ENPP1 INHIBITORS AND THEIR USE FOR THE TREATMENT OF CANCER - Patent application

Abstract

To provide methods for treating diseases.SOLUTION: The disclosure provides compounds, compositions and methods for inhibiting ENPP1. An aspect of the subject methods comprises contacting a sample with an ENPP1 inhibitor to inhibit cGAMP-hydrolyzing activity of ENPP1. In some cases, the ENPP1 inhibitor is not cell-permeating. Also provided are compositions and methods for treating cancer. An aspect of the method comprises administering to a subject a therapeutically effective amount of an ENPP1 inhibitor to treat cancer in the subject. In certain cases, the cancer is a solid tumor cancer. Also provided is a method comprising applying radiotherapy to a subject either before or after the administration of an ENPP1 inhibitor. The radiotherapy may be applied at an effective dose and / or frequency to reduce the radiation damage to the subject. In certain cases, the method is carried out in combination with a chemotherapeutic agent or a checkpoint inhibitor or both.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] cross reference This application claims the benefit of U.S. Provisional Patent Application No. 62 / 556,117, filed Sep. 8, 2017, which is incorporated herein by reference in its entirety.

[0002] Government Rights 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 the invention. [Background technology]

[0003] Introduction Cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) activates the stimulator of interferon genes (STING) pathway, an important anti-cancer innate immune pathway. The cGAS-cGAMP-STING pathway is activated in the presence of cytoplasmic DNA, either due to microbial infection or pathophysiological conditions including cancer and autoimmune diseases. Cyclic GMP-AMP synthase (cGAS) belongs to the nucleotidyl transferase family and is a versatile DNA sensor that is activated upon binding to cytoplasmic dsDNA to produce the signaling molecule (2'-5',3'-5') cyclic GMP-AMP (or 2',3'-cGAMP or cyclic guanosine monophosphate-adenosine monophosphate, cGAMP). 2',3'-cGAMP, which functions as a second messenger during microbial infection, binds to and activates STING, which is responsible for 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 promising new target for immunotherapy of cancer and autoimmune diseases.

[0004] Ectonucleotide pyrophosphatase / phosphodiesterase 1 (ENPP1) is the main hydrolase of cGAMP that can degrade cGAMP. ENPP1 is a member of the ectonucleotide pyrophosphatase / phosphodiesterase (ENPP) family. The encoded protein is a type II transmembrane glycoprotein that contains two identical disulfide-linked subunits. The ENPP1 protein has broad specificity and can cleave a variety of substrates, including the phosphodiester bond of nucleotides and nucleotide sugars, as well as the pyrophosphate bond of nucleotides and nucleotide sugars. The protein can function to hydrolyze nucleoside 5'-triphosphates to the corresponding monophosphates, as well as diadenosine polyphosphates. Summary of the Invention

[0005] Compounds, compositions and methods are provided for the inhibition of ENPP1. ENPP1 inhibitor compounds can act extracellularly to block the degradation of cGAMP. An embodiment of the subject method includes contacting a sample with an ENPP1 inhibitor to inhibit the cGAMP hydrolysis activity of ENPP1. In some cases, the ENPP1 inhibitor is cell impermeable. Compositions and methods for treating cancer are also provided. An embodiment of the method includes administering to a subject a therapeutically effective amount of an ENPP1 inhibitor to treat the cancer in the subject. In certain cases, the cancer is a solid tumor cancer. Also provided is a method of administering radiation therapy to the subject either before or after administration of an ENPP1 inhibitor. The radiation therapy can be administered at a dose and / or frequency effective to reduce radiation damage to the subject, but can also ... It can also provoke answers.

[0006] These and other advantages and features of the present disclosure will become apparent to those skilled in the art upon reading the details of the compositions and methods of use that are more fully described below.

[0007] The present invention is best understood by reading the following detailed description in conjunction with the accompanying drawings. The 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. Rather, the dimensions of the various features have been arbitrarily expanded or reduced for clarity. The following figures are included in the drawings. It is understood that the figures described below are for illustrative purposes only. These figures are not intended to limit the scope of the present disclosure in any way. [Brief description of the drawings]

[0008] [Figure 1] 1A-1C present data showing that an exemplary ENPP1 inhibitor can increase the amount of extracellular cGAMP present in a cell line. [Diagram 2] 2A-2B show that exemplary ENPP1 inhibitors can increase cGAMP-stimulated interferon transcription. [Diagram 3] 3A-3B present data showing that an exemplary ENPP1 inhibitor can increase tumor-associated dendritic cell numbers in a mouse tumor model. [Figure 4A] 4A-4C show that ENPP1 inhibition exerts anti-tumor effects in synergy with IR therapy and anti-CTLA-4. [Figure 4B-C] 4A-4C show that ENPP1 inhibition exerts anti-tumor effects in synergy with IR therapy and anti-CTLA-4. [Diagram 5] FIG. 1 shows a schematic diagram showing that ENPP1 is an innate immune checkpoint that regulates the immune mediator cGAMP. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] definition Before the embodiments of the present disclosure are further described, it is to be understood that the present disclosure is not limited to particular embodiments described, as such may, of course, vary, and the scope of the present disclosure will be limited only by the appended claims, and 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.

[0010] 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 may also be used in the practice or testing of embodiments of the present disclosure.

[0011] It should be noted that, as used in this specification and the appended claims, the singular forms "a," "and," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a compound" includes not only a single compound but also a combination of two or more compounds, reference to "a substituent" includes a single substituent as well as two or more substituents, and so forth.

[0012] 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, some chemical moieties may be included in more than one definition.

[0013] As used herein, "for example," "for example," The phrases "in one instance," "such as," or "including" are intended to introduce examples that further clarify a more general subject matter. These examples are provided merely as an aid in understanding the disclosure and are not intended to be limiting in any way.

[0014] 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 publication dates provided may be different from the actual publication dates, which may need to be independently confirmed.

[0015] The terms "active agent," "antagonist," "inhibitor," "drug," and "pharmacologically active agent" are used interchangeably herein and refer to chemical compounds or compounds that, when administered to an organism (human or animal), induce a desired pharmacological and / or physiological effect through local and / or systemic action.

[0016] As used herein, the terms "treatment", "treating" and the like refer to obtaining a desired pharmacological and / or physiological effect, e.g., reduction of tumor burden. This effect may be prophylactic, in that it completely or partially prevents the disease or its symptoms, and / or it may be therapeutic, in that it partially or completely cures the disease and / or side effects caused by the disease. As used herein, "treatment" refers to any treatment of a disease in a mammal, particularly a human, and includes: (a) preventing a subject who may be susceptible to the disease but has not yet been diagnosed as suffering from it from developing the disease or symptoms of the disease (e.g., a disease that may be associated with or caused by the primary disease, such as in the case of liver fibrosis that may occur in the context of chronic HCV infection); (b) inhibiting the disease, i.e., halting its development; and (c) alleviating the disease, i.e., causing regression of the disease (e.g., reduction of tumor burden).

[0017] The term "pharmaceutical acceptable salt" refers to a salt that is acceptable for administration to a patient, e.g., a mammal (a salt with a counterion that has acceptable mammalian safety for a given dosing regimen). Such salts can be derived from pharmaceutical acceptable inorganic or organic bases, and from pharmaceutical acceptable inorganic or organic acids. "Pharmaceutical acceptable salt" refers to pharmaceutical acceptable salts of a compound, which can be 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, and the like, and, when the molecule contains a basic functional group, an organic or inorganic acid salt, e.g., hydrochloride, hydrobromide, formate, tartrate, besylate, mesylate, acetate, maleate, oxalate, and the like.

[0018] 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, cows, horses, sheep, cats, dogs, etc. "Mammal" means a member or members of any mammalian species, including, by way of example, dogs, cats, horses, cows, sheep, rodents, etc., as well as primates, e.g., non-human primates, and humans. Non-human animal models, e.g., mammals, e.g., non-human primates, mice, lagomorphs, etc., may be used in experimental studies.

[0019] As used herein, the terms "determining," "measuring," "assessing," and "assaying" are used interchangeably and include both quantitative and qualitative determination.

[0020] As used interchangeably herein, the terms "polypeptide" and "protein" refers to polymeric forms of amino acids of any length and can include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides with modified peptide backbones. The term includes fusion proteins, including, but not limited to, fusion proteins containing heterologous amino acid sequences, fusions containing heterologous and native leader sequences, with or without an N-terminal methionine residue, immunologically labeled proteins, fusion proteins containing a detectable fusion partner, such as fusion proteins containing a fluorescent protein, β-galactosidase, luciferase, etc. as a fusion partner.

[0021] 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 analogs thereof. 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, control regions, isolated RNA of any sequence, nucleic acid probes, and primers. The nucleic acid molecule may be linear or circular.

[0022] "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.

[0023] As used herein, the term "unit dosage form" refers to physically discrete units suitable as single doses 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 the desired effect in association with a pharma- ceutically acceptable diluent, carrier, or vehicle. The specifications for the unit dosage forms depend on the particular compound employed and the effect to be achieved, as well as the pharmacodynamics associated with each compound in the host.

[0024] "Pharmaceutically acceptable excipient," "pharmaceutically acceptable diluent," "pharmaceutically acceptable carrier," and "pharmaceutically acceptable adjuvant" refer to excipients, diluents, carriers, and adjuvants that are generally safe, non-toxic, and not biologically or otherwise harmful and are useful in preparing pharmaceutical compositions, and include excipients, diluents, carriers, and adjuvants that are acceptable for veterinary and human pharmaceutical use. As used in the specification and claims, "pharmaceutically acceptable excipients, diluents, carriers, and adjuvants" includes both one and more than one such excipients, diluents, carriers, and adjuvants.

[0025] As used herein, "pharmaceutical composition" is meant to encompass compositions suitable for administration to a subject, e.g., a mammal, particularly a human. Generally, a "pharmaceutical composition" is sterile and preferably free of contaminants that may cause an undesirable response in the subject (e.g., the compound(s) in the pharmaceutical composition are of pharmaceutical grade). A pharmaceutical composition can be designed for administration to a subject or patient in need thereof via many different routes of administration, including oral, buccal, rectal, parenteral, intraperitoneal, intradermal, intratracheal, intramuscular, subcutaneous, etc.

[0026] As used herein, the terms "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" refers to any of the recited elements, e.g., any R group or any combination thereof. "Similar" or "similar" is used herein to indicate that things may be the same or different.

[0027] As used herein, the terms "may," "optionally," "optionally," or "optionally may" mean that the subsequently described circumstance may or may not occur, and thus the description includes examples where the circumstance occurs and examples where it does not occur. For example, the phrase "optionally substituted" means that a non-hydrogen substituent may or may not be present at a given atom, and thus the description includes structures where the non-hydrogen substituent is present and structures where the non-hydrogen substituent is not present.

[0028] "Acyl" means 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 It refers to the groups 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 CHC(O)-.

[0029] The term "alkyl" refers to a branched or unbranched saturated hydrocarbon group (i.e., a monoradical), usually, but not necessarily, containing 1 to about 24 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, octyl, decyl, and the like, and cycloalkyl groups, such as cyclopentyl, cyclohexyl, and the like. Generally, but not necessarily, alkyl groups herein may contain 1 to about 18 carbon atoms, and such groups may contain 1 to about 12 carbon atoms. The term "lower alkyl" contemplates an alkyl group of 1 to 6 carbon atoms. "Substituted alkyl" refers to an alkyl group substituted with one or more substituents, including, for example, in a carbonyl group, where two hydrogen atoms of the same carbon atom of the alkyl substituent are replaced (i.e., the substituted alkyl group may 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 described in more detail below. Unless otherwise specified, the terms "alkyl" and "lower alkyl" include straight-chain, branched, cyclic, unsubstituted, substituted, and / or heteroatom-containing alkyl or lower alkyl, respectively.

[0030] The term "substituted alkyl" refers to an alkyl group, as defined herein, in which one or more carbon atoms of 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, carboxy, etc. and -NRaRb, wherein R is an alkyl group selected from the group consisting of aryl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, 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, 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.

[0031] 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, and the like. Generally, but again not necessarily, an alkenyl group herein may contain from 2 to about 18 carbon atoms, such as from 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 has been replaced with a heteroatom. Unless otherwise specified, the terms "alkenyl" and "lower alkenyl" include straight-chain, branched, cyclic, unsubstituted, substituted, and / or heteroatom-containing alkenyl and lower alkenyl, respectively.

[0032] The term "alkynyl" refers to straight-chain or branched hydrocarbon groups of 2 to 24 carbon atoms containing at least one triple bond, e.g., ethynyl, n-propynyl, and the like. Generally, but again not necessarily, alkynyl groups herein may contain from 2 to about 18 carbon atoms, and such groups may further contain from 2 to 12 carbon atoms. The term "lower alkynyl" contemplates alkynyl groups of 2 to 6 carbon atoms. The term "substituted alkynyl" refers to alkynyl substituted with one or more substituents, and the terms "heteroatom-containing alkynyl" and "heteroalkynyl" refer to alkynyl in which at least one carbon atom has been replaced with 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.

