent bond, and L 2 In some cases, L 1 is a linking group, and L 2 Any convenient linking group can be used to connect A to X and / or A to Z. 3 (e.g., as described herein). In some cases, A is linked to X by a covalent bond. In some cases, A is linked to X by a linear linker having a length of 1-12 atoms, such as 1-10, 1-8, or 1-6 atoms, such as 1, 2, 3, 4, 5, or 6 atoms. The linker L 2 Can be (C 1-6 ) alkyl linker or substituted (C 1-6 ) alkyl linker, which is optionally substituted with a heteroatom or a linking functional group such as an ester (-CO2-), an amide (CONH), a carbamate (OCONH), an ether (-O-), a thioether (-S-) and / or an amino group (-NR-, wherein R is H or an alkyl group). In some cases, A is covalently linked to Z. 3In some cases, A is linked to Z via a linear linker of 1-12 atoms in length, such as 1-10, 1-8, or 1-6 atoms in length, such as 1, 2, 3, 4, 5, or 6 atoms in length. 3 The linking group L 1 Can be (C 1-6 ) alkyl linker or substituted (C 1-6 ) alkyl linker, which is optionally substituted with heteroatoms or linking functional groups such as keto (CO), ester (-CO2-), amido (CONH), carbamate (OCONH), ether (-O-), thioether (-S-) and / or amino group (-NR-, wherein R is H or alkyl). 3 NR 22 When the linker L 1 It may include a terminal keto group (C=O) which is 3 Together provide an acylamino group (NR 22 CO) connection. When Z 31 When L is O or S, the linking group 1 It may include a terminal keto group (C=O) which is 31 Together they provide an ester or thioester group linkage.

[0118] In certain embodiments of Formula (I), Z 3 It is a phosphorus-containing group capable of binding zinc ions, or a prodrug form thereof.

[0119] In certain cases of Formula (I), Z 3 Selected from NR 22 , O and S. Therefore, the subject ENPP1 inhibitor compound of formula (I) can be described by formula (II):

[0120]

[0121] Where Z 31 Selected from NR 22 , O and S.

[0122] In certain embodiments of Formula (II), Z 31 NR 22 , where R 22 Selected from H, C (1-6) Alkyl and substituted C (1-6) In some cases, Z 31 is NH. In some cases, Z 31 NR 22 , and R 22 C (1-6) In some cases, Z 31 NR 22, and R 22 To replace C (1-6) In certain cases of formula (I), Z 31 is O. In certain cases of formula (I), Z 31 For S.

[0123] In some cases of Formula (II), Z 1 CR 11 , and R 11 is selected from hydrogen, cyano, trifluoromethyl, halogen, alkyl and substituted alkyl hydrogen. In some cases, the alkyl or substituted alkyl is C 1-5 In some cases of formula (II), Z 1 CR 11 , and R 11 is hydrogen. In some cases, R 11 It is cyano.

[0124] In some cases, R 11 is trifluoromethyl. In some cases, R 11 is a halogen, such as Br, I, Cl or F. In some cases, R 11 is an alkyl group, such as C 1-5 In some cases, R 11 is a substituted alkyl group, such as a substituted C 1-5 alkyl.

[0125] In some cases of Formula (II), Z 2 CR 12 , and R 12 is selected from hydrogen, cyano, trifluoromethyl, halogen, alkyl and substituted alkyl hydrogen. In some cases, the alkyl or substituted alkyl is C 1-5 In some cases of formula (II), Z 2 CR 12 , and R 12 is hydrogen. In some cases, R 12 In some cases, R 12 is trifluoromethyl. In some cases, R 12 is a halogen, such as Br, I, Cl or F. In some cases, R 12 is an alkyl group, such as C 1-5 In some cases, R 12 is a substituted alkyl group, such as a substituted C 1-5 alkyl.

[0126] In certain embodiments of Formula (II), Z 1 and Z 2 At least one of is N. In certain embodiments of Formula (I), Z1 CR 11 , and Z 2 is N. In certain cases of formula (I), Z 1 is N, and Z 2 CR 12 In certain cases of Formula (I), Z 1 CR 11 , and Z 2 CR 12 In certain cases of Formula (I), Z 1 is N, and Z 2 is N.

[0127] In certain embodiments of Formula (II), L 1 and L 2 Each is a covalent bond. In some cases, L 1 and L 2 Each is a linker. In some cases, L 1 is a covalent bond, and L 2 In some cases, L 1 is a linking group, and L 2 Any convenient linking group can be used to connect A to X and / or A to Z. 3 (e.g., as described herein). In some cases, A is linked to X by a covalent bond. In some cases, A is linked to X by a linear linker having a length of 1-12 atoms, such as 1-10, 1-8, or 1-6 atoms, such as 1, 2, 3, 4, 5, or 6 atoms. The linker L 2 Can be (C 1-6 ) alkyl linker or substituted (C 1-6 ) alkyl linker, which is optionally substituted with a heteroatom or a linking functional group such as a keto group (CO), an ester (-CO2-), an amide group (CONH), a carbamate group (OCONH), an ether (-O-), a thioether (-S-) and / or an amino group (-NR-, wherein R is H or an alkyl group). In some cases, A is covalently linked to Z. 3 In some cases, A is linked to Z via a linear linker of 1-12 atoms in length, such as 1-10, 1-8, or 1-6 atoms in length, such as 1, 2, 3, 4, 5, or 6 atoms in length. 3 The linking group L 1 Can be (C 1-6 ) alkyl linker or substituted (C 1-6) alkyl linker, which is optionally substituted with heteroatoms or linking functional groups such as keto (CO), ester (-CO2-), amido (CONH), carbamate (OCONH), ether (-O-), thioether (-S-) and / or amino group (-NR-, wherein R is H or alkyl). 31 NR 22 When the linker L 1 It may include a terminal keto group (C=O) which is 31 Together provide an acylamino group (NR 22 CO) connection. When Z 31 When L is O or S, the linking group 1 It may include a terminal keto group (C=O) which is 31 Together they provide an ester or thioester group linkage.

[0128] In some instances of Formula (II), the subject ENPP1 inhibitor compound is a compound of Formula (III):

[0129]

[0130] in:

[0131] R 31 To R 34 are each independently selected from H, halogen, alkyl and substituted alkyl, or R 31 and R 32 or R 33 and R 34 are cyclically attached and, taken together with the carbon atoms to which they are attached, provide a cycloalkyl, substituted cycloalkyl, heterocyclyl, or substituted heterocyclyl ring; and

[0132] n and m are each independently an integer from 0 to 6 (eg, 0-3).

[0133] In certain embodiments of Formula (III), Z 31 NR 22 , where R 22 Selected from H, C (1-6) Alkyl and substituted C (1-6) In some cases, Z 31 is NH. In some cases, Z 31 NR 22 , and R 22 C (1-6) In some cases, Z 31 NR 22 , and R 22 To replace C (1-6) In certain cases of formula (III), Z 31is O. In certain cases of formula (III), Z 31 For S.

[0134] In formula (II), when Z 31 NR 22 When the linker L 1 It may include a terminal keto group (C=O) which is 31 Together provide an acylamino group (NR 22 Thus, in some cases of formula (II), the subject ENPP1 inhibitor compound is a compound of formula (IIIa):

[0135]

[0136] in:

[0137] Z 41 For-NR 22 C(=O)-;

[0138] R 31 To R 34 are each independently selected from H, halogen, alkyl and substituted alkyl, or R 31 and R 32 or R 33 and R 34 are cyclically attached and, taken together with the carbon atoms to which they are attached, provide a cycloalkyl, substituted cycloalkyl, heterocyclyl, or substituted heterocyclyl ring; and

[0139] n and m are each independently an integer from 0 to 6 (eg, 0-3).

[0140] In some cases of Formulas (III)-(IIIa), Z 1 CR 11 , and R 11 is selected from hydrogen, cyano, trifluoromethyl, halogen, alkyl and substituted alkyl hydrogen. In some cases, the alkyl or substituted alkyl is C 1-5 In some cases of Formula (III)-(IIIa), Z 1 CR 11 , and R 11 is hydrogen. In some cases, R 11 In some cases, R 11 is trifluoromethyl. In some cases, R 11 is a halogen, such as Br, I, Cl or F. In some cases, R 11 is an alkyl group, such as C 1-5 In some cases, R 11 is a substituted alkyl group, such as a substituted C 1-5 alkyl.

[0141] In some cases of Formulas (III)-(IIIa), Z 2 CR 12 , and R 12 is selected from hydrogen, cyano, trifluoromethyl, halogen, alkyl and substituted alkyl hydrogen. In some cases, the alkyl or substituted alkyl is C 1-5 In some cases of Formula (III)-(IIIa), Z 2 CR 12 , and R 12 is hydrogen. In some cases, R 12 In some cases, R 12 is trifluoromethyl. In some cases, R 12 is a halogen, such as Br, I, Cl or F. In some cases, R 12 is an alkyl group, such as C 1-5 In some cases, R 12 is a substituted alkyl group, such as a substituted C 1-5 alkyl.

[0142] In certain embodiments of Formula (III)-(IIIa), Z 1 and Z 2 At least one of is N. In certain embodiments of Formula (III)-(IIIa), Z 1 CR 11 , and Z 2 is N. In certain cases of Formula (III)-(IIIa), Z 1 is N, and Z 2 CR 12 In certain cases of Formula (III)-(IIIa), Z 1 CR 11 , and Z 2 CR 12 In certain cases of Formula (III)-(IIIa), Z 1 is N, and Z 2 is N.

[0143] In certain embodiments of Formula (III)-(IIIa), R 31 To R 34 In certain embodiments, R 31 To R 34 In certain embodiments, at least one of R 31 To R 34 In certain embodiments, at least one of R 31 To R 34In some cases, at least one of R 31 To R 34 One of them is halogen, and the rest are selected from hydrogen, halogen, alkyl and substituted alkyl. 31 To R 34 One of them is alkyl, and the others are selected from hydrogen, halogen, alkyl and substituted alkyl. 31 To R 34 One of them is substituted alkyl, and the rest are selected from hydrogen, halogen, alkyl and substituted alkyl. 31 To R 34 One of them is halogen, and the rest are hydrogen. In certain cases, R 31 To R 34 One of them is alkyl, and the rest are hydrogen. 31 To R 34 One of them is substituted alkyl and the rest are hydrogen.

[0144] In certain embodiments of Formula (III)-(IIIa), n is an integer from 0 to 3. In certain cases, n is 0. In certain cases, n is 1. In certain cases, n is 2. In certain cases, n is 3. In certain embodiments of Formula (III)-(IIIa), m is an integer from 0 to 3. In certain cases, m is 0. In certain cases, m is 1. In certain cases, m is 2. In certain cases, m is 3. In certain cases, n is 0 and m is 1. In certain cases, n is 0 and m is 2. In certain cases, n is 0 and m is 3. In certain cases, n is 1 and m is 0. In certain cases, n is 1 and m is 1. In certain cases, n is 1 and m is 2. In certain cases, n is 1 and m is 3. In certain cases, n is 2 and m is 0. In certain cases, n is 2 and m is 1. In certain cases, n is 2 and m is 2. In certain cases, n is 2 and m is 3. In certain cases, n is 3 and m is 0. In some cases, n is 3 and m is 1. In some cases, n is 3 and m is 2. In some cases, n is 3 and m is 3. In some cases, n+m is an integer from 0 to 3. In some cases, n+m is 0. In some cases, n+m is 1. In some cases, n+m is 2. In some cases, n+m is 3.

[0145] In some embodiments of any one of Formulas (I) to (IIIa), the ring system A is selected from phenyl, substituted phenyl, pyridyl, substituted pyridyl, pyrimidine, substituted pyrimidine, piperidine, substituted piperidine, piperazine, substituted piperazine, pyridazine, substituted pyridazine, cyclohexyl, and substituted cyclohexyl. In some cases, the ring system A is phenyl or substituted phenyl. In some cases, the ring system A is pyridyl or substituted pyridyl. In some cases, the ring system A is pyrimidine or substituted pyrimidine. In some cases, the ring system A is piperidine or substituted piperidine. In some cases, the ring system A is piperazine or substituted piperazine. In some cases, the ring system A is cyclohexyl or substituted cyclohexyl.

[0146] In some embodiments, the ring system A is described by formula (A1):

[0147]

[0148] in:

[0149] R 6 are each selected from the group consisting of hydrogen, alkyl, substituted alkyl, hydroxy, alkoxy, substituted alkoxy, trifluoromethyl, halogen, acyl, substituted acyl, carboxyl, carboxamide, substituted carboxamide, sulfonyl, substituted sulfonyl, sulfonamide, and substituted sulfonamide; and

[0150] p is an integer from 0 to 4.

[0151] In some cases, A1 is phenylene. In some cases, A1 is monosubstituted phenylene. In some cases, A1 is disubstituted phenylene. In some cases, A1 is trisubstituted phenylene. In some cases, A1 is tetrasubstituted phenylene. In some cases, the substituents of phenylene are selected from lower alkyl (e.g., methyl, ethyl, propyl, butyl, pentyl and hexyl) and halogen (e.g., F, Cl, I or Br).

[0152] In some embodiments, the A1 ring is described by formula (A1a):

[0153]

[0154] In some embodiments, the ring system A is described by formula (A2):

[0155]

[0156] in:

[0157] Z 5 Selected from N and CR 6 ;

[0158] R 6are each selected from the group consisting of hydrogen, alkyl, substituted alkyl, hydroxy, alkoxy, substituted alkoxy, trifluoromethyl, halogen, acyl, substituted acyl, carboxyl, carboxamide, substituted carboxamide, sulfonyl, substituted sulfonyl, sulfonamide, and substituted sulfonamide; and

[0159] q is an integer from 0 to 2.

[0160] In some cases, A2 is pyridyl. In some cases, A2 is substituted pyridyl. In some cases, the pyridyl is monosubstituted pyridyl. In other cases, the pyridyl is disubstituted pyridyl. In other cases, the pyridyl is trisubstituted pyridyl. In some cases, Z 5 is N, such that A is pyrimidinyl. In some cases, A is a substituted pyrimidinyl. In some cases, the pyrimidinyl is monosubstituted. In some cases, the pyrimidinyl is disubstituted. In certain embodiments of A, the substituent is selected from lower alkyl (e.g., methyl, ethyl, propyl, butyl, pentyl and hexyl), trifluoromethyl and halogen (e.g., F, Cl, I or Br).

[0161] In some embodiments, the ring system A is described by formula (A3):

[0162]

[0163] in:

[0164] Z 5 Selected from N and CR 6 ;

[0165] R 6 are each selected from the group consisting of hydrogen, alkyl, substituted alkyl, hydroxy, alkoxy, substituted alkoxy, trifluoromethyl, halogen, acyl, substituted acyl, carboxyl, carboxamide, substituted carboxamide, sulfonyl, substituted sulfonyl, sulfonamide, and substituted sulfonamide; and

[0166] q is an integer from 0 to 2.

[0167] In some cases, A3 is pyridyl. In some cases, A3 is substituted pyridyl. In some cases, the pyridyl is monosubstituted pyridyl. In other cases, the pyridyl is disubstituted pyridyl. In other cases, the pyridyl is trisubstituted pyridyl. In some cases, Z 5 is N, such that A is pyrimidinyl. In some cases, A is a substituted pyrimidinyl. In some cases, the pyrimidinyl is monosubstituted. In some cases, the pyrimidinyl is disubstituted. In certain embodiments of A, the substituent is selected from lower alkyl (e.g., methyl, ethyl, propyl, butyl, pentyl and hexyl), trifluoromethyl and halogen (e.g., F, Cl, I or Br).

[0168] In some embodiments, the ring system A is described by formula (A4):

[0169]

[0170] in:

[0171] Z 5 is N;

[0172] R 6 are each selected from the group consisting of hydrogen, alkyl, substituted alkyl, hydroxy, alkoxy, substituted alkoxy, trifluoromethyl, halogen, acyl, substituted acyl, carboxyl, carboxamide, substituted carboxamide, sulfonyl, substituted sulfonyl, sulfonamide, and substituted sulfonamide; and

[0173] q is an integer from 0 to 2.

[0174] In some cases, A4 is a substituted pyrimidinyl. In some cases, the pyrimidinyl is monosubstituted. In some cases, the pyrimidinyl is disubstituted. In certain embodiments of A4, the substituent is selected from lower alkyl (e.g., methyl, ethyl, propyl, butyl, pentyl and hexyl), trifluoromethyl and halogen (e.g., F, Cl, I or Br).

[0175] In some cases of Formula (III)-(IIIa), the ENPP1 inhibitor compound is a compound of Formula (IV)-(IVa):

[0176]

[0177] in:

[0178] Z 31 Selected from NR 22 , O and S;

[0179] Z 41 For-NR 22 C(=O)-;

[0180] Z 11 and Z 21 independently selected from N and C(CN);

[0181] R 31 To R 34 are each independently selected from H, halogen, alkyl and substituted alkyl, or R 31 and R 32 or R 33 and R 34 are cyclically attached and, taken together with the carbon atoms to which they are attached, provide a cycloalkyl, substituted cycloalkyl, heterocyclyl, or substituted heterocyclyl ring;

[0182] R6 are each independently selected from H, alkyl, substituted alkyl, hydroxy, alkoxy, substituted alkoxy, trifluoromethyl and halogen;

[0183] p is an integer from 0 to 4; and

[0184] n and m are each independently an integer from 0 to 6 (eg, 0-3).

[0185] In certain embodiments of Formulas (IV)-(IVa), Z 31 NR 22 , where R 22 Selected from H, C (1-6) Alkyl and substituted C (1-6) In some cases, Z 31 is NH. In some cases, Z 31 NR 22 , and R 22 C (1-6) In some cases, Z 31 NR 22 , and R 22 To replace C (1-6) In certain cases of Formulas (IV)-(IVa), Z 31 is O. In certain cases of Formulas (IV)-(IVa), Z 31 For S.

[0186] In certain embodiments of Formulas (IV)-(IVa), Z 11 and Z 21 At least one of is N. In certain embodiments of Formulas (IV)-(IVa), Z 11 is C(CN), and Z 21 is N. In certain cases of Formulas (IV)-(IVa), Z 11 is N, and Z 21 is C(CN). In certain cases of Formulas (IV)-(IVa), Z 11 is C(CN), and Z 21 is C(CN). In certain cases of Formulas (IV)-(IVa), Z 11 is N, and Z 21 is N.

[0187] In certain embodiments of Formula (IV)-(IVa), R 31 To R 34 In certain embodiments, R 31 To R 34 In certain embodiments, at least one of R 31To R 34 In certain embodiments, at least one of R 31 To R 34 In some cases, at least one of R 31 To R 34 One of them is halogen, and the rest are selected from hydrogen, halogen, alkyl and substituted alkyl. 31 To R 34 One of them is alkyl, and the others are selected from hydrogen, halogen, alkyl and substituted alkyl. 31 To R 34 One of them is substituted alkyl, and the rest are selected from hydrogen, halogen, alkyl and substituted alkyl. 31 To R 34 One of them is halogen, and the rest are hydrogen. In certain cases, R 31 To R 34 One of them is alkyl, and the rest are hydrogen. 31 To R 34 One of them is substituted alkyl and the rest are hydrogen.

[0188] In certain embodiments of Formulae (IV)-(IVa), n is an integer from 0 to 3. In certain cases, n is 0. In certain cases, n is 1. In certain cases, n is 2. In certain cases, n is 3. In certain embodiments of Formulae (IV)-(IVa), m is an integer from 0 to 3. In certain cases, m is 0. In certain cases, m is 1. In certain cases, m is 2. In certain cases, m is 3. In certain cases, n is 0 and m is 1. In certain cases, n is 0 and m is 2. In certain cases, n is 0 and m is 3. In certain cases, n is 1 and m is 0. In certain cases, n is 1 and m is 1. In certain cases, n is 1 and m is 2. In certain cases, n is 1 and m is 3. In certain cases, n is 2 and m is 0. In certain cases, n is 2 and m is 1. In certain cases, n is 2 and m is 2. In certain cases, n is 2 and m is 3. In certain cases, n is 3 and m is 0. In some cases, n is 3 and m is 1. In some cases, n is 3 and m is 2. In some cases, n is 3 and m is 3. In some cases, n+m is an integer from 0 to 3. In some cases, n+m is 0. In some cases, n+m is 1. In some cases, n+m is 2. In some cases, n+m is 3.

[0189] In some instances of Formula (IVa), n is 0, and m is 0-2, eg, m is 1 or 2.

[0190] In some instances of Formula (IV)-(IVa), the ENPP1 inhibitor compound is a compound of Formula (V)-(Va):

[0191]

[0192]

[0193] in:

[0194] R 41 To R 44 Independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, hydroxy, alkoxy, substituted alkoxy, trifluoromethyl, halogen, acyl, substituted acyl, carboxyl, carboxamide, substituted carboxamide, sulfonyl, substituted sulfonyl, sulfonamide, and substituted sulfonamide.

[0195] In certain embodiments of Formulas (V)-(Va), Z 11 and Z 21 At least one of is N. In certain embodiments of Formulas (V)-(Va), Z 11 is C(CN), and Z 21 is N. In certain cases of Formulas (V)-(Va), Z 11 is N, and Z 21 In certain cases of Formulas (V)-(Va), Z 11 is C(CN), and Z 21 In certain cases of Formulas (V)-(Va), Z 11 is N, and Z 21 is N.

[0196] In some instances of Formula (V)-(Va), the subject ENPP1 inhibitor compound is a compound of one of Formula (VIa)-(VId):

[0197]

[0198] In certain embodiments of Formulas (VIa)-(VId), R 41 To R 44 In certain embodiments, R 41 To R 44 In certain embodiments, at least one of R 41 To R 44 In certain embodiments, at least one of R 41 To R 44 At least one of R is alkoxy or substituted alkoxy. 41 To R 44In some cases, at least one of R 41 To R 44 At least one of is halogen. In some cases, R 41 To R 44 At least one of is acyl or substituted acyl. In some cases, R 41 To R 44 In some cases, at least one of R 41 To R 44 At least one of is formamide or substituted formamide. In certain cases, R 41 To R 44 In some cases, at least one of R 41 To R 44 At least one of is sulfonamide and substituted sulfonamide. In some cases, R 31 To R 34 One of them is hydrogen, and the others are selected from hydrogen, alkyl, substituted alkyl, hydroxy, alkoxy, substituted alkoxy, trifluoromethyl, halogen, acyl, substituted acyl, carboxyl, carboxamide, substituted carboxamide, sulfonyl, substituted sulfonyl, sulfonamide, and substituted sulfonamide. In certain cases, R 31 To R 34 Two of them are hydrogen, and the remaining are selected from hydrogen, alkyl, substituted alkyl, hydroxy, alkoxy, substituted alkoxy, trifluoromethyl, halogen, acyl, substituted acyl, carboxyl, formamide, substituted formamide, sulfonyl, substituted sulfonyl, sulfonamide, and substituted sulfonamide. In some cases, R 31 To R 34 Three of them are hydrogen, and the rest are selected from the group consisting of hydrogen, alkyl, substituted alkyl, hydroxy, alkoxy, substituted alkoxy, trifluoromethyl, halogen, acyl, substituted acyl, carboxyl, formamide, substituted formamide, sulfonyl, substituted sulfonyl, sulfonamide, and substituted sulfonamide.

[0199] In certain embodiments of Formulae (VIa)-(VId), n is an integer from 0 to 3. In certain cases, n is 0. In certain cases, n is 1. In certain cases, n is 2. In certain cases, n is 3. In certain embodiments of Formulae (VIa)-(VId), m is an integer from 0 to 3. In certain cases, m is 0. In certain cases, m is 1. In certain cases, m is 2. In certain cases, m is 3. In certain cases, n is 0 and m is 1. In certain cases, n is 0 and m is 2. In certain cases, n is 0 and m is 3. In certain cases, n is 1 and m is 0. In certain cases, n is 1 and m is 1. In certain cases, n is 1 and m is 2. In certain cases, n is 1 and m is 3. In certain cases, n is 2 and m is 0. In certain cases, n is 2 and m is 1. In certain cases, n is 2 and m is 2. In certain cases, n is 2 and m is 3. In certain cases, n is 3 and m is 0. In some cases, n is 3 and m is 1. In some cases, n is 3 and m is 2. In some cases, n is 3 and m is 3. In some cases, n+m is an integer from 0 to 3. In some cases, n+m is 0. In some cases, n+m is 1. In some cases, n+m is 2. In some cases, n+m is 3.

[0200] In certain embodiments of Formulas (VIa)-(VId), R 22 is hydrogen. In some cases, R 22 In some cases, R 22 In some cases, the alkyl or substituted alkyl is C (1-6) alkyl.

[0201] In certain embodiments of any one of Formulas (I)-(VId), R 1 is selected from the group consisting of hydrogen, alkylaryl, substituted alkylaryl, alkylheteroaryl, substituted alkylheteroaryl, alkenylaryl (e.g., vinylaryl), substituted alkenylaryl, alkenylheteroaryl (e.g., vinylheteroaryl), substituted alkenylheteroaryl, aryl, substituted aryl, heteroaryl, and substituted heteroaryl.

[0202] In certain instances of Formulas (I)-(VId), R 1 is hydrogen. In some cases, R 1 is aryl or substituted aryl. In some cases, R 1 is heteroaryl or substituted heteroaryl. In some cases, R 1 is alkylaryl or substituted alkylaryl. In some cases, R 1 is alkylheteroaryl or substituted alkylheteroaryl. 1is alkenyl aryl or substituted alkenyl aryl. In some cases, R 1 In some cases, R 1 is a substituted vinyl aryl. In some cases, R 1 is a vinyl heteroaryl group. In some cases, R 1 is alkenylheteroaryl or substituted alkenylheteroaryl. In some cases, R 1 is a substituted vinyl heteroaryl.

[0203] In some cases of Formula (VIa)-(VId), the ENPP1 inhibitor compound is a compound of one of Formula (VIIa)-(VIIb):

[0204]

[0205] In certain embodiments of any one of Formulas (I)-(VIIb), R 2 To R 5 Independently selected from H, OH, alkyl, substituted alkyl, alkoxy, substituted alkoxy, -OCF3, halogen, cyano, amine, substituted amine, amide, heterocycle and substituted heterocycle.

[0206] In certain embodiments of any one of Formulas (I)-(VIIb), R 2 To R 5 independently selected from hydrogen, OH, C (1-6) Alkoxy, -OCF3, C (1-6) Alkylamino, di-C (1-6) Alkylamino, F, Cl, Br and CN.

[0207] In some cases, R 2 To R 5 At least one of is hydrogen. In certain cases, R 2 To R 5 At least two of R are hydrogen. 2 To R 5 Each is hydrogen. In certain cases, R 2 To R 5 At least one of R is a hydroxyl group. 2 To R 5 At least one of R is alkyl or substituted alkyl. 2 To R 5 At least one of them is an alkoxy group or a substituted alkoxy group.

[0208] In some cases, the alkoxy or substituted alkoxy is C (1-6)Alkoxy, for example, methoxy, ethoxy, propoxy, butoxy, pentyloxy, hexyloxy. In some cases, R 2 To R 5 In some cases, at least one of R 2 To R 5 At least one of them is -OCF3. In some cases, R 2 To R 5 In some cases, at least one of is a halogen. In some cases, the halogen is fluorine. In some cases, the halogen is chlorine. In some cases, the halogen is bromine. In some cases, R 2 To R 5 In some cases, at least one of R 2 To R 5 At least one of is an amine or a substituted amine. In some cases, R 2 To R 5 At least one of them is C (1-6) In some cases, R 2 To R 5 At least one of them is di-C (1-6) In some cases, R 2 To R 5 At least one of is an amide. In some cases, R 2 To R 5 At least one of them is a heterocycle or a substituted heterocycle.

[0209] In some cases of Formula (I)-(VIIb), R 3 and R 4 are independently alkoxy; and R 2 and R 5 are all hydrogen. In some cases, the alkoxy group is methoxy. In some cases, R 3 is an alkoxy group; and R 2 , R 4 and R 5 is hydrogen. In some cases, R 4 is an alkoxy group; and R 2 , R 3 and R 5 Each is hydrogen. In certain cases, R 2 , R 3 and R 4 is hydrogen, and R 5 In some cases, the alkoxy group is C (1-6)In some cases, the alkoxy group is methoxy. In some cases, the alkoxy group is ethoxy. In some cases, the alkoxy group is propoxy. In some cases, the alkoxy group is butoxy. In some cases, the alkoxy group is pentoxy. In some cases, the alkoxy group is hexoxy.

[0210] In some cases of Formulas (VIc)-(VId), R 41 -R 44 are independently H, halogen, C (1-6) Alkyl or C (1-6) In some cases of Formula (VIc)-(VId), m is 1 or 2. In some cases of Formula (VIc)-(VId), R 2 is H, and R 3 To R 5 are independently selected from hydrogen, C (1-6) Alkoxy, F, Cl and C (1-6) alkyl.

[0211] In some instances of Formula (VIIa)-(VIIb), the subject ENPP1 inhibitor compound is a compound of one of Formula (VIIc)-(VIIl):

[0212]

[0213]

[0214] In some instances of Formula (VIa), the ENPP1 inhibitor compound is a compound of Formula (VIIm):

[0215]

[0216] In certain embodiments of Formula (VIIm), R 2 To R 5 is independently selected from H, OH, alkyl, substituted alkyl, alkoxy, substituted alkoxy, -OCF3, halogen, cyano, amine, substituted amine, amide, heterocycle, and substituted heterocycle. In certain embodiments of Formula (VIIm), R 2 To R 5 independently selected from hydrogen, OH, C (1-6) Alkoxy, -OCF3, C (1-6) Alkylamino, di-C (1-6) In certain embodiments of Formula (VIIm), n+m=1. In certain embodiments of Formula (VIIm), n+m=2. In certain embodiments of Formula (VIIm), n is 1, and m is 0.

[0217] In certain instances of Formula (VIIm), R 3 To R 5 At least one of is hydrogen. In certain cases, R 3 To R 5 At least two of R are hydrogen. 3 To R 5 Each is hydrogen. In certain cases, R 3 To R 5 At least one of R is a hydroxyl group. 3 To R 5 At least one of R is alkyl or substituted alkyl. 3 To R 5 In some cases of Formula (VIIm), the alkoxy or substituted alkoxy is C (1-6) Alkoxy, for example, methoxy, ethoxy, propoxy, butoxy, pentyloxy, hexyloxy. In some cases, R 3 To R 5 In certain cases of Formula (VIIm), R 3 To R 5 At least one of them is -OCF3. In some cases, R 3 To R 5 In some cases, at least one of is a halogen. In some cases, the halogen is fluorine. In some cases, the halogen is chlorine. In some cases, the halogen is bromine. In some cases, R 3 To R 5 In some cases, at least one of R 3 To R 5 At least one of is an amine or a substituted amine. In some cases, R 3 To R 5 At least one of them is C (1-6) In some cases, R 3 To R 5 At least one of them is di-C (1-6) In certain cases of Formula (VIIm), R 3 To R 5 At least one of is an amide. In some cases, R 3 To R 5 At least one of them is a heterocycle or a substituted heterocycle.

[0218] In some cases of Formula (VIIm), R 3 and R 4 are independently alkoxy; and R 2 and R 5are all hydrogen. In some cases, the alkoxy group is methoxy. In some cases, R 3 is an alkoxy group; and R 2 , R 4 and R 5 is hydrogen. In some cases, R 4 is an alkoxy group; and R 2 , R 3 and R 5 Each is hydrogen. In certain cases of Formula (VIIm), R 2 , R 3 and R 4 is hydrogen, and R 5 In some cases, the alkoxy group is C (1-6) In some cases, the alkoxy group is methoxy. In some cases, the alkoxy group is ethoxy. In some cases, the alkoxy group is propoxy. In some cases, the alkoxy group is butoxy. In some cases, the alkoxy group is pentoxy. In some cases, the alkoxy group is hexoxy.

[0219] In certain embodiments of Formula (VIIm), n is 0-3, and m is 0-3. In some cases of Formula (VIIm), m is 0. In certain cases, m is 1. In certain cases, m is 2. In certain cases, m is 3. In certain cases, n is 0, and m is 1. In certain cases, n is 0, and m is 2. In certain cases, n is 0, and m is 3. In certain cases, n is 1, and m is 0. In certain cases, n is 1, and m is 1. In certain cases, n is 1, and m is 2. In certain cases, n is 1, and m is 3. In certain cases, n is 2, and m is 0. In certain cases, n is 2, and m is 1. In certain cases, n is 2, and m is 2. In certain cases, n is 2, and m is 3. In certain cases, n is 3, and m is 0. In certain cases, n is 3, and m is 1. In certain cases, n is 3, and m is 2. In certain cases, n is 3, and m is 3. In some cases, n+m is an integer from 0 to 3. In some cases, n+m is 0. In some cases, n+m is 1. In some cases, n+m is 2. In some cases, n+m is 3.

[0220] In certain instances of the ENPP1 inhibitor compounds of formula (I), Z 3 In certain embodiments of Formula (I), Z 3 Does not exist, Z 2 CR 12 , R 12 is cyano, and the compound is described by formula (X):

[0221]

[0222] Where L 11 and L 12 are independently a covalent bond or a linker. In some cases of Formula (X), L 11 It is a covalent bond.

