ENPP1 inhibitors and methods for modulating immune responses

Abstract

Compounds, compositions, and methods for inhibiting ENPP1 are provided. Embodiments of the subject methods 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 and block the degradation of cGAMP. Similarly, pharmaceutical compositions and methods for treating cancer are provided. Embodiments of the method include administering a therapeutically effective amount of an ENPP1 inhibitor to a subject to treat the cancer in the subject. In certain cases, the cancer is a solid tumor cancer. Similarly, methods are provided in which radiation therapy is administered to a subject in conjunction with administering an ENPP1 inhibitor to the subject. In the subject methods, radiation therapy can be administered at a dosage and / or frequency effective to reduce radiation damage to the subject while still eliciting an immune response.

Description

[Technical Field]

[0001] This application claims priority to and the benefit of U.S. Provisional Patent Application No. 62 / 800,283, filed February 1, 2019, and U.S. Provisional Patent Application No. 62 / 814,745, filed March 6, 2019, each of which is incorporated by reference in its entirety. Government Rights

[0002] 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 technology]

[0003] Introduction Cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) activates the stimulator of interferon genes (STING) pathway, a key anti-cancer innate immune pathway. The cGAS-cGAMP-STING pathway is activated in the presence of cytosolic DNA due to either microbial infection or pathophysiological conditions, including cancer and innate immune disorders. Cyclic GMP-AMP synthase (cGAS), a member of the nucleotidyl transferase family, is a versatile DNA sensor that is activated upon binding to cytosolic dsDNA to produce the signaling molecule (2'-5',3'-5') cyclic GMP-AMP (or 2',3'-cGAMP, or cyclic guanosine monophosphate-adenosine monophosphate, cGAMP). Acting as a second messenger during microbial infection, 2',3'-cGAMP binds to and activates STING, leading to the production of type I interferons (IFNs) and other costimulatory 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.

[0004] Ecto-nucleotide pyrophosphatase / phosphodiesterase 1 (ENPP1) is the major hydrolase of cGAMP that can degrade cGAMP. ENPP1 is a member of the ecto-nucleotide pyrophosphatase / phosphodiesterase (ENPP) family and is a type II transmembrane glycoprotein containing two identical disulfide-linked subunits. ENPP1 has broad specificity, cleaving various substrates, including the phosphodiester bond of nucleotides and nucleotide sugars, as well as the pyrophosphate bond of nucleotides and nucleotide sugars. ENPP1 can hydrolyze nucleoside 5' triphosphates to their corresponding monophosphates and can also hydrolyze diadenosine polyphosphates. Summary of the Invention [Means for solving the problem]

[0005] Compounds, compositions, and methods for inhibiting ENPP1 are provided. Embodiments of the subject methods 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 and block the degradation of cGAMP. Similarly, pharmaceutical compositions and methods for treating cancer are provided. Embodiments of the method include administering a therapeutically effective amount of an ENPP1 inhibitor to a subject to treat the cancer in the subject. In certain cases, the cancer is a solid tumor cancer. Similarly, methods are provided in which radiation therapy is administered to a subject in conjunction with administering an ENPP1 inhibitor to the subject. In the subject methods, radiation therapy can be administered at a dosage and / or frequency effective to reduce radiation damage to the subject while still eliciting an immune response.

[0006] These and other advantages and features of the present disclosure will become apparent to those skilled in the art upon reading the detailed description of the compositions and methods of use, which is set forth more fully below.

[0007] The invention is best understood from the following detailed description when read in connection with the accompanying drawings. This patent or application file contains at least one drawing executed in color. It is emphasized that, according to common practice, the various features of the drawings are not to scale. To the contrary, the dimensions of the various features have been arbitrarily enlarged or reduced for clarity. The following figures are included in the drawings: It is understood that the drawings described below are for illustrative purposes only. The figures are not intended to limit the scope of the present teachings in any way. [Brief explanation of the drawings]

[0008] [Figure 1-1] Panels A-J of Figure 1 show experimental results demonstrating that cGAMP is exported from 293T cGAS ENPP1 − / − cells as a soluble factor. [Figure 1-2] Panels A-J of Figure 1 show experimental results demonstrating that cGAMP is exported from 293T cGAS ENPP1 − / − cells as a soluble factor. [Figure 1-3] Panels A-J of Figure 1 show experimental results demonstrating that cGAMP is exported from 293T cGAS ENPP1 − / − cells as a soluble factor.

[0009] [Figure 2] Panels A to C of Figure 2 show experimental results demonstrating that ENPP1 can regulate extracellular cGAMP.

[0010] [Figure 3] Panels A-F of Figure 3 illustrate the structure of an exemplary ENPP1 inhibitor (compound 1) and its activity in various cellular assays.

[0011] [Figure 4] Panels A-E of Figure 4 show experimental results demonstrating that cancer cells express cGAS in culture and continuously excrete cGAMP.

[0012] [Figure 5-1] Panels A-I of Figure 5 show experimental results demonstrating that sequestration of extracellular cGAMP reduces tumor-associated dendritic cells in a tumor cGAS- and host STING-dependent manner. [Figure 5-2] Panels A-I of Figure 5 show experimental results demonstrating that sequestration of extracellular cGAMP reduces tumor-associated dendritic cells in a tumor cGAS- and host STING-dependent manner. [Figure 5-3] Panels A-I of Figure 5 show experimental results demonstrating that sequestration of extracellular cGAMP reduces tumor-associated dendritic cells in a tumor cGAS- and host STING-dependent manner.

[0013] [Figure 6-1] Figure 6, panels A-D, show experimental results demonstrating that ENPP1- / - tumor-mobilized innate immune infiltrates are less aggressive and more sensitive to IR and anti-CTLA-4 (cytotoxic T-lymphocyte-associated antigen 4) therapy. [Figure 6-2] Figure 6, panels A-D, show experimental results demonstrating that ENPP1- / - tumor-mobilized innate immune infiltrates are less aggressive and more sensitive to IR and anti-CTLA-4 (cytotoxic T-lymphocyte-associated antigen 4) therapy. [Figure 6-3] Figure 6, panels A-D, show experimental results demonstrating that ENPP1- / - tumor-mobilized innate immune infiltrates are less aggressive and more sensitive to IR and anti-CTLA-4 (cytotoxic T-lymphocyte-associated antigen 4) therapy.

[0014] [Figure 7] Panels AC of Figure 7 show experimental results demonstrating that ENPP1 inhibition synergizes with IR treatment and anti-CTLA-4 to exert an anti-tumor effect.

[0015] [Figure 8-1] Panels A-D of Figure 8 illustrate the LC-MS / MS method for assessing ENPP1 hydrolytic activity and cGAMP levels and the use of 293T cGAS ENPP1low and 293T cGAS ENPP1- / - cell lines. [Figure 8-2]Panels A-D of Figure 8 illustrate the LC-MS / MS method for assessing ENPP1 hydrolytic activity and cGAMP levels and the use of 293T cGAS ENPP1low and 293T cGAS ENPP1- / - cell lines.

[0016] [Figure 9] Panels A-B of Figure 9 show an experimental schematic and results illustrating that CD14+ primary human peripheral blood mononuclear cells (PBMCs) respond to extracellular cGAMP.

[0017] [Figure 10] Panels AB of FIG. 10 show experimental results comparing the ENPP1 inhibitory activity of compound 1 and compound QS1 and demonstrating the activity of QS1 in a cellular assay.

[0018] [Figure 11-1] Panels A-F of Figure 11 show experimental results demonstrating that an exemplary ENPP1 inhibitor, Compound 1 (STF-1084), is cell-impermeable, specific for ENPP1, and non-toxic. [Figure 11-2] Panels A-F of Figure 11 show experimental results demonstrating that an exemplary ENPP1 inhibitor, Compound 1 (STF-1084), is cell-impermeable, specific for ENPP1, and non-toxic.

[0019] [Figure 12] Panels A-E of Figure 12 show experimental results demonstrating that cancer cells continuously excrete cGAMP in culture.

[0020] [Figure 13] Panels A to D of Figure 13 show experimental results demonstrating that sequestration of extracellular cGAMP reduces tumor-associated dendritic cells in a tumor cGAS- and host STING-dependent manner.

[0021] [Figure 14-1]Figure 14, panels A-F, show experimental results demonstrating that established ENPP1- / - tumors develop an increase in tumor-associated dendritic cells, are less invasive, and are more sensitive to IR and anti-CTLA-4 therapy. [Figure 14-2] Figure 14, panels A-F, show experimental results demonstrating that established ENPP1- / - tumors develop an increase in tumor-associated dendritic cells, are less invasive, and are more sensitive to IR and anti-CTLA-4 therapy.

[0022] [Figure 15] FIG. 15 shows a graph of data demonstrating that ENPP1 inhibition (eg, using Compound 1; STF-1084) synergizes with IR treatment to increase tumor-associated dendritic cells.

[0023] [Figure 16] FIG. 16 shows a schematic diagram illustrating various modes of cGAMP transfer from the synthesizing cell to the target cell.

[0024] [Figure 17] FIG. 17 shows a schematic illustrating that cGAMP is a cancer danger signal secreted by cancer cells in vivo.

[0025] [Figure 18] 18A-18C show data illustrating that an exemplary ENPP1 inhibitor (compound 1) can increase the amount of extracellular cGAMP present in a cell line.

[0026] [Figure 19] 19A-19B show experimental schematics and results illustrating that an exemplary ENPP1 inhibitor (compound 1) can increase cGAMP-stimulated interferon transcription.

[0027] [Figure 20]20A-20B show data illustrating that an exemplary ENPP1 inhibitor (compound 1) can increase the number of tumor-associated dendritic cells in a mouse tumor model.

[0028] [Figure 21-1] 21A-21C show experimental results illustrating that ENPP1 inhibition synergizes with IR treatment and anti-CTLA-4 to exert an anti-tumor effect. [Figure 21-2] 21A-21C show experimental results illustrating that ENPP1 inhibition synergizes with IR treatment and anti-CTLA-4 to exert an anti-tumor effect.

[0029] [Figure 22] FIG. 22 shows a schematic illustrating that ENPP1 is an innate immune checkpoint that regulates the immune mediator cGAMP. DETAILED DESCRIPTION OF THE INVENTION

[0030] definition Before further describing the embodiments of the present disclosure, it is to be understood that this disclosure is not limited to particular embodiments described, which may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. 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.

[0032] It should be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, a reference to "a compound" includes not only a single compound but also a combination of two or more compounds; a reference to "a substituent" includes a single substituent and two or more substituents, and so forth.

[0033] In describing and claiming the present invention, certain terms will be used in accordance with the definitions set forth below. It will be understood that the definitions provided herein are not intended to be mutually exclusive. Thus, a chemical moiety may fall within the definition of more than one term.

[0034] The phrases "for example," "for instance," "such as," or "including" are intended to incorporate examples that further clarify the more general subject matter. These examples are presented solely to aid in understanding the disclosure and are not intended to be limiting in any way.

[0035] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such publications by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates, which may need to be independently confirmed.

[0036] The terms "active agent," "antagonist," "inhibitor," "drug," and "pharmacologically active agent" are used interchangeably herein to refer to chemical substances or compounds that, when administered to an organism (human or animal), elicit a desired pharmacological and / or physiological effect through local and / or systemic action.

[0037] The terms "treatment," "treating," and the like refer to obtaining a desired pharmacological and / or physiological effect, such as a reduction in tumor burden. This effect can be prophylactic, in that a disease or its symptoms are completely or partially prevented, and / or therapeutic, in that a partial or complete cure is achieved for the disease and / or adverse effects that may result from the disease. "Treatment" is intended to encompass any treatment of disease in a mammal, particularly a human, and includes: (a) preventing the occurrence of a disease or a symptom of the disease (including, for example, a disease that may be associated with or caused by a primary disease (such as in liver fibrosis, which may result in the setting of chronic HCV infection)) in a subject who may be susceptible to the disease but has not yet been diagnosed with the disease; (b) inhibiting the disease, i.e., halting its development; and (c) palliating the disease, i.e., causing regression of the disease (e.g., a reduction in tumor burden).

[0038] The term "pharmaceutically acceptable salt" refers to a salt that is acceptable for administration to a patient, such as a mammal (a salt with a counterion that is safe for the mammal for a given dosage regimen). Such salts can be derived from pharmaceutically acceptable inorganic or organic bases and from pharmaceutically acceptable inorganic or organic acids. "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, including, by way of example only, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, etc., and, if the molecule contains a basic functional group, salts of organic or inorganic acids such as hydrochloride, hydrobromide, formate, tartrate, besylate, mesylate, acetate, maleate, oxalate, etc.

[0039] 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. "Mammals" refers to members or members of any mammalian species, including, for example, canines; felines; equines; bovines; ovines; rodents, etc., and 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.

[0040] The terms "determining," "measuring," "assessing," and "assaying" are used interchangeably and include both quantitative and qualitative determinations.

[0041] The terms "polypeptide" and "protein," used interchangeably herein, refer to polymeric forms of amino acids of any length, including coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides with modified peptide backbones. The terms include, but are not limited to, fusion proteins with heterologous amino acid sequences, which are fusions with heterologous sequences and native leader sequences, with or without an N-terminal methionine residue; immunologically tagged proteins; and fusion proteins with a detectable fusion partner, such as fusion proteins containing a fluorescent protein, β-galactosidase, luciferase, etc., as a fusion partner.

[0042] 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. Polynucleotides may have any three-dimensional structure and may perform any function, known or unknown. 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, regulatory regions, isolated RNA of any sequence, nucleic acid probes, and primers. Nucleic acid molecules can be linear or circular.

[0043] A "therapeutically effective amount" or "effective amount" means the amount of a compound that, when administered to a mammal or other subject for treating a disease, condition, or disorder, is sufficient to effect such treatment for the disease, condition, or disorder. The "therapeutically effective amount" will vary depending on the compound, the disease and its severity, and the age, weight, etc., of the subject being treated.

[0044] The term "unit dosage form," as used herein, refers to a physically discrete unit suitable as a unitary administration for human and animal subjects, each unit containing a predetermined amount of a compound (e.g., an aminopyrimidine compound described herein) calculated in an amount sufficient to produce a desired effect, in association with a pharmaceutically acceptable diluent, carrier, or vehicle. The specifications for the unit dosage form depend on the particular compound used and the effect to be achieved, and the pharmacodynamics associated with each compound in the host.

[0045] The terms "pharmaceutically acceptable excipient," "pharmaceutically acceptable diluent," "pharmaceutically acceptable carrier," and "pharmaceutically acceptable adjuvant" refer to generally safe, non-toxic, and not biologically or otherwise undesirable excipients, diluents, carriers, or adjuvants useful in preparing pharmaceutical compositions, and include excipients, diluents, carriers, and adjuvants acceptable for veterinary and human pharmaceutical use. As used herein and in the claims, "pharmaceutically acceptable excipients, diluents, carriers, and adjuvants" includes both one and more than one such excipients, diluents, carriers, and adjuvants.

[0046] The term "pharmaceutical composition" is intended to encompass compositions suitable for administration to a subject, such as a mammal, especially a human. Generally, a "pharmaceutical composition" is sterile and preferably free of contaminants capable of eliciting an undesired response in a subject (e.g., the compound(s) in a pharmaceutical composition are pharmaceutical grade). Pharmaceutical compositions can be designed to be administered to a subject or patient in need thereof via several different routes of administration, including oral, buccal, rectal, parenteral, intraperitoneal, intradermal, intratracheal, intramuscular, subcutaneous, etc.

[0047] The phrases "having the formula" or "having the structure" are not intended to be limiting and are used in the same manner as the term "comprising" is generally used. The term "independently selected from" is used herein to indicate that the elements being described, such as R groups, can be the same or different.

[0048] The terms "may," "optional," "optionally," or "optionally may" mean that the subsequently described circumstance may or may not occur, and such that the description includes instances where the circumstance occurs and instances where the circumstance does not occur. For example, the phrase "optionally substituted" means that a non-hydrogen substituent may or may not be present on a given atom, and such that the description includes structures where the non-hydrogen substituent is present and structures where the non-hydrogen substituent is not present.

[0049] "Acyl" refers to the groups 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)-, where alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein. For example, acyl includes the "acetyl" group CH3C(O)-.

[0050] The term "alkyl" refers to a branched or unbranched saturated hydrocarbon group (i.e., a monoradical) typically, but not necessarily, containing 1 to about 24 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, octyl, decyl, etc., and cycloalkyl groups such as cyclopentyl, cyclohexyl, etc. Generally, although not necessarily, alkyl groups herein can contain 1 to about 18 carbon atoms, and such groups can contain 1 to about 12 carbon atoms. The term "lower alkyl" refers to an alkyl group of 1 to 6 carbon atoms. "Substituted alkyl" refers to an alkyl that is substituted with one or more substituents, including examples in which two hydrogen atoms are replaced from the same carbon atom in an alkyl substituent, such as in a carbonyl group (i.e., a substituted alkyl group can contain 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 with a heteroatom, as further detailed below. The terms "alkyl" and "lower alkyl", unless otherwise indicated, include straight-chain, branched, cyclic, unsubstituted, substituted and / or heteroatom-containing alkyl or lower alkyl, respectively.

[0051] The term "substituted alkyl" refers to an alkyl group, as defined herein, wherein one or more carbon atoms in the alkyl chain are optionally replaced with a heteroatom such as -O-, -N-, -S-, -S(O)n- (n is 0-2), -NR- (R is hydrogen or alkyl), and includes alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thio It is intended to include alkyl groups having from 1 to 5 substituents selected from the group consisting of all, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-aryl, -SO2-heteroaryl and -NRaRb (where R' and R" may be the same or different and are selected from hydrogen, optionally substituted alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, heteroaryl and heterocyclic).

[0052] 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 ethenyl, n-propenyl, isopropenyl, n-butenyl, isobutenyl, octenyl, decenyl, tetradecenyl, hexadecenyl, eicosenyl, tetracosenyl, etc. Generally, although not necessarily, an alkenyl group herein can contain 2 to about 18 carbon atoms, e.g., 2 to 12 carbon atoms. The term "lower alkenyl" contemplates an alkenyl group of 2 to 6 carbon atoms. The term "substituted alkenyl" refers to an alkenyl substituted with one or more substituents, and the terms "heteroatom-containing alkenyl" and "heteroalkenyl" refer to an alkenyl in which at least one carbon atom is replaced by a heteroatom. The terms "alkenyl" and "lower alkenyl", unless otherwise indicated, include straight-chain, branched, cyclic, unsubstituted, substituted and / or heteroatom-containing alkenyl and lower alkenyl, respectively.

[0053] The term "alkynyl" refers to a straight-chain or branched hydrocarbon group of 2 to 24 carbon atoms containing at least one triple bond, such as ethynyl, n-propynyl, etc. Generally, although not necessarily, alkynyl groups herein can contain from 2 to about 18 carbon atoms, and such groups can further contain from 2 to 12 carbon atoms. The term "lower alkynyl" contemplates an alkynyl group of 2 to 6 carbon atoms. The term "substituted alkynyl" refers to an alkynyl group substituted with one or more substituents, and the terms "heteroatom-containing alkynyl" and "heteroalkynyl" refer to an alkynyl group in which at least one carbon atom has been replaced by a heteroatom. Unless otherwise specified, the terms "alkynyl" and "lower alkynyl" include straight-chain, branched, unsubstituted, substituted, and / or heteroatom-containing alkynyl and lower alkynyl, respectively.

[0054] The term "alkoxy" refers to an alkyl group attached through a single, terminal ether linking group. That is, an "alkoxy" group can be represented as -O-alkyl, where 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, t-butyloxy, and the like. Substituents identified herein as "C1-C6 alkoxy" or "lower alkoxy" can contain, for example, 1 to 3 carbon atoms; as a further example, such substituents can contain 1 or 2 carbon atoms (i.e., methoxy and ethoxy). The designations "-OMe" and "MeO-" refer to a methoxy group.

[0055] The term "substituted alkoxy" refers to the groups substituted alkyl-O-, substituted alkenyl-O-, substituted cycloalkyl-O-, substituted cycloalkenyl-O-, and substituted alkynyl-O-, where substituted alkyl, substituted alkenyl, substituted cycloalkyl, substituted cycloalkenyl, and substituted alkynyl are as defined herein.

[0056] The term "aryl," unless otherwise specified, generally refers to an aromatic substituent containing, but not necessarily, 5 to 30 carbon atoms and containing a single aromatic ring or multiple aromatic rings fused together, directly linked, or indirectly linked (thus different aromatic rings are bonded to a common group such as a methylene or ethylene moiety). An aryl group can contain, for example, 5 to 20 carbon atoms; as a further example, an aryl group can contain 5 to 12 carbon atoms. For example, an aryl group can contain one aromatic ring or two or more fused or linked aromatic rings (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 with one or more substituents, and the terms "heteroatom-containing aryl" and "heteroaryl" refer to an aryl substituent in which at least one carbon atom is replaced with a heteroatom, as described in more detail below. Aryl includes stable cyclic, heterocyclic, polycyclic, and heteropolycyclic unsaturated C-C aryls exemplified by, but not limited to, phenyl, biphenyl, naphthyl, pyridyl, furyl, thiophenyl, imidazoyl, pyrimidinyl, and oxazoyl. 14 moieties, which may be further substituted with 1 to 5 members selected from the group consisting of hydroxy, C1-C8 alkoxy, branched or straight chain C1-C8 alkyl, acyloxy, carbamoyl, amino, N-acylamino, nitro, halogen, trifluoromethyl, cyano, and carboxyl (see, e.g., Katritzky, Handbook of Heterocyclic Chemistry). Unless otherwise indicated, the term "aryl" includes unsubstituted, substituted, and / or heteroatom-containing aromatic substituents.

[0057] 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, where "alkyl" and "aryl" are as defined above. Generally, aralkyl and alkaryl groups, as used herein, contain 6 to 30 carbon atoms. Aralkyl and alkaryl groups can, for example, contain 6 to 20 carbon atoms; as a further example, such groups can contain 6 to 12 carbon atoms.

[0058] The term "alkylene" refers to the diradical alkyl 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. "Lower alkylene" refers to alkylene linking groups containing 1 to 6 carbon atoms. Examples include methylene (--CH--), ethylene (--CHCH--), propylene (--CHCHCH--), 2-methylpropylene (--CH--CH(CH)--CH--), hexylene (--(CH)--), and the like.

[0059] Similarly, the terms "alkenylene," "alkynylene," "arylene," "aralkylene," and "alkarylene" refer to the diradical alkenyl, alkynyl, aryl, aralkyl, and alkaryl groups, respectively.

[0060] The term "amino" refers to the group -NRR', where R and R' are independently hydrogen or non-hydrogen substituents, including, for example, alkyl, aryl, alkenyl, aralkyl, and substituted and / or heteroatom-containing variants thereof.

[0061] The terms "halo" and "halogen" are used in the conventional sense to refer to a chloro, bromo, fluoro, or iodo substituent.

[0062] "Carboxyl", "carboxy" or "carboxylate" refers to -CO2H or salts thereof.

[0063] "Cycloalkyl" refers to cyclic alkyl groups of 3 to 10 carbon atoms having monocyclic or polycyclic rings, including fused, bridged, and spirocyclic systems. Examples of suitable cycloalkyl groups include, for example, adamantyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, and the like. Such cycloalkyl groups include, for example, single ring structures such as cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, and the like, or multiple ring structures such as adamantanyl, and the like.

[0064] The term "substituted cycloalkyl" refers to a cycloalkyl group having from one to five substituents or from one to three 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, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl, and -SO2-heteroaryl.

[0065] The term "heteroatom-containing," as in "heteroatom-containing alkyl group" (also referred to as "heteroalkyl" group) or "heteroatom-containing aryl group" (also referred to as "heteroaryl" group), refers to a molecule, linking group, or substituent in which one or more carbon atoms have been replaced with an atom other than carbon, such as nitrogen, oxygen, sulfur, phosphorus, or silicon, usually nitrogen, oxygen, or sulfur. Similarly, the term "heteroalkyl" refers to an alkyl substituent that contains a heteroatom; the term "heterocycloalkyl" refers to a cycloalkyl substituent that contains a heteroatom; the terms "heterocyclic" or "heterocycle" refer to cyclic substituents that contain a heteroatom; the terms "heteroaryl" and "heteroaromatic" refer to "aryl" and "aromatic" substituents, respectively, that contain a heteroatom; and so forth. Examples of heteroalkyl groups include alkoxyaryl, alkylsulfanyl-substituted alkyls, N-alkylated aminoalkyls, and the like. Examples of heteroaryl substituents include pyrrolyl, pyrrolidinyl, pyridinyl, quinolinyl, indolyl, furyl, pyrimidinyl, imidazolyl, 1,2,4-triazolyl, tetrazolyl, and the like; examples of hetero-containing alicyclic groups are pyrrolidino, morpholino, piperazino, piperidino, tetrahydrofuranyl, and the like.

[0066] "Heteroaryl" refers to an aromatic group consisting of 1 to 15 carbon atoms, such as 1 to 10 carbon atoms, within the ring, and 1 to 10 heteroatoms selected from the group consisting of oxygen, nitrogen, and sulfur. Such heteroaryl groups can have a single ring (such as pyridinyl, imidazolyl, or furyl) or multiple fused rings in the ring system (e.g., as in groups such as indolizinyl, quinolinyl, benzofuran, benzimidazolyl, or benzothienyl), where at least one ring in the ring system is aromatic, provided that the point of attachment is through an atom in the aromatic ring. In certain embodiments, the nitrogen and / or sulfur ring atom(s) of a heteroaryl group are optionally oxidized to provide N-oxide (N→O), sulfinyl, or sulfonyl moieties. The term includes, by way of example, pyridinyl, pyrrolyl, indolyl, thiophenyl, and furanyl. Unless otherwise constrained by a definition with respect to heteroaryl substituents, such heteroaryl groups may be optionally substituted with from 1 to 5 substituents or from 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, heterocyclooxy, 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.

[0067] The terms "heterocycle," "heterocyclic," and "heterocyclyl" refer to saturated or unsaturated groups having a single ring or multiple fused rings, including fused, bridged, and spiro ring systems, and having 3 to 15 ring atoms, including 1 to 4 heteroatoms. These ring heteroatoms are selected from nitrogen, sulfur, and oxygen, where in fused ring systems, one or more of the rings can be cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, provided that the point of attachment is through a non-aromatic ring. In certain embodiments, the nitrogen atom(s) and / or sulfur atom(s) of a heterocyclic group are optionally oxidized to provide an N-oxide, -S(O)-, or -SO2- moiety.

[0068] Examples of heterocycles and heteroaryls include, but are not limited to, azetidine, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, dihydroindole, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, phenanthroline, isothiazole, phenazine, benzophenone ... Examples of suitable amines include benzo[b]thiophene, ...

[0069] Unless otherwise constrained by a definition of a heterocyclic substituent, such heterocyclic groups may be optionally substituted with from 1 to 5 substituents or from 1 to 3 substituents selected from alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl, -SO2-heteroaryl, and fused heterocycle.

[0070] "Hydrocarbyl" refers to a monovalent hydrocarbyl radical, such as an alkyl group, an alkenyl group, an aryl group, or the like, 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 species. The hydrocarbyl may be substituted with one or more substituents. The term "heteroatom-containing hydrocarbyl" refers to a hydrocarbyl in which at least one carbon atom has been replaced by a heteroatom. Unless otherwise indicated, the term "hydrocarbyl" should be interpreted as including substituted and / or heteroatom-containing hydrocarbyl moieties.

[0071] By "substituted," as in "substituted hydrocarbyl," "substituted alkyl," "substituted aryl," etc., as implicit in some of the definitions above, it is intended that at least one hydrogen atom bonded to a carbon (or other) atom in a 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 the hydrocarbyl 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-mentioned hydrocarbyl moieties may be further substituted with one or more functional groups or additional hydrocarbyl moieties, such as those specifically described. Unless otherwise indicated, any group described herein should be interpreted to include substituted and / or heteroatom-containing moieties in addition to unsubstituted moieties.

[0072] "Sulfonyl" refers to the groups SO-alkyl, SO-substituted alkyl, SO-alkenyl, SO-substituted alkenyl, SO-cycloalkyl, SO-substituted cylcoalkyl, SO-cycloalkenyl, SO-substituted cylcoalkenyl, SO-aryl, SO-substituted aryl, SO-heteroaryl, SO-substituted heteroaryl, SO-heterocyclic, and SO-substituted heterocyclic, where alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein. Sulfonyl includes, by way of example, methyl-SO-, phenyl-SO-, and 4-methylphenyl-SO-.

[0073] The term "functional group" includes halo, hydroxyl, sulfhydryl, C1-C24 alkoxy, C2-C24 alkenyloxy, C2-C24 alkynyloxy, C5-C20 aryloxy, acyl (including C2-C24 alkylcarbonyl (-CO-alkyl) and C6-C20 arylcarbonyl (-CO-aryl)), acyloxy (-O-acyl), C2-C24 alkoxycarbonyl (-(CO)-O-alkyl), C6-C20 aryloxycarbonyl (-(CO)-O-aryl), halocarbonyl (-CO)-X (wherein X is halo), C2 to C24 alkylcarbonate (-O-(CO)-O-alkyl), C6 to C20 arylcarbonate (-O-(CO)-O-aryl), carboxy (-COOH), carboxylato (-COO-), carbamoyl (-(CO)-NH2), monosubstituted C1 to C24 alkylcarbamoyl (-(CO)-NH(C1 to C24 alkyl)), disubstituted alkylcarbamoyl (-(CO)-N(C1 to C24 alkyl)2), monosubstituted arylcarbamoyl (-(CO)-NH-aryl), thiocarbamoyl (-(CS)-NH2), Carbamide (-NH-(CO)-NH2), cyano (-C≡N), isocyano (-N≡C-), cyanato (-OC≡N), isocyanato (-O-N≡C-), isothiocyanato (-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)-aryl), imino (-CR=NH (R=hydrogen, C1-C24 alkyl, C5-C20 aryl, C6-C20 alkaryl, C6-C20 aralkyl, etc.)), alkylimino (-CR=N(alkyl) (R=hydrogen, alkyl, aryl, alkaryl, etc.)), arylimino (-CR=N(aryl) (R=hydrogen, alkyl, aryl, alkaryl, etc.)), nitro (-NO2), nitroso (-NO), sulfo (-SO2-OH), sulfonato (-SO2-O-), C1-C24 alkylsulfanyl (-S-alkyl;Chemical groups such as 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), phosphinato (-P(O)(O-)), phospho (-PO2) and phosphino (-PH2), mono- and di-(C1-C24 alkyl)-substituted phosphino, mono- and di-(C5-C20 aryl)-substituted phosphines are contemplated. Additionally, the aforementioned functional groups may, where the particular group allows, be further substituted with one or more additional functional groups, such as those specifically described above, or one or more hydrocarbyl moieties;

[0074] By "linking" or "linker," as in "linking group," "linker moiety," etc., is intended a linking moiety that connects two groups by a covalent bond. The linker can be linear, branched, cyclic, or single atom. Examples of such linking groups include alkyl, alkenylene, alkynylene, arylene, alkarylene, aralkylene, and linking moieties containing functional groups, including, but not limited to, amide (-NH-CO-), ureylene (-NH-CO-NH-), imide (-CO-NH-CO-), epoxy (-O-), epithio (-S-), epidioxy (-OO-), carbonyldioxy (-O-CO-O-), alkyldioxy (-O-(CH2)nO-), epoxyimino (-O-NH-), epiimino (-NH-), carbonyl (-CO-), and the like. In certain cases, one, two, three, four, five, or more carbon atoms of the linker backbone may be optionally substituted with a sulfur, nitrogen, or oxygen heteroatom. 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 linker backbone. The linker may include one or more substituents, for example, with an alkyl, aryl, or alkenyl group. Linkers include, but are not limited to, poly(ethylene glycol) unit(s) (e.g., —(CH—CH—O)—); ether, thioether, amine, alkyl (e.g., (C1-C 12 ) alkyl), which may be linear or branched, e.g., methyl, ethyl, n-propyl, 1-methylethyl (iso-propyl), n-butyl, n-pentyl, 1,1-dimethylethyl (t-butyl), etc. The linker backbone may include a cyclic group, e.g., an aryl, heterocycle, or cycloalkyl group, in which case two or more atoms, e.g., two, three, or four atoms, of the cyclic group are included in the backbone. The linker may be cleavable or non-cleavable. Any convenient orientation and / or attachment of the linker to the linked group may be used.

[0075] When the term "substituted" appears before a list of possible substituted groups, it is intended that the term apply to each and every member of that group. For example, the phrase "substituted alkyl and aryl" should be interpreted as "substituted alkyl and substituted aryl."

[0076] Further to the disclosure herein, the term "substituted," when used to modify a specified group or radical, can also mean that one or more hydrogen atoms of the specified group or radical are each, independently of one another, replaced with the same or different substituents, as defined below.