[0033] The term "alkoxy" refers to an alkyl group attached through a single, terminal ether bond. That is, an "alkoxy" group may 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" may contain, for example, 1 to 3 carbon atoms, and as a further example, such substituents may contain 1 or 2 carbon atoms (i.e., methoxy and ethoxy).

[0034] The term "substituted alkoxy" refers to substituted alkyl-O-, substituted alkenyl-O-, substituted cycloalkyl-O-, substituted cycloalkenyl-O-, and substituted alkynyl-O- groups, where substituted alkyl, substituted alkenyl, substituted cycloalkyl, substituted cycloalkenyl, and substituted alkynyl are as defined herein.

[0035] The term "aryl", unless otherwise specified, generally, but not necessarily, refers to an aromatic substituent containing 5-30 carbon atoms and containing a single aromatic ring or multiple aromatic rings that are fused, directly linked, or indirectly linked (such that different aromatic rings are bonded to a common group, such as a methylene or ethylene moiety). An aryl group may, for example, contain 5-20 carbon atoms, and as a further example, an aryl group may contain 5-12 carbon atoms. For example, an aryl group may 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 aryl substituents in which at least one carbon atom has been replaced with a heteroatom, as described in more detail below. Aryl includes stable cyclic, heterocyclic, polycyclic, and polyheterocyclic unsaturated C3-C 14 Examples of aryl groups are intended to include, but are not limited to, phenyl, biphenyl, naphthyl, pyridyl, furyl, thiophenyl, imidazoyl, pyrimidinyl, and oxazoyl, which may be further substituted with 1 to 5 members selected from the group consisting of hydroxy, C1-C8 alkoxy, C1-C8 branched or straight chain alkyl, acyloxy, carbamoyl, amino, N-acylamino, nitro, halogen, trifluoromethyl, cyano, and carboxyl (see, e.g., Katritzky, Handbook of Heterocyclic Chemistry).Unless otherwise indicated, the term "aryl" includes unsubstituted, substituted, and / or heteroatom-containing aromatic substituents.

[0036] 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 defined above. Typically, aralkyl and alkaryl groups herein contain from 6 to 30 carbon atoms. Aralkyl and alkaryl groups can, for example, contain from 6 to 20 carbon atoms, and, as a further example, such groups can contain from 6 to 12 carbon atoms.

[0037] The term "alkylene" refers to a diradical alkyl group. Unless otherwise specified, such groups include saturated hydrocarbon chains containing 1 to 24 carbon atoms, which may be substituted or unsubstituted, may include one or more alicyclic groups, and may be heteroatom-containing. "Lower alkylene" refers to an alkylene linkage containing 1 to 6 carbon atoms. Examples include methylene (--CH2--), ethylene (--CH2CH2--), propylene (--CH2CH2CH2--), 2-methylpropylene (--CH2--CH(CH3)--CH2--), hexylene (--(CH2)6--), and the like.

[0038] Similarly, the terms "alkenylene," "alkynylene," "arylene," "aralkylene," and "alkarylene" refer to the diradical alkenyl, alkynyl, aryl, aralkyl, and alkaryl groups, respectively.

[0039] 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.

[0040] The terms "halo" and "halogen" are used in the conventional sense to refer to a chloro, bromo, fluoro, or iodo substituent.

[0041] "Carboxyl", "carboxy" or "carboxylate" refers to -COH or a salt thereof.

[0042] "Cycloalkyl" refers to cyclic alkyl groups of from 3 to 10 carbon atoms having a single or multiple cyclic rings, including fused, bridged, and spiro ring systems. Examples of suitable cycloalkyl groups include, for example, adamantyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, and the like. Such cycloalkyl groups include, by way of example, single ring structures, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, and the like, or multiple ring structures, such as adamantanyl, and the like.

[0043] The term "substituted cycloalkyl" refers to a cycloalkyl group having from 1 to 5 substituents, or from 1 to 3 substituents, selected from alkyl, substituted alkyl, alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, 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.

[0044] 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, bond, 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 alkyl substituents that are heteroatom-containing, the term "heterocycloalkyl" refers to cycloalkyl substituents that are heteroatom-containing, the terms "heterocyclic" or "heterocycle" refer to cyclic substituents that are heteroatom-containing, the terms "heteroaryl" and "heteroaromatic" refer to "aryl" and "aromatic" substituents, respectively, that are heteroatom-containing, 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 heteroatom-containing alicyclic groups are pyrrolidino, morpholino, piperazino, piperidino, tetrahydrofuranyl, and the like.

[0045] "Heteroaryl" refers to an aromatic group containing 1-15 carbon atoms, e.g., 1-10 carbon atoms and 1-10 heteroatoms selected from the group consisting of oxygen, nitrogen, and sulfur, in the ring. Such heteroaryl groups may have a single ring (e.g., pyridinyl, imidazolyl, or furyl) or may contain multiple condensed rings in the ring system (e.g., indolizinyl, quinolinyl, benzofuran, benzimidazolyl, or benzothienyl groups), provided that at least one ring in the ring system is aromatic and the point of attachment is through an atom of the aromatic ring. In certain embodiments, the nitrogen and / or sulfur atom(s) of the heteroaryl group are optionally oxidized to provide an N-oxide (N→O), sulfinyl, or sulfonyl moiety. The term includes, by way of example, pyridinyl, pyrrolyl, indolyl, thiophenyl, and furanyl. Unless otherwise constrained by the definition of a heteroaryl substituent, such heteroaryl groups can be any of the following: 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. The alkyl group may be optionally substituted with 1 to 5 substituents or 1 to 3 substituents selected from haloaryl, trihalomethyl, and trihalomethyl.

[0046] 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, and 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 and / or sulfur atom(s) of the heterocyclic group are optionally oxidized to provide an N-oxide, -S(O)-, or -SO2- moiety.

[0047] Examples of heterocycles and heteroaryls include 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, and isoxazole. , phenoxazine, phenothiazine, imidazolidine, imidazoline, piperidine, piperazine, indoline, phthalimide, 1,2,3,4-tetrahydroisoquinoline, 4,5,6,7-tetrahydrobenzo[b]thiophene, thiazole, thiazolidine, thiophene, benzo[b]thiophene, morpholinyl, thiomorpholinyl (also called thiamorpholinyl), 1,1-dioxothiomorpholinyl, piperidinyl, pyrrolidine, tetrahydrofuranyl, and the like.

[0048] Unless otherwise constrained by the definition of a heterocyclic substituent, such heterocyclic groups may be optionally substituted with 1 to 5 substituents or 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.

[0049] "Hydrocarbyl" refers to monovalent hydrocarbyl radicals containing 1 to about 30 carbon atoms, such as 1 to about 24 carbon atoms, further such as 1 to about 18 carbon atoms, and further such as about 1 to about 12 carbon atoms, including linear, branched, cyclic, saturated and unsaturated species, such as alkyl, alkenyl, aryl, and the like. 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 is replaced with a heteroatom. Unless otherwise indicated, the term "hydrocarbyl" is intended to include substituted and / or heteroatom-containing hydrocarbyl moieties.

[0050] As alluded to in some of the definitions above, "substituted" as in "substituted hydrocarbyl," "substituted alkyl," "substituted aryl," etc. means that at least one hydrogen atom bonded to a carbon (or other) atom in the hydrocarbyl, alkyl, aryl, or other moiety is replaced with It means that it is replaced by one or more non-hydrogen substituents. Examples of such substituents include, but are not limited to, functional groups and hydrocarbyl moieties C1-C24 alkyl (C1-C18 alkyl, C1-C12 alkyl, and C1-C6 alkyl), C2-C24 alkenyl (C2-C18 alkenyl, C2-C12 alkenyl, and C2-C6 alkenyl), C2-C24 alkynyl (C2-C18 alkynyl, C2-C12 alkynyl, and C2-C6 alkynyl), C5-C30 aryl (C5-C20 aryl, and C5-C12 aryl), and C6-C30 aralkyl (C6-C20 aralkyl, and C6-C12 aralkyl). The hydrocarbyl moieties described above may be further substituted with one or more functional groups or additional hydrocarbyl moieties such as those specifically enumerated. Unless otherwise indicated, any of the groups described herein are intended to include substituted and / or heteroatom-containing moieties in addition to unsubstituted groups.

[0051] "Sulfonyl" refers to SO2-alkyl, SO2-substituted alkyl, SO2-alkenyl, SO2-substituted alkenyl, SO2-cycloalkyl, SO2-substituted cycloalkyl, SO2-cycloalkenyl, SO2-substituted cycloalkenyl, SO2-aryl, SO2-substituted aryl, SO2-heteroaryl, SO2-substituted heteroaryl, SO2-heterocyclic, and SO2-substituted heterocyclic groups, 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-SO2-, phenyl-SO2-, and 4-methylphenyl-SO2-.

[0052] The term "functional group" refers to a chemical group, such as, for example, halo, hydroxyl, sulfhydryl, C1-C24 alkoxy, C2-C24 alkenyloxy, C2-C24 alkynyloxy, C5-C20 aryloxy, acyl (such as 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 where X is halo), C2-C24 alkylcarbonato (-O-(CO)-O-alkyl), C6-C20 arylcarbonato (-O-(CO)-O-aryl), carboxy (-COOH), carboxylato (-COO-), carbamoyl (-(CO)-NH2), monosubstituted C1-C24 alkylcarbamoyl (-(CO)-NH(C1-C24 alkyl)), disubstituted alkylcarbamoyl (-(CO)-N(C1-C24 alkyl)2), monosubstituted arylcarbamoyl (-(CO)-NH-aryl), thiocarbamoyl (-(CS)-NH2), carbamide (-(CS)-NH2), carbazinyl ... 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 alkylamide (-NH-(CO)-alkyl), C5-C20 arylamide (-NH-(CO)-aryl), imino (- CR=NH, where R=hydrogen, C1-C24 alkyl, C5-C20 aryl, C6-C20 alkaryl, C6-C20 aralkyl, etc.), alkylimino (-CR=N(alkyl), where R=hydrogen, alkyl, aryl, alkaryl, etc.), arylimino (-CR=N(aryl), where R=hydrogen, alkyl, aryl, alkaryl, etc.), nitro (-NO2), nitroso (-NO), sulfo (-SO2-OH), sulfonato (-SO2-O-), C1-C24 alkylsulfanyl (-S-alkyl, also known as "alkylthio");Arylsulfanyl (-S-aryl, also known as "arylthio"), C1-C24, It means 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. Furthermore, the aforementioned functional groups may be further substituted with one or more additional functional groups, or with one or more hydrocarbyl moieties, such as those specifically listed above, if the particular group allows.

[0053] "Linkage" or "linker" as in "linking group", "linker moiety" and the like, refers to a linking moiety that connects two groups via a covalent bond. The linker can be linear, branched, cyclic, or a single atom. Examples of such linking groups include alkyl, alkenylene, alkynylene, arylene, alkarylene, aralkylene, and linking moieties that contain 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-), epimino (-NH-), carbonyl (-CO-), and the like. In certain cases, one, two, three, four, or five or more carbon atoms of the linker backbone may be optionally substituted with sulfur, nitrogen, or oxygen heteroatoms. The bonds between the backbone atoms may be saturated or unsaturated, but usually there are at most one, two, or three unsaturated bonds in the linker backbone. The linker may include one or more substituents with, for example, an alkyl, aryl, or alkenyl group. The linker may include a poly(ethylene glycol) unit(s) (e.g., -(CH2-CH2-O)-), an ether, a thioether, an amine, an alkyl that may be linear or branched (e.g., (C1-C 12 ) alkyl), such as, but not limited to, methyl, ethyl, n-propyl, 1-methylethyl (iso-propyl), n-butyl, n-pentyl, 1,1-dimethylethyl (t-butyl), and the like. The linker backbone may include a cyclic group, such as an aryl, heterocycle, or cycloalkyl group, and more than one atom of the cyclic group, such as 2, 3, or 4 atoms, is included in the backbone. Linkers may be cleavable or non-cleavable. Any convenient orientation and / or connection of the linker to the groups being linked may be used.

[0054] When the term "substituted" appears before a list of groups that may be substituted, it is intended that the term apply to every member of that group. For example, the phrase "substituted alkyl and aryl" is interpreted as "substituted alkyl and substituted aryl."

[0055] Further to the disclosure herein, when used to modify a particular group or radical, the term "substituted" can also mean that one or more hydrogen atoms of the particular group or radical are replaced, each independently of the other, with the same or different substituents defined below.