[0223] In some embodiments of formula (X), the ring system A is selected from phenyl, substituted phenyl, pyridyl, substituted pyridyl, pyrimidine, substituted pyrimidine, piperidine, substituted piperidine, piperazine, substituted piperazine, pyridazine, substituted pyridazine, cyclohexyl and substituted cyclohexyl. In some cases, the ring system A is phenyl or substituted phenyl. In some cases, the ring system A is pyridyl or substituted pyridyl. In some cases, the ring system A is pyrimidine or substituted pyrimidine. In some cases, the ring system A is piperidine or substituted piperidine. In some cases, the ring system A is piperazine or substituted piperazine. In some cases, the ring system A is cyclohexyl or substituted cyclohexyl.

[0224] In some embodiments, the ring system A is described by one of Formulae (A1)-(A4) (eg, as described herein):

[0225]

[0226] in:

[0227] Z 5 Selected from N and CR 6 ;

[0228] R 6 are each selected from the group consisting of hydrogen, alkyl, substituted alkyl, hydroxy, alkoxy, substituted alkoxy, trifluoromethyl, halogen, acyl, substituted acyl, carboxyl, formamide, substituted formamide, sulfonyl, substituted sulfonyl, sulfonamide, and substituted sulfonamide;

[0229] p is an integer from 0 to 4; and

[0230] q is an integer from 0 to 2.

[0231] In some embodiments, the A ring is described by formula (A5):

[0232]

[0233] in:

[0234] Z 5 Each independently selected from N and CR 16 ;

[0235] R 16each independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, hydroxy, alkoxy, substituted alkoxy, trifluoromethyl, halogen, acyl, substituted acyl, carboxyl, carboxamide, substituted carboxamide, sulfonyl, substituted sulfonyl, sulfonamide, and substituted sulfonamide; and

[0236] r is an integer from 0 to 8.

[0237] In some cases, A5 is piperidine or substituted piperidine. In some cases, A5 is piperazine or substituted piperazine. In some cases, A5 is cyclohexyl or substituted cyclohexyl. In certain embodiments of A5, r is greater than 0, such as 1, 2, 3, 4, 5, 6, 7 or 8. In some cases, A5 includes one R 16 In some cases, A5 includes two R 16 In some cases, A5 includes three R 16 In some cases, A5 includes four R 16 In certain embodiments, the substituent is selected from lower alkyl (eg, methyl, ethyl, propyl, butyl, pentyl, and hexyl), trifluoromethyl, and halogen (eg, F, Cl, I, or Br).

[0238] In certain embodiments, the A ring has any one of Formulae (A5a)-(A5c):

[0239]

[0240] In certain embodiments, the A ring is a cyclohexyl group having a relative configuration of formula (A5d) or (A5e):

[0241]

[0242] In certain instances of Formula (X), the subject ENPP1 inhibitor compound is a compound of Formula (XI):

[0243]

[0244] in:

[0245] Z 5 Each independently selected from N and CR 16 ;

[0246] R 16 each independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, hydroxy, alkoxy, substituted alkoxy, trifluoromethyl, halogen, acyl, substituted acyl, carboxyl, carboxamide, substituted carboxamide, sulfonyl, substituted sulfonyl, sulfonamide, and substituted sulfonamide; and

[0247] r is an integer from 0 to 8.

[0248] In certain embodiments of Formula (XI), at least one Z 5 is N. In certain embodiments of Formula (XI), one Z 5 is N, and another Z 5 CR 16 In certain cases of Formula (XI), two Z 5 The groups are all CR 16 In certain cases of Formula (XI), two Z 5 The groups are all N.

[0249] In certain embodiments of the compound of any one of Formula (X)-(XI), L 11 and L 12 Each is a covalent bond. In some cases, L 11 and L 12 Each is a linker. In some cases, L 11 is a covalent bond, and L 12 In some cases, L 11 is a linking group, and L 12 Any convenient linking group may be used as L 11 and L 12 In some cases, L 11 is a covalent bond. In some cases, L 11 is a linear linker of 1-12 atoms in length, such as 1-10, 1-8 or 1-6 atoms in length, such as 1, 2, 3, 4, 5 or 6 atoms in length. 11 Can be (C 1-6 ) alkyl linker or substituted (C 1-6 ) alkyl linker, which is optionally substituted with a heteroatom or a linking functional group such as an ester (-CO2-), an amide (CONH), a carbamate (OCONH), an ether (-O-), a thioether (-S-) and / or an amino group (-NR-, wherein R is H or an alkyl group). In some cases, L 12 is a covalent bond. In some cases, L 12 is a linker having a length of 1-12 atoms, such as 1-10, 1-8 or 1-6 atoms, such as 1, 2, 3, 4, 5 or 6 atoms. 12 Can be (C 1-6 ) alkyl linker or substituted (C 1-6 ) alkyl linker, which is optionally substituted with heteroatoms or linking functional groups such as ester (-CO2-), amido (CONH), carbamate (OCONH), ether (-O-), thioether (-S-) and / or amino group (-NR-, wherein R is H or alkyl).

[0250] In some instances of Formula (XI), the subject ENPP1 inhibitor compound is a compound of Formula (XII):

[0251]

[0252] In certain embodiments of the compound of formula (XII), Z 5 CR 16 , where R 16 is selected from the group consisting of hydrogen, alkyl, substituted alkyl, hydroxy, alkoxy, substituted alkoxy, trifluoromethyl, halogen, acyl, substituted acyl, carboxyl, carboxamide, substituted carboxamide, sulfonyl, substituted sulfonyl, sulfonamide, and substituted sulfonamide. In certain instances of the compound of formula (XII), Z 5 is N.

[0253] In certain embodiments of the compound of Formula (XII), L 12 is a covalent bond. In some cases, L 12 Any convenient linking group may be used as L 12 In some cases, L 12 is a linear linker of 1-12 atoms in length, such as 1-10, 1-8 or 1-6 atoms in length, such as 1, 2, 3, 4, 5 or 6 atoms in length. 12 Can be (C 1-6 ) alkyl linker or substituted (C 1-6 ) alkyl linker, which is optionally substituted with heteroatoms or linking functional groups such as ester (-CO2-), amido (CONH), carbamate (OCONH), ether (-O-), thioether (-S-) and / or amino group (-NR-, wherein R is H or alkyl).

[0254] In some instances of Formula (XII), the subject ENPP1 inhibitor compound is a compound of Formula (XIII):

[0255]

[0256] in:

[0257] R 35 and R 36 are each independently selected from H, halogen, alkyl and substituted alkyl, or R 35 and R 36 are cyclically attached and, taken together with the carbon atoms to which they are attached, provide a cycloalkyl, substituted cycloalkyl, heterocyclyl, or substituted heterocyclyl ring; and

[0258] s is an integer from 0 to 6 (eg, 0 to 3).

[0259] In certain embodiments of Formula (XIII), R 35 and R 36 In certain embodiments, R 35 or R 36 In certain embodiments, at least one of R 35 or R 36 In certain embodiments, at least one of R 35 or R 36 In some cases, at least one of R 35 is a halogen, and R 36 is selected from the group consisting of hydrogen, halogen, alkyl and substituted alkyl. 35 is an alkyl group, and R 36 is selected from the group consisting of hydrogen, halogen, alkyl and substituted alkyl. 35 is a substituted alkyl group, and R 36 is selected from the group consisting of hydrogen, halogen, alkyl and substituted alkyl. 35 is a halogen, and R 36 is hydrogen. In some cases, R 35 is an alkyl group, and R 36 is hydrogen. In some cases, R 35 is a substituted alkyl group, and R 36 For hydrogen.

[0260] In certain embodiments of Formula (XIII), s is an integer from 0 to 3. In certain cases, s is 0. In certain cases, s is 1. In certain cases, s is 2. In certain cases, s is 3.

[0261] In some instances of Formula (XIII), the subject ENPP1 inhibitor compound is a compound of Formula (XIV):

[0262]

[0263] wherein s is an integer from 0 to 6 (eg, 0 to 3).

[0264] In certain embodiments of Formula (XIII), s is an integer from 0 to 3. In certain cases, s is 0. In certain cases, s is 1. In certain cases, s is 2. In certain cases, s is 3.

[0265] In certain embodiments of any of Formulas (X)-(XIV), R 2 To R 5 Independently selected from H, OH, alkyl, substituted alkyl, alkoxy, substituted alkoxy, -OCF3, halogen, cyano, amine, substituted amine, amide, heterocycle and substituted heterocycle.

[0266] In certain embodiments of any of Formulas (X)-(XIV), R 2 To R 5 independently selected from hydrogen, OH, C (1-6) Alkoxy, -OCF3, C (1-6) Alkylamino, di-C (1-6) Alkylamino, F, Cl, Br and CN.

[0267] In certain cases of any of Formulas (X)-(XIV), R 2 To R 5 At least one of is hydrogen. In certain cases, R 2 To R 5 At least two of R are hydrogen. 2 To R 5 At least three of them are hydrogen. In certain cases, R 2 To R 5 Each is hydrogen. In certain cases, R 2 To R 5 At least one of R is a hydroxyl group. 2 To R 5 At least one of R is alkyl or substituted alkyl. 2 To R 5 At least one of is an alkoxy or substituted alkoxy. In some cases, the alkoxy or substituted alkoxy is C (1-6) Alkoxy, for example, methoxy, ethoxy, propoxy, butoxy, pentyloxy, hexyloxy. In some cases, R 2 To R 5 In some cases, at least one of R 2 To R 5 At least one of them is -OCF3. In some cases, R 2 To R 5 In some cases, at least one of is a halogen. In some cases, the halogen is fluorine. In some cases, the halogen is chlorine. In some cases, the halogen is bromine. In some cases, R 2 To R 5 In some cases, at least one of R 2 To R 5 At least one of is an amine or a substituted amine. In some cases, R 2 To R 5 At least one of them is C (1-6) In some cases, R 2 To R 5 At least one of them is di-C (1-6) In some cases, R2 To R 5 At least one of is an amide. In some cases, R 2 To R 5 At least one of them is a heterocycle or a substituted heterocycle.

[0268] In some cases of any of Formulas (X)-(XIV), R 3 and R 4 are independently alkoxy; and R 2 and R 5 are all hydrogen. In some cases, R 3 is an alkoxy group; and R 2 , R 4 and R 5 is hydrogen. In some cases, R 4 is an alkoxy group; and R 2 , R 3 and R 5 Each is hydrogen. In certain cases, R 2 , R 3 and R 4 is hydrogen, and R 5 In some cases, the alkoxy group is C (1-6) In some cases, the alkoxy group is methoxy. In some cases, the alkoxy group is ethoxy. In some cases, the alkoxy group is propoxy. In some cases, the alkoxy group is butoxy. In some cases, the alkoxy group is pentoxy. In some cases, the alkoxy group is hexoxy.

[0269] In some instances of Formula (XIV), the subject ENPP1 inhibitor compound is a compound of one of Formulas (XIVa)-(XIVe):

[0270]

[0271] wherein s is an integer from 0 to 6 (eg, 0 to 3).

[0272] In some instances of Formula (I), the subject ENPP1 inhibitor compound is a compound of Formula (XVa) or (XVb):

[0273]

[0274] in:

[0275] s is 0 to 3;

[0276] R 21 C (1-6) Alkyl or substituted C (1-6) Alkyl; and

[0277] R3 and R 4 Selected from Cl and F.

[0278] In some cases of Formulas (XVa)-(XVb), R 21 is selected from the group consisting of methyl, ethyl, n-propyl and isopropyl. 21 In some cases of Formulas (XVa)-(XVb), R 3 and R 4 For Cl.

[0279] In some cases, R 3 and R 4 is F. In some instances of Formula (XVa)-(XVb), s is 2. In some instances, s is 1. In some embodiments of Formula (XVa)-(XVb), s is 2; R 21 is methyl or isopropyl; and R 3 and R 4 Selected from Cl and F.

[0280] In some instances of Formulae (XVa)-(XVb), the subject ENPP1 inhibitor compound is a compound of one of the following structures, or a prodrug thereof (eg, as described herein):

[0281]

[0282] As mentioned above, X 1 Any embodiment of the hydrophilic head group described herein may be incorporated into any embodiment of Formula (I)-(XVb) described herein. In some embodiments of Formula (I)-(XVb), X 1 It is a hydrophilic head group comprising a charged group capable of binding zinc ions, or a prodrug form thereof. In some cases, the hydrophilic head group capable of binding zinc ions is a phosphorus-containing functional group (e.g., as described herein).

[0283] In some embodiments of Formulae (I)-(XVb), the hydrophilic head group (X 1) is selected from phosphonic acid or phosphonate, phosphonate, phosphate, phosphate ester, thiophosphate, thiophosphate, aminophosphoramate, thiophosphoramate, sulfonate, sulfonic acid, sulfate, hydroxamic acid, ketoacid, amide and carboxylic acid. In some embodiments of any one of formula (I)-(XVb), the hydrophilic head group is phosphonic acid, phosphonate, or a salt thereof. In some embodiments of any one of formula (I)-(XVb), the hydrophilic head group is phosphate, or a salt thereof. In some embodiments of any one of formula (I)-(XVb), the hydrophilic head group is phosphonate or phosphate. In some embodiments of any one of formula (I)-(XVb), the hydrophilic head group is thiophosphate. In some embodiments of any one of formula (I)-(XVb), the hydrophilic head group is thiophosphate. In some embodiments of any one of formula (I)-(XVb), the hydrophilic head group is aminophosphoramate. In some embodiments of any of Formulae (I)-(XVb), the hydrophilic head group is phosphorothioate.

[0284] Specific examples of target hydrophilic head groups that may be incorporated into any of the embodiments of Formulae (I)-(XVb) described herein include, but are not limited to, head groups comprising a first portion selected from: phosphate (RPO4H - ), phosphonate (RPO3H - ), boric acid (RBO2H2), carboxylate (RCO2 - ), sulfate (RSO4 - ), sulfonate (RSO3 - ), amine (RNH3 + ), glycerol, sugars such as lactose or derived from hyaluronic acid, polar amino acids, polyethylene oxide and oligoethylene glycol, the first part is optionally conjugated to a residue of the second part selected from the group consisting of choline, ethanolamine, glycerol, nucleic acids, sugars, inositols, amino acids or amino acid esters (e.g., serine) and lipids (e.g., fatty acids or hydrocarbon chains, such as C8-C30 saturated or unsaturated hydrocarbons). The head group may contain various other modifications, such as in the case of oligoethylene glycol and polyethylene oxide (PEG) containing head groups, such PEG chains may be terminated with methyl groups or have distal functional groups for further modification. Examples of hydrophilic head groups also include, but are not limited to, thiophosphocholine, phosphorylcholine, phosphoglycerol, phosphoethanolamine, phosphoserine, phosphoinositol, ethylphosphosphorylcholine, polyethylene glycol, polyglycerol, melamine, glucosamine, trimethylamine, spermine, spermidine and conjugated carboxylates, sulfates, boric acid, sulfonates, sulfates and carbohydrates.

[0285] In some cases of any of Formulas (I)-(XVb), the hydrophilic head group X1 Formula (XVI):

[0286]

[0287] in:

[0288] Z 6 Not present or selected from O and CH2;

[0289] Z 7 and Z 9 Each independently selected from O and NR 10 , where R 10 is H, alkyl or substituted alkyl;

[0290] Z 8 selected from O and S; and

[0291] R 8 and R 9 Each is independently selected from H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, acyl, substituted acyl, non-aromatic heterocycle, substituted non-aromatic heterocycle, cycloalkyl, substituted cycloalkyl, and a promoiety.

[0292] In some embodiments of Formula (XVI), Z 6 does not exist. In other cases, Z 6 is CH2. In other cases, Z 6 In some embodiments of Formula (XVI), Z 7 is oxygen, and Z 9 NR 10 In some cases, Z 7 NR 10 , and Z 9 In some cases, Z 7 and Z 9 In other cases, Z 7 and Z 9 All are NR 10 In some cases, Z 8 In other cases, Z 8 For sulfur.

[0293] In some embodiments of Formula (XVI), Z 7 , Z 8 and Z 9 are all oxygen atoms, and Z 6 In other cases, Z 8 is a sulfur atom, Z 7 and Z 9 are all oxygen atoms, and Z 6In other cases, Z 8 is a sulfur atom, Z 6 , Z 7 and Z 9 are all oxygen atoms. In some cases, Z 8 is an oxygen atom, Z 7 NR 10 , Z 9 is an oxygen atom, and Z 6 In other cases, Z 8 is an oxygen atom, Z 7 NR 10 , Z 6 and Z 9 In other cases, Z 8 is an oxygen atom, Z 7 and Z 9 Each independently is NR 10 , and Z 6 In other cases, Z 8 is an oxygen atom, Z 7 and Z 9 Each independently is NR 10 , and Z 6 is absent or is CH2. In some cases, Z 7 and Z 9 Each is the same. In other cases, Z 7 and Z 9 It is to be understood that the radical of formula (XVI) may include one or more tautomeric forms of the depicted structure and is intended to include all such forms and salts thereof.

[0294] In some embodiments of Formula (XVI), Z 7 and Z 9 At least one of them is NR 10 In some cases, R 10 is hydrogen. In some cases, R 10 In some other cases, R 10 is a substituted alkyl. 7 and Z 9 All are NR 10 In some cases, Z 7 and Z 9 All are NR 10 , and R 10 , R 8 and R 9 are each independently hydrogen. 7 and Z 9 All are NR 10 , R10 Each is an alkyl group, and R 8 and R 9 Each is hydrogen. In some cases, Z 7 and Z 9 All are NR 10 , R 10 are each substituted alkyl (eg, alkyl substituted by an ester or carboxyl group), and R 8 and R 9 Each is hydrogen.

[0295] In some embodiments of Formula (XVI), R 8 and R 9 are all hydrogen atoms. In some cases, R 8 and R 9 At least one of is a substituent other than hydrogen. 8 and R 9 are all substituents other than hydrogen. 8 and R 9 In some cases, at least one of R 8 and R 9 In some other cases, at least one of R 8 and R 9 In some cases, at least one of R 8 and R 9 At least one of is acyl or substituted acyl. In some cases, R 8 and R 9 In some cases, at least one of R 8 and R 9 In some cases, at least one of R 8 and R 9 In some cases, R 8 and R 9 are all substituted alkyl groups (e.g., C (1-6) alkyl, which is substituted with alkoxy, substituted alkoxy, ester or carboxyl). In some cases, R 8 and R 9 In some cases, at least one of R 8 and R 9 In some cases, R 8 and R 9 are the same. In other cases, R 8 and R 9 are different.

[0296] In some cases of any of Formulas (I)-(XVb), the hydrophilic head group X 1 Any one selected from formula (XVIa) to (XVIf):

[0297]

[0298] in:

[0299] R 10 and R 11 Each is independently selected from H, alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, acyl, substituted acyl, carboxyl, substituted carboxyl, and a promoiety (e.g., as described herein).

[0300] In some embodiments of Formulas (XVIa) to (XVIf), R 10 and R 11 are all hydrogen atoms. In some cases, R 10 and R 11 At least one of R is a substituent other than hydrogen. 10 and R 11 are all substituents other than hydrogen. In some cases, R 10 and R 11 are the same. In other cases, R 10 and R 11 are different. In some cases, R 10 and R 11 In some cases, at least one of R 10 and R 11 In some cases, at least one of R 10 and R 11 are all alkyl or substituted alkyl. 10 and R 11 are all aryl or substituted aryl. In some cases, R 10 and R 11 are acyl or substituted acyl. 10 and R 11 In some cases, R 10 and R 11 are all substituted alkyl groups (e.g., C (1-6) alkyl, which is substituted with alkoxy, substituted alkoxy, ester or carboxyl). In some cases, R 10 and R 11 In some cases, at least one of R10 and R 11 All are phenyl.

[0301] In certain cases of Formulas (XVIa) to (XVId), R 10 and R 11 At least one of the R comprises a cleavable group or a self-immolative promoiety. The self-immolative group may be a disulfide-linked promoiety or a self-immolative ester containing a promoiety. In some cases, R 10 and / or R 11 Including formula -CH2CH2-SS-R 12 disulfide-bonded precursor moiety, wherein R 12 is alkyl or substituted alkyl. 12 is a C8-C30 saturated or unsaturated hydrocarbon chain. 10 and / or R 11 Including formula -CH2OCOR 13 The precursor part, wherein R 13 is H, alkyl or substituted alkyl. In some cases, R 10 and / or R 11 Including the formula -CH2C(R 14 )2CO2R 14 The precursor part, wherein R 14 Each is independently H, alkyl or substituted alkyl.

[0302] In some cases of any of Formulas (I)-(XVb), the hydrophilic head group X 1 , or a prodrug form thereof selected from:

[0303]

[0304] or a pharmaceutically acceptable salt thereof.

[0305] In some cases of any of Formulas (I)-(XVb), the hydrophilic head group X 1 is a group of formula (XVI):

[0306]

[0307] in:

[0308] R 81 and R 91 are each independently selected from H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, aryl, substituted aryl, acyl, ester, amide, heterocycle, substituted heterocycle, cycloalkyl and substituted cycloalkyl, or R 81 and R 91Together with the atoms to which they are attached they form a group selected from heterocycle and substituted heterocycle.

[0309] In some embodiments of Formula (XVI), R 81 and R 91 are all hydrogen atoms. In other cases, R 81 and R 91 are all substituents other than hydrogen.

[0310] In some cases of any of Formulas (I)-(XVb), the hydrophilic head group X 1 is a group of formula (XVII):

[0311]

[0312] In some cases of any of Formulas (I)-(XVb), the hydrophilic head group X 1 is a group of formula (XVIII):

[0313]

[0314] in:

[0315] Z 61 Absent or selected from O and CH2.

[0316] In some embodiments of Formula (XVIII), the hydrophilic head group is selected from one of the following groups:

[0317]

[0318] In some cases of any of Formulas (I)-(XVb), the hydrophilic head group X 1 is a group of formula (XIX):

[0319]

[0320] In some cases of any of Formulas (I)-(XVb), the hydrophilic head group X 1 A group of formula (XX):

[0321]

[0322] in:

[0323] R 92 is selected from the group consisting of H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, aryl, substituted aryl, acyl, ester, amide, heterocycle, substituted heterocycle, cycloalkyl, and substituted cycloalkyl.

[0324] In some embodiments of Formula (XX), R92 is hydrogen. In other cases, R 92 is a substituent other than hydrogen. 92 is an alkyl group or a substituted alkyl group. In certain embodiments of formula (XX), the hydrophilic head group is a group of the following structure:

[0325]

[0326] In some cases of any of Formulas (I)-(XVb), the hydrophilic head group X 1 is a group of formula (XXI):

[0327]

[0328] It should be understood that the group X in any of formulas (I)-(XVb) 1 Any hydroxyl and amine groups in the formula (I) may be further optionally substituted with any convenient group (e.g., alkyl, substituted alkyl, phenyl, substituted phenyl, ester, etc.). It should be understood that any convenient replaceable hydrophilic group may be used as the group X in any of the compounds of formula (I)-(XVb). 1 .

[0329] In certain embodiments, the ENPP1 inhibitor compound is described by one of the structures of Table 1 or a prodrug thereof (eg, as described herein), or a pharmaceutically acceptable salt thereof.

[0330] Table 1: ENPP1 inhibitor compounds

[0331]

[0332]

[0333]

[0334]

[0335]

[0336]

[0337] In certain embodiments, the ENPP1 inhibitor compound is described by one of the structures of Table 2, or a prodrug thereof (eg, as described herein), or a pharmaceutically acceptable salt thereof.

[0338] Table 2: ENPP1 inhibitor compounds

[0339]

[0340]

[0341]

[0342]

[0343]

[0344] In certain embodiments, the ENPP1 inhibitor compound is described by one of the structures of Table 3 or a prodrug thereof (eg, as described herein), or a pharmaceutically acceptable salt thereof.

[0345] Table 3: ENPP1 inhibitor compounds

[0346]

[0347]

[0348]

[0349] In certain embodiments, the compound is described by the structure of one of the compounds of Tables 1-3 (herein, reference to Tables 1-3 includes Table 3a). It should be understood that any of the compounds shown in Tables 1-3 may exist in the form of a salt. In some cases, the salt form of the compound is a pharmaceutically acceptable salt. It should be understood that any of the compounds shown in Tables 1-3 may exist in the form of a prodrug.

[0350] In some embodiments, the compound is described by the structure of one of the compounds of Table 3a.

[0351] Table 3a

[0352]

[0353]

[0354]

[0355]

[0356]

[0357]

[0358]

[0359]

[0360] Aspects of the present disclosure include ENPP1 inhibitor compounds (e.g., as described herein), salts thereof (e.g., pharmaceutically acceptable salts), and / or solvates, hydrates, and / or prodrug forms thereof. In addition, it is understood that in any compound described herein having one or more chiral centers, if the absolute stereochemistry is not explicitly indicated, each center may independently have an R-configuration or an S-configuration or a mixture thereof. It is understood that all permutations of salts, solvates, hydrates, prodrugs, and stereoisomers are intended to be encompassed by the present disclosure.

[0361] In some embodiments, the subject ENPP1 inhibitor compound or its prodrug form is provided in the form of a pharmaceutically acceptable salt. Compounds containing amine or nitrogen-containing heteroaryl groups can be basic in nature and can therefore react with many inorganic and organic acids to form pharmaceutically acceptable acid addition salts. Acids commonly used to form such salts include inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid and phosphoric acid, and organic acids such as p-toluenesulfonic acid, methanesulfonic acid, oxalic acid, p-bromobenzenesulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid and acetic acid, as well as related inorganic and organic acids. Thus, such pharmaceutically acceptable salts include sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, phosphates, monohydrogenphosphates, dihydrogenphosphates, metaphosphates, pyrophosphates, chlorides, bromides, iodides, acetates, propionates, decanoates, octanoates, acrylates, formates, isobutyrates, decanoates, heptanoates, propionates, oxalates, malonates, succinates, suberates, sebacates, fumarates, maleates, butyne-1,4-dioates, hexyne-1 Pharmaceutically acceptable acid addition salts include benzoates, chlorobenzoates, methylbenzoates, dinitrobenzoates, hydroxybenzoates, methoxybenzoates, phthalates, terephthalates, sulfonates, xylenesulfonates, phenylacetates, phenylpropionates, phenylbutyrates, citrates, lactates, β-hydroxybutyrates, glycolates, maleates, tartrates, methanesulfonates, propanesulfonates, naphthalene-1-sulfonates, naphthalene-2-sulfonates, mandelates, hippurates, gluconates, lactobionates, and the like. In certain specific embodiments, pharmaceutically acceptable acid addition salts include those formed with inorganic acids such as hydrochloric acid and hydrobromic acid, and those formed with organic acids such as fumaric acid and maleic acid.

[0362] In some embodiments, the subject compound is provided in a prodrug form. "Prodrug" refers to a derivative of an active agent, which needs to be converted in vivo to release the active agent. In certain embodiments, the conversion is an enzymatic conversion. Prodrugs are often (although not necessarily) pharmacologically inactive until they are converted into an active agent. "Promoiety" is a form of protective group that converts an active agent into a prodrug when it is used to cover the functional group in the active agent. In some cases, the promoiety is attached to the drug via one or more bonds cut by enzymatic or non-enzymatic means in vivo. For example, according to the strategies and methods described by Rautio et al. ("Prodrugs:design and clinical applications", Nature Reviews Drug Discovery7, 255-270 (February 2008)), any convenient subject compound prodrug form can be prepared. In some cases, the promoiety is attached to the hydrophilic head group of the subject compound. In some cases, the promoiety is attached to the hydroxyl or carboxylic acid group of the subject compound. In some cases, the promoiety is an acyl group or a substituted acyl group. In certain cases, the promoiety is an alkyl or substituted alkyl group, for example, that forms an ester functionality (eg, phosphonate, phosphate, etc.) when attached to the hydrophilic head group of the subject compound.

[0363] In some embodiments, the subject compounds are phosphonate or phosphate prodrugs, which can be converted to compounds that include a phosphonic acid or phosphonate or phosphate head group.

[0364] In some embodiments, the subject compound, its prodrug, stereoisomer or salt is provided in the form of a solvate (such as a hydrate). As used herein, the term "solvate" refers to a complex or aggregate formed by molecules of one or more solutes (such as a prodrug or a pharmaceutically acceptable salt thereof) and one or more solvent molecules. Such solvates are typically crystalline solids with a substantially fixed molar ratio of solute to solvent. Representative solvents include, for example, water, methanol, ethanol, isopropanol, acetic acid, etc. When the solvent is water, the solvate formed is a hydrate.

[0365] In some embodiments, the subject compound is provided by oral administration and absorbed into the bloodstream. In some embodiments, the oral bioavailability of the subject compound is 30% or higher. Any convenient method can be used to modify the subject compound or its preparation to increase absorption through the intestinal lumen or their bioavailability.

[0366] In some embodiments, the subject compound is metabolically stable (e.g., remains substantially intact in vivo during the half-life of the compound). In certain embodiments, the half-life of the compound (e.g., half-life in vivo) is 5 minutes or longer, such as 10 minutes or longer, 12 minutes or longer, 15 minutes or longer, 20 minutes or longer, 30 minutes or longer, 60 minutes or longer, 2 hours or longer, 6 hours or longer, 12 hours or longer, 24 hours or longer, or even longer.

[0367] Methods to inhibit ENPP1

[0368] As outlined above, aspects of the present disclosure include ENPP1 inhibitors, and inhibition methods using the ENPP1 inhibitors. ENPP1 is a member of the ectonucleotide pyrophosphatase / phosphodiesterase (ENPP) family. Thus, aspects of the subject method include inhibiting the hydrolase activity of ENPP1 against cGAMP. The inventors have found that cGAMP can have important extracellular biological functions, which can be enhanced by preventing the extracellular degradation of cGAMP, such as hydrolysis caused by its degrading enzyme ENPP1. In some examples, the ENPP1 inhibition target is located extracellularly, and the subject ENPP1 inhibition compound is cell-impermeable and therefore cannot diffuse into the cell. Therefore, the subject method can provide selective extracellular inhibition of ENPP1 hydrolase activity and increase the extracellular level of cGAMP. Therefore, in some cases, the ENPP1 inhibition compound is a compound that inhibits ENPP1 activity extracellularly. Experiments performed by the inventors show that inhibiting the activity of ENPP1 increases extracellular cGAMP and can therefore enhance the STING pathway.

[0369] Inhibiting ENPP1 means that the activity of the enzyme (e.g., relative to a control in any convenient in vitro inhibition assay) is reduced by 10% or more, such as 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more. In some cases, inhibiting ENPP1 means that the activity of the enzyme is reduced by a factor of 2 or less, such as a factor of 3 or less, a factor of 5 or less, a factor of 10 or less, a factor of 100 or less, or a factor of 1000 or less relative to its normal activity (e.g., relative to a control as measured by any convenient assay).

[0370] In some cases, the method is a method of inhibiting ENPP1 in a sample.The term "sample" as used herein refers to a material or mixture of materials containing one or more components of interest, typically but not necessarily in fluid form.

[0371] In some embodiments, a method for inhibiting ENPP1 is provided, the method comprising contacting a sample with a cell-impermeable ENPP1 inhibitor to inhibit the cGAMP hydrolysis activity of ENPP1. In some cases, the sample is a cell sample. In some cases, the sample comprises cGAMP. In some cases, the cGAMP level increases in the cell sample (e.g., relative to a control sample not contacted with the inhibitor). The subject method can provide an increased cGAMP level. "Increased cGAMP level" means the level of cGAMP in a cell sample contacted with a subject compound, wherein the cGAMP level in the sample increases by 10% or more relative to a control sample not contacted with the agent, such as 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 100% or more, or even more.

[0372] In certain embodiments, the ENPP1 inhibitor is an inhibitor as defined herein. In some embodiments, the ENPP1 inhibitor is an inhibitor according to any one of Formulas (I)-(XVb) (e.g., as described herein). In some cases, the ENPP1 inhibitor is a compound of any one of Tables 1-3 (e.g., as described herein). In some cases, the ENPP1 inhibitor is cell impermeable.

[0373] In some embodiments, the ENPP1 inhibitor is configured to be cell permeable. In some embodiments, a method of inhibiting ENPP1 is provided, the method comprising contacting a sample with a cell permeable ENPP1 inhibitor to inhibit ENPP1.

[0374] In some embodiments, the subject compounds have ENPP1 inhibitory properties that reflect activity against additional enzymes. In some embodiments, the subject compounds specifically inhibit ENPP1 without undesirably inhibiting one or more other enzymes.

[0375] In some embodiments, the compounds of the present disclosure interfere with the interaction of cGAMP and ENPP1. For example, the subject compounds can act to increase extracellular cGAMP by inhibiting the hydrolase activity of ENPP1 against cGAMP. Without being bound by any particular theory, it is believed that increasing extracellular cGAMP can activate the STING pathway.

[0376] In some embodiments, the subject compounds inhibit ENPP1 as determined by an inhibition assay, e.g., an assay by determining the level of activity of the enzyme in a cell-free system or in cells after treatment with the subject compounds relative to a control, by measuring the IC 50 or EC 50In certain embodiments, the subject compounds have an IC of 10 μM or less, such as 3 μM or less, 1 μM or less, 500 nM or less, 300 nM or less, 200 nM or less, 100 nM or less, 50 nM or less, 30 nM or less, 10 nM or less, 5 nM or less, 3 nM or less, 1 nM or less, or even less. 50 Value (or EC 50 value).

[0377] As summarized above, aspects of the present disclosure include methods of inhibiting ENPP1. The subject compounds (e.g., as described herein) can inhibit the activity of ENPP1 by 10% to 100%, such as by 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more. In certain assays, the subject compounds can be used at 1 x 10 -6 M or less (such as 1x 10 -6 M or less, 1x 10 -7 M or less, 1x10 -8 M or less, 1x 10 -9 M or less, 1x 10 -10 M or less, or 1x 10 -11 M or less) IC 50 Inhibit its target.