[0077] In addition to the groups disclosed for each individual term herein, one or more hydrogens on a saturated carbon atom in the specified group or radical (any two hydrogens on a single carbon can be replaced by ═O, ═NR 70 , =N-OR 70 , ═N2 or ═S), unless otherwise specified, is substituted with -R 60 , halo, =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 )O - 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 and R 60 is selected from the group consisting of optionally substituted alkyl, cycloalkyl, heteroalkyl, heterocycloalkylalkyl, cycloalkylalkyl, aryl, arylalkyl, heteroaryl, and heteroarylalkyl; R 70 are each independently hydrogen or R 60 and R 80 are each independently R 70 or alternatively, two R 80together with the nitrogen atom to which they are attached form a 5-, 6-, or 7-membered heterocycloalkyl, which optionally contains 1 to 4 of the same or different additional heteroatoms selected from the group consisting of O, N, and S, where N of the heteroatoms may have —H or C1-C3 alkyl substitution; M + are counterions each with a net single positive charge. + are each independently, e.g., K + , Na + , Li + Alkaline ions such as; + N(R 60 ) 4; or [Ca 2+ ] 0.5 , [Mg 2+ ] 0.5 or [Ba 2+ ] 0.5 (The subscript 0.5 means that one of the counterions to such divalent alkaline earth ions can be the ionized form of a compound of the invention, and that other typical counterions such as chloride or the two ion compounds disclosed herein can serve as a counterion to such divalent alkaline earth ions, or that a doubly ionized compound of the invention can serve as a counterion to such divalent alkaline earth ions.) Specific examples include -NR 80 R 80 is intended to include -NH2, -NH-alkyl, N-pyrrolidinyl, N-piperazinyl, N-methyl-piperazin-1-yl, and N-morpholinyl.

[0078] Further to the disclosure herein, the substituents for hydrogens on unsaturated carbon atoms in "substituted" alkene, alkyne, aryl, and heteroaryl groups are, unless otherwise specified, -R 60 , halo, -O - M + , -OR 70 , -SR 70 , -S - M+ 、-NR 80 R 80 、トリハロメチル、-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 )O - 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)R 70 、-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 および-NR 70 C(NR70 )NR 80 R 80 and R 60 , R 70 , R 80 and M + is as previously defined, except that in the case of a substituted alkene or alkyne, the substituent is -O - M + , -OR 70 , -SR 70 or -S - M + The condition is that it is not.

[0079] In addition to the groups disclosed for each individual term herein, the substituents for the hydrogen on the nitrogen atom in "substituted" 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 )O - 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(NR70 )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 and R 60 , R 70 , R 80 and M + is as already defined.

[0080] Further 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.

[0081] Unless otherwise indicated, naming of substituents not explicitly defined herein is accomplished by naming the terminal portion of that functionality and then the adjacent functionality, in the direction toward the point of attachment. For example, the substituent "arylalkyloxycarbonyl" refers to the group (aryl)-(alkyl)-OC(O)-.

[0082] With respect to any of the groups disclosed herein that contain one or more substituents, it is of course understood that such groups do not include any substitutions or substitution patterns that are sterically impractical and / or synthetically impractical. Furthermore, the subject compounds include all stereochemical isomers arising from the substitution of these compounds.

[0083] In certain embodiments, substituents may contribute to the optical isomerism and / or stereoisomerism of compounds.Salts, solvates, hydrates and prodrug forms of compounds are also of interest.All such forms are encompassed by the present disclosure.Therefore, the compounds described herein include their salts, solvates, hydrates, prodrugs and isomers, including their pharmaceutically acceptable salts, solvates, hydrates, prodrugs and isomers.In certain embodiments, compounds can be metabolized into pharmaceutically active derivatives.

[0084] Unless otherwise specified, a reference to an atom is intended to include isotopes of that atom. For example, a reference to H 1 H, 2 H (i.e., D) and 3 It is intended to include H (i.e., T), and when referring to C, 12 C and all isotopes of carbon ( 13 C, etc.

[0085] As will be apparent to those skilled in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has distinct components and features that can be readily separated from, or combined with, the features of any of the other several embodiments without departing from the scope or spirit of the invention. Any of the methods described can be carried out in the order of events recited or in any other order that is logically possible.

[0086] Although the apparatus and methods have been or will be described for the purpose of grammatical fluidity with functional descriptions, unless expressly reasoned under 35 U.S.C. 112, the claims are in no way to be construed as necessarily limited by constituting "means" or "step" limitations, but rather are to be expressly understood to conform to the full range of meanings and equivalents of the definitions set forth by the claims under the doctrine of judicial equivalents, and, if the claims are expressly reasoned under 35 U.S.C. 112, to conform to their full legal equivalents under 35 U.S.C. 112.

[0087] Definitions of other terms and concepts appear throughout the detailed description. Detailed Description

[0088] As summarized above, embodiments of the present disclosure include compounds, compositions, and methods for inhibiting ENPP1. Embodiments 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 find use in a variety of applications in which inhibition of ENPP1 is desirable.

[0089] Also provided are pharmaceutical compositions and methods for treating cancer using the subject ENPP1 inhibitor compounds.Embodiments of the method include administering to a subject a therapeutically effective amount of an ENPP1 inhibitor compound to inhibit cGAMP hydrolysis and treat the cancer in the subject. ENPP1 inhibitor compounds

[0090] The target ENPP1 inhibitor compounds may comprise a core structure based on an aryl or heteroaryl ring system, such as a quinazoline or quinoline group, linked to a hydrophilic head group. The linker between the aryl or heteroaryl ring system and the hydrophilic head group may comprise a monocyclic aryl, heteroaryl, carbocyclic or heterocyclic ring, and one or more acyclic linking moieties. The quinazoline or quinoline core structure may be substituted at the 4-position with a linker. The aryl or heteroaryl ring system may be further substituted as needed. The present disclosure includes compounds having a quinoline core structure substituted at the 4-position with a linker and at the 3-position with a cyano group. In some cases, the linker comprises a 6-membered 1,4-disubstituted aryl or heteroaryl ring group, such as phenyl or substituted phenyl. In certain cases, the linker comprises a 6-membered 1,4-disubstituted saturated heterocyclic or carbocyclic ring, such as an N1,4-disubstituted piperidine ring or an N1,N4-disubstituted piperazine ring. Further embodiments of ENPP1 inhibitor compounds of interest are described below and by Li et al. in PCT Application No. PCT / US2018 / 050018, filed September 7, 2018, the disclosure of which is incorporated herein by reference in its entirety.

[0091] The term "hydrophilic head group" refers to a group linked to a core aryl or heteroaryl ring system that is hydrophilic and that is well solvated in an aqueous environment, e.g., physiological conditions, and has low permeability through cell membranes. In some cases, low permeability through cell membranes refers to a permeability coefficient of less than 10, as measured by any convenient method of passive diffusion for the isolated hydrophilic head group across a membrane (e.g., a cell monolayer such as a colorectal Caco-2 or renal MDCK cell line). -5 cm / s or less, 10 -6 cm / s or less, 10 -7 cm / s or less, 10 -8 cm / s or less, 10 -9 10 cm / s or less, or even lower -4It is intended that the viscosity be 0.01 cm / s or less. See, for example, Yang and Hinner, Methods Mol Biol. 2015; 1266: 29-53. The hydrophilic head group can provide improved aqueous 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 membrane permeability. In certain examples, the hydrophilic group is a discrete functional group (e.g., as described herein) or a substituted version thereof. Generally, charged groups or larger uncharged polar groups have low permeability. In some cases, the hydrophilic head group is charged, e.g., positively or negatively charged. In some embodiments, the hydrophilic head group is not itself cell-permeable but provides cell impermeability to the compound of interest. It is understood that the hydrophilic head group or its prodrug form can be selected to provide the desired cell permeability of the compound of interest. In certain cases, the hydrophilic head group is a neutral hydrophilic group. In some cases, the hydrophilic head group is included in a prodrug form and thus includes a promoiety that can be removed in vivo. In certain instances, the subject compounds are cell-permeable.

[0092] The hydrophilic head group can be any convenient group that can bind to or form a chelate with zinc ion, or its prodrug form.In certain cases, the hydrophilic head group is a phosphorus-containing group.The phosphorus-containing group of interest that can be used in the target ENPP1 inhibitor includes, but is not limited to, phosphonic acid or phosphonate, phosphonate ester, phosphate, phosphoric acid ester, thiophosphate, thiophosphate ester, phosphoramidate and thiophosphoramidate or their salt, or their prodrug form (for example, as described herein).

[0093] Exemplary ENPP1 inhibitor compounds of interest containing quinazoline and isoquinoline ring systems are illustrated in Formulas (I)-(XVb) and the compound structures in Tables 1-2.

[0094] In some cases, the ENPP1 inhibitor compounds of interest have the formula (I): [ka] (In the formula, X 1 is a hydrophilic head group (e.g., as described herein), A is a ring system selected from aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic, and substituted heterocyclic; L 1 and L 2 are independently a covalent bond or a linker; Z 3 is absent or NR 22 , O and S; Z 2 is CR 12 or N, Z 1 is CR 11 or N, R 1 is selected from H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkylaryl, substituted alkylaryl, alkylheteroaryl, substituted alkylheteroaryl, alkenylaryl (e.g., ethenylaryl), substituted alkenylaryl, alkenylheteroaryl (e.g., ethenylheteroaryl), substituted alkenylheteroaryl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocycle, and substituted heterocycle; R 11 and R 12 is independently selected from H, cyano, trifluoromethyl, halogen, alkyl, and substituted alkyl; R 22 is selected from H, alkyl and substituted alkyl; R 2 ~R 5are 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 R 2 and R 3 , R 3 and R 4 Or R 4 and R 5 taken together with the carbon atoms to which they are attached form a fused ring (e.g., a 5- or 6-membered monocyclic ring) selected from heterocycle, substituted heterocycle, cycloalkyl, substituted cycloalkyl, aryl, and substituted aryl). or a prodrug, pharmaceutically acceptable salt or solvate thereof.

[0095] In certain embodiments of Formula (I), Z 3 is absent. In certain embodiments of Formula (I), Z 3 is NR 22 and R 22 is H, C (1~6) Alkyl and Substituted C (1~6) In certain cases, Z is selected from alkyl. 3 is NH. In certain cases, Z 3 is NR 22 and R 22 is C (1~6) alkyl, for example, methyl, ethyl, propyl, pentyl, or hexyl. 3 is NR 22 and R 22 is a substitution C (1~6) In certain cases of formula (I), Z 3 is O. In certain cases of formula (I), Z 3 is S.

[0096] In some examples of formula (I), Z 1 is CR 11 and R 11is selected from hydrogen, cyano, trifluoromethyl, halogen, alkyl, and substituted alkyl. In some cases, alkyl or substituted alkyl is selected from C 1~5 In some examples of formula (I), Z 1 is CR 11 and R 11 is hydrogen. In some cases, R 11 is cyano. In some cases, R 11 is trifluoromethyl. In some cases, R 11 is a halogen, for example, Br, I, Cl, or F. In some cases, R 11 is an alkyl, e.g., C 1~5 In some cases, R 11 is a substituted alkyl, e.g., substituted C 1~5 It is alkyl.

[0097] In some examples of formula (I), Z 2 is CR 12 and R 12 is selected from hydrogen, cyano, trifluoromethyl, halogen, alkyl, and substituted alkyl hydrogen. In some cases, alkyl or substituted alkyl is selected from C 1~5 In some examples of formula (I), Z 2 is CR 12 and R 12 is hydrogen. In some cases, R 12 is cyano. In some cases, R 12 is trifluoromethyl. In some cases, R 12 is a halogen, for example, Br, I, Cl, or F. In some cases, R 12 is an alkyl, e.g., C 1~5 In some cases, R 12 is a substituted alkyl, e.g., substituted C 1~5 It is alkyl.

[0098] 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 is CR11 and Z 2 is N. In certain cases of formula (I), Z 1 is N and Z 2 is CR 12 In certain examples of formula (I), Z 1 is CR 11 and Z 2 is CR 12 In certain cases of formula (I), Z 1 is N and Z 2 is N.

[0099] In certain embodiments of Formula (I), L 1 and L 2 are covalent bonds. In certain cases, L 1 and L 2 are each linkers. In certain cases, L 1 is a covalent bond, and L 2 is a linker. In certain cases, L 1 is a linker, and L 2 is a covalent bond. Any convenient linker may be utilized 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 certain cases, A is linked to X via a linear linker 1 to 12 atoms in length, such as 1 to 10, 1 to 8, or 1 to 6 atoms in length, e.g., 1, 2, 3, 4, 5, or 6 atoms in length. The linker L 2 is (C 1~6 ) alkyl linkers, or substituted (C) alkyl linkers, optionally substituted with heteroatoms or linking functional groups such as esters (—CO—), amides (CONH), carbamates (—OCONH), ethers (—O—), thioethers (—S—), and / or amino groups (—NR—, where R is H or alkyl). 1~6 ) alkyl linker. In some cases, A is covalently linked to Z 3In certain cases, A is linked to Z via a linear linker of 1 to 10, 1 to 8, or 1 to 6 atoms in length, e.g., 1 to 12 atoms in length, such as 1, 2, 3, 4, 5, or 6 atoms in length. 3 Linker L 1 is (C 1~6 ) alkyl linkers, or substituted (C) alkyl linkers optionally substituted with heteroatoms or linking functional groups such as keto (CO), ester (—CO—), amide (CONH), carbamate (OCONH), ether (—O—), thioether (—S—), and / or amino groups (—NR—, where R is H or alkyl). 1~6 ) alkyl linker. Z 3 NR 22 If the linker L 1 is Z 3 together with the amide group (NR 22 It may contain a terminal keto (C=O) group, providing a (CO) linking group. Z 31 is O or S, the linker L 1 is Z 31 may contain a terminal keto (C=O) group which together provide an ester or thioester linking group.

[0100] In certain embodiments of Formula (I), Z 3 is a phosphorus-containing group or a prodrug form thereof capable of binding to a zinc ion.

[0101] In certain examples of formula (I), Z 3 is NR 22 , O and S. Accordingly, the ENPP1 inhibitor compounds of formula (I) of interest are those of formula (II): [ka] (In the formula, Z 31 is NR 22 , O and S) It can be described by:

[0102] In certain embodiments of Formula (II), Z 31 is NR 22 and R 22 is H, C (1~6) Alkyl and Substituted C (1~6) In certain cases, Z is selected from alkyl. 31 is NH. In certain cases, Z 31 is NR 22 and R 22 is C (1~6) alkyl, for example, methyl, ethyl, propyl, pentyl, or hexyl. 31 is NR 22 and R 22 is a substitution C (1~6) In certain cases of formula (I), Z 31 is O. In certain cases of formula (I), Z 31 is S.

[0103] In some examples of formula (II), Z 1 is CR 11 and R 11 is selected from hydrogen, cyano, trifluoromethyl, halogen, alkyl, and substituted alkyl hydrogen. In some cases, alkyl or substituted alkyl is selected from C 1~5 In some examples of formula (II), Z 1 is CR 11 and R 11 is hydrogen. In some cases, R 11 is cyano. In some cases, R 11 is trifluoromethyl. In some cases, R 11 is a halogen, for example, Br, I, Cl, or F. In some cases, R 11 is an alkyl, e.g., C 1~5 In some cases, R 11 is a substituted alkyl, e.g., substituted C 1~5 It is alkyl.

[0104] In some examples of formula (II), Z 2 is CR 12 and R 12is selected from hydrogen, cyano, trifluoromethyl, halogen, alkyl, and substituted alkyl hydrogen. In some cases, alkyl or substituted alkyl is selected from C 1~5 In some examples of formula (II), Z 2 is CR 12 and R 12 is hydrogen. In some cases, R 12 is cyano. In some cases, R 12 is trifluoromethyl. In some cases, R 12 is a halogen, for example, Br, I, Cl, or F. In some cases, R 12 is an alkyl, e.g., C 1~5 In some cases, R 12 is a substituted alkyl, e.g., substituted C 1~5 It is alkyl.

[0105] In certain embodiments of Formula (II), Z 1 and Z 2 At least one of is N. In certain embodiments of Formula (I), Z 1 is CR 11 and Z 2 is N. In certain cases of formula (I), Z 1 is N and Z 2 is CR 12 In certain examples of formula (I), Z 1 is CR 11 and Z 2 is CR 12 In certain cases of formula (I), Z 1 is N and Z 2 is N.

[0106] In certain embodiments of Formula (II), L 1 and L 2 are covalent bonds. In certain cases, L 1 and L 2 are each linkers. In certain cases, L 1 is a covalent bond, and L 2 is a linker. In certain cases, L 1is a linker, and L 2 is a covalent bond. Any convenient linker may be utilized 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 certain cases, A is linked to X via a linear linker 1 to 12 atoms in length, such as 1 to 10, 1 to 8, or 1 to 6 atoms in length, e.g., 1, 2, 3, 4, 5, or 6 atoms in length. The linker L 2 is (C 1~6 ) alkyl linkers, or substituted (C) alkyl linkers optionally substituted with heteroatoms or linking functional groups such as keto (CO), ester (—CO—), amide (CONH), carbamate (OCONH), ether (—O—), thioether (—S—), and / or amino groups (—NR—, where R is H or alkyl). 1~6 ) alkyl linker. In some cases, A is covalently linked to Z 3 In certain cases, A is linked to Z via a linear linker of 1 to 10, 1 to 8, or 1 to 6 atoms in length, e.g., 1 to 12 atoms in length, such as 1, 2, 3, 4, 5, or 6 atoms in length. 3 Linker L 1 is (C 1~6 ) alkyl linkers, or substituted (C) alkyl linkers optionally substituted with heteroatoms or linking functional groups such as keto (C=O), ester (-CO2-), amide (CONH), carbamate (OCONH), ether (-O-), thioether (-S-) and / or amino groups (-NR-, where R is H or alkyl). 1~6 ) alkyl linker. Z 31 NR 22 If the linker L 1 is Z 31 together with the amide group (NR 22 It may contain a terminal keto (C=O) group, providing a (CO) linking group. Z 31 is O or S, the linker L1 is Z 31 may contain a terminal keto (C=O) group which together provide an ester or thioester linking group.

[0107] In some cases of formula (II), the subject ENPP1 inhibitor compounds have the formula (III): [ka] (In the formula, R 31 ~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 linked and, together with the carbon atom to which they are attached, result in a cycloalkyl, substituted cycloalkyl, heterocyclyl, or substituted heterocyclyl ring; n and m are each independently an integer of 0 to 6 (e.g., 0 to 3). It is of the type.

[0108] In certain embodiments of Formula (III), Z 31 is NR 22 and R 22 is H, C (1~6) Alkyl and Substituted C (1~6) In certain cases, Z is selected from alkyl. 31 is NH. In certain cases, Z 31 is NR 22 and R 22 is C (1~6) alkyl, for example, methyl, ethyl, propyl, pentyl, or hexyl. 31 is NR 22 and R 22 is a substitution C (1~6) In certain cases of formula (III), Z 31 is O. In certain cases of formula (III), Z 31 is S.

[0109] In formula (II), when Z 31 is NR 22 , linker L 1 can, together with Z 31 , form an amide group (NR 22 CO) linking group and contain a terminal keto (C=O) group. Thus, in some cases of formula (II), the ENPP1 inhibitor compound of interest is of formula (IIIa):

Chemical formula

[0110] In some examples of formulas (III) to (IIIa), Z 1 is 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 alkyl. In some examples of formulas (III) to (IIIa), Z 1 is CR 11 , and R 11 is hydrogen. In some cases, R 11 is cyano. In some cases, R 11 is trifluoromethyl. In some cases, R 11is a halogen, for example, Br, I, Cl, or F. In some cases, R 11 is an alkyl, e.g., C 1~5 In some cases, R 11 is a substituted alkyl, e.g., substituted C 1~5 It is alkyl.

[0111] In some examples of formulas (III) to (IIIa), Z 2 is CR 12 and R 12 is selected from hydrogen, cyano, trifluoromethyl, halogen, alkyl, and substituted alkyl hydrogen. In some cases, alkyl or substituted alkyl is selected from C 1~5 In some examples of formulas (III) to (IIIa), Z 2 is CR 12 and R 12 is hydrogen. In some cases, R 12 is cyano. In some cases, R 12 is trifluoromethyl. In some cases, R 12 is a halogen, for example, Br, I, Cl, or F. In some cases, R 12 is an alkyl, e.g., C 1~5 In some cases, R 12 is a substituted alkyl, e.g., substituted C 1~5 It is alkyl.

[0112] In certain embodiments of Formulas (III)-(IIIa), Z 1 and Z 2 At least one of is N. In certain embodiments of Formulas (III)-(IIIa), Z 1 is CR 11 and Z 2 is N. In certain cases of formulas (III) to (IIIa), Z 1 is N and Z 2 is CR 12 In certain examples of formulas (III)-(IIIa), Z 1 is CR 11 and Z 2 is CR 12In certain cases of formulas (III) to (IIIa), Z 1 is N and Z 2 is N.

[0113] In certain embodiments of Formulas (III)-(IIIa), R 31 ~R 34 Each is hydrogen. In certain embodiments, R 31 ~R 34 At least one of R is halogen. 31 ~R 34 At least one of R is alkyl. 31 ~R 34 At least one of R is substituted alkyl. 31 ~R 34 One of R is halogen and the remainder are selected from hydrogen, halogen, alkyl and substituted alkyl. 31 ~R 34 One of R is alkyl and the rest are selected from hydrogen, halogen, alkyl, and substituted alkyl. 31 ~R 34 is substituted alkyl, the remainder being selected from hydrogen, halogen, alkyl, and substituted alkyl. 31 ~R 34 One of the R is halogen and the rest are hydrogen. 31 ~R 34 One of R is alkyl and the rest are hydrogen. 31 ~R 34 is a substituted alkyl and the remainder are hydrogen.

[0114] In certain embodiments of Formulas (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 Formulas (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 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 certain cases, n+m is an integer between 0 and 3. In certain cases, n+m is 0. In certain cases, n+m is 1. In certain cases, n+m is 2. In certain cases, n+m is 3.

[0115] In some embodiments of any of Formulas (I) to (IIIa), 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 certain cases, ring system A is phenyl or substituted phenyl. In some cases, ring system A is pyridyl or substituted pyridyl. In some cases, ring system A is pyrimidine or substituted pyrimidine. In some cases, ring system A is piperidine or substituted piperidine. In some cases, ring system A is piperazine or substituted piperazine. In some cases, ring system A is cyclohexyl or substituted cyclohexyl.

[0116] In some embodiments, ring system A has formula (A1): [ka] (In the formula, R 6 are each selected from hydrogen, alkyl, substituted alkyl, hydroxy, alkoxy, substituted alkoxy, trifluoromethyl, halogen, acyl, substituted acyl, carboxy, carboxamido, substituted carboxamido, sulfonyl, substituted sulfonyl, sulfonamido, and substituted sulfonamido; p is an integer from 0 to 4. It is described by:

[0117] In certain cases, A1 is phenylene. In certain cases, A1 is monosubstituted phenylene. In certain cases, A1 is disubstituted phenylene. In certain cases, A1 is trisubstituted phenylene. In certain cases, A1 is tetrasubstituted phenylene. In certain cases, the phenylene substitutent is selected from lower alkyl (e.g., methyl, ethyl, propyl, butyl, pentyl, and hexyl) and halogen (e.g., F, Cl, I, or Br).

[0118] In some embodiments, the A1 ring has the formula (A1a): [ka] It is described by:

[0119] In some embodiments, ring system A has formula (A2): [ka] (In the formula, Z 5 are N and CR 6 is selected from R 6are each selected from hydrogen, alkyl, substituted alkyl, hydroxy, alkoxy, substituted alkoxy, trifluoromethyl, halogen, acyl, substituted acyl, carboxy, carboxamido, substituted carboxamido, sulfonyl, substituted sulfonyl, sulfonamido, and substituted sulfonamido; q is an integer from 0 to 2. It is described by:

[0120] In certain instances, A2 is pyridyl. In certain instances, A2 is substituted pyridyl. In some instances, the pyridyl is monosubstituted pyridyl. In other instances, the pyridyl is disubstituted pyridyl. In other instances, the pyridyl is trisubstituted pyridyl. In certain instances, Z 5 is N, thus making A pyrimidyl. In some cases, A is a substituted pyrimidyl. In some cases, the pyrimidyl is monosubstituted. In some cases, the pyrimidyl is disubstituted. In certain embodiments of A, the substituents are selected from lower alkyl (e.g., methyl, ethyl, propyl, butyl, pentyl, and hexyl), trifluoromethyl, and halogen (e.g., F, Cl, I, or Br).

[0121] In some embodiments, ring system A has formula (A3): [ka] (In the formula, Z 5 are N and CR 6 is selected from R 6 are each selected from hydrogen, alkyl, substituted alkyl, hydroxy, alkoxy, substituted alkoxy, trifluoromethyl, halogen, acyl, substituted acyl, carboxy, carboxamido, substituted carboxamido, sulfonyl, substituted sulfonyl, sulfonamido, and substituted sulfonamido; q is an integer from 0 to 2. It is described by:

[0122] In certain instances, A3 is pyridyl. In certain instances, A3 is substituted pyridyl. In some instances, the pyridyl is monosubstituted pyridyl. In other instances, the pyridyl is disubstituted pyridyl. In other instances, the pyridyl is trisubstituted pyridyl. In certain instances, Z 5 is N, thus making A pyrimidyl. In some cases, A is a substituted pyrimidyl. In some cases, the pyrimidyl is monosubstituted. In some cases, the pyrimidyl is disubstituted. In certain embodiments of A, the substituents are selected from lower alkyl (e.g., methyl, ethyl, propyl, butyl, pentyl, and hexyl), trifluoromethyl, and halogen (e.g., F, Cl, I, or Br).

[0123] In some embodiments, ring system A has formula (A4): [ka] (In the formula, Z 5 is N, R 6 are each selected from hydrogen, alkyl, substituted alkyl, hydroxy, alkoxy, substituted alkoxy, trifluoromethyl, halogen, acyl, substituted acyl, carboxy, carboxamido, substituted carboxamido, sulfonyl, substituted sulfonyl, sulfonamido, and substituted sulfonamido; q is an integer from 0 to 2. It is described by:

[0124] In some cases, A4 is substituted pyrimidyl. In some cases, the pyrimidyl is monosubstituted. In some cases, the pyrimidyl is disubstituted. In certain embodiments of A4, the substituents are selected from lower alkyl (e.g., methyl, ethyl, propyl, butyl, pentyl, and hexyl), trifluoromethyl, and halogen (e.g., F, Cl, I, or Br).

[0125] In some cases of formula (III) to (IIIa), the ENPP1 inhibitor compound may be represented by formula (IV) to (IVa): [ka] (In the formula, Z 31 is NR 22 , O and S; Z 41 is -NR 22 C(=O)-, Z 11 and Z 21 is independently selected from N and C(CN), R 31 ~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 linked and, together with the carbon atom to which they are attached, result in a cycloalkyl, substituted cycloalkyl, heterocyclyl, or substituted heterocyclyl ring; R 6 are each independently selected from H, alkyl, substituted alkyl, hydroxy, alkoxy, substituted alkoxy, trifluoromethyl, and halogen; p is an integer from 0 to 4, n and m are each independently an integer of 0 to 6 (e.g., 0 to 3). It is of the type.

[0126] In certain embodiments of Formulas (IV)-(IVa), Z 31 is NR 22 and R 22 is H, C (1~6) Alkyl and Substituted C (1~6) In certain cases, Z is selected from alkyl. 31 is NH. In certain cases, Z 31 is NR 22 and R 22 is C (1~6) alkyl, for example, methyl, ethyl, propyl, pentyl, or hexyl. 31 is NR 22 and R22 is a substitution C (1~6) In certain cases of formulas (IV) to (IVa), Z 31 is O. In certain cases of formulas (IV) to (IVa), Z 31 is S.

[0127] 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) to (IVa), Z 11 is N and Z 21 is C(CN). In certain examples of formulas (IV)-(IVa), Z 11 is C(CN) and Z 21 is C(CN). In certain cases of formulas (IV) to (IVa), Z 11 is N and Z 21 is N.

[0128] In certain embodiments of Formulas (IV)-(IVa), R 31 ~R 34 Each is hydrogen. In certain embodiments, R 31 ~R 34 At least one of R is halogen. 31 ~R 34 At least one of R is alkyl. 31 ~R 34 At least one of R is substituted alkyl. 31 ~R 34 One of R is halogen and the remainder are selected from hydrogen, halogen, alkyl and substituted alkyl. 31 ~R 34 is alkyl and the remainder are selected from hydrogen, halogen, alkyl, and substituted alkyl. 31 ~R 34is substituted alkyl and the remainder are selected from hydrogen, halogen, alkyl and substituted alkyl. 31 ~R 34 One of the R is halogen and the rest are hydrogen. 31 ~R 34 One of R is alkyl and the rest are hydrogen. 31 ~R 34 One of the groups is a substituted alkyl and the rest are hydrogen.

[0129] In certain embodiments of Formulas (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 Formulas (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 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 certain cases, n+m is an integer between 0 and 3. In certain cases, n+m is 0. In certain cases, n+m is 1. In certain cases, n+m is 2. In certain cases, n+m is 3.

[0130] In some cases of formula (IVa), n is 0 and m is 0-2, such as 1 or 2.

[0131] In some cases of formula (IV) to (IVa), the ENPP1 inhibitor compound may be represented by formula (V) to (Va): [ka] (In the formula, R 41 ~R 44 are independently selected from hydrogen, alkyl, substituted alkyl, hydroxy, alkoxy, substituted alkoxy, trifluoromethyl, halogen, acyl, substituted acyl, carboxy, carboxamido, substituted carboxamido, sulfonyl, substituted sulfonyl, sulfonamido, and substituted sulfonamido. It is of the type.

[0132] 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) to (Va), Z 11 is N and Z 21 is C(CN). In certain examples of formulas (V)-(Va), Z 11 is C(CN) and Z 21 is C(CN). In certain cases of formulas (V) to (Va), Z 11 is N and Z 21 is N.

[0133] In some cases of formula (V)-(Va), the ENPP1 inhibitor compound of interest is one of formulas (VIa)-(VId): [ka] It is of the type.

[0134] In certain embodiments of Formulas (VIa)-(VId), R 41 ~R 44 Each is hydrogen. In certain embodiments, R 41 ~R 44At least one of R is alkyl or substituted alkyl. 41 ~R 44 At least one of R is hydroxy. 41 ~R 44 At least one of R is alkoxy or substituted alkoxy. 41 ~R 44 At least one of R is trifluoromethyl. 41 ~R 44 At least one of R is halogen. 41 ~R 44 At least one of R is acyl or substituted acyl. 41 ~R 44 At least one of R is carboxy. 41 ~R 44 At least one of R is a carboxamide or a substituted carboxamide. 41 ~R 44 At least one of R is sulfonyl or substituted sulfonyl. 41 ~R 44 At least one of R is a sulfonamide and a substituted sulfonamide. 31 ~R 34 is hydrogen and the remainder are selected from hydrogen, alkyl, substituted alkyl, hydroxy, alkoxy, substituted alkoxy, trifluoromethyl, halogen, acyl, substituted acyl, carboxy, carboxamido, substituted carboxamido, sulfonyl, substituted sulfonyl, sulfonamido, and substituted sulfonamido. 31 ~R 34 Two of R are hydrogen and the remainder are selected from hydrogen, alkyl, substituted alkyl, hydroxy, alkoxy, substituted alkoxy, trifluoromethyl, halogen, acyl, substituted acyl, carboxy, carboxamido, substituted carboxamido, sulfonyl, substituted sulfonyl, sulfonamido, and substituted sulfonamido.31 ~R 34 are hydrogen and the remainder are selected from hydrogen, alkyl, substituted alkyl, hydroxy, alkoxy, substituted alkoxy, trifluoromethyl, halogen, acyl, substituted acyl, carboxy, carboxamido, substituted carboxamido, sulfonyl, substituted sulfonyl, sulfonamido, and substituted sulfonamido.

[0135] In certain embodiments of Formulas (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 any of Formulas (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 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 certain cases, n+m is an integer between 0 and 3. In certain cases, n+m is 0. In certain cases, n+m is 1. In certain cases, n+m is 2. In certain cases, n+m is 3.

[0136] In certain embodiments of any of Formulas (VIa)-(VId), R 22 is hydrogen. In certain cases, R 22 is alkyl. In certain cases, R 22is a substituted alkyl. In certain cases, the alkyl or substituted alkyl is C (1~6) It is alkyl.

[0137] In certain embodiments of any of Formulas (I)-(VId), R 1 is selected from hydrogen, alkylaryl, substituted alkylaryl, alkylheteroaryl, substituted alkylheteroaryl, alkenylaryl (e.g., ethenylaryl), substituted alkenylaryl, alkenylheteroaryl (e.g., ethenylheteroaryl), substituted alkenylheteroaryl, aryl, substituted aryl, heteroaryl, and substituted heteroaryl.

[0138] In certain cases of formulas (I)-(VId), R 1 is hydrogen. In certain cases, R 1 is aryl or substituted aryl. In certain cases, R 1 is heteroaryl or substituted heteroaryl. In certain cases, R 1 is alkylaryl or substituted alkylaryl. In certain cases, R 1 is alkylheteroaryl or substituted alkylheteroaryl. In certain cases, R 1 is alkenylaryl or substituted alkenylaryl. In certain cases, R 1 is ethenylaryl. In certain cases, R 1 is a substituted ethenylaryl. In some cases, R 1 is ethenylheteroaryl. In certain cases, R 1 is alkenylheteroaryl or substituted alkenylheteroaryl. In some cases, R 1 is a substituted ethenylheteroaryl.