[0056] In addition to the groups disclosed for each individual term herein, one or more hydrogens on a saturated carbon atom of the specified group or radical (any two hydrogens on a single carbon can be ═O, ═NR 70 , =N-OR 70 , =N2 or =S) is substituted with -R, unless otherwise specified. 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 7 0 , -C(NR 70 )R 70 , -C(O)O - M + , -C(O)OR70 , -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 , -O-C(O)O - M + , -OC(O)OR 70 , -OC(S)OR 70 , -NR 70 C(O)R 70 , -NR 70 C(S)R 70 , -NR 70 CO2 - M + , -NR 70 CO2R 70 , -NR 70 C(S)OR 70 , -NR 70 C(O)NR 80 R 80 , -NR 70 C(NR 70 )R 70 and -NR 70 C(NR 70 )NR 80 R 80 where R 60 is selected from the group consisting of optionally substituted alkyl, cycloalkyl, heteroalkyl, heterocycloalkylalkyl, cycloalkylalkyl, aryl, arylalkyl, heteroaryl, and heteroarylalkyl; each R 70 are independently hydrogen or R 60 And each R 80 is independently R 70 or, alternatively, two R 80 together 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, wherein N may have -H or C1-C3 alkyl substitution; + is a counterion with a net single positive charge.+ are independently, for example, alkali ions, e.g., K + , Na + , Li + , ammonium ions, e.g. + N(R 60 )4, or alkaline earth ions, e.g., [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 the compounds of the invention and the other is a normal counterion, e.g., chloride, or a two ionized compound disclosed herein can be the counterion to such divalent alkaline earth ions, or a doubly ionized compound of the invention can be the 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, 4N-methyl-piperazin-1-yl, and N-morpholinyl.

[0057] Further to the disclosure herein, the substitution of hydrogen on an unsaturated carbon atom of a "substituted" alkene, alkyne, aryl, and heteroaryl group is, unless otherwise specified, -R 60 , halo, -O - M + , -OR 70 , -SR 70 , -S - M + , -NR 80 R 80 , trihalomethyl, -CF3, -CN, -OCN, -SCN, -NO, -NO2, -N3, -SO2R 70 , -SO3 - M + , -SO3R 70 , -OSO2R 70 , -OSO3 - M + , -OSO3R 70 , -PO3 -2 (M+ )2, -P(O)(OR 70 )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 and -NR 70 C(NR 70 )NR 80 R 80 where 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 + isn't it.

[0058] In addition to the groups disclosed for each individual term herein, the substituents of the hydrogen on the nitrogen atom of "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)O - 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(NR 70 )NR 80 R 80 , -OC(O)R 70 , -OC(S)R 70 , -OC(O)OR 70 , -OC(S)OR 70 , -NR 70 C(O)R 70 , -NR 70 C(S)R 70 , -NR 70 C(O)OR 70 , -NR 70 C(S)OR70 , -NR 70 C(O)NR 80 R 80 , -NR 70 C(NR 70 )R 70 and -NR 70 C(NR 70 )NR 80 R 80 where R 60 , R 70 , R 80 and M + is as already defined.

[0059] 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.

[0060] Unless otherwise stated, the naming of substituents not explicitly defined herein is obtained by naming the terminal portion of the functional group and then naming the adjacent functional group toward the point of attachment. For example, the substituent "arylalkyloxycarbonyl" refers to the group (aryl)-(alkyl)-OC(O)-.

[0061] 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 substitutions or patterns that are sterically impractical and / or synthetically infeasible. Moreover, the subject compounds include all stereochemical isomers arising from the substitution of these compounds.

[0062] In certain embodiments, the substituents may contribute to the optical isomerism and / or stereoisomerism of the compound. The salts, solvates, hydrates, and prodrug forms of the compound are also of interest. All such forms are included in the present disclosure. Thus, the compounds described herein include their salts, solvates, hydrates, prodrugs, and isomers, including their pharma- ceutically acceptable salts, solvates, hydrates, prodrugs, and isomers. In certain embodiments, the compounds may be metabolized to pharma- ceutically active derivatives.

[0063] Unless otherwise specified, a reference to an atom is intended to include isotopes of that atom. For example, a reference to H is 1 H, 2 H (i.e., D) and 3 is intended to include H (i.e., T) and any reference to C is 12 C and all isotopes of carbon ( 13 C, etc.)

[0064] Definitions of other terms and concepts appear throughout the detailed description.

[0065] Detailed Description As discussed above, embodiments of the present disclosure include compounds, compositions, and methods for inhibiting ENPP1. The method embodiments include contacting a sample with a cell-impermeable ENPP1 inhibitor to inhibit the cGAMP hydrolysis activity of ENPP1.

[0066] Also provided are compositions and methods for treating cancer. An embodiment of the method includes administering to a subject a therapeutically effective amount of an ENPP1 inhibitor to treat the cancer in the subject. An embodiment of the method includes administering to a subject a therapeutically effective amount of a cell-impermeable ENPP1 inhibitor to inhibit cGAMP hydrolysis and treat the cancer in the subject.

[0067] These compounds and methods find use in a variety of applications in which inhibition of ENPP1 is desirable.

[0068] ENPP1 inhibitors As mentioned above, embodiments of the present disclosure include ENPP1 inhibitor compounds. The subject compounds include a core structure based on an aryl or heteroaryl ring system, such as quinazoline, isoquinoline or can include a pyrimidine group, which is linked to a hydrophilic head group. The linker between the aryl or heteroaryl ring system and the hydrophilic head group can include a monocyclic carbocycle or heterocycle and an acyclic linker. In some cases, the linker includes a 1,4-disubstituted 6-membered ring, such as cyclohexyl, piperidinyl, or piperazinyl. The aryl or heteroaryl ring system is optionally further substituted. Exemplary ENPP1 inhibitor compounds of interest that include quinazoline, isoquinoline, or pyrimidine ring systems are shown in formulas I, IV, V, VI, and VII, and in the following structures 1-106.

[0069] Optionally, the subject ENPP1 inhibitor compound has formula (I): YALX (I) wherein: Y is selected from aryl, substituted aryl, heteroaryl, substituted heteroaryl, carbocycle, substituted carbocycle, heterocycle, and substituted heterocycle; A is selected from carbocycle, substituted carbocycle, heterocycle, and substituted heterocycle; L is a covalent bond or a linker; X is a hydrophilic head group; or a prodrug, pharma- ceutically acceptable salt or solvate thereof.

[0070] The term "hydrophilic head group" refers to a linked group of a subject compound that is hydrophilic, is well solvated in an aqueous environment, e.g., a physiological environment, and has low permeability through cell membranes. In some cases, low permeability through cell membranes refers to a permeability coefficient of 10 or less, as measured by any convenient method of passive diffusion of an isolated hydrophilic head group through a membrane (e.g., a cell monolayer such as a colorectal Caco-2 or renal MDCK cell line). -4 cm / s or less, e.g., 10 -5 cm / s or less, 10 -6 cm / s or less, 10 -7 cm / s or less, 10 -8 cm / s or less, 10 -9cm / s or less, or even lower. See, e.g., Yang and Hinner, Methods Mol Biol. 2015;1266:29-53.

[0071] The hydrophilic head group can improve the water solubility and reduce cell permeability of 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 instances, the hydrophilic group is a separate functional group (e.g., as described herein) or a substituted version thereof. Generally, larger, uncharged polar groups or charged groups have low permeability. In some instances, the hydrophilic head group has a charge, e.g., a positive or negative charge. In some embodiments, the hydrophilic head group confers cell impermeability to the subject compounds rather than cell permeability. It is understood that the hydrophilic head group, or a prodrug form thereof, can be selected to impart the desired cell permeability of the subject compounds. In certain instances, the hydrophilic head group is a neutral hydrophilic group. In some instances, the hydrophilic head group includes a promoiety. In certain instances, the subject compounds are cell permeable.

[0072] In some embodiments of formula (I), the hydrophilic head group (X) is selected from phosphonic acid or phosphonate, phosphonate ester, phosphate, phosphate ester, thiophosphate, thiophosphate ester, phosphoramidate, thiophosphoramidate, sulfonate, sulfonic acid, sulfate, hydroxamic acid, keto acid, amide, and carboxylic acid. In some embodiments of formula (I), the hydrophilic head group is phosphonic acid, phosphonate, or a salt thereof. In some embodiments of formula (I), the hydrophilic head group is phosphate or a salt thereof. In some embodiments of formula (I), the hydrophilic head group is a phosphonate ester or a phosphate ester.

[0073] Specific examples of hydrophilic head groups of interest include phosphate (RPO4H - ), phosphonates (RPO3H -), boric acid (RBO2H2), carboxylate (RCO2 - ), sulfate (RSO4 - ), sulfonates (RSO3 - ), amines (RNH3 + Examples of head groups include, but are not limited to, head groups comprising a first molecule selected from glycerol, sugars such as those derived from lactose or hyaluronic acid, polar amino acids, polyethylene oxides, and oligoethylene glycols, which are optionally conjugated to a residue of a second molecule selected from choline, ethanolamine, glycerol, nucleic acids, sugars, inositol, and serine. The head groups may include a variety of other modifications, for example, in the case of oligoethylene glycol and polyethylene oxide (PEG) containing head groups, such PEG chains may be methyl terminated or may have distal functional groups for further modification. Examples of hydrophilic head groups 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.

[0074] Any convenient linker can be used to link A to X. In some cases, A is linked to X via a covalent bond. In certain cases, A is linked to X via a linear linker that is 1-12 atoms in length, e.g., 1-10, 1-8, or 1-6 atoms in length, e.g., 1, 2, 3, 4, 5, or 6 atoms in length. The linker L is optionally substituted with heteroatoms or linking functional groups, e.g., esters (-CO2-), amides (CONH), carbamates (OCONH), ethers (-O-), thioethers (-S-), and / or amino groups (-NR-, where R is H or alkyl) (C 1-6 ) alkyl linker or substituted (C 1-6) alkyl linker.

[0075] In some instances of formula (I), L is selected from alkyl, substituted alkyl, alkyloxy, and substituted alkoxy, and X is selected from phosphonic acid, phosphonate, phosphate, thiophosphate, phosphoramidate, and thiophosphoramidate. In some embodiments of formula (I), LX comprises a group of formula (XI): [ka] During the ceremony, Z 12 is selected from O and S; Z 13 and Z 14 are each independently selected from O and NR′; Z 15 is selected from O and CH2; R 15 and R 16 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; R' is H, alkyl or substituted alkyl; q 1 is an integer from 0 to 6.

[0076] In some embodiments of formula (XI), Z 12 , Z 13 and Z 14 are all oxygen atoms, and Z 15 is CH2. In other cases, Z 12 is a sulfur atom, Z 13 and Z 14 are both oxygen atoms, and Z 15 is CH2. In other cases, Z 12 is a sulfur atom, Z 13 , Z 14 , Z 15 are all oxygen atoms. In some cases, Z 12is an oxygen atom, and Z 13 is NR', and Z 14 is an oxygen atom, and Z 15 is a carbon atom. In other cases, Z 12 is an oxygen atom, and Z 13 is a nitrogen atom, Z 14 and Z 15 are both oxygen atoms. In other cases, Z 12 is an oxygen atom, and Z 13 and Z 14 are each independently NR'; Z 15 is an oxygen atom. In still other cases, Z 12 is an oxygen atom, and Z 13 and Z 14 are each independently NR'; Z 15 is CH2. It is understood that groups of formula (XI) may include one or more tautomeric forms of the structure shown, and that all such forms and salts thereof are intended to be included.

[0077] In some embodiments of formula (XI), R 15 and R 16 are both hydrogen atoms. In other cases, R 15 and R 16 are both non-hydrogen substituents. 15 and R 16 are each independently an alkyl or substituted alkyl group. 15 and R 16 are each independently an aryl group. 15 and R 16 are each independently an alkyl group. 15 and R 16 are both alkyl groups substituted with an ester. 15 and R 16 are both alkyl groups substituted with an ester. In certain cases, R 15 and R 16 are both phenyl groups. In some cases, R 15 and R 16are each the same substituent. In other cases, R 15 and R 16 are different substituents.

[0078] In some embodiments of formula (XI), Z 15 is a carbon atom, and q 1 is 0. In other cases, Z 15 is a carbon atom, and q 1 is greater than 0, e.g., 1, 2, 3, 4, 5, or 6. In some cases, Z 15 is a carbon atom, and q 1 is 1. In another embodiment, Z 15 is an oxygen atom, and q 1 is 1. In other cases, Z 15 is an oxygen atom, and q 1 is greater than 1, e.g., 2, 3, 4, 5, or 6. In some cases, Z 15 is an oxygen atom, and q 1 is 2.

[0079] In some embodiments of Formula (XI), LX is selected from one of the following groups: [ka]

[0080] In some embodiments of Formula (I), LX comprises a group of formula (XII): [ka] During the ceremony, R 17 and R 18 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 17 and R 18together with the atom to which they are attached form a group selected from heterocycles and substituted heterocycles; q 2 is an integer from 1 to 6.