[0378] The protocols that can be used to determine ENPP1 activity are numerous and include, but are not limited to, cell-free assays, such as binding assays; assays using purified enzymes, cellular assays in which cellular phenotype is measured, such as gene expression assays; and in vivo assays involving specific animals (which, in certain embodiments, may be an animal model for a disease associated with the pathogen of interest).

[0379] In some embodiments, the subject methods are in vitro methods comprising contacting a sample with a subject compound that specifically inhibits ENPP1. In certain embodiments, the sample is suspected of containing ENPP1, and the subject methods further comprise evaluating whether the compound inhibits ENPP1.

[0380] In certain embodiments, the subject compound is a modified compound that includes a label, such as a fluorescent label, and the subject methods further include detecting the label, if present, in the sample, such as using optical detection.

[0381] In certain embodiments, the compound is modified with a support or with an affinity group bound to a support (e.g., biotin) so that any sample not bound to the compound can be removed (e.g., by washing). Specifically bound ENPP1, if present, can be detected using any convenient method, such as using binding of a labeled target-specific probe or using a fluorescent protein reagent.

[0382] In another embodiment of the subject methods, the sample is known to contain ENPP1.

[0383] In some embodiments, the method is a method of reducing cancer cell proliferation, wherein the method comprises contacting the cell with an effective amount of a subject ENPP1 inhibitor compound (e.g., as described herein) to reduce cancer cell proliferation. In some cases, the subject ENPP1 inhibitor compound may act intracellularly. The method may be performed in combination with a chemotherapeutic agent (e.g., as described herein). The cancer cell may be in vitro or in vivo. In some examples, the method comprises contacting the cell with an ENPP1 inhibitor compound (e.g., as described herein) and contacting the cell with a chemotherapeutic agent. Any convenient cancer cell may be a target.

[0384] Treatment

[0385] Aspects of the present disclosure include methods for inhibiting the hydrolase activity of ENPP1 against cGAMP to provide increased cGAMP levels and / or downstream regulation (e.g., activation) of the STING pathway. The inventors have found that cGAMP can be present in the extracellular space, and ENPP1 can control the extracellular level of cGAMP. The inventors have also found that cGAMP can have important extracellular biological functions in vivo. The results described and confirmed herein show that ENPP1 inhibition according to the subject method can regulate STING activity in vivo, and can therefore be used to treat a variety of diseases, such as as a target for cancer immunotherapy. Therefore, the subject method can provide selective extracellular inhibition of ENPP1 activity (e.g., cGAMP hydrolase activity) to increase the extracellular level of cGAMP and activate the interferon gene stimulator (STING) pathway. In some examples, the subject method is a method for enhancing a STING-mediated response in a subject. In some examples, the subject method is a method for regulating an immune response in a subject.

[0386] "STING-mediated response" refers to any response mediated by STING, including but not limited to, for example, immune responses to bacterial pathogens, viral pathogens, and eukaryotic pathogens. See, e.g., Ishikawa et al. Immunity 29:538-550 (2008); Ishikawa et al. Nature 461:788-792 (2009); and Sharma et al. Immunity 35:194-207 (2011). STING may also play a role in certain autoimmune diseases caused by inappropriate recognition of self DNA (see, e.g., Gall et al. Immunity 36:120-131 (2012), and may also play a role in inducing adaptive immunity in response to DNA vaccines (see, e.g., Ishikawa et al. Nature 461:788-792 (2009). Increasing a STING-mediated response in a subject means that the STING-mediated response in the subject is increased compared to a control subject (e.g., a subject not administered a subject compound). In some cases, the subject is a human and the subject compounds and methods provide activation of human STING. In some cases, the STING-mediated response includes modulation of an immune response. In some examples, the subject methods are methods of modulating an immune response in a subject.

[0387] In some cases, the STING-mediated response includes increasing the production of interferons (e.g., type I interferons (IFNs), type III interferons (IFNs)) in a subject. Interferons (IFNs) are proteins with a variety of biological activities, such as antiviral, immunomodulatory, and antiproliferative. IFNs are relatively small, species-specific single-chain polypeptides produced by mammalian cells in response to exposure to a variety of inducers, such as viruses, peptides, mitogens, etc. Interferons protect animal tissues and cells from viral invasion and are an important host defense mechanism. Interferons can be divided into type I, type II, and type III interferons. Mammalian type I interferons of interest include IFN-α (alpha), IFN-β (beta), IFN-κ (kappa), IFN-δ (delta), IFN-ε (epsilon), IFN-τ (tau), IFN-ω (omega), and IFN-ζ (zeta, also known as limitin).

[0388] Interferon can be used to treat a variety of cancers because these molecules have anticancer activity that works at multiple levels. Interferon protein can directly inhibit the proliferation of human tumor cells. In some cases, antiproliferative activity also synergizes with various approved chemotherapeutic agents such as cisplatin, 5FU and paclitaxel. The immunomodulatory activity of interferon protein can also lead to the induction of antitumor immune response. This response includes the activation of NK cells, the stimulation of macrophage activity and the induction of MHC class I surface expression, thereby leading to the induction of antitumor cytotoxic T lymphocyte activity. In addition, interferon also works in the cross presentation of antigens in the immune system. In addition, some studies further show that IFN-β protein can have anti-angiogenic activity. Angiogenesis (neovascularization) is essential for the growth of solid tumors. IFN-β can inhibit angiogenesis by inhibiting the expression of pro-angiogenic factors such as bFGF and VEGF. Interferon protein can also inhibit the invasion of tumors by regulating the expression of important enzymes such as collagenase and elastase in tissue remodeling.

[0389] Aspects of the method include administering a therapeutically effective amount of an ENPP1 inhibitor to a subject with cancer to treat the subject's cancer. In some instances, the subject is a subject diagnosed with or suspected of having cancer. Any convenient ENPP1 inhibitor can be used in the subject method for treating cancer. In some cases, the ENPP1 inhibitor compound is a compound as described herein. In some cases, the ENPP1 inhibitor is a cell-impermeable compound. In some cases, the ENPP1 inhibitor is a cell-permeable compound. In some cases, the cancer is a solid tumor cancer. In certain embodiments, the cancer is selected from adrenal cancer, liver cancer, kidney cancer, bladder cancer, breast cancer, colon cancer, gastric cancer, ovarian cancer, cervical cancer, uterine cancer, esophageal cancer, colorectal cancer, prostate cancer, pancreatic cancer, lung cancer (small cell carcinoma and non-small cell carcinoma), thyroid cancer, malignant epithelial tumors, sarcomas, glioblastomas, melanomas, and various head and neck tumors. In some cases, cancer is breast cancer. In certain embodiments, cancer is lymphoma.

[0390] Aspects of the method include administering to a subject a therapeutically effective amount of a cell-impermeable ENPP1 inhibitor to inhibit the hydrolysis of cGAMP and treat the subject's cancer. In some cases, cancer is a solid tumor cancer. In certain embodiments, cancer is selected from adrenal cancer, liver cancer, kidney cancer, bladder cancer, breast cancer, colon cancer, gastric cancer, ovarian cancer, cervical cancer, uterine cancer, esophageal cancer, colorectal cancer, prostate cancer, pancreatic cancer, lung cancer (small cell carcinoma and non-small cell carcinoma), thyroid cancer, malignant epithelial tumors, sarcomas, glioblastomas, melanomas, and various head and neck tumors. In certain embodiments, cancer is breast cancer. In some cases, cancer is lymphoma.

[0391] In some embodiments of the methods disclosed herein, the cell-impermeable ENPP1 inhibitor is an inhibitor of any one of Formula (I)-(XVb) (e.g., as described herein). In some cases, the ENPP1 inhibitor is a compound in Tables 1-3 or a prodrug form thereof (e.g., as described herein).

[0392] In some embodiments of the methods disclosed herein, the ENPP1 inhibitor is cell permeable.

[0393] Thus, aspects of the method include contacting the sample with a subject compound (e.g., as described above) under conditions where the compound inhibits ENPP1. Any convenient protocol for contacting the compound with the sample may be employed. The specific protocol employed may vary, for example, depending on whether the sample is in vitro or in vivo. For in vitro protocols, any convenient protocol may be used to achieve contact of the sample with the compound. In some examples, the sample includes cells maintained in a suitable culture medium, and the complex is introduced into the culture medium. For in vivo protocols, any convenient administration protocol may be employed. Various protocols may be employed depending on the potency of the compound, the target cells, the mode of administration, and the number of cells present.

[0394] In some embodiments, the subject method is a method for treating cancer in a subject. In some embodiments, the subject method includes administering an effective amount of the subject compound (e.g., as described herein) or a pharmaceutically acceptable salt thereof to the subject. The subject compound can be administered as a part of a pharmaceutical composition (e.g., as described herein). In some examples of the method, the compound administered is a compound of one of formula (I)-(XVb) (e.g., as described herein). In some examples of the method, the compound administered is described by one of the compounds in Table 1-3.

[0395] In some embodiments, an "effective amount" is an amount of a subject compound that, when administered to an individual in one or more doses, in monotherapy or in combination therapy, is effective to inhibit ENPP1 by about 20% (20% inhibition), at least about 30% (30% inhibition), at least about 40% (40% inhibition), at least about 50% (50% inhibition), at least about 60% (60% inhibition), at least about 70% (70% inhibition), at least about 80% (80% inhibition), or at least about 90% (90% inhibition), as compared to the activity of ENPP1 in the individual in the absence of treatment with the compound, or alternatively, as compared to the activity of ENPP1 in the individual before or after treatment with the compound.

[0396] In some embodiments, a "therapeutically effective amount" is an amount of a subject compound that, when administered to an individual in one or more doses, in monotherapy or combination therapy, is effective to reduce the tumor burden in the subject by about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% compared to the tumor burden in the individual in the absence of treatment with the compound, or alternatively compared to the tumor burden in the subject before or after treatment with the compound. As used herein, the term "tumor burden" refers to the total mass of tumor tissue carried by a subject with cancer.

[0397] In some embodiments, a "therapeutically effective amount" is an amount of a subject compound that, when administered to an individual in one or more doses, in monotherapy or in combination therapy, is effective to reduce the dose of radiation therapy required to observe tumor reduction in the subject by about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% as compared to the dose of radiation therapy required to observe tumor reduction in the individual in the absence of treatment with the compound.

[0398] In some embodiments, a "therapeutically effective amount" of a compound is an amount effective to achieve a 1.5-log, 2-log, 2.5-log, 3-log, 3.5-log, 4-log, 4.5-log, or 5-log reduction in tumor size when administered in one or more doses to an individual suffering from cancer.

[0399] In some embodiments, the effective amount of the compound is in the range of about 50 ng / ml to about 50 μg / ml (e.g., about 50 ng / ml to about 40 μg / ml, about 30 ng / ml to about 20 μg / ml, about 50 ng / ml to about 10 μg / ml, about 50 ng / ml to about 1 μg / ml, about 50 ng / ml to about 800 ng / ml, about 50 ng / ml to about 700 ng / ml, about 50 ng / ml to about 600 ng / ml, about 50 ng / ml to about 500 ng / ml, about 50 ng / ml to about 400 ng / ml, about 60 ng / ml to about 400 ng / ml, or about 50 ng / ml to about 600 ng / ml. / ml, about 70ng / ml to about 300ng / ml, about 60ng / ml to about 100ng / ml, about 65ng / ml to about 85ng / ml, about 70ng / ml to about 90ng / ml, about 200ng / ml to about 900ng / ml, about 200ng / ml to about 800ng / ml, about 200ng / ml to about 700ng / ml, about 200ng / ml to about 600ng / ml, about 200ng / ml to about 500ng / ml, about 200ng / ml to about 400ng / ml or about 200ng / ml to about 300ng / ml).

[0400] In some embodiments, the effective amount of the compound is in the range of about 10 pg to about 100 mg, for example, about 10 pg to about 50 pg, about 50 pg to about 150 pg, about 150 pg to about 250 pg, about 250 pg to about 500 pg, about 500 pg to about 750 pg, about 750 pg to about 1 ng, about 1 ng to about 10 ng, about 10 ng to about 50 ng, about 50 ng to about 150 ng, about 150 ng to about 250 ng, about 2 The amount of about 50 ng to about 500 ng, about 500 ng to about 750 ng, about 750 ng to about 1 μg, about 1 μg to about 10 μg, about 10 μg to about 50 μg, about 50 μg to about 150 μg, about 150 μg to about 250 μg, about 250 μg to about 500 μg, about 500 μg to about 750 μg, about 750 μg to about 1 mg, about 1 mg to about 50 mg, about 1 mg to about 100 mg, or about 50 mg to about 100 mg. The amount can be the amount of a single dose or can be a daily total amount. The daily total amount can be in the range of 10 pg to 100 mg, or can be in the range of 100 mg to about 500 mg, or can be in the range of 500 mg to about 1000 mg.

[0401] In some embodiments, a single dose of the compound is administered. In other embodiments, multiple doses are administered. In the case of multiple doses administered over a period of time, the compound can be administered twice a day (qid), every day (qd), every other day (qod), every three days, three times a week (tiw) or twice a week (biw) over a period of time. For example, the compound is administered qid, qd, qod, tiw or biw from one day to about 2 years or longer. For example, depending on various factors, the compound is administered with any of the above frequencies for one week, two weeks, one month, two months, six months, one year or two years or longer.

[0402] Administration of a therapeutically effective amount of a subject compound to an individual with cancer may result in one or more of the following: 1) a reduction in tumor burden; 2) a reduction in the dose of radiation therapy required to achieve tumor shrinkage; 3) a reduction in the spread of cancer from one cell to another in an individual; 4) a reduction in the morbidity or mortality of clinical outcomes; 5) a reduction in the total time of treatment when used in combination with other anticancer agents; and 6) an improvement in disease response indicators (e.g., one or more symptoms of cancer are reduced). Any of a variety of methods can be used to determine whether a treatment method is effective. For example, a biological sample obtained from an individual who has been treated with the subject method can be assayed.

[0403] Any of the compounds described herein can be used in the subject methods of treatment. In some instances, the compound is one of Formulas (I)-(XVb) (e.g., as described herein). In some cases, the compound is one of the compounds in Tables 1-3 or a prodrug form thereof. In some cases, the compound used in the subject methods is not cell permeable. In some cases, the compound used in the subject methods has poor cell permeability.

[0404] In some embodiments, the compound specifically inhibits ENPP1. In some embodiments, the compound modulates the activity of cGAMP. In some embodiments, the compound interferes with the interaction of ENPP1 and cGAMP. In some embodiments, the compound results in activation of the STING pathway.

[0405] In some embodiments, the subject is a mammal. In some examples, the subject is a human. Other subjects may include domestic pets (e.g., dogs and cats), livestock (e.g., cattle, pigs, goats, horses, etc.), rodents (e.g., mice, guinea pigs and rats, for example, as in animal disease models), and non-human primates (e.g., chimpanzees and monkeys). The subject may need to treat cancer. In some examples, the subject method includes diagnosing cancer including any cancer described herein. In some embodiments, the compound is administered as a pharmaceutical product.

[0406] In certain embodiments, the ENPP1 inhibitor compound is a modified compound comprising a label, and the method further comprises detecting the label in the subject. The selection of the label depends on the detection means. Any convenient label and detection system can be used in the subject method, see, e.g., Baker, "The whole picture," Nature, 463, 2010, pp. 977-980. In certain embodiments, the compound comprises a fluorescent label suitable for optical detection. In certain embodiments, the compound comprises a radioactive label so as to be detected using positron emission tomography (PET) or single photon emission computed tomography (SPECT). In some cases, the compound comprises a paramagnetic label suitable for tomography detection. As described above, the subject compound can be labeled, but in some methods, the compound is not labeled and a second labeling agent is used for imaging.

[0407] Combination therapy

[0408] The subject compound can be administered to the subject alone or in combination with another active agent, i.e., a second active agent. Combination therapy methods, wherein the subject ENPP1 inhibitor compound can be used in combination with a second active agent or another therapy (e.g., radiotherapy). The terms "agent", "compound" and "drug" are used interchangeably herein. For example, the ENPP1 inhibitor compound can be administered alone or in combination with one or more other drugs, such as drugs for treating target diseases (including but not limited to immunomodulatory diseases and disorders and cancer). In some embodiments, the subject method also includes concomitantly or sequentially co-administering a second agent, such as a small molecule, a chemotherapeutic agent, an antibody, an antibody fragment, an antibody-drug conjugate, an aptamer, a protein or a checkpoint inhibitor. In some embodiments, the method also includes performing radiotherapy on the subject.

[0409] The terms "co-administered" and "in combination with..." include administering two or more therapeutic agents simultaneously, during the same period, or sequentially without specific time limits. In one embodiment, the agents are present in a cell or in the subject's body at the same time, or exert their biological or therapeutic effects at the same time. In one embodiment, the therapeutic agents are in the same composition or unit dosage form. In other embodiments, the therapeutic agents are in separate compositions or unit dosage forms. In certain embodiments, the first dose can be administered prior to (e.g., minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concomitantly with, or subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) administration of the second therapeutic agent.

[0410] "Concomitant administration" of a known therapeutic drug or another therapy with a pharmaceutical composition of the present disclosure means that the compound and the second agent or another therapy are administered at a time such that both the known drug and the composition of the present invention will have a therapeutic effect. With respect to the administration of the subject compound, such concomitant administration may involve administration of the drug in the same period (i.e., at the same time), before, or after. The route of administration of the two agents may vary, with representative routes of administration being described in more detail below. For a particular drug or therapy and compound of the present disclosure, one of ordinary skill in the art will have no difficulty determining the appropriate administration schedule, order of administration, and dosage of administration.

[0411] In some embodiments, the compound (e.g., subject compound and at least one additional compound or therapy) is administered to the subject within twenty-four hours of each other, such as within 12 hours of each other, within 6 hours of each other, within 3 hours of each other, or within 1 hour of each other. In certain embodiments, the compound is administered within 1 hour of each other. In certain embodiments, the compound is administered substantially simultaneously. Substantially simultaneously means administering the compound to the subject within about 10 minutes or less of each other, such as 5 minutes or less of each other, or 1 minute or less of each other.

[0412] Also provided are pharmaceutical preparations of the subject compound and a second active agent. In pharmaceutical dosage forms, the compounds may be administered in the form of their pharmaceutically acceptable salts, or they may be administered alone or in appropriate combination with other pharmaceutically active compounds, or may be used in combination with other pharmaceutically active compounds.

[0413] In conjunction with any of the subject methods, an ENPP1 inhibitor compound (e.g., as described herein) (or a pharmaceutical composition comprising such a compound) may be administered in combination with another drug designed to reduce or prevent inflammation, treat or prevent chronic inflammation or fibrosis, or treat cancer. In each case, the ENPP1 inhibitor compound may be administered before, simultaneously with, or after the administration of the other drug. In some cases, the cancer is selected from adrenal cancer, liver cancer, kidney cancer, bladder cancer, breast cancer, colon cancer, stomach cancer, ovarian cancer, cervical cancer, uterine cancer, esophageal cancer, colorectal cancer, prostate cancer, pancreatic cancer, lung cancer (both small cell and non-small cell), thyroid cancer, carcinomas, sarcomas, gliomas, glioblastomas, melanomas, and various head and neck tumors.

[0414] For the treatment of cancer, the ENPP1 inhibitor compound can be administered in combination with a chemotherapeutic agent selected from the group consisting of an alkylating agent, a nitrosourea, an antimetabolite, an oncobiotic, a plant (vinca) alkaloid, a steroid hormone, a taxane, a nucleoside analog, a steroid, an anthracycline, a thyroid hormone replacement drug, a thymidylate-targeted drug, a chimeric antigen receptor / T cell therapy, a chimeric antigen receptor / NK cell therapy, an inhibitor of apoptosis regulators (e.g., B cell CLL / lymphoma 2 (BCL-2) BCL-2-like 1 (BCL-XL) inhibitors), a CARP-1 / CCAR1 (cell division cycle and apoptosis regulator 1) inhibitor, a colony stimulating factor-1 receptor (CSF1R) inhibitor, a CD47 inhibitor, a cancer vaccine (e.g., a Th17-induced dendritic cell vaccine or a genetically modified tyrosinase such as ) and other cell therapies.

[0415] Specific chemotherapeutic agents of interest include, but are not limited to, gemcitabine, docetaxel, bleomycin, erlotinib, gefitinib, lapatinib, imatinib, dasatinib, nilotinib, bosutinib, crizotinib, ceritinib, trametinib, bevacizumab, sunitinib, sorafenib, trastuzumab, Ado-trastuzumab emtansine, rituximab, ipilimumab, rapamycin, sirolimus, everolimus, methotrexate, doxorubicin, Abraxane, Folfirinox, cisplatin, carboplatin, 5-fluorouracil, Teysumo, paclitaxel, prednisone, levothyroxine, pemetrexed, navitoclax, and ABT-199. Peptide compounds may also be used. Target cancer chemotherapeutic agents include, but are not limited to, dolastatin and its active analogs and derivatives; and auristatin and its active analogs and derivatives (such as monomethyl auristatin D (MMAD), monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), etc.). See, for example, WO 96 / 33212, WO 96 / 14856, and US6,323,315. Suitable cancer chemotherapeutic agents also include maytansinoids and their active analogs and derivatives (see, for example, EP 1391213; and Liu et al. (1996) Proc. Natl. Acad. Sci. USA 93: 8618-8623); duocarmycins and their active analogs and derivatives (e.g., including synthetic analogs, KW-2189 and CB 1-TM1); and benzodiazepines and their active analogs and derivatives (such as pyrrolobenzodiazepines (PBD).

[0416] In some embodiments, the ENPP1 inhibitor compound can be administered in combination with a chemotherapeutic agent to treat cancer. In some cases, the chemotherapeutic agent is gemcitabine. In some cases, the chemotherapeutic agent is docetaxel. In some cases, the chemotherapeutic agent is Abraxane.

[0417] To treat cancer (e.g., solid tumor cancer), ENPP1 inhibitor compounds can be administered in combination with an immunotherapeutic agent. An immunotherapeutic agent is any convenient agent used to treat a disease by inducing, enhancing, or suppressing an immune response. In some cases, the immunotherapeutic agent is an immune checkpoint inhibitor. For example, Figure 21A-4C shows that exemplary ENPP1 inhibitors can work synergistically with immune checkpoint inhibitors in a mouse model. Any convenient checkpoint inhibitor can be used, including but not limited to cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) inhibitors, programmed death 1 (PD-1) inhibitors, and PD-L1 inhibitors. In some examples, the checkpoint inhibitor is selected from cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) inhibitors, programmed death 1 (PD-1) inhibitors, and PD-L1 inhibitors. Exemplary target checkpoint inhibitors include but are not limited to ipilimumab, pembrolizumab, and nivolumab. In certain embodiments, one or more immunomodulatory polypeptides can be administered in combination with colony stimulating factor-1 receptor (CSF1R) inhibitors for the treatment of cancer and / or inflammatory diseases. Target CSF1R inhibitors include but are not limited to emactuzumab.

[0418] Any convenient cancer vaccine therapy and agent can be used in combination with the subject ENPP1 inhibitor compounds, compositions and methods. To treat cancer (such as ovarian cancer), ENPP1 inhibitor compounds can be administered in combination with vaccine therapies (e.g., dendritic cell (DC) vaccination agents that promote Th1 / Th17 immunity). Th17 cell infiltration is associated with a significant increase in the overall survival of ovarian cancer patients. In some cases, ENPP1 inhibitor compounds can be used as adjuvant therapy in combination with Th17-induced vaccination.

[0419] Also of interest are agents that are inhibitors of CARP-1 / CCARI (cell division cycle and apoptosis regulator 1), including but not limited to those described by Rishi et al., Journal of Biomedical Nanotechnology, Vol. 11, No. 9, September 2015, pp. 1608-1627 (20) and CD47 inhibitors (including but not limited to anti-CD47 antibody agents such as Hu5F9-G4).

[0420] In some examples, the combination provides an enhanced effect relative to any single component; in some cases, the combination provides a super-additive or synergistic effect relative to the combination or additive effect of each component. A variety of combinations of the subject compound and chemotherapeutic agents used sequentially or simultaneously can be adopted. For multiple administrations, for example, two agents can be directly alternated, or two doses or more doses of one agent can be alternated with a single dose of another agent. The simultaneous administration of these two agents can also be alternated with the administration of a single agent or interspersed with the administration of a single agent in another way. In some cases, the time between two administrations can be from about 1-6 hours to about 6-12 hours, to about 12-24 hours, to about 1-2 days, to about 1-2 weeks or longer time periods after starting treatment.

[0421] Combination with cGAMP-inducing chemotherapy

[0422] Aspects of the present disclosure include methods for treating cancer, wherein ENPP1 inhibitor compounds (or pharmaceutical compositions comprising such compounds) can be administered in combination with chemotherapeutic agents capable of inducing the production of cGAMP in vivo. When a subject is exposed to an effective amount of a particular chemotherapeutic agent, 2'3'-cGAMP can be induced in the subject. When the subject ENPP1 inhibitor compound is co-administered to prevent cGAMP degradation, the level of induction of cGAMP can be maintained and / or enhanced, for example, enhanced compared to the level achieved by using either agent alone. Any convenient chemotherapeutic agent that can cause DNA damage and induce dying cells to produce cGAMP due to overwhelmed repair or degradation mechanisms can be used in the subject combination treatment methods, such as alkylating agents, nucleic acid analogs, and intercalating agents. In some cases, the chemotherapeutic agent that induces cGAMP is an anti-mitotic agent. An anti-mitotic agent is an agent that acts by damaging DNA or binding to microtubules. In some cases, the chemotherapeutic agent that induces cGAMP is an anti-tumor agent.

[0423] Target cancers that can be treated using the subject combination therapy include, but are not limited to, adrenal cancer, liver cancer, kidney cancer, bladder cancer, breast cancer, colon cancer, gastric cancer, ovarian cancer, cervical cancer, uterine cancer, esophageal cancer, colorectal cancer, prostate cancer, pancreatic cancer, lung cancer (both small cell carcinoma and non-small cell carcinoma), thyroid cancer, malignant epithelial tumors, sarcomas, gliomas, glioblastomas, melanomas, and various head and neck tumors. In some cases, the cancer is breast cancer. In some instances, the cancer is a glioma or a glioblastoma.

[0424] Chemotherapeutic agents of interest include, but are not limited to, uracil analogs, fluorouracil prodrugs, thymidylate synthase inhibitors, deoxycytidine analogs, DNA synthesis inhibitors (e.g., causing apoptosis in S phase cells), folate analogs, dehydrofolate reductase inhibitors, anthracyclines, intercalators (e.g., causing double-strand breaks), topoisomerase IIa inhibitors, taxanes, microtubule disassembly inhibitors (e.g., causing G2 / M phase arrest / apoptosis), microtubule assembly inhibitors, microtubule function stabilizers (e.g., causing apoptosis in G2 / M-phase cells), tubulin polymerization promoters, tubulin binding agents (e.g. causing apoptosis by M-phase arrest), epothilone B analogs, vinca alkaloids, nitrogen mustards, nitrosoureas, DNA alkylating agents (e.g. causing interstrand crosslinks, apoptosis via p53), VEGF inhibitors, anti-angiogenic antibodies, HER2 inhibitors, quinazoline HER2 inhibitors, EGFR inhibitors, tyrosine kinase inhibitors, sirolimus analogs, mTORC1 inhibitors (e.g. in combination with exemestane = aromatase inhibitors that inhibit estrogen production in breast cancer), triazenes, dacarbazine prodrugs, methylhydrazines.

[0425] Exemplary target breast cancer chemotherapeutic agents include but are not limited to capecitabine, carmofur, fluorouracil, tegafur, gemcitabine, methotrexate, doxorubicin, epirubicin, docetaxel, ixabepilone, vindesine, vinorelbine, cyclophosphamide, bevacizumab, pertuzumab, trastuzumab, lapatinib and everolimus. Exemplary glioma / glioblastoma related antineoplastic drugs include but are not limited to carmustine, lomustine, temozolomide, procarbazine, vincristine and bevacizumab. Exemplary target DNA damage chemotherapeutic agents include but are not limited to melphalan, cisplatin and etoposide, fluorouracil, gemcitabine.

[0426] Combination radiation therapy

[0427] Alternatively, for methods of treating cancer, ENPP1 inhibitor compounds (or pharmaceutical compositions comprising such compounds) may be administered in combination with radiotherapy. In certain embodiments, the method comprises administering radiotherapy to the subject. Likewise, the ENPP1 inhibitor compound may be administered before or after the administration of radiotherapy. Thus, the subject method may also comprise administering radiotherapy to the subject. The combination of radiotherapy and administration of the subject compound may provide a synergistic therapeutic effect. When a subject is exposed to radiation at a suitable dose and / or frequency during radiotherapy (RT), the production of 2'3'-cGAMP may be induced in the subject. When the subject ENPP1 inhibitor compound is co-administered to prevent cGAMP degradation, these induced cGAMP levels may be maintained and / or enhanced, for example, enhanced compared to levels achieved using RT alone. For example, Fig.21AIt is shown that exemplary ENPP1 inhibitors can act synergistically with radiotherapy (RT) to reduce the tumor burden in the mouse model. Therefore, compared with the effective dose and / or frequency / regimen of radiotherapy alone, aspects of the subject method include administering a reduced dose and / or frequency / regimen of radiotherapy. In some cases, radiotherapy is combined with the subject compound to effectively reduce the radiation damage to the subject (e.g., the radiation damage expected to occur under the single radiotherapy of the effective dose and / or frequency / regimen) and / or frequency.

[0428] In some cases, the method comprises administering an ENPP1 inhibitor to a subject prior to radiotherapy. In some cases, the method comprises administering an ENPP1 inhibitor to a subject after exposing the subject to radiotherapy. In some cases, the method comprises sequentially administering radiotherapy, followed by an ENPP1 inhibitor, and then a checkpoint inhibitor to a subject in need thereof.

[0429] Practicality

[0430] For example, the compounds and methods of the invention as described herein can be used in a variety of applications. Target applications include, but are not limited to, research applications and therapeutic applications. The methods of the invention can be used in a variety of different applications including any convenient application requiring inhibition of ENPP1.

[0431] The subject compounds and methods can be used in a variety of research applications. The subject compounds and methods can be used to optimize the bioavailability and metabolic stability of compounds.

[0432] The subject compounds and methods can be used in a variety of therapeutic applications. Targeted therapeutic applications include those in the treatment of cancer. Thus, the subject compounds can be used to treat a variety of different conditions that require suppression and / or treatment of cancer in a host. For example, the subject compounds and methods can be used to treat solid tumor cancers (e.g., as described herein).

[0433] Pharmaceutical composition

[0434] The compounds discussed herein can be formulated using any convenient excipients, reagents, and methods. Compositions configured with one or more pharmaceutically acceptable excipients are provided. Various pharmaceutically acceptable excipients are known in the art and need not be discussed in detail herein. Pharmaceutically acceptable excipients have been fully described in many publications, including, for example, A. Gennaro (2000) "Remington: The Science and Practice of Pharmacy," 20th edition, Lippincott, Williams, & Wilkins; Pharmaceutical Dosage Forms and Drug Delivery Systems (1999) HC Ansel et al., ed., 7th edition, Lippincott, Williams, & Wilkins; and Handbook of Pharmaceutical Excipients (2000) AK Kibbe et al., ed., 3rd edition Amer. Pharmaceutical Assoc.

[0435] Pharmaceutically acceptable excipients such as vehicles, adjuvants, carriers or diluents are readily available to the public. In addition, pharmaceutically acceptable auxiliary substances, such as pH adjusters and buffers, tonicity adjusters, stabilizers, wetting agents, etc. are readily available to the public.

[0436] In some embodiments, the subject compound is formulated in an aqueous buffer. Suitable aqueous buffers include, but are not limited to, acetate, succinate, citrate, and phosphate buffers, with strengths ranging from 5mM to 100mM. In some embodiments, the aqueous buffer includes an agent that provides an isotonic solution. Such agents include, but are not limited to, sodium chloride; and sugars, such as mannitol, dextrose, sucrose, etc. In some embodiments, the aqueous buffer also includes a nonionic surfactant such as polysorbate 20 or 80. Optionally, the formulation may also include a preservative. Suitable preservatives include, but are not limited to, benzyl alcohol, phenol, chlorobutanol, benzalkonium chloride, etc. In many cases, the formulation is stored at about 4°C. The formulation may also be lyophilized, in which case they typically include cryoprotectants, such as sucrose, trehalose, lactose, maltose, mannitol, etc. Lyophilized formulations may be stored for a long time even at ambient temperature. In some embodiments, the subject compound is formulated for sustained release.

[0437] In some embodiments, the subject compound and a second active agent (e.g., as described herein), such as a small molecule, a chemotherapeutic agent, an antibody, an antibody fragment, an antibody-drug conjugate, an aptamer, or a protein, etc., are administered to an individual in the form of a formulation containing one or more pharmaceutically acceptable excipients (e.g., in the same formulation or in separate formulations). In some embodiments, the second active agent is a checkpoint inhibitor, e.g., a cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) inhibitor, a programmed death 1 (PD-1) inhibitor, or a PD-L1 inhibitor.