[0139] In some cases of formula (VIa)-(VId), the ENPP1 inhibitor compound may be one of formulas (VIIa)-(VIIb): [ka] It is of the type.

[0140] In certain embodiments of any of Formulas (I)-(VIIb), R 2 ~R 5 are independently selected from H, OH, alkyl, substituted alkyl, alkoxy, substituted alkoxy, -OCF3, halogen, cyano, amine, substituted amine, amide, heterocycle, and substituted heterocycle.

[0141] In certain embodiments of any of Formulas (I)-(VIIb), R 2 ~R 5 is hydrogen, OH, C (1~6) Alkoxy, -OCF3, C (1~6) Alkylamino, Di-C (1~6) alkylamino, independently selected from F, Cl, Br and CN.

[0142] In certain cases, R 2 ~R 5 At least one of R is hydrogen. 2 ~R 5 At least two of R are hydrogen. 2 ~R 5 are hydrogen. In certain cases, R 2 ~R 5 At least one of R is hydroxy. 2 ~R 5 At least one of R is alkyl or substituted alkyl. 2 ~R 5 At least one of is alkoxy or substituted alkoxy. In certain cases, the alkoxy or substituted alkoxy is C (1~6) Alkoxy, for example, methoxy, ethoxy, propoxy, butoxy, pentoxy, hexoxy. In certain cases, R 2 ~R 5 At least one of R is methoxy. 2 ~R 5 At least one of R is -OCF3.2 ~R 5 At least one of R is a halogen. In certain cases, the halogen is a fluoride. In certain cases, the halogen is a chloride. In certain cases, the halogen is a bromide. In certain cases, R 2 ~R 5 At least one of R is cyano. 2 ~R 5 At least one of R is an amine or a substituted amine. 2 ~R 5 At least one of the (1~6) In certain cases, R 2 ~R 5 At least one of them is di-C (1~6) In certain cases, R 2 ~R 5 At least one of R is an amide. 2 ~R 5 At least one of is a heterocycle or a substituted heterocycle.

[0143] In some examples of formulas (I)-(VIIb), R 3 and R 4 are independently alkoxy, and R 2 and R 5 are both hydrogen. In certain cases, alkoxy is methoxy. In some cases, R 3 is alkoxy and R 2 , R 4 and R 5 is hydrogen. In some cases, R 4 is alkoxy 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 is an alkoxy. In certain cases, the alkoxy is C (1~6)In certain cases, the alkoxy is methoxy. In certain cases, the alkoxy is ethoxy. In certain cases, the alkoxy is propoxy. In certain cases, the alkoxy is butoxy. In certain cases, the alkoxy is pentoxy. In certain cases, the alkoxy is hexyloxy.

[0144] In some cases of formulas (VIc) to (VId), R 41 ~R 44 are each independently H, halogen, or C (1~6) Alkyl or C (1~6) In some of the formulae (VIc) to (VId), m is 1 or 2. In some of the formulae (VIc) to (VId), R2 is H and R 3 ~R 5 is hydrogen, C (1~6) Alkoxy, F, Cl and C (1~6) alkyl.

[0145] In some cases of formula (VIIa)-(VIIb), the ENPP1 inhibitor compound of interest is one of formulas (VIIc)-(VIIl): [ka] It is of the type.

[0146] In some cases of formula (VIa), the ENPP1 inhibitor compound may be represented by formula (VIIm): [ka] It is of the type.

[0147] In certain embodiments of Formula (VIIm), R 2 ~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 ~R 5is 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.

[0148] In certain cases of formula (VIIm), R 3 ~R 5 At least one of R is hydrogen. 3 ~R 5 At least two of R are hydrogen. 3 ~R 5 are hydrogen. In certain cases, R 3 ~R 5 At least one of R is hydroxy. 3 ~R 5 At least one of R is alkyl or substituted alkyl. 3 ~R 5 In certain cases of formula (VIIm), the alkoxy or substituted alkoxy is C (1~6) Alkoxy, for example, methoxy, ethoxy, propoxy, butoxy, pentoxy, hexoxy. In certain cases, R 3 ~R 5 In certain cases of formula (VIIm), at least one of R 3 ~R 5 At least one of R is -OCF3. 3 ~R 5 At least one of R is a halogen. In certain cases, the halogen is a fluoride. In certain cases, the halogen is a chloride. In certain cases, the halogen is a bromide. In certain cases, R 3 ~R 5At least one of R is cyano. 3 ~R 5 At least one of R is an amine or a substituted amine. 3 ~R 5 At least one of the (1~6) In certain cases, R 3 ~R 5 At least one of them is di-C (1~6) In certain cases of formula (VIIm), R 3 ~R 5 At least one of R is an amide. 3 ~R 5 At least one of is a heterocycle or a substituted heterocycle.

[0149] In some examples of formula (VIIm), R 3 and R 4 are independently alkoxy, and R 2 and R 5 are both hydrogen. In certain cases, alkoxy is methoxy. In some cases, R 3 is alkoxy and R 2 , R 4 and R 5 is hydrogen. In some cases, R 4 is alkoxy and R 2 , R 3 and R 5 are each hydrogen. In certain cases of formula (VIIm), R 2 , R 3 and R 4 is hydrogen and R 5 is an alkoxy. In certain cases, the alkoxy is C (1~6) In certain cases, the alkoxy is methoxy. In certain cases, the alkoxy is ethoxy. In certain cases, the alkoxy is propoxy. In certain cases, the alkoxy is butoxy. In certain cases, the alkoxy is pentoxy. In certain cases, the alkoxy is hexyloxy.

[0150] In certain embodiments of Formula (VIIm), n is 0-3 and m is 0-3. In some examples 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 certain cases, n+m is an integer between 0 and 3. In certain cases, n+m is 0. In certain cases, n+m is 1. In certain cases, n+m is 2. In certain cases, n+m is 3.

[0151] In certain examples of ENPP1 inhibitor compounds of formula (I), Z 3 is absent. In certain embodiments of Formula (I), Z 3 does not exist, and Z 2 is CR 12 and R 12 is cyano, and the compound has the formula (X): [ka] (In the formula, L 11 and L 12 are independently a covalent bond or a linker. In some examples of formula (X), L 11 is a covalent bond.

[0152] In some embodiments of formula (X), 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 certain cases, ring system A is phenyl or substituted phenyl. In some cases, ring system A is pyridyl or substituted pyridyl. In some cases, ring system A is pyrimidine or substituted pyrimidine. In some cases, ring system A is piperidine or substituted piperidine. In some cases, ring system A is piperazine or substituted piperazine. In some cases, ring system A is cyclohexyl or substituted cyclohexyl.

[0153] In some embodiments, ring system A is any one of formulas (A1)-(A4): [ka] (In the formula, Z 5 are N and CR 6 is selected from R 6 are each selected from hydrogen, alkyl, substituted alkyl, hydroxy, alkoxy, substituted alkoxy, trifluoromethyl, halogen, acyl, substituted acyl, carboxy, carboxamido, substituted carboxamido, sulfonyl, substituted sulfonyl, sulfonamido, and substituted sulfonamido; p is an integer from 0 to 4, q is an integer from 0 to 2. (e.g., as described herein).

[0154] In some embodiments, the A ring has the formula (A5): [ka] (In the formula, Z 5 are N and CR, respectively. 16 are independently selected from R 16are each independently selected from hydrogen, alkyl, substituted alkyl, hydroxy, alkoxy, substituted alkoxy, trifluoromethyl, halogen, acyl, substituted acyl, carboxy, carboxamido, substituted carboxamido, sulfonyl, substituted sulfonyl, sulfonamido, and substituted sulfonamido; r is an integer from 0 to 8. It is described by:

[0155] In certain instances, A5 is piperidine or substituted piperidine. In certain instances, A5 is piperazine or substituted piperazine. In certain instances, 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 instances, A5 is one R 16 In some cases, A5 contains two R 16 In some cases, A5 contains three R 16 In some cases, A5 contains four R 16 In certain embodiments, the substituents are selected from lower alkyl (e.g., methyl, ethyl, propyl, butyl, pentyl, and hexyl), trifluoromethyl, and halogen (e.g., F, Cl, I, or Br).

[0156] In certain embodiments, the A ring is any one of formulas (A5a)-(A5c): [ka] It has.

[0157] In certain embodiments, the A ring is a cyclohexyl having the relative configuration of formula (A5d) or (A5e): [ka] is.

[0158] In certain instances of formula (X), the ENPP1 inhibitor compound of interest has the formula (XI): [ka] (In the formula, Z 5 are N and CR, respectively. 16 are independently selected from R 16 are each independently selected from hydrogen, alkyl, substituted alkyl, hydroxy, alkoxy, substituted alkoxy, trifluoromethyl, halogen, acyl, substituted acyl, carboxy, carboxamido, substituted carboxamido, sulfonyl, substituted sulfonyl, sulfonamido, and substituted sulfonamido; r is an integer from 0 to 8. It is of the type.

[0159] In certain embodiments of Formula (XI), at least one Z 5 is N. In certain embodiments of formula (XI), Z 5 One of them is N and the other is Z 5 is CR 16 In certain cases of formula (XI), Z 5 Both bases are CR 16 In certain cases of formula (XI), Z 5 Both groups are N.

[0160] In certain embodiments of compounds of any one of Formulas (X)-(XI), L 11 and L 12 are covalent bonds. In certain cases, L 11 and L 12 are each linkers. In certain cases, L 11 is a covalent bond, and L 12 is a linker. In certain cases, L 11 is a linker, and L 12 is a covalent bond. Any convenient linker may be used as the linker for L 11 and L 12 In some cases, L 11 is a covalent bond. In certain cases, L 11is a linear linker of 1 to 12 atoms in length, such as 1, 2, 3, 4, 5, or 6 atoms in length. 11 is (C 1~6 ) alkyl linkers, or substituted (C) alkyl linkers, optionally substituted with heteroatoms or linking functional groups such as esters (—CO—), amides (CONH), carbamates (—OCONH), ethers (—O—), thioethers (—S—), and / or amino groups (—NR—, where R is H or alkyl). 1~6 ) alkyl linker. In some cases, L 12 is a covalent bond. In certain cases, L 12 is a linker 1 to 12 atoms in length, such as 1 to 10, 1 to 8, or 1 to 6 atoms in length, e.g., 1, 2, 3, 4, 5, or 6 atoms in length. 12 is (C 1~6 ) alkyl linkers, or substituted (C) alkyl linkers, optionally substituted with heteroatoms or linking functional groups such as esters (—CO—), amides (CONH), carbamates (—OCONH), ethers (—O—), thioethers (—S—), and / or amino groups (—NR—, where R is H or alkyl). 1~6 ) alkyl linker.

[0161] In some cases of formula (XI), the ENPP1 inhibitor compound of interest is represented by formula (XII): [ka] It is of the type.

[0162] In certain embodiments of compounds of Formula (XII), Z 5 is CR 16 and R 16are each selected from hydrogen, alkyl, substituted alkyl, hydroxy, alkoxy, substituted alkoxy, trifluoromethyl, halogen, acyl, substituted acyl, carboxy, carboxamido, substituted carboxamido, sulfonyl, substituted sulfonyl, sulfonamido, and substituted sulfonamido. 5 is N.

[0163] In certain embodiments of compounds of Formula (XII), L 12 is a covalent bond. In certain cases, L 12 is a linker. Any convenient linker can be used for L 12 In certain cases, L 12 is a linear linker of 1 to 12 atoms in length, such as 1, 2, 3, 4, 5, or 6 atoms in length. 12 is (C 1~6 ) alkyl linkers, or substituted (C) alkyl linkers, optionally substituted with heteroatoms or linking functional groups such as esters (—CO—), amides (CONH), carbamates (—OCONH), ethers (—O—), thioethers (—S—), and / or amino groups (—NR—, where R is H or alkyl). 1~6 ) alkyl linker.

[0164] In some cases of formula (XII), the ENPP1 inhibitor compound of interest is of formula (XIII): [ka] (In the formula, R 35 and R 36 are each independently selected from H, halogen, alkyl and substituted alkyl, or R 35 and R 36 are cyclically linked and, together with the carbon atom to which they are attached, result in a cycloalkyl, substituted cycloalkyl, heterocyclyl, or substituted heterocyclyl ring; s is an integer from 0 to 6 (e.g., 0 to 3) It is of the type.

[0165] In certain embodiments of Formula (XIII), R 35 and R 36 Each is hydrogen. In certain embodiments, R 35 or R 36 At least one of R is halogen. 35 or R 36 At least one of R is alkyl. 35 or R 36 At least one of R is substituted alkyl. 35 is a halogen and R 36 is selected from hydrogen, halogen, alkyl, and substituted alkyl. In certain cases, R 35 is alkyl, and R 36 is selected from hydrogen, halogen, alkyl, and substituted alkyl. In certain cases, R 35 is a substituted alkyl, and R 36 is selected from hydrogen, halogen, alkyl, and substituted alkyl. In certain cases, R 35 is a halogen and R 36 is hydrogen. In certain cases, R 35 is alkyl, and R 36 is hydrogen. In certain cases, R 35 is a substituted alkyl, and R 36 is hydrogen.

[0166] 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.

[0167] In some cases of formula (XIII), the ENPP1 inhibitor compound of interest is represented by formula (XIV): [ka] (wherein s is an integer of 0 to 6 (e.g., 0 to 3)) It is of the type.

[0168] 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.

[0169] In certain embodiments of any of Formulas (X)-(XIV), R 2 ~R 5 are independently selected from H, OH, alkyl, substituted alkyl, alkoxy, substituted alkoxy, -OCF3, halogen, cyano, amine, substituted amine, amide, heterocycle, and substituted heterocycle.

[0170] In certain embodiments of any of Formulas (X)-(XIV), R 2 ~R 5 is hydrogen, OH, C (1~6) Alkoxy, -OCF3, C (1~6) Alkylamino, Di-C (1~6) alkylamino, independently selected from F, Cl, Br and CN.

[0171] In certain cases of any of formulas (X)-(XIV), R 2 ~R 5 At least one of R is hydrogen. 2 ~R 5 At least two of R are hydrogen. 2 ~R 5 At least three of R are hydrogen. 2 ~R 5 are hydrogen. In certain cases, R 2 ~R 5 At least one of R is hydroxy. 2 ~R 5 At least one of R is alkyl or substituted alkyl. 2~R 5 At least one of is alkoxy or substituted alkoxy. In certain cases, the alkoxy or substituted alkoxy is C (1~6) Alkoxy, for example, methoxy, ethoxy, propoxy, butoxy, pentoxy, hexoxy. In certain cases, R 2 ~R 5 At least one of R is methoxy. 2 ~R 5 At least one of R is -OCF3. 2 ~R 5 At least one of R is a halogen. In certain cases, the halogen is a fluoride. In certain cases, the halogen is a chloride. In certain cases, the halogen is a bromide. In certain cases, R 2 ~R 5 At least one of R is cyano. 2 ~R 5 At least one of R is an amine or a substituted amine. 2 ~R 5 At least one of the (1~6) In certain cases, R 2 ~R 5 At least one of them is di-C (1~6) In certain cases, R 2 ~R 5 At least one of R is an amide. 2 ~R 5 At least one of is a heterocycle or a substituted heterocycle.

[0172] In some examples of any of formulas (X)-(XIV), R 3 and R 4 are independently alkoxy, and R 2 and R 5 are both hydrogen. In some cases, R 3 is alkoxy and R 2 , R 4 and R 5is hydrogen. In some cases, R 4 is alkoxy 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 is an alkoxy. In certain cases, the alkoxy is C (1~6) In certain cases, the alkoxy is methoxy. In certain cases, the alkoxy is ethoxy. In certain cases, the alkoxy is propoxy. In certain cases, the alkoxy is butoxy. In certain cases, the alkoxy is pentoxy. In certain cases, the alkoxy is hexyloxy.

[0173] In some cases of formula (XIV), the ENPP1 inhibitor compound of interest is one of formulas (XIVa) to (XIVe): [ka] (wherein s is an integer of 0 to 6 (e.g., 0 to 3)) It is of the type.

[0174] In some cases of formula (I), the subject ENPP1 inhibitor compounds may be represented by formula (XVa) or (XVb): [ka] (In the formula, s is 0 to 3, R 21 is C (1~6) Alkyl or substituted C (1~6) is alkyl, R 3 and R 4 is selected from Cl and F It is of the type.

[0175] In some cases of formulas (XVa) to (XVb), R 21is selected from methyl, ethyl, n-propyl, and isopropyl. 21 is methyl. In some cases of formulas (XVa) to (XVb), R 3 and R 4 is Cl. In certain instances, R 3 and R 4 is F. In some cases of Formulas (XVa)-(XVb), s is 2. In certain instances, s is 1. In some embodiments of Formulas (XVa)-(XVb), s is 2 and R 21 is methyl or isopropyl, and R 3 and R 4 is selected from Cl and F.

[0176] In some examples of formulas (XVa)-(XVb), the subject ENPP1 inhibitor compound has one of the following structures: [ka] or a prodrug thereof (e.g., as described herein).

[0177] As mentioned above, X 1 is a hydrophilic head group or a prodrug form thereof. Any embodiment of a hydrophilic head group described herein can be incorporated into any one of the embodiments of Formulas (I)-(XVb) described herein. In some embodiments of Formulas (I)-(XVb), X 1 is a hydrophilic head group or a prodrug form thereof that includes a charged group capable of binding to a zinc ion. In certain cases, the hydrophilic head group capable of binding to a zinc ion is a phosphorus-containing functional group (e.g., as described herein).

[0178] In some embodiments of Formulas (I)-(XVb), the hydrophilic head group (X 1) is selected from phosphonic acid or phosphonate, phosphonate ester, phosphate, phosphoric acid ester, thiophosphate, thiophosphate ester, phosphoramidate, thiophosphoramidate, sulfonate, sulfonic acid, sulfate, hydroxamic acid, keto acid, amide, and carboxylic acid. In some embodiments of any one of Formulas (I)-(XVb), the hydrophilic head group is phosphonic acid, phosphonate, or a salt thereof. In some embodiments of any one of Formulas (I)-(XVb), the hydrophilic head group is phosphate or a salt thereof. In some embodiments of any one of Formulas (I)-(XVb), the hydrophilic head group is a phosphonic acid ester or a phosphoric acid ester. In some embodiments of any one of Formulas (I)-(XVb), the hydrophilic head group is a thiophosphate. In some embodiments of any one of Formulas (I)-(XVb), the hydrophilic head group is a thiophosphate ester. In some embodiments of any one of Formulas (I)-(XVb), the hydrophilic head group is a phosphoramidate. In some embodiments of any one of Formulas (I)-(XVb), the hydrophilic head group is a thiophosphoramidate.

[0179] Specific examples of hydrophilic head groups of interest that may be incorporated into any one of the embodiments of Formulas (I)-(XVb) described herein include, but are not limited to, phosphate (RPOH), - ), phosphonate (RPO3H - ), boric acid (RBO2H2), carboxylate (RCO2 - ), sulfate (RSO4 - ), sulfonate (RSO3 - ), amines (RNH3 +The headgroups include a headgroup comprising a first moiety selected from a sugar such as glycerol, lactose, or a sugar derived from hyaluronic acid, a polar amino acid, polyethylene oxide, and an oligoethylene glycol, optionally conjugated to a residue of a second moiety selected from a choline, ethanolamine, glycerol, a nucleic acid, a sugar, inositol, an amino acid or amino acid ester (e.g., serine), and a lipid (e.g., a fatty acid, or a hydrocarbon chain such as a C8-C30 saturated or unsaturated hydrocarbon). The headgroups may contain a variety of other modifications; for example, in the case of oligoethylene glycol and polyethylene oxide (PEG)-containing headgroups, such PEG chains may be terminated with a methyl group or may have distal functional groups for further modification. Examples of hydrophilic head groups also include, but are not limited to, thiophosphate, phosphocholine, phosphoglycerol, phosphoethanolamine, phosphoserine, phosphoinositol, ethylphosphosphorylcholine, polyethylene glycol, polyglycerol, melamine, glucosamine, trimethylamine, spermine, spermidine, and conjugated carboxylates, sulfates, borates, sulfonates, sulfates, and carbohydrates.

[0180] In some examples of any one of formulas (I)-(XVb), the hydrophilic head group X 1 is represented by formula (XVI): [ka] (In the formula, Z 6 is absent or selected from O and CH2; Z 7 and Z 9 O and NR 10 are independently selected from R 10 is H, alkyl or substituted alkyl, Z 8 is selected from O and S; R 8 and R 9are each 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. It is of the type.

[0181] In some embodiments of Formula (XVI), Z 6 does not exist. Otherwise, Z 6 is CH2. Otherwise, Z 6 is oxygen. In some embodiments of Formula (XVI), Z 7 is oxygen, and Z 9 is NR 10 In some cases, Z 7 is NR 10 and Z 9 is oxygen. In some cases, Z 7 and Z 9 are both oxygen atoms. Otherwise, Z 7 and Z 9 Both are NR 10 In some cases, Z 8 is oxygen. Otherwise, Z 8 is sulfur.

[0182] In some embodiments of Formula (XVI), Z 7 , Z 8 and Z 9 are all oxygen atoms, and Z 6 is absent or is CH2. Otherwise, Z 8 is a sulfur atom, and Z 7 and Z 9 are both oxygen atoms, and Z 6 is absent or is CH2. Otherwise, Z 8 is a sulfur atom, and Z 6 , Z 7 and Z 9 are all oxygen atoms. In some cases, Z 8 is an oxygen atom, and Z 7 is NR 10 and Z 9is an oxygen atom, and Z 6 is absent or is CH2. Otherwise, Z 8 is an oxygen atom, and Z 7 is NR 10 and Z 6 and Z 9 are both oxygen atoms. In other cases, Z 8 is an oxygen atom, and Z 7 and Z 9 are each independently, NR 10 and Z 6 is an oxygen atom. In still other cases, Z 8 is an oxygen atom, and Z 7 and Z 9 are each independently, NR 10 and Z 6 is absent or is CH2. In some cases, Z 7 and Z 9 are the same for each other. Otherwise, Z 7 and Z 9 is different. It is understood that the group of formula (XVI) may include one or more tautomeric forms of the structure shown, and that all such forms and salts thereof are intended to be included.

[0183] In some embodiments of Formula (XVI), Z 7 and Z 9 At least one of the 10 In some cases, R 10 is hydrogen. In some cases, R 10 is alkyl. In some other cases, R 10 is a substituted alkyl. In some cases, Z 7 and Z 9 Both are NR 10 In some cases, Z 7 and Z 9 Both are NR 10 and R 10 , R 8 and R 9 are each independently hydrogen. 7 and Z 9Both are NR 10 and R 10 are each an alkyl group, and R 8 and R 9 are hydrogen. In some cases, Z 7 and Z 9 Both are NR 10 and R 10 are each a substituted alkyl group (e.g., an alkyl group substituted with an ester group or a carboxyl group), and R 8 and R 9 are hydrogen atoms.

[0184] In some embodiments of Formula (XVI), R 8 and R 9 are both hydrogen atoms. In some cases, R 8 and R 9 At least one of R is a substituent other than hydrogen. 8 and R 9 are both non-hydrogen substituents. 8 and R 9 At least one of R is alkyl or substituted alkyl. 8 and R 9 At least one of R is alkenyl or substituted alkenyl. 8 and R 9 At least one of R is aryl or substituted aryl. 8 and R 9 At least one of R is acyl or substituted acyl. 8 and R 9 At least one of R is heteroaryl or substituted heteroaryl. 8 and R 9 At least one of R is cycloalkyl or substituted cycloalkyl. 8 and R 9 and R are both alkyl groups (e.g., lower alkyl). 8 and R 9are both substituted alkyl groups (e.g., C substituted with alkoxy, substituted alkoxy, ester, or carboxyl groups). (1~6) In some cases, R 8 and R 9 At least one of R contains a promoiety. 8 and R 9 are both phenyl groups. In some cases, R 8 and R 9 are the same. Otherwise, R 8 and R 9 is different.

[0185] In some examples of any one of formulas (I)-(XVb), the hydrophilic head group X 1 is any one of formulas (XVIa) to (XVIf): [ka] (In the formula, R 10 and R 11 are each 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). is selected from.

[0186] In some embodiments of Formulas (XVIa)-(XVIf), R 10 and R 11 are both 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 both non-hydrogen substituents. 10 and R 11 are the same. Otherwise, R 10 and R 11 are different. In some cases, R 10 and R 11At least one of R is alkyl or substituted alkyl. 10 and R 11 At least one of R is aryl or substituted aryl. 10 and R 11 are both alkyl or substituted alkyl. In some cases, R 10 and R 11 Both of R are aryl or substituted aryl. 10 and R 11 Both of R are acyl or substituted acyl. 10 and R 11 are both lower alkyl groups. In some cases, R 10 and R 11 are both substituted alkyl groups (e.g., C substituted with alkoxy, substituted alkoxy, ester, or carboxyl groups). (1~6) In some cases, R 10 and R 11 At least one of R contains a promoiety. 10 and R 11 are both phenyl groups.

[0187] In certain cases of formulas (XVIa) to (XVId), R 10 and R 11 At least one of R comprises a cleavable group or a self-immolative promoiety. The self-immolative group can be a disulfide-linked promoiety or a self-immolative ester-containing promoiety. In some cases, R 10 and / or R 11 is a disulfide-linked promoiety of the formula: -CHCH-SS-R 12 (R 12 is alkyl or substituted alkyl). In certain instances, R 12 is a C8 to C30 saturated or unsaturated hydrocarbon chain. In some cases, R 10 and / or R 11 is the promoiety with the following formula: -CH2OCOR 13 (R 13is H, alkyl, or substituted alkyl. 10 and / or R 11 is a promoiety having the following formula: -CHC(R 14 )2CO2R 14 (R 14 are each independently H, alkyl, or substituted alkyl.

[0188] In some examples of any one of formulas (I)-(XVb), the hydrophilic head group X 1 or a prodrug form thereof: [ka] [ka] or a pharmaceutically acceptable salt thereof.

[0189] In some examples of any one of formulas (I)-(XVb), the hydrophilic head group X 1 is represented by formula (XVI): [ka] (In the formula, 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 91 together with the atom to which they are attached form a group selected from heterocycles and substituted heterocycles It is of the type.

[0190] In some embodiments of Formula (XVI), R 81 and R 91 are both hydrogen atoms. Otherwise, R 81 and R 91 are both non-hydrogen substituents.

[0191] In some examples of any one of formulas (I)-(XVb), the hydrophilic head group X 1 is represented by formula (XVII): [ka] It is of the type.

[0192] In some examples of any one of formulas (I)-(XVb), the hydrophilic head group X 1 is represented by formula (XVIII): [ka] (In the formula, Z 61 is absent or selected from O and CH It is of the type.

[0193] In some embodiments of Formula (XVIII), the hydrophilic head group is one of the following groups: [ka] is selected from.

[0194] In some examples of any one of formulas (I)-(XVb), the hydrophilic head group X 1 is represented by formula (XIX): [ka] It is of the type.

[0195] In some examples of any one of formulas (I)-(XVb), the hydrophilic head group X 1 is the formula (XX): [ka] (In the formula, R 92is selected from H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, aryl, substituted aryl, acyl group, ester, amide, heterocycle, substituted heterocycle, cycloalkyl, and substituted cycloalkyl It is of the type.

[0196] In some embodiments of Formula (XX), R 92 is hydrogen. Otherwise, R 92 is a substituent other than hydrogen. In certain embodiments, R 92 is alkyl or substituted alkyl. In certain embodiments of Formula (XX), the hydrophilic head group has the following structure: [ka] It is of the type.

[0197] In some examples of any one of formulas (I)-(XVb), the hydrophilic head group X 1 is represented by formula (XXI): [ka] It is of the type.

[0198] Group X of any of formulae (I) to (XVb) 1 It will be understood that any of the hydroxyl and amine groups in may be optionally further substituted with any convenient group, for example, alkyl groups, substituted alkyl groups, phenyl groups, substituted phenyl groups, ester groups, etc. Any of the convenient alternative hydrophilic groups may be substituted with the group X in any of the compounds of formulae (I) to (XVb). 1 It will be understood that it can be utilized as

[0199] In certain embodiments, the ENPP1 inhibitor compound is described by one of the structures in Table 1 or a prodrug thereof (e.g., as described herein), or a pharmaceutically acceptable salt thereof. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6]

[0200] In certain embodiments, the ENPP1 inhibitor compound is described by one of the structures in Table 2 or a prodrug thereof (e.g., as described herein), or a pharmaceutically acceptable salt thereof. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5]

[0201] In certain embodiments, the ENPP1 inhibitor compound is described by one of the structures in Table 3 or a prodrug thereof (e.g., as described herein), or a pharmaceutically acceptable salt thereof. [Table 3-1] [Table 3-2] In certain embodiments, the compound is described by the structure of one of the compounds in Tables 1-3 (when Tables 1-3 are mentioned herein, Table 3a is included). It is understood that any of the compounds shown in Tables 1-3 can exist in a salt form. In some cases, the salt form of the compound is a pharmaceutically acceptable salt. It is understood that any of the compounds shown in Tables 1-3 can exist in a prodrug form.

[0202] In some embodiments, the compound is described by the structure of one of the compounds in Table 3a. [Table 3A-1] [Table 3A-2] [Table 3A-3] [Table 3A-4] [Table 3A-5] [Table 3A-6] [Table 3A-7] [Table 3A-8] [Table 3A-9]

[0203] Embodiments of the present disclosure include ENPP1 inhibitor compounds (e.g., as described herein), their salts (e.g., pharmaceutically acceptable salts), and / or solvates, hydrates, and / or prodrug forms thereof. Furthermore, in any of the compounds described herein that have one or more chiral centers, if the absolute stereochemistry is not explicitly indicated, it is understood that each center can independently be in the R or S configuration or a mixture thereof. It will be recognized that all variations of salts, solvates, hydrates, prodrugs, and stereoisomers are intended to be encompassed by the present disclosure.

[0204] 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 are basic in nature, and therefore can react with various inorganic and organic acids to form pharmaceutically acceptable acid addition salts.The 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 para-toluenesulfonic acid, methanesulfonic acid, oxalic acid, para-bromophenylsulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid and acetic acid, and related inorganic and organic acids. Accordingly, such pharmaceutically acceptable salts include sulfate, pyrosulfate, hydrogen sulfate, sulfite, bisulfite, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butyne-1,4-dioate, hexyne-1,6-dioate, benzoate, benzoyl esters, benzoates, benzoyl esters ... benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, terephthalate, sulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, β-hydroxybutyrate, glycolate, maleate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, mandelate, hippurate, gluconate, lactobionate, and similar salts. In certain 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.

[0205] In some embodiments, the subject compound is provided in a prodrug form. "Prodrug" refers to a derivative of an active agent that requires conversion in the body to release the active agent. In certain embodiments, the conversion is enzymatic. Prodrugs are often, but not necessarily, pharmacologically inactive until converted to the active agent. "Promoiety" refers to a form of protecting group that, when used to mask a functional group in an active agent, converts the active agent into a prodrug. In some cases, the promoiety is attached to the drug via a bond(s) that is cleaved in vivo by enzymatic or non-enzymatic means. Any convenient prodrug form of the subject compound can be prepared following the strategies and methods described, for example, by Rautio et al. ("Prodrugs: design and clinical applications," Nature Reviews Drug Discovery 7, 255-270 (February 2008)). In some cases, the promoiety is attached to a hydrophilic head group of the subject compound. In some cases, the promoiety is attached to a hydroxy group or a carboxylic acid group of the subject compound. In certain cases, the promoiety is an acyl group or a substituted acyl group. In certain cases, the promoiety is, for example, an alkyl or substituted alkyl group that, when attached to a hydrophilic head group of the compound of interest, forms an ester functionality, e.g., a phosphonate, phosphate, or the like.

[0206] In some embodiments, the subject compounds are phosphonate or phosphate ester prodrugs that can be converted to compounds containing a phosphonic acid or phosphonate or phosphate head group.

[0207] In some embodiments, the subject compound, its prodrug, stereoisomer, or salt is provided in the form of a solvate (e.g., hydrate). As used herein, the term "solvate" refers to a complex or aggregate formed by one or more molecules of a solute, such as a prodrug or a pharmaceutically acceptable salt thereof, and one or more molecules of a solvent. Such a solvate is usually a crystalline solid having 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.

[0208] In some embodiments, the subject compound is delivered by oral administration and absorbed into the bloodstream. In some embodiments, the oral bioavailability of the subject compound is 30% or higher. The subject compound or its formulation may be modified using any convenient method to increase its absorption or bioavailability through the intestinal lumen.