[0081] In some embodiments of Formula (XII), R 17 and R 18 are both hydrogen atoms. In other cases, R 17 and R 18 are both substituents other than hydrogen. In certain embodiments of formula (XII), q 2 is 1. In a particular case, q 2 is greater than 1, for example, 2, 3, 4, 5 or 6. In some cases of formula (XII), q 2 is 2. In certain embodiments of formula (XII), the hydrophilic head group is of the structure: [ka]

[0082] In some embodiments of Formula (I), LX comprises a group of formula (XIII): [ka] In the formula, q3 is an integer from 1 to 6. In certain embodiments, q 3 is 1. In certain embodiments, q 3 is greater than 1, e.g., 2, 3, 4, 5, or 6. In certain embodiments, q 3 is 2. In certain embodiments of Formula (XIII), the hydrophilic head group is of the structure: [ka] or [ka]

[0083] In some embodiments of Formula (I), LX comprises a group of formula (XIV): [ka] In the formula, Z 16 is selected from O and CH2; q 1 is an integer from 0 to 6 (e.g., 0 to 5). In some embodiments of Formula (XIV), Z 16 is CH2, and q 4 is 0. In other cases, Z 16 is CH2, and q 1 is greater than 0, e.g., 1, 2, 3, 4, 5, or 6. In some cases, Z 16 is CH2, and q 1 is 1. In another embodiment, Z 16 is an oxygen atom, and q 1 is 1. In other cases, Z 16 is an oxygen atom, and q 1 is greater than 1, e.g., 2, 3, 4, 5, or 6. In some cases, Z 16 is an oxygen atom, and q 1 is 2.

[0084] In some embodiments of Formula (XIV), the hydrophilic head group is selected from one of the following groups: [ka] or [ka]

[0085] In some embodiments of Formula (I), LX comprises a group of formula (XV): [ka] In the formula, q5 is an integer from 1 to 6. In certain embodiments, q 5is 1. In certain embodiments, q 5 is greater than 1, e.g., 2, 3, 4, 5, or 6. In certain embodiments, q 5 is 2. In certain embodiments of formula (XV), the hydrophilic head group is of the structure: [ka] or [ka]

[0086] In some embodiments of Formula (I), LX comprises a group of formula (XVI): [ka] During the ceremony, R 19 is 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; q 6 is an integer from 1 to 6.

[0087] In some embodiments of Formula (XVI), R 19 is hydrogen. In other cases, R 19 is a substituent other than hydrogen. In certain embodiments, R 19 is alkyl or substituted alkyl. In certain embodiments of formula (XVI), q 6 is 1. In a particular case, q 6 is greater than 1, for example, 2, 3, 4, 5 or 6. In some cases of formula (XVI), q 6 is 2. In certain embodiments of formula (XVI), LX is of the structure: [ka]

[0088] In some embodiments of Formula (I), LX is of formula (XVII): [ka] In the formula, q7 is an integer from 1 to 6. In certain embodiments, q 7 is 1. In certain embodiments, q 7 is greater than 1, e.g., 2, 3, 4, 5, or 6. In certain embodiments, q 7 is 2. In certain embodiments of formula (XVII), LX is of the structure: [ka]

[0089] In some embodiments of formula (I), A is a heterocycle or a substituted heterocycle. In some cases, A is a saturated heterocycle or a substituted saturated heterocycle. The heterocycle can be a 5-, 6-, or 7-membered monocyclic heterocycle. Heterocycles of interest include, but are not limited to, piperidine, piperazine, morpholine, tetrahydropyran, dioxane, imidazolidine, pyrazolidine, oxazolidine, isoxazolidine, and the like. In certain cases, the heterocycle is a 6-membered ring connected to Y and L in a 1,4-configuration. In certain cases, the heterocycle is a 5- or 6-membered ring connected to Y and L in a 1,3-configuration. In certain cases, the heterocycle is piperidine, substituted piperidine, piperazine, or substituted piperazine. When the linking atom of the ring is C, the heterocycle can include a chiral center. In some cases, A is selected from one of the following heterocyclic groups: [ka] or [ka]

[0090] In some embodiments of formula (I), A is a carbocycle. Optionally, A is a saturated carbocycle or a substituted saturated carbocycle. The carbocycle can be a 5-, 6-, or 7-membered monocyclic carbocycle, such as a cycloalkyl ring. Carbocycles of interest include, but are not limited to, cyclopentane, cyclohexane, cycloheptane, and the like. In certain cases, the carbocycle is a 6-membered ring connected to Y and L in a 1,4-configuration. In certain cases, the carbocycle is a 5- or 6-membered ring connected to Y and L in a 1,3-configuration. In certain cases, the carbocycle is cyclohexane or a substituted cyclohexane. The cyclohexane can include a chiral center. Optionally, A is of the following structure: [ka]

[0091] In certain other cases, A is an aromatic carbocycle, i.e., aryl. The aryl ring can be monocyclic. In some cases, A is phenylene or substituted phenylene. In some cases, A is 1,4-phenylene of the following structure: [ka]

[0092] In certain other cases, A is an aromatic heterocycle, i.e., heteroaryl or substituted heteroaryl. The heteroaryl ring may be monocyclic. Heteroaryls of interest include, but are not limited to, pyridine, pyridazine, pyrimidine, and pyrazine.

[0093] In some embodiments of formula (I), L is -(CH2)n-. In certain cases, n is 1-8, e.g., 1-5. In some cases, n is 1-3, e.g., 2 or 3. In some cases, n is less than 8, e.g., 7, 6, 5, 4, 3, 2 or 1. In some cases, n is 1-6, e.g., 1-4 or 1-3. In some cases, n is 1. In some other cases, n is 2. In some cases, L is an ethylene or substituted ethylene group. In some other cases, L is a methylene or substituted methylene group. In certain other cases, L is a covalent bond.

[0094] In some embodiments of formula (I), Y is selected from quinazoline, substituted quinazoline, quinoline, substituted quinoline, naphthalene, substituted naphthalene, isoquinoline, and substituted isoquinoline. In certain examples, Y is selected from quinazoline and substituted quinazoline. In certain examples, Y is selected from quinoline and substituted quinoline. In certain examples, Y is selected from naphthalene and substituted naphthalene. In certain examples, Y is selected from isoquinoline and substituted isoquinoline. In some embodiments of formula (I), Y is a group of formula (II): [ka] During the ceremony, Z 1 and Z 2 are each independently 1 and N, Each R 1 is independently selected from H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, heterocycle, and substituted heterocycle; R 2 and R 5 are each independently selected from H, OH, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, -OCF3, amine, substituted amine, amide, heterocycle, and substituted heterocycle; R 3 and R 4are each independently selected from H, OH, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, -OCF3, amine, substituted amine, amide, heterocycle, and substituted heterocycle; or R 3 and R 4 together with the carbon atom to which they are attached form a fused ring selected from heterocycle, substituted heterocycle, cycloalkyl, substituted cycloalkyl, aryl, and substituted aryl.

[0095] In certain embodiments of formula (II), Z 1 and Z 2 At least one of Z is N. In certain embodiments of formula (II), Z 1 is C and Z 2 is N. In certain cases of formula (II), Z 1 is N and Z 2 is C. In certain examples of formula (IIa), Z 1 is C and Z 2 is C. In certain cases of formula (II), Z 1 is N and Z 2 is N. In some cases of formula (II), R 1 and R 4 is not hydrogen. In some cases of formula (II), R 1 , R 3 and R 4 is not hydrogen. In some cases of formula (II), R 1 , R 3 , R 4 and R 5 is not hydrogen.

[0096] In some cases of formula (II), R 1 is hydrogen, C 1-5 In certain instances of formula (IIa), R is selected from the group consisting of alkyl, vinyl heterocycle (e.g., -CH=CH-heterocycle). In certain instances, the -vinyl heterocycle is vinylpyridine (e.g., -CH=CH-pyridine). 1 is hydrogen. In some cases, R 1 is C 1-5 In other cases, R 1is a vinyl heterocycle. In certain cases, R 1 is vinylpyridine. In some cases, R 2 and R 5 are both hydrogen. In some cases, R 5 is C 1-5 Selected from alkyl, amine, triazole, imidazole, amide, alkoxy, OCF3, and hydroxy. In certain cases, R 5 is alkoxy, e.g., methoxy. In some cases, R 3 and R 4 are each independently hydrogen, C 1-5 alkyl, triazole, imidazole, amine, amide, alkoxy, OCF3, hydroxy, or R 3 and R 4 taken together with the carbons to which they are attached form a heterocycle. 3 and R 4 is alkoxy, for example, in some cases, R 3 and R 4 are both methoxy. In some cases, R 5 is methoxy and R 1 ~R 4 Each of R is hydrogen. 5 is methoxy and R 1 is a -CH=CH-heterocycle, and R 2 ~R 4 Each of is hydrogen.

[0097] In some embodiments of Formula (II), Y is a group of formula (IIA): [ka] During the ceremony, R 7 is selected from the group consisting of H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, heterocycle, and substituted heterocycle; R 8is selected from the group consisting of OH, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, -OCF3, amine, substituted amine, amide, heterocycle, and substituted heterocycle.

[0098] In some cases of formula (IIA), R 7 is hydrogen, C 1-5 Alkyl, substituted C 1-5 In some cases of formula (IIA), R is selected from alkyl, vinyl-heterocycle and substituted vinyl-heterocycle. 7 is hydrogen, C 1-5 In certain instances of formula (IIA), R is selected from the group consisting of alkyl, vinyl heterocycle (e.g., -CH=CH-heterocycle). In certain instances, the -vinyl heterocycle is vinylpyridine (e.g., -CH=CH-pyridine). 7 is hydrogen. In some cases, R 7 is C 1-5 In other cases, R 7 is a vinyl heterocycle. In certain cases, R 7 is vinylpyridine. In some cases, R 8 is hydrogen, C 1-5 Optionally, R is selected from alkyl, triazole, imidazole, amine, amide, alkoxy, OCF3, and hydroxyl. 8 is alkoxy, e.g., methoxy. 8 is methoxy and R 7 is hydrogen. In some cases, R 8 is methoxy and R 7 is -CH=CH-heterocycle. In some embodiments of Formula (II), Y is a group of formula (IIB): [ka] During the ceremony, R 7 is selected from the group consisting of H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, heterocycle, and substituted heterocycle; R 8 and R 9are each independently selected from the group consisting of OH, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, -OCF3, amine, substituted amine, amide, heterocycle, and substituted heterocycle; or R 8 and R 9 together with the carbon atom to which they are attached form a fused ring selected from heterocycle, substituted heterocycle, cycloalkyl, substituted cycloalkyl, aryl, and substituted aryl.

[0099] In some cases of formula (IIB), R 7 is hydrogen, C 1-5 In certain instances of formula (IIB), R is selected from the group consisting of alkyl, vinyl heterocycle (e.g., -CH=CH-heterocycle). In certain instances, the -vinyl heterocycle is vinylpyridine (e.g., -CH=CH-pyridine). 7 is hydrogen. In some cases, R 7 is C 1-5 In other cases, R 7 is a vinyl heterocycle. In certain cases, R 7 is vinylpyridine. In some cases, R 8 and R 9 are each independently hydrogen, C 1-5 Alkyl, triazo or R is selected from the group consisting of aryl, imidazole, amine, amide, alkoxy, OCF3 and hydroxy; 8 and R 9 together with the carbon atoms to which they are attached form a fused heterocycle. 8 and R 9 is alkoxy, for example, in some cases, R 8 and R 9 are both methoxy. In some embodiments of Formula (II), Y is a group of Formula (IIC): [ka] [ka] During the ceremony, R 7 is selected from the group consisting of H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, heterocycle, and substituted heterocycle; R 10 is selected from the group consisting of OH, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, -OCF3, amine, substituted amine, amide, heterocycle, and substituted heterocycle; R 8 and R 9 are each independently selected from the group consisting of OH, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, -OCF3, amine, substituted amine, amide, heterocycle, and substituted heterocycle; or R 8 and R 9 together with the carbon atom to which they are attached form a fused ring selected from heterocycle, substituted heterocycle, cycloalkyl, substituted cycloalkyl, aryl, and substituted aryl.