[0438] In another aspect of the present invention, a pharmaceutical composition is provided, which comprises the following or is substantially composed of the following: a compound of the present invention, or a pharmaceutically acceptable salt, isomer, tautomer or prodrug thereof, and further comprises one or more additional target active agents. Any convenient active agent can be used in combination with the subject compound in the subject method. In some examples, the other agent is a checkpoint inhibitor. The subject compound and the checkpoint inhibitor, as well as other therapeutic agents for combination therapy as described herein, can be administered orally, subcutaneously, intramuscularly, intranasally, parenterally or by other routes. The subject compound and the second active agent (if present) can be administered by the same route of administration or by different routes of administration. The therapeutic agent can be administered by any suitable means, including, but not limited to, for example, oral administration, rectal administration, nasal administration, topical (including transdermal, aerosol, buccal and sublingual) administration, vaginal administration, parenteral (including subcutaneous, intramuscular, intravenous and intradermal) administration, intravesical administration or injection into the affected organ. In some cases, the therapeutic agent can be administered intranasally. In some cases, the therapeutic agent can be administered intratumorally.

[0439] In some embodiments, the subject compound and the chemotherapeutic agent are administered to an individual in the form of a formulation (e.g., in the same formulation or in separate formulations) together with one or more pharmaceutically acceptable excipients. Chemotherapeutic agents include, but are not limited to, alkylating agents, nitrosoureas, antimetabolites, antitumor antibiotics, plant (vinca) alkaloids, and steroid hormones. Peptide compounds may also be used. Suitable cancer chemotherapeutic agents include dolastatin and its active analogs and derivatives; and auristatin and its active analogs and derivatives (e.g., monomethyl auristatin D (MMAD), monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), etc.). See, for example, WO 96 / 33212, WO 96 / 14856, and US6,323,315. Suitable cancer chemotherapeutic agents also include maytansinoids and their active analogs and derivatives (see, e.g., EP 1391213; and Liu et al. (1996) Proc. Natl. Acad. Sci. USA 93:8618-8623); duocarmycins and their active analogs and derivatives (e.g., including synthetic analogs, KW-2189 and CB 1-TM1); and benzodiazepines. and their active analogs and derivatives (e.g., pyrrolobenzodiazepines (PBD).

[0440] The subject compound and the second chemotherapeutic agent and other therapeutic agents for combination therapy as described herein can be administered orally, subcutaneously, intramuscularly, parenterally or by other routes. The subject compound and the second chemotherapeutic agent can be administered by the same route of administration or by different routes of administration. The therapeutic agent can be administered by any suitable means, including but not limited to, for example, oral administration, rectal administration, nasal administration, topical (including transdermal, aerosol, buccal and sublingual) administration, vaginal administration, parenteral (including subcutaneous, intramuscular, intravenous and intradermal) administration, intravesical administration or injection into the affected organ.

[0441] The subject compound can be administered in unit dosage form and can be prepared by any method known in the art. Such methods include combining the subject compound with a pharmaceutically acceptable carrier or diluent constituting one or more auxiliary components. A pharmaceutically acceptable carrier is selected according to the selected route of administration and standard pharmaceutical practice. Each carrier must be "pharmaceutically acceptable" in the sense that it is compatible with the other ingredients of the preparation and harmless to the subject. The carrier can be solid or liquid, and the type is usually selected based on the type of administration used.

[0442] The example of suitable solid carrier comprises lactose, sucrose, gelatin, agar and loose powder.The example of suitable liquid carrier comprises water, pharmaceutically acceptable fat and oil, alcohol or other organic solvents, emulsion, syrup or elixir, suspension, solution and / or suspension including ester, and solution or suspension by non-effervescent granule redissolution, and the effervescent product from effervescent granule redissolution.This type of liquid carrier can contain for example suitable solvent, preservative, emulsifier, suspending agent, diluent, sweetener, thickening agent and melting agent.Preferred carrier is edible oil, for example corn oil or canola oil.Polyethylene glycol, as PEG, is also a good carrier.

[0443] Any drug delivery device or system that provides the dosing regimen of the present disclosure can be used.A variety of delivery devices and systems are known to those skilled in the art. Example

[0444] The following examples are presented to provide a person of ordinary skill in the art with a complete disclosure and description of how to make and use the embodiments of the present disclosure, and are not intended to limit the scope of the invention that the inventors regard as their invention, nor are they intended to represent that the following experiments are all or the only experiments performed. Every effort has been made to ensure the accuracy of the numbers (such as amounts, temperatures, etc.) used, but some experimental errors and deviations should be considered. Unless otherwise specified, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is atmospheric pressure or near atmospheric pressure.

[0445] Although the present invention has been described with reference to specific embodiments of the present invention, it will be appreciated by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the present invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps to the purpose, spirit and scope of the present disclosure. All such modifications are intended to fall within the scope of the appended claims.

[0446] Example 1: Synthesis of compounds

[0447] The compounds may be synthesized using any convenient method. Methods applicable to the preparation of the compounds of the present disclosure include the exemplary synthesis methods described in Examples 1a-1c, and those described in PCT Application No. PCT / US2018 / 050018 filed by Li et al. on September 7, 2018, the disclosure of which is incorporated herein by reference in its entirety. Many general references providing known chemical synthesis schemes and conditions that can be used to synthesize the disclosed compounds are also available (see, for example, Smith and March, March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, Fifth Edition, Wiley-Interscience, 2001; or Vogel, A Textbook of Practical Organic Chemistry, Including Qualitative Organic Analysis, Fourth Edition, New York: Longman, 1978). The reaction can be analyzed by thin layer chromatography (TLC), LC / MS, and by LC / MS and 1 The reaction products were monitored by HNMR characterization. Intermediates and final products could be purified by silica gel chromatography or by HPLC.

[0448] Example 1a: Exemplary Synthesis Scheme of Compound 1

[0449] The synthesis of compound 1 (which may be suitable for preparing the compounds of the present disclosure) is shown below:

[0450]

[0451] Preparation of dimethyl (2-(piperidin-4-yl)ethyl)phosphonate

[0452]

[0453] Sodium hydride (2.16 g, 54.11 mmol) was carefully added to a stirred solution of bis(dimethoxyphosphoryl)methane (11.42 g, 49.19 mmol) in toluene (100 mL) at room temperature. The reaction mixture was then placed under a nitrogen atmosphere and a solution of 1-benzylpiperidine-4-carboxaldehyde (10 g, 49.19 mmol) in toluene (50 mL) was slowly added, keeping the temperature below 40°C. The resulting mixture was stirred at room temperature for 16 hours and then quenched by the addition of saturated aqueous ammonium chloride. The organic phase was separated, washed with brine, dried (MgSO4) and evaporated to dryness. Chromatography (120 g SiO2; 5 to 100% gradient of EtOAc in hexanes) gave dimethyl (E)-(2-(1-benzylpiperidin-4-yl)vinyl)phosphonate (6.2 g, 16%) as a colorless oil.

[0454] To a mixture of dimethyl (E)-(2-(1-benzylpiperidin-4-yl)vinyl)phosphonate (3.7 g, 12.0 mmol) in ethanol (40 mL) was added Pd / C (1.1 g, 10.3 mmol). The mixture was placed under a hydrogen atmosphere and stirred at room temperature for 12 hours, filtered and evaporated to dryness under reduced pressure to give dimethyl (2-(piperidin-4-yl)ethyl)phosphonate (2.7 g, 100%) as a colorless oil.

[0455] Preparation of dimethyl (2-(1-(6,7-dimethoxyquinazolin-4-yl)piperidin-4-yl)ethyl)phosphonate

[0456]

[0457] Diisopropylethylamine (0.6 g, 8.9 mmol) was added to a mixture of dimethyl (2-(piperidin-4-yl)ethyl)phosphonate (1.1 g, 4.9 mmol) and 4-chloro-6,7-dimethoxyquinazoline (1.0 g, 4.5 mmol) in isopropanol (20 mL). After stirring at 90 °C for 3 hours, the reaction mixture was cooled and evaporated to dryness. Purification on silica gel (5% MeOH in dichloromethane) gave dimethyl (2-(1-(6,7-dimethoxyquinazolin-4-yl)piperidin-4-yl)ethyl)phosphonate (755 mg, 37%) as an oil.

[0458] LC-MS: m / z=410.25 [M+H] +

[0459] 1H NMR (500MHz, CDCl3) δ8.65(s,1H),7.23(s,1H),7.09(s,1H),4.19(dq,J=14.0,2.9,2.4Hz,2H),4.02(s,3H),3.99(s,3H),3.77(s,3 H),3.75(s,3H),3.05(td,J=12.8,2.3Hz,2H),1.93–1.77(m,4H),1.67(ddd,J=14.1,9.5,5.9Hz,3H),1.46(qd,J=12.2,3.7Hz,2H).

[0460] Preparation of dimethyl (2- (1- (6,7-dimethoxyquinazolin-4-yl) piperidin-4-yl) ethyl) phosphonic acid (Compound 1) Preparation

[0461]

[0462] Bromotrimethylsilane (3.67 g, 24 mmol) was added to a cooled solution of dimethyl (2-(1-(6,7-dimethoxyquinazolin-4-yl)piperidin-4-yl)ethyl)phosphonate (3.25 g, 7.94 mmol) in chloroform (60 mL) (which was cooled by an ice bath). The reaction mixture was allowed to warm to room temperature and quenched by the addition of methanol (20 mL) after 90 minutes. The mixture was evaporated to dryness under reduced pressure and then solvated in methanol (100 mL). The reaction mixture was concentrated to half volume, filtered to remove the precipitate, and then evaporated to dryness. The residue was crystallized from dichloromethane, filtered and dried in vacuo to give dimethyl (2-(1-(6,7-dimethoxyquinazolin-4-yl)piperidin-4-yl)ethyl)phosphonic acid (2.1 g, 69%).

[0463] LC-MS: m / z=381.8[M+H] +

[0464] 1 H NMR(500MHz,DMSO-d6)δ8.77(s,1H),7.34(s,1H),7.23(s,1H),4.71(d,J=13.1Hz,2H),3.99(s,3H),3.9 7(s,3H),3.48(t,J=12.7Hz,2H),3.18(s,1H),1.97–1.90(m,2H),1.62–1.43(m,4H),1.40–1.27(m,2H).

[0465] Example 1b: Synthesis of Compound 5 (Table 1)

[0466] The synthetic scheme listed below was used to prepare compound 5:

[0467]

[0468]

[0469] Example 1c: Synthesis of Compound 6 (Table 1)

[0470] The synthetic scheme listed below was used to prepare compound 6:

[0471]

[0472] Chemical synthesis: Unless otherwise specified, the reactions were carried out under ambient atmosphere. Qualitative TLC analysis was performed on glass-backed F254 silica (Silicycle, Quebec City, Canada). Visualization was accomplished using UV light and exposure to p-anisaldehyde or KMnO4 staining solutions followed by heating. All solvents used were ACS grade Sure-Seal, and all other reagents were used as received unless otherwise noted. The syntheses of non-commercially available 4-chloroquinazoline and 4-chloro-3-quinolinecarbonitrile and amine building blocks are described in the Supplementary Information. Silica gel flash columns ( SiliaSep TM 40-63mm, ) were subjected to flash chromatography on a Teledyne Isco purification system. HPLC was performed on an Agilent 1260 Infinity preparative-scale purification system using an Agilent PrepHT Zorbax Eclipse XDB-C18 reverse phase column (21.2 x 250 mm). The HPLC was performed using a Bruker AV-500 spectrometer. 1 Structural determination was performed using H spectroscopy and low-resolution mass spectra (ESI-MS) collected on a Shimadzu 20-20ESI LCMS instrument. 1 The structure was determined by H spectroscopy and low-resolution mass spectra (ESI-MS) collected on a Shimadzu 20-20ESI LCMS instrument. The final compounds were >95% pure according to HPLC-MS. All final compounds 1 The H spectra were consistent with the expected structure.

[0473] Synthesis of ureas 4 and 5.

[0474]

[0475] a)DIPEA, i-PrOH.

[0476] Preparation of 1-(2-(1-(6,7-dimethoxyquinazolin-4-yl)piperidin-4-yl)ethyl)urea 4 (in Table 3a)

[0477] To a solution of 1-(2-(piperidin-4-yl)ethyl)urea 64 (173 mg, 1.01 mmol) in isopropanol (5 mL) was added 4-chloro-6,7-dimethoxyquinazoline 63 (181 mg, 0.81 mmol) and N,N-diisopropylethylamine (391 mg, 3.03 mmol) under nitrogen atmosphere. The mixture was stirred at room temperature for 2 hours and then evaporated to dryness under reduced pressure. Purification (preparative HPLC) gave the title compound 4 (172 mg, 47%) as pale yellow crystals.

[0478] LCMS: [M+H] + m / z 360. 1 H NMR(400MHz,DMSO-d6)δ8.49(s,1H),7.17(s,1H),7.07(s,1H),5.92–5.90(m,1H),5.36(br s,2H),4.13–4.09(m,2H),3.90(s,3H),3.88(s,3H),3.04–2.94(m,4H),1.81–1.78(m,2H),1.62–1.56(m,1H) and 1.38–1.33(m,4H).

[0479] Preparation of 1-((1-(6,7-dimethoxyquinazolin-4-yl)piperidin-4-yl)methyl)urea 5 (in Table 3a)

[0480] To a solution of 1-(piperidin-4-ylmethyl)urea 65 (155 mg, 0.97 mmol) in isopropanol (10 mL) was added 4-chloro-6,7-dimethoxyquinazoline 63 (174 mg, 0.78 mmol) and N,N-diisopropylethylamine (394 mg, 2.9 mmol) under nitrogen atmosphere. The mixture was stirred at 10 °C for 3 hours and then evaporated to dryness under reduced pressure. Chromatography (SiO2: 0 to 6% MeOH in dichloromethane) gave the desired product 5 (150 mg, 44%) as a white solid.

[0481] LCMS: [M+H] + m / z 346.0 1H NMR (400 MHz, methanol-d4) δ 8.44 (s, 1H), 7.14 (s, 1H), 7.12 (s, 1H), 4.28–4.24 (m, 2H), 3.96 (s, 3H), 3.94 (s, 3H), 3.13–3.07 (m, 4H), 1.94–1.87 (m, 3H) and 1.50–1.41 (m, 2H).

[0482] Preparation of 3-(1-(6,7-dimethoxyquinazolin-4-yl)piperidin-4-yl)propanoic acid 6 (in Table 3a)

[0483]

[0484] 4-Chloro-6,7-dimethoxy-quinazoline 63 (3.14 g, 13.98 mmol) and 3-(4-piperidinyl)propionic acid (2.0 g, 12.72 mmol) were suspended in isopropanol (100 mL) and stirred at 90 °C for 3 hours. Once cooled, the mixture was evaporated to dryness under reduced pressure. The residue was then triturated with CH2Cl2 (20 mL) to give the title compound 6 (1.87 g, 42%) as a white solid.

[0485] 1 H NMR (400 MHz, methanol-d4)δ

[0486] Preparation of (2-(1-(6,7-dimethoxyquinolin-4-yl)piperidin-4-yl)ethyl)boronic acid 7 (in Table 3a)

[0487] a) Cp2ZrCl2, PhMe60℃; b) Pd / C, H2, MeOH; c) HCI(aq), MeOH / hexane;; d) 63, DIEA, THF80℃.

[0488] A solution of tert-butyl 4-ethynylpiperidine-1-carboxylate 66 (2.92 g, 13.95 mmol), bis(cyclopentadienyl)zirconium chloride hydride (150 mg, 0.518 mmol) and 4,4,5,5-tetramethyl-1,3,2-dioxaborolane 67 (1.49 g, 11.63 mmol) in solvent was stirred at 60 °C for 16 h, then diluted with ether and evaporated to dryness under reduced pressure. Chromatography (SiO2; 2–5% ethyl acetate in petroleum ether) afforded 68 (4.2 g, 89%). A mixture of (E)-tert-butyl 4-(2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)vinyl)piperidine-1-carboxylate 68 (4.2 g, 12.46 mmol) and palladium / carbon (840 mg, 20% w / w) in MeOH (500 mL) was placed under a hydrogen atmosphere and stirred at room temperature for 16 hours. The mixture was then passed through The mixture was filtered through a pad of 4% paraformaldehyde and then evaporated to dryness under reduced pressure to afford 69 (4.2 g, 92%). 1 M aqueous HCl solution (4 mL) was added to a cooled (0 °C) mixture of 73 (460 mg, 1.36 mmol) in MeOH / hexane (5 mL / 5 mL). The mixture was warmed to room temperature and stirred for 3 hours and then evaporated to dryness under reduced pressure to afford (2-(piperidin-4-yl)ethyl)boronic acid 70 (180 mg, 68%) as the hydrochloride salt. To a solution of 70 (140 mg, 1.04 mmol) in THF (5 mL) was added 4-chloro-6,7-dimethoxyquinazoline 63 (180 mg, 0.935 mmol) followed by N,N-diisopropylethylamine (360 mg, 1.87 mmol). The mixture was stirred at 80 °C for 16 hours and then evaporated to dryness under reduced pressure. Purification (preparative HPLC) afforded the title compound as a pale yellow solid (105 mg; 37%).

[0489] LCMS: [M+H] + m / z 346.3. 1 H NMR(400MHz,DMSO-d6)δ8.67(s,1H),7.26(s,1H),7.25(s,1H),4.62–4.59(m,2H),3.92(s,3H),3.9 0(s,3H), 3.42–3.36(m,4H), 2.46(s,1H), 1.88–1.86(m,2h), 1.29–1.14(m,3H) and 0.60–0.56(m,2H).

[0490] Preparation of hydroxamic acids 8 and 9

[0491]

[0492] a) 63, i-PrOH, 100℃; b) NaOH, THF, H2O; c) NH2OH.HCI, DIPEA, BOP, THF.

[0493] Preparation of 2-(1-(6,7-dimethoxyquinazolin-4-yl)piperidin-4-yl)-N-hydroxyacetamide 8 (in Table 3a)

[0494] A mixture of 4-chloro-6,7-dimethoxyquinazoline 63 (600 mg, 2.68 mmol) and ethyl 2-(piperidin-4-yl)acetate 71 (504 mg, 2.95 mmol) in i-PrOH (6 mL) was stirred at 100 °C in a sealed tube for 16 hours. The reaction mixture was then concentrated under reduced pressure and the residue was purified by silica gel chromatography to give ethyl 2-(1-(6,7-dimethoxyquinazoline-4-yl)piperidin-4-yl)acetate (750 mg, 77%). A 2M NaOH solution in H2O (1 mL) was added to a mixture of ethyl 2-(1-(6,7-dimethoxyquinazoline-4-yl)piperidin-4-yl)acetate (250 mg, 0.696 mmol) in THF (10 mL). The mixture was stirred at room temperature for 16 hours and then quenched by the addition of 1M HCl solution. The organic phase was extracted with ethyl acetate, washed with brine, dried (Na2SO4), and evaporated to dryness under reduced pressure to give acid 72 (200 mg, 86%) as a white solid.

[0495] To a mixture of acid 72 (300 mg, 0.906 mmol) in THF (10 mL) were added NH2OH·HCl (76 mg, 1.09 mmol), DIEA (468 mg, 3.63 mmol) and (benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP) (481 mg, 1.09 mmol). The mixture was stirred at room temperature for 16 hours and then diluted with water, extracted with ethyl acetate, washed with brine solution, dried (Na2SO4), and evaporated to dryness under reduced pressure. Chromatography (SiO2, solvent) gave the title product 8 (180 mg, 77%) as a white solid. LCMS: [M+H] + m / z 347.10. 1 H NMR(400MHz,D2O)δ8.42(s,1H),7.13(s,1H),7.00(s,1H),4.68–4.62(m,2H),3.95(s,3H) ,3.91(s,3H),3.51–3.45(m,2H),2.21–2.15(m,3H),1.93–1.0(m,2H) and 1.45–1.36(m,2H).

[0496] Preparation of 1-(6,7-dimethoxyquinazolin-4-yl)-N-hydroxypiperidine-4-carboxamide 9 (in Table 3a).

[0497] Synthesized according to the procedure of 8 but using ethyl piperidine-4-carboxylate 73 .

[0498] LCMS: [M+H] + m / z 333.25. 1 H NMR(400MHz,D2O)δ8.39(s,1H),7.04(s,1H),6.94(s,1H),4.62–4.58(m,2H),3.91(s,3H) ,3.86(s,3H),3.47–3.41(m,2H),2.65–2.60(m,1H),1.97–1.94(m,2H) and 1.82–1.77(m,2H).

[0499] General procedure used for compounds 10, 11, 12, 13 and 16 (in Table 3a).

[0500]

[0501] a) i-PrOH, 100°C; b) pyridine, PSCl3 then H2O; or c) pyridine, POCl3, 然后 H2O; d) PPh3, I2 imidazole, CH2Cl2; e) PO(OBn)2,

[0502] DBU, MeCN; f) Pd / C, H2, MeOH.

[0503] Preparation of 2-(1-(6,7-dimethoxyquinolin-4-yl)piperidin-4-yl)ethan-1-ol 77

[0504] A mixture of 4-chloro-6,7-dimethoxyquinazoline 63 (1.0 g, 4.46 mmol) and piperidin-4-ylethanol 79 (633 mg, 4.91 mmol) in isopropanol (10 mL) was stirred in a sealed tube at 100° C. for 16 h. After cooling, the reaction mixture was concentrated under reduced pressure and the residue was purified by silica gel chromatography (SiO2; EtOAc in petroleum ether) to give 2-(1-(6,7-dimethoxyquinolin-4-yl)piperidin-4-yl)ethan-1-ol 75 (1.3 g, 91%).

[0505] Preparation of (1-(6,7-dimethoxyquinolin-4-yl)piperidin-4-yl)methanol 78

[0506] A mixture of 4-chloro-6,7-dimethoxyquinazoline 63 (900 mg, 4.02 mmol) and piperidin-4-ylmethanol 76 (508 mg, 4.42 mmol) in i-PrOH (10 mL) was stirred in a sealed tube at 100 °C for 16 hours. After cooling, the reaction mixture was evaporated to dryness under reduced pressure. Purification by chromatography (SiO2; 10 to 80% ethyl acetate in petroleum ether) gave (1-(6,7-dimethoxyquinolin-4-yl)piperidin-4-yl)methanol 78 (1 g, 82%).

[0507] Preparation of 2-(1-(6,7-dimethoxyquinazolin-4-yl)piperidin-4-yl)ethyl dihydrogen phosphate 10 (in Table 3a)

[0508] 2-(1-(6,7-dimethoxyquinazolin-4-yl)piperidin-4-yl)ethan-1-ol 77 (340 mg, 1.07 mmol) was dissolved in 10 mL of anhydrous pyridine, which was then cooled to -15 °C and stirred for 10 min. POCl3 (821 mg, 5.4 mmol) was added dropwise under N2 atmosphere, the reaction temperature was slowly raised to 0 °C, and then stirred for another 30 min. The mixture was poured into sodium bicarbonate solution (800 mg in 250 mL of water) at 0 °C. The desired compound was extracted with dichloromethane. The organic phase was concentrated and purified by preparative HPLC to give 2-(1-(6,7-dimethoxyquinazolin-4-yl)piperidin-4-yl)ethyl dihydrogen phosphate 10 (52 mg, 12%) as a white solid.

[0509] LCMS: [M+H] + m / z 398. 1 H NMR(400MHz,DMSO-d6)δ8.54(s,1H),7.17(s,1H),7.15(s,1H),4.28–4.16(m,2H),3.93(s,8H) ,3.13–3.04(m,2H),1.90–1.80(m,2H),1.75(s,1H),1.59(d,J=6.4Hz,2H) and 1.44–1.32(m,2H).

[0510] Preparation of (1-(6,7-dimethoxyquinolin-4-yl)piperidin-4-yl)methyl dihydrogen phosphate 11 (in Table 3a)

[0511] (1-(6,7-dimethoxyquinolin-4-yl)piperidin-4-yl)methanol 78 (100 mg, 0.33 mmol) was dissolved in anhydrous pyridine (3 mL), which was then cooled to -15 °C and stirred for 10 minutes. POCl3 (253 mg, 1.65 mmol) was added dropwise under a nitrogen atmosphere. The reaction temperature was slowly raised to 0 °C and then stirred for another 30 minutes. The mixture was poured into an aqueous NaHCO3 solution (160 mg in 50 mL of water) at 0 °C. The desired compound was extracted with dichloromethane and then evaporated to dryness under reduced pressure. After freeze-drying, it was purified by preparative HPLC to give (1-(6,7-dimethoxyquinolin-4-yl)piperidin-4-yl)methyl dihydrogen phosphate 11 (70 mg, 55%) as a white powder. LCMS: [M+H] + m / z 384.20. 1 H NMR (400MHz, DMSO-d6) δ8.74(d,J=1.7Hz,1H),7.31(s,1H),7.20(s,1H),4.66(d,J=13.0Hz,1H),3.97(m,J=12. 6,1.6Hz,8H),3.76(t,J=6.6Hz,3H),2.19–2.00(m,1H),1.92(d,J=13.5Hz,2H),1.45(dd,J=14.2,10.7Hz,1H).

[0512] Preparation of O-(2-(1-(6,7-dimethoxyquinazolin-4-yl)piperidin-4-yl)ethyl)O,O-dihydrogenphosphorothioate 12 (in Table 3a)

[0513] To a solution of 2-(1-(6,7-dimethoxyquinolin-4-yl)piperidin-4-yl)ethan-1-ol 77 (150 mg, 0.473 mmol) in anhydrous pyridine (5 mL) was added P(S)Cl3 (477 mg, 2.84 mmol) at –15°C. After stirring at 0°C for 0.5 h, the mixture was poured into a solution of NaHCO3 (238 mg, 2.84 mmol) in H2O (50 mL). The mixture was stirred at 0°C for 2 h. The progress of the reaction mixture was monitored by LCMS. The mixture was then concentrated under reduced pressure, and the residue was purified by preparative HPLC to give compound 12 (16 mg, 8%) as a light yellow solid. LCMS: [M+H] + m / z 414.05. 1H NMR (400MHz, DMSO-d6) δ8.62(s,1H),7.19(d,J=7.7Hz,2H),4.45(d,J=12.3Hz,2H),3.91( d,J=11.3Hz,10H),1.86(d,J=12.2Hz,3H),1.56(d,J=6.4Hz,2H),1.34(d,J=10.7Hz,2H).

[0514] Preparation of O-((1-(6,7-dimethoxyquinazolin-4-yl)piperidin-4-yl)methyl)O,O-dihydrogenphosphorothioate 13 (in Table 3a)

[0515] To a solution of (1-(6,7-dimethoxyquinolin-4-yl)piperidin-4-yl)methanol 78 (100 mg, 0.330 mmol) in anhydrous pyridine (5 mL) was added P(S)Cl3 (280 mg, 1.98 mmol) at -15°C. After stirring at 0°C for 0.5 h, the mixture was poured into a solution of NaHCO3 (116 mg, 1.98 mmol) in H2O (50 mL). The mixture was stirred at 0°C for 2 h. The mixture was evaporated to dryness under reduced pressure and the residue was purified by preparative HPLC to give compound 13 (10 mg, 7.6%) as a yellow solid. LCMS: [M+H] + m / z 400.15. 1 H NMR (400MHz, DMSO-d6) δ8.54(s,1H),7.18(s,1H),7.11(s,1H),4.25(d,J=13.4Hz,2H),3.89(d,J=9.1Hz ,6H),3.76(s,2H),3.10(d,J=11.8Hz,3H),1.94(s,1H),1.81(d,J=12.7Hz,2H),1.39(d,J=11.4Hz,1H).

[0516] Preparation of ((1-(6,7-dimethoxyquinazolin-4-yl)piperidin-4-yl)methyl)phosphonic acid 16

[0517] A solution of PPh3 (3.39 g, 15 mmol) and imidazole (1.02 g, 15 mmol) in anhydrous CH2Cl2 (40 mL) was stirred at 0°C for 10 min, and then I2 (3.8 g, 15 mmol) was added. The crude reaction mixture was placed under a nitrogen atmosphere and stirred for an additional 10 min, and then (1-(6,7-dimethoxyquinazolin-4-yl)piperidin-4-yl)methanol 78 (3.03 g, 10 mmol) was added. The reaction mixture was stirred at room temperature overnight. The reaction was terminated by the addition of aqueous Na2S2O3. The crude mixture was extracted with CH2Cl2, washed with water, brine, dried (Na2SO4), and evaporated to dryness under reduced pressure. Recrystallization from methanol gave 4-(4-(iodomethyl)piperidin-1-yl)-6,7-dimethoxyquinazoline (2.28 g, 56%) as a light yellow solid. LCMS: [M+H] + m / z 414.3. 1 H NMR(400MHz, CDCl3)δ8.63(d,J=1.3Hz,1H),7.28(s,1H),7.07(s,1H),4.23(s,2H),4.00(s,6H),3 .19(d,J=6.5Hz,2H),3.08(s,2H),2.11–2.00(m,2H),1.82(s,1H),1.49(s,2H),1.29–1.20(m,1H).

[0518] 1,8-Diazabicyclo(5.4.0)undec-7-ene (DBU) (9.2 g, 60.5 mmol) was added to a cooled (0°C) solution of bis(benzyloxy)(oxo)-λ4-phosphine (9.5 g, 36.3 mmol) in anhydrous MeCN (40 mL). After 10 minutes, 4-(4-(iodomethyl)piperidin-1-yl)-6,7-dimethoxyquinazoline (5.0 g, 12.1 mmol) was added. The resulting mixture was stirred overnight and then evaporated to dryness under reduced pressure. The residue was dissolved in ethyl acetate, washed with water, brine, dried (MgSO4), and evaporated to dryness under reduced pressure. Purification by FCC [CH2Cl2:MeOH (50:1)] gave dibenzyl ((1-(6,7-dimethoxyquinazolin-4-yl)piperidin-4-yl)methyl)phosphonate (1.1 g, 18%) as a colorless viscous oil. LCMS: [M+H] + m / z 548.20. 1HNMR(400MHz, CDCl3)δ8.57(s,1H),7.89(s,1H),7.39–7.33(m,10H),6.99(s,1H),5.08(m,3H),4.96(m,2H),4.64(d,J=13 .5Hz,2H),4.09(s,3H),3.93(s,3H),3.27(d,J=12.9Hz,2H),2.05(d,J=13.9Hz,5H),1.76(m,4H),1.42(d,J=12.5Hz,2H).

[0519] A mixture of dibenzyl ((1-(6,7-dimethoxyquinazolin-4-yl)piperidin-4-yl)methyl)phosphonate (660 mg, 1.2 mmol) and Pd / C (132 mg, 20% w / w) in MeOH (20 mL) was placed under H2 atmosphere and stirred at room temperature. After 4 hours, the crude mixture was purified by The mixture was filtered through a pad of 4% paraformaldehyde and the filtrate was evaporated to dryness under reduced pressure. Purification by preparative HPLC gave ((1-(6,7-dimethoxyquinazolin-4-yl)piperidin-4-yl)methyl)phosphonic acid 16 (125 mg, 28%) as a light yellow solid. LCMS: [M+H] + m / z 368.10. 1 H NMR(400MHz,DMSO-d6)δ8.72(s,1H),7.29(s,2H),4.60(d,J=12.8Hz,2H),3 .95(d,J=11.2Hz,6H),3.46(s,2H),2.09(s,3H),1.61(s,2H),1.42(s,2H).

[0520] Preparation of (2-(1-(6,7-dimethoxyquinazolin-4-yl)piperidin-4-yl)propyl)phosphonic acid 14 (in Table 3a)

[0521]

[0522] a)PPh3, I2, imidazole, CH2Cl2; b) P(O)H(OEt)2, Cs2CO3, DMF; c) TFA, CH2Cl2; d) 63, DIPEA,

[0523] CH2Cl2; e) TMSBr, MeCN, 60℃

[0524] Iodine (1.35 g, 5.34 mmol) was added to a solution of PPh3 (1.4 g, 5.34 mmol) and imidazole (0.36 g, 5.34 mmol) in CH2Cl2 (20 mL). The mixture was stirred at room temperature for 0.5 h, and then a solution of 79 (1.0 g, 4.11 mmol) in CH2Cl2 (5 mL) was added dropwise. The reaction mixture was stirred at room temperature for 4 h, and then quenched with saturated Na2SO3 solution and extracted with CH2Cl2. The organic phase was washed with water, brine, dried (Na2SO4), and evaporated to dryness under reduced pressure. Chromatography (SiO2; 5% EtOAc in petroleum ether) gave tert-butyl 4-(3-iodopropyl)piperidine-1-carboxylate (1.0 g, 68% yield) as a light yellow oil.

[0525] To a mixture of tert-butyl 4-(3-iodopropyl)piperidine-1-carboxylate (1.0 g, 2.83 mmol) in DMF (50 mL) was added diethyl phosphonate (0.58 g, 4.24 mmol) and Cs2CO3 (1.84 g, 5.66 mmol). The reaction mixture was stirred overnight at room temperature under nitrogen atmosphere and then terminated by the addition of water. The organic phase was washed with water, brine, dried (Na2SO4), and evaporated to dryness under reduced pressure. Chromatography (SiO2; 20% EtOAc in petroleum ether) afforded 80 (0.78 g, 76%) as a light yellow oil. LCMS: [M+H] + m / z 364.30.

[0526] To a solution of 80 (0.78 g, 2.14 mmol) in CH2Cl2 (8 mL) was added TFA (1.5 mL, 21.4 mmol). The mixture was stirred at room temperature for 4 hours and then evaporated to dryness under reduced pressure to give crude 81 as an oil, which was used in the next step without further purification. LCMS: [M+H] + m / z 264.25.