[0209] In some embodiments, the subject compounds are metabolically stable (e.g., remain substantially intact in vivo during the half-life of the compound). In certain embodiments, the compounds have a half-life (e.g., in vivo half-life) of 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. Methods for inhibiting ENPP1

[0210] As summarized above, embodiments of the present disclosure include ENPP1 inhibitors and inhibitory methods using the same. ENPP1 is a member of the ecto-nucleotide pyrophosphatase / phosphodiesterase (ENPP) family. Accordingly, embodiments of the subject methods include inhibiting the hydrolase activity of ENPP1 against cGAMP. The inventors have discovered that cGAMP can have important extracellular biological functions, which can be enhanced by blocking the extracellular degradation of cGAMP, e.g., hydrolysis by its degradative enzyme, ENPP1. In certain instances, the ENPP1 target of inhibition is extracellular, and the targeted ENPP1-inhibiting compound is cell-impermeable and therefore cannot diffuse into cells. Thus, the subject methods can achieve selective extracellular inhibition of the hydrolase activity of ENPP1 and increase the extracellular level of cGAMP. Thus, in some cases, the ENPP1-inhibiting compound is a compound that inhibits the activity of ENPP1 extracellularly. Experiments performed by the present inventors have shown that inhibiting the activity of ENPP1 can increase extracellular cGAMP and, as a result, promote the STING pathway.

[0211] By inhibiting ENPP1 is intended a reduction in the activity of the enzyme 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 (e.g., compared to a control in any convenient in vitro inhibition assay). In some cases, inhibiting ENPP1 means reducing the activity of the enzyme by 2-fold or less, such as 3-fold or less, 5-fold or less, 10-fold or less, 100-fold or less, or 1000-fold or less, compared to its normal activity (e.g., compared to a control measured by any convenient assay).

[0212] In some cases, the method is a method of inhibiting ENPP1 in a sample. The term "sample", as used herein, relates to a substance or mixture of substances, usually, but not necessarily, in fluid form, containing one or more components of interest.

[0213] In some embodiments, a method for inhibiting ENPP1 is provided, 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 contains cGAMP. In certain cases, the cGAMP level is increased in the cell sample (e.g., compared to a control sample not contacted with the inhibitor). The subject method can result in an increase in the cGAMP level. By "increased cGAMP level" is intended the cGAMP level in a cell sample contacted with a subject compound, wherein the cGAMP level in the sample is increased 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, 100% or more, or even more, compared to a control sample not contacted with the agent.

[0214] 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) to (XVb) (e.g., as described herein). In some cases, the ENPP1 inhibitor is any one of the compounds in Tables 1 to 3 (e.g., as described herein). In some cases, the ENPP1 inhibitor is cell-impermeable.

[0215] In some embodiments, the ENPP1 inhibitor is configured to be cell-permeable. In some embodiments, a method of inhibiting ENPP1 is provided, comprising contacting a sample with a cell-permeable ENPP1 inhibitor to inhibit ENPP1.

[0216] In some embodiments, the subject compounds have an ENPP1 inhibition profile that reflects their activity against additional enzymes, hi some embodiments, the subject compounds specifically inhibit ENPP1 without undesirable inhibition of one or more other enzymes.

[0217] In some embodiments, the compound of the present disclosure disrupts the interaction of cGAMP and ENPP1.For example, the target compound can inhibit the hydrolase activity of ENPP1 on cGAMP, thereby increasing extracellular cGAMP.Without being bound by any particular theory, it is believed that the increase in extracellular cGAMP activates STING pathway.

[0218] In some embodiments, the subject compounds are evaluated by an inhibition assay, e.g., an assay that determines the level of enzyme activity in either a cell-free system or cells after treatment with the subject compound compared to a control, to determine an IC 50 or EC 50 In certain embodiments, the subject compounds inhibit ENPP1 as determined by measuring IC values ​​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 lower. 50 value (or EC 50 value).

[0219] As summarized above, embodiments of the present disclosure include methods for inhibiting ENPP1. A subject compound (e.g., as described herein) can inhibit the activity of ENPP1 in the range of 10% to 100%, for example, 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, a subject compound is administered at a concentration of 1×10 -6 M or less (e.g., 1 x 10 -6 M or less, 1 x 10 -7 M or less, 1 x 10 -8 M or less, 1 x 10 -9 M or less, 1 x 10 -10 M or less, or 1 x 10 -11 M or less) 50 can inhibit its target.

[0220] There are numerous protocols that can be used in determining ENPP1 activity, including, but not limited to, cell-free assays, e.g., binding assays; assays using purified enzymes; cellular assays in which a cellular phenotype is measured, e.g., gene expression assays; and in vivo assays involving certain animals, which in certain embodiments may be animal models of a condition associated with the target pathogen.

[0221] In some embodiments, a subject method is an in vitro method that includes contacting a sample with a subject compound that specifically inhibits ENPP1. In certain embodiments, the sample is suspected of containing ENPP1, and the subject method further includes assessing whether the compound inhibits ENPP1.

[0222] In certain embodiments, the subject compound is a modified compound that includes a label, e.g., a fluorescent label, and the subject method further includes detecting the label, if present, in the sample, e.g., using optical detection.

[0223] In certain embodiments, the compound is modified with a support or an affinity group (e.g., biotin) that binds to the support, so that any sample that does not bind to the compound can be removed (e.g., by washing). If present, specifically bound ENPP1 can then be detected using any conventional means, such as using binding of a labeled target-specific probe or using a fluorescent protein-reactive reagent.

[0224] In another embodiment of the subject method, the sample is known to contain ENPP1.

[0225] In some embodiments, the method is a method for reducing cancer cell proliferation, comprising contacting cells with an effective amount of a subject ENPP1 inhibitor compound (e.g., as described herein) to reduce cancer cell proliferation. In certain cases, the subject ENPP1 inhibitor compound can act intracellularly. The method can be performed in combination with a chemotherapeutic agent (e.g., as described herein). The cancer cells can be in vitro or in vivo. In certain examples, the method includes contacting cells with an ENPP1 inhibitor compound (e.g., as described herein) and contacting cells with a chemotherapeutic agent. Any convenient cancer cell can be targeted. Treatment method

[0226] Embodiments of the present disclosure include a method for inhibiting the hydrolase activity of ENPP1 on cGAMP to increase the level of cGAMP and / or modulate (e.g., activate) the STING pathway downstream. The inventors have discovered that cGAMP can exist in the extracellular space and that ENPP1 can control the extracellular level of cGAMP. The inventors have also discovered that cGAMP can have important extracellular biological functions in vivo. The results described and demonstrated herein demonstrate that ENPP1 inhibition by the subject method can modulate STING activity in vivo, and thus find use in the treatment of various diseases, for example, as a target for cancer immunotherapy. Thus, the subject method can selectively inhibit ENPP1 activity (e.g., cGAMP hydrolase activity) extracellularly to increase the extracellular level of cGAMP and activate the stimulator of interferon genes (STING) pathway. In some examples, the subject method is a method for increasing a STING-mediated response in a subject. In some examples, a subject method is a method of modulating an immune response in a subject.

[0227] " STING-mediated response " refers to any response mediated by STING, including, but not limited to, immune responses to bacterial pathogens, viral pathogens and eukaryotic pathogens.See, for example, 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 also plays a role in certain autoimmune diseases initiated by inappropriate recognition of self-DNA (see, for example, Gall et al. Immunity 36: 120-131 (2012)), and in inducing adaptive immunity in response to DNA vaccines (see, for example, Ishikawa et al. Nature 461: 788-792 (2009)). By increasing the STING-mediated response in a subject, it is meant that the STING-mediated response in the subject is increased compared to a control subject (e.g., a subject that has not received the subject compound).In some cases, the subject is a human, and the subject compound and method result in the activation of human STING.In some cases, the STING-mediated response includes modulating immune response.In some examples, the subject method is a method of modulating immune response in a subject.

[0228] In some cases, the STING-mediated response includes an increase in the production of interferon (e.g., type I interferon (IFN), type III interferon (IFN)) in a subject. Interferon (IFN) is a protein with various biological activities, such as antiviral, immunomodulatory, and antiproliferative properties. IFN is a relatively small, species-specific, single-chain polypeptide produced by mammalian cells in response to exposure to various inducers, such as viruses, polypeptides, and mitogens. Interferon protects animal tissues and cells from viral attacks and is an important host defense mechanism. Interferons can be classified as 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).

[0229] Interferons have found use in the treatment of various cancers because these molecules possess anticancer activity that acts at multiple levels. Interferon proteins can directly inhibit the growth of human tumor cells. In some cases, their antiproliferative activity is also synergistic with various approved chemotherapeutic agents, such as cisplatin, 5FU, and paclitaxel. The immunomodulatory activity of interferon proteins can also lead to the induction of antitumor immune responses. This response includes the activation of NK cells, stimulation of macrophage activity, and induction of MHC class I surface expression, leading to the induction of antitumor cytotoxic T lymphocyte activity. Furthermore, interferons play a role in antigen cross-presentation in the immune system. Some studies have further indicated that IFN-β proteins can possess antiangiogenic activity. Angiogenesis, the formation of new blood vessels, is important for the growth of solid tumors. IFN-β can inhibit angiogenesis by inhibiting the expression of proangiogenic factors such as bFGF and VEGF. Interferon proteins can also inhibit tumor invasiveness by modulating the expression of enzymes such as collagenase and elastase, which are important in tissue remodeling.

[0230] Aspects of the method include administering a therapeutically effective amount of an ENPP1 inhibitor to a subject having cancer to treat the cancer in the subject. In some examples, the subject has been diagnosed with cancer or is suspected of having cancer. Any convenient ENPP1 inhibitor can be used in the subject methods of treating cancer. In certain cases, the ENPP1 inhibitor compound is a compound described herein. In certain cases, the ENPP1 inhibitor is a cell-impermeable compound. In certain cases, the ENPP1 inhibitor is a cell-permeable compound. In certain cases, the cancer is a solid tumor cancer. In certain embodiments, the cancer is selected from adrenal, liver, kidney, bladder, breast, colon, stomach, ovarian, cervical, uterine, esophageal, colorectal, prostate, pancreatic, lung (both small cell and non-small cell), thyroid, carcinoma, sarcoma, glioblastoma, melanoma, and various head and neck tumors. In some cases, the cancer is breast cancer. In some embodiments, the cancer is lymphoma.

[0231] An aspect of this method includes administering a therapeutically effective amount of a cell-impermeable ENPP1 inhibitor to a subject to inhibit cGAMP hydrolysis and treat the subject's cancer. In certain cases, the cancer is a solid tumor cancer. In certain embodiments, the cancer is selected from adrenal gland, liver, kidney, bladder, breast, colon, stomach, ovary, cervix, uterus, esophagus, colorectum, prostate, pancreas, lung (both small cell and non-small cell), thyroid, carcinoma, sarcoma, glioblastoma, melanoma, and various head and neck tumors. In certain embodiments, the cancer is breast cancer. In some examples, the cancer is lymphoma.

[0232] In some embodiments of the methods disclosed herein, the cell-impermeable ENPP1 inhibitor is an inhibitor of any one of Formulas (I)-(XVb) (e.g., as described herein). In some cases, the ENPP1 inhibitor is a compound of Tables 1-3, or a prodrug form thereof (e.g., as described herein).

[0233] In some embodiments of the methods disclosed herein, the ENPP1 inhibitor is cell-permeable.

[0234] Thus, an embodiment of the method includes contacting a sample with a compound of interest (e.g., as described above) under conditions in which the compound inhibits ENPP1. Any convenient protocol for contacting the sample with the compound can be used. The particular protocol used can vary, for example, depending on whether the sample is in vitro or in vivo. For in vitro protocols, contacting the sample with the compound can be achieved using any convenient protocol. In some examples, the sample contains cells maintained in a suitable culture medium, and the complex is introduced into the culture medium. For in vivo protocols, any convenient administration protocol can be used. Various protocols can be used depending on the potency of the compound, the cells of interest, the method of administration, and the number of cells present.

[0235] In some embodiments, a subject method is a method of treating cancer in a subject. In some embodiments, a subject method includes administering to a subject an effective amount of a subject compound (e.g., as described herein) or a pharmaceutically acceptable salt thereof. The subject compound may be administered as part of a pharmaceutical composition (e.g., as described herein). In certain examples of the method, the administered compound is a compound of one of Formulas (I)-(XVb) (e.g., as described herein). In certain examples of the method, the administered compound is described by one of the compounds in Tables 1-3.

[0236] In some embodiments, an "effective amount" refers to the amount of a subject compound that, when administered to an individual in one or more doses in monotherapy or 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), compared to ENPP1 activity in the individual in the absence of treatment with the compound, or alternatively, compared to ENPP1 activity in the individual before or after treatment with the compound.

[0237] In some embodiments, a "therapeutically effective amount" refers to an amount of a subject compound that, when administered to an individual in one or more doses as a monotherapy or combination therapy, is effective to reduce the tumor burden in a 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 borne by a subject with cancer.

[0238] 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 as a monotherapy or in combination therapy, is effective to reduce the dose of radiation therapy required to observe tumor shrinkage in a 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 dose of radiation therapy required to observe tumor shrinkage in the individual in the absence of treatment with the compound.

[0239] In some embodiments, a "therapeutically effective amount" of a compound is an amount that, when administered in one or more doses to an individual with cancer, is 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.

[0240] In some embodiments, an effective amount of the compound is from about 50 ng / ml to about 50 μg / ml (e.g., from about 50 ng / ml to about 40 μg / ml, from about 30 ng / ml to about 20 μg / ml, from about 50 ng / ml to about 10 μg / ml, from about 50 ng / ml to about 1 μg / ml, from about 50 ng / ml to about 800 ng / ml, from about 50 ng / ml to about 700 ng / ml, from about 50 ng / ml to about 600 ng / ml, from about 50 ng / ml to about 500 ng / ml, from about 50 ng / ml to about 400 ng / ml, from about 60 ng / ml to about 400 ng / ml, The amount is in the range of 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.

[0241] In some embodiments, the effective amount of the compound is from about 10 pg to about 100 mg, e.g., from about 10 pg to about 50 pg, from about 50 pg to about 150 pg, from about 150 pg to about 250 pg, from about 250 pg to about 500 pg, from about 500 pg to about 750 pg, from about 750 pg to about 1 ng, from about 1 ng to about 10 ng, from about 10 ng to about 50 ng, from about 50 ng to about 150 ng, from about 150 ng to about 250 ng, or from about 250 ng The dose ranges from about 500 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. This amount can be a single dose or a total daily dose. The total daily dose can be in the range of 10 pg to 100 mg, 100 mg to about 500 mg, or 500 mg to about 1000 mg.

[0242] In some embodiments, a single dose of the compound is administered. In other embodiments, multiple doses are administered. When multiple doses are administered over a period of time, the compound can be administered twice a day (qid), daily (qd), every other day (qod), every third day, 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 for a period of one day to about two years, or longer. For example, the compound is administered at any of the above frequencies for one week, two weeks, one month, two months, six months, one year, two years, or longer, depending on various factors.

[0243] When administered to an individual with cancer, a therapeutically effective amount of the subject compound can result in one or more of the following: 1) reduction in tumor burden; 2) reduction in the dose of radiation therapy required to cause tumor shrinkage; 3) reduction in the spread of cancer from one cell to another in the individual; 4) reduction in the morbidity or mortality rate in clinical outcomes; 5) reduction in the total treatment period when combined with other anti-cancer drugs; and 6) improvement in indicators of disease response (for example, reduction in one or more symptoms of cancer).Any of a variety of methods can be used to determine whether a treatment method is effective.For example, biological samples obtained from individuals treated by the subject method can be assayed.

[0244] Any of the compounds described herein can be utilized in the subject methods of treatment. In certain instances, the compound is one of Formulas (I)-(XVb) (e.g., as described herein). In certain instances, the compound is one of the compounds in Tables 1-3, or a prodrug form thereof. In some instances, the compound utilized in the subject methods is not cell-permeable. In some instances, the compound utilized in the subject methods is poorly cell-permeable.

[0245] 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 between ENPP1 and cGAMP. In some embodiments, the compound causes the activation of the STING pathway.

[0246] In some embodiments, the subject is a mammal. In certain examples, the subject is a human. Other subjects can include domestic pets (e.g., dogs and cats), poultry (e.g., cows, pigs, goats, horses, etc.), rodents (e.g., mice, guinea pigs, and rats, such as in animal models of disease), and non-human primates (e.g., chimpanzees and monkeys). The subject may require treatment for cancer. In some examples, the subject method includes diagnosing cancer, including any one of the cancers described herein. In some embodiments, the compound is administered as a pharmaceutical preparation.

[0247] In certain embodiments, the ENPP1 inhibitor compound is a modified compound containing a label, and the method further comprises detecting the label in the subject. The choice of label depends on the detection means. Any convenient labeling and detection system can be used in the subject method. See, for example, Baker, "The whole picture," Nature, 463, 2010, p977-980. In certain embodiments, the compound contains a fluorescent label suitable for optical detection. In certain embodiments, the compound contains a radiolabel for detection using positron emission tomography (PET) or single photon emission computed tomography (SPECT). In some cases, the compound contains a paramagnetic label suitable for tomographic detection. The subject compound may be labeled as described above, but in some methods, the compound is unlabeled, and a secondary labeling agent is used for imaging. Combination therapy

[0248] The subject compound may be administered to a subject alone or in combination with an additional, i.e., second, active agent. Combination treatment methods may involve using the subject ENPP1 inhibitor compound in combination with a second active agent or additional therapy, such as radiation therapy. The terms "agent," "compound," and "drug" are used interchangeably herein. For example, the ENPP1 inhibitor compound may be administered alone or in conjunction with one or more other drugs, such as drugs used to treat a disease of interest, including, but not limited to, immunomodulatory diseases and conditions and cancer. In some embodiments, the subject method further includes simultaneously or sequentially 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 further includes administering radiation therapy to the subject.

[0249] The terms "co-administration" and "in combination with" include either simultaneous, concurrent, or sequential administration of two or more therapeutic agents, without specific time limitations. In one embodiment, the agents are present in a cell or in a subject's body at the same time or exert their biological or therapeutic effect at the same time. In one embodiment, the therapeutic agents are present in the same composition or unit dosage form. In other embodiments, the therapeutic agents are present in separate compositions or unit dosage forms. In certain embodiments, the first agent 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), concurrently 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) the administration of the second therapeutic agent.

[0250] "Co-administration" of a known therapeutic agent or additional therapy with a pharmaceutical composition of the present disclosure means that the compound and the second agent or additional therapy are administered at a time such that both the known drug and the composition of the present disclosure have a therapeutic effect. Such co-administration can include simultaneous (i.e., at the same time) administration, prior administration, or subsequent administration of the drug with respect to the administration of the subject compound. The routes of administration of the two agents can vary, and in this case, representative routes of administration are described in more detail below. Those skilled in the art will have no difficulty in determining the appropriate timing, sequence, and dosage of administration for a particular drug or therapy and a compound of the present disclosure.

[0251] In some embodiments, the compounds (e.g., the subject compound and at least one additional compound or therapy) are administered to a subject within 24 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 compounds are administered within 1 hour of each other. In certain embodiments, the compounds are administered substantially simultaneously. By administered substantially simultaneously, it is intended that the compounds are administered to a subject within about 10 minutes or less of each other, such as within 5 minutes or less of each other, or within 1 minute or less of each other.

[0252] Similarly, pharmaceutical preparations of the subject compound and a second active agent are provided.In pharmaceutical dosage forms, the subject compound can be administered in the form of its pharmaceutically acceptable salt, or they can be used alone or in suitable association and in combination with other pharmaceutically active compounds.

[0253] In conjunction with any of the subject methods, the ENPP1 inhibitor compound (e.g., as described herein) (or a pharmaceutical composition comprising such a compound) can be administered in combination with another drug designed to reduce or prevent infection, treat or prevent chronic inflammation or fibrosis, or treat cancer. In each case, the ENPP1 inhibitor compound can be administered prior to, simultaneously with, or after the administration of the other drug. In certain cases, the cancer is selected from adrenal, liver, kidney, bladder, breast, colon, stomach, ovary, cervix, uterus, esophagus, colorectum, prostate, pancreas, lung (both small cell and non-small cell), thyroid, carcinoma, sarcoma, glioma, glioblastoma, melanoma, and various head and neck tumors.

[0254] For the treatment of cancer, the ENPP1 inhibitor compounds may be administered in combination with chemotherapeutic agents selected from the group consisting of alkylating agents, nitrosoureas, antimetabolites, antitumor antibiotics, plant (vinca) alkaloids, steroid hormones, taxanes, nucleoside analogs, steroids, anthracyclines, thyroid hormone replacement drugs, thymidylate-targeted drugs, chimeric antigen receptor / T cell therapy, chimeric antigen receptor / NK cell therapy, apoptosis regulator inhibitors (e.g., B-cell CLL / lymphoma 2 (BCL-2) BCL-2-like 1 (BCL-XL) inhibitors), CARP-1 / CCAR1 (cell division cycle and apoptosis regulator 1) inhibitors, colony-stimulating factor 1 receptor (CSF1R) inhibitors, CD47 inhibitors, cancer vaccines (e.g., Th17-inducing dendritic cell vaccines or genetically modified tyrosinase (such as Oncept®)), and other cellular therapies.

[0255] 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, temsirolimus, everolimus, methotrexate, doxorubicin, Abraxane, Forfirinox, cisplatin, carboplatin, 5-fluorouracil, Teysumo, paclitaxel, prednisone, levothyroxine, pemetrexed, navitoclax, and ABT-199. Peptide compounds can also be used.Cancer chemotherapy agents of interest include but are not limited to dolastatin and its active analogue and derivative; and auristatin and its active analogue and derivative (for example, monomethyl auristatin D (MMAD), monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF) etc.).For example, see WO96 / 33212, WO96 / 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 (including, e.g., the synthetic analogs, KW-2189 and CB1-TM1); and benzodiazepines and their active analogs and derivatives (e.g., pyrrolobenzodiazepines (PBDs)).

[0256] In some embodiments, the ENPP1 inhibitor compound can be administered in combination with a chemotherapy agent for treating cancer. In certain cases, the chemotherapy agent is gemcitabine. In some cases, the chemotherapy agent is docetaxel. In some cases, the chemotherapy agent is abraxane.

[0257] For the treatment of cancer (e.g., solid tumor cancer), the ENPP1 inhibitor compound can be administered in combination with an immunotherapeutic agent. An immunotherapeutic agent is any convenient agent found to be used in the treatment of disease by inducing, enhancing, or suppressing an immune response. In some cases, the immunotherapeutic agent is an immune checkpoint inhibitor. For example, Figures 21A-21C illustrate that an exemplary ENPP1 inhibitor can act synergistically with an immune checkpoint inhibitor in a mouse model. Any convenient checkpoint inhibitor can be utilized, including, but not limited to, a cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) inhibitor, a programmed death 1 (PD-1) inhibitor, and a PD-L1 inhibitor. In certain examples, the checkpoint inhibitor is selected from a cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) inhibitor, a programmed death 1 (PD-1) inhibitor, and a PD-L1 inhibitor. Exemplary checkpoint inhibitors of interest include, but are not limited to, ipilimumab, pembrolizumab, and nivolumab. In certain embodiments, when treating cancer and / or inflammatory diseases, the immunomodulatory polypeptide(s) can be administered in combination with a colony-stimulating factor 1 receptor (CSF1R) inhibitor. CSF1R inhibitors of interest include, but are not limited to, emactuzumab.

[0258] Any advantageous cancer vaccine therapy and drug can be used in combination with the target ENPP1 inhibitor compound, composition and method.For the treatment of cancer, such as ovarian cancer, the ENPP1 inhibitor compound can be administered in combination with vaccination therapy, for example, dendritic cell (DC) vaccination agent that promotes Th1 / Th17 immunity.Th17 cell infiltration is associated with a significant increase in overall survival time in ovarian cancer patients.In some cases, the ENPP1 inhibitor compound is found to be used as an adjuvant therapy in combination with Th17-inducing vaccination.

[0259] Also of interest are agents that are CARP-1 / CCAR1 (cell cycle and apoptosis regulator 1) inhibitors, including but not limited to those described by Rishi et al., Journal of Biomedical Nanotechnology, Volume 11, Number 9, September 2015, pp. 1608-1627(20), and CD47 inhibitors, including but not limited to anti-CD47 antibody agents such as Hu5F9-G4.

[0260] In certain instances, the combination provides enhanced efficacy compared to either component alone. In some cases, the combination achieves greater than additive or synergistic effects compared to the combined or additive effects of the components. Various combinations of the subject compound and chemotherapeutic agent may be used, either sequentially or simultaneously. In the case of multiple doses, for example, the two agents may be staggered directly, or two or more doses of one agent may be staggered sequentially with a single dose of the other agent. Simultaneous administration of both agents may also be staggered sequentially or otherwise interspersed with the doses of the individual agents. In some cases, the time between doses may 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, after the start of treatment. Combination with cGAMP-inducing chemotherapeutic agents

[0261] Embodiments of the present disclosure include methods for treating cancer, in which the present ENPP1 inhibitor compound (or a pharmaceutical composition comprising such a compound) can be administered in combination with a chemotherapeutic agent capable of inducing cGAMP production in vivo. Exposing a subject to an effective amount of a particular chemotherapeutic agent can induce the production of 2'3'-cGAMP in the subject. Co-administration of the subject ENPP1 inhibitor compound to prevent the degradation of cGAMP can maintain and / or enhance the induced level of cGAMP, for example, compared to the level achieved with either agent alone. Any convenient chemotherapeutic agent capable of causing DNA damage and inducing cGAMP production by cell death through overwhelming repair or degradation mechanisms, such as alkylating agents, nucleic acid analogs, and intercalating agents, can be used in the subject combination therapy. In some cases, the cGAMP-inducing chemotherapeutic agent is an antimitotic agent. An antimitotic agent is an agent that acts by damaging DNA or binding to microtubules. In some cases, the cGAMP-inducing chemotherapeutic agent is an antitumor agent.

[0262] The cancers that can be treated using the target combination therapy include, but are not limited to, adrenal gland, liver, kidney, bladder, breast, colon, stomach, ovary, cervix, uterus, esophagus, colorectal, prostate, pancreas, lung (both small cell and non-small cell), thyroid, carcinoma, sarcoma, glioma, glioblastoma, melanoma and various head and neck tumors.In some cases, cancer is breast cancer.In certain examples, cancer is glioma or glioblastoma.

[0263] Chemotherapeutic agents of interest include, but are not limited to, uracil analogs, fluorouracil prodrugs, thymidylate synthase inhibitors, deoxycytidine analogs, DNA synthesis inhibitors (e.g., resulting in S-phase apoptosis), folic acid analogs, dehydrofolate reductase inhibitors, anthracyclines, intercalating agents (e.g., resulting in double-strand breaks), topoisomerase IIa inhibitors, taxanes, microtubule disassembly inhibitors (e.g., resulting in G2 / M arrest / apoptosis), microtubule assembly inhibitors, microtubule function stabilizers (e.g., resulting in G2 / M apoptosis), tubulin polymerization promoters, tubulin binding agents (e.g., These include, for example, agents that cause apoptosis by M-phase arrest), epothilone B analogs, vinca alkaloids, nitrogen mustards, nitrosoureas, DNA alkylating agents (e.g., interstrand crosslinks, causing apoptosis by p53), VEGF inhibitors, anti-angiogenic antibodies, HER2 inhibitors, quinazoline HER2 inhibitors, EGFR inhibitors, tyrosine kinase inhibitors, sirolimus analogs, mTORC1 inhibitors (e.g., in breast cancer, in combination with exemestane, an aromatase inhibitor that blocks estrogen production), triazenes, dacarbazine prodrugs, and methylhydrazine.

[0264] Exemplary breast cancer chemotherapy agents of interest include, but are not limited to, capecitabine, carmofur, fluorouracil, tegafur, gemcitabine, methotrexate, doxorubicin, epirubicin, docetaxel, ixabepilone, vindesine, vinorelbine, cyclophosphamide, bevacicumab, pertuzumab, trastuzumab, lapatinib, and everolimus. Exemplary antineoplastic drugs related to glioma / glioblastoma include, but are not limited to, carmustine, lomustine, temozolomide, procarbazine, vincristine, and bevacicumab. Exemplary DNA-damaging chemotherapy agents of interest include, but are not limited to, melphalan, cisplatin, etoposide, fluorouracil, and gemcitabine. Concomitant radiation therapy

[0265] Alternatively, in the case of methods for treating cancer, the subject ENPP1 inhibitor compound (or a pharmaceutical composition comprising such a compound) can be administered in combination with radiation therapy. In certain embodiments, the method includes administering radiation therapy to a subject. Again, the subject ENPP1 inhibitor compound can be administered before or after radiation therapy. Thus, the subject method can further include administering radiation therapy to a subject. Combining radiation therapy with the administration of a subject compound can achieve a synergistic therapeutic effect. When a subject is exposed to a suitable dose and / or frequency of radiation during radiation therapy (RT), the production of 2'3'-cGAMP can be induced in the subject. When a subject ENPP1 inhibitor compound is co-administered to prevent the degradation of cGAMP, the induction of such levels of cGAMP can be maintained and / or enhanced, e.g., compared to the levels achieved with RT alone. For example, Figure 21A illustrates that an exemplary ENPP1 inhibitor can act synergistically with radiation therapy (RT) to reduce tumor burden in a mouse model. Thus, embodiments of the subject methods include administering a reduced dose and / or frequency / regimen of radiation treatment compared to a therapeutically effective dose and / or frequency / regimen of radiation treatment alone. In some cases, radiation therapy is administered in combination with a subject compound at a dose and / or frequency effective to reduce the subject's risk of radiation damage, e.g., radiation damage that would be expected to occur under a therapeutically effective dose and / or frequency / regimen of radiation treatment alone.

[0266] In some cases, the method includes administering an ENPP1 inhibitor to the subject before radiation therapy. In some cases, the method includes administering an ENPP1 inhibitor to the subject after exposing the subject to radiation therapy. In certain cases, the method includes sequentially administering radiation therapy, then an ENPP1 inhibitor, and then a checkpoint inhibitor to the subject in need thereof. usefulness

[0267] For example, the compounds and methods of the present invention described herein find use in a variety of applications.Targeted applications include, but are not limited to, research applications and therapeutic applications.The methods of the present invention find use in a variety of different applications, including any convenient application where inhibition of ENPP1 is desired.

[0268] The subject compounds and methods find use in a variety of research applications: The subject compounds and methods can be used to optimize the bioavailability and metabolic stability of compounds.

[0269] The subject compounds and methods find use in various therapeutic applications.The intended therapeutic applications include use in cancer treatment.Therefore, the subject compounds find use in treating a variety of different conditions, where it is desirable to inhibit and / or treat cancer in the host.For example, the subject compounds and methods can find use in treating solid tumor cancer (for example, as described herein). Pharmaceutical Composition

[0270] The compounds discussed herein can be formulated using any convenient excipients, reagents and methods. The compositions are provided in formulations containing pharmaceutically acceptable excipient(s). A wide variety of pharmaceutically acceptable excipients are known in the art and need not be discussed in detail herein. Pharmaceutically acceptable excipients are, for example, those described in A. Gennaro (2000) "Remington: The Science and Practice of Pharmacy," 20th edition, Lippincott, Williams, & Wilkins; Pharmaceutical Dosage Forms and Drug Delivery Systems (1999) H.C. Ansel et al., eds., 7 thed., Lippincott, Williams, & Wilkins; and Handbook of Pharmaceutical Excipients (2000) AH Kibbe et al., eds., 3 rd ed. Amer. Pharmaceutical Assoc.

[0271] Pharmaceutically acceptable excipients, such as vehicles, adjuvants, carriers, or diluents, are readily available to the public. Additionally, pharmaceutically acceptable auxiliary substances, such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents, and the like, are readily available to the public.

[0272] 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, ranging in strength from 5 mM to 100 mM. In some embodiments, the aqueous buffer contains an agent that provides an isotonic solution. Such agents include, but are not limited to, sodium chloride; and sugars, such as mannitol, dextrose, and sucrose. In some embodiments, the aqueous buffer further contains a non-ionic surfactant, such as polysorbate 20 or 80. If necessary, the formulation may further contain a preservative. Suitable preservatives include, but are not limited to, benzyl alcohol, phenol, chlorobutanol, and benzalkonium chloride. In many cases, the formulation is stored at about 4°C. The formulation may also be lyophilized, in which case the formulation generally contains a cryoprotectant, such as sucrose, trehalose, lactose, maltose, or mannitol. Lyophilized formulations can be stored for extended periods, even at ambient temperatures. In some embodiments, the subject compounds are formulated for sustained release.

[0273] In some embodiments, the subject compound and a second active agent (e.g., as described herein), such as a small molecule, chemotherapeutic agent, antibody, antibody fragment, antibody-drug conjugate, aptamer, or protein, are administered to an individual in a formulation (e.g., in the same formulation or separate formulations) comprising a pharmaceutically acceptable excipient(s). In some embodiments, the second active agent is a checkpoint inhibitor, such as a cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) inhibitor, a programmed death 1 (PD-1) inhibitor, or a PD-L1 inhibitor.

[0274] In another aspect of the present invention, a pharmaceutical composition is provided comprising or consisting essentially of a compound of the present invention, or a pharmaceutically acceptable salt, isomer, tautomer, or prodrug thereof, further comprising one or more additional active agents of interest. Any convenient active agent can be used in conjunction with the subject compound in the subject method. In some examples, the additional agent is a checkpoint inhibitor. For combination therapy, the subject compound and checkpoint inhibitor, as well as the additional therapeutic agent described herein, can be administered orally, subcutaneously, intramuscularly, nasally, parenterally, or by other routes. The subject compound and the second active agent (if present) can be administered by the same or different routes of administration. The therapeutic agent can be administered by any suitable means, including, but not limited to, oral, rectal, nasal, topical (including transdermal, aerosol, buccal, and sublingual), vaginal, parenteral (including subcutaneous, intramuscular, intravenous, and intradermal), intravesically, or by injection into the affected organ. In certain cases, the therapeutic agent can be administered intranasally. In some cases, the therapeutic agent can be administered intratumorally.