[0100] In some cases of formula (IIC), R 7 is hydrogen, C 1-5 In certain instances of formula (IIC), R is selected from the group consisting of alkyl, vinyl heterocycle (e.g., -CH=CH-heterocycle). In certain instances, the -vinyl heterocycle is vinylpyridine (e.g., -CH=CH-pyridine). 7 is hydrogen. In some cases, R 7 is C 1-5 In some cases, R 7 is a vinyl heterocycle. In certain cases, R 7 is vinylpyridine. In some cases, R 10 is hydrogen, C 1-5 Optionally, R is selected from alkyl, amine, triazole, imidazole, amide, alkoxy, OCF3, and hydroxy. 10 is hydrogen. In certain cases, R 10 is alkoxy, e.g., methoxy. In some cases, R 8 and R 9are each independently hydrogen, C 1-5 alkyl, triazole, imidazole, amine, amide, alkoxy, OCF3, hydroxy, or R 8 and R 9 together with the carbon atoms to which they are attached form a fused heterocycle. 8 and R 9 is alkoxy, for example, in some cases, R 8 and R 9 are both methoxy. In some cases, R 10 is methoxy and R 7 ~R 9 Each of R is hydrogen. 10 is methoxy and R 7 is a -CH=CH-heterocycle, and R 8 and R 9 is hydrogen. In some embodiments of Formula (II), Y is a group of Formula (IID): [ka] During the ceremony, R 7 is selected from the group consisting of H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, heterocycle, and substituted heterocycle; R 11 and R 12 are each independently selected from the group consisting of H, OH, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, -OCF3, amine, substituted amine, amide, heterocycle, and substituted heterocycle; or R 11 and R 12 together with the carbon atom to which they are attached form a fused ring selected from heterocycle, substituted heterocycle, cycloalkyl, substituted cycloalkyl, aryl, and substituted aryl.

[0101] In the case of formula (IID), R 7 is hydrogen, C 1-5In certain instances of formula (IID), R is selected from the group consisting of alkyl, vinyl heterocycle (e.g., -CH=CH-heterocycle). In certain instances, the -vinyl heterocycle is vinylpyridine (e.g., -CH=CH-pyridine). 7 is hydrogen. In some cases, R 7 is C 1-5 In some cases, R 7 is a vinyl heterocycle. In certain cases, R 7 is vinylpyridine. In some cases, R 11 and R 12 are each independently hydrogen, C 1-5 or R is selected from alkyl, triazole, imidazole, amine, amide, alkoxy, OCF3, and hydroxy; 11 and R 12 together with the carbon atoms to which they are attached form a fused heterocycle. 11 and R 12 is alkoxy, for example, in some cases, R 11 and R 12 are both methoxy.

[0102] In some embodiments of Formula (II), Y is a group of formula (IIE): [ka] During the ceremony, R 7 is selected from the group consisting of H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, heterocycle, and substituted heterocycle; R 11 and R 12 are each independently selected from the group consisting of H, OH, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, -OCF3, amine, substituted amine, amide, heterocycle, and substituted heterocycle; or R 11 and R 12together with the carbon atom to which they are attached form a fused ring selected from heterocycle, substituted heterocycle, cycloalkyl, substituted cycloalkyl, aryl, and substituted aryl.

[0103] In some cases of formula (IIE), R 7 is hydrogen, C 1-5 In one particular example, the -vinyl heterocycle is vinylpyridine (e.g., -CH=CH-pyridine). In one particular example of formula (IIE), In this case, R 7 is hydrogen. In some cases, R 7 is C 1-5 In other cases, R 7 is a vinyl heterocycle. In certain cases, R 7 is vinylpyridine. In some cases, R 11 and R 12 are each independently hydrogen, C 1-5 or R is selected from alkyl, triazole, imidazole, amine, amide, alkoxy, OCF3, and hydroxy; 11 and R 12 taken together with the carbons to which they are attached form a heterocycle. 11 and R 12 is alkoxy, for example, in some cases, R 11 and R 12 are both methoxy.

[0104] In some embodiments of Formula (II), Y is a group selected from the following: [ka] [ka] [ka] [ka] and [ka]

[0105] In some embodiments of Formula (II), R 1 ~R 5 Any of R can be halogen, e.g., F, Cl, Br, or I. In some embodiments of Formula (II), R 1 ~R 5 At least one of R is a halogen atom. 1 ~R 5 At least one of R is fluoride. In another embodiment of formula (II), R 1 ~R 5 In another embodiment of formula (II), at least one of R 1 ~R 5 In yet another embodiment of formula (II), at least one of R 1 ~R 5 At least one of the is iodide.

[0106] In some embodiments of Formula (II), Y is a group selected from the following: [ka] and [ka]

[0107] In some embodiments of Formula (I), Y is a group of formula (XI): [ka] During the ceremony, Z 21 CR 1 and N, R 1 , R21 and R 22 is independently selected from H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, heterocycle, and substituted heterocycle; R 2 and R 5 is independently selected from H, OH, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, -OCF3, amine, substituted amine, amide, heterocycle, and substituted heterocycle; R 3 and R 4 is independently selected from H, OH, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, -OCF3, amine, substituted amine, amide, heterocycle, and substituted heterocycle; or R 3 and R 4 taken together with the carbons to which they are attached form a fused ring selected from heterocycle, substituted heterocycle, cycloalkyl, substituted cycloalkyl, aryl, and substituted aryl.

[0108] In some cases of formula (XI), R 1 and R 4 is not hydrogen. In some cases of formula (XI), R 1 , R 3 and R 4 is not hydrogen. In some cases of formula (XI), R 1 , R 3 , R 4 and R 5 is not hydrogen.

[0109] In some cases of formula (XI), Z 21 is CR 1 and R 1 is hydrogen, C 1-5 In certain instances of formula (XI), Z is selected from the group consisting of alkyl, vinyl heterocycle (e.g., -CH=CH-heterocycle). In certain instances, the -vinyl heterocycle is vinylpyridine (e.g., -CH=CH-pyridine). 21 is CR 1 and R 1 is hydrogen. In some cases, R 1 is C1-5 In other cases, Z is an alkyl group. 21 is CR 1 and R 1 is a vinyl heterocycle. In certain cases, R 1 is vinylpyridine. In some cases, R 2 and R 5 are both hydrogen. In some cases, R 5 is C 1-5 Alkyl, amine In certain cases, R is selected from the group consisting of aryl, triazole, imidazole, amide, alkoxy, OCF3, and hydroxy. 5 is alkoxy, e.g., methoxy. In some cases, R 3 and R 4 are each independently hydrogen, C 1-5 alkyl, triazole, imidazole, amine, amide, alkoxy, OCF3, hydroxy, or R 3 and R 4 taken together with the carbons to which they are attached form a heterocycle. 3 and R 4 is alkoxy, for example, in some cases, R 3 and R 4 are both methoxy. In some cases, R 5 is methoxy and R 1 ~R 4 Each of R is hydrogen. 5 is methoxy and R 1 is a -CH=CH-heterocycle, and R 2 ~R 4 Each of is hydrogen.

[0110] In some embodiments of Formula (I), Y is a group of formula (III): [ka] [ka] During the ceremony, Z 1 and Z 2 are each independently 1 and N, Each R 1 is independently selected from the group consisting of H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, heterocycle, and substituted heterocycle; R 6 is selected from the group consisting of heterocycle, substituted heterocycle, cycloalkyl, substituted cycloalkyl, aryl, and substituted aryl. In certain embodiments of formula (III), Z 1 and Z 2 At least one of Z is N. In certain embodiments of formula (III), Z 1 is CH and Z 2 is N. In certain cases of formula (III), Z 1 is N and Z 2 is CH. In certain examples of formula (III), Z 1 is CH and Z 2 is CH. In certain cases of formula (III), Z 1 is N and Z 2 is N.

[0111] In some embodiments of Formula (III), Y is a group of formula (IIIA): [ka] During the ceremony, Z 5 , Z 6 , Z 7 and Z 8 are each independently 14 and N, R 13 is selected from the group consisting of H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, heterocycle, and substituted heterocycle; Each R 14is independently selected from the group consisting of H, OH, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, -OCF3, amine, substituted amine, amide, heterocycle, and substituted heterocycle; m is 0 to 5.

[0112] In some cases of formula (IIIA), Z 5 , Z 6 , Z 7 and Z 8 and only one of Z is N. In some cases of formula (IIIA), 5 , Z 6 , Z 7 and Z 8 Only two of the groups are N. In some cases of formula (IIIA), Z 5 is N. In some cases of formula (IIIA), Z 6 is N. In some cases of formula (IIIA), Z 7 is N. In some cases of formula (IIIA), Z 8 is N. In some cases of formula (IIIA), Z 5 and Z 7 is N. In some cases of formula (IIIA), Z 7 and Z 8 are each N.

[0113] In some embodiments of Formula (III), Y is a group of formula (IIIB): [ka] During the ceremony, Z 9 , Z 10 and Z 11 are each independently 14 and N, R 13 is selected from the group consisting of H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, heterocycle, and substituted heterocycle; Each R 14is independently selected from the group consisting of H, OH, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, -OCF3, amine, substituted amine, amide, heterocycle, and substituted heterocycle; p is 0 to 4.

[0114] In some cases of formula (IIIB), Z 9 , Z 10 and Z 11 and only one of Z is N. In some cases of formula (IIIB), 9 , Z 10 and Z 11 Only two of the groups are N. In some cases of formula (IIIB), Z 9 is N. In some cases of formula (IIIA), Z 10 is N. In some cases of formula (IIIB), Z 11 is N. In some cases of formula (IIIB), R 14 is selected from alkyl and substituted alkyl. In some instances of formula (IIIB), p is 0. In some instances of formula (IIIB), p is 1. In some instances of formula (IIIB), p is 2.

[0115] In some embodiments of Formula (III), Y is a group selected from the following: [ka] and [ka] , or their replacement types.

[0116] In some embodiments of Formula (I), Y is a group of formula (IIIC): [ka] [ka] During the ceremony, Z 1, Z 2 , Z 17 , Z 18 and Z 19 are each independently 20 and N, Each R 20 is independently selected from the group consisting of H, OH, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, -OCF3, amine, substituted amine, amide, heterocycle, and substituted heterocycle; p 1 is an integer from 0 to 4. In some cases of formula (IIIC), Z 1 , Z 2 , Z 17 and Z 19 are N and Z 18 is CR 20 In some embodiments of formula (IIIC), Y is of the following structure: [ka]

[0117] In some embodiments of formula (I), the structure is represented by formula (IV): [ka] wherein Z 1 and Z 2 are each independently 1 and N, Z 3 and Z 4 are each independently selected from CR and N, where R is H, alkyl or substituted alkyl; R 1 is selected from the group consisting of H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, heterocycle, and substituted heterocycle; R 2 and R 5are each independently selected from the group consisting of H, OH, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, -OCF3, amine, substituted amine, amide, heterocycle, and substituted heterocycle; R 3 and R 4 are each independently selected from the group consisting of H, OH, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, -OCF3, amine, substituted amine, amide, heterocycle, and substituted heterocycle; Or R 3 and R 4 together with the carbon to which they are attached form a group selected from heterocycle, substituted heterocycle, cycloalkyl, substituted cycloalkyl, aryl and substituted aryl; or a prodrug, pharma- ceutically acceptable salt or solvate thereof.

[0118] In certain embodiments of formula (IV), Z 1 and Z 2 At least one of Z is N. In certain embodiments of formula (IV), Z 1 is C and Z 2 is N. In certain cases of formula (IV), Z 1 is N and Z 2 is C. In certain examples of formula (IV), Z 1 is C and Z 2 is C. In certain cases of formula (IV), Z 1 is N and Z 2 is N. In certain embodiments of formula (IV), Z 3 and Z 4 At least one of Z is N. In certain cases of formula (IV), 3 is N and Z 4 is N. In certain cases of formula (IV), Z 3 is N and Z 4 is CH. In certain cases of formula (IV), Z 3 is CH and Z 4 is N. In certain cases of formula (VI), Z3 is CH and Z 4 is CH.

[0119] In some cases of formula (IV), R 1 is hydrogen, C 1-5 In certain instances of formula (IV), R is selected from the group consisting of alkyl, vinyl heterocycle (e.g., -CH=CH-heterocycle). In certain instances, the -vinyl heterocycle is vinylpyridine (e.g., -CH=CH-pyridine). 1 is hydrogen. In some cases, R 1 is C 1-5 In other cases, R 1 is a vinyl heterocycle. In certain cases, R 1 is vinylpyridine. In some cases, R 2 and R 5 are both hydrogen. In some cases, R 5 is C 1-5 Optionally, R is selected from alkyl, amine, triazole, imidazole, amide, alkoxy, OCF3, and hydroxy. 5 is an alkoxy, e.g., methoxy; Yes. In some cases, R 3 and R 4 are each independently hydrogen, C 1-5 alkyl, triazole, imidazole, amine, amide, alkoxy, OCF3, hydroxy, or R 3 and R 4 taken together with the carbons to which they are attached form a heterocycle. 3 and R 4 is alkoxy, for example, in some cases, R 3 and R 4 are both methoxy. In some cases, R 5 is methoxy and R 1 ~R 4 Each of R is hydrogen. 5 is methoxy and R 1 is a -CH=CH-heterocycle, and R 2 ~R 4Each of is hydrogen.