[0527] DIPEA (1.37 g, 10.63 mmol) was added to a solution of diethyl phosphonate (597 mg, 2.65 mmol) and crude 81 in CH2Cl2 (10 mL). The mixture was stirred at room temperature overnight and then quenched with saturated aqueous NH4Cl and extracted with CH2Cl2. The organic phase was washed with water, brine, dried (Na2SO4), and evaporated to dryness under reduced pressure. Chromatography (SiO2, 5% MeOH in CH2Cl2) gave the diethyl phosphonate (0.5 g, 39%) intermediate as a yellow oil. This was solvated in MeCN (10 mL) and TMSBr (1.46 mL, 11.07 mmol) was added. The resulting mixture was stirred at 60 °C for 6 h and then cooled to room temperature and evaporated to dryness under reduced pressure. Chromatography (preparative HPLC) gave 14 (220 mg, 50%) as a white solid. LCMS: [M+H] + m / z 396.20. 1 H NMR (400 MHz, methanol-d4) δ 8.51 (s, 1H), 7.33 (s, 1H), 7.14 (s, 1H), 4.02 (s, 3H), 3.97 (s, 3H), 3.49 (t, J = 12 Hz, 2H), 2.00-1.97 (m, 3H), 1.75-1.66 (m, 5H) and 1.45-1.37 (m, 5H).

[0528] Preparation of (2-(1-(6,7-dimethoxyquinazolin-4-yl)piperidin-4-yl)ethyl)phosphinothioate O,O-acid 17

[0529]

[0530] To a stirred solution of (2-(piperidin-4-yl)ethyl)phosphonothioic acid O,O-diethyl ester (400 mg, 1.51 mmol) and DIPEA (927 mg, 7.19 mmol) in DMSO (10 mL) was added 4-chloro-6,7-dimethoxy-quinazoline 66 (403 mg, 1.80 mmol). The reaction mixture was placed under a nitrogen atmosphere and then stirred at 80 °C for 16 hours. The reaction mixture was cooled to room temperature, diluted with water, and extracted with ethyl acetate. The organic phase was dried (Na2SO4) and evaporated to dryness under reduced pressure. Purification (SiO2, 0-100% EtOAc in hexanes) gave (2-(1-(6,7-dimethoxyquinazolin-4-yl)piperidin-4-yl)ethyl)phosphonothioic acid O,O-diethyl ester (380 mg, 46%). A stirred solution of (2-(1-(6,7-dimethoxyquinazolin-4-yl)piperidin-4-yl)ethyl)phosphinothioic acid O,O-diethyl ester (45 mg, 0.099 mmol) in TMSI (7 mL) was stirred at 60 ° C for 16 hours and then cooled to room temperature. The mixture was diluted with water and extracted with ethyl acetate. The organic phase was dried (Na2SO4) and then evaporated to dryness under reduced pressure. Purification by preparative HPLC gave (2-(1-(6,7-dimethoxyquinazolin-4-yl)piperidin-4-yl)ethyl)phosphinothioic acid O,O-acid 17 (13 mg, 32%) as a white solid. LCMS: [M+H] + m / z 396.25. 1 H NMR (400MHz, DMSO-d6) δ8.51(s,1H),7.33(s,1H),7.16(s,1H),4.02(s,3H),3.97(s,3H),3.58(d,J=10.4Hz,3H),3.48 (t,J=12.0Hz,2H),2.00(d,J=11.7Hz,2H),1.81(s,1H),1.64(d,J=17.9Hz,2H),1.61–1.51(m,2H),1.45–1.32(m,2H).

[0531] (2-(4-(6,7-dimethoxyquinazolin-4-yl)piperidin-1-yl)ethyl)phosphonic acid 18 (in Table 3a)

[0532]

[0533] LCMS: [M+H] + m / z 382.25 1H NMR(400MHz,D2O)δ8.65(s,1H),6.93(s,1H),6.76(s,1H),3.86(s,3H),3.84(s,3H),3.30 –3.22(m,1H), 3.18–3.15(m,2H), 2.73–2.66(m,2H), 2.37–2.32(m,2H) and 1.79–1.63(m,6H).

[0534] (2-(4-(6,7-dimethoxyquinazolin-4-yl)piperazin-1-yl)ethyl)phosphonic acid hydrobromide 19 (in Table 3a).

[0535]

[0536] a) iPrOH, Δ; b) H20, 50℃; c) TMSBr, CHCI s， DMF

[0537] A mixture containing pyrazine 82 and compound 63 in propan-2-ol was heated to reflux for 30 minutes and then cooled to room temperature. The reaction mixture was quenched with water and extracted into chloroform. The organic phase was separated, washed with water, brine, dried (Na2SO4), and evaporated to dryness under reduced pressure. Recrystallization from ether gave 83 as a white solid (1.65 g, 84%). Piperazine 83 (0.31 g, 1.1 mmol) was dissolved in water (20 mL) and vinyl phosphonate 84 (0.19 g, 1.2 mmol) was added. The resulting mixture was heated at 50°C for 1 hour and then cooled to room temperature. Extraction with chloroform, drying (Na2SO4), and evaporation to dryness under reduced pressure. Chromatography (SiO2 12 g; 15% MeOH in CH2Cl2) followed by recrystallization from ether gave the ethyl ester 85 (0.23 g; 49%) as a white solid. LCMS: [M+H] + m / z 410.10 1H NMR (500 MHz, chloroform-d) δ 8.67 (s, 1H), 7.25 (s, 1H), 7.09 (s, 1H), 4.01 (d, J = 18.8 Hz, 6H), 3.77 (d, J = 10.9 Hz, 6H), 3.68 (t, J = 4.9 Hz, 4H), 3.49 (s, 2H), 2.81-2.66 (m, 6H), 2.11-2.00 (m, 2H). Trimethylsilyl bromide (198 mg, 1.3 mmol) was added to a solution of 4-[4-(2-diethoxyphosphorylethyl)piperazin-1-yl]-6,7-dimethoxy-quinazoline 85 (600 mg, 3.8 mmol) in chloroform (20 mL) and DMF (5 mL). The resulting solution was stirred at room temperature for 3 hours and then quenched by the addition of methanol. The mixture was evaporated to dryness under reduced pressure and recrystallized from methanol-ether to give the desired product 19 (0.23 g, 89%) as HBr salt. LCMS: [M+H] + m / z 382.8. 1 H NMR(500MHz,DMSO-d6)δ8.97(s,1H),7.96(s,1H),7.42(s,1H),7.35(s,1H),4.02(s,3H),4. 00(s,3H),3.34(t,J=8.6Hz,2H),3.17(s,2H),2.90(s,2H),2.74(s,2H),2.20–2.08(m,2H).

[0538] Preparation of (4-(6,7-dimethoxyquinazolin-4-yl)phenethyl)phosphonic acid 20 (in Table 3a)

[0539]

[0540] a) nBuli, triisopropyl borate, THF; b) Pd(PPh3)4, K2CO3, H2O, THF, 65°C; c) PPh3, imidazole, I2, CH2Cl2; d) (BnO)2P(O)H, Cs2CO3, DMF; θ) Pd / C, H2, MeOH.

[0541] 2.5 M n-butyl lithium (24 mL) was added to a solution of 2-(4-bromophenyl)ethan-1-ol 86 (5.0 g, 24.8 mmol) in anhydrous THF (100 mL) at –78 °C under nitrogen atmosphere. After stirring for 1 h, triisopropyl borate (8.6 mL) was added to the mixture. The reaction mixture was stirred at room temperature for 1 h and then quenched by the addition of 2 M HCl solution (100 mL) and stirred for 1 h. The mixture was extracted with dichloromethane (3×100 mL), dried (Na2SO4), and evaporated to dryness under reduced pressure. Chromatography (SiO2; dichloromethane:methanol, 1:0 to 20:0) afforded boronic acid 87 (1.34 g, 33%) as a pale yellow solid. This material was then dissolved in a solution of THF (30 mL) and water (10 mL). 4-Chloro-6,7-dimethoxyquinazoline 63 (2.24 g, 10.0 mmol) and potassium carbonate (2.76 g, 20.0 mmol) were added to the solution followed by tetrakis(triphenylphosphine)palladium (0.5 g, 0.43 mmol). The resulting mixture was stirred at 65°C for 16 hours and then diluted with ethyl acetate, washed with brine, dried (Na2SO4), and evaporated to dryness under reduced pressure. Chromatography (SiO2: (0 to 10% methanol in dichloromethane) afforded 88 (1.43 g, 60%) as a pale yellow solid.

[0542] To a solution of triphenylphosphine (2.36 g, 9.0 mmol) in dichloromethane (24 mL) at 0°C was added imidazole (700 mg, 10.28 mmol). After stirring for 10 minutes, I2 (2.3 g, 9.0 mmol) was added. After stirring for another 10 minutes, a solution of compound 89 (1.5 g, 4.8 mmol) in dichloromethane (12 mL) was added. The mixture was warmed to room temperature and stirred for 5 hours. The mixture was then diluted with dichloromethane (36 mL), washed with brine, dried (Na2SO4), and evaporated to dryness under reduced pressure. Chromatography (SiO2: petroleum ether: ethyl acetate 10:1) gave 89 (4.0 g) as a colorless oil.

[0543] Cesium carbonate (1.426 g, 4.4 mmol) was added to a mixture of crude 89 (930 mg, 2.2 mmol) and dibenzyl phosphonate (884 mg, 3.37 mmol) in DMF (20 mL). The mixture was placed under a nitrogen atmosphere and stirred at room temperature for 3 hours. Once complete, the reaction mixture was filtered and evaporated to dryness under reduced pressure. Chromatography (C18 column: water: acetonitrile, 1:0 to 80:1) followed by lyophilization afforded the dibenzyl intermediate (750 mg, 79%) as an off-white solid. Dibenzyl (4-(6,7-dimethoxyquinazolin-4-yl)phenethyl)phosphonate (230 mg, 0.41 mmol) was dissolved in MeOH (20 mL). Pd / C (46 mg, 20% w / w) was added and the mixture was stirred at room temperature for 24 hours under a hydrogen atmosphere and then purified by Filtration. Chromatography (preparative HPLC under acidic conditions) gave compound 20 (55.4 mg, 36%) as a yellow solid. LCMS: [M+H] + m / z 375.0. 1 H NMR (400MHz, DMSO-d6)): δ9.09(s,1H),7.75-7.73(d,J=8.0Hz,2H),7.45–7.43(m,2H),7.41(s,1H) ,7.32(s,1H),4.08(s,1H),3.98(s,3H),3.91(s,1H),3.81(s,3H),2.89–2.87(m,3H) and 1.89(m,2H).

[0544] Synthesis of (4-((6,7-dimethoxyquinolin-4-yl)amino)phenyl)phosphonic acid sodium salt 21 (in Table 3a)

[0545]

[0546] A mixture of 4-chloro-6,7-dimethoxy-quinazoline 67 (0.67 g, 3.0 mmol) and diethyl (4-aminophenyl)phosphonate (0.69 g, 3.0 mmol) in iPrOH (10 mL) was heated to reflux overnight. The solid precipitate was filtered, washed with EtOAc and dried to give diethyl (4-((6,7-dimethoxyquinazoline-4-yl)amino)phenyl)phosphonate (0.92 g, 73% yield) as a white solid. The product was dissolved in MeCN (20 mL) and trimethylsilyl bromide (2.8 mL, 22 mmol) was added thereto. The resulting mixture was stirred at 60 °C for 6 h, cooled, and then evaporated to dryness under reduced pressure. The crude residue was terminated by the addition of saturated aqueous NaHCO3 solution (adjusted to pH 8). The resulting mixture was purified by preparative HPLC (under neutral conditions) and then lyophilized to give the desired product 21 (300 mg, 35% yield) as an off-white solid. LCMS: [M+H] + m / z 362.10. 1 H NMR (400MHz, D2O) δ7.73 (s, 1H), 7.53 (t, J = 9.8Hz, 2H), 7.22 (d, J = 7.4Hz, 2H), 6.39 (s, 1H), 6.16 (s, 1H) and 3.39 (s, 6H).

[0547] Synthesis of (4-((6,7-dimethoxyquinolin-4-yl)amino)benzyl)phosphonic acid sodium salt 22 (in Table 3a)

[0548]

[0549] A mixture of 4-chloro-6,7-dimethoxy-quinazoline 67 (0.34 g, 1.5 mmol) and diethyl (4-aminobenzyl)phosphonate (0.36 g, 3.0 mmol) in iPrOH (10 mL) was heated to reflux overnight. The precipitate was filtered, washed with EtOAc, evaporated to dryness under reduced pressure, and then dissolved in acetonitrile (20 mL). Trimethylsilyl bromide (0.58 mL, 4.6 mmol) was added thereto. The mixture was stirred at 60 °C for 6 hours. After concentration, the residue was treated with saturated aqueous NaHCO3 solution until the solution reached a pH of 8. The mixture was purified by preparative HPLC (neutral) to give 22 (104 mg, 57%) as an off-white solid. LCMS: [M+H] + m / z 376.10. 1H NMR (400MHz, D2O) δ7.77 (s, 1H), 7.15 (s, 4H), 6.49 (s, 1H), 6.21 (s, 1H), 3.47 (s, 6H) and 2.70 (d, J = 19.5Hz, 2H).

[0550] (4-(((6,7-dimethoxyquinolin-4-yl)amino)methyl)phenyl)phosphonic acid sodium salt 23 (also referred to as 4 in Table 1).

[0551]

[0552] a) iPrOH, Δ; b) Et3N, KOAc, Pd(OAc)2, dppf, (EtO)2PH, THF; c) TMSBr, MeCN, 60°C.

[0553] A mixture of 4-chloro-6,7-dimethoxy-quinazoline 63 (0.93 g, 4.14 mmol) and (4-bromophenyl)methanamine 90 (0.77 g, 4.14 mmol) in iPrOH (10 mL) was heated to reflux overnight. The solid precipitate was filtered; washed with ethyl acetate and evaporated to dryness under reduced pressure to give N-(4-bromobenzyl)-6,7-dimethoxyquinazoline-4-amine hydrochloride 91 (1.5 g, 88%) as a white solid. Triethylamine (0.37 mL, 2.68 mmol) was added to a mixture of KOAc (11 mg, 0.112 mmol), Pd(OAc)2 (5.5 mg, 0.025 mmol), dppf (27 mg, 0.049 mmol) in THF (10 mL) and purged with nitrogen. Triethylamine (0.37 mL, 2.68 mmol) was added. After stirring at 70°C for 15 minutes, a solution of N-(4-bromobenzyl)-6,7-dimethoxyquinazolin-4-amine hydrochloride (0.5 g, 1.22 mmol) and diethyl phosphonate (0.16 g, 1.22 mmol) in THF (10 mL) was added. The reaction was stirred at reflux for 6 hours and then partitioned between EtOAc (30 mL) and water (20 mL). The organic phase was separated, washed with water, brine, dried (Na2SO4), and evaporated to dryness under reduced pressure. Purification by column chromatography (SiO2; 50% petroleum ether in ethyl acetate) gave diethyl (4-(((6,7-dimethoxyquinazolin-4-yl)amino)methyl)phenyl)phosphonate (0.2 g, 38%) as a yellow solid.

[0554] To a solution of diethyl (4-(((6,7-dimethoxyquinazolin-4-yl)amino)methyl)phenyl)phosphonate (0.5 g, 1.16 mmol) in MeCN (20 mL) was added TMSBr (1.45 mL, 11.5 mmol). The mixture was stirred at 60 °C for 6 h, cooled to room temperature, and then evaporated under reduced pressure. The residue was quenched with saturated aqueous NaHCO3 solution (pH 9), and the resulting mixture was purified by preparative HPLC (neutral) to give the title product 23 as an off-white solid (102 mg, 22%). LCMS: [M–H] + m / z: 374.00. 1 H NMR (400MHz, D2O) δ8.00(s,1H),7.61(s,2H),7.29(d,J=7.6Hz,2H),6.70(s,1H),6.55(s,1H),4.62(s,2H),3.75(d,J=18.2Hz,6H).

[0555] General procedure for the synthesis of dimethyl (2-(piperidin-4-yl)ethyl)phosphonate 92 and diethyl (2-(piperidin-4-yl)ethyl)phosphonate 93

[0556]

[0557] a) NaH, PhMe; b) Pd / C, H2, EtOH.

[0558] Sodium hydride (1.1 mol. equiv) was carefully added to a stirred toluene solution of bis(dimethoxyphosphoryl)methane 92 or bis(diethoxyphosphoryl)methane 93 (1 mol. equiv) at room temperature. The reaction mixture was then placed under a nitrogen atmosphere and a toluene solution of 1-benzylpiperidine-4-carboxaldehyde 94 (1 mol. equiv) was slowly added, keeping the temperature below 40°C. The resulting mixture was stirred at room temperature for 16 hours and then quenched by the addition of saturated aqueous NH4Cl. The organic phase was separated, washed with brine, dried (MgSO4) and evaporated to dryness. Chromatography (120 g SiO2; 5 to 100% gradient of EtOAc in hexanes) gave dimethyl (E)-(2-(1-benzylpiperidin-4-yl)vinyl)phosphonate or diethyl (E)-(2-(1-benzylpiperidin-4-yl)vinyl)phosphonate as a colorless oil. To a mixture of dimethyl (E)-(2-(1-benzylpiperidin-4-yl)vinyl)phosphonate or diethyl (E)-(2-(1-benzylpiperidin-4-yl)vinyl)phosphonate (1 mol. equiv.) in ethanol was added a catalytic amount of Pd / C. The mixture was placed under a hydrogen atmosphere and stirred at room temperature for 12 h, filtered, and evaporated to dryness under reduced pressure to give dimethyl (2-(piperidin-4-yl)ethyl)phosphonate 95 and diethyl (2-(piperidin-4-yl)ethyl)phosphonate 96 as colorless oils.

[0559] General Procedure for the Synthesis of Dibenzyl (2-(Piperidin-4-yl)ethyl)phosphonate 100

[0560]

[0561] a) PPh3, I 2. Imidazole, CH2Cl2; b) P(O)OBn2, DBU, MeCN; c) TFA, CH2Cl2

[0562] Iodine (1.5 mol. equivalent) was added to a CH2Cl2 solution of PPh3 (1.5 mol. equivalent) and imidazole (1.5 mol. equivalent). After stirring for 10 minutes, a CH2Cl2 solution of 97 (1.0 mol. equivalent) was added dropwise. The mixture was stirred at room temperature for 2 hours. The mixture was filtered through a pad of Celite® and treated with 5% sodium thiosulfate solution. The mixture was extracted with ethyl acetate, washed with brine, dried (Na2SO4), and evaporated to dryness under reduced pressure. Chromatography afforded 98 as an oil.

[0563] DBU (5.0 mol. equiv.) was added to a MeCN solution of compound 98 (3.0 mol. equiv.) at 40°C. After stirring for 10 minutes, a MeCN solution of dibenzyl phosphonate (1.0 mol equiv.) was added dropwise. After stirring for 2 hours, the reaction mixture was evaporated to dryness under reduced pressure and purified by chromatography to give 99.

[0564] A solution of compound 99 (1.0 mol. equiv.) in TFA / DCM was stirred at room temperature for 1 hour and then evaporated to dryness under reduced pressure to afford 100 as an oil.

[0565] General method for synthesizing (2-(1-(quinazolin-4-yl)piperidin-4-yl)ethyl)phosphonic acid, (2-(1-(quinolin-4-yl)piperidin-4-yl)ethyl)phosphonic acid and (2-(1-(isoquinolin-1-yl)piperidin-4-yl)ethyl)phosphonic acid

[0566] Method A:

[0567] Diisopropylethylamine (2 mol. equiv) is added to a mixture of dimethyl (2-(piperidin-4-yl)ethyl)phosphonate 95 or diethyl (2-(piperidin-4-yl)ethyl)phosphonate 96 (1.1 mol. equiv) and 4-chloroquinazoline, 4-chloroquinoline or 1-chloroisoquinoline (1 mol. equiv) in isopropanol (0.1 M reaction concentration). After stirring at 90° C. for 3 hours, the reaction mixture is cooled and evaporated to dryness. Purification on silica gel (5% MeOH in dichloromethane) gives the dimethyl or diethyl phosphonate. To a cooled (0° C.) solution of the phosphonate (1 mol. equiv) in chloroform or dichloromethane (0.5 M reaction concentration) is added trimethylsilyl bromide (3 mol. equiv). The reaction mixture is warmed to room temperature and, after 90 minutes, quenched by the addition of methanol. The mixture is evaporated to dryness under reduced pressure and then solvated in methanol. The reaction mixture was concentrated to half volume, filtered to remove the precipitate, and then evaporated to dryness. The residue was crystallized from dichloromethane, filtered and dried under reduced pressure to give the desired hydrobromide salt of the phosphonic acid.

[0568] Method B:

[0569] Diisopropylethylamine (3 mol. equiv.) is added to a mixture of dimethyl (2-(piperidin-4-yl)ethyl)phosphonate 95 or diethyl (2-(piperidin-4-yl)ethyl)phosphonate 96 (1.1 mol. equiv.) and 4-chloroquinazoline, 4-chloroquinoline or 1-chloroisoquinoline (1 mol. equiv.) in dichloromethane (0.1 M reaction concentration). After stirring overnight at room temperature, the reaction mixture is quenched by the addition of saturated aqueous NH4Cl solution. The organic phase is separated and washed with water and brine, dried (Na2SO4), and evaporated to dryness under reduced pressure. Purification on silica gel (5% MeOH in dichloromethane) gives the dimethyl or diethyl phosphonate. To a cooled (0°C) solution of dimethyl or diethyl phosphonate (7 mol. equiv.) in acetonitrile (0.1 M reaction concentration) is added trimethylsilyl bromide (3 mol. equiv.). The reaction mixture was stirred at 60° C. for 6 hours, cooled, and evaporated to dryness under reduced pressure, and the crude residue was quenched by addition of saturated aqueous NaHCO3 solution (until pH 8 to 9 was observed). The crude residue was purified by preparative HPLC (neutral) to give the sodium salt of the phosphonic acid.

[0570] Method C:

[0571] Diisopropylethylamine (3 mol. equiv.) was added to a mixture of dibenzyl (2-(piperidin-4-yl)ethyl)phosphonate 100 (1.1 mol. equiv.) and 4-chloroquinazoline, 4-chloroquinoline or 1-chloroisoquinoline (1 mol. equiv.) in dichloromethane (0.1 M reaction concentration). After stirring overnight at room temperature, the reaction mixture was quenched by the addition of saturated aqueous NH4Cl solution. The organic phase was separated and washed with water and brine, dried (Na2SO4), and evaporated to dryness under reduced pressure. Purification on silica gel (5% MeOH in dichloromethane) gave dibenzyl phosphonate. A mixture of dibenzyl phosphonate (1 mol. equiv.) and Pd / C in MeOH was placed under a hydrogen atmosphere and stirred for 2 hours at room temperature. The mixture was then passed through Filtration and evaporation to dryness under reduced pressure gave the phosphonic acid.

[0572] Preparation of (2-(1-(6,7-dimethoxyquinazolin-4-yl)piperidin-4-yl)ethyl)phosphonic acid (or compound 1)

[0573]

[0574] Prepared according to method A to give 15 (2.1 g, 69%) as an off-white solid. LCMS: [M+H] + m / z 381.8. 1HNMR(500MHz,DMSO-d6)δ8.77(s,1H),7.34(s,1H),7.23(s,1H),4.71(d,J=13.1Hz,2H),3.99(s,3H),3. 97(s,3H),3.48(t,J=12.7Hz,2H),3.18(s,1H),1.97–1.90(m,2H),1.62–1.43(m,4H),1.40–1.27(m,2H).

[0575] Preparation of (4-(((6,7-dimethoxyquinazolin-4-yl)amino)methyl)benzyl)phosphonic acid 24 (also referred to as 5 in the table herein)

[0576]

[0577] Prepared according to method B. The product was isolated by preparative HPLC (9% yield) as an off-white solid. LCMS: [M+H] + m / z 390.15. 1 H NMR(400MHz,D2O)δ8.12(s,1H),7.22(s,4H),7.11(s,1H),6.91(s,1H),4. 79(s,2H),4.76(s,2H),3.98(s,3H),3.91(s,3H),2.79(s,1H),2.74(s,1H)

[0578] Preparation of (2-(1-(6-methoxyquinazolin-4-yl)piperidin-4-yl)ethyl)phosphonic acid 25

[0579]

[0580] Prepared according to method A to give 25 (50% yield) as an off-white solid. LCMS: [M+H] + m / z 352.10. 1 H NMR (400 MHz, methanol-d4) δ 8.57 (s, 1H), 7.74–7.73 (m, 1H), 7.68–7.66 (m, 1H), 7.46 (d, 1H), 4.96 (br s, 2H), 3.98, (s, 3H), 3.57 (br s, 2H), 2.65 (s, 2H), 2.07–2.04 (m, 2H), 1.81 (m, 1H), 1.79–1.75 (m, 2H), 1.66–1.63 (m, 2H) and 1.46–1.44 (m, 2H).

[0581] Preparation of (2-(1-(6-hydroxyquinazolin-4-yl)piperidin-4-yl)ethyl)phosphonic acid 26

[0582]

[0583] Prepared according to method C to give 26 (7% yield) as an off-white solid. LCMS: [M+H] + m / z 338.15. 1 H NMR (400MHz, DMSO-d6) δ8.48(s,1H),7.65(d,J=8.8Hz,1H),7.32(d,J=8.8Hz,1H),7.18(s,1H), 4.21–4.17(m,2H), 2.99–2.95(m,2H), 1.82–1.79(m,2H), 1.53–1.49(m,5H) and 1.30–1.19(m,2H).

[0584] Preparation of (2-(1-(7-methoxyquinazolin-4-yl)piperidin-4-yl)ethyl)phosphonic acid 27

[0585]

[0586] Prepared according to method A to give 27 (95% yield) as an off-white solid. LCMS: [M+H] + m / z 352.0 1 H NMR (500 MHz, methanol-d4) δ 8.55 (s, 1H), 8.10 (d, J = 10 Hz, 1H), 7.30 (dd, J = 10 Hz and 5 Hz, 1H), 7.10 (d, J = 5 Hz, 1H), 4.01 (s, 3H), 3.57–3.48 (m, 2H), 2.65 (s, 1H), 2.05–2.02 (m, 2H), 1.94–1.90 (m, 1H), 1.80–1.74 (m, 2H), 1.65–1.60 (m, 2H) and 1.46–1.41 (m, 2H).

[0587] Preparation of (2-(1-(7-ethoxyquinazolin-4-yl)piperidin-4-yl)ethyl)phosphonic acid 28

[0588]

[0589] Prepared according to method B to give 28 as an off-white solid.

[0590] Preparation of (2-(1-(7-hydroxyquinazolin-4-yl)piperidin-4-yl)ethyl)phosphonic acid 29

[0591]

[0592] Prepared according to method B to give 29 (4% yield) as a light yellow solid. LCMS: [M+H] + m / z 338.25. 1 H NMR (400MHz, DMSO-d6) δ11.49(s,1H),8.64(s,1H),7.96(d,J=9.2Hz,1H),7.10(dd,J=9.2and2.2Hz,1H),7.00(d,J=2.2Hz,1H),4.62(br s,2H),3.38(br s,2H),1.87(d,J=12.7Hz,2H),1.72(br s,1H),1.58–1.38(m,4H) and 1.28–1.22(m,2H).

[0593] Preparation of (2-(1-(7-aminoquinazolin-4-yl)piperidin-4-yl)ethyl)phosphonic acid 30

[0594]

[0595] 30 was prepared according to method C (32% yield) as a light yellow solid. LCMS: [M+H] + m / z 337.10. 1 HNMR (400MHz, DMSO-d6) δ8.35(s,1H),7.62(d,J=8.8Hz,1H),6.81(d,J=8.8Hz,1H),6.61(br s,1H),6.30(br s,2H), 4.26–4.20(m,2H), 3.10–2.90(m,2H), 1.79–1.76(m,2H), 1.60–1.30(m,5H) and 1.25–1.20(m,2H).

[0596] Preparation of (2-(1-(7-isopropoxyquinazolin-4-yl)piperidin-4-yl)ethyl)phosphonic acid 31

[0597]

[0598] 31 was prepared according to method B as a light yellow solid. LCMS: [M+H] + m / z 337.10. 1H NMR (400MHz, DMSO-d6) δ8.35(s,1H),7.62(d,J=8.8Hz,1H),6.81(d,J=9.2Hz,1H),6.66(s,1H),6.30(br s,2H), 4.25–4.21(m,2H), 3.08–2.96(m,2H), 1.81–1.75(m,2H), 1.65–1.31(m,5H) and 1.27–1.18(m,2H).

[0599] Preparation of (2-(1-(8-methoxyquinazolin-4-yl)piperidin-4-yl)ethyl)phosphonic acid 32

[0600]

[0601] Prepared according to method B to give 32 (32% yield) as an off-white solid. LCMS: [M+H] + m / z 352.15. 1 HNMR(400MHz,DMSO-d6)δ7.92(s,1H),6.92–6.88(m,1H),6.80(d,J=7.6Hz,1H),6.71(d,J=8.4Hz,1H),3.76 –3.70(m,2H),3.71(s,3H),2.72(t,J=12Hz,2H),1.64(d,J=12Hz,2H),1.51–1.28(m,5H) and 1.02–0.94(m,2H).

[0602] Preparation of (2-(1-(8-ethoxyquinazolin-4-yl)piperidin-4-yl)ethyl)phosphonic acid 33 (also referred to as 226 in the tables herein)

[0603]

[0604] Prepared according to method B to give 33 as a white solid. LCMS: [M+H] + m / z 366.20. 1 H NMR (400 MHz, DMSO-d6)δ 1 H NMR(400MHz,D2O)δ8.17(s,1H),7.21–7.09(m,2H),4.13–3.98(m,4H),2.97(t,J =12.4Hz,2H),1.82(d,J=13.0Hz,2H),1.56–1.35(m,8H),1.26(q,J=11.4Hz,2H).

[0605] Preparation of (2-(1-(8-isopropoxyquinazolin-4-yl)piperidin-4-yl)ethyl)phosphonic acid 34 (in Table 3a)

[0606]

[0607] Prepared according to method B to give 34 (43% yield) as an off-white solid. LCMS: [M+H] + m / z 1 H NMR (400 MHz, DMSO-d6)δ 1 H NMR(400MHz,D2O)δ8.10(s,1H),7.11(d,J=7.5Hz,2H),7.03(d,J=7.1Hz,1H),3.94(d ,J=13.0Hz,2H),2.86(t,J=12.6Hz,2H),1.67(d,J=13.1Hz,2H),1.40–1.07(m,13H).

[0608] Preparation of (2-(1-(8-hydroxyquinazolin-4-yl)piperidin-4-yl)ethyl)phosphonic acid 36 (in Table 3a)

[0609]

[0610] Prepared according to Method B to give 35 as an off-white solid.

[0611] LCMS: [M+H] + m / z 1 H NMR (400 MHz, DMSO-d6)δ 1 H NMR (400MHz, D2O)

[0612] Preparation of (2-(1-(5,8-dimethoxyquinazolin-4-yl)piperidin-4-yl)ethyl)phosphonic acid 36

[0613]

[0614] Prepared according to method B to give 36 as an off-white solid. LCMS: [M+H] + m / z 382.15. 1 H NMR (400MHz, DMSO-d6) δ8.47(s,1H),7.54(d,J=8.9Hz,1H),7.15(d,J=8.8Hz,1H), 3.95(d,J=12.0Hz,8H),1.82(s,2H),1.67(s,1H),1.59–1.30(m,6H),1.21(s,2H).

[0615] Preparation of (2-(1-(6,8-dimethoxyquinazolin-4-yl)piperidin-4-yl)ethyl)phosphonic acid 37

[0616]

[0617] Prepared according to method C to give 37 (15% yield) as an off-white solid. LCMS: [M+H] + m / z 382.15 1 H NMR (400MHz, DMSO-d6) δ8.54(s,1H),7.11(s,1H),6.86-6.82(m,1H),4.50(d,J=12.4Hz,1H),4.27(m,1H) ,3.85(m,6H),3.74(m,2H),3.27(m,2H),1.88–1.85(m,2H),1.66(m,1H),1.54–1.48(m,4H) and 1.29(m,2H).

[0618] Preparation of (2-(1-(7,8-dimethoxyquinazolin-4-yl)piperidin-4-yl)ethyl)phosphonic acid 38

[0619]

[0620] Prepared according to method C to give 38 (16% yield) as an off-white solid. LCMS: [M+H] + m / z 382.15 1 HNMR(400MHz,DMSO-d6)δ8.60(s,1H),7.90(d,J=9.6Hz,1H),7.49(d,J=9.2Hz,1H),4.69(m,2H),4.02(s, 3H), 3.89 (s, 3H), 3.46 (m, 2H), 1.90 (d, J = 12.8Hz, 2H), 1.75 (m, 1H), 1.53–1.49 (m, 4H) and 1.31–1.28 (m, 2H).

[0621] Preparation of (2-(1-(7,8-dihydro-[1,4]dioxino[2,3-g]quinazolin-4-yl)piperidin-4-yl)ethyl)phosphonic acid 39 (in Table 3a)

[0622]

[0623] Prepared according to method C to give 39 (7% yield) as an off-white solid. LCMS: [M+H] + m / z 380.15. 1H NMR (400MHz, DMSO-d6) δ8.64(s,1H),7.48(s,1H),7.19(s,1H),4.56(d,J=11.8Hz,2H),4.45(d,J=3.0Hz,2H),4.38(d,J =3.3Hz,2H),3.39(s,1H),3.33(s,1H),1.87(d,J=12.2Hz,2H),1.71(s,1H),1.58–1.38(m,4H),1.26(d,J=10.2Hz,2H).