[0275] In some embodiments, the subject compound and the chemotherapeutic agent are administered to an individual in a formulation (e.g., in the same formulation or separate formulations) containing a pharmaceutically acceptable excipient(s). 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 dolastatins and their active analogs and derivatives; and auristatins and their active analogs and derivatives (e.g., monomethylauristatin D (MMAD), monomethylauristatin E (MMAE), monomethylauristatin F (MMAF), etc.). See, for example, WO96 / 33212, WO96 / 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 (including, e.g., the synthetic analogs, KW-2189 and CB1-TM1); and benzodiazepines and their active analogs and derivatives (e.g., pyrrolobenzodiazepines (PBDs)).

[0276] For combination therapy, the subject compound and the second chemotherapeutic agent, as well as the additional therapeutic agent 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 or different routes of administration. The therapeutic agents can be administered by any suitable means, including, but not limited to, oral, rectal, nasal, topical (including transdermal, aerosol, buccal, and sublingual), vaginal, parenteral (including subcutaneous, intramuscular, intravenous, and intradermal), intravesical, or by injection into the affected organ.

[0277] The subject compounds may be provided in unit dosage form and may be prepared by any method known in the art. Such methods include combining the subject compounds with pharmaceutically acceptable carriers or diluents, which constitute one or more accessory ingredients. Pharmaceutically acceptable carriers are selected based on the selected route of administration and standard pharmaceutical practice. Each carrier must be "pharmaceutically acceptable" in the sense of being compatible with the other ingredients of the formulation and not harmful to the subject. The carrier can be solid or liquid, and the type is generally selected based on the type of administration used.

[0278] Examples of suitable solid carriers include lactose, sucrose, gelatin, agar, and bulk powder.Examples of suitable liquid carriers include water, pharmaceutically acceptable fats and oils, alcohols, or other organic solvents, including esters, emulsions, syrups or elixirs, suspensions, solutions and / or suspensions, and solutions and / or suspensions reconstituted from non-foaming granules, and foaming preparations reconstituted from foaming granules.Such liquid carriers can contain, for example, suitable solvents, preservatives, emulsifiers, suspending agents, diluents, sweeteners, thickeners, and melting agents.Preferred carriers are edible oils, such as corn oil or canola oil.Polyethylene glycol, such as PEG, is also a good carrier.

[0279] Any drug delivery device or system that achieves the dosing regimen of the present disclosure may be used. A wide variety of delivery devices and systems are known to those skilled in the art. [Example]

[0280] The following examples are put forward so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use embodiments of the present disclosure, and are not intended to limit the scope of what the inventors regard as their invention, nor are they intended to represent that the following experiments are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric.

[0281] While the present invention has been described with reference to specific embodiments thereof, it should be understood 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 invention. Moreover, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step(s), to the objective, spirit and scope of the present disclosure. All such modifications are intended to be within the scope of the appended claims.

[0282] Example 1 Compound synthesis

[0283] The compounds may be synthesized using any convenient method. Methods that can be adapted for use in preparing the compounds of the present disclosure include the exemplary synthetic methods described in Examples 1a-1c and the method described by Li et al. in PCT Application No. PCT / US2018 / 050018, filed September 7, 2018, the entire disclosure of which is incorporated herein by reference. Numerous general references presenting generally known chemical synthetic schemes and conditions useful for synthesizing 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). Reactions can be monitored by thin layer chromatography (TLC), LC / MS, and reaction products can be analyzed by LC / MS and 1 The compounds may be characterized by H NMR. Intermediates and final products may be purified by silica gel chromatography or by HPLC.

[0284] Example 1a Exemplary Synthesis Scheme Compound 1

[0285] The synthesis of compound 1, which may be suitable for use in preparing compounds of this disclosure, is described below; [ka]

[0286] Preparation of dimethyl(2-(piperidin-4-yl)ethyl)phosphonate [ka]

[0287] To a stirred solution of bis(dimethoxyphosphoryl)methane (11.42 g, 49.19 mmol) in toluene (100 mL) was carefully added sodium hydride (2.16 g, 54.11 mmol) at room temperature. The reaction mixture was then placed under a nitrogen atmosphere, and a solution of 1-benzylpiperidine-4-carbaldehyde (10 g, 49.19 mmol) in toluene (50 mL) was slowly added while maintaining the temperature below 40 °C. The resulting mixture was stirred at room temperature for 16 h and then quenched by the addition of saturated aqueous ammonium chloride. The organic phase was separated, washed with brine, dried (MgSO ), and evaporated to dryness. Chromatography (120 g SiO ; 5–100% EtOAc gradient in hexanes) afforded dimethyl (E)-(2-(1-benzylpiperidin-4-yl)vinyl)phosphonate (6.2 g, 16%) as a colorless oil.

[0288] 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, stirred at room temperature for 12 hours, filtered, and evaporated to dryness under reduced pressure to give dimethyl (2-(piperidin-4yl)ethyl)phosphonate (2.7 g, 100%) as a colorless oil.

[0289] Preparation of dimethyl(2-(1-(6,7-dimethoxyquinazolin-4-yl)piperidin-4-yl)ethyl)phosphonate [ka]

[0290] 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 isopropyl alcohol (20 mL) was added diisopropylethylamine (0.6 g, 8.9 mmol). 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) afforded dimethyl (2-(1-(6,7-dimethoxyquinazolin-4-yl)piperidin-4-yl)ethyl)phosphonate (755 mg, 37%) as an oil. LC-MS: m / z=410.25[M+H] + 1 H NMR (500 MHz, CDCl3) δ 8.65 (s, 1H), 7.23 (s, 1H), 7.09 (s, 1H), 4.19 (dq, J = 14.0,2.9, 2.4 Hz, 2H), 4.02 (s, 3H), 3.99 (s, 3H), 3.77 (s, 3H), 3.75 (s, 3H), 3.05 (td, J = 12.8,2.3 Hz, 2H), 1.93 - 1.77 (m, 4H), 1.67 (ddd, J = 14.1, 9.5, 5.9 Hz, 3H), 1.46 (qd, J =12.2, 3.7 Hz, 2H).

[0291] Preparation of dimethyl(2-(1-(6,7-dimethoxyquinazolin-4-yl)piperidin-4yl)ethyl)phosphonic acid (compound 1) [ka]

[0292] 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) cooled with an ice bath was added bromotrimethylsilane (3.67 g, 24 mmol). The reaction mixture was warmed to room temperature and quenched after 90 minutes by the addition of methanol (20 mL). The mixture was evaporated to dryness under reduced pressure and then solvated in methanol (100 mL). The reaction mixture was concentrated to half its volume, filtered to remove the precipitate, and evaporated to dryness. The residue was crystallized from dichloromethane, filtered, and dried under vacuum to give dimethyl (2-(1-(6,7-dimethoxyquinazolin-4-yl)piperidin-4-yl)ethyl)phosphonic acid (2.1 g, 69%). LC-MS: m / z=381.8[M+H] + 1 H NMR (500 MHz, DMSO-d6) δ 8.77 (s, 1H), 7.34 (s, 1H), 7.23 (s, 1H), 4.71 (d, J =13.1 Hz, 2H), 3.99 (s, 3H), 3.97 (s, 3H), 3.48 (t, J = 12.7 Hz, 2H), 3.18 (s, 1H), 1.97-1.90 (m, 2H), 1.62-1.43 (m, 4H), 1.40-1.27 (m, 2H).

[0293] Example 1b Synthesis of Compound 5 (Table 1)

[0294] Compound 5 is prepared using the synthetic scheme illustrated below: [ka]

[0295] Example 1c Synthesis of Compound 6 (Table 1)

[0296] Compound 6 is prepared using the synthetic scheme illustrated below: [ka]

[0297] Chemical synthesis: Reactions were performed under ambient atmosphere unless otherwise noted. Qualitative TLC analysis was performed on 250 mm thick, 60 Å, glass-backed, F254 silica (Silicycle, Quebec City, Canada). Visualization was performed by exposure to UV light and p-anisaldehyde or KMnO4 staining solution followed by heating. All solvents used were ACS-grade, Sure-Sealed, and all other reagents were used as received unless otherwise noted. The synthesis of commercially unavailable 4-chloroquinazoline and 4-chloro3-quinone nitrile, along with the amine building blocks, is described in the Supplementary Information. Flash chromatography was performed on a Teledyne Isco purification system using silica gel flash cartridges (SiliCycle®, SiliaSep™ 40-63 μm, 60 Å). HPLC was performed on an Agilent 1260 Infinity preparative-scale purification system using an Agilent PrepHT Zorbax Eclipse XDB-C18 reversed-phase column (21.2 × 250 mm). Structure determinations were recorded on a Bruker AV-500 spectrometer. 1 H spectra and low-resolution mass spectra (ESI-MS) collected on a Shimadzu 20-20 ESI LCMS instrument. Structural determinations were recorded on either a Bruker AV-500 or AV-400 spectrometer. 1 H spectra and low-resolution mass spectra (ESI-MS) collected on a Shimadzu 20-20 ESI LCMS instrument. The purity of the final compound was >95% as determined by HPLC-MS. 1 All H spectra were consistent with the expected structure.

[0298] Synthesis of ureas 4 and 5.

[0299] [ka]

[0300] Preparation of 1-(2-(1-(6,7-dimethoxyquinazolin-4-yl)piperidin-4-yl)ethyl)urea 4 (in Table 3a)

[0301] 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 a nitrogen atmosphere. The mixture was stirred at room temperature for 2 h and then evaporated to dryness under reduced pressure. Purification (preparative HPLC) afforded the title compound 4 (172 mg, 47%) as light yellow crystals.

[0302] LCMS: [M+H] + m / z 360. 1 H NMR (400 MHz, DMSO-d6) δ 8.49 (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).

[0303] Preparation of 1-((1-(6,7-dimethoxyquinazolin-4-yl)piperidin-4-yl)methyl)urea 5 (in Table 3a)

[0304] 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 a nitrogen atmosphere. The mixture was stirred at 10 °C for 3 h and then evaporated to dryness under reduced pressure. Chromatography (SiO: 0-6% MeOH in dichloromethane) afforded the desired product 5 (150 mg, 44%) as a white solid.

[0305] LCMS: [M+H] + m / z346.0 1 H 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).

[0306] Preparation of 3-(1-(6,7-dimethoxyquinazolin-4-yl)piperidin-4-yl)propanoic acid 6 (in Table 3a)

[0307] [ka]

[0308] 4-Chloro-6,7-dimethoxy-quinazoline 63 (3.14 g, 13.98 mmol) and 3-(4-piperidyl)propanoic acid (2.0 g, 12.72 mmol) were suspended in isopropanol (100 mL) and stirred for 3 h at 90 °C. Once cooled, the mixture was evaporated to dryness under reduced pressure. The residue was then triturated with CHCl (20 mL) to give the title compound 6 (1.87 g, 42%) as a white solid.

[0309] 1 H NMR (400 MHz, methanol-d4) δ

[0310] Preparation of (2-(1-(6,7-dimethoxyquinolin-4-yl)piperidin-4-yl)ethyl)boronic acid 7 (in Table 3a)

[0311] [ka]

[0312] 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 (SiO; 2–5% ethyl acetate in petroleum ether) gave 68 (4.2 g, 89%). A mixture of tert-butyl (E)-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 on carbon (840 mg, 20% w / w) in MeOH (500 mL) was placed under a hydrogen atmosphere and stirred at room temperature for 16 h. The mixture was then filtered through a pad of Celite® and evaporated to dryness under reduced pressure to give 69 (4.2 g, 92%). A solution of 1 M aqueous HCl (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, stirred for 3 h, and then evaporated to dryness under reduced pressure to give (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 h and then evaporated to dryness under reduced pressure. Purification (preparative HPLC) afforded the title compound as a light yellow solid (105 mg; 37%).

[0313] LCMS: [M+H] + m / z 346.3. 1H NMR (400 MHz, DMSO-d6) δ 8.67 (s, 1H), 7.26 (s, 1H), 7.25 (s, 1H), 4.62-4.59 (m, 2H), 3.92 (s, 3H), 3.90 (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).

[0314] Preparation of hydroxamic acids 8 and 9.

[0315] [ka]

[0316] Preparation of 2-(1-(6,7-dimethoxyquinazolin-4-yl)piperidin-4-yl)-N-hydroxyacetamide 8 (in Table 3a)

[0317] 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. for 16 hours in a sealed tube. 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-dimethoxyquinazolin-4-yl)piperidin-4-yl)acetate (750 mg, 77%). To a mixture of ethyl 2-(1-(6,7-dimethoxyquinazolin-4-yl)piperidin-4-yl)acetate (250 mg, 0.696 mmol) in THF (10 mL) was added 2 M NaOH solution in HO (1 mL). The mixture was stirred at room temperature for 16 h and then quenched by the addition of 1 M HCl solution. The organic phase was extracted with ethyl acetate, washed with brine, dried (NaSO), and evaporated to dryness under reduced pressure to give acid 72 (200 mg, 86%) as a white solid.

[0318] To a mixture of acid 72 (300 mg, 0.906 mmol) in THF (10 mL) was added NHOH·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 h, then diluted with water, extracted with ethyl acetate, washed with brine solution, dried (NaSO), and evaporated to dryness under reduced pressure. Chromatography (SiO, solvent) afforded the title product 8 (180 mg, 77%) as a white solid. LCMS: [M+H] + m / z 347.10. 1 H NMR (400 MHz, D2O) δ 8.42 (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). Preparation of 1-(6,7-dimethoxyquinazolin-4-yl)-N-hydroxypiperidine-4-carboxamide 9 (in Table 3a)

[0319] Synthesized according to the procedure of 8, except that ethyl piperidine-4-carboxylate 73 was used.

[0320] LCMS: [M+H] + m / z 333.25. 1 H NMR (400 MHz, D2O) δ 8.39 (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).

[0321] General procedure for compounds 10, 11, 12, 13 and 16 (in Table 3a).

[0322] [ka]

[0323] Preparation of 2-(1-(6,7-dimethoxyquinolin-4-yl)piperidin-4-yl)ethan-1-ol 77

[0324] 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) in a sealed tube was stirred for 16 h at 100° C. Upon cooling, the reaction mixture was concentrated under reduced pressure, and the residue was purified by silica gel chromatography (SiO; EtOAc in petroleum ether) to give 2-(1-(6,7-dimethoxyquinolin-4-yl)piperidin-4-yl)ethan-1-ol 75 (1.3 g, 91%).

[0325] Preparation of (1-(6,7-dimethoxyquinolin-4-yl)piperidin-4-yl)methanol 78

[0326] 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 at 100 °C for 16 h in a sealed tube. Upon cooling, the reaction mixture was evaporated to dryness under reduced pressure. Purification by chromatography (SiO; 10–80% ethyl acetate in petroleum ether) gave (1-(6,7-dimethoxyquinolin-4-yl)piperidin-4-yl)methanol 78 (1 g, 82%).

[0327] Preparation of 2-(1-(6,7-dimethoxyquinazolin-4-yl)piperidin-4-yl)ethyl dihydrogen phosphate 10 (in Table 3a)

[0328] 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 dry pyridine, which was then cooled to −15° C. and stirred for 10 min. POCl (821 mg, 5.4 mmol) was added dropwise under a N atmosphere. The reaction temperature was slowly raised to 0° C. and then stirred again for an additional 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 using 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.

[0329] LCMS: [M+H] + m / z 398. 1 H NMR (400 MHz, DMSO-d6) δ 8.54 (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 = 6.4 Hz, 2H) and 1.44-1.32 (m, 2H).

[0330] Preparation of (1-(6,7-dimethoxyquinolin-4-yl)piperidin-4-yl)methyl dihydrogen phosphate 11 (in Table 3a)

[0331] (1-(6,7-Dimethoxyquinolin-4-yl)piperidin-4-yl)methanol 78 (100 mg, 0.33 mmol) was dissolved in dry pyridine (3 mL), which was then cooled to -15 °C and stirred for 10 min. POCl (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 an additional 30 min. The mixture was poured into aqueous NaHCO (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. Purification by preparative HPLC afforded (1-(6,7-dimethoxyquinolin-4-yl)piperidin-4-yl)methyl dihydrogen phosphate 11 (70 mg, 55%) as a white powder after lyophilization. LCMS: [M+H] + m / z 384.20. 1 H NMR (400 MHz, DMSO-d6) δ 8.74 (d, J = 1.7 Hz, 1H), 7.31 (s, 1H), 7.20 (s, 1H), 4.66 (d, J = 13.0 Hz, 1H), 3.97 (m, J = 12.6, 1.6 Hz, 8H), 3.76 (t, J = 6.6 Hz, 3H), 2.19-2.00 (m, 1H), 1.92 (d, J = 13.5 Hz, 2H), 1.45 (dd, J = 14.2, 10.7 Hz, 1H).

[0332] Preparation of O-(2-(1-(6,7-dimethoxyquinazolin-4-yl)piperidin-4-yl)ethyl) O,O-dihydrogen phosphorothioate 12 (in Table 3a)

[0333] To a solution of 2-(1-(6,7-dimethoxyquinolin-4-yl)piperidin-4-yl)ethan-1-ol 77 (150 mg, 0.473 mmol) in dry pyridine (5 mL) was added P(S)Cl (477 mg, 2.84 mmol) at −15° C. The mixture was stirred at 0° C. for 0.5 h and then poured into a solution of NaHCO (238 mg, 2.84 mmol) in HO (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. 1 H NMR (400 MHz, DMSO-d6) δ 8.62 (s, 1H), 7.19 (d, J = 7.7 Hz, 2H), 4.45 (d, J = 12.3 Hz, 2H), 3.91 (d, J = 11.3 Hz, 10H), 1.86 (d, J = 12.2 Hz, 3H), 1.56 (d, J = 6.4 Hz, 2H), 1.34 (d, J = 10.7 Hz, 2H).

[0334] Preparation of O-((1-(6,7-dimethoxyquinazolin-4-yl)piperidin-4-yl)methyl) O,O-dihydrogen phosphorothioate 13 (in Table 3a)

[0335] To a solution of (1-(6,7-dimethoxyquinolin-4-yl)piperidin-4-yl)methanol 78 (100 mg, 0.330 mmol) in dry pyridine (5 mL) was added P(S)Cl (280 mg, 1.98 mmol) at −15° C. The mixture was stirred at 0° C. for 0.5 h and then poured into a solution of NaHCO (116 mg, 1.98 mmol) in HO (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. 1H NMR (400 MHz, DMSO-d6) δ 8.54 (s, 1H), 7.18 (s, 1H), 7.11 (s, 1H), 4.25 (d, J = 13.4 Hz, 2H), 3.89 (d, J = 9.1 Hz, 6H), 3.76 (s, 2H), 3.10 (d, J = 11.8 Hz, 3H), 1.94 (s, 1H), 1.81 (d, J = 12.7 Hz, 2H), 1.39 (d, J = 11.4 Hz, 1H).

[0336] Preparation of ((1-(6,7-dimethoxyquinazolin-4-yl)piperidin-4-yl)methyl)phosphonic acid 16

[0337] PPh3 (3.39 g, 15 mmol) and imidazole (1.02 g, 15 mmol) in anhydrous CHCl2 (40 mL) were stirred at 0 °C for 10 min, 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, 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 quenched by adding aqueous NaSO3. The crude mixture was extracted with CHCl2, washed with water, brine, dried (NaSO4), 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. 1H NMR (400 MHz, CDCl3) δ 8.63 (d, J = 1.3 Hz, 1H), 7.28 (s, 1H), 7.07 (s, 1H), 4.23 (s, 2H), 4.00 (s, 6H), 3.19 (d, J = 6.5 Hz, 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). To a cooled (0°C) solution of bis(benzyloxy)(oxo)-λ-phosphane (9.5 g, 36.3 mmol) in anhydrous MeCN (40 mL) was added 1,8-diazabicyclo(5.4.0)undec-7-ene (DBU) (9.2 g, 60.5 mmol). After 10 min, 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. 1 H NMR (400 MHz, CDCl3) δ 8.57 (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 = 13.5 Hz, 2H), 4.09 (s, 3H), 3.93 (s, 3H), 3.27 (d, J = 12.9 Hz, 2H), 2.05 (d, J = 13.9 Hz, 5H), 1.76 (m, 4H), 1.42 (d, J = 12.5 Hz, 2H).

[0338] A mixture containing 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 an atmosphere of H and stirred at room temperature. After 4 h, the crude mixture was filtered through a Celite® pad and the filtrate was evaporated to dryness under reduced pressure. Purification by preparative HPLC afforded ((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 (400 MHz, DMSO-d6) δ 8.72 (s, 1H), 7.29 (s, 2H), 4.60 (d, J = 12.8 Hz, 2H), 3.95 (d, J = 11.2 Hz, 6H), 3.46 (s, 2H), 2.09 (s, 3H), 1.61 (s, 2H), 1.42 (s, 2H).

[0339] Preparation of (2-(1-(6,7-dimethoxyquinazolin-4-yl)piperidin-4-yl)propyl)phosphonic acid 14 (in Table 3a)

[0340] [ka]

[0341] To a solution of PPh3 (1.4 g, 5.34 mmol) and imidazole (0.36 g, 5.34 mmol) in CHCl2 (20 mL) was added iodine (1.35 g, 5.34 mmol). The mixture was stirred at room temperature for 0.5 h, and then a solution of 79 (1.0 g, 4.11 mmol) in CHCl2 (5 mL) was added dropwise. The reaction mixture was stirred at room temperature for 4 h, then quenched with saturated NaSO3 solution and extracted with CHCl2. The organic phase was washed with water, brine, dried (NaSO4), and evaporated to dryness under reduced pressure. Chromatography (SiO2; 5% EtOAc in petroleum ether) afforded tert-butyl 4-(3-iodopropyl)piperidine-1-carboxylate (1.0 g, 68% yield) as a light yellow oil.

[0342] To a mixture of tert-butyl 4-(3-iodopropyl)piperidine-1-carboxylate (1.0 g, 2.83 mmol) in DMF (50 mL) was added diethylphosphonate (0.58 g, 4.24 mmol) and CsCO (1.84 g, 5.66 mmol). The reaction mixture was stirred overnight at room temperature under a nitrogen atmosphere and then quenched by the addition of water. The organic phase was washed with water, brine, dried (NaSO), and evaporated to dryness under reduced pressure. Chromatography (SiO; 20% EtOAc in petroleum ether) afforded 80 (0.78 g, 76%) as a light yellow oil. LCMS: [M+H] + m / z 364.30.

[0343] 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 h 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.

[0344] To a solution of diethyl phosphonate (597 mg, 2.65 mmol) and crude 81 in CHCl (10 mL) was added DIPEA (1.37 g, 10.63 mmol). The mixture was stirred overnight at room temperature, then quenched with saturated aqueous NHCl and extracted with CHCl. ​​The organic phase was washed with water, brine, dried (NaSO), and evaporated to dryness under reduced pressure. Chromatography (SiO, 5% MeOH in CHCl) afforded the diethyl phosphonate intermediate (0.5 g, 39%) 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, then cooled to room temperature and evaporated to dryness under reduced pressure. Chromatography (preparative HPLC) afforded 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).

[0345] Preparation of (2-(1-(6,7-dimethoxyquinazolin-4-yl)piperidin-4-yl)ethyl)phosphonothio O,O-acid 17

[0346] [ka]

[0347] To a stirred solution of O,O-diethyl (2-(piperidin-4-yl)ethyl)phosphonothioate (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 h. The reaction mixture was cooled to room temperature, diluted with water, and extracted with ethyl acetate. The organic phase was dried (NaSO) and evaporated to dryness under reduced pressure. Purification (SiO, 0–100% EtOAc in hexanes) gave O,O-diethyl (2-(1-(6,7-dimethoxyquinazolin-4-yl)piperidin-4-yl)ethyl)phosphonothioate (380 mg, 46%). A stirred solution of O,O-diethyl (2-(1-(6,7-dimethoxyquinazolin-4-yl)piperidin-4-yl)ethyl)phosphonothioate (45 mg, 0.099 mmol) in TMSI (7 mL) was stirred at 60° C. for 16 h and then cooled to room temperature. The mixture was diluted with water and extracted with ethyl acetate. The organic phase was dried (NaSO) and then evaporated to dryness under reduced pressure. Purification by preparative HPLC afforded (2-(1-(6,7-dimethoxyquinazolin-4-yl)piperidin-4-yl)ethyl)phosphonothio O,O-acid 17 (13 mg, 32%) as a white solid. LCMS: [M+H] + m / z 396.25. 1 H NMR (400 MHz, DMSO-d6) δ 8.51 (s, 1H), 7.33 (s, 1H), 7.16 (s, 1H), 4.02 (s, 3H), 3.97 (s, 3H), 3.58 (d, J = 10.4 Hz, 3H), 3.48 (t, J = 12.0 Hz, 2H), 2.00 (d, J = 11.7 Hz, 2H), 1.81 (s, 1H), 1.64 (d, J = 17.9 Hz, 2H), 1.61-1.51 (m, 2H), 1.45-1.32 (m, 2H).

[0348] (2-(4-(6,7-dimethoxyquinazolin-4-yl)piperidin-1-yl)ethyl)phosphonic acid 18 (in Table 3a)

[0349] [ka] LCMS: [M+H] + m / z 382.25 1 H NMR (400 MHz, D2O) δ 8.65 (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).

[0350] (2-(4-(6,7-Dimethoxyquinazolin-4-yl)piperazin-1-yl)ethyl)phosphonic acid hydrobromide 19 (in Table 3a).

[0351] [ka]

[0352] 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 (NaSO), and evaporated to dryness under reduced pressure. Crystallization from diethyl ether afforded 83 (1.65 g, 84%) as a white solid. 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 (NaSO), and evaporation to dryness under reduced pressure were performed. Chromatography (12 g of SiO; 15% MeOH in CH2Cl2) followed by crystallization from diethyl ether gave the ethyl ester 85 (0.23 g; 49%) as a white solid. LCMS: [M+H] + m / z 410.10 1 H 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). 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) was added trimethylsilyl bromide (198 mg, 1.3 mmol). The resulting solution was stirred at room temperature for 3 h and then quenched by the addition of methanol. The mixture was evaporated to dryness under reduced pressure and crystallized from methanol-diethyl ether to give the desired product 19 (0.23 g, 89%) as the HBr salt. LCMS: [M+H] + m / z 382.8. 1H NMR (500 MHz, DMSO-d6) δ 8.97 (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 = 8.6 Hz, 2H), 3.17 (s, 2H), 2.90 (s, 2H), 2.74 (s, 2H), 2.20-2.08 (m, 2H).

[0353] Preparation of (4-(6,7-dimethoxyquinazolin-4-yl)phenethyl)phosphonic acid 20 (in Table 3a)

[0354] [ka]

[0355] To a solution of 2-(4-bromophenyl)ethan-1-ol 86 (5.0 g, 24.8 mmol) in anhydrous THF (100 mL) was added 2.5 M n-butyllithium (24 mL) under a nitrogen atmosphere at −78 °C. 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 adding 2 M HCl solution (100 mL) and stirring for 1 h. The mixture was extracted with dichloromethane (3 × 100 mL), dried (NaSO), and evaporated to dryness under reduced pressure. Chromatography (SiO; dichloromethane:methanol, 1:0 to 20:0) afforded boronic acid 87 (1.34 g, 33%) as a light yellow solid. This material was then dissolved in a solution of THF (30 mL) and water (10 mL). To this solution were added 4-chloro-6,7-dimethoxyquinazoline 63 (2.24 g, 10.0 mmol) and potassium carbonate (2.76 g, 20.0 mmol), followed by tetrakis(triphenylphosphine)palladium (0.5 g, 0.43 mmol). The resulting mixture was stirred at 65 °C for 16 h, then diluted with ethyl acetate, washed with brine, dried (NaSO), and evaporated to dryness under reduced pressure. Chromatography (SiO: 0–10% methanol in dichloromethane) afforded 88 (1.43 g, 60%) as a light yellow solid.

[0356] To a solution of triphenylphosphine (2.36 g, 9.0 mmol) in dichloromethane (24 mL) was added imidazole (700 mg, 10.28 mmol) at 0 °C. After stirring for 10 min, I2 (2.3 g, 9.0 mmol) was added. After stirring for an additional 10 min, compound 89 (1.5 g, 4.8 mmol) in dichloromethane (12 mL) was added. The mixture was allowed to warm to room temperature and stirred for 5 h. 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) afforded 89 (4.0 g) as a colorless oil.

[0357] To a mixture of crude 89 (930 mg, 2.2 mmol) and dibenzyl phosphonate (884 mg, 3.37 mmol) in DMF (20 mL) was added cesium carbonate (1.426 g, 4.4 mmol). The mixture was placed under a nitrogen atmosphere and stirred at room temperature for 3 h. 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 under hydrogen atmosphere at room temperature for 24 hours, then filtered through Celite®. Chromatography (preparative HPLC, acidic conditions) afforded compound 20 (55.4 mg, 36%) as a yellow solid. LCMS: [M+H] + m / z 375.0. 1 H NMR (400 MHz, DMSO-d6)): δ 9.09 (s, 1H), 7.75-7.73 (d, J = 8.0 Hz, 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).

[0358] Synthesis of (4-((6,7-dimethoxyquinolin-4-yl)amino)phenyl)phosphonic acid sodium salt 21 (in Table 3a)

[0359] [ka]

[0360] A mixture of 4-chloro-6,7-dimethoxyquinazoline 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-dimethoxyquinazolin-4-yl)amino)phenyl)phosphonate (0.92 g, 73% yield) as a white solid. This product was dissolved in MeCN (20 mL) to which trimethylsilyl bromide (2.8 mL, 22 mmol) was added. The resulting mixture was stirred at 60 °C for 6 h, cooled, and then evaporated to dryness under reduced pressure. The crude residue was quenched by the addition of saturated aqueous NaHCO (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 (400 MHz, D2O) δ 7.73 (s, 1H), 7.53 (t, J = 9.8 Hz, 2H), 7.22 (d, J = 7.4 Hz, 2H), 6.39 (s, 1H), 6.16 (s, 1H) and 3.39 (s, 6H).

[0361] Synthesis of (4-((6,7-dimethoxyquinolin-4-yl)amino)benzyl)phosphonic acid sodium salt 22 (in Table 3a)

[0362] [ka]

[0363] A mixture of 4-chloro-6,7-dimethoxyquinazoline 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). To this was added trimethylsilyl bromide (0.58 mL, 4.6 mmol). The mixture was stirred at 60 °C for 6 h. After concentration, the residue was treated with saturated aqueous NaHCO3 until the solution reached pH 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. 1 H NMR (400 MHz, D2O) δ 7.77 (s, 1H), 7.15 (s, 4H), 6.49 (s, 1H), 6.21 (s, 1H), 3.47 (s, 6H) and 2.70 (d, J = 19.5 Hz, 2H).

[0364] (4-(((6,7-Dimethoxyquinolin-4-yl)amino)methyl)phenyl)phosphonic acid sodium salt 23 (also referred to as 4 in Table 1).

[0365] [ka]

[0366] A mixture of 4-chloro-6,7-dimethoxyquinazoline 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 precipitated solid was filtered, washed with ethyl acetate, and evaporated to dryness under reduced pressure to give N-(4-bromobenzyl)-6,7-dimethoxyquinazolin-4-amine hydrochloride 91 (1.5 g, 88%) as a white solid. To a mixture of KOAc (11 mg, 0.112 mmol), Pd(OAc) (5.5 mg, 0.025 mmol), and dppf (27 mg, 0.049 mmol) in THF (10 mL) was added triethylamine (0.37 mL, 2.68 mmol) 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 10 mL of THF was added. The reaction was stirred at reflux for 6 hours and then partitioned between 30 mL of EtOAc and 20 mL of water. The organic phase was separated, washed with water, brine, dried (NaSO), and evaporated to dryness under reduced pressure. Purification by column chromatography (SiO; 50% petroleum ether in ethyl acetate) afforded diethyl (4-(((6,7-dimethoxyquinazolin-4-yl)amino)methyl)phenyl)phosphonate (0.2 g, 38%) as a yellow solid.

[0367] 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 NaHCO (pH 9), and the resulting mixture was purified by preparative HPLC (neutral) to give the title product 23 (102 mg, 22%) as an off-white solid. LCMS: [MH] + m / z:374.00.1 H NMR (400 MHz, D2O) δ 8.00 (s, 1H), 7.61 (s, 2H), 7.29 (d, J = 7.6 Hz, 2H), 6.70 (s, 1H), 6.55 (s, 1H), 4.62 (s, 2H), 3.75 (d, J = 18.2 Hz, 6H).