[0120] In some embodiments of formula (I), the structure is of formula (V): [ka] wherein Z 1 and Z 2 are each independently 1 and N, Z 3 and Z 4 are each independently selected from CR and N, where R is H, alkyl or substituted alkyl; Each R 1 is independently selected from H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, heterocycle, and substituted heterocycle; R 6 is selected from heterocycle, substituted heterocycle, cycloalkyl, substituted cycloalkyl, aryl, and substituted aryl; or a prodrug, pharma- ceutically acceptable salt or solvate thereof.

[0121] In certain embodiments of formula (V), Z 1 and Z 2 At least one of Z is N. In certain embodiments of formula (V), Z 1 is CH and Z 2 is N. In certain cases of formula (IV), Z 1 is N and Z 2 is CH. In certain examples of formula (V), Z 1 is CH and Z 2 is CH. In certain cases of formula (IV), Z 1 is N and Z 2 is N. In certain embodiments of formula (V), Z 3 and Z 4 At least one of Z is N. In certain cases of formula (V), 3 is N and Z 4is N. In certain cases of formula (V), Z 3 is N and Z 4 is CH. In certain cases of formula (V), Z 3 is CH and Z 4 is N. In certain cases of formula (V), Z 3 is CH and Z 4 is CH.

[0122] In some embodiments of Formula (I), the inhibitor has the formula (VI): [ka] wherein X is a hydrophilic head group selected from phosphonic acid, phosphonate, phosphonate ester, phosphate, phosphate ester, thiophosphate, thiophosphate ester, phosphoramidate, and thiophosphoramidate; L is a linker, Z 1 and Z 2 are each independently 1 and N, Z 3 and Z 4 are each independently selected from CR and N, where R is H, alkyl or substituted alkyl; Each R 1 is independently selected from H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, heterocycle, and substituted heterocycle; R 2 and R 5 are each independently selected from H, OH, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, -OCF3, halogen, amine, substituted amine, amide, heterocycle, and substituted heterocycle; R 3 and R 4are each independently selected from H, OH, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, -OCF3, halogen, amine, substituted amine, amide, heterocycle, and substituted heterocycle; Or R 3 and R 4 taken together with the carbon atom to which they are attached form a fused ring selected from a heterocycle, a substituted heterocycle, a cycloalkyl, a substituted cycloalkyl, an aryl, and a substituted aryl; or a prodrug, pharma- ceutically acceptable salt or solvate thereof.

[0123] In some embodiments of formula (I), the structure is represented by formula (VI): [ka] wherein L is selected from the group consisting of -CH2-, -(CH2)2-, -(CH2)3-, -(CH2)4-, -(CH2)5- and -(CH2)6-; X is [ka] [ka] [ka] and [ka] wherein R a and R b each independently represents an aryl, an alkyl, or -CHOC(O)R e , -CH2OC(O)OR e Selected from R c and R d are each independently selected from -C(CH3)C(O)ORe, alkyl; R eis alkyl, Z 1 , Z 2 , Z 3 and Z 4 are each independently 1 and N, R 1 is selected from the group consisting of H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, heterocycle, and substituted heterocycle; R 2 and R 5 are each independently selected from the group consisting of H, OH, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, -OCF3, amine, substituted amine, amide, heterocycle, and substituted heterocycle; R 3 and R 4 are each independently selected from the group consisting of H, OH, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, -OCF3, amine, substituted amine, amide, heterocycle, and substituted heterocycle; Or R 3 and R 4 together with the carbon to which they are attached form a group selected from heterocycle, substituted heterocycle, cycloalkyl, substituted cycloalkyl, aryl and substituted aryl; or a prodrug, pharma- ceutically acceptable salt or solvate thereof.

[0124] In certain embodiments of formula (VI), Z 1 and Z 2 At least one of Z is N. In certain embodiments of formula (VI), Z 1 is C and Z 2 is N. In certain cases of formula (VI), Z 1 is N and Z 2 is C. In certain examples of formula (VI), Z 1 is C and Z 2 is C. In certain cases of formula (VI), Z 1 is N Yes, Z 2is N. In certain embodiments of formula (VI), Z 3 and Z 4 At least one of Z is N. In certain cases of formula (VI), 3 is N and Z 4 is N. In certain cases of formula (IV), Z 3 is N and Z 4 is C. In certain cases of formula (VI), Z 3 is C and Z 4 is N. In certain cases of formula (VI), Z 3 is C and Z 4 is C.

[0125] In some cases of formula (VI), R 1 is hydrogen, C 1-5 In certain instances of formula (VI), R is selected from the group consisting of alkyl, vinyl heterocycle (e.g., -CH=CH-heterocycle). In certain instances, the -vinyl heterocycle is vinylpyridine (e.g., -CH=CH-pyridine). 1 is hydrogen. In some cases, R 1 is C 1-5 In other cases, R 1 is a vinyl heterocycle. In certain cases, R 1 is vinylpyridine. In some cases, R 2 and R 5 are both hydrogen. In some cases, R 5 is C 1-5 Selected from alkyl, amine, triazole, imidazole, amide, alkoxy, OCF3, and hydroxy. In certain cases, R 5 is alkoxy, e.g., methoxy. In some cases, R 3 and R 4 are each independently hydrogen, C 1-5 alkyl, triazole, imidazole, amine, amide, alkoxy, OCF3, hydroxy, or R 3 and R 4 taken together with the carbons to which they are attached form a heterocycle. 3 and R4 is alkoxy, for example, in some cases, R 3 and R 4 are both methoxy. In some cases, R 5 is methoxy and R 1 ~R 4 Each of R is hydrogen. 5 is methoxy and R 1 is a -CH=CH-heterocycle, and R 2 ~R 4 Each of is hydrogen.

[0126] In certain embodiments of formula (VI), L is -CH2-. In certain other instances of formula (VI), L is -(CH2)2-.

[0127] In certain embodiments of Formula (VI), X is [ka] In certain cases of formula (VI), X is [ka] In certain other instances of formula (VI), X is [ka] In certain cases of formula (VI), X is [ka] In certain other instances of formula (VI), X is [ka] In certain embodiments of formula (VI), X is [ka] In certain cases of formula (VI), X is [ka] In certain other instances of formula (VI), X is [ka] In certain other instances of formula (VI), X is [ka] In certain other instances of formula (VI), X is [ka] In certain cases of formula (VI), X is [ka] In certain other instances of formula (VI), X is [ka] In certain other instances of formula (VI), X is [ka] where R a and R b each independently represents an aryl, an alkyl, or -CHOC(O)R e , -CH2OC(O)OR e where R e is alkyl. In certain cases of formula (VI), X is [ka] where R c and R d are each independently selected from -C(CH3)C(O)Ore and alkyl, where R e is alkyl. In certain other instances of formula (VI), X is [ka] where R a is aryl, alkyl, -CH2OC(O)R e , -CH2OC(O)OR e Selected from R c is selected from -C(CH3)C(O)Ore and alkyl, where R e is alkyl.

[0128] It will be appreciated that any of the hydroxyl and amine groups in group X of formula (VI) may optionally be further substituted with any convenient group such as alkyl groups, substituted alkyl groups, phenyl groups, substituted phenyl groups, ester groups, etc. It will be appreciated that any convenient alternative hydrophilic group may be utilized as group X in compounds of formula (VI).

[0129] In some embodiments of formula (I), the structure has formula (VII): [ka] wherein L is selected from the group consisting of -CH2-, -(CH2)2-, -(CH2)3-, -(CH2)4-, -(CH2)5- and -(CH2)6-; X is [ka] [ka] [ka] and [ka] wherein R a and R b each independently represents an aryl, an alkyl, or -CHOC(O)R e , -CH2OC(O)OR e Selected from Rc and R d are each independently selected from -C(CH3)C(O)ORe, alkyl, where R e is alkyl, Z 1 and Z 2 are each independently selected from C and N; R 1 is selected from the group consisting of H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, heterocycle, and substituted heterocycle; R 2 and R 5 are each independently selected from the group consisting of H, OH, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, -OCF3, amine, substituted amine, amide, heterocycle, and substituted heterocycle; R 3 and R 4 are each independently selected from the group consisting of H, OH, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, -OCF3, amine, substituted amine, amide, heterocycle, and substituted heterocycle; Or R 3 and R 4 together with the carbon to which they are attached form a group selected from heterocycle, substituted heterocycle, cycloalkyl, substituted cycloalkyl, aryl and substituted aryl; or a prodrug, pharma- ceutically acceptable salt or solvate thereof.

[0130] In certain embodiments of formula (VII), Z 1 and Z 2 At least one of Z is N. In certain embodiments of formula (VII), Z 1 is C and Z 2 is N. In certain cases of formula (VII), Z 1 is N and Z 2 is C. In certain examples of formula (VII), Z 1 is C and Z 2 is C. In certain cases of formula (VII), Z 1is N and Z 2 is N.

[0131] In some cases of formula (VII), R 1 is hydrogen, C 1-5 In certain instances, the -vinyl heterocycle is selected from the group consisting of alkyl, vinyl heterocycle (e.g., -CH=CH-heterocycle). The ring is a vinylpyridine (e.g., -CH=CH-pyridine). In some cases of formula (VII), R 1 is hydrogen. In some cases, R 1 is C 1-5 In other cases, R 1 is a vinyl heterocycle. In certain cases, R 1 is vinylpyridine. In some cases, R 2 and R 5 are both hydrogen. In some cases, R 5 is C 1-5 Selected from alkyl, amine, triazole, imidazole, amide, alkoxy, OCF3, and hydroxy. In certain cases, R 5 is alkoxy, e.g., methoxy. In some cases, R 3 and R 4 are each independently hydrogen, C 1-5 alkyl, triazole, imidazole, amine, amide, alkoxy, OCF3, hydroxy, or R 3 and R 4 taken together with the carbons to which they are attached form a heterocycle. 3 and R 4 is alkoxy, for example, in some cases, R 3 and R 4 are both methoxy. In some cases, R 5 is methoxy and R 1 ~R 4 Each of R is hydrogen. 5 is methoxy and R 1 is a -CH=CH-heterocycle, and R 2 ~R 4 Each of is hydrogen.

[0132] In certain embodiments of formula (VII), L is -CH2-. In certain other instances of formula (VII), L is -(CH2)2-.

[0133] In certain embodiments of formula (VII), X is [ka] In certain cases of formula (VII), X is [ka] In certain other cases of formula (VII), X is [ka] In certain cases of formula (VII), X is [ka] In certain other cases of formula (VII), X is [ka] In certain embodiments of formula (VII), X is [ka] In certain cases of formula (VII), X is [ka] In certain other cases of formula (VII), X is [ka] In certain cases of formula (VII), X is [ka] In certain other cases of formula (VII), X is [ka] In certain cases of formula (VII), X is [ka] In certain other cases of formula (VII), X is [ka] In certain other instances of formula (VI), X is [ka] where R a and R b each independently represents an aryl, an alkyl, or -CHOC(O)R e , -CH2OC(O)OR e where R e is alkyl. In certain other instances of formula (VI), X is [ka] where R c and R d are each independently selected from -C(CH3)C(O)Ore and alkyl, where R e is alkyl. In certain other instances of formula (VI), X is [ka] and R a is aryl, alkyl, -CH2OC(O)R e , -CH2OC(O)OR e Selected from R c is selected from -C(CH3)C(O)Ore and alkyl, where R e is alkyl.

[0134] It will be appreciated that any of the hydroxyl and amine groups in group X of formula (VII) may optionally be further substituted with any convenient group such as alkyl groups, substituted alkyl groups, phenyl groups, substituted phenyl groups, ester groups, etc. It will be appreciated that any convenient alternative hydrophilic group may be utilized as group X in compounds of formula (VII).

[0135] In certain embodiments, the compound is represented by the structure of one of the compounds in Table 1 or Table 2. Table 1: Compounds [Table 1-1] Table 1 continued [Table 1-2] Table 1 continued [Table 1-3] Table 1 continued [Table 1-4] Table 1 continued [Table 1-5] Table 1 continued [Table 1-6] Table 1 continued [Table 1-7] Table 2: Compounds [Table 2-1] Table 2 continued [Table 2-2]

[0136] In certain embodiments, the compound is represented by the structure of one of the compounds in Table 1 or Table 2. It is understood that any of the compounds shown in Table 1 or Table 2 can exist in salt form. In some cases, the salt form of the compound is a pharma- ceutically acceptable salt. It is understood that any of the compounds shown in Table 1 or Table 2 can exist in prodrug form.

[0137] Aspects of the present disclosure include ENPP1 inhibitor compounds (e.g., those described herein), their salts (e.g., pharma- ceutically acceptable salts), and / or solvates, hydrates, and / or prodrug forms thereof. Additionally, for any compounds described herein having one or more chiral centers, unless the absolute stereochemistry is explicitly shown, it is understood that each center may be independently in the R or S configuration, or a mixture thereof. It is understood that all permutations of salts, solvates, hydrates, prodrugs, and stereoisomers are intended to be included in the present disclosure.