[0624] Preparation of (2-(1-(5-fluoro-8-methoxyquinazolin-4-yl)piperidin-4-yl)ethyl)phosphonic acid 40 (in Table 3a)

[0625]

[0626] 40 was prepared according to method C (7% yield) as a light yellow solid. LCMS: [M+H] + m / z 370.10. 1 H NMR(400MHz,DMSO-d6)δ8.55(s,1H),7.44–7.38(m,2H),4.28–4.23(m,2H),3.94(s,3H),3.2 8–3.18(m,2H), 1.82–1.78(m,2H), 1.70–1.66(m,1H), 1.49–1.23(m,4H) and 1.26–1.09(m,2H).

[0627] Preparation of (2-(1-(6-fluoro-8-methoxyquinazolin-4-yl)piperidin-4-yl)ethyl)phosphonic acid 41 (in Table 3a)

[0628]

[0629] Prepared according to method B to give 41 (44% yield) as a white solid. LCMS: [M+H] + m / z 366.15. 1 H NMR (400 MHz, DMSO-d6) δ 8.02 (d, J = 9.2 Hz, 1H), 7.21 (d, J = 9.2 Hz, 1H), 7.04 (s, 1H), 3.93 (s, 3H), 2.49 (s, 3H), 1.91-1.88 (m, 2H), 1.74 (m, 1H), 1.53-1.49 (m, 5H) and 1.29-1.27 (m, 3H).

[0630] Preparation of (2-(1-(6-chloro-8-methoxyquinazolin-4-yl)piperidin-4-yl)ethyl)phosphonic acid 42 (in Table 3a)

[0631]

[0632] Prepared according to method B to give 42 (42% yield) as a light yellow solid. LCMS: [M+H] + m / z 386.10. 1 HNMR(400MHz,D2O)δ8.03(s,1H),6.91–6.88(m,2H),3.92–3.89(m,2H),3.77(s,3H),2.93–2.87(m,2H),1.70–1.67(m,2H) and 1.41–1.12(m,7H).

[0633] Preparation of (2-(1-(7-chloro-8-methoxyquinazolin-4-yl)piperidin-4-yl)ethyl)phosphonic acid 43 (in Table 3a)

[0634]

[0635] Prepared according to method B to give 43 (50% yield) as a white solid. LCMS: [M+H] + m / z 386.05. 1 H NMR(400MHz,D2O)δ8.07(s,1H),7.10–7.06(m,2H),3.95–3.91(m,2H),3.71(s,3H),2.96–2.90(m,2H),1.71–1.68(m,2H) and 1.42–1.01(m,7H).

[0636] Preparation of 2-[1-[6,7-dimethoxy-2-[(E)-2-(3-pyridyl)vinyl]quinazolin-4-yl]-4-piperidinyl]ethyl-hydroxy-phosphinic acid 44

[0637]

[0638] Prepared according to method B to give 44 (49% yield) as a light yellow solid. LCMS: [M+H] + m / z 485.25. 1HNMR(400MHz,D2O)δ8.09(s,1H),7.94(s,1H),7.36(d,J=8Hz,1H),7.06(s,1H),6.74(d,J=16.8Hz 1H),6.58(d,J=3.2Hz,1H),6.40(d,J=3.2Hz,1H),6.24(d,J=16.8Hz,1H),3.94–3.91(m, 2H), 3.84(s,3H), 3.67(s,3H), 2.96–2.90(m,2H), 1.96–1.93(m,2H) and 1.56–1.32(m,7H).

[0639] Preparation of (E)-(2-(1-(8-methoxy-2-(2-(pyridin-3-yl)vinyl)quinazolin-4-yl)piperidin-4-yl)ethyl)phosphonic acid 45

[0640]

[0641] Prepared according to method B to give 45 (79% yield) as a yellow solid. LCMS: [M+H] + m / z 455.20. 1 H NMR (400 MHz, methanol-d4) δ 9.07 (br s, 1H), 8.70 (br s, 1H), 8.62–8.60 (m, 1H), 8.31 (d, 1H), 7.89–7.88 (m, 1H), 7.70–7.54 (m, 4H), 5.20–5.00 (m, 2H) 4.13 (s, 3H), 3.58–3.50 (m, 2H), 2.07–2.02 (m, 2H), 1.88–1.82 (m, 1H), 1.78–1.64 (m, 4H) and 1.51–1.46 (m, 2H).

[0642] Preparation of (2-(1-(6,7-dimethoxyquinolin-4-yl)piperidin-4-yl)ethyl)phosphonic acid 46

[0643]

[0644] Prepared according to method C to give 46 (22% yield) as a white solid. LCMS: [M+H] + m / z 381.30. 1H NMR (400 MHz, methanol-d4) δ 8.35 (d, J = 6.8 Hz, 1H), 7.29-7.27 (m, 2H), 7.11 (d, J = 6.8 Hz, 1H), 4.27-4.23 (m, 2H), 4.03 (s, 3H), 4.02 (s, 3H), 3.40-3.32 (m, 2H), 2.06-2.03 (m, 4H) 1.82-1.79 (m, 3H) and 1.62-1.48 (m, 2H).

[0645] Preparation of (2-(1-(3-cyano-6,7-dimethoxyquinolin-4-yl)piperidin-4-yl)ethyl)phosphonic acid 47 (also referred to as 42 in Table 2)

[0646]

[0647] Prepared according to method B to give 47 (47% yield) as an off-white solid. LCMS: [M+H] + m / z 406.20. 1 HNMR(400MHz,D2O)δ8.00(s,1H),6.62(s,1H),6.40(s,1H),3.75(s,3H),3.66(s,3H),3.18(d,J=1 2.3Hz,2H),2.94(t,J=12.2Hz,2H),1.72(d,J=12.7Hz,2H),1.43–1.30(m,6H),1.17–1.04(m,2H).

[0648] Preparation of (2-(1-(3-cyano-6-methoxyquinolin-4-yl)piperidin-4-yl)ethyl)phosphonic acid 48 (also referred to as 44 in Table 2)

[0649]

[0650] Prepared according to method B to give 48 (16% yield) as an off-white solid. LCMS: [M+H] + m / z 376.20. 1 HNMR(400MHz,D2O)δ8.15(s,1H),7.51(s,1H),7.18(s,1H),6.88(s,1H),3.71(s, 3H), 3.60–3.51(m,2H), 3.15–3.08(m,2H), 1.81–1.74(m,2H) and 1.41–1.15(m,7H).

[0651] Preparation of (2-(1-(3-cyano-7-methoxyquinolin-4-yl)piperidin-4-yl)ethyl)phosphonic acid 49 (also referred to as 43 in Table 2)

[0652]

[0653] Prepared according to method B to give 49 (23% yield) as an off-white solid. LCMS: [M+H] + m / z 376.20. 1 HNMR(400MHz,D2O)δ7.94(s,1H),7.39(d,J=9.4Hz,1H),6.84–6.64(m,2H),3.90(s,3H),3.59(d,J =12.4Hz,2H), 3.22(t,J=12Hz,2H), 1.89(d,J=12.8Hz,2H), 1.62–1.45(m,5H) and 1.33–1.25(m,2H).

[0654] Preparation of (2-(1-(3-cyano-8-methoxyquinolin-4-yl)piperidin-4-yl)ethyl)phosphonic acid 50 (also referred to as 45 in Table 1)

[0655]

[0656] 50 was prepared according to method B as an off-white solid. LCMS: [M+H] + m / z 376.20. 1 H NMR(400MHz,D2O)δ8.03(s,1H),7.27–7.23(m,1H),7.11–7.09(m,1H),7.04–7.02(m,1H),3.90(s,3H),3.43(br d,J=12.4Hz,2H),3.06(br t,J=12Hz,2H), 1.80(br d,J=12.8Hz,2H), 1.50–1.47(m,5H) and 1.31–1.24(m,2H).

[0657] Preparation of (2-(1-(6,7-dimethoxyisoquinolin-1-yl)piperidin-4-yl)ethyl)phosphonic acid 51

[0658]

[0659] Prepared according to method B to give 51 (30% yield) as an off-white solid. LCMS: [M+H] + m / z 381.10. 1 HNMR (400MHz, D2O)δ 1H NMR (400MHz, DMSO-d6): δ7.79(d,J=6.4Hz,1H),7.51–7.44(m,2H),7.31(s,1H),3.9 6(d,J=4.8Hz,6H), 3.23(m,4H), 1.88(m,2H), 1.55(m,2H), 1.48(m,1H) and 1.45(m,4H).

[0660] Preparation of (2-(1-(4-cyano-6,7-dimethoxyisoquinolin-1-yl)piperidin-4-yl)ethyl)phosphonic acid 52 (in Table 3a)

[0661]

[0662] Prepared according to method B to give 52 (50% yield) as an off-white solid. LCMS: [M+H] + m / z 1 H NMR (400MHz, D2O)δ

[0663] O-((1-(8-methoxyquinazolin-4-yl)piperidin-4-yl)methyl)O,O-dihydrogenphosphorothioate 53 (in

[0664] Preparation of Table 3a)

[0665]

[0666] To a solution of (1-(8-methoxyquinazolin-4-yl)piperidin-4-yl)methanol (prepared using the same method as compound 80) (500 mg, 1.83 mmol) in pyridine (5 mL) was added trichlorothiophosphorus (1.6 g, 9.45 mmol) dropwise at -15 °C. After stirring at 0 °C for 1 hour, the reaction mixture was added to a solution of sodium bicarbonate (923 mg, 10.98 mmol) in water (20 mL) at 0 °C. The resulting mixture was stirred at 0 °C for 2 hours and then evaporated to dryness under reduced pressure. Purification (preparative HPLC) gave 53 (83 mg, 12%) as a white solid. LCMS: [M+H] + m / z 354.10 1 H NMR(400MHz,,DMSO-d6)δ8.59(s,1H),7.59–7.50(m,2H),7.45(dd,J=6.5,2.4Hz,1H),4.53(d,J=12.7 Hz,2H),3.97(s,3H),3.80–3.74(m,4H),2.03(s,1H),1.86(d,J=13.5Hz,2H),1.41(q,J=11.8Hz,2H).

[0667] Preparation of (((1-(8-methoxyquinazolin-4-yl)piperidin-4-yl)oxy)methyl)phosphonic acid 54 (in Table 3a)

[0668]

[0669] Prepared using the same procedure as compounds 10 and 11. The mixture was purified (preparative HPLC 0.1% TFA) to afford 54 as an off-white solid.

[0670] LCMS: [M+H] + m / z 353.3. 1 H NMR(400MHz,D2O)δ8.40(s,1H),7.54(s,2H),7.40(d,J=7.0Hz,1H),4.37(s,3H), 4.01–3.88(m,9H),3.71(d,J=9.3Hz,4H),3.63(s,1H),2.11(s,2H),1.81(s,2H).

[0671] Preparation of (4-(8-methoxyquinazolin-4-yl)phenethyl)phosphonic acid 55 (in Table 3a)

[0672]

[0673] Prepared using the same method as compound 20 as a white solid.

[0674] LCMS: [M+H] + m / z 345.10. 1 H NMR (400MHz, D2O)δ

[0675] Preparation of (4-(((8-methoxyquinazolin-4-yl)amino)methyl)phenyl)phosphonic acid 56

[0676]

[0677] Prepared according to the same method as compound 23. LCMS: [M+H] + m / z 346.10. 1 H NMR (400 MHz, D2O) δ 8.20 (s, 1H), 7.62–7.57 (m, 2H), 7.43–7.41 (m, 2H), 7.31–7.28 (m, 2H), 7.23–7.21 (m, 1H) and 2.93 (s, 3H).

[0678] Preparation of (4-(((3-cyano-8-methoxyquinolin-4-yl)amino)methyl)phenyl)phosphonic acid 57 (also referred to as 52 in Table 1)

[0679]

[0680] Prepared according to the same method as compound 23, 57 was obtained as an off-white solid. LCMS: [M+H] + m / z 370.10. 1 H NMR (400 MHz, D2O) δ 8.02 (s, 1H), 7.48–7.43 (m, 2H, 7.36–7.30 (m, 2H), 7.15–7.09 (m, 3H), 4.77 (s, 2H) and 3.81 (s, 3H).

[0681] Preparation of (4-(((3-cyano-8-methoxyquinolin-4-yl)amino)methyl)benzyl)phosphonic acid 58 (also referred to as 211 in Table 2)

[0682]

[0683] Prepared according to the same method as compound 23, 58 was obtained as a white solid. LCMS: [M+H] + m / z 384.15. 1 H NMR (400 MHz, methanol-d4) δ 8.32 (s, 1H), 7.77 (d, J = 8.4 Hz, 1H), 7.50 (t, J = 8.4 Hz, 1H), 7.35-7.33 (m, 2H), 7.26 (d, J = 7.6 Hz, 1H), 7.20 (d, J = 8.4 Hz, 2H), 5.02 (s, 2H), 3.99 (s, 3H) and 2.85 (d, J = 20 Hz, 2H).

[0684] Preparation of (3-(1-(3-cyano-8-methoxyquinolin-4-yl)piperidin-4-yl)propyl)phosphonic acid 59 (also referred to as 210 in Table 2)

[0685]

[0686] Prepared according to the same method as compound 14, 59 was obtained as a white solid. LCMS: [M+H] + m / z 390.20. 1 H NMR(400MHz,D2O)δ8.30(s,1H),7.39(br s,2H),7.19(br s,1H),3.98(s,3H),3.73–3.70(m,2H),3.30(t,J=12Hz,2H),1.92–1.88(m,2H),1.70–1.45(m,3H) and 1.40–1.27(m,6H).

[0687] Preparation of (4-(((3-cyano-8-methoxyquinolin-4-yl)amino)methyl)phenyl)boronic acid 60 (also referred to as 214 in Table 2)

[0688]

[0689] a) Et3N, CH3OCH2CH2OH; b) PdCl2 (dppf), KOAc, B2Pin2, DMSO, 80℃; c) HCl, EtOAc

[0690] To a solution of compound 101 (0.97 g, 5.0 mmol) in 2-methoxyethanol (10 mL) was added (4-bromophenyl)methylamine 90 (1.74 g, 10.0 mmol) and Et3N (1.51 g, 15 mmol). The mixture was heated at 100°C overnight, cooled to room temperature, and then evaporated to dryness under reduced pressure. Chromatography (35% EtOAc in petroleum ether) afforded 102 (1.5 g, 88%) as a white solid. To a solution of compound 102 (69 mg, 0.2 mmol) in DMSO (3 mL) was added bis(pinacolato)diboron (61.0 mg, 0.24 mmol), potassium acetate (58.8 mg, 3.0 mmol), Pd(dppf)Cl2 (7.4 mg, 0.05 mmol). The reaction was degassed by purging with nitrogen and then heated at 80°C for 48 hours. The mixture was cooled to room temperature and diluted with ethyl acetate and then washed with The product was filtered through a pad of 4% paraffin wax. The filtrate was evaporated to dryness under reduced pressure. The residue was dissolved in EtOAc (10 mL) and a solution of HCl (4M, 0.2 mL, 4.0 mmol) in EtOAc was added. The mixture was stirred at room temperature overnight and then evaporated to dryness under reduced pressure. Chromatography (preparative HPLC (TFA)) gave 60 (40.5 mg, 65% in two steps) as a white solid. LCMS: [M+H] + m / z 334.15. 1 H NMR (400 MHz, methanol-d4) δ 8.69 (s, 1H), 7.90 (d, J = 8.4 Hz, 1H), 7.73 (t, J = 8.3 Hz, 2H), 7.61 (d, J = 8.1 Hz, 2H), 7.41 (dd, J = 17.3, 7.8 Hz, 2H), 5.05 (s, 2H), 4.13 (s, 3H).

[0691] Preparation of (2-(1-(3-cyano-8-methoxyquinolin-4-yl)piperidin-4-yl)ethyl)boronic acid 61 (also referred to as 216 in Table 2)

[0692]

[0693] Prepared according to the same procedure as compound 7. Compound 61 was isolated as a yellow solid. LCMS: [M+H] + m / z 340.20. 1 H NMR (400 MHz, methanol-d4) δ 8.81 (s, 1H), 7.83 (d, J = 12 Hz, 1H), 7.72 (t, J = 8 Hz, 1H), 7.60 (d, J = 8 Hz, 1H), 4.51 (d, J = 12 Hz, 2H), 3.81 (t, J = 12 Hz, 2H), 3.31 (s, 3H), 2.66 (s, 1H). 2.05 (br d, J = 12 Hz, 2H), 1.72-1.30 (m, 4H) and 0.91-0.85 (m, 2H).

[0694] Preparation of 4-(((3-cyano-8-methoxyquinolin-4-yl)amino)methyl)-N-hydroxybenzamide 62 (also referred to as 220 in Table 2)

[0695]

[0696] a)CH3OCH2CH2O; b)NaOH, THF / H2O; c) NH2OH.HCl, BOP, DIPEA, THF

[0697] A solution of 101 (2.0 g, 8.9 mmol) and 103 (1.5 g 8.9 mmol) in 2-methoxyethanol (40 mL) was heated to reflux overnight and then cooled to room temperature. The reaction mixture was evaporated to dryness under reduced pressure and then triturated with EtOAc, filtered and dried to give a crude compound 104 (1.7 g) as a light yellow solid.

[0698] To a solution of compound 104 (0.5 g, 1.55 mmol) in THF (20 mL) was added NaOH (0.17 g, 4.65 mmol, dissolved in 2 mL of water). The mixture was heated at 45 °C overnight. The cooled solution was concentrated under reduced pressure, and the residue was treated with aqueous HCl (2N) until a pH of 5.5 was reached. The resulting precipitate was filtered and dried to give the crude acid intermediate (0.3 g, 62% yield) as a light yellow solid. The crude acid was dissolved in DMF (10 mL) and then cooled to 0 °C and placed under nitrogen. BOP (0.48 g, 1.06 mmol) and DIPEA (0.50 g, 3.88 mmol) were added, followed by HONH 2-HCl (0.09 g, 1.26 mmol). The mixture was stirred at room temperature overnight, quenched with water (50 mL), and extracted with EtOAc. The organic phase was washed with water and brine, dried (Na2SO3), and evaporated to dryness under reduced pressure. Chromatography (5% MeOH in CH2Cl2) and then preparative HPLC (H + , 0.1% TFA) to give 62 (34 mg, 10%) as an off-white solid. LCMS: [M+H] +1 HNMR(400MHz,DMSO-d6)δ11.19(s,1H),9.06(s,1H),8.55(s,1H),8.45(d,J=8.7Hz,1H),7.72(d,J =7.4Hz,2H),7.39(dd,J=4.6,2.9Hz,2H),7.29(dd,J=12.3,5.0Hz,2H),5.11(s,2H),3.93(s,3H).

[0699] Example 2: Evaluation of compound activity

[0700] Selected compounds and other derivatives of Tables 1-3 were prepared and evaluated in an ENPP1 activity assay using thymidine monophosphate p-nitrophenol (TMP-pNP) as a substrate. Enzyme reactions were prepared at room temperature with TMP-pNP (2 μM), a 5-fold dilution of the ENPP1 inhibitor, and purified recombinant mouse ENPP1 (0.5 nM) in 100 mM Tris, 150 mM NaCl, 2 mM CaCl2, 200 μM ZnCl2, pH 7.5. The progress of the reaction was monitored for 20 minutes by measuring the absorbance of the p-nitrophenol ester produced by the reaction at 400 nm. The slope of product formation was calculated, and the IC was obtained by plotting and fitting using GraphpadPrism 7.03. 50 value.

[0701] The compounds were also evaluated in an ENPP1 enzyme activity assay using cGAMP as a substrate. Methods that can be used to evaluate the subject compounds include those described in Li et al., PCT Application No. PCT / US2018 / 050018, filed September 7, 2018. Exemplary methods are described below.

[0702] Material:

[0703] Mouse ENPP1: Expression and purification according to Kato et al. PNAS (2012) 109(42): 16876-8. cGAMP: Synthesized and purified according to Li et al. Nat. Chem. Biol. (2014) 10: 1043-8. Polyphosphate AMP phosphotransferase (PAP): The PAP gene (GenBank: AB092983.1) was synthesized (Integrated DNA Technologies) and cloned into the pTB146 vector with a His-SUMO C-terminal tag. BL21 (DE3) cells transformed with the plasmid were grown and induced overnight at 16°C with 0.75 mM IPTG at OD600 = 1. The cells were resuspended in a buffer containing 50 mM Tris pH 7.5, 400 mM NaCl, 10 mM imidazole, 2 mM DTT, protease inhibitors (Roche) and lysed with two freeze-thaw cycles and sonication. All subsequent steps were performed at 4°C. Lysates were cleared by centrifugation at 40,000 rcf for 1 hour and the supernatant was incubated with HisPur cobalt resin (Thermo Fisher Scientific) for 2 hours. The resin was washed twice with 30 mL buffer containing 50 mM Tris pH 7.5, 150 mM NaCl and the protein was eluted with 50 mM Tris pH 7.5, 150 mM NaCl, 600 mM imidazole. Anion exchange chromatography (HiTrap Q HP) was performed. Myokinase (MilliporeSigma). CellTiterGlo (Promega)

[0704] Exemplary protocol for ENPP1 enzyme activity assay:

[0705] 3nM mouse ENPP1 was incubated with 5uM cGAMP and 5-fold serial dilutions of the compound in a buffer containing 50mMTris pH 7.6, 250nM NaCl, 500uM CaCl2 and 1uM ZnCl2 (total reaction volume = 10mL) for 3 hours at room temperature, and the reaction was then heat-inactivated at 95°C for 10 minutes. AMP degradation products were converted to ATP, which was detected using luciferase. To this end, an enzyme mixture of polyphosphate:AMP phosphotransferase (PAP) and myokinase was prepared according to Goueli et al. in EP2771480. Briefly, PAP was diluted to 2mg / mL in a buffer containing 50mM Tris pH 7.5, 0.1% NP-40. Myokinase was diluted to 2KU / mL in a buffer containing 3.2mM ammonium sulfate pH 6.0, 1mM EDTA and 4mM polyphosphate. Heat-inactivated ENPP1 reactions were incubated with PAP (0.01 mg / mL) and myokinase (0.0075 U / mL) in a buffer containing 40 mM Tris pH 7.5, 0.05 mg / mL Prionex, 5 mM MgCl2, 20 mM polyphosphate, and 0.15 g / L phenol red (for ease of pipetting) for 3 hours (total reaction volume = 20 mL). CellTiterGlo (20 uL) was added to the reaction and luminescence was measured according to the manufacturer's protocol. Data were normalized to 100% enzyme activity (no compound) and 0% enzyme activity (no enzyme) before fitting to the function 100 / (1+([compound] / IC50)).

[0706] The IC50 values ​​fall within the range denoted by the letters AC, where A represents an IC50 value less than 50 nM, B represents an IC50 value between 50 nM and 100 nM, and C represents an IC50 value greater than 100 nM.

[0707] Table 4: ENPP1 enzyme activity. IC50 values: A (<50nM); B (50nM–100nM); C (>100nM).

[0708]

[0709]

[0710]

[0711]

[0712] Table 2 Compounds

[0713]

[0714]

[0715]

[0716]

[0717]

[0718]

[0719]

[0720]

[0721] Example 3: Display of extracellular ENPP1 and inhibition of extracellular ENPP1

[0722] refer to Figures 18A to 18C , observed that ENPP1 controls extracellular cGAMP levels and that cGAMP levels can be restored by treating cells with ENPP1 inhibitors (e.g., compound 1).

[0723] 293T cGAS ENPP1 - / - Cells were transfected with human ENPP1 expression plasmid, and cGAMP hydrolase activity was confirmed in whole cell lysates ( Fig.18A ). 293T cells were purchased from ATCC and transfected with a virus to stably express mouse cGAS. 293TmcGAS ENPP1 - / - It was generated by viral transfection of CRISPR sgRNA targeting human ENPP1 (5'CACCGCTGGTTCTATGCACGTCTCC-3') (SEQ ID NO: 1). - / -Cells were plated in tissue culture treated plates coated with PurCol (Advanced BioMatrix) in DMEM (Corning Cellgro) supplemented with 10% FBS (Atlanta Biologics) (v / v) and 100 U / mL penicillin-streptomycin (ThermoFisher). 12-24 hours after plating, cells were transfected with Fugene 6 (Promega) according to the manufacturer's instructions plus the specified concentration of pcDNA3 plasmid DNA (empty or containing human ENPP1). 24 hours after transfection, cells were lysed to analyze ENPP1 expression by Western blotting (using antibodies rabbit anti-ENPP1 (L520, 1:1000) and mouse anti-tubulin (DM1A, 1:2,000, Cell Signaling Technologies). 1x10 6 Whole cell lysate was produced by 32 P-cGAMP (5 μM) was incubated with whole cell lysates and the same conditions were used as in Example 2 above ( Fig.18A Degradation was monitored as described in ).

[0724] In intact cells, ENPP1 expression depletes extracellular cGAMP but does not affect intracellular cGAMP concentration ( Fig.18B ). 293T mcGAS ENPP1 was transfected with pcDNA3 (empty or containing human ENPP1) - / - After 24 hours, the culture medium was removed and replaced with serum-free DMEM supplemented with 1% insulin-transferrin-seleno-pyruvate (ThermoFisher) and 100U / mL penicillin-streptomycin. After 12-24 hours of replacing the culture medium, the culture medium was removed and the cells were washed off the plate with cold PBS. Both the culture medium and the cells were centrifuged at 1000rcf for 10 minutes at 4°C and prepared for cGAMP concentration measurement by liquid chromatography-tandem mass spectrometry (LC-MS / MS). The cells were supplemented with 500nM cyclic GMP- 13 C 10 , 15 N5-AMP was cleaved in 50:50 acetonitrile:water and centrifuged at 15,000 rcf for 20 minutes at 4°C to remove the insoluble fraction. 13 C 10 , 15N5-AMP and 20% formic acid in the culture medium. The cGAMP, ATP and GTP content in the samples were analyzed on a Shimadzu HPLC (SanFrancisco, CA) with an autosampler set to 4°C and connected to an AB Sciex 4000QTRAP (Foster City, CA). A volume of 10 μL was injected onto a Biobasic AX LC column, 5 μm, 50 x 3 mm (Thermo Scientific). The mobile phase consisted of an acetonitrile solution (B) containing 100 mM ammonium carbonate (A) and 0.1% formic acid. The initial condition was 90% B for 0.5 min. The mobile phase was ramped up to 30% A from 0.5 min to 2.0 min, maintained at 30% A from 2.0 min to 3.5 min, ramped up to 90% B from 3.5 min to 3.6 min, and maintained at 90% B from 3.6 min to 5 min. The flow rate was set to 0.6 mL / min. The mass spectrometer was operated in electrode spray positive ion mode with the source temperature set to 500 °C. Declustering and collision-induced dissociation were achieved using nitrogen. Declustering potentials and collision energies were optimized by direct infusion of standards. For each molecule, the MRM transitions (s) (m / z), DP (V) and CE (V) were as follows: ATP (508>136, 341, 55), GTP (524>152, 236, 43), cGAMP (675>136, 121, 97; 675>312, 121, 59; 675>152, 121, 73), internal standard cyclic GMP- 13 C 10 , 15 N5-AMP (690>146,111,101; 690>152,111,45; 690>327,111,47), extraction standard ring 13 C 10 , 15 N5-GMP- 13 C 10 , 15 N5-AMP (705>156,66,93; 705>162,66,73).

[0725] Inhibition of ENPP1 can block the degradation of extracellular cGAMP ( Fig.18C ). The same experiment was performed as above, this time also including 50 μM of ENPP1 inhibitor (Compound 1) when changing the medium. In the presence of this inhibitor, the extracellular cGAMP concentration in the medium returned to the previous level.

[0726] Fig.18ATransfection with empty vector and vector containing human ENPP1 is shown and ENPP1 protein expression was analyzed by Western blot after 24 hours (top) and ENPP1 was analyzed by thin layer chromatography (TLC) 32 P-cGAMP hydrolysis activity (bottom) of 293T cGAS ENPP1 - / - cell. Fig.18B Intracellular and extracellular cGAMP concentrations using LC-MS / MS are shown. BQL = below limit of quantitation. Mean ± SEM (n = 2). **P = 0.005 (Student's t-test). Fig.18C Shown are 293T cGAS ENPP1 cells transfected with empty vector or vector containing human ENPP1 in the presence or absence of 50 μM compound 1 - / - Intracellular and extracellular cGAMP concentrations of cells. BQL = below limit of quantitation. Mean ± SEM (n = 2). **P = 0.0013 (Student's t-test).

[0727] Example 4: ENPP1 inhibition increases cGAMP activation of primary CD14+ monocytes

[0728] Using an ENPP1 inhibitor (compound 1), we tested the expression of ENPP1 in 293T cGAS. 低 Can cGAMP exported by cell lines be expressed by, for example, human CD14 + Antigen presenting cells (APC) of monocytes detect ( Fig.19A ). 293T cGAS ENPP1 低 Cells were transfected with pcDNA (empty or containing human ENPP1). Primary human peripheral blood mononuclear cells (PBMCs) were isolated by subjecting enriched buffy coats from whole blood to a Percoll density gradient. CD14 + MicroBeads (Miltenyi) to separate CD14 + Monocytes. CD14 were cultured in RMPI supplemented with 2% human serum and 100 U / mL penicillin-streptomycin. + In monocytes, 293T cells were transfected with cGAS ENPP1 低 Eight hours after the cells were grown, the medium was changed to RMPI supplemented with 2% human serum and 100 U / mL penicillin-streptomycin with or without the addition of the exemplary ENPP1 inhibitor Compound 1. 24 hours after the medium change, cells from 293T cGAS ENPP1 低 2. Transfer of cell supernatant to CD14 + Monocytes ( Fig.19A). Total RNA was extracted using Trizol (Thermo Fisher Scientific) 24-26 hours after supernatant transfer and reverse transcribed using Maxima H Minus reverse transcriptase (Thermo Fisher Scientific). Real-time RT-PCR was performed in duplicate using AccuPower 2XGreenstar qPCR Master Mix (Bioneer) on a 7900HT fast real-time PCR system (Applied Biosystems). For each sample, data were normalized for CD14 expression. Induction fold was calculated using ΔΔCt. Primers of human IFNB1: fwd(5'-AAACTCATGAGCAGTCTGCA-3')(SEQ ID NO:2), rev(5'-AGGAGATCTTCAGTTTCGGAGG-3')(SEQ ID NO:3); Primers of human CD14: fwd(5'-GCCTTCCGTGTCCCCACTGC-3')(SEQ ID NO: 4), rev (5'-TGAGGGGGCCCTCGACG-3') (SEQ ID NO: 5).

[0729] cGAS ENPP1 from 293T expressing cGAS 低 Supernatants from 293T cells induced CD14+IFNB1 expression, whereas supernatants from cGAS-null 293T cells did not, suggesting that extracellular cGAMP exported by cancer cells can be regulated by CD14+IFNB1. + Cell detection as signal transduction factor ( Fig.19B ). ENPP1 in 293T cGAS ENPP1 低 Transient overexpression on cells leads to degradation of extracellular cGAMP and CD14 + IFNB1 expression was reduced, but the addition of compound 1 rescued the extracellular cGAMP level and induced CD14 + IFNB1 expression ( Fig.19B ).

[0730] refer to Fig.19A A schematic diagram of the supernatant transfer experiment is shown. Fig.19B cGAS-free 293T cells or 293TcGAS ENPP1 transfected with DNA and incubated in the presence or absence of compound 1 are shown. 低 The supernatant from these cells was transferred to primary CD14 + IFNB1 mRNA levels were normalized to CD14 and compared to untreated CD14 +The induction fold of cells was calculated. Mean ± SEM (n = 2). *P < 0.05, ***P < 0.001 (one-way ANOVA).

[0731] Example 5: ENPP1 inhibition synergizes with ionizing radiation (IR) treatment to increase tumor-associated dendritic cells Cell

[0732] It has been tested whether cancer cell lines export cGAMP and whether ionizing radiation (IR) affects the level of extracellular cGAMP produced. Ionizing radiation (IR) has been shown to increase cytoplasmic DNA in tumor cells and activate cGAS-dependent IFN-β production in tumor cells (Bakhoum et al. Nat. Commun. (2015) 6:1-10; and Vanpouille Nat. Commun. (2017) 8:15618). 24 hours after plating, 4T1 cells were treated with 20Gy IR using a cesium source, and the medium was replaced and supplemented with 50uM of an ENPP1 inhibitor (Compound 1) to inhibit ENPP1 present in the cell culture. The culture medium was collected at the indicated times, centrifuged at 1000x g to remove residual cells, acidified with 0.5% acetic acid, and supplemented with cyclohexane as an extraction standard. 13 C 10 , 15 5-GMP- 13 C 10 , 15 N5-AMP (appropriate amount for a final concentration of 2 μM in 100 μL). The culture medium was applied to a HyperSepAminopropyl SPE column (ThermoFisher Scientific) to enrich for cGAMP as previously described (Gao et al., Proc. Natl. Acad. Sci. USA (2015) 112: E5699-705). The eluate was evaporated to dryness and reconstituted in 50:50 acetonitrile: water supplemented with 500 nM internal standard. The culture medium was submitted for mass spectrometry quantification of cGAMP.

[0733] Continuous cGAMP export was detected in 4T1 cells within 48 hours. At 48 hours, cells treated with IR had significantly higher extracellular cGAMP levels compared to untreated cells.