[0368] General procedure for synthesis of dimethyl(2-(piperidin-4-yl)ethyl)phosphonate 92 and diethyl(2-(piperidin-4-yl)ethyl)phosphonate 93 [ka]

[0369] To a stirred solution of bis(dimethoxyphosphoryl)methane 92 or bis(diethoxyphosphoryl)methane 93 (1 mol.eq.) in toluene was carefully added sodium hydride (1.1 mol.eq.) at room temperature. The reaction mixture was then placed under a nitrogen atmosphere, and a toluene solution of 1-benzylpiperidine-4-carbaldehyde 94 (1 mol.eq.) was slowly added, maintaining the temperature below 40°C. The resulting mixture was stirred at room temperature for 16 h 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; 5-100% EtOAc gradient in hexanes) afforded the dimethyl or diethyl (E)-(2-(1-benzylpiperidin-4-yl)vinyl)phosphonate as a colorless oil. To a mixture of dimethyl or diethyl (E)-(2-(1-benzylpiperidin-4-yl)vinyl)phosphonate (1 mol. equivalent) in ethanol is added catalytic Pd / C. The mixture is placed under a hydrogen atmosphere, stirred at room temperature for 12 hours, filtered, and evaporated to dryness under reduced pressure to give either dimethyl or diethyl (2-(piperidin-4-yl)ethyl)phosphonate 95 and 96 as colorless oils.

[0370] General procedure for the synthesis of dibenzyl(2-(piperidin-4-yl)ethyl)phosphonate 100

[0371] [ka]

[0372] To a solution of PPh3 (1.5 mol. equiv.) and imidazole (1.5 mol. equiv.) in CHCl was added iodine (1.5 mol. equiv.). After stirring for 10 min, a solution of 97 (1.0 mol. equiv.) in CHCl was added dropwise. The mixture was stirred at room temperature for 2 h, filtered through a Celite® pad, and treated with 5% sodium thiosulfate solution. The mixture was extracted with ethyl acetate, washed with brine, dried (NaSO), and evaporated to dryness under reduced pressure. Chromatography afforded 98 as an oil.

[0373] To a solution of compound 98 (3.0 mol.eq.) in MeCN was added DBU (5.0 mol.eq.) at 40°C. After stirring for 10 min, a solution of dibenzylphosphonate (1.0 mol.eq.) in MeCN was added dropwise. After stirring for 2 h, the reaction mixture was evaporated to dryness under reduced pressure and purified by chromatography to give 99.

[0374] A solution of compound 99 (1.0 mol. equiv.) in TFA / DCM is stirred at room temperature for 1 hour and then evaporated to dryness under reduced pressure to give 100 as an oil.

[0375] General method for the synthesis of (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.

[0376] Method A

[0377] 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 either 4-chloroquinazoline, 4-chloroquinoline, or 1-chloroisoquinoline (1 mol. equiv.) in isopropyl alcohol (0.1 M reaction concentration) was added diisopropylethylamine (2 mol. equiv.). After stirring at 90 °C for 3 h, the reaction mixture was cooled and evaporated to dryness. Purification on silica gel (5% MeOH in dichloromethane) afforded 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) was added trimethylsilyl bromide (3 mol. equiv.). The reaction mixture was warmed to room temperature and quenched after 90 min by the addition of methanol. The mixture was evaporated to dryness under reduced pressure and then solvated in methanol. The reaction mixture was concentrated to half its 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 phosphonic acid as the bromide salt.

[0378] Method B:

[0379] To a mixture of dimethyl (2-(piperidin-4-yl)ethyl)phosphonate 95 or diethyl (2-(piperidin-4-yl)ethyl)phosphonate 96 (1.1 mol. eq.) and either 4-chloroquinazoline, 4-chloroquinoline, or 1-chloroisoquinoline (1 mol. eq.) in dichloromethane (0.1 M reaction concentration) was added diisopropylethylamine (3 mol. eq.). After stirring overnight at room temperature, the reaction mixture was quenched by the addition of saturated aqueous NH4Cl solution. The organic phase was separated, washed with water and brine, dried (Na2SO4), and evaporated to dryness under reduced pressure. Purification on silica gel (5% MeOH in dichloromethane) afforded 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) was added trimethylsilyl bromide (3 mol. equiv.). The reaction mixture was stirred at 60 °C for 6 h, cooled, evaporated to dryness under reduced pressure, and the crude residue was quenched by the addition of saturated aqueous NaHCO3 (until a pH of 8-9 was observed). The crude residue was purified by preparative HPLC (neutral) to give the phosphonic acid as the sodium salt.

[0380] Method C:

[0381] To a mixture of dibenzyl (2-(piperidin-4-yl)ethyl)phosphonate 100 (1.1 mol. eq.) and either 4-chloroquinazoline, 4-chloroquinoline, or 1-chloroisoquinoline (1 mol. eq.) in dichloromethane (0.1 M reaction concentration) was added diisopropylethylamine (3 mol. eq.). After stirring overnight at room temperature, the reaction mixture was quenched by the addition of saturated aqueous NH4Cl. The organic phase was separated, washed with water and brine, dried (Na2SO4), and evaporated to dryness under reduced pressure. Purification on silica gel (5% MeOH in dichloromethane) afforded the dibenzylphosphonate. A mixture of dibenzylphosphonate (1 mol. eq.) and Pd / C in MeOH was placed under a hydrogen atmosphere and stirred at room temperature for 2 hours. The mixture was then filtered through Celite® and evaporated to dryness under reduced pressure to afford the phosphonic acid.

[0382] Preparation of (2-(1-(6,7-dimethoxyquinazolin-4-yl)piperidin-4-yl)ethyl)phosphonic acid (or compound 1) [ka]

[0383] Prepared according to Method A to give 15 (2.1 g, 69%) as an off-white solid. LCMS: [M+H] + m / z 381.8. 1 H NMR (500 MHz, DMSO-d6) δ 8.77 (s, 1H), 7.34 (s, 1H), 7.23 (s, 1H), 4.71 (d, J = 13.1 Hz, 2H), 3.99 (s, 3H), 3.97 (s, 3H), 3.48 (t, J = 12.7 Hz, 2H), 3.18 (s, 1H), 1.97-1.90 (m, 2H), 1.62-1.43 (m, 4H), 1.40-1.27 (m, 2H).

[0384] Preparation of (4-(((6,7-dimethoxyquinazolin-4-yl)amino)methyl)benzyl)phosphonic acid 24 (also referred to as 5 in the tables herein) [ka]

[0385] Prepared according to Method B. The product was isolated by preparative HPLC as an off-white solid (9% yield). LCMS: [M+H] + m / z 390.15. 1 H NMR (400 MHz, D2O) δ 8.12 (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)

[0386] Preparation of (2-(1-(6-methoxyquinazolin-4-yl)piperidin-4-yl)ethyl)phosphonic acid 25

[0387] [ka]

[0388] 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).

[0389] Preparation of (2-(1-(6-hydroxyquinazolin-4-yl)piperidin-4-yl)ethyl)phosphonic acid 26

[0390] [ka]

[0391] 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 (400 MHz, DMSO-d6) δ 8.48 (s, 1H), 7.65 (d, J = 8.8 Hz, 1H), 7.32 (d, J = 8.8 Hz, 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). Preparation of (2-(1-(7-methoxyquinazolin-4-yl)piperidin-4-yl)ethyl)phosphonic acid 27

[0392] [ka]

[0393] Prepared according to Method A to give 27 (95% yield) as an off-white solid. LCMS: [M+H] + m / z 352.0 1H 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).

[0394] Preparation of (2-(1-(7-ethoxyquinazolin-4-yl)piperidin-4-yl)ethyl)phosphonic acid 28. [ka]

[0395] Prepared according to Method B to give 28 as an off-white solid.

[0396] Preparation of (2-(1-(7-hydroxyquinazolin-4-yl)piperidin-4-yl)ethyl)phosphonic acid 29

[0397] [ka]

[0398] Prepared according to Method B to give 29 (4% yield) as a pale yellow solid. LCMS: [M+H] + m / z 338.25. 1H NMR (400 MHz, DMSO-d6) δ 11.49 (s, 1H), 8.64 (s, 1H), 7.96 (d, J = 9.2 Hz, 1H), 7.10 (dd, J = 9.2 and 2.2 Hz, 1H), 7.00 (d, J = 2.2 Hz, 1H), 4.62 (br s, 2H), 3.38 (br s, 2H), 1.87 (d, J = 12.7 Hz, 2H), 1.72 (br s, 1H), 1.58-1.38 (m, 4H) and 1.28-1.22 (m, 2H). Preparation of (2-(1-(7-aminoquinazolin-4-yl)piperidin-4-yl)ethyl)phosphonic acid 30

[0399] [ka]

[0400] Prepared according to Method C to give 30 (32% yield) as a light yellow solid. LCMS: [M+H] + m / z 337.10. 1 H NMR (400 MHz, DMSO-d6) δ 8.35 (s, 1H), 7.62 (d, J = 8.8 Hz, 1H), 6.81 (d, J = 8.8 Hz, 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).

[0401] Preparation of (2-(1-(7-isopropoxyquinazolin-4-yl)piperidin-4-yl)ethyl)phosphonic acid 31

[0402] [ka]

[0403] Prepared according to Method B to give 31 as a light yellow solid. LCMS: [M+H] + m / z 337.10. 1 H NMR (400 MHz, DMSO-d6) δ 8.35 (s, 1H), 7.62 (d, J = 8.8 Hz, 1H), 6.81 (d, J = 9.2 Hz, 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).

[0404] Preparation of (2-(1-(8-methoxyquinazolin-4-yl)piperidin-4-yl)ethyl)phosphonic acid 32

[0405] [ka]

[0406] Prepared according to Method B to give 32 (32% yield) as an off-white solid. LCMS: [M+H] + m / z 352.15. 1 H NMR (400 MHz, DMSO-d6) δ 7.92 (s, 1H), 6.92-6.88 (m, 1H), 6.80 (d, J = 7.6 Hz, 1H), 6.71 (d, J = 8.4 Hz, 1H), 3.76-3.70 (m, 2H), 3.71 (s, 3H), 2.72 (t, J = 12 Hz, 2H), 1.64 (d, J = 12 Hz, 2H), 1.51-1.28 (m, 5H) and 1.02-0.94 (m, 2H).

[0407] Preparation of (2-(1-(8-ethoxyquinazolin-4-yl)piperidin-4-yl)ethyl)phosphonic acid 33 (also referred to as 226 in the tables herein)

[0408] [ka]

[0409] 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 (400 MHz, D2O) δ 8.17 (s, 1H), 7.21-7.09 (m, 2H), 4.13-3.98 (m, 4H), 2.97 (t, J = 12.4 Hz, 2H), 1.82 (d, J = 13.0 Hz, 2H), 1.56-1.35 (m, 8H), 1.26 (q, J = 11.4 Hz, 2H). Preparation of (2-(1-(8-isopropoxyquinazolin-4-yl)piperidin-4-yl)ethyl)phosphonic acid 34 (in Table 3a)

[0410] [ka]

[0411] 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 (400 MHz, D2O) δ 8.10 (s, 1H), 7.11 (d, J = 7.5 Hz, 2H), 7.03 (d, J = 7.1 Hz, 1H), 3.94 (d, J = 13.0 Hz, 2H), 2.86 (t, J = 12.6 Hz, 2H), 1.67 (d, J = 13.1 Hz, 2H), 1.40-1.07 (m, 13H). Preparation of (2-(1-(8-hydroxyquinazolin-4-yl)piperidin-4-yl)ethyl)phosphonic acid 36 (in Table 3a)

[0412] [ka] Prepared according to Method B to give 35 as an off-white solid.

[0413] LCMS: [M+H] + m / z 1 H NMR (400 MHz, DMSO-d6) δ 1 H NMR (400 MHz, DO) Preparation of (2-(1-(5,8-dimethoxyquinazolin-4-yl)piperidin-4-yl)ethyl)phosphonic acid 36

[0414] [ka]

[0415] Prepared according to Method B to give 36 as an off-white solid. LCMS: [M+H] + m / z 382.15. 1 H NMR (400 MHz, DMSO-d6) δ8.47 (s, 1H), 7.54 (d, J = 8.9 Hz, 1H), 7.15 (d, J = 8.8 Hz, 1H), 3.95 (d, J = 12.0 Hz, 8H), 1.82 (s, 2H), 1.67 (s, 1H), 1.59-1.30 (m, 6H), 1.21 (s, 2H).

[0416] Preparation of (2-(1-(6,8-dimethoxyquinazolin-4-yl)piperidin-4-yl)ethyl)phosphonic acid 37

[0417] [ka]

[0418] Prepared according to Method C to give 37 (15% yield) as an off-white solid. LCMS: [M+H] + m / z 382.15 1H NMR (400 MHz, DMSO-d6) δ8.54 (s, 1H), 7.11 (s, 1H), 6.86-6.82 (m, 1H), 4.50 (d, J = 12.4 Hz, 1H), 4.27 (m, 1 H), 3.85 (m, 6 H), 3.74 (m, 2 H), 3.27 (m, 2 H), 1.88-1.85 (m, 2 H), 1.66 (m, 1 H), 1.54-1.48 (m, 4 H) and 1.29 (m, 2 H).

[0419] Preparation of (2-(1-(7,8-dimethoxyquinazolin-4-yl)piperidin-4-yl)ethyl)phosphonic acid 38

[0420] [ka]

[0421] Prepared according to Method C to give 38 (16% yield) as an off-white solid. LCMS: [M+H] + m / z 382.15 1 H NMR (400 MHz, DMSO-d6) δ8.60 (s, 1H), 7.90 (d, J = 9.6 Hz, 1H), 7.49 (d, J = 9.2 Hz, 1H), 4.69 (m, 2H), 4.02 (s, 3H), 3.89 (s, 3H), 3.46 (m, 2H), 1.90 (d, J = 12.8 Hz, 2H), 1.75 (m, 1H), 1.53-1.49 (m, 4 H) and 1.31-1.28 (m, 2H). 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)

[0422] [ka]

[0423] Prepared according to Method C to give 39 (7% yield) as an off-white solid. LCMS: [M+H] + m / z 380.15. 1 H NMR (400 MHz, DMSO-d6) δ8.64 (s, 1H), 7.48 (s, 1H), 7.19 (s, 1H), 4.56 (d, J = 11.8 Hz, 2H), 4.45 (d, J = 3.0 Hz, 2H), 4.38 (d, J = 3.3 Hz, 2H), 3.39 (s, 1H), 3.33 (s, 1H), 1.87 (d, J = 12.2 Hz, 2H), 1.71 (s, 1H), 1.58-1.38 (m, 4H), 1.26 (d, J = 10.2 Hz, 2H). Preparation of (2-(1-(5-fluoro-8-methoxyquinazolin-4-yl)piperidin-4-yl)ethyl)phosphonic acid 40 (in Table 3a)

[0424] [ka]

[0425] Prepared according to Method C to give 40 (7% yield) as a light yellow solid. LCMS: [M+H] + m / z 370.10. 1 H NMR (400 MHz, DMSO-d6) δ8.55 (s, 1H), 7.44-7.38 (m, 2H), 4.28-4.23 (m, 2H), 3.94 (s, 3H), 3.28-3.18 (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).

[0426] Preparation of (2-(1-(6-fluoro-8-methoxyquinazolin-4-yl)piperidin-4-yl)ethyl)phosphonic acid 41 (in Table 3a)

[0427] [ka]

[0428] 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) δ8.02 (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, 2) H), 1.74(m, 1 H), 1.53-1.49(m, 5 H), and 1.29-1.27 (m, 3 H).

[0429] Preparation of (2-(1-(6-chloro-8-methoxyquinazolin-4-yl)piperidin-4-yl)ethyl)phosphonic acid 42 (in Table 3a)

[0430] [ka]

[0431] Prepared according to Method B to give 42 (42% yield) as a light yellow solid. LCMS: [M+H] + m / z 386.10. 1 H NMR (400 MHz, 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).

[0432] Preparation of (2-(1-(7-chloro-8-methoxyquinazolin-4-yl)piperidin-4-yl)ethyl)phosphonic acid 43 (in Table 3a)

[0433] [ka]

[0434] Prepared according to Method B to give 43 (50% yield) as a white solid. LCMS: [M+H] + m / z 386.05. 1 H NMR (400 MHz, 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).

[0435] Preparation of 2-[1-[6,7-dimethoxy-2-[(E)-2-(3-pyridyl)vinyl]quinazolin-4-yl]-4-piperidyl]ethyl-hydroxy-phosphinate 44

[0436] [ka]

[0437] Prepared according to Method B to give 44 (49% yield) as a light yellow solid. LCMS: [M+H] + m / z 485.25. 1 H NMR (400 MHz, D2O) δ8.09 (s, 1H), 7.94 (s, 1H), 7.36 (d, J = 8 Hz, 1H), 7.06 (s, 1H), 6.74 (d, J = 16.8 Hz 1H), 6.58 (d, J = 3.2 Hz, 1H), 6.40 (d, J = 3.2 Hz, 1H), 6.24 (d, J = 16.8 Hz, 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). Preparation of (E)-(2-(1-(8-methoxy-2-(2-(pyridin-3-yl)vinyl)quinazolin-4-yl)piperidin-4-yl)ethyl)phosphonic acid 45

[0438] [ka]

[0439] 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) δ9.07 (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).

[0440] Preparation of (2-(1-(6,7-dimethoxyquinolin-4-yl)piperidin-4-yl)ethyl)phosphonic acid 46

[0441] [ka]

[0442] Prepared according to Method C to give 46 (22% yield) as a white solid. LCMS: [M+H] + m / z 381.30. 1 H NMR (400 MHz, methanol-d4) δ8.35 (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).

[0443] 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)

[0444] [ka]

[0445] Prepared according to Method B to give 47 (47% yield) as an off-white solid. LCMS: [M+H] + m / z 406.20. 1 H NMR (400 MHz, 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 = 12.3 Hz, 2H), 2.94 (t, J = 12.2 Hz, 2H), 1.72 (d, J = 12.7 Hz, 2H), 1.43-1.30 (m, 6H), 1.17-1.04 (m, 2H).

[0446] 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)

[0447] [ka]

[0448] Prepared according to Method B to give 48 (16% yield) as an off-white solid. LCMS: [M+H] + m / z 376.20. 1H NMR (400 MHz, 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).

[0449] 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)

[0450] [ka]

[0451] Prepared according to Method B to give 49 (23% yield) as an off-white solid. LCMS: [M+H] + m / z 376.20. 1 H NMR (400 MHz, D2O) δ7.94 (s, 1H), 7.39 (d, J = 9.4 Hz, 1H), 6.84-6.64 (m, 2H), 3.90 (s, 3H), 3.59 (d, J = 12.4 Hz, 2H), 3.22 (t, J = 12 Hz, 2H), 1.89 (d, J = 12.8 Hz, 2H), 1.62-1.45 (m, 5H) and 1.33-1.25 (m, 2H).

[0452] 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)

[0453] [ka]

[0454] Prepared according to Method B to give 50 as an off-white solid. LCMS: [M+H] +m / z 376.20. 1 H NMR (400 MHz, 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 = 12.4 Hz, 2H), 3.06 (br t, J = 12 Hz, 2H), 1.80 (br d, J = 12.8 Hz, 2H), 1.50-1.47 (m, 5H) and 1.31-1.24 (m, 2H). Preparation of (2-(1-(6,7-dimethoxyisoquinolin-1-yl)piperidin-4-yl)ethyl)phosphonic acid 51

[0455] [ka]

[0456] Prepared according to Method B to give 51 (30% yield) as an off-white solid. LCMS: [M+H] + m / z 381.10. 1 H NMR (400 MHz, DO) δ 1 H NMR (400 MHz, DMSO-d): δ 7.79 (d, J = 6.4 Hz, 1H), 7.51-7.44 (m, 2H), 7.31 (s, 1H), 3.96 (d, J = 4.8 Hz, 6H), 3.23 (m, 4H), 1.88 (m, 2H), 1.55 (m, 2H), 1.48 (m, 1H) and 1.45 (m, 4H).

[0457] Preparation of (2-(1-(4-cyano-6,7-dimethoxyisoquinolin-1-yl)piperidin-4-yl)ethyl)phosphonic acid 52 (in Table 3a)

[0458] [ka]

[0459] Prepared according to Method B to give 52 (50% yield) as an off-white solid. LCMS: [M+H] + m / z 1 H NMR (400 MHz, DO) δ

[0460] Preparation of O-((1-(8-methoxyquinazolin-4-yl)piperidin-4-yl)methyl) O,O-dihydrogen phosphorothioate 53 (in Table 3a)

[0461] [ka]

[0462] To a solution of (1-(8-methoxyquinazolin-4-yl)piperidin-4-yl)methanol (prepared using the same method as for compound 80) (500 mg, 1.83 mmol) in pyridine (5 mL) was added dropwise phosphorothioyl trichloride (1.6 g, 9.45 mmol) at -15°C. The reaction mixture was stirred at 0°C for 1 h and then 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 h. It was then evaporated to dryness under reduced pressure. Purification (preparative HPLC) afforded 53 (83 mg, 12%) as a white solid. LCMS: [M+H] + m / z 354.10 1 H NMR (400 MHz, , DMSO-d6) δ8.59 (s, 1H), 7.59-7.50 (m, 2H), 7.45 (dd, J = 6.5, 2.4 Hz, 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 = 13.5 Hz, 2H), 1.41 (q, J = 11.8 Hz, 2H). Preparation of (((1-(8-methoxyquinazolin-4-yl)piperidin-4-yl)oxy)methyl)phosphonic acid 54 (in Table 3a)

[0463] [ka]

[0464] Prepared using the same method as compounds 10 and 11. Purification of this mixture (preparative HPLC, 0.1% TFA) gave 54 as an off-white solid.

[0465] LCMS: [M+H] + m / z 353.3. 1 H NMR (400 MHz, D2O) δ 8.40 (s, 1H), 7.54 (s, 2H), 7.40 (d, J = 7.0 Hz, 1H), 4.37 (s, 3H), 4.01-3.88 (m, 9H), 3.71 (d, J = 9.3 Hz, 4H), 3.63 (s, 1H), 2.11 (s, 2H), 1.81 (s, 2H).

[0466] Preparation of (4-(8-methoxyquinazolin-4-yl)phenethyl)phosphonic acid 55 (in Table 3a)

[0467] [ka]

[0468] Prepared using the same method as compound 20 as a white solid.

[0469] LCMS: [M+H] + m / z 345.10. 1 H NMR (400 MHz, DO) δ

[0470] Preparation of (4-(((8-methoxyquinazolin-4-yl)amino)methyl)phenyl)phosphonic acid 56

[0471] [ka]

[0472] Prepared according to the same method as compound 23. LCMS: [M+H] + m / z 346.10. 1 H NMR (400 MHz, D2O) δ8.20 (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). Preparation of (4-(((3-cyano-8-methoxyquinolin-4-yl)amino)methyl)phenyl)phosphonic acid 57 (also referred to as 52 in Table 1)

[0473] [ka]

[0474] Prepared following the same method as compound 23 to give 57 as an off-white solid. LCMS: [M+H] + m / z 370.10. 1 H NMR (400 MHz, D2O) δ8.02 (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).

[0475] Preparation of (4-(((3-cyano-8-methoxyquinolin-4-yl)amino)methyl)benzyl)phosphonic acid 58 (also referred to as 211 in Table 2)

[0476] [ka]

[0477] Prepared following the same method as compound 23 to give 58 as a white solid. LCMS: [M+H] + m / z 384.15. 1H NMR (400 MHz, methanol-d4) δ8.32 (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).

[0478] 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)

[0479] [ka]

[0480] Prepared following the same method as compound 14 to give 59 as a white solid. LCMS: [M+H] + m / z 390.20. 1 H NMR (400 MHz, 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 = 12 Hz, 2H), 1.92-1.88 (m, 2H), 1.70-1.45 (m, 3H) and 1.40-1.27 (m, 6H).

[0481] Preparation of (4-(((3-cyano-8-methoxyquinolin-4-yl)amino)methyl)phenyl)boronic acid 60 (also referred to as 214 in Table 2)

[0482] [ka]

[0483] To a solution of compound 101 (0.97 g, 5.0 mmol) in 2-methoxyethanol (10 mL) was added (4-bromophenyl)methanamine 90 (1.74 g, 10.0 mmol) and EtN (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% EtOH 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), and Pd(dppf)Cl (7.4 mg, 0.05 mmol). The reaction was degassed by purging with nitrogen and then heated at 80° C. for 48 h. The mixture was cooled to room temperature, diluted with ethyl acetate, and then filtered through a Celite® pad. The filtrate was evaporated to dryness under reduced pressure. The residue was dissolved in EtOAc (10 mL), and a solution of HCl (4 M, 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)] afforded 60 (40.5 mg, 65% overall over 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).

[0484] 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)

[0485] [ka]

[0486] Prepared according to the same procedure as compound 7. Compound 61 was isolated as a yellow solid.

[0487] LCMS: [M+H] + m / z 340.20. 1 H NMR (400 MHz, methanol-d4) δ8.81 (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).

[0488] Preparation of 4-(((3-cyano-8-methoxyquinolin-4-yl)amino)methyl)-N-hydroxybenzamide 62 (also referred to as 220 in Table 2)

[0489] [ka]

[0490] 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, then triturated with EtOAc, filtered, and dried to give crude compound 104 (1.7 g) as a light yellow solid.

[0491] 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 to 45° C. overnight. The cooled solution was concentrated under reduced pressure, and the residue was treated with aqueous HCl (2 N) until a pH of 5.5 was achieved. 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), 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) was added. 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) followed by preparative HPLC (H + , 0.1% TFA) gave 62 (34 mg, 10%) as an off-white solid. LCMS: [M+H] + 1 H NMR (400 MHz, DMSO-d6) δ 11.19 (s, 1H), 9.06 (s, 1H), 8.55 (s, 1H), 8.45 (d, J = 8.7 Hz, 1H), 7.72 (d, J = 7.4 Hz, 2H), 7.39 (dd, J = 4.6, 2.9 Hz, 2H), 7.29 (dd, J = 12.3, 5.0 Hz, 2H), 5.11 (s, 2H), 3.93 (s, 3H).

[0492] Example 2 Evaluation of compound activity

[0493] Select compounds and other derivatives listed in Tables 1-3 are prepared and evaluated in an ENPP1 activity assay using thymidine monophosphate paranitrophenol (TMP-pNP) as the substrate. Enzyme reactions are set up at room temperature in 100 mM Tris, 150 mM NaCl, 2 mM CaCl2, 200 μM ZnCl2 (pH 7.5) with TMP-pNP (2 μM), 5-fold dilutions of ENPP1 inhibitors, and purified recombinant mouse ENPP1 (0.5 nM). Reaction progress is monitored by measuring the absorbance of the resulting paranitrophenolate at 400 nm for 20 minutes. The slopes of product formation are extracted, plotted, and fitted using Graphpad Prism 7.03 to determine IC values. 50 value can be obtained.

[0494] Compounds are also evaluated by ENPP1 enzyme activity assay using cGAMP as substrate.Methods that can be used to evaluate target compounds include those described by Li et al. in PCT application number PCT / US2018 / 050018, filed September 7, 2018.Exemplary methods are described below.

[0495] material:

[0496] Mouse ENPP1: Expressed and purified 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 (GeneBank: 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 overnight at 16°C and induced with 0.75 mM IPTG to an OD600 of 1. Cells were resuspended in a buffer containing 50 mM Tris (pH 7.5), 400 mM NaCl, 10 mM imidazole, 2 mM DTT, and protease inhibitors (Roche) and lysed by two freeze-thaw cycles and sonication. All subsequent steps were performed at 4°C. The lysate was clarified 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 of 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, and 600 mM imidazole. Anion exchange chromatography (HiTrap Q HP) was performed. Myokinase (MilliporeSigma) and CellTiterGlo (Promega) were used.

[0497] Exemplary Procedure for ENPP1 Enzyme Activity Assay:

[0498] 3 nM mouse ENPP1 was incubated with 5 μM cGAMP and 5-fold serial dilutions of compounds in a buffer containing 50 mM Tris (pH 7.6), 250 nM NaCl, 500 μM CaCl2, and 1 μM ZnCl2 (total reaction volume = 10 μL) at room temperature for 3 hours, after which the reaction was heat-inactivated at 95°C for 10 minutes. AMP degradation products were converted to ATP, which was detected using luciferase. To achieve this, an enzyme mixture consisting of polyphosphate:AMP phosphotransferase (PAP) and myokinase was prepared according to EP 2771480 by Goueli et al. Briefly, PAP was diluted to 2 mg / mL in a buffer containing 50 mM Tris (pH 7.5), 0.1% NP-40. Myokinase was diluted to 2 KU / mL in a buffer containing 3.2 mM ammonium sulfate (pH 6.0), 1 mM EDTA, and 4 mM polyphosphate. Heat-inactivated ENPP1 reactions were incubated with PAP (0.01 μg / μL) and myokinase (0.0075 U / μL) for 3 hours (total reaction volume = 20 μL) in a buffer containing 40 mM Tris (pH 7.5), 0.05 mg / mL Prionex, 5 mM MgCl2, 20 μM polyphosphate, and 0.15 g / L phenol red (to facilitate pipetting). CellTiterGlo (20 μL) was added to the reactions 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) and then fitted with the function 100 / (1 + ([compound] / IC50)).

[0499] The IC50 values ​​fall within a range designated by the letters A to C, 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.

[0500] [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4] [Table 4-5] [Table 4-6] [Table 4-7] [Table 4-8] [Table 4-9] [Table 4-10] [Table 4-11]

[0501] Example 3 Demonstration of extracellular ENPP1 and inhibition of extracellular ENPP1

[0502] Referring to Figures 18A-18C, it was observed that ENPP1 controls the extracellular levels of cGAMP, and that the cGAMP levels could be restored by treating cells with an ENPP1 inhibitor (e.g., Compound 1).

[0503] 293T cGAS ENPP1 - / - Cells were transfected with a human ENPP1 expression plasmid, and cGAMP hydrolase activity was confirmed in whole cell lysates (Figure 18A). 293T cells were purchased from ATCC and transfected with a virus to stably express mouse cGAS. 293T mcGAS ENPP1 - / -was generated by viral transfection of CRISPR sgRNA targeting human ENPP1 (5'CACCGCTGGTTCTATGCACGTCTCC-3') (SEQ ID NO: 1). 293T mcGAS ENPP1 was grown 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). - / - Cells were plated. 12–24 h after plating, cells were transfected with Fugene6 (Promega) by adding the indicated concentrations of pcDNA3 plasmid DNA (empty or containing human ENPP1) according to the manufacturer's instructions. 24 h after transfection, cells were lysed for analysis of ENPP1 expression by Western blotting using rabbit anti-ENPP1 (L520, 1:1000) and mouse anti-tubulin (DM1A, 1:2,000) antibodies from Cell Signaling Technologies. Whole cell lysates were collected at 1 × 10 ng / ml in 10 mM Tris, 150 mM NaCl, 1.5 mM MgCl2, 1% NP-40 (pH 9.0). 6 The cells were lysed. 32 P-cGAMP (5 μM) was incubated with whole cell lysates and degradation was monitored as described in Example 2 above (FIG. 18A).

[0504] In intact cells, ENPP1 expression depletes extracellular cGAMP but does not affect intracellular cGAMP concentrations (Figure 18B). - / -Twenty-four hours after transfection with pcDNA3 (empty or containing human ENPP1), the medium was removed and replaced with serum-free DMEM supplemented with 1% insulin-transferrin-selenium-sodium pyruvate (ThermoFisher) and 100 U / mL penicillin-streptomycin. 12–24 hours after medium change, the medium was removed and the cells were washed off the plate with cold PBS. Both the medium and cells were centrifuged at 1000 rcf for 10 minutes at 4°C, and the cGAMP concentration was measured by liquid chromatography-tandem mass spectrometry (LC-MS / MS). 500 nM cyclic GMP-1 was used as an internal standard. 13 C 10 , 15 Cells were lysed in 30-100 μL of 50:50 acetonitrile:water supplemented with N5-AMP and centrifuged at 15,000 rcf for 20 minutes at 4°C to remove the insoluble fraction. The medium was removed and 500 nM cyclic GMP-1 was added as an internal standard. 13 C 10 , 15The samples were supplemented with N5-AMP and 20% formic acid. The cGAMP, ATP, and GTP contents of the samples were analyzed using a Shimadzu HPLC (San Francisco, CA) equipped with an autosampler set at 4°C and connected to an AB Sciex 4000 QTRAP (Foster City, CA). A 10 μL aliquot was injected onto a Biobasic AX LC column, 5 μm, 50 × 3 mm (Thermo Scientific). The mobile phase consisted of 100 mM ammonium carbonate (A) and 0.1% formic acid (B) in acetonitrile. The initial condition was 90% B, held for 0.5 min. The mobile phase was increased to 30% A from 0.5 to 2.0 min, maintained at 30% A from 2.0 to 3.5 min, increased to 90% B from 3.5 to 3.6 min, and maintained at 90% B from 3.6 to 5 min. The flow rate was set at 0.6 mL / min. The mass spectrometer was operated in electrospray positive ion mode with the source temperature set at 500 °C. Declustering and collision-induced dissociation were performed using nitrogen gas. 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), and the internal standard cyclic GMP- 13 C 10 , 15 N5-AMP (690>146, 111, 101; 690>152, 111, 45; 690>327, 111, 47), extracted standard cyclic 13 C 10 , 15 N5-GMP- 13 C 10 , 15 N5-AMP (705>156, 66, 93; 705>162, 66, 73).