[0138] In some embodiments, the subject ENPP1 inhibitor compound, or its prodrug form, is provided in the form of a pharmaceutically acceptable salt.Compounds that contain amine or nitrogen-containing heteroaryl groups may be basic in nature, and accordingly can react with any number of inorganic and organic acids to form pharmaceutically acceptable acid addition salts.The acids that are 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. Such pharma- ceutically acceptable salts thus include, but are not limited to, sulfate, pyrosulfate, bisulfate, 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, chlorate, chloram ... Included are salts such as benzoate, 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, etc. In certain specific embodiments, pharma- ceutically acceptable acid addition salts include those formed with mineral acids, such as hydrochloric acid and hydrobromic acid, as well as those formed with organic acids, such as fumaric acid and maleic acid.

[0139] In some embodiments, the subject compounds are 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 an enzymatic conversion reaction. 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 of 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 compounds can be prepared according to 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 or carboxylic acid group of the subject compound. In certain instances, the promoiety is an acyl or substituted acyl group. In certain instances, the promoiety is an alkyl or substituted alkyl group, e.g., an alkyl or substituted alkyl group that, when attached to a hydrophilic head group of the subject compound, forms an ester functionality, e.g., a phosphonate ester, a phosphate ester, and the like.

[0140] In some embodiments, the subject compounds are phosphonate ester or phosphate ester prodrugs that can be converted to compounds containing a phosphonic acid or phosphonate, or phosphate head group. In certain embodiments, the prodrug compound is one of compounds 74, 77, and 78 in Table 1.

[0141] 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 solute, e.g., a molecule of a prodrug or its pharma- ceutically acceptable salt, and one or more solvent molecules. Such solvates are usually crystalline solids with a substantially fixed molar ratio of solute and solvent. Representative solvents include, by way of example, water, methanol, ethanol, isopropanol, acetic acid, and the like. When the solvent is water, the solvate formed is a hydrate.

[0142] In some embodiments, the subject compounds are delivered orally and absorbed into the bloodstream. In some embodiments, the oral bioavailability of the subject compounds is 30% or greater. The subject compounds or their formulations may be modified in any convenient manner to enhance absorption across the intestinal lumen or their bioavailability.

[0143] 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 more, such as 10 minutes or more, 12 minutes or more, 15 minutes or more, 20 minutes or more, 30 minutes or more, 60 minutes or more, 2 hours or more, 6 hours or more, 12 hours or more, 24 hours or more, or even more.

[0144] Methods for inhibiting ENPP1 As mentioned above, embodiments of the present disclosure include ENPP1 inhibitors and inhibition methods using same. ENPP1 is a member of the ectonucleotide pyrophosphatase / phosphodiesterase (ENPP) family. Thus, embodiments of the subject methods include inhibiting the hydrolase activity of ENPP1 on cGAMP. The inventors have discovered that cGAMP can have significant extracellular biofunctions that can be enhanced by blocking the extracellular degradation of cGAMP, e.g., hydrolysis by its degradative enzyme ENPP1. In certain instances, the ENPP1 inhibition target is extracellular, and the subject ENPP1 inhibitor compounds are cell-impermeable, Thus, it cannot diffuse into cells. Therefore, the subject method can selectively inhibit the hydrolase activity of ENPP1 extracellularly, and increase extracellular cGAMP levels. Therefore, in some cases, the ENPP1 inhibitor compound is a compound that inhibits the activity of ENPP1 extracellularly. The experiments performed by the present inventors show that inhibiting the activity of ENPP1 can increase extracellular cGAMP, thereby enhancing the STING pathway.

[0145] Inhibiting ENPP1 means that the activity of the enzyme is reduced by 10% or more, for example, 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., relative to a control in any convenient in vitro inhibition assay). In some cases, inhibiting ENPP1 means that the activity of the enzyme is reduced by 2-fold or more, for example, 3-fold or more, 5-fold or more, 10-fold or more, 100-fold or more, or 1000-fold or more, relative to its normal activity (e.g., relative to a control measured in any convenient assay).

[0146] In some cases, the method is a method of inhibiting ENPP1 in a sample. As used herein, the term "sample" refers to a material or mixture of materials, usually, but not necessarily, in a fluid state, containing one or more components of interest.

[0147] 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. Optionally, the sample is a cell sample. Optionally, the sample contains cGAMP. In certain cases, the cGAMP level is high in the cell sample (e.g., relative to a control sample that is not contacted with the inhibitor). The subject method can provide an increase in cGAMP level. "Increased cGAMP level" refers to 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, for example, 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, relative to a control sample that is not contacted with the agent.

[0148] In certain embodiments, the cell-impermeable ENPP1 inhibitor is an inhibitor as defined herein. In some embodiments, the cell-impermeable ENPP1 inhibitor is represented by Formula I, IV In some cases, the cell-impermeable ENPP1 inhibitor is any one of compounds 1-106.

[0149] In some embodiments, the ENPP1 inhibitor is cell-permeable. In some embodiments, a method of inhibiting ENPP1 is provided, the method comprising contacting a sample with a cell-permeable ENPP1 inhibitor to inhibit ENPP1.

[0150] In some embodiments, the subject compounds have an ENPP1 inhibition profile that reflects activity against additional enzymes, hi some embodiments, the subject compounds specifically inhibit ENPP1 without undesired inhibition of one or more other enzymes.

[0151] In some embodiments, the compounds of the present disclosure prevent the interaction of cGAMP and ENPP1.For example, the subject compounds can act to increase extracellular cGAMP by inhibiting the hydrolase activity of ENPP1 on cGAMP.Without being bound by any particular theory, it is believed that the increase in extracellular cGAMP activates STING pathway.

[0152] In some embodiments, the subject compounds are assayed in inhibition assays, e.g., by treatment with a subject compound. IC 50 or EC 50 In certain embodiments, the subject compounds inhibit ENPP1 relative to a control as measured in an assay that determines the activity level of the enzyme by measuring IC values. 50 Value (or EC 50 value) is 10 μM or less, e.g., 3 μM or less, 1 μM or less, 500 nM or less, 300 nM or less, 200 nM or less, 100 nM or less, 50 nM or less, 30 nM or less, 10 nM or less, 5 nM or less, 3 nM or less, 1 nM or less, or even less.

[0153] As mentioned above, embodiments of the present disclosure include methods of inhibiting ENPP1. The subject compounds (e.g., those described herein) may inhibit the activity of ENPP1 in the range of 10% to 100%, e.g., 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more. In certain assays, the subject compounds inhibit their targets at IC 50 1x10 -6 M or less (e.g. 1x10 -6 M or less, 1x10 -7 M or less, 1x10 -8 M or less, 1x10 -9 M or less, 1x10 -10 M or smaller, or 1x10 -11 It can be inhibited by 500 M or less.

[0154] There are many protocols that can be used to measure 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 using particular animals, which in certain embodiments may be animal models for a condition occurring in association with the target pathogen.

[0155] In some embodiments, the subject methods are in vitro methods, which include contacting a sample with a subject compound that specifically inhibits ENPP1. In certain embodiments, the sample is suspected of containing ENPP1, and the subject methods further include evaluating whether the compound inhibits ENPP1.

[0156] In certain embodiments, the subject compounds are modified compounds that include a label, e.g., a fluorescent label, and the subject methods further include detecting the label in the sample, if present, e.g., using optical detection.

[0157] In certain embodiments, the compound is modified with a support or with an affinity group (e.g., biotin) that binds to the support, and any sample that does not bind to the compound can be removed (e.g., by washing).Specifically bound ENPP1, if present, can then be detected using any convenient means, for example, using the binding of a labeled target-specific probe or using a fluorescent protein-reactive reagent.

[0158] In another embodiment of the subject method, the sample is known to contain ENPP1.

[0159] In some embodiments, the method is a method of reducing the proliferation of cancer cells, the method comprising contacting the cells with an effective amount of a subject ENPP1 inhibitory compound (e.g., as described herein) to reduce the proliferation of cancer cells. In certain cases, the subject ENPP1 inhibitory 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 comprises contacting the cells with an ENPP1 inhibitory compound (e.g., as described herein) and contacting the cells with a chemotherapeutic agent. Any convenient cancer cells can be targeted.

[0160] Treatment method An embodiment of the present disclosure provides a method for inhibiting the hydrolase activity of ENPP1 on cGAMP. The subject method includes the inhibition of ENPP1, which increases cGAMP levels and / or provides downstream regulation (e.g., activation) of the STING pathway. The inventors have discovered that cGAMP is present in the extracellular space and that ENPP1 can control extracellular cGAMP levels. The inventors have also discovered that cGAMP can have significant extracellular biofunctions in vivo (see, e.g., Figures 3A-4C). The results described and demonstrated herein indicate that inhibition of ENPP1 according to the subject method can regulate STING activity in vivo, which in turn has applications in the treatment of various diseases, e.g., as a target for cancer immunotherapy. Thus, the subject method can inhibit selective extracellular ENPP1 activity (e.g., cGAMP hydrolase activity) to increase extracellular cGAMP levels and activate the stimulator of interferon genes (STING) pathway. In some cases, the subject method is a method of increasing a STING-mediated response in a subject. In some cases, the subject method is a method of regulating an immune response in a subject.

[0161] "STING-mediated response" refers to any response mediated by STING, including, but not limited to, immune responses to bacterial, viral, and eukaryotic pathogens. See, e.g., Ishikawa et al. Immunity 29:538-550 (2008), Ishikawa et al. Nature 461:788-792 (2009), and Sharma et al. Immunity 35:194-207 (2011). STING also functions in certain autoimmune diseases initiated by inappropriate recognition of self-DNA (see, e.g., Gall et al. Immunity 36:120-131 (2012)) and to induce adaptive immunity in response to DNA vaccines (see, e.g., Ishikawa et al. al. Nature 461:788-792 (2009). Increasing a STING-mediated response in a subject means increasing the STING-mediated response in a subject compared to a control subject (e.g., a subject not administered the subject compound). In some cases, the subject is a human, and the subject compounds and methods activate human STING. In some cases, the STING-mediated response includes modulating an immune response. In some cases, the subject methods are methods of modulating an immune response in a subject.

[0162] In some cases, the STING-mediated response includes increased production of interferon (e.g., type I interferon (IFN), type III interferon (IFN)) in the 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 that is produced in mammalian cells in response to exposure to various inducers, such as viruses, polypeptides, mitogens, etc. Interferon protects animal tissues and cells from viral attack and is an important host defense mechanism. Interferon can be classified into type I, type II and type III interferon. Type I interferons of mammalian 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).

[0163] Interferons are used in the treatment of various cancers. This is because these molecules have anti-cancer activity that acts at multiple levels. Interferon proteins can directly inhibit the proliferation of human tumor cells. In some cases, the anti-proliferative 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 anti-tumor 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 anti-tumor cytotoxic T lymphocyte activity. In addition, interferons play a role in the tolerance presentation of antigens in the immune system. Moreover, some studies have further shown that IFN-β protein may have antiangiogenic activity. Angiogenesis, i.e., the formation of new blood vessels, is essential for the growth of solid tumors. IFN-β may inhibit angiogenesis by inhibiting the expression of angiogenic factors, such as bFGF and VEGF. Interferon protein may also inhibit tumor invasiveness by regulating the expression of enzymes, such as collagenase and elastase, which are important for tissue remodeling.

[0164] An aspect of the method includes administering a therapeutically effective amount of an ENPP1 inhibitor to a subject having cancer to treat the cancer in the subject. In some cases, the subject has been diagnosed with cancer or is suspected of having cancer. Any convenient ENPP1 inhibitor can be used in the subject method 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, 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. In some cases, the cancer is breast cancer. In some embodiments, the cancer is lymphoma.

[0165] The method aspect includes administering a therapeutically effective amount of a cell-impermeable ENPP1 inhibitor to a subject, inhibiting the hydrolysis of cGAMP and treating the cancer of the subject.In some cases, the cancer is solid tumor cancer.In some 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 some embodiments, the cancer is breast cancer.In some cases, the cancer is lymphoma.

[0166] In some embodiments of the methods disclosed herein, the cell-impermeable ENPP1 inhibitor is an inhibitor of any one of formulas I, IV, V, VI, or VII. In some cases, the cell-impermeable ENPP1 inhibitor is any one of compounds 1-106.

[0167] In some embodiments of the methods disclosed herein, the ENPP1 inhibitor is cell-permeable.

[0168] Thus, the method embodiment includes contacting a sample with a subject compound (e.g., as described above) under conditions in which the compound inhibits ENPP1. Any convenient protocol for contacting the compound with the sample can be used. The specific 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 cases, the sample includes cells maintained in a suitable medium, and the complex is introduced into the medium. For in vivo protocols, any suitable administration protocol can be used. Depending on the potency of the compound, the cells of interest, the method of administration, and the number of cells present, various protocols can be used.