[0734] Next, we investigated the effect of the combination of IR and the exemplary ENPP1 inhibitor Compound 1 on the number of tumor-associated dendritic cells in the mouse 4T1 tumor model ( Fig. 20B ). In 7- to 9-week-old female Balb / c mice (Jackson Laboratories), 1 x 10 64T1-luciferase tumor cells were inoculated into the mammary fat pad. Two days after injection, the tumor was irradiated with 20Gy using a 225kVp cabinet X-ray irradiator (IC 250, Kimtron Inc., CT) with a 0.5mm Cu filter. The anesthetized animals were shielded with a 3.2mm lead shield with a 15x 20mm hole in which the tumor was placed. The mice were injected intratumorally with 100μL 1mM compound 1 in PBS or PBS alone. The next day, the tumor was extracted and incubated at 37°C for 30min in RPMI+10% FBS containing 20μg / mL DNA I enzyme type IV (Sigma-Aldrich) and 1mg / mL collagenase (Sigma-Aldrich) from Clostridium histolyticum. Tumors are passed through a 100 μm cell strainer (Sigma-Aldrich), and erythrocytes are lysed at room temperature using red blood cell lysis buffer (155 mM NH4Cl, 12 mM NaHCO3, 0.1 mMEDTA) for 5 minutes. Cells are stained with a fixable near-infrared dead cell staining kit (ThermoFisher Scientific) for live / dead, Fc blocking is performed using TruStain fcX for 10 min, and then antibody staining is performed using CD11c, CD45 and IA / IE (all from Biolegend). Cells are analyzed using SH800S cell sorter (Sony) or LSR II (BD Biosciences). Data were analyzed by statistical analysis using FlowJo V10 software (Treestar) and Prism 7.04 software (Graphpad), and statistical significance was assessed using unpaired t-test and Welch correction.

[0735] Intratumoral injection of compound 1 did not alter the composition of tumor-associated leukocytes compared with PBS control ( Fig. 20B ), indicating that ENPP1 does not play a significant role in clearing basal levels of extracellular cGAMP in this tumor model. However, when tumors were pretreated with IR, compound 1 was observed to increase tumor-associated CD11c + group( Fig. 20B ).

[0736] The results are Fig. 20A and Fig. 20B Shown in. Fig. 20A Shown is the extracellular cGAMP production by 4T1 cells over 48 hours. At time 0, cells were either left untreated or treated with 20 Gy IR and refreshed with medium supplemented with 50 μM Compound 1. Mean ± SEM (n=2). **P=0.004 (Student's t-test). Fig. 20BShown are 4T1 cells (1×10 6 ). Tumors were left untreated or treated with 20 Gy IR and injected intratumorally with PBS (n=5 for IR (0 Gy); n=4 for IR (20 Gy)) or compound 1 (n=5) on day 2. Tumors were harvested on day 3 and analyzed by FACS. *P=0.047 (Welch t test).

[0737] Example 6: ENPP1 inhibition synergizes with IR treatment and anti-CTLA-4 to exert anti-tumor effects

[0738] We have investigated whether further increasing extracellular cGAMP in vivo by using ionizing radiation (IR) and exemplary ENPP1 inhibitors such as compound 1 could improve tumor immune detection and clearance.

[0739] In 7- to 9-week-old female Balb / c mice (Jackson Laboratories), 5 x 10 4 4T1-luciferase cells were inoculated into the mammary fat pad. 2 x width / 2) up to 80mm 3 Up to 120mm 3 Tumors were irradiated with 20 Gy using a 225 kVp cabinet X-ray irradiator (IC 250, Kimtron Inc., CT) with a 0.5 mm Cu filter. Anesthetized animals were shielded with a 3.2 mm lead shield with a 15 x 20 mm hole in which the tumor was placed. On days 2, 4, and 7 after IR, 100 μL of 100 μM compound 1 and / or 10 μg cGAMP in PBS, or PBS alone, were injected intratumorally. Alternatively, on days 2, 5, and 7 after IR, 1 mM compound 1 in PBS or PBS alone was injected intratumorally, and 200 μg of anti-CTLA-4 antibody or Syrian hamster IgG antibody (both from BioXCell) was injected intraperitoneally. Mice from different treatment groups were co-housed in each cage to eliminate cage effects. Throughout the study, the experimenter was blinded. Tumor volume was recorded every other day. Tumor volume was analyzed using generalized estimating equations to account for correlation within mice. Treatment groups were compared pairwise at each time point using post hoc tests, and multiple comparisons were made using Tukey adjustments. Animal deaths were plotted in Kaplan Meier curves using Graphpad Prism 7.03, and statistical significance was assessed using the Logrank Mantel-Cox test. All animal procedures were approved by the Laboratory Animal Care Regulatory Team.

[0740] Administration of compound 1 enhanced the tumor shrinkage effect of IR treatment, but not significantly ( Fig.21A Although intratumoral injection of cGAMP had no effect on IR treatment, injection of compound 1 in addition to cGAMP synergistically reduced tumor size, prolonged survival, and achieved a 10% cure rate ( Fig.21A and Fig.21B ).

[0741] Synergy with the adaptive immune checkpoint blocker anti-CTLA-4 was also tested. In the absence of IR, treatment with anti-CTLA-4 and compound 1 had no effect on prolonging survival ( Fig. 21C ). However, combining IR pretreatment with compound 1 and anti-CTLA-4 exerted a significant synergistic effect and achieved a 10% cure rate. Taken together, these results suggest that enhancing extracellular cGAMP by combining IR treatment with ENPP1 inhibition can increase tumor immunogenicity and exert antitumor effects.

[0742] The results are shown in Fig.21A The figure shows the tumor shrinkage effect of the combination of compound 1 and IR. 3 ) were treated once with 20 Gy IR, followed by 3 intratumoral injections of PBS or therapeutic agents on days 2, 4, and 7 after IR (n=9 / treatment group). Mice from different treatment groups were housed together and the experimenters were blinded. Tumor volumes were analyzed in generalized estimating equations to account for intra-mouse correlations. Pairwise comparisons of treatment groups were performed at each time point using post hoc tests, and multiple comparisons were performed using Tukey adjustments. Fig.21B Shows Fig.21A Kaplan Meier curves were used and P values ​​were determined by log-rank Mantel-Cox test. Fig. 21C In addition to showing Fig.21B In addition to the same procedure, anti-CTLA4 or IgG isotype control antibodies injected intraperitoneally on days 2, 5, and 7 after IR are also shown (n=8 for IR(0)+Compound 1+CTLA-4 treatment group; n=17-19 for all other treatment groups). Fig.21B Statistical analyses were performed as indicated.

[0743] Taken together, these results demonstrate that cGAMP exists extracellularly and that the subject ENPP1 inhibitors can act extracellularly; thus, indicating that extracellular inhibition of ENPP1 is sufficient to achieve a therapeutic effect. ENPP1 qualifies as an innate immune checkpoint. These experiments demonstrate that extracellular inhibition of ENPP1 enables cGAMP to enhance anticancer immunity and can be combined synergistically with immune checkpoint blockade drugs that are already available as therapeutics ( Fig. 22 ).

[0744] Example 7: 2'3'-cGAMP is an immunotransmitter produced by cancer cells and regulated by ENPP1

[0745] introduce

[0746] 2'3'-Cyclic GMP-AMP (cGAMP) is characterized as an intracellular second messenger that is responsive to cytoplasmic dsDNA synthesis and activates the innate immune STING pathway. Its extracellular hydrolase ENPP1 implies the presence of extracellular cGAMP. Using mass spectrometry, cGAMP was detected to be continuously exported by engineered cell lines as a soluble factor, but then efficiently cleared by ENPP1. By developing a potent, specific, and cell-impermeable ENPP1 inhibitor, cGAMP export was detected in cancer cell lines commonly used in mouse tumor models. In tumors, depletion of extracellular cGAMP using neutralizing proteins reduced tumor-associated dendritic cells. Promoting extracellular cGAMP by genetic knockout and pharmacological inhibition of ENPP1 increased tumor-associated dendritic cells, shrank tumors, and synergized with ionizing radiation and anti-CTLA-4 treatment of tumors. In summary, cGAMP is an anticancer immunotransmitter released by tumors and detected by host innate immunity.

[0747] This second messenger 2'3'-cyclic GMP-AMP (cGAMP) plays a key role in antiviral and anticancer innate immunity. It is synthesized by the enzyme cyclic-GMP-AMP synthase (cGAS) in response to double-stranded DNA (dsDNA) in the cytosol, a danger signal for intracellular pathogens and damaged or cancerous cells. cGAMP binds and activates its endoplasmic reticulum (ER) surface receptor stimulator of interferon genes (STING) to activate the production of type 1 interferons (IFNs). These potent cytokines trigger downstream innate and adaptive immune responses to clear the threat.

[0748] In addition to activating STING within its cell of origin, cGAMP can also diffuse to bystander cells via gap junctions in epithelial cells. This intercellular communication mechanism alerts neighboring cells of damaged cells and, unfortunately, is also responsible for the spread of drug-induced hepatotoxicity and brain metastasis. In addition, cytosolic cGAMP can be packaged into budding viral particles and spread during the next round of infection. In both modes of transmission, cGAMP is never exposed to the extracellular space.

[0749] The enzyme responsible for the only detectable cGAMP hydrolase activity is ectonucleotide pyrophosphatase phosphodiesterase 1 (ENPP1) (see, e.g., Li, L. et al. Hydrolysis of 2'3'-cGAMP by ENPP1 and design of nonhydrolyzable analogs. Nat. Chem. Biol. 10, 1043–8 (2014)). This is surprising because ENPP1 is annotated as an extracellular enzyme, both as a membrane-bound form anchored by a single-pass transmembrane domain and as a soluble protein that is cleaved in serum. cGAMP (which has two negative charges and is presumed to be unable to passively cross the cell membrane) can enter cells to activate STING (see, e.g., Gao, P. et al. Structure-function analysis of STING activation by c[G(2′,5′)pA(3′,5′)p]and targeting by antiviral DMXAA. Cell 154, 748–762 (2013); and Corrales, L. et al. Direct Activation of STING in the TumorMicroenvironment Leads to Potent and Systemic Tumor Regression andImmunity. Cell Rep. 11, 1018–1030 (2015)), indicating the existence of a transport channel for cGAMP. Because it can enter cells, cGAMP analogs are currently being tested in clinical trials to treat metastatic solid tumors by intratumoral injection. Knowing that extracellular cGAMP can be imported and have anticancer effects and that the dominant cGAMP hydrolase is extracellular, we hypothesized that cGAMP is exported to the extracellular space to signal to other cells and is regulated by extracellular degradation.

[0750] Here we demonstrate that cancer exports cGAMP and that extracellular cGAMP plays a role in anticancer immune sensing. Using genetic knockout and pharmacological inhibition, we also investigated the role of ENPP1 in controlling extracellular cGAMP concentrations, immune infiltration, and tumor progression. In conclusion, cGAMP is characterized as an immune transmitter regulated by ENPP1.

[0751] Materials and methods

[0752] Reagents, antibodies, and cell lines

[0753] [α- 32 P]ATP (800Ci / mmol, 10mCi / mL, 250μCi) and [35 S]ATPαS (1250 Ci / mmol, 12.5 mCi / mL, 250 μCi) was purchased from Perkin Elmer. Adenosine triphosphate, guanosine triphosphate, adenosine- 13 C 10 , 15 N5,5'-triphosphate,guanosine- 13 C 10 , 15 N5-triphosphate, 4-nitrophenyl phosphate, and bis(4-nitrophenyl) phosphate were purchased from Sigma-Aldrich and were >98% atomic purity. 2'3'-cGAMP was purchased from Invivogen. Caco-2 kit was purchased from Cyprotex. Kinome screening was performed by Eurofins. PAMPA and MDCK permeability assays were performed by Quintara Discovery. Total protein content was quantified using the BCA assay (ThermoFisher). Cell viability was quantified using the CellTiterGlo assay (Promega). Full-length human ENPP1 was cloned into the pcDNA3 vector. A set of 4 ON-TARGETplusENPP1 siRNAs (LQ-003809-00-0002) were purchased from Dharmacon. QS1 was synthesized as previously described. 25 The following monoclonal antibodies were used for immunoblotting: rabbit anti-cGAS (D1D3G Cell Signaling, 1:1,000), rabbit anti-mouse cGAS (D2O8OCell Signaling, 1:1,000), mouse anti-tubulin (DM1A Cell Signaling, 1:2,000), and rabbit anti-STING (D2P2F Cell Signaling, 1:1,000), IRDye 800CW goat anti-rabbit (LI-COR, 1:15,000), and IRDye 680RD goat anti-mouse (LI-COR, 1:15,000).

[0754] 293T cells were purchased from ATCC and stably expressed mouse cGAS by viral transfection. 293T cGAS ENPP1 low Cells were generated by viral transfection with a CRISPR sgRNA targeting human ENPP1 (5'-CACCGCTGGTTCTATGCACGTCTCC-3'), and single cells were cloned from this pool and selected for 293T mcGAS ENPP1 - / - Cells. 4T1 and E0771 cGAS - / -Cells were generated by viral transfection with CRISPR sgRNA targeting mouse Mb21d1 (5'-CACCGGAAGGGGCGCGCGCTCCACC-3') using lentiCRISPRv2-blast, Addgene plasmid #83480. Cells were selected after single cell cloning. 4T1-LucENPP1 - / - Cells were generated by viral transfection with CRISPR sgRNA targeting mouse Enpp1 (5'-GCTCGCGCCCATGGACCT-3' and 5'-ATATGACTGTACCCTACGGG-3') or scrambled sequences (using lentiCRISPRv2-blast) (Sanjana, NE, Shalem, O. & Zhang, F. Improved vectors and genome-wide libraries for CRISPR screening. Nat. Methods 11, 783–784 (2014)). 4T1-Luc shcGAS cells were generated by viral transfection of shRNA (5'-CAGGATTGAGCTACAAGAATAT-3') using plasmid pGH188. Cells with shRNA were selected with blasticidin and sorted for GFP expression and used as experimental pools. MDA-MB-231 was purchased from ATCC, E0771 was purchased from CH3 BioSystems, 4T1-luciferase and HEK293S GnT1 expressing secreted mENPP1 were obtained - cell.

[0755] Cell culture

[0756] Cell lines were maintained in DMEM (Corning Cellgro) (293T, MC38) or RPMI (Corning Cellgro) (4T1-Luc, E0771, MDA-MD-231) supplemented with 10% FBS (Atlanta Biologics) (v / v) and 100 U / mL penicillin-streptomycin (ThermoFisher). Primary human peripheral blood mononuclear cells (PBMCs) were isolated by subjecting enriched buffy coats from whole blood to a Percoll density gradient. CD14 + MicroBeads (Miltenyi) to separate CD14 + PBMC. CD14 were cultured in RMPI supplemented with 2% human serum and 100 U / mL penicillin-streptomycin. + PBMCs.

[0757] Expression and purification of recombinant proteins

[0758] sscGAS: The DNA sequence encoding porcine cGAS (residues 135-497) was amplified from a porcine cDNA library using the primer pair fwd: (5'-CTGGAAGTTCTGTTCCAGGGGCCCCATATGGGCGCCTGGAAGCTCCAG AC-3') and rev: (5'-GATCTCAGTGGTGGTGGTGGTGGTGCTCGAGCCAAAAAACTGGAAATCCATTGT-3'). The PCR product was inserted into pDB-His-MBP by Gibson assembly and expressed in Rosetta cells. The cells were grown in 2xYT medium containing kanamycin (100 μg / ml) and the OD was set at 4 °C. 600 When it reaches 1, it is induced with 0.5mM IPTG and grown overnight at 16°C. All the following operations involving proteins and cell lysates are performed at 4°C. Cells are precipitated and lysed in 20mM HEPES pH 7.5, 400mM NaCl, 10% glycerol, 10mM imidazole, 1mM DTT and a mixture of protease inhibitors (cOmplete, EDTA-free tablets, Roche). Cell extracts are cleared by ultracentrifugation at 50,000xg for 1 hour. The clarified supernatant is incubated with HisPur cobalt resin (ThermoFisher Scientific; 1mL resin per liter of bacterial culture). The cobalt resin is washed with 20mM HEPES pH 7.5, 1M NaCl, 10% glycerol, 10mM imidazole, 1mM DTT. The protein was eluted from the resin with 300 mM imidazole in 20 mM HEPES pH 7.5, 1 M NaCl, 10% glycerol, and 1 mM DTT. The fractions containing His-MBP-sscGAS were pooled, concentrated, and dialyzed against 20 mM HEPES pH 7.5, 400 mM NaCl, 1 mM DTT. The protein was snap frozen in aliquots for future use.

[0759] STING: Mouse STING (residues 139-378) was inserted into the pTB146 His-SUMO vector and expressed in Rosetta cells. The cells were grown in 2xYT medium containing 100 μg / mL ampicillin and the OD 600The lysate was precipitated and lysed by ultracentrifugation at 50000 rcf for 1 hour. The supernatant was incubated with HisPur cobalt resin (ThermoFisher Scientific; 1 mL resin per 1 L bacterial culture) for 30 minutes. The resin-bound protein was washed with 50 column volumes of 50 mM Tris pH 7.5, 150 mM NaCl, 2% triton X-114, 50 CV of 50 mM Tris pH 7.5, 1 M NaCl (the drip rate was set to 1 drop / 2-3 seconds for each wash, which took 2-3 hours), and 20 CV of 50 mM Tris pH 7.5, 150 mM NaCl. The protein was eluted from the resin with 600 mM imidazole in 50 mM Tris pH 7.5, 150 mM NaCl. The fractions containing His-SUMO-STING were combined, concentrated, and dialyzed against 50 mM Tris pH 7.5, 150 mM NaCl while incubating with the SUMOlase enzyme His-ULP1 overnight to remove the His-SUMO tag. The solution was incubated again with HisPur cobalt resin to remove the His-SUMO tag, and STING was collected from the effluent. The protein was dialyzed against 50 mM Tris pH 7.5 using FPLC (GE Healthcare) was loaded onto a HitrapQ anion exchange column (GE Healthcare) and eluted with a NaCl gradient. Fractions containing STING were pooled and the buffer was exchanged into PBS and stored at 4°C until use.

[0760] ENPP1: mENPP1 was prepared as described in Kato, K. et al. (Expression, purification, crystallization and preliminary X-ray crystallographic analysis of Enpp1. ActaCrystallogr. Sect. F Struct. Biol. Cryst. Commun. 68, 778–782 (2012); and Crystal structure of Enpp1, an extracellular glycoprotein involved in bonemineralization and insulin signaling. Proc. Natl. Acad. Sci. USA 109, 16876–81 (2012)).

[0761] Liquid chromatography-tandem mass spectrometry

[0762] Cyclic GMP- 13 C 10 , 15 N5-AMP was used as an internal standard, and cyclic 13 C 10 , 15 5-GMP- 13 C 10 , 15 N5-AMP was used as an extraction standard. Isotope-labeled cGAMP standards were synthesized by incubating 1 mM ATP (isotope-labeled), 1 mM GTP (isotope-labeled), 20 mM MgCl2, 0.1 mg / mL herring testis DNA (Sigma) and 2 μM sscGAS in 100 mM Tris, pH 7.5 overnight. The reaction was heated at 95 ° C and filtered through a 3 kDa centrifugal filter. Water was removed on a rotary evaporator. On a preparative HPLC (1260 Infinity LC system; Agilent Technologies) connected to a UV-vis detector (ProStar; Agilent Technologies) and a fraction collector (440-LC; Agilent Technologies), a PLRP-S polymer reverse phase preparative column ( cGAMP was purified from the crude reaction mixture using a 50 μm, 8 μm, 300 x 25 mm, Agilent Technologies. The flow rate was set to 25 mL / min. The mobile phase consisted of 10 mM triethylammonium acetate in water and acetonitrile. The mobile phase for the first 5 minutes was 2% acetonitrile. The acetonitrile was then increased to 30% from 5-20 minutes, increased to 90% from 20-22 minutes, maintained at 90% from 22-25 minutes, and then decreased to 2% from 25-28 minutes. The fractions containing cGAMP were lyophilized and resuspended in water. The concentration was determined by measuring the absorbance at 280 nm. The samples were analyzed for cGAMP, ATP, and GTP content on a Shimadzu HPLC (San Francisco, CA), with the autosampler set to 4 °C and connected to an AB Sciex 4000 QTRAP (Foster City, CA). A volume of 10 μL was injected into a Biobasic AX LC column, 5 μm, 50 x 3 mm (ThermoScientific). The mobile phase consisted of 100 mM ammonium carbonate (A) and 0.1% formic acid in acetonitrile (B). The initial condition was 90% B, held for 0.5 min. The mobile phase was increased to 30% A from 0.5 min to 2.0 min, held at 30% A from 2.0 min to 3.5 min, increased to 90% B from 3.5 min to 3.6 min, and held at 90% B from 3.6 min to 5 min. The flow rate was set to 0.6 mL / min. The mass spectrometer was operated in electrode spray positive ion mode with the source temperature set to 500 °C. Declustering and collision-induced dissociation were achieved using nitrogen. The declustering potential and collision energy were optimized by direct injection of standards. For each molecule, the MRM transitions (m / z), DP (V ), and CE (V ) were as follows: ATP (508>136, 341, 55), GTP (524>152, 236, 43), cGAMP (675>136, 121, 97; 675>312, 121, 59; 675>152, 121, 73), internal standard cyclic GMP- 13 C 10 , 15 N5-AMP (690>146,111,101; 690>152,111,45; 690>327,111,47), extraction standard ring 13 C 10 , 15 N5-GMP- 13 C 10 , 15 N5-AMP (705>156,66,93; 705>162,66,73).

[0763] 293TcGAS ENPP1 - / - Export assays in cells

[0764] 293T cGAS ENPP1 - / - Cells are seeded in the plates treated with tissue culture coated with PurCol (Advanced BioMatrix). After 24 hours, the culture medium is gently removed and replaced with the serum-free DMEM supplemented with 1% insulin-transferrin-seleno-pyruvate (ThermoFisher) and 100U / mL penicillin-streptomycin. At the specified time, the culture medium is removed and the cells are washed off from the plate with cold PBS. Both culture medium and cells are centrifuged at 4°C for 10 minutes with 1000rcf. Cells are supplemented with 50:50 acetonitrile: water of 500nM internal standard in 30 to 100 μL and are centrifuged at 4°C for 20 minutes with 15,000rcf to remove insoluble fractions. If it is not necessary to concentrate, aliquots of culture medium are removed and the internal standard and 20% formic acid of 500nM are supplemented. If it is necessary to concentrate, the culture medium is acidified with 0.5% acetic acid and supplemented with extraction standards (the final concentration in 100 μL is the appropriate amount of 2 μM). As described in Gao, D. et al. (Activation of cyclic GMP-AMP synthase by self-DNA causes autoimmune diseases. Proc. Natl. Acad. Sci. USA 112, E5699-705 (2015)), the culture medium was applied to a HyperSep Aminopropyl SPE column (ThermoFisher Scientific) to enrich cGAMP. The eluate was evaporated to dryness and reconstituted in 50:50 acetonitrile: water supplemented with 500 nM internal standard. The culture medium and cell extracts were provided for mass spectrometry quantification of cGAMP, ATP and GTP.

[0765] 293T cGAS ENPP1 - / - Transfection stimulation of cells

[0766] 293T cGAS ENPP1 cells were transfected with Fugene 6 (Promega) according to the manufacturer's instructions plus the indicated concentrations of pcDNA3 plasmid DNA (empty or containing human ENPP1). - / - 24 h after transfection, export assays were performed as described above.

[0767] Conditioned medium transfer

[0768] As described above, 293T cGAS ENPP1 低Cells were plated and transfected with plasmid DNA. 24 hours after transfection, the medium was changed to RPMI + 2% human serum + 1% penicillin-streptomycin, + / - 2 μM cGAMP, + / - 20 nM recombinant mENPP1 or + / - 50 uM compound 1. 24 hours after the medium change, the cells were transfected with plasmid DNA from 293T cGAS ENPP1 低 Conditioned medium was removed from cells and mixed with freshly isolated CD14 + PBMC incubation. CD14 was analyzed 14-16 hours later + Gene expression of PBMCs.

[0769] RT-PCR analysis

[0770] Total RNA was extracted using Trizol (Thermo Fisher Scientific) and reverse transcribed using Maxima HMinus reverse transcriptase (Thermo Fisher Scientific). Real-time RT-PCR was performed in duplicate using AccuPower 2X Greenstar qPCR Master Mix (Bioneer) on a 7900HT Fast Real-Time PCR System (Applied Biosystems). Data for each sample were normalized to CD14, ACTB, or GAPDH expression. Fold induction was calculated using ΔΔCt. Primers for human IFNB1: fwd (5'-AAACTCATGAGCAGTCTGCA-3'), rev (5'-AGGAGATCTTCAGTTTCGGAGG-3'); primers for human CD14: fwd (5'-GCCTTCCGTGTCCCCACTGC-3'), rev (5'-TGAGGGGGCCCTCGACG-3'); primers for human ACTB: fwd (5'-GGCATCCTCACCCTGAAGTA-3'), rev (5'-AGAGGCGTACAGGGATAGCA-3'); primers for human GAPDH: fwd (5'-CCAAGGTCATCCATGACAAC-3'); rev (5'-CAGTGAGCTTCCCGTTCAG-3').

[0771] 32 TLC Assay for P-cGAMP Degradation

[0772] By mixing unlabeled ATP (1 mM) and 32 Radiolabeled ATP was synthesized by incubating GTP (1 mM) with 2 μM purified recombinant porcine cGAS in 20 mM Tris pH 7.5, 2 mM MgCl2, and 100 μg / mL herring testis DNA overnight at room temperature. 32P cGAMP, and the remaining nucleotide raw materials were degraded with alkaline phosphatase at 37°C for 4 hours. Cell lysates were prepared by scraping and lysing 1x10 cells in 100 μL of 10 mM Tris, 150 mM NaCl, 1.5 mM MgCl2, 1% NP-40, pH 9.0. 6 cells (293T) or 10x10 6 The total protein concentration of the lysate was measured using the BCA assay (Pierce, Thermo Fisher) for 4T1-Luc, E0771, and MDA-MB-231, and the samples were normalized so that the same amount of protein was used for each lysate reaction. 32 P-cGAMP (5 μM) was incubated with mENPP1 (20 nM) or whole cell lysate in 100 mM Tris, 150 mM NaCl, 2 mM CaCl2, 200 μM ZnCl2, pH 7.5 or pH 9.0 for the indicated times. To generate inhibition curves, a 5-fold dilution of ENPP1 inhibitor was included in the reaction. Degradation was assessed by TLC (see, e.g., Li, L. et al. Hydrolysis of 2'3'-cGAMP by ENPP1 and designof nonhydrolyzable analogs. Nat. Chem. Biol. 10, 1043–8 (2014)). The plates were exposed to a phosphor screen (Molecular Dynamics) and imaged on a Typhoon 9400 and analyzed using ImageJ 32 The P signal was quantified. The inhibition curve was fitted using Graphpad Prism 7.03 to obtain the IC 50 Using the Cheng-Prusoff equation K i,app =IC 50 / (1+[S] / K m ) 50 Convert value to K i,app value.

[0773] ALPL and ENPP2 inhibition assays

[0774] Inhibition assays for other ectonucleotidases were performed by incubating the reaction components at room temperature in a 96-well plate format and monitoring the production of 4-nitrophenol by measuring the absorbance at 400 nM in a plate reader (Tecan). ALPL: 0.1 nM ALPL, 2 μM 4-nitrophenyl phosphate and varying concentrations of inhibitors in buffer pH 9.0 (containing 50 mM Tris, 20 μM ZnCl2, 1 mM MgCl2) at room temperature. ENPP2: 2 nM ENPP2, 500 μM bis(4-nitrophenyl)phosphate and varying concentrations of inhibitors in buffer pH 9.0 (containing 100 mM Tris, 150 mM NaCl, 200 μM ZnCl2, 2 mM CaCl2).

[0775] Output assays in cancer cell lines

[0776] 4T1-Luc, E0771, and MC38 cells were replaced with new medium supplemented with 50 μM compound 1. At the indicated times, the medium was collected; the cells were washed off the plates with PBS, pelleted at 1000 rcf, lysed with 4 mL of 50:50 acetonitrile:water, and centrifuged at 15,000 rcf. cGAMP was enriched from the medium and cell supernatant using HyperSep Aminopropyl SPE columns as described above and submitted for mass spectrometry quantification.

[0777] 4T1-Luc tumor mouse model

[0778] In 7- to 9-week-old female BALB / c mice (Jackson Laboratories), 5 x 10 4 or 5x 10 5 4T1-Luc-luciferase cells were inoculated into the mammary fat pad. 2 x width / 2) up to 80mm 3 Up to 120mm 3When irradiated, the tumor was irradiated with 20 Gy using a 225 kVp cabinet X-ray radiator (IC-250, Kimtron Inc., CT) with a 0.5 mm Cu filter. The anesthetized animals were shielded with a 3.2 mm lead shield with a 15 x 20 mm hole in which the tumor was placed. On the 2nd, 4th and 7th days after IR, 100 μL of 100 μM compound 1 in PBS and / or 10 μg cGAMP, or PBS alone, was injected into the tumor. Alternatively, on the 2nd, 5th and 7th days after IR, 1 mM compound 1 in PBS or PBS alone was injected into the tumor, and 200 μg anti-CTLA-4 antibody or Syrian hamster IgG antibody (both from BioXCell) was injected intraperitoneally. Mice from different treatment groups were co-housed in each cage to eliminate cage effects. Throughout the study, the experimenter was blinded. Tumor volume was recorded every other day. Tumor volume was analyzed using generalized estimating equations to account for correlation within mice. Treatment groups were compared pairwise at each time point using post hoc tests, and multiple comparisons were made using Tukey adjustments. Animal mortality was plotted in Kaplan Meier curves using Graphpad Prism 7.03, and statistical significance was assessed using the Log-rank Mantel-Cox test. All mice were housed at Stanford University in accordance with the regulations of the Stanford University Institutional Animal Care and Use Committee, and procedures were approved by the Stanford University Laboratory Animal Care Management Group.

[0779] FACS analysis of tumors

[0780] In 7- to 9-week-old female BALB / c WT (4T1-Luc tumors) or C57BL / 6 (E0771 tumors) WT, cGAS - / - or STING gt / gt (called STING - / - ) mice (Jackson Laboratories), 1x 10 6Tumor cells were inoculated into mammary fat pads. Two days after injection, tumors were irradiated as described and injected intratumorally with 100 μL of 1 mM compound 1 in PBS, or PBS alone. For experiments using STING and mENPP1, 100 μL of 100 μM neutralized STING or unbound STING (R237A) or 700 nM mENPP1 or PBS was injected intratumorally. The next day, tumors were extracted and incubated in RPMI + 10% FBS containing 20 μg / mL DNase type IV (Sigma-Aldrich) and 1 mg / mL collagenase from Clostridium histolyticum (Sigma-Aldrich) at 37 ° C for 30 min. The tumor was passed through a 100 μm cell strainer (Sigma-Aldrich) and erythrocytes were lysed using erythrocyte lysis buffer (155 mM NH4Cl, 12 mM NaHCO3, 0.1 mM EDTA) for 5 minutes at room temperature. Cells were stained with a live / dead fixable near-infrared dead cell staining kit (ThermoFisher Scientific), Fc blocking was performed for 10 min using TruStain fcX, and then antibody staining was performed using CD11c, CD45, and IA / IE (all from Biolegend). Cells were analyzed using SH800S cell sorter (Sony) or LSR II (BD Biosciences). Data were analyzed by statistical analysis using FlowJo V10 software (Treestar) and Prism 7.04 software (Graphpad), and statistical significance was assessed using unpaired t-test and Welch correction.

[0781] In vivo imaging

[0782] Mice were injected intraperitoneally with 3 mg of XenoLight D-luciferin (Perkin-Elmer) in 200 μl of water and imaged using the Lago X in vivo imaging system (Spectral Instruments Imaging). The target height was set to 1.5 cm, binning was 4, FStop was 1.2, and the exposure time was 120 s. Images were analyzed using aura 2.0.1 software (Spectral Instruments Imaging).

[0783] result

[0784] cGAMP as a soluble factor from 293T cGAS ENPP1 - / - Output in cells

[0785] To test the hypothesis that cGAMP exists outside cells, we first developed a liquid chromatography-tandem mass spectrometry (LC-MS / MS) method to detect cGAMP in complex mixtures. Two isotopically labeled cGAMP standards ( Figure 1 , Panel A), we can quantify cGAMP concentrations as low as 0.5 nM in basal cell culture medium and serum-containing medium, and we can quantify intracellular cGAMP concentrations in cell extracts in the same experiment ( Figure 1 , small picture B and Figure 8 , panels A and B). We chose to use 293T cells that do not express cGAS or STING. By stably expressing mouse cGAS and knocking out ENPP1 using CRISPR, we created 293T cGAS ENPP1 低 Cell lines ( Figure 8 , Panel C). We then isolated a clone to create the 293T cGAS ENPP1 - / - Cell lines ( Figure 8 , Panel C). We also used serum-free medium because serum contains the proteolytically soluble form of ENPP1. Using this ENPP1-free cell culture system, we detected ENPP1 in 293TcGAS without any stimulation. - / - A constant low micromolar basal intracellular cGAMP concentration was detected in cells ( Figure 1 , panel C). This is not surprising, as faulty DNA segregation results in abundant cytoplasmic dsDNA in cancer cells (see, e.g., Mackenzie, KJ et al. cGAS surveillance of micronuclei links genome instability to innate immunity. Nature 548, 461–465 (2017); Harding, SM Metatic progression following DNA damage enables pattern recognition within micronuclei. Nature 548, 466–470 (2017); and Bakhoum, SF et al. Chromosomal instability drives metastasis through acytosolic DNA response. Nature 553, 467–472 (2018)). After replenishing cells with fresh medium, we measured a linear increase in extracellular cGAMP concentration to 100 nM after 30 h ( Figure 1, Panel D). At 30 hours, the number of cGAMP molecules outside the cell is equal to the number inside the cell ( Figure 1 , Panel E). We detected negligible cell death based on extracellular lactose dehydrogenate (LDH) activity, suggesting that cGAMP in the culture medium is exported by living cells ( Figure 1 , Panel E). We calculate the output rate (v export ) is 220 molecular cells -1 s -1 ( Figure 1 , Panel F). Finally, cGAMP in the culture medium can pass through a 10 kDa filter without any retention (which should retain extracellular vesicles and proteins), indicating that cGAMP is exported as a freely soluble molecule ( Figure 1 , small picture H).