[0505] Inhibition of ENPP1 blocks the degradation of extracellular cGAMP (Figure 18C). The same experiment was performed as above, but this time the medium change also included 50 μM of an ENPP1 inhibitor (Compound 1). With the inhibitor, the extracellular cGAMP concentration in the medium returned to previous levels.

[0506] FIG. 18A shows 293T cGAS ENPP1 transfected with an empty vector and a vector containing human ENPP1. - / - 293T cGAS ENPP1 cells analyzed after 24 hours for ENPP1 protein expression using Western blot - / - Cells (top), ENPP1 using thin layer chromatography (TLC) 32 Figure 18B shows the intracellular and extracellular cGAMP concentrations using LC-MS / MS. BQL = below the limit of quantification. Mean ± standard error (n = 2). ** P=0.005 (Student's t-test). Figure 18C shows the effect of 293T cGAS ENPP1 transfected with empty vector or vector containing human ENPP1 in the presence or absence of 50 μM Compound 1. - / - Intracellular and extracellular cGAMP concentrations for cells are shown. BQL = below limit of quantification. Mean ± standard error (n = 2). ** P = 0.0013 (Student's t test).

[0507] Example 4 Inhibition of ENPP1 enhances cGAMP activation of primary CD14+ monocytes

[0508] Using an ENPP1 inhibitor (compound 1), we investigated the ENPP1 expression in 293T cGAS. low cGAMP secreted by the cell line interacts with human CD14 + We tested whether 293T cGAS ENPP1 could be detected by antigen-presenting cells (APCs) such as monocytes (Figure 19A). lowCells were transfected with pcDNA (empty or containing human ENPP1). Primary human peripheral blood mononuclear cells (PBMCs) were isolated by applying a Percoll density gradient to concentrated buffy coats from whole blood. CD14 + Monocytes are CD14 + CD14 cells were isolated using microbeads (Miltenyi) in RMPI supplemented with 2% human serum and 100 U / mL penicillin-streptomycin. + Mononuclear cells were cultured. 293T cGAS ENPP1 low Eight hours after transfection of the cells, the medium was changed to RMPI supplemented with 2% human serum and 100 U / mL penicillin-streptomycin, with or without the exemplary ENPP1 inhibitor Compound 1. Twenty-four hours after the medium change, 293T cGAS ENPP1 low The supernatant from the cells was analyzed by CD14 + The supernatant was transferred to monocytes (Figure 19A). 24–26 h after transfer, total RNA was extracted using Trizol (Thermo Fisher Scientific) and reverse transcribed using Maxima H Minus 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 were normalized to CD14 expression levels for each sample. Fold induction was calculated using ΔΔCt. Primers for human IFNB1: forward (5'-AAACTCATGAGCAGTCTGCA-3') (SEQ ID NO: 2), reverse (5'-AGGAGATCTTCAGTTTCGGAGG-3') (SEQ ID NO: 3); human CD14: forward (5'-GCCTTCCGTGTCCCCACTGC-3') (SEQ ID NO: 4), reverse (5'-TGAGGGGGCCCTCGACG-3') (SEQ ID NO: 5).

[0509] 293T cGAS-ENPP1 expressing cGAS lowSupernatants derived from CD14+ cells induced IFNB1 expression, whereas supernatants derived from cGAS-null 293T cells did not. This suggests that extracellular cGAMP secreted by cancer cells acts as a signaling factor for CD14. + This suggests that 293T cGAS ENPP1 can be detected by the cells (Figure 19B). low Transient overexpression of cell surface ENPP1 inhibits the degradation of extracellular cGAMP and CD14 + Although the expression of IFNB1 was decreased, the addition of compound 1 rescued the extracellular cGAMP level and increased the CD14 + IFNB1 expression was induced (Fig. 19B).

[0510] Referring to Figure 19A, a schematic of the supernatant transfer experiment is shown. Figure 19B shows the supernatant transfer experiment in cGAS-null 293T cells or 293T cGAS ENPP1 cells transfected with DNA and incubated in the presence or absence of Compound 1. low The supernatant from these cells was cultured as primary CD14 + IFNB1 mRNA levels were normalized to CD14 and fold induction was compared with untreated CD14. + Calculated relative to cells. Mean ± standard error (n=2). * P<0.05, *** P<0.001 (one-way ANOVA).

[0511] Example 5 ENPP1 inhibition synergizes with ionizing radiation (IR) treatment to expand tumor-associated dendritic cells.

[0512] We tested whether cancer cell lines excrete cGAMP and whether ionizing radiation (IR) affects the levels of extracellular cGAMP produced. Ionizing radiation (IR) has been shown to increase cytosolic DNA 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). After 24 hours of plating, 4T1 cells were treated with 20 Gy IR using a cesium source and then supplemented with 50 μM of the ENPP1 inhibitor (compound 1) to inhibit ENPP1 present in the cell culture. The medium was collected at the indicated times, centrifuged at 1000 × g to remove residual cells, and acidified with 0.5% acetic acid to extract cyclic α-GAMP as a standard. 13 C 10 , 15 5-GMP- 13 C 10 , 15 The medium was supplemented with N5-AMP (an appropriate amount to achieve a final concentration of 2 μM in 100 μL). cGAMP was enriched by applying the medium to a HyperSep aminopropyl SPE column (ThermoFisher Scientific) 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 medium was subjected to mass spectrometry quantification of cGAMP.

[0513] Continuous cGAMP excretion was detected in 4T1 cells over 48 hours, at which time cells treated with IR had significantly higher extracellular cGAMP levels than untreated cells.

[0514] Next, we examined the effect of IR in combination with the exemplary ENPP1 inhibitor Compound 1 on tumor-associated dendritic cell numbers in a murine 4T1 tumor model (Figure 20B). 1 x 10 dendritic cells suspended in 50 μL of PBS were injected into the mammary fat pads of 7-9 week-old female Balb / c mice (Jackson Laboratories). 6 Mice were inoculated with 4T1-luciferase tumor cells. Two days after injection, tumors were irradiated with 20 Gy using a 225 kVp cabinet X-ray irradiator (IC250, 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 gap where the tumor was located. Mice were injected intratumorally with 100 μL of 1 mM Compound 1 in PBS or PBS alone. The following day, tumors were extracted and incubated for 30 minutes at 37°C in RPMI + 10% FBS containing 20 μg / mL DNase I type IV (Sigma-Aldrich) and 1 mg / mL collagenase from Clostridium histolyticum (Sigma-Aldrich). Tumors were passed through a 100 μm cell strainer (Sigma-Aldrich), and red blood cells were lysed using red blood cell lysis buffer (155 mM NH4Cl, 12 mM NaHCO3, 0.1 mM EDTA) for 5 minutes at room temperature. Cells were stained using the Live / Dead Fixed Near-Infrared Dead Cell Staining Kit (Thermo Fisher Scientific), Fc-blocked using TruStain fcX for 10 minutes, and then antibody stained for CD11c, CD45, and IA / IE (all Biolegend). Cells were analyzed using an SH800S cell sorter (Sony) or an LSR II (BD Biosciences). For statistical analysis, data were analyzed using FlowJo V10 software (Treestar) and Prism 7.04 software (Graphpad), and statistical significance was assessed using an unpaired t-test with Welch's correction.

[0515] Compared with the PBS control, intratumoral injection of compound 1 did not alter the composition of tumor-associated leukocytes (Figure 20B), suggesting that ENPP1 does not play a major role in clearing basal levels of extracellular cGAMP in this tumor model. However, when tumors were pretreated with IR, compound 1 significantly downregulated tumor-associated CD11c. + An increase in population was observed (Figure 20B).

[0516] These results are illustrated in Figures 20A and 20B. Figure 20A shows extracellular cGAMP produced by 4T1 cells over a 48-hour period. At time 0, cells were left untreated or treated with 20 Gy IR and refreshed with medium supplemented with 50 μM Compound 1. Mean ± standard error (n=2). ** P = 0.004 (Student's t-test). Figure 20B shows the effect of 4T1 cells (1 x 10) orthotopically injected into BALB / cJ mice on day 0. 6 On day 2, 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 3, tumors were harvested and analyzed by FACS. * P = 0.047 (Welch's t test).

[0517] Example 6 ENPP1 inhibition synergizes with IR treatment and anti-CTLA-4 to exert antitumor effects

[0518] We investigated whether tumor immune detection and clearance could be enhanced by further increasing extracellular cGAMP in vivo using ionizing radiation (IR) and an exemplary ENPP1 inhibitor, such as Compound 1.

[0519] 5 × 10 cells suspended in 50 μL of PBS were injected into the mammary fat pads of 7- to 9-week-old female Balb / c mice (Jackson Laboratories). 4 4T1-luciferase cells were inoculated. The tumor volume (length2 × width / 2) is 80mm 3 ~120mm 3 Once the tumor reached IR, 20 Gy of radiation was administered to the tumor using a 225 kVp cabinet X-ray irradiator (IC250, Kimtron Inc., CT) with a 0.5 mm Cu filter. Anesthetized animals were shielded using a 3.2 mm lead shield with a 15 x 20 mm gap where the tumor was located. On days 2, 4, and 7 after IR, 100 μL of 100 μM Compound 1 and / or 10 μg of 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 were injected intratumorally, and 200 μg of anti-CTLA-4 antibody or Syrian hamster IgG antibody (both BioXCell) was injected intraperitoneally. Mice from different treatment groups were co-housed in individual cages to eliminate cage effects. Experimenters were blinded throughout the study. Tumor volumes were recorded every other day. To account for intra-mouse correlation, tumor volumes were analyzed using generalized estimating equations. Pairwise comparisons of treatment groups at each time point were performed using post-hoc tests with Tukey's adjustment for multiple comparisons. Animal mortality was plotted on Kaplan-Meier curves using Graphpad Prism 7.03, and statistical significance was assessed using the log-rank Mantel-Cox test. All animal procedures were approved by the Institutional Animal Care Committee.

[0520] Administration of compound 1 enhanced the tumor shrinkage effect of IR treatment, but not significantly (Figure 21A). Intratumoral injection of cGAMP was not as effective as IR treatment, but injection of compound 1 in addition to cGAMP synergistically reduced tumors, prolonged survival, and achieved a 10% cure rate (Figures 21A and 21B).

[0521] We also tested the synergistic effect of anti-CTLA-4, an adaptive immune checkpoint blocker. In the absence of IR, treatment with anti-CTLA-4 and Compound 1 was ineffective in extending survival (Figure 21C). However, combining IR pretreatment with Compound 1 and anti-CTLA-4 exerted significant synergistic effects, achieving a 10% cure rate. Together, these results demonstrate that increasing extracellular cGAMP by combining IR treatment with ENPP1 inhibition enhances tumor immunogenicity and exerts anti-tumor effects.

[0522] These results are illustrated in Figure 21A, which shows the tumor-reducing effect of Compound 1 in combination with IR. Established tumors (100±20 mm 3 ) were treated once with 20 Gy IR, followed by three intratumoral injections or treatments with PBS on days 2, 4, and 7 after IR (n = 9 per treatment group). Mice from different treatment groups were co-housed, and the experimenters were blinded. To account for intra-mouse correlation, tumor volumes were analyzed using generalized estimating equations. Pairwise comparisons of treatment groups at each time point were performed using post-hoc tests with Tukey's adjustment for multiple comparisons. Figure 21B shows the Kaplan-Meier curves for Figure 21A, and P values ​​were determined by the log-rank Mantel-Cox test. Figure 21C shows the same procedure as in Figure 21B, with the addition of anti-CTLA4 or IgG isotype control antibodies injected intraperitoneally on days 2, 5, and 7 after IR (n = 8 for the IR(0) + Compound 1 + CTLA-4 treatment group; n = 17–19 for all other treatment groups). Statistical analysis was performed as in Figure 21B.

[0523] In summary, these results demonstrate that cGAMP exists extracellularly and that targeted ENPP1 inhibitors can act extracellularly, thus demonstrating that extracellular inhibition of ENPP1 is sufficient for therapeutic efficacy. ENPP1 is a suitable innate immune checkpoint. These experiments demonstrate that extracellular inhibition of ENPP1 enhances anti-cancer immunity and can be synergistically combined with already available immune checkpoint blockers as a therapeutic approach (Figure 22). Example 7 2'3'-cGAMP is an immune mediator produced by cancer cells and regulated by ENPP1 Introduction

[0524] 2'3'-cyclic GMP-AMP (cGAMP) is synthesized in response to cytosolic dsDNA and is characterized as an intracellular second messenger that activates the innate immune STING pathway. Its extracellular hydrolase, ENPP1, implies the presence of extracellular cGAMP. Using mass spectrometry, we detected that cGAMP is continuously excreted as a soluble factor by engineered cell lines, but is then efficiently cleared by ENPP1. By developing a potent, specific, and cell-impermeable ENPP1 inhibitor, cGAMP excretion was detected in cancer cell lines commonly used in mouse tumor models. In tumors, depletion of extracellular cGAMP using a neutralizing protein reduced tumor-associated dendritic cells. Increasing extracellular cGAMP by genetic knockout and pharmacological inhibition of ENPP1 increased tumor-associated dendritic cells, leading to tumor regression and synergizing with ionizing radiation and anti-CTLA-4 to cure tumors. In conclusion, cGAMP is an anti-cancer immune mediator released by tumors and detected by the host's innate immunity.

[0525] The second messenger 2'3'-cyclic GMP-AMP (cGAMP) plays a pivotal role in antiviral and anticancer innate immunity. cGAMP is synthesized by the enzyme cyclic-GMP-AMP synthase (cGAS) in response to double-stranded DNA (dsDNA) in the cytosol and is a danger signal for intracellular pathogens and damaged or cancerous cells. cGAMP binds to and activates its endoplasmic reticulum (ER) surface receptor, stimulator of interferon genes (STING), which activates the production of type 1 interferons (IFNs). These potent cytokines trigger downstream innate and adaptive immune responses to eliminate the threat.

[0526] In addition to activating STING in its cell of origin, cGAMP can diffuse to bystander cells through gap junctions in epithelial cells. This intercellular communication mechanism alerts neighboring cells of damaged cells and, unfortunately, may also be a major cause of the spread of drug-induced liver toxicity and brain metastasis. Furthermore, cytosolic cGAMP can be packaged and delivered by budding virus particles during the next stage of infection. In both modes of delivery, cGAMP is never exposed to the extracellular space.

[0527] The only enzyme with 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 transmembrane domain and as a cleaved, soluble protein in serum. cGAMP, which has two negative charges and presumably cannot passively pass through the cell membrane, can enter cells and 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 Tumor Microenvironment Leads to Potent and Systemic Tumor Regression and Immunity. Cell Rep. 11, 1018-1030 (2015)), suggesting the existence of a transport channel for cGAMP. Because cGAMP can enter cells, cGAMP analogs are currently being tested in clinical trials for the treatment of metastatic solid tumors via intratumoral injection. Recognizing that extracellular cGAMP is influxed and has anticancer effects, and that the major cGAMP hydrolase is extracellular, we hypothesized that cGAMP is exported into the extracellular space to signal other cells and be regulated by extracellular degradation.

[0528] The role of extracellular cGAMP in cancer-induced cGAMP excretion and anti-cancer immune detection is demonstrated herein. Using genetic knockout and pharmacological inhibition, we also investigated the role of ENPP1 in controlling extracellular cGAMP levels, immune infiltration, and tumor progression. Collectively, we characterized cGAMP as an immune mediator regulated by ENPP1. Materials and Methods

[0529] Reagents, antibodies and cell lines

[0530] [α- 32 P]ATP (800 Ci / mmol, 10 mCi / mL, 250 μCi) and [ 35 [S]ATPαS (1250 Ci / mmol, 12.5 mCi / mL, 250 μCi) was purchased from Perkin Elmer. 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% atomically pure. 2'3'-cGAMP was purchased from Invivogen. Caco-2 assays were 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 series of 4ON-TARGETplus ENPP1 siRNAs (LQ-003809-00-0002) was purchased from Dharmacon. QS1 was synthesized as previously described. 25The following monoclonal antibodies were used for Western blotting: rabbit anti-cGAS (D1D3G Cell Signaling, 1:1,000), rabbit anti-mouse cGAS (D2O8O Cell 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).

[0531] 293T cells were purchased from ATCC and transfected with a virus to stably express mouse cGAS. 293T cGAS ENPP1 low Cells were generated by viral transfection of CRISPR sgRNA targeting human ENPP1 (5'-CACCGCTGGTTCTATGCACGTCTCC-3') and transfected with 293T mcGAS ENPP1. - / - Cells were sorted after single cell cloning from this pool. 4T1 and E0771 cGAS - / - Cells were generated by viral transfection of mouse Mb21d1 (5'-CACCGGAAGGGGCGCGCGCTCCACC-3') targeting CRISPR sgRNA (using lentiCRISPRv2-blast, Addgene plasmid, #83480). Cells were sorted after single-cell cloning. 4T1-Luc ENPP1 - / -Cells were generated by viral transfection of CRISPR sgRNA (using lentiCRISPRv2-blast) (Sanjana, N.E., Shalem, O. & Zhang, F. Improved vectors and genome-wide libraries for CRISPR screening. Nat. Methods 11, 783-784 (2014)) targeting mouse ENPP1 (5'-GCTCGCGCCCATGGACCT-3' and 5'-ATATGACTGTACCCTACGGG-3') or a scrambled sequence. 4T1-Luc shcGAS cells were generated by viral transfection of shRNA (5'-CAGGATTGAGCTACAAGAATAT-3') using the plasmid pGH188. Cells harboring shRNA were selected using blasticidin, sorted for GFP expression, and pooled for experiments. MDA-MB-231 was purchased from ATCC, E0771 was purchased from CH3 BioSystems, and HEK293S GnT1 expressing 4T1-luciferase and secreted mENPP1 was used. - cells were obtained.

[0532] cell culture

[0533] 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 applying a Percoll density gradient to enriched buffy coats from whole blood. CD14 + PBMCs are CD14 + CD14 cells were isolated using microbeads (Miltenyi) in RMPI supplemented with 2% human serum and 100 U / mL penicillin-streptomycin. + PBMCs were cultured.

[0534] Recombinant protein expression and purification

[0535] sscGAS: The primer pair forward: (5'-CTGGAAGTTCTGTTCCAGGGGCCCCATATGGGCGCCTGGAAGCTCCAGAC-3') and reverse: (5'-GATCTCAGTGGTGGTGGTGGTGGTGCTCGAGCCAAAAAACTGGAAATCCATTGT-3') was used to amplify the DNA sequence encoding porcine cGAS (residues 135-497) from a porcine cDNA library. The PCR product was inserted into pDB-His-MBP by Gibson assembly and expressed in Rosetta cells. Cells were grown in 2xYT medium containing kanamycin (100 μg / ml) to reach an OD 600 Once the RI reached 1, the cells were induced with 0.5 mM IPTG and grown overnight at 16°C. All of the following procedures, including protein and cell lysates, were performed at 4°C. Cells were pelleted and lysed in 20 mM HEPES (pH 7.5), 400 mM NaCl, 10% glycerol, 10 mM imidazole, 1 mM DTT, and a protease inhibitor cocktail (cOmplete EDTA-free tablets, Roche). The cell extract was clarified by ultracentrifugation at 50,000 × g for 1 hour. The clear supernatant was incubated with HisPur cobalt resin (ThermoFisher Scientific; 1 mL of resin per liter of bacterial culture). The cobalt resin was washed with 20 mM HEPES (pH 7.5), 1 M NaCl, 10% glycerol, 10 mM imidazole, and 1 mM 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. Fractions containing His-MBP-sscGAS were pooled, concentrated, and dialyzed against 20 mM HEPES (pH 7.5), 400 mM NaCl, and 1 mM DTT. The protein was quickly frozen in aliquots for future use.

[0536] STING: Mouse STING (residues 139-378) was inserted into the pTB146 His-SUMO vector and expressed in Rosetta cells. Cells were grown in 2xYT medium containing 100 μg / mL ampicillin and incubated at OD 600Once the RI reached 1, the cells were induced with 0.75 mM IPTG overnight at 16°C. All of the following procedures using protein and cell lysates were performed at 4°C. Cells were pelleted and lysed in 50 mM Tris (pH 7.5), 400 mM NaCl, 10 mM imidazole, 2 mM DTT, and protease inhibitors (cOmplete, an EDTA-free protease inhibitor cocktail, Roche). Cells were lysed by sonication, and the lysate was clarified by ultracentrifugation at 50,000 rcf for 1 hour. The clear supernatant was incubated with HisPur cobalt resin (ThermoFisher Scientific; 1 mL of resin per 1 L of 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 (each wash took 2–3 h at a drip rate of 1 drop per 2–3 seconds), 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. Fractions containing His-SUMO-STING were pooled, concentrated, and dialyzed against 50 mM Tris (pH 7.5), 150 mM NaCl while incubating with the SUMOase enzyme His-ULP1 overnight to remove the His-SUMO tag. This solution was reincubated with HisPur cobalt resin to remove the His-SUMO tag, and STING was collected from the flow-through. The protein was dialyzed against 50 mM Tris (pH 7.5) and loaded onto a HitrapQ anion-exchange column (GE Healthcare) using an Aekta FPLC (GE Healthcare) and eluted with a NaCl gradient. STING-containing fractions were pooled, buffer-exchanged into PBS, and stored at 4°C until use.

[0537] ENPP1:mENPP1 was produced as described by Kato, K. et al. (Expression, purification, crystallization and preliminary X-ray crystallographic analysis of Enpp1. Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 68, 778-782 (2012); and Crystal structure of Enpp1, an extracellular glycoprotein involved in bone mineralization and insulin signaling. Proc. Natl. Acad. Sci. USA 109, 16876-81 (2012)).

[0538] Liquid chromatography-tandem mass spectrometry

[0539] 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 , 15N5-AMP was used as the extraction standard. Isotopically labeled cGAMP standards were synthesized by overnight incubation of 1 mM ATP (isotopically labeled), 1 mM GTP (isotopically labeled), 20 mM MgCl2, 0.1 mg / mL herring testis DNA (Sigma), and 2 μM sscGAS in 100 mM Tris (pH 7.5). The reaction was heated to 95°C and filtered through a 3 kDa centrifugal filter. Water was removed by rotary evaporation. cGAMP was purified from the crude reaction mixture using a PLRP-S polymer reversed-phase preparative column (100 Å, 8 μm, 300 × 25 mm; Agilent Technologies) on a preparative HPLC (1260 Infinity LC system; Agilent Technologies) connected to a UV-visible detector (ProStar; Agilent Technologies) and a fraction collector (440-LC; Agilent Technologies). The flow rate was set at 25 mL / min. The mobile phase consisted of 10 mM triethylammonium acetate and acetonitrile in water. The mobile phase started at 2% acetonitrile for the first 5 min. The acetonitrile was then increased to 30% from 5 to 20 min, increased to 90% from 20 to 22 min, maintained at 90% from 22 to 25 min, and then decreased to 2% from 25 to 28 min. The cGAMP-containing fractions were lyophilized and resuspended in water. Concentrations were determined by measuring absorbance at 280 nm. The cGAMP, ATP, and GTP contents of the samples were analyzed using a Shimadzu HPLC (San Francisco, CA) connected to an AB Sciex 4000 QTRAP (Foster City, CA) equipped with an autosampler set at 4 °C. 10 μL aliquots were injected onto a Biobasic AX LC column, 5 μm, 50 × 3 mm (Thermo Scientific). The mobile phase consisted of 100 mM ammonium carbonate (A) and 0.1% formic acid (B) in acetonitrile. The initial condition was 90% B and maintained for 0.5 min.The mobile phase was ramped to 30% A over 0.5 to 2.0 min, maintained at 30% A from 2.0 to 3.5 min, ramped to 90% B from 3.5 to 3.6 min, and maintained at 90% B from 3.6 to 5 min. The flow rate was set to 0.6 mL / min. The mass spectrometer was operated in electrospray positive ion mode with the source temperature set to 500 °C. Declustering and collision-induced dissociation were performed using nitrogen gas. 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) are 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), and the internal standard cyclic GMP-. 13 C 10 , 15 N5-AMP (690>146, 111, 101; 690>152, 111, 45; 690>327, 111, 47), extracted standard cyclic 13 C 10 , 15 N5-GMP- 13 C 10 , 15 N5-AMP (705>156, 66, 93; 705>162, 66, 73).

[0540] 293T cGAS ENPP1 - / - Efflux assay in cells

[0541] 293T cGAS ENPP1 - / -Cells were plated on PurCol-coated, tissue culture-treated plates (Advanced BioMatrix). After 24 hours, the medium was gently removed and replaced with serum-free DMEM supplemented with 1% insulin-transferrin-selenium-sodium pyruvate (ThermoFisher) and 100 U / mL penicillin-streptomycin. At the indicated times, the medium was removed and the cells were washed off the plate with cold PBS. Both the medium and cells were centrifuged at 1000 rcf for 10 minutes at 4°C. Cells were lysed in 30-100 μL of 50:50 acetonitrile:water supplemented with 500 nM internal standard and centrifuged at 15,000 rcf for 20 minutes at 4°C to remove the insoluble fraction. If no concentration was required, an aliquot of the medium was removed and supplemented with 500 nM internal standard and 20% formic acid. If enrichment was required, the medium was acidified with 0.5% acetic acid and supplemented with an extract standard (appropriate volume to achieve a final concentration of 2 μM in 100 μL). cGAMP was enriched by applying the medium to a HyperSep aminopropyl SPE column (ThermoFisher Scientific) as described by 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 eluate was evaporated to dryness and reconstituted in 50:50 acetonitrile:water supplemented with 500 nM internal standard. The medium and cell extracts were subjected to mass spectrometry quantification of cGAMP, ATP, and GTP.

[0542] 293T cGAS ENPP1 - / - Transfected stimulation of cells

[0543] Following the manufacturer's instructions, add the indicated concentrations of pcDNA3 plasmid DNA (empty or containing human ENPP1) to 293T cGAS ENPP1. - / -Cells were transfected with Fugene6 (Promega). 24 hours after transfection, efflux assays were performed as described above.

[0544] Transfer of conditioned medium

[0545] 293T cGAS ENPP1 low Cells were plated and transfected with plasmid DNA as described above. 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 μM Compound 1. 24 hours after the medium change, 293T cGAS ENPP1 low Remove the conditioned medium from the cells and resuspend in freshly isolated CD14 + Incubated with PBMCs. CD14 + Gene expression levels in PBMCs were analyzed 14-16 hours later.

[0546] RT-PCR analysis

[0547] Total RNA was extracted using Trizol (Thermo Fisher Scientific) and reverse transcribed using Maxima H Minus 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 were normalized to CD14, ACTB, or GAPDH expression levels for each sample. Fold induction was calculated using ΔΔCt. Primers for human IFNB1: forward (5'-AAACTCATGAGCAGTCTGCA-3'), reverse (5'-AGGAGATCTTCAGTTTCGGAGG-3'); human CD14: forward (5'-GCCTTCCGTGTCCCCACTGC-3'), reverse (5'-TGAGGGGGCCCTCGACG-3'); human ACTB: forward (5'-GGCATCCTCACCCTGAAGTA-3'), reverse (5'-AGAGGCGTACAGGGATAGCA-3'); human GAPDH: forward (5'-CCAAGGTCATCCATGACAAC-3'); reverse (5'-CAGTGAGCTTCCCGTTCAG-3').

[0548] 32 P-cGAMP degradation TLC assay

[0549] Radiolabel 32 P cGAMP contains unlabeled ATP (1 mM) and 2 μM purified recombinant porcine cGAS in 20 mM Tris (pH 7.5), 2 mM MgCl2, 100 μg / mL herring testis DNA. 32 P-ATP was synthesized by incubating 1 mM GTP doped with ATP overnight at room temperature, and the remaining nucleotide starting material was degraded with alkaline phosphatase for 4 hours at 37°C. Cell lysates were diluted to 1 × 10 in 100 μL of 10 mM Tris, 150 mM NaCl, 1.5 mM MgCl, 1% NP-40 (pH 9.0). 6 cells (293T) or 10 × 10 6Cells (4T1-Luc, E0771, and MDA-MB-231) were disrupted and lysed to generate lysates. For 4T1-Luc, E0771, and MDA-MB-231, the total protein concentration of the lysates was measured using a BCA assay (Pierce, Thermo Fisher Scientific). Samples were normalized so that the same amount of protein was used for each lysate reaction. The probe was incubated in 100 mM Tris, 150 mM NaCl, 2 mM CaCl, 200 μM ZnCl (pH 7.5 or 9.0). 32 P-cGAMP (5 μM) was incubated with mENPP1 (20 nM) or whole cell lysate for the indicated amount of time. Five-fold dilutions of ENPP1 inhibitors were included in the reaction to generate inhibition curves. Degradation was assessed by TLC (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)). Plates were exposed to a fluorescent screen (Molecular Dynamics) and imaged on a Typhoon 9400. 32 P signals were quantified using ImageJ. Inhibition curves were fitted and IC values ​​were calculated using Graphpad Prism 7.03. 50 The value obtained was IC 50 The value is calculated using the Cheng-Prusoff formula K i,app =IC 50 / (1+[S] / K m ) to find K i,app converted to a value.

[0550] ALPL and ENPP2 inhibition assays

[0551] Inhibition assays for other ectonucleotidases were performed by incubating reaction components at room temperature in a 96-well plate format and monitoring the production of 4-nitrophenolate by measuring absorbance at 400 nM in a plate reader (Tecan). ALPL: 0.1 nM ALPL, 2 μM 4-nitrophenyl phosphate, and various concentrations of inhibitor in a buffer containing 50 mM Tris, 20 μM ZnCl, and 1 mM MgCl (pH 9.0) at room temperature. ENPP2: 2 nM ENPP2, 500 μM bis(4-nitrophenyl)phosphate, and various concentrations of inhibitor in a buffer containing 100 mM Tris, 150 mM NaCl, 200 μM ZnCl, and 2 mM CaCl (pH 9.0).

[0552] Efflux assay in cancer cell lines

[0553] 4T1-Luc, E0771, and MC38 cells were changed to fresh medium supplemented with 50 μM Compound 1. At the indicated times, the medium was collected. Cells were detached from the plate using PBS, pelleted at 1000 rcf, dissolved in 4 mL of 50:50 acetonitrile:water, and centrifuged at 15,000 rcf. cGAMP was enriched from the medium and cell supernatants using a HyperSep aminopropyl SPE column as described above and subjected to mass spectrometry quantification.

[0554] 4T1-Luc tumor mouse model

[0555] 5 × 10 cells suspended in 50 μL of PBS were added to the mammary fat pads of 7- to 9-week-old female BALB / c mice (Jackson Laboratories). 4 pcs or 5 x 10 5 4T1-Luc-luciferase cells were inoculated. The tumor volume (length 2 × width / 2) is 80mm 3 ~120mm 3Once the tumor reached IR, 20 Gy of radiation was administered to the tumor using a 225 kVp cabinet X-ray irradiator (IC250, Kimtron Inc., CT) with a 0.5 mm Cu filter. Anesthetized animals were shielded using a 3.2 mm lead shield with a 15 x 20 mm gap where the tumor was located. On days 2, 4, and 7 after IR, 100 μL of 100 μM Compound 1 and / or 10 μg of 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 were injected intratumorally, and 200 μg of anti-CTLA-4 antibody or Syrian hamster IgG antibody (both BioXCell) was injected intraperitoneally. Mice from different treatment groups were co-housed in individual cages to eliminate cage effects. Experimenters were blinded throughout the study. Tumor volumes were recorded every other day. To account for intra-mouse correlation, tumor volumes were analyzed using generalized estimating equations. Pairwise comparisons of treatment groups at each time point were performed using post-hoc tests with Tukey's adjustment for multiple comparisons. Animal deaths were plotted on Kaplan-Meier curves using Graphpad Prism 7.03, and statistical significance was assessed using the log-rank Mantel-Cox test. All mice were maintained at Stanford University in compliance with the regulations of the Stanford University Institutional Animal Care Committee, and procedures were approved by the Stanford University Committee on Laboratory Animal Care.

[0556] FACS analysis of tumors

[0557] 7-9 week old female BALB / c WT (4T1-Luc tumor) or C57BL / 6 WT (E0771 tumor) cGAS - / - or STING gt / gt (STING - / - ) mice (Jackson Laboratories) were injected with 1 × 10 cells suspended in 50 μL of PBS into the mammary fat pads of the mice. 6Tumor cells were inoculated into tumors. Two days after injection, tumors were irradiated as described and injected with 100 μL of 1 mM Compound 1 in PBS or PBS alone. For experiments using STING and mENPP1, 100 μL of 100 μM neutralizing STING or non-binding STING (R237A), or 700 nM mENPP1 or PBS was injected intratumorally. The following day, tumors were extracted and incubated for 30 minutes at 37°C in RPMI + 10% FBS containing 20 μg / mL DNase I type IV (Sigma-Aldrich) and 1 mg / mL collagenase from Clostridium histolyticum (Sigma-Aldrich). Tumors were passed through a 100 μm cell strainer (Sigma-Aldrich), and red blood cells were lysed using red blood cell lysis buffer (155 mM NH4Cl, 12 mM NaHCO3, 0.1 mM EDTA) for 5 minutes at room temperature. Cells were stained using the Live / Dead Fixed Near-Infrared Dead Cell Staining Kit (Thermo Fisher Scientific), Fc-blocked using TruStain fcX for 10 minutes, and then antibody stained for CD11c, CD45, and IA / IE (all Biolegend). Cells were analyzed using an SH800S cell sorter (Sony) or an LSR II (BD Biosciences). For statistical analysis, data were analyzed using FlowJo V10 software (Treestar) and Prism 7.04 software (Graphpad), and statistical significance was assessed using an unpaired t-test with Welch's correction.

[0558] In vivo imaging

[0559] Mice were injected i.p. with 3 mg of XenoLight D-luciferin (Perkin-Elmer) in 200 μl of water and imaged using a Lago X in vivo imaging system (Spectral Instruments Imaging). The objective height was set to 1.5 cm, binning to 4, f-stop to 1.2, and exposure time was 120 seconds. Images were analyzed using aura 2.0.1 software (Spectral Instruments Imaging). result

[0560] cGAMP, 293T cGAS ENPP1 - / - Excreted from cells as soluble factors To test the hypothesis that cGAMP exists extracellularly, we first developed a liquid chromatography-tandem mass spectrometry (LC-MS / MS) method to detect cGAMP from complex mixtures. Using two isotopically labeled cGAMP standards (Figure 1, panel A), we were able to quantify cGAMP concentrations down to 0.5 nM in both basal cell culture medium and serum-containing medium. In the same experiment, we were able to quantify intracellular cGAMP concentrations from cell extracts (Figure 1, panel B, and Figure 8, panels A and B). We chose to use 293T cells, which do not express cGAS or STING. We stably expressed mouse cGAS and knocked out ENPP1 using CRISPR, thereby detecting cGAMP levels in 293T cells. low A cell line was generated (Figure 8, panel C). Next, we isolated a single clone and expressed the 293T cGAS ENPP1 - / - We generated a cell line (Figure 8, panel C). Because serum contains a proteolytically cleaved soluble form of ENPP1, we also used serum-free medium. We used this ENPP1-free cell culture system to express 293T cGAS ENPP1 without any stimulation. - / -We detected a consistent, low-micromolar basal intracellular cGAMP concentration in cancer cells (Figure 1, Panel C). This is not surprising, as cytosolic dsDNA is abundant in cancer cells as a result of mis-segregation of DNA (see, e.g., Mackenzie, KJ et al. cGAS surveillance of micronuclei links genome instability to innate immunity. Nature 548, 461-465 (2017); Harding, SM Mitotic progression following DNA damage enables pattern recognition within micronuclei. Nature 548, 466-470 (2017); and Bakhoum, SF et al. Chromosomal instability drives metastasis through a cytosolic DNA response. Nature 553, 467-472 (2018)). We measured a linear increase in extracellular cGAMP concentration to 100 nM 30 hours after replenishing the cells with fresh medium (Figure 1, Panel D). At 30 hours, the number of cGAMP molecules outside the cells was equal to the number inside (Figure 1, Panel E). We detected negligible amounts of cell death based on extracellular lactose dehydrogenate (LDH) activity, suggesting that cGAMP in the medium is excreted by viable cells (Figure 1, Panel E). We calculated the excretion rate (v export ) but 220 molecules cells -1 s -1 (Figure 1, Panel F). Finally, cGAMP in the medium was able to pass through a 10 kDa filter without any retention, which should retain extracellular vesicles and proteins, suggesting that cGAMP is excreted as a freely soluble molecule (Figure 1, Panel H).

[0561] To further confirm that the extracellular cGAMP secreted by 293T cells is mainly in a soluble form and not present in extracellular vesicles, we used CD14 as a reporter. + Human peripheral blood mononuclear cells (PBMCs) were used. These cells have previously been shown to take up soluble cGAMP, which leads to IFN-β production. 17 The present inventors have investigated the CD14 + We observed that PBMCs responded to submicromolar concentrations of soluble cGAMP by upregulating IFNB1 (Figure 9). 293T cells expressing cGAS DNA transfected with cGAS ENPP1 low Conditioned medium from cells was identified as CD14 + IFNB1 expression was induced in 293T cells, but not in conditioned medium from DNA-transfected cGAS-null 293T cells, suggesting that this activity is the result of extracellular cGAMP produced by 293T cells (Figure 1, panels H and I). Addition of purified soluble recombinant mouse ENPP1 (mENPP1) (Figure 8, panel D) depleted detectable cGAMP in the conditioned medium and also abolished this activity (Figure 1, panels H and J). Because soluble ENPP1 (MW = approximately 100 kDa) cannot penetrate the membrane and therefore is accessible only to soluble extracellular cGAMP, we conclude that 293T cells secrete soluble cGAMP. Together, our data demonstrate that this artificial cancer cell line maintains its intracellular cGAMP at steady state by excreting it into the extracellular medium as a soluble factor.

[0562] Panels A–J of Figure 1: cGAMP is a 293T cGAS ENPP1 - / -It is excreted from cells as a soluble factor. a) Chemical structures of cGAMP and monoisotopically labeled cGAMP. b) cGAMP is detected by LC-MS / MS. Lower limit of quantification = 4 nM. (Left) Liquid chromatography traces of cGAMP at 0, 4, and 10 nM, and monoisotopically labeled cGAMP (15 Da heavy) at 500 nM as an internal standard; (Right) External standard curve of cGAMP, R 2 =0.996. Data are representative of more than 10 independent experiments. c-d, 293T cGAS ENPP1 without exogenous stimulation measured using LC-MS / MS. - / - Intracellular and extracellular concentrations of cGAMP from cells. At time 0, cells were supplemented with serum-free medium. Mean ± standard error (n=2) with some error bars too small to visualize. Data are representative of three independent experiments. e, The ratio of extracellular / total cGAMP molecules (left y-axis) calculated from the data in (c) and (d) compared to the ratio of extracellular / total lactose dehydrogenate (LDH) activity (right y-axis). f, The amount of cGAMP excreted per cell over time calculated from the data in (d). The excretion rate was found using linear regression. g, 293T cGAS ENPP1 measured before and after passing the medium through a 10 kDa filter. - / - Intracellular and extracellular cGAMP concentrations produced by cells. Mean ± standard error (n=2). Data are representative of two independent experiments. h, Schematic of conditioned medium transfer experiments for (i) and (j). cGAS-null 293T or 293T cGAS ENPP1 low Cells were transfected with an empty pcDNA vector and treated with + / - 20 nM recombinant mouse ENPP1 (mENPP1). Conditioned medium from these cells was transferred to primary CD14 + i, IFNB1 mRNA levels were normalized to CD14 and fold induction was calculated using naive CD14 + Calculated relative to cells. Mean ± standard error (n=4). *** P = 0.0003 (one-way ANOVA). cGAMP concentrations were measured in conditioned medium. Mean ± standard error (n = 2). ****P=0.0002 (one-way ANOVA). Data are representative of two independent experiments. j, IFNB1 mRNA levels were normalized to CD14 and fold induction was compared with untreated CD14 + Calculated relative to cells. Mean ± standard error (n=2). * P = 0.04 (one-way ANOVA). cGAMP concentrations were measured in the conditioned medium. Mean ± standard error (n = 2). ** P=0.002 (one-way ANOVA). Data are representative of two independent experiments.

[0563] Panels A-D of Figure 8: LC-MS / MS method development and 293T cGAS ENPP1 low and 293T cGAS ENPP1 - / - Cell line construction. a, Liquid chromatography traces of cGAMP at 0, 20, and 80 nM; monoisotopically labeled internal standard cGAMP (15 Da heavy) at 500 nM; and 2 μM dual-isotopically labeled extracted standard cGAMP (30 Da heavy). Chemical structures of all analytes. b, Calibration of cell number to ATP concentration measured by LC-MS / MS. Mean ± standard error (n=2). c, 293T, 293T cGAS ENPP1 analyzed by Western blot. - / - and 293T cGAS ENPP1 low cGAS expression levels in cell lines (left): 293T cGAS, 293T cGAS ENPP1, as determined by TLC and autoradiography. - / - and 293T cGAS ENPP1 low in whole cell lysates from 1 million cells each 32 ENPP1 hydrolysis activity of P-cGAMP (right). Lysate data are representative of two independent experiments. d, Coomassie gel of recombinant mouse ENPP1 purified from culture medium; elution fractions were pooled before use (left). Mouse ENPP1 analyzed by TLC. 32 P-cGAMP degradation (right).

[0564] Panel A of Figure 9: CD14 +PBMCs respond to extracellular cGAMP. a) Schematic of stimulation of CD14+ PBMCs with extracellular cGAMP. b) Human CD14+ PBMCs stimulated with increasing concentrations of extracellular cGAMP for 16 hours. + IFNB1 induction measured by RT-qPCR on PMBCs. Mean ± SEM (n=2 technical qPCR replicates).

[0565] ENPP1 regulates only extracellular cGAMP

[0566] Since we first observed extracellular cGAMP by knocking out ENPP1 from 293T cells and culturing the cells in ENPP1-free medium, we next investigated whether only extracellular cGAMP is regulated by ENPP1. Despite its extracellular annotation, ENPP1 may be able to reverse its membrane surface orientation, as in the case of 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)). Alternatively, ENPP1 may be activated when synthesized in the ER lumen, and cGAMP may be able to cross the ER membrane (Figure 2, panel A). To investigate the localization of ENPP1 activity, we analyzed 293T cGAS ENPP1 - / - Cells were transfected with a human ENPP1 expression plasmid and its activity in whole cell lysates was confirmed (Figure 2, Panel B). In intact cells, ENPP1 expression depletes extracellular cGAMP but does not affect intracellular cGAMP concentrations (Figure 2, Panel C). Thus, in these cells, extracellular cGAMP is regulated by ENPP1, but intracellular cGAMP is not.

[0567] Panels A–C of Figure 2: ENPP1 regulates only extracellular cGAMP. a, Three possible cellular locations of ENPP1 activity. b, 293T cGAS ENPP1 - / - Cells were transfected with either an empty vector or a vector containing human ENPP1, and 24 hours later, Western blot was used to measure ENPP1 protein expression (top) and thin-layer chromatography (TLC) to measure ENPP1. 32 P-cGAMP hydrolysis activity was analyzed (bottom). Data are representative of two independent experiments. c, Intracellular and extracellular cGAMP concentrations measured using LC-MS / MS. BQL = below the limit of quantification. Mean ± standard error (n = 2). ** P=0.002 (Student's t-test). Data are representative of three independent experiments.

[0568] Development of cell-impermeable ENPP1 inhibitors

[0569] To address the physiological relevance of extracellular cGAMP and why it must be regulated by specific hydrolases, we attempted to manipulate its concentration by pharmacologically inhibiting ENPP1. We first tested the nonspecific ENPP1 inhibitor QS1 (Figure 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)). QS1 can inhibit extracellular cGAMP degradation in cells overexpressing ENPP1, but it also partially blocked cGAMP efflux in ENPP1 knockout cells (Figure 10, Panel B). QS1-treated cells increased intracellular cGAMP, again demonstrating that efflux is an important mechanism for maintaining cGAMP homeostasis in cancer cells. Due to its efflux blocking activity, our efflux studies exclude QS1 as a means to investigate extracellular cGAMP. Compound 1, a phosphonate analog, binds Zn at the ENPP1 catalytic site. 2+ Compound 1 was designed to chelate with , minimize cell permeability, and avoid intracellular off-targets (Figure 3, Panel A). Compound 1 exhibited a K of 110 ± 10 nM. i,app (Figure 3, panel B), which is approximately 60-fold more potent than QS1 (Figure 10, panel A).

[0570] We confirmed that Compound 1 is cell impermeable by performing three independent permeability assays: a parallel artificial membrane permeability assay (PAMPA) (Panel A of Figure 11); an intestinal cell Caco-2 permeability assay (Panel B of Figure 11); and an epithelial cell MDCK permeability assay (Panel C of Figure 11). Compared with control compounds with high and low cell permeability, Compound 1 falls into the category of impermeable compounds in all three assays. Furthermore, Compound 1 inhibits the activity of alkaline phosphatase (K), a closely related ectonucleotidase. i,app >100 μM) and ENPP2 (K i,app = 5.5 μM (Panel D of Figure 11). Although we do not expect Compound 1 to have intracellular off-targets due to its low cell permeability, we tested its binding to a panel of 468 kinases to further determine its specificity. Despite structural similarity to AMP, Compound 1 only binds to two kinases at 1 μM (Panel E of Figure 11). Compound 1 also exhibits high stability (t 1 / 2 In summary, we have demonstrated that compound 1 is a potent, cell-impermeant, specific, and stable ENPP1 inhibitor.

[0571] Next, we measured the efficacy of compound 1 in maintaining extracellular cGAMP concentrations in 293T cGAS cells overexpressing ENPP1, finding an IC of 340 ± 160 nM. 50 The values ​​obtained were (Figure 3, Panel C), and 10 μM was sufficient to completely block extracellular cGAMP degradation (Figure 3, Panel D). Unlike QS1, Compound 1 did not affect intracellular cGAMP, demonstrating that Compound 1 did not affect cGAMP efflux (Figure 3, Panel D). Therefore, Compound 1 is a potent ENPP1 inhibitor that specifically increases extracellular cGAMP concentrations.

[0572] Finally, we investigated the CD14 +The effectiveness of Compound 1 in enhancing detectable extracellular cGAMP signals in PBMCs was tested. We first confirmed that Compound 1 was not toxic to PBMCs at the concentrations used (Figure 11, panel F). Conditioned medium from 293T cGAS cells overexpressing ENPP1 increased CD14 + Compound 1 failed to induce IFNB1 expression in CD14 cells (Figure 3, panels E and F). However, it significantly increased the extracellular cGAMP levels in the culture medium and CD14 expression. + ENPP1 rescued the induction of IFNB1 expression in CD14 cells (Figure 3, Panel F). These results demonstrate that the enzymatic activity of ENPP1, without the potential scaffolding effect of a transmembrane protein, was significantly enhanced by CD14 + We demonstrate that ENPP1 suppresses the response to extracellular cGAMP by PBMCs. Collectively, our data suggest that extracellular cGAMP levels can be reduced by ENPP1 expression and enhanced by ENPP1 inhibition, suggesting that CD14 + Affects the in vitro activation of PBMCs.

[0573] Panels A-F of Figure 3: Activity of cell-impermeable ENPP1 inhibitors. a, Chemical structure of compound 1. b, ENPP1 activity as a substrate at pH 7.5. 32 Inhibitory activity of compound 1 against purified mouse ENPP1 with P-cGAMP (K i,app =110±10 nM). Mean ± standard error (n=3 independent experiments) with some error bars too small to visualize. c, 293T cGAS ENPP1 - / - Inhibitory activity (IC) of compound 1 against human ENPP1 transiently expressed in cells 50 = 340 ± 160 nM). Mean ± standard error (n = 2). d, 293T cGAS ENPP1 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 for cells. BQL = below limit of quantification. Mean ± standard error (n = 3). ****P<0.0001 (one-way ANOVA). Data are representative of two independent experiments. e, Schematic of the conditioned medium experiment. 293T cGAS ENPP1 low Cells were transfected with a vector containing human ENPP1 and incubated in the presence or absence of compound 1. Conditioned medium from these cells was cultured in primary CD14 + f, IFNB1 mRNA levels were normalized to CD14 and fold induction was calculated using naive CD14 + Calculated relative to cells. Mean ± standard error (n=2). ** P = 0.007 (one-way ANOVA). cGAMP concentrations were measured in conditioned medium. Mean ± standard error (n = 2). ** P=0.006 (one-way ANOVA). Data are representative of two independent experiments.

[0574] Figure 10, Panels A-B: Improvement of Compound 1 over QS1. a, Structure of QS1 and its substrate at pH 7.5 32 Its inhibitory activity against purified mouse ENPP1 using P-cGAMP (compared to compound 1) (QS1 K i,app = 6.4 ± 3.2 μM). Mean ± standard error (n = 2 independent experiments). b, 293T cGAS ENPP1 transfected with empty vector or vector containing human ENPP1 in the presence or absence of QS1. - / - Intracellular, extracellular and total cGAMP for cells. Mean ± standard error (n=2). * P<0.05. ** P<0.01 (one-way ANOVA).

[0575] Panels A-F of Figure 11: Compound 1 is cell impermeant, specific for ENPP1, and non-toxic. a, Permeability of Compound 1 in an artificial membrane permeability assay (PAMPA). b, Permeability of Compound 1 in an enterocyte 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 side of Caco-2 monolayers for 2 hours. The concentration of compounds on the basolateral side was monitored by LC-MS / MS. The apparent permeation rate (P app ) was calculated from the slope. Data are representative of two independent experiments. c, Permeability of compound 1 in epithelial cell MDCK permeability assay. d, Inhibitory activity of compound 1 against alkaline phosphatase (ALPL) and ENPP2. Mean ± standard error (n=2). e, Kinome interaction map for compound 1 illustrating kinase inhibition as a percentage of control (468 kinases tested). Image generated using TREEspot™ software tool and reproduced with permission from KINOMEscan®, a division of DiscoverRx Corporation. Copyright © 2010 DiscoverRx Corporation. f, Cell viability measured by CellTiterGlo. Total PBMC and CD14 + PBMCs were incubated with Compound 1 for 16 hours and then assayed for ATP levels using CellTiterGlo. Data was normalized to the absence of Compound 1 and % cell viability was calculated.

[0576] Cancer cells express cGAS in culture and continuously excrete cGAMP.

[0577] To determine whether extracellular cGAMP can function as a danger signal secreted by cancer cells in vivo, we first sought to identify tumor models that excrete cGAMP. We tested one human (MDA-MB-231) and three mouse cancer cell lines (E0771, MC38, and 4T1-Luc, a 4T1 cell line expressing luciferase for in vivo imaging) in culture, all of which express cGAS (Figure 4, panel A). It is difficult to detect intracellular cGAMP concentrations in these cells. However, using extra concentration and purification steps, we found that 5.8 × 10 cGAMP was expressed in 4T1-Luc cells. -10 We were able to detect intracellular cGAMP at nmol / cell (approximately 150 nM) (Figure 4, panel B). Knocking down cGAS using shRNA resulted in a decrease in cGAS protein levels, which in turn reduced intracellular cGAMP levels, demonstrating that cGAS expression controls the amount of cGAMP present in 4T1-Luc cells (Figure 12, panels A and B). Using compound 1, which inhibits cell surface and soluble ENPP1 in the cell culture medium, we detected continuous cGAMP excretion in all of these cell lines, with extracellular cGAMP levels reaching approximately 6 x 10 over 48 hours. -9The cGAMP concentration reached approximately 10 nmol / cell (approximately 10 nM when diluted in culture medium) (Figure 4, Panels C and D, and Figure 12, Panels C and D). Surprisingly, this is approximately 10 times the amount of cGAMP present in cells, suggesting that cancer cells efficiently clear their cGAMP by excretion. Ionizing radiation (IR) can increase cytosolic DNA and activate cGAS-dependent IFN-β production in tumor cells (see, e.g., Bakhoum, S. F. 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 tumor immunogenicity. Nat. Commun. 8, 15618 (2017)). Indeed, IR treatment also increased extracellular cGAMP production in 4T1-Luc cells after 2 days (Figure 4, Panel E and Figure 12, Panel E). Collectively, our data demonstrate that these cancer cell lines continuously produce and efficiently excrete cGAMP and can be stimulated by IR to produce even more extracellular cGAMP.

[0578] Figure 4, panels A-E: Cancer cells express cGAS in culture and continuously excrete cGAMP. a) cGAS expression in 4T1-Luc, E0771, MDA-MB-231, and MC38 analyzed by Western blot. b) Estimated intracellular cGAMP concentration in 4T1-Luc cells in the absence of exogenous stimulation. Mean ± standard error (n = 2). c) Extracellular cGAMP produced by MC38 cells over 48 hours. At time 0, cells were refreshed with medium supplemented with 50 μM Compound 1. Mean ± standard error (n = 2). Data are representative of two independent experiments. d) Extracellular cGAMP produced by 4T1-Luc, E0771, and MDA-MB-231 cells measured after 48 hours in the presence of 50 μM Compound 1. BQL = below the limit of quantification. Mean ± standard error (n = 2). e, Extracellular cGAMP produced by 4T1-Luc cells over 48 hours. At time 0, cells were left untreated or treated with 20 Gy IR and refreshed with medium supplemented with 50 μM Compound 1. Mean ± standard error (n=2). * P = 0.04 (Student's t test).

[0579] Figure 12, Panels A-E: Cancer cells continuously excrete cGAMP in culture. a) cGAS expression levels 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 ± standard error (n = 2). Data for 4T1-Luc WT are reproduced from panel b of Figure 4 for comparison. c) Extracellular cGAMP (shown in media concentrati...

Claims

1. Compounds of formula (I): 【Chemistry 101】 [In the formula, X １ teeth, (a) 【Chemistry 101-1】 or the group consisting of (b) 【Chemistry 101-2】 (In the formula, R ８１ and R ９１ are each independently selected from H, alkyl, alkenyl, alkoxy, aryl, acyl, ester, amide, heterocycle, and cycloalkyl, or R ８１ and R ９１ together with the atoms to which they are attached form a group selected from heterocycles) is a hydrophilic head group selected from A is a ring system selected from aryl, heteroaryl, cycloalkyl, and heterocycle; L １ and L ２ are independently a covalent bond or C １～６ alkyl; Z ３ is absent or NR ２２ , O and S; Z ２ is CR １２ and Z １ is CR １１ or N, R １ is selected from H, alkyl, alkenyl, alkylaryl, alkylheteroaryl, alkenylaryl, alkenylheteroaryl, aryl, heteroaryl, and heterocycle; R １１ is selected from H, cyano, trifluoromethyl, halogen, and alkyl; R １２ is cyano, R ２２ is selected from H and alkyl; R ２ ~R ５ is H, OH, alkyl, alkenyl, alkoxy, -OCF ３ , halogen, cyano, amine, amide, and heterocycle, or R ２ and R ３ , R ３ and R ４ Or R ４ and R ５ taken together with the carbon atom to which they are attached form a fused ring selected from heterocycle, cycloalkyl, and aryl. or a pharmaceutically acceptable salt or solvate thereof.

2. The compound has the formula (II): 【Chemical Engineering 102】 [In the formula, Z ３１ is NR ２２ , O and S] 2. The compound of claim 1, wherein

3. Z ３１ But NR ２２ and R ２２ But H, and C （１～６） selected from alkyl, The compound of claim 2.

4. The compound has the formula (III): 【Chemistry 103】 [In the formula, R ３１ ~R ３４ are H, respectively, n and m are each independently an integer of 0 to 6.

4. The compound of claim 2 or 3,

5. The compound of claim 4, wherein n+m is 0 to 3.

6. 6. The compound of any one of claims 1 to 5, wherein the ring system A is selected from phenyl, pyridyl, pyrimidine, piperidine, piperazine, pyridazine, and cyclohexyl.

7. The ring system A is: 【Chemical 104】 [In the formula, Z ５ is N and CR ６ is selected from R ６ are hydrogen, p is an integer from 0 to 4, and q is an integer from 0 to 2.

7. The compound of claim 6, selected from:

8. The compound has the formula (IV): 【Chemistry 105】 [In the formula, Z １１ is selected from N and C(CN), Z ２１ is C(CN), R ６ are H, respectively, p is an integer from 0 to 4.

8. The compound according to any one of claims 4 to 7, wherein

9. The compound has the formula (V): 【Chemistry 106】 [In the formula, R ４１ ~R ４４ is hydrogen] 9. The compound of claim 8, wherein

10. The compound has the formula (VIb): 【Chemistry 107】 10. The compound of claim 9, wherein

11. R １ 11. The compound of claim 1, wherein is selected from H, alkylaryl, alkylheteroaryl, alkenylaryl, alkenylheteroaryl, aryl, and heteroaryl.

12. R １ 12. The compound of claim 11, wherein is selected from H, ethenylaryl, and ethenylheteroaryl.

13. The compound has the formula (VIIb): 【Chemistry 108】 11. The compound of claim 10, wherein

14. R ２ ~R ５ is H, OH, alkyl, alkoxy, -OCF ３ 14. The compound of claim 1, wherein each of the groups is independently selected from: halogen, cyano, amine, amide, and heterocycle.

15. R ２ ~R ５ But H, OH, C （１～６） Alkoxy, -OCF ３ , C （１～６） Alkylamino, di-C （１～６） 15. The compound of claim 14, wherein each is independently selected from alkylamino, F, Cl, Br, and CN.

16. R ３ and R ４ are independently alkoxy; R ２ and R ５ is hydrogen, 15. The compound of claim 14.

17. R ３ is an alkoxy; R ２ , R ４ and R ５ is hydrogen, 15. The compound of claim 14.

18. R ４ is an alkoxy; R ２ , R ３ and R ５ is hydrogen, 15. The compound of claim 14.

19. R ２ , R ３ and R ４ is H, R ５ is an alkoxy; 15. The compound of claim 14.

20. 20. The compound of any one of claims 4 to 19, wherein n is 0 and m is 1.

21. 20. The compound of any one of claims 4 to 19, wherein n is 1 and m is 0.

22. 20. The compound of any one of claims 4 to 19, wherein n and m are both 1.

23. 20. The compound of any one of claims 4 to 19, wherein n and m are both 0.

24. Z ３ does not exist, and Z ２ is CR １２ and R １２ is cyano, and the compound has formula (X): 【Chemistry 109】 [In the formula, L １１ and L １２ are independently a covalent bond or C １～６ is an alkyl linker] 2. The compound of claim 1, wherein

25. 25. The compound of claim 24, wherein said ring system A is selected from phenyl, pyridyl, pyrimidine, piperidine, piperazine, pyridazine, and cyclohexyl.

26. The compound has the formula (XI): 【Chemical 110】 [In the formula, Z ５ are N and CR, respectively. １６ are independently selected from R １６ are hydrogen, and r is an integer from 0 to 8.

26. The compound of claim 24 or 25,

27. The compound has the formula (XII): 【Chemistry 111】 27. The compound of claim 26, wherein

28. Z ５ 28. The compound of claim 27, wherein is N.

29. The compound has the formula (XIII): 【Chemistry 112】 [R ３５ and R ３６ is H, wherein s is an integer from 0 to 6.

29. The compound of claim 28, wherein

30. The compound has the formula (XIV): 【Chemistry 113】 30. The compound of claim 29, wherein

31. R ２ ~R ５ is H, OH, alkyl, alkoxy, -OCF ３ 31. The compound of any one of claims 24 to 30, wherein each of the groups is independently selected from: halogen, cyano, amine, amide, and heterocycle.

32. R ２ ~R ５ But H, OH, C （１～６） Alkoxy, -OCF ３ , C （１～６） Alkylamino, di-C （１～６） 32. The compound of claim 31, wherein each is independently selected from alkylamino, F, Cl, Br and CN.

33. R ３ and R ４ are independently alkoxy; R ２ and R ５ is hydrogen, 32. The compound of claim 31.

34. R ３ is an alkoxy; R ２ , R ４ and R ５ is hydrogen, 32. The compound of claim 31.

35. R ４ is an alkoxy; R ２ , R ３ and R ５ is hydrogen, 32. The compound of claim 31.

36. R ２ , R ３ and R ４ is H, R ５ is an alkoxy; 32. The compound of claim 31.

37. 37. The compound of any one of claims 29 to 36, wherein s is 1.

38. 37. The compound of any one of claims 29 to 36, wherein s is 2.

39. 37. The compound of any one of claims 29 to 36, wherein s is 3.

40. The compound is 【Chemistry 118】 【Chemical 119】 10. The compound of claim 1 selected from the group consisting of: or a pharmaceutically acceptable salt thereof.

41. The compound has the following structure: 【Chemistry 119-1】 2. The compound of claim 1 selected from:

42. A compound according to any one of claims 1 to 41, and pharmaceutically acceptable excipients A pharmaceutical composition comprising:

43. 1. A pharmaceutical composition for use in the treatment of cancer, comprising: A compound according to any one of claims 1 to 41, and pharmaceutically acceptable excipients A pharmaceutical composition comprising:

44. 42. A composition comprising a compound of any one of claims 1 to 41 for use in a method of inhibiting ENPP1, the method comprising the step of contacting a sample containing ENPP1 with the composition to inhibit the cGAMP hydrolysis activity of the ENPP1. A composition comprising:

45. 45. The composition of claim 44, wherein the compound is cell-impermeable.

46. 45. The composition of claim 44, wherein the compound is cell-permeable.

47. 47. The composition of any one of claims 44 to 46, wherein the sample is a cell sample.

48. 48. The composition of claim 47, wherein the sample comprises cGAMP.

49. 49. The composition of claim 48, wherein contacting the sample with the composition increases the cGAMP level in the cell sample compared to a control sample not contacted with the composition.

50. 1. A composition for treating cancer, comprising:

42. A composition comprising a compound according to any one of claims 1 to 41.

51. 51. The composition of claim 50, wherein the cancer is a solid tumor cancer.

52. 52. The composition of claim 50 or 51, wherein the cancer is selected from adrenal gland, liver, kidney, bladder, breast, colon, stomach, ovary, cervix, uterus, esophagus, colorectal, prostate, pancreas, lung (both small cell and non-small cell), thyroid, carcinoma, sarcoma, glioblastoma, melanoma, and various head and neck tumors.

53. 53. The composition of claim 52, wherein the cancer is breast cancer.

54. 51. The composition of claim 50, wherein the cancer is lymphoma.

55. 53. The composition of claim 52, wherein the cancer is glioblastoma.

56. 56. The composition of any one of claims 50 to 55, wherein the composition is administered in combination with one or more additional active agents.

57. 57. The composition of claim 56, wherein the one or more additional active agents is a chemotherapeutic agent or an immunotherapeutic agent.

58. 58. The composition of claim 56 or 57, wherein the one or more additional active agents is a small molecule, an antibody, an antibody fragment, an antibody-drug conjugate, an aptamer, or a protein.

59. 59. The composition of any one of claims 56-58, wherein the one or more additional active agents comprises a checkpoint inhibitor.

60. 60. The composition of claim 59, wherein the checkpoint inhibitor is selected from a cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) inhibitor, a programmed death 1 (PD-1) inhibitor, and a PD-L1 inhibitor.

61. 59. The composition of any one of claims 56 to 58, wherein the one or more additional active agents comprises a chemotherapeutic agent.

62. 62. The composition of claim 61, wherein the chemotherapeutic agent is a cGAMP-inducing chemotherapeutic agent.

63. 63. The composition of claim 62, wherein the cGAMP-inducing chemotherapeutic agent is an antimitotic or antitumor agent administered in an amount effective to induce the production of cGAMP.

64. 64. The composition of any one of claims 50 to 63, wherein the composition is administered in combination with radiation therapy.

65. 65. The composition of claim 64, wherein the compound is administered prior to radiation therapy.

66. 65. The composition of claim 64, wherein the compound is administered after exposure to radiation therapy.

67. 67. The composition of claim 65 or 66, wherein the radiation therapy induces the production of cGAMP.

68. 68. The composition of any one of claims 50 to 67, wherein the compound is cell-impermeable.

69. 68. The composition of any one of claims 50 to 67, wherein the compound is cell-permeable.

70. 42. A composition comprising a compound of any one of claims 1 to 41 for use in treating cancer.

71. 42. Use of a compound according to any one of claims 1 to 41 in the manufacture of a medicament for treating cancer.

72. 42. A composition comprising a compound of any one of claims 1 to 41 for modulating an immune response in a subject, wherein the composition is administered to the subject to treat an inflammatory condition in the subject.

73. Z ３ does not exist, and Z ２ is CR １２ and R １２ is cyano, and the compound has formula (X): 【Chemical 120】 [In the formula, L １１ and L １２ are independently a covalent bond or C １～６ is an alkyl linker] 2. The compound of claim 1, wherein

74. 74. The compound of claim 73, wherein said ring system A is selected from phenyl, pyridyl, pyrimidine, piperidine, piperazine, pyridazine, and cyclohexyl.

75. The compound has the formula (XI): 【Chemistry 121】 [In the formula, Z ５ are N and CR, respectively. １６ are independently selected from R １６ are hydrogen, and r is an integer from 0 to 8.

75. The compound of claim 73 or 74,

76. The compound has the formula (XII): 【Chemistry 122】 74. The compound of claim 73, wherein

77. Z ５ 77. The compound of claim 76, wherein is N.

78. The compound has the formula (XIII): 【Chemical 123】 [R ３５ and R ３６ is H, wherein s is an integer from 0 to 6.

76. The compound of claim 75,

79. The compound has the formula (XIV): 【Chemistry 124】 76. The compound of claim 75,

80. R ２ ~R ５ is H, OH, alkyl, alkoxy, -OCF ３ 79. The compound of any one of claims 73 to 78, wherein each of the groups is independently selected from: halogen, cyano, amine, amide, and heterocycle.

81. R ２ ~R ５ But H, OH, C （１～６） Alkoxy, -OCF ３ , C （１～６） Alkylamino, di-C （１～６） 81. The compound of claim 80, wherein each is independently selected from alkylamino, F, Cl, Br, and CN.

82. Structural formula: 【Chemistry 125】 or a pharmaceutically acceptable salt thereof.

83. Structural formula: 【Chemistry 126】 A compound represented by:

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