[0169] In some embodiments, the subject method is a method of treating cancer in a patient. In some embodiments, the subject method comprises administering to the subject an effective amount of a subject compound (e.g., as described herein) or a pharma- ceutically 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 compound administered is a compound of one of formulas (I), (IV), (V), (VI) or (VII). In certain examples of the method, the compound administered is a compound of formula (I), (IV), (V), (VII), or (VII). The compound is represented by one of the compounds in Tables 1 or 2.

[0170] In some embodiments, an "effective amount" is an amount of a subject compound that, when administered one or more times to an individual, either in monotherapy or in combination therapy, is effective to inhibit ENPP1 by about 20% (20% inhibition), at least about 30% (30% inhibition), at least about 40% (40% inhibition), at least about 50% (50% inhibition), at least about 60% (60% inhibition), at least about 70% (70% inhibition), at least about 80% (80% inhibition), or at least about 90% (90% inhibition), compared to ENPP1 activity in an individual not treated with the compound, or alternatively, compared to ENPP1 activity in an individual before or after treatment with the compound.

[0171] In some embodiments, a "therapeutically effective amount" is an amount of a subject compound that, when administered one or more times to an individual, either as a monotherapy or in combination therapy, is effective to reduce tumor burden by about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%, as compared to the tumor burden in an individual not treated with the compound, or alternatively, as 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.

[0172] In some embodiments, a "therapeutically effective amount" is an amount of a subject compound that, when administered one or more times to an individual, either as a monotherapy or in combination therapy, is effective to reduce the radiation therapy dose required to effect tumor shrinkage in the subject by about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%, as compared to the radiation therapy dose required to effect tumor shrinkage in an individual not treated with the compound.

[0173] In some embodiments, a "therapeutically effective amount" of a compound is an amount that is effective when administered one or more times to an individual with cancer to achieve a 1.5, 2, 2.5, 3, 3.5, 4, 4.5, or 5 log reduction in tumor size.

[0174] In some embodiments, the effective amount of the compound is in the range of about 50 ng / ml to about 50 μg / ml (e.g., about 50 ng / ml to about 40 μg / ml, about 30 ng / ml to about 20 μg / ml, about 50 ng / ml to about 10 μg / ml, about 50 ng / ml to about 1 μg / ml, about 50 ng / ml to about 800 ng / ml, about 50 ng / ml to about 700 ng / ml, about 50 ng / ml to about 600 ng / ml, about 50 ng / ml to about 500 ng / ml, about 50 ng / ml to about 400 ng / ml, about 60 ng / ml to about 400 ng / ml, / ml, about 70ng / ml to about 300ng / ml, about 60ng / ml to about 100ng / ml, about 65ng / ml to about 85ng / ml, about 70ng / ml to about 90ng / ml, about 200ng / ml to about 900ng / ml, about 200ng / ml to about 800ng / ml, about 200ng / ml to about 700ng / ml, about 200ng / ml to about 600ng / ml, about 200ng / ml to about 500ng / ml, about 200ng / ml to about 400ng / ml, or about 200ng / ml to about 300ng / ml).

[0175] 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, from about 250 ng to about 500 ng, from about 500 ng to about 750 ng, from about 750 ng to about 1 μg, from about 1 μg to about 10 μg, from about 10 μg to about 50 μg, from about 50 μg to about 15 The amount is in the range of about 0 μg, about 150 μg to about 250 μg, about 250 μg to about 500 μg, about 500 μg to about 750 μg, about 750 μg to about 1 mg, about 1 mg to about 50 mg, about 1 mg to about 100 mg, or about 50 mg to about 100 mg. The amount may be a single dose or a total amount per day. The total amount per day may be in the range of 10 pg to 100 mg, 100 mg to about 500 mg, or 500 mg to about 1000 mg.

[0176] 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), every day (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 time ranging from 1 day to about 2 years or more. For example, the compound is administered at any of the aforementioned frequencies for 1 week, 2 weeks, 1 month, 2 months, 6 months, 1 year, or 2 years or more, depending on various factors.

[0177] Administration of a therapeutically effective amount of a subject compound to an individual having cancer can result in one or more of the following: 1) a reduction in tumor burden, 2) a reduction in the radiation therapy dose required to cause tumor shrinkage, 3) a reduction in the spread of cancer from one cell in the individual to another, 4) a reduction in morbidity or mortality in clinical outcomes, 5) a reduction in the overall duration of treatment when combined with other anti-cancer drugs, and 6) an improvement in indicators of disease response (e.g., a reduction in one or more cancer symptoms). Any of a variety of methods can be used to determine whether a treatment method is effective. For example, a biological sample from an individual treated with a subject method can be assayed.

[0178] Any of the compounds described herein can be used in the subject treatment method.In certain examples, the compound is one of formulas I, IV or V.In certain cases, the compound is one of the compounds in table 1 or 2.In some cases, the compound used in the subject method is not cell permeable.In some cases, the compound used in the subject method has low cell permeability.

[0179] In some embodiments, the compound specifically inhibits ENPP1. In some embodiments, the compound regulates the activity of cGAMP. In some embodiments, the compound prevents the interaction between ENPP1 and cGAMP. In some embodiments, the compound causes the activation of the STING pathway.

[0180] 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), livestock (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 be in need of cancer treatment. In some cases, the subject method includes diagnosing cancer, including any one of the cancers described herein. In some embodiments, the compound is administered as a pharmaceutical formulation.

[0181] In certain embodiments, the ENPP1 inhibitory compound is a modified compound comprising a label, and the method further comprises detecting the label in the subject. The choice of the label depends on the detection method. 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 comprises a fluorescent label suitable for optical detection. In certain embodiments, the compound comprises a radioactive label for detection using positron emission tomography (PET) or single photon emission computed tomography (SPECT). In some cases, the compound comprises a radioactive label suitable for tomographic detection. The subject compounds can be labeled as described above, although in some methods the compounds are unlabeled and a secondary imaging agent is used for imaging.

[0182] Combination therapy The subject compound can be administered to a subject alone or in combination with an additional, i.e., second, active agent. In combination therapy, the subject ENPP1 inhibitor compound can be used in combination with a second active agent or additional treatment, such as radiation therapy. The terms "agent", "compound" and "drug" are used interchangeably herein. For example, the ENPP1 inhibitor compound can be administered alone or in combination with one or more other drugs, such as drugs used to treat a disease of interest, including, but not limited to, immune-modulating diseases and conditions and cancer. In some embodiments, the subject method further comprises administering a second agent, such as a small molecule, a chemotherapeutic drug, an antibody, an antibody fragment, an antibody-drug conjugate, an aptamer, a protein, or a checkpoint inhibitor, either in combination or in sequence. In some embodiments, the method further comprises administering radiation therapy to the subject.

[0183] The terms "co-administration" and "in combination with" include administration of two or more therapeutic agents simultaneously, concurrently, or sequentially without a specified time period. In one embodiment, the agents are present in a cell or in a subject at the same time or exert their biological or therapeutic effects at the same time. In one embodiment, the therapeutic agents are in the same composition or unit dosage form. In other embodiments, the therapeutic agents are in separate compositions or unit dosage forms. In certain embodiments, the first 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), concomitantly with, or subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) administration of the second therapeutic agent.

[0184] "Concurrent administration" of a known therapeutic agent or additional treatment and a pharmaceutical composition of the present disclosure means administering the compound and a second agent or additional treatment at a time when both the known agent and the composition of the present invention would have a therapeutic effect. Such concurrent administration may include administering the agent simultaneously (i.e., at the same time), prior to, or subsequent to administration of the subject compound. The routes of administration of the two agents may be different, and representative routes of administration are described in more detail below. A person skilled in the art would have no difficulty in determining the appropriate timing, sequence, and dosages of administration of a particular agent or treatment and a compound of the present disclosure.

[0185] In some embodiments, the compounds (e.g., the subject compound and the at least one additional compound or treatment) are administered to the subject within 24 hours of each other, e.g., 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 meant that the compounds are administered to the subject within about 10 minutes, within 5 minutes, or within 1 minute of each other.

[0186] Also provided are pharmaceutical formulations of the subject compounds and the second active agent, in which the compounds may be administered in the form of pharma- ceutically acceptable salts or in which the compounds are used alone or in appropriate association and combination with other pharma- ceutical active compounds.

[0187] In conjunction with any of the subject methods, the ENPP1 inhibitory 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 inflammation, treat or prevent chronic inflammation or fibrosis, or treat cancer. In any case, the ENPP1 inhibitory compound can be administered before, simultaneously with, or after the administration of the other drug. In certain cases, 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, glioma, glioblastoma, melanoma, and various head and neck tumors.

[0188] In the case of treating cancer, the ENPP1 inhibitor compound may be administered in combination with a chemotherapeutic agent selected from the group consisting of alkylating agents, nitrosoureas, antimetabolites, antitumor antibiotics, plant (vinca) alkaloids, steroid hormones, taxanes, nucleoside analogues, steroids, anthracyclines, thyroid hormone replacement drugs, thymidylate targeting 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., Thl7-induced dendritic cell vaccines, or genetically modified tyrosinase such as Oncept®), and other cell therapies.

[0189] Specific chemotherapeutic agents of interest include gemcitabine, docetaxel, bleomycin, erlotinib, gefitinib, lapatinib, imatinib, dasatinib, nilotinib, bosutinib, crizotinib, ceritinib, trametinib, bevacizumab, sunitinib, sorafenib, trastuzumab, trastuzumab emtansine (Ado-trastuzumab emtansine), rituximab, ipilimumab, rapamycin, temsirolimus, everolimus, methotrexate, doxorubicin, abraxane, forfirinox, cisplatin, carboplatin, 5-fluorouracil, teismo, paclitaxel, prednisone, levothyroxine, pemetrexed, navitoclax, and ABT-199. Peptide compounds can also be used. Cancer chemotherapeutic agents of interest include, but are not limited to, 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 analogues and derivatives (see, e.g., EP 1391213, and Liu et al (1996) Proc. Natl. Acad. Sci. USA 93:8618-8623), duocarmycins and their active analogues and derivatives (including, e.g., synthetic analogues, KW-2189 and CB1-TM1), and benzodiazepines and their active analogues and derivatives (e.g., pyrrolobenzodiazepines (PBDs)).

[0190] In some embodiments, the ENPP1 inhibitor compound can be administered in combination with a chemotherapeutic agent for treating cancer.In some cases, the chemotherapeutic agent is gemcitabine.In some cases, the chemotherapeutic agent is docetaxel.In some cases, the chemotherapeutic agent is abraxane.

[0191] In the case of treating 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 drug used to treat disease by inhibiting, enhancing, or suppressing the immune response. , the immunotherapeutic agent is an immune checkpoint inhibitor. For example, Figures 4A-4C show that an exemplary ENPP1 inhibitor can act synergistically with an immune checkpoint inhibitor in a mouse model. Any convenient checkpoint inhibitor can be used, 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, for the treatment of 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.

[0192] Any convenient cancer vaccine therapy and drug can be used in combination with the subject ENPP1 inhibitory compound, composition and method.For the treatment of cancer, for example, ovarian cancer, the ENPP1 inhibitory 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 correlated with a significant prolongation of overall survival of ovarian cancer patients.In some cases, the ENPP1 inhibitory compound is used as an adjuvant therapy in combination with Th17-inducing vaccination.

[0193] Also of interest are agents that are CD47 inhibitors, including but not limited to CARP-1 / CCAR1 (cell cycle and apoptosis regulator 1) inhibitors, including but not limited to those described in Rishi et al., Journal of Biomedical Nanotechnology, Volume 11, Number 9, September 2015, pp. 1608-1627(20), and anti-CD47 antibody agents, e.g., Hu5F9-G4.

[0194] In certain instances, the combination provides improved efficacy compared to either component alone, and in some cases, the combination provides synergistic or additive effects on the combined or additive effects of the components. Various combinations of the subject compounds and the chemotherapeutic agents may be used, and may be used sequentially or simultaneously. In the case of multiple doses, the two agents may be alternated as is, or, for example, two or more doses of one agent may be alternated with a single dose of the other agent. The simultaneous administration of both agents may also be alternated with or interleaved with the administration of the individual agents. In some cases, the interval between doses may be about 1-6 hours, about 6-12 hours, about 12-24 hours, about 1-2 days, about 1-2 weeks, or longer, after the start of treatment.

[0195] Combination with cGAMP-inducing chemotherapy agents An embodiment of the present disclosure includes a method of treating cancer in which the ENPP1 inhibitory compound (or pharmaceutical composition comprising such compound) can be administered in combination with a chemotherapeutic agent capable of inducing the production of cGAMP in vivo. When a subject is exposed to