[0786] To further confirm that the extracellular cGAMP secreted by 293T cells is mainly present in soluble form but not in extracellular vesicles, we used CD14 + Human peripheral blood mononuclear cells (PBMCs) were used as reporters. These cells have previously been shown to take up soluble cGAMP, leading to the production of IFN-β 17 We observed that CD14 + PBMCs respond to submicromolar concentrations of soluble cGAMP by upregulating IFNB1 ( Fig. 9 Conditioned medium (obtained from DNA-transfected 293TcGASENPP1 expressing cGAS) 低 cells, but not from DNA-transfected cGAS-null 293T cells) in CD14 + induced IFNB1 expression in 293T cells, suggesting that this activity is a result of extracellular cGAMP produced by 293T cells ( Figure 1 , panels H and I). Purified soluble recombinant mouse ENPP1 (mENPP1) ( Figure 8 , Panel D) depleted the conditioned medium of detectable cGAMP and also abolished this activity ( Figure 1 , panels H and J). Because soluble ENPP1 (MW = ∼100 kDa) is membrane-impermeable and therefore only accessible to soluble extracellular cGAMP, we conclude that 293T cells secrete soluble cGAMP. In summary, our data suggest that this artificial cancer cell line maintains its intracellular cGAMP in a stable state by exporting it as a soluble factor into the extracellular medium.

[0787] Figure 1, Panels A to J: cGAMP as a soluble factor from 293T cGAS ENPP1 - / - Output in cells. a, Chemical structures of cGAMP and single isotope-labeled cGAMP. b, cGAMP detected by LC-MS / MS, lower limit of quantification = 4 nM. (Left) Liquid chromatograms of 0, 4 and 10 nM cGAMP and 500 nM monoisotope-labeled cGAMP (weight 15 Da) as internal standard; (Right) External standard curve of cGAMP, R 2 = 0.996. Data represent >10 independent experiments. cd, measured by LC-MS / MS from 293T cGAS ENPP1 - / - Intracellular and extracellular concentrations of cGAMP in cells (without exogenous stimulation). At time 0, cells were supplemented with serum-free medium. Mean ± SEM (n = 2), some error bars are too small to visualize. Data represent three independent experiments. e, The ratio of extracellular cGAMP molecules / total cGAMP molecules (left y-axis) calculated from the data in (c) and (d) is compared with the ratio of extracellular lactose dehydrogenase / total lactose dehydrogenase (LDH) activity (right y-axis). f, The amount of cGAMP exported by each cell over time calculated from the data in (d). The export rate was found using linear regression. g, 293T cGAS ENPP1 measured before and after the medium passed through a 10 kDa filter - / - Intracellular and extracellular cGAMP concentrations produced by cells. Mean ± SEM (n = 2). Data are representative of two independent experiments. h, Schematic diagram of conditioned medium transfer experiments in (i) and (j). cGAS-null293T or 293T cGAS ENPP1 低 Cells were transfected with empty pcDNA vector and treated with + / - 20 nM recombinant mouse ENPP1 (mENPP1). Conditioned media from these cells were transferred to primary CD14 + i. IFNB1 mRNA levels were normalized to CD14 and compared to untreated CD14 + Fold induction was calculated for cells. Mean ± SEM (n = 4). ***P = 0.0003 (one-way ANOVA). cGAMP concentration was measured in conditioned medium. Mean ± SEM (n = 2). ***P = 0.0002 (one-way ANOVA). Data represent two independent experiments. j, IFNB1 mRNA levels were normalized to CD14 and compared to untreated CD14 +The fold induction of cells was calculated. Mean ± SEM (n = 2). *P = 0.04 (one-way ANOVA). cGAMP concentration was measured in the conditioned medium. Mean ± SEM (n = 2). **P = 0.002 (one-way ANOVA). Data represent two independent experiments.

[0788] Figure 8 , Panels A to D: Development of LC-MS / MS method and construction of 293T cGAS ENPP1 低 and 293T cGASENPP1 - / - Cell lines. a, LC chromatograms of cGAMP at 0, 20 and 80 nM; LC chromatograms of monoisotopically labeled internal standard cGAMP (weight 15 Da) at 500 nM; and LC chromatograms of doubly isotopically labeled extraction standard cGAMP (weight 30 Da) at 2 μM. Chemical structures of all analytes. b, Cell number was calibrated to ATP concentration measured by LC-MS / MS. Mean ± SEM (n = 2). c, Western blot analysis of 293T, 293T cGAS ENPP1 - / - and 293TcGAS ENPP1 低 cGAS expression in cell lines (left). 293T cGAS, 293T cGAS ENPP1 - / - and 293T cGAS ENPP1 低 In whole cell lysate of 1 million cells 32 ENPP1 hydrolysis activity of P-cGAMP was measured by TLC and autoradiography (right). Lysate data are representative of two independent experiments. d, Coomassie gel of recombinant mouse ENPP1 purified from culture medium; elution fractions were combined before use (left). TLC analysis of mouse ENPP1 on 32 Degradation of P-cGAMP (right).

[0789] Fig. 9 ,Small picture A: CD14 + PBMCs respond to extracellular cGAMP. a, CD14 cells stimulated with extracellular cGAMP + Schematic diagram of PBMC. b, Human CD14 measured by RT-qPCR + PMBCs induced by IFNB1, human CD14 + PMBCs were stimulated with increasing concentrations of extracellular cGAMP for 16 h. Mean ± SEM (n = 2 technical qPCR replicates).

[0790] ENPP1 regulates only extracellular cGAMP

[0791] Since we were able to observe extracellular cGAMP for the first time when we knocked out ENPP1 from 293T cells and cultured them in ENPP1-free medium, we investigated whether only extracellular cGAMP is regulated by ENPP1. Despite the extracellular annotation, ENPP1 might flip orientation on the membrane like the enzyme CD38 (see, e.g., Zhao, YJ, Lam, CMC & Lee, HC The membrane-bound enzyme CD38 exists in two opposing orientations. Sci. Signal. 5, ra67 (2012)), or it might be active when synthesized in the ER lumen and cGAMP might cross the ER membrane ( Figure 2 , Panel A). To investigate the localization of ENPP1 activity, we transfected 293T cGAS ENPP1 with human ENPP1 expression plasmid. - / - cells and confirmed its activity in whole cell lysates ( Figure 2 , Panel B). In intact cells, ENPP1 expression depletes extracellular cGAMP but does not affect intracellular cGAMP concentration ( Figure 2 , Panel C). Thus, in these cells, extracellular, but not intracellular, cGAMP is regulated by ENPP1.

[0792] Figure 2 ,Panels A to C: ENPP1 regulates only extracellular cGAMP. a, Three possible cellular locations of ENPP1 activity. b, 293T cGAS ENPP1 - / - Cells were transfected with empty vector or vector containing human ENPP1, and ENPP1 protein expression was analyzed 24 hours later by Western blotting (top) and ENPP1 by thin layer chromatography (TLC). 32 P-cGAMP hydrolysis activity (bottom). Data represent two independent experiments. c, Intracellular and extracellular cGAMP concentrations were measured using LC-MS / MS. BQL = below limit of quantification. Mean ± SEM (n = 2). **P = 0.002 (Student's t test). Data represent three independent experiments.

[0793] Development of cell-impermeable ENPP1 inhibitors

[0794] To investigate the physiological relevance of extracellular cGAMP and why it needs to be regulated by a specific hydrolase, we sought to modulate its concentration by pharmacologically inhibiting ENPP1. We first tested the nonspecific ENPP1 inhibitor QS1 ( Fig.10, Panel A) (Patel, SD et al. Quinazolin-4-piperidin-4-methyl sulfamide PC-1 inhibitors: Alleviating hERG interactions through structure based design. Bioorganic Med. Chem. Lett. 19, 3339–3343 (2009); and Shayhidin, EE et al. Quinazoline-4-piperidine sulfamides are specific inhibitors of human NPP1 and prevent pathological mineralization of valve interstitial cells. Br. J. Pharmacol. 172, 4189–4199 (2015)). Although QS1 can inhibit extracellular cGAMP degradation in cells overexpressing ENPP1, it also partially blocks cGAMP output in ENPP1 knockout cells ( Fig.10 , Panel B). QS1-treated cells have elevated intracellular cGAMP, again demonstrating that export is an important mechanism for maintaining cGAMP homeostasis in cancer cells. In our export studies, export-blocking activity excludes QS1 as a tool for studying extracellular cGAMP. Phosphonate analogs (compound 1) are designed to chelate Zn at the catalytic site of ENPP1. 2+ , and minimize cell permeability and avoid intracellular off-target ( Figure 3 , Panel A). K of compound 1 i,app 110±10nM( Figure 3 , panel B), which is about 60 times more effective than QS1 ( Fig.10 , panel A).

[0795] We confirmed that compound 1 is cell-impermeable by performing three independent permeability assays: Parallel Artificial Membrane Permeability Assay (PAMPA) ( Fig.11 , panel A); intestinal cell Caco-2 permeability assay ( Fig.11 , panel B); and epithelial cell MDCK permeability assay ( Fig.11 , Panel C). Compared with control compounds with high and low cell permeability, compound 1 fell into the category of impermeable compounds in all three assays. In addition, it was active against the closely related ectonucleotidase alkaline phosphatase (K i,app >100 μM) and ENPP2 (K i,app=5.5 μM) had lower activity ( Fig.11 , Panel D). Although we did not expect compound 1 to have off-target effects in cells due to its low cell permeability, we tested its binding to a panel of 468 kinases to further determine its specificity. Despite its structural similarity to AMP, compound 1 bound only to two kinases at 1 μM ( Fig.11 , Panel E). Compound 1 also showed high stability in human and mouse liver microsomes (t 1 / 2 >159min). In conclusion, we demonstrated that compound 1 is a potent, cell-impermeable, specific, and stable ENPP1 inhibitor.

[0796] We then measured the efficacy of compound 1 in maintaining extracellular cGAMP concentrations in 293T cGAS cells overexpressing ENPP1 and obtained an IC of 340 ± 160 nM 50 value( Figure 3 , panel C), where 10 μM is sufficient to completely block the degradation of extracellular cGAMP ( Figure 3 , Panel D). Unlike QS1, compound 1 had no effect on intracellular cGAMP, indicating that it does not affect cGAMP export ( Figure 3 , Panel D). Therefore, compound 1 is an excellent tool compound for ENPP1 inhibitors that specifically increases extracellular cGAMP concentration.

[0797] Finally, we tested the ability of compound 1 to enhance the expression of CD14 + We first confirmed that compound 1 was not toxic to PBMC at the concentrations used ( Fig.11 , Panel F). Conditioned medium from 293T cGAS cells overexpressing ENPP1 failed to express in CD14 + Induction of IFNB1 expression in cells Figure 3 , Panels E and F). However, compound 1 restored the extracellular cGAMP level and CD14 + Induction of IFNB1 expression in cells Figure 3 , Panel F). These results suggest that the enzymatic activity of ENPP1 (rather than its potential scaffolding effect as a transmembrane protein) inhibits CD14 + In summary, our data suggest that extracellular cGAMP levels can be decreased by ENPP1 expression and increased by ENPP1 inhibition, which affects CD14 + Activation of PBMCs.

[0798] Figure 3, Panels A to F: Activity of cell-impermeable ENPP1 inhibitors. a, Chemical structure of compound 1. b, 32 Inhibitory activity of compound 1 against purified mouse ENPP1 at pH 7.5 using P-cGAMP as substrate (K i,app =110±10 nM). Mean ± SEM (n=3 independent experiments), some error bars are too small to be visualized. c, Effect of compound 1 on 293T cGAS ENPP1 - / - The inhibitory activity of human ENPP1 transiently expressed in cells (IC 50 =340±160 nM). Mean±SEM (n=2). d, 293T cGAS ENPP1 cells transfected with empty pcDNA vector or vector containing human ENPP1 in the presence or absence of 10 μM compound 1 - / - Intracellular and extracellular cGAMP concentrations of cells. BQL = below limit of quantitation. Mean ± SEM (n = 3). ****P < 0.0001 (one-way ANOVA). Data represent two independent experiments. e, Schematic diagram of conditioned medium experiment. 293T cGAS ENPP1 低 Cells were transfected with a vector containing human ENPP1 and incubated in the presence or absence of compound 1. Conditioned media from these cells were transferred to primary CD14 + Human PBMC. f, IFNB1 mRNA levels were normalized to CD14 and compared to untreated CD14 + The fold induction of cells was calculated. Mean ± SEM (n = 2). **P = 0.007 (one-way ANOVA). The cGAMP concentration was measured in the conditioned medium. Mean ± SEM (n = 2). **P = 0.006 (one-way ANOVA). The data represent two independent experiments.

[0799] Fig.10 , Panels A to B: Improvement of compound 1 relative to QS1

[0800] a, Structure of QS1 and its activity at pH 7.5 32 P-cGAMP as a substrate for the inhibitory activity of purified mouse ENPP1 (compared with compound 1) (QS1 K i,app =6.4±3.2 μM). Mean±SEM (n=2 independent experiments). b, 293T cGAS ENPP1 cells transfected with empty vector or vector containing human ENPP1 in the presence or absence of QS1 - / - Intracellular, extracellular and total cGAMP of cells. Mean ± SEM (n = 2). *P < 0.05. **P < 0.01 (one-way ANOVA).

[0801] Fig.11 ,Panels A to F: Compound 1 is cell-impermeable, specific for ENPP1, and non-toxic. a, Permeability of compound 1 in the artificial membrane permeability assay (PAMPA).

[0802] b, Permeability of compound 1 in the intestinal cell Caco-2 assay. PA = peak area, IS = internal standard. Compounds (including compound 1), atenolol (low passive permeability negative control) and propranolol (high passive permeability positive control) were incubated on the apical surface of the Caco-2 monolayer for 2 h. The basolateral compound concentration was monitored by LC-MS / MS. The apparent permeability (P app ). Data are representative of two independent experiments. c, Permeability of compound 1 in epithelial MDCK permeability assay. d, Inhibitory activity of compound 1 against alkaline phosphatase (ALPL) and ENPP2. Mean ± SEM (n = 2). e, Kinome interaction map of compound 1 (468 kinases tested), kinase inhibition is depicted as a percentage of control. Using TREEspot TM Software tools generate images, KINOME (a division of DiscoveRx, Inc.) DiscoveRX, Inc. 2010. f, Cell viability as determined by CellTiterGlo. Total PBMC and CD14 + PBMCs were incubated with compound 1 for 16 hours, and then ATP levels were detected using CellTiterGlo. Data were normalized to the absence of compound 1 to calculate the percentage of cell viability.

[0803] Cancer cells express cGAS and continuously export cGAMP in culture

[0804] To determine whether extracellular cGAMP could serve as a danger signal secreted by cancer cells in vivo, we first sought to identify tumor models that export cGAMP. We tested one human cancer cell line (MDA-MB-231) and three mouse cancer cell lines (E0771, MC38, and 4T1-Luc, a luciferase-expressing 4T1 cell line used for in vivo imaging) in culture, all of which express cGAS ( Figure 4 , Panel A). Intracellular cGAMP concentrations in these cells were difficult to detect. However, with additional concentration and purification steps, we were able to detect 5.8 x 10 -10 nmol / cell (~150nM) intracellular cGAMP ( Figure 4, Panel B). Knockdown of cGAS using shRNA resulted in decreased cGAS protein levels and decreased intracellular cGAMP levels, demonstrating that cGAS expression controls the amount of cGAMP present in 4T1-Luc cells ( Fig.12 , Panels A and B). Using compound 1 to inhibit both cell surface and soluble ENPP1 in the cell culture medium, we detected sustained cGAMP export in all these cell lines, with extracellular cGAMP levels reaching approximately 6 x 10 -9 nmol / cell (approximately 10 nM when diluted into culture medium) Figure 4 , panels C and D and Fig.12 , panels C and D). Notably, this is approximately 10 times the amount of cGAMP present in the cell, suggesting that cancer cells effectively clear their cGAMP by exporting it. Ionizing radiation (IR) increases cytoplasmic DNA and activates cGAS-dependent IFN-β production in tumor cells (see, e.g., Bakhoum, SF et al. Numerical chromosomal instability mediates susceptibility to radiation treatment. Nat. Commun. 6, 1–10 (2015); and Vanpouille-Box, C et al. DNA exonuclease Trex1 regulates radiotherapy-induced tumour immunogenicity. Nat. Commun. 8, 15618 (2017)). In fact, IR treatment also increased the production of extracellular cGAMP in 4T1-Luc cells 2 days later ( Figure 4 , small picture E and Fig.12 , Panel E). In summary, our data indicate that these cancer cell lines continuously produce and efficiently export cGAMP and can be stimulated with IR to produce more extracellular cGAMP.

[0805] Figure 4,Panels A to E: Cancer cells express cGAS and continuously export cGAMP in culture. a, Analysis of cGAS expression by 4T1-Luc, E0771, MDA-MB-231, and MC38 by Western blot. b, Estimation of intracellular cGAMP concentration in 4T1-Luc cells without exogenous stimulation. Mean ± SEM (n = 2). c, Extracellular cGAMP produced by MC38 cells over 48 h. At time 0, cells were refreshed with medium supplemented with 50 μM compound 1. Mean ± SEM (n = 2). Data represent two independent experiments. d, Extracellular cGAMP produced by 4T1-Luc, E0771, and MDA-MB-231 cells was measured after 48 h in the presence of 50 μM compound 1. BQL = below the limit of quantification. Mean ± SEM (n = 2). e, Extracellular cGAMP produced by 4T1-Luc cells over 48 h. At time 0, cells were either untreated or treated with 20 Gy IR and refreshed with medium supplemented with 50 μM Compound 1. Mean ± SEM (n=2). *P=0.04 (Student's t-test).

[0806] Fig.12 , Panels A to E: Cancer cells continuously export cGAMP in culture. a, cGAS expression in 4T1-Luc WT and 4T1-Luc shcGAS cell lines analyzed by Western blot. b, Intracellular cGAMP in 4T1-Luc WT and 4T1-Luc shcGAS cell lines without exogenous stimulation. Mean ± SEM (n = 2). 4T1-Luc WT data from Figure 4 , repeated in panel b for comparison. c, Figure 4 ,Extracellular cGAMP (expressed in medium concentration units) for the experiment shown in panel c. d, Figure 4 ,Extracellular cGAMP (expressed in medium concentration units) for the experiment shown in panel d. BQL = below limit of quantification. Mean ± SEM (n = 2). e, Figure 4 , Extracellular cGAMP (expressed in medium concentration units) from the experiment shown in panel c. Mean ± SEM (n = 2). **P = 0.004 (Student's t test).

[0807] Sequestration of extracellular cGAMP reduces tumor dependence on tumor cGAS and host STING Related dendritic cells

[0808] In tumors, the extracellular space is estimated to be 0.3-0.8 times the volume of the intracellular space. 28 However, in cell culture, the volume of the extracellular space is approximately 250-1000 times the volume of the intracellular space. 6We used 1 mL of culture medium per cell and estimated that approximately 1–4 pL of cell volume was used to make this calculation. Therefore, our cell culture system dilutes the extracellular space 300–3000-fold compared to the tumor microenvironment. Given this dilution factor and our measurements of nanomolar extracellular cGAMP exported by cancer cells in vitro, we predict that extracellular cGAMP in the tumor microenvironment could reach the micromolar range, which could result in innate immune recognition of tumor cells. Recognizing the limitations of our in vitro cell experiments, we turned to in vivo experiments to investigate the role of extracellular cGAMP ( Figure 5 First, we attempted to knock down cGAS in tumor cells ( Fig.13 , Panel A) and in the C57BL / 6 background using cGAS - / - and STING - / - mice to determine the importance of tumor-relative host cGAMP. We also developed a neutralizing agent as a tool to specifically sequester extracellular cGAMP ( Figure 5 , Panel A). We used the soluble cytoplasmic domain of STING ( Figure 5 , panel B), with a K of 73±14 nM d Combined with cGAMP ( Figure 5 , Panel C). We also generated the R237A mutant STING (see, e.g., Gao, P. et al., Cell 154, 748–762 (2013)) as a non-binding STING control ( Figure 5 To test the neutralizing efficacy of these proteins in cell culture, we used CD14 + PBMCs. Wild-type (WT) STING (neutralizing STING) was able to neutralize extracellular cGAMP at a predicted 2:1 stoichiometry, whereas unbound STING had no effect even at 200-fold higher concentrations ( Figure 5 ,panel E).

[0809] We established E0771 orthotopic tumors in mice, then injected neutralizing STING intratumorally to deplete extracellular cGAMP, and excised the tumors to stain tumor-associated leukocytes. In WT E0771 tumors, neutralizing STING significantly reduced total CD45 + / MHC-II + CD11c in tumor-associated antigen presenting cell (APC) populations + dendritic cell populations, suggesting that the immune system can detect extracellular cGAMP ( Figure 5 , small pictures F and G and Fig.13 , Panel B). When the tumor is in cGAS - / - Depletion of extracellular cGAMP also reduced CD11c when grown in mice+ This suggests that host cells do not contribute significantly to the production of extracellular cGAMP ( Figure 5 , thumbnail G and Fig.13 , Panel B). In contrast, when cGAS - / - E0771 cells (pool multiple clones to achieve clean knockout but minimize clonal effects) or STING - / - Depletion of extracellular cGAMP does not affect CD11c in mice + This suggests that tumor cells, rather than host cells, are the primary producers of extracellular cGAMP, which is then sensed by the host STING ( Figure 5 , thumbnail G and Fig.13 , Panel B). We also tested an orthotopic 4T1-Luc tumor model in a BALB / c background. Although cGAS and STING knockout strains have not been established in this background, we knocked out cGAS in 4T1-Luc tumors. Intratumoral injection of neutralized STING into WT 4T1-Luc tumors significantly reduced CD45 + / MHC II + Tumor-associated CD11c + group( Figure 5 , thumbnail H and Fig.13 , Panel C). In contrast, depletion of extracellular cGAMP had an effect on cGAS - / - 4T1-Luc tumors had no effect ( Figure 5 , thumbnail H and Fig.13 , Panel C). We also depleted extracellular cGAMP by intratumoral injection of mENPP1 protein ( Figure 8 , panel D), and CD45 was observed again + / MHC II + CD11c + Cell reduction ( Figure 5 , small picture I and Fig.13 , Panel D). Together, our results from the E0771 and 4T1-Luc models demonstrate that extracellular cGAMP produced by tumor cells activates innate immune responses in a manner that is dependent on host STING but independent of host cGAS. In summary, our data suggest that extracellular cGAMP produced by cancer cells is a danger signal that triggers innate immune responses.

[0810] Figure 5Panels A to I: Sequestration of extracellular cGAMP reduces tumor-associated dendritic cells in a tumor cGAS- and host STING-dependent manner. a, Experimental setup to evaluate the effects of extracellular cGAMP in vivo. b, Coomasie gels of recombinant mouse WT STING and R237A STING. c, Binding curves of the cytoplasmic domains of mouse WT STING (neutralized) and R237A STING (non-binding) using radiolabeled 35 Determined by membrane binding assay using S-cGAMP as probe. Mean ± SEM (n = 2, obtained from two independent experiments). d, Crystal structure of mouse WT STING in complex with cGAMP, with R237 highlighted in pink (PDB ID 4LOJ). e, CD14 treated with 2 μM cGAMP in the presence of neutralizing or non-binding STING (2 μM to 100 μM, 2.5-fold dilution). + Fold induction of IFNB1 mRNA in PBMCs. Mean ± SEM (n = 2 qPCR...

Claims

1. A compound of formula (I), or a pharmaceutically acceptable salt thereof: in, X 1 is a hydrophilic head group selected from (a) or (b) Where R 81 and R 91 each independently is hydrogen; A is a ring system selected from the group consisting of phenyl, piperidine and piperazine; L 1 and L 2 independently covalently bonded or C 1-6 alkyl; Z 3 Does not exist or is NR 22 ; Z 2 CR 12 ; Z 1 is N; R 1 Selected from H, C 1-6 Alkyl and C2-C6 alkenyl substituted by pyridyl; R 12 is cyano; R 22 Selected from H and C 1-6 Alkyl; and R 2 To R 5 Each independently selected from H, C 1-6 Alkoxy and halogen.

2. The compound according to claim 1, wherein the compound is a compound of formula (II): Where Z 31 NR 22 .

3. The compound according to claim 2, wherein the compound is a compound of formula (III): Where R 31 To R 34 each independently is H; and n and m are each independently an integer of 0 to 6. The compound according to claim 3 , wherein n+m is 0 to 3.

5. The compound according to any one of claims 1 to 4, wherein the ring system A is: in: Each R 6 is hydrogen; and p is an integer from 0 to 4.

6. The compound according to claim 3, wherein the compound is a compound of formula (IV): in: Z 11 is N; Z 21 is C(CN); Each R 6 are independently H; and p is an integer from 0 to 4.

7. The compound according to claim 6, wherein the compound is a compound of formula (VIb):

8. A compound according to any one of claims 1 to 4, wherein R 1 is selected from H and vinylpyridyl.

9. The compound according to claim 7, wherein the compound is a compound of formula (VIIb):

10. A compound according to any one of claims 1 to 4, wherein R 2 To R 5 Each independently selected from H, C (1-6) Alkoxy, F, Cl and Br.

11. A compound according to any one of claims 1 to 4, wherein: R 3 and R 4 Independently for C 1-6 Alkoxy; and R 2 and R 5 For hydrogen.

12. A compound according to any one of claims 1 to 4, wherein: R 3 C 1-6 Alkoxy; and R 2 , R 4 and R 5 For hydrogen.

13. A compound according to any one of claims 1 to 4, wherein: R 4 C 1-6 Alkoxy; and R 2 , R 3 and R 5 For hydrogen.

14. A compound according to any one of claims 1 to 4, wherein: R 2 , R 3 and R 4 is H; and R 5 C 1-6 Alkoxy.

15. The compound according to claim 3, wherein n is 0, and m is 1.

16. The compound according to claim 3, wherein n is 1, and m is 0.

17. The compound according to claim 3, wherein n and m are both 1.

18. The compound according to claim 3, wherein n and m are both 0.

19. The compound according to claim 1, wherein Z 3 does not exist, and Z 2 CR 12 , where R 12 is a cyano group, wherein the compound is a compound of formula (X): Where L 11 and L 12 independently covalently bonded or C 1-6 alkyl.

20. The compound according to claim 19, wherein the compound is a compound of formula (XI): in: Each Z 5 Independently selected from N and CR 16 , where at least one Z 5 is N; Each R 16 are independently hydrogen; and r is an integer from 0 to 8.

21. The compound according to claim 20, wherein the compound is a compound of formula (XII):

22. A compound according to claim 21, wherein Z 5 is N.

23. The compound according to claim 22, wherein the compound is a compound of formula (XIII): R 35 and R 36 each independently is H; and Here, s is an integer from 0 to 6.

24. The compound according to claim 23, wherein the compound is a compound of formula (XIV):

25. A compound according to claim 19, wherein R 2 To R 5 Independently selected from H, C (1-6) Alkoxy, F, Cl and Br.

26. A compound according to claim 19, wherein: R 3 and R 4 Independently for C 1-6 Alkoxy; and R 2 and R 5 For hydrogen.

27. A compound according to claim 19, wherein: R 3 C 1-6 Alkoxy; and R 2 , R 4 and R 5 For hydrogen.

28. A compound according to claim 19, wherein: R 4 C 1-6 Alkoxy; and R 2 , R 3 and R 5 For hydrogen.

29. A compound according to claim 19, wherein: R 2 , R 3 and R 4 is H; and R 5 C 1-6 Alkoxy.

30. A compound according to claim 23 or 24, wherein s is 1.

31. A compound according to claim 23 or 24, wherein s is 2.

32. A compound according to claim 23 or 24, wherein s is 3.

33. A compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein the compound is selected from:

34. A compound according to claim 1, wherein Z 3 does not exist, and Z 2 CR 12 , where R 12 is a cyano group, wherein the compound is a compound of formula (X): Where L 11 and L 12 independently covalently bonded or C 1-6 alkyl.

35. The compound according to claim 34, wherein the compound is a compound of formula (XI): in: Each Z 5 Independently selected from N and CR 16 , where at least one Z 5 is N; Each R 16 are independently hydrogen; and r is an integer from 0 to 8.

36. A compound according to claim 34, wherein the compound is a compound of formula (XII):

37. A compound according to claim 36, wherein Z 5 is N.

38. The compound according to claim 35, wherein the compound is a compound of formula (XIII): R 35 and R 36 each independently is H; and Here, s is an integer from 0 to 6.

39. The compound according to claim 35, wherein the compound is a compound of formula (XIV):

40. A compound according to any one of claims 34 to 38, wherein R 2 To R 5 Each independently selected from H, C (1-6) Alkoxy, F, Cl and Br.

41. A pharmaceutical composition comprising: Means for inhibiting ENPP1 function, wherein the means for inhibiting ENPP1 function is an ENPP1 inhibitor according to any one of claims 1 to 3, 19 and 33; and Pharmaceutically acceptable excipients.

42. Use of the ENPP1 inhibitor of any one of claims 1-3, 19 and 33 for the preparation of a medicament for inhibiting ENPP1 in a sample comprising ENPP1.

43. The use according to claim 42, wherein the ENPP1 inhibitor is cell-impermeable.

44. Use according to claim 42, wherein the ENPP1 inhibitor is cell permeable.

45. Use according to claim 42, wherein the sample is a cell sample.

46. ​​Use according to claim 45, wherein the sample comprises cGAMP.

47. The use according to claim 46, wherein cGAMP levels are elevated in the cell sample relative to a control sample not contacted with the ENPP1 inhibitor.

48. Use of an ENPP1 inhibitor according to any one of claims 1-3, 19 and 33 in the preparation of a medicament for treating an ENPP1 -associated cancer in a subject.

49. Use according to claim 48, wherein the cancer is a solid tumor cancer.

50. The method according to claim 48, wherein the cancer is selected from the group consisting of adrenal cancer, liver cancer, kidney cancer, bladder cancer, breast cancer, colon cancer, stomach cancer, ovarian cancer, cervical cancer, uterine cancer, esophageal cancer, colorectal cancer, prostate cancer, pancreatic cancer, lung cancer, thyroid cancer, malignant epithelial tumors, sarcomas, glioblastomas, melanomas, and various head and neck tumors.

51. The use according to claim 50, wherein the lung cancer is small cell lung cancer.

52. The use according to claim 50, wherein the lung cancer is non-small cell lung cancer.

53. Use according to claim 50, wherein the cancer is breast cancer.

54. Use according to claim 48, wherein the cancer is lymphoma.

55. Use according to claim 50, wherein the cancer is glioblastoma.

56. The use according to claim 48, wherein the medicament is administered to a subject together with one or more additional active agents.

57. Use according to claim 56, wherein the one or more additional active agents are chemotherapeutic agents or immunotherapeutic agents.

58. The use according to claim 56, wherein the one or more additional active agents are small molecules, antibodies, antibody fragments, antibody-drug conjugates, aptamers or proteins.

59. The use according to claim 56, wherein the one or more additional active agents include a checkpoint inhibitor.

60. The use according to claim 59, wherein the checkpoint inhibitor is selected from a cytotoxic T-lymphocyte-associated antigen 4 inhibitor, a programmed death 1 inhibitor and a PD-L1 inhibitor.

61. The use according to claim 56, wherein the one or more additional active agents comprises a chemotherapeutic agent.

62. Use according to claim 61, wherein the chemotherapeutic agent is a cGAMP inducing chemotherapeutic agent.

63. The use according to claim 62, wherein the cGAMP-inducing chemotherapeutic agent is an anti-mitotic agent or an anti-tumor agent administered in an amount effective to induce cGAMP production in the subject.

64. The use according to claim 48, wherein the medicament is to be administered to a subject together with radiation therapy.

65. The use according to claim 64, wherein the medicament is administered to the subject prior to radiotherapy.

66. The use according to claim 64, wherein the medicament is administered after the subject has been exposed to radiation therapy.

67. The use according to claim 65, wherein the radiation therapy is administered at a dose and / or frequency effective to reduce radiation damage to the subject.

68. The use according to claim 48, wherein the ENPP1 inhibitor is cell-impermeable.

69. The use according to claim 48, wherein the ENPP1 inhibitor is cell permeable.

70. Use of an ENPP1 inhibitor according to any one of claims 1-3, 19 and 33 for the preparation of a medicament for the treatment of an inflammatory disorder associated with ENPP1.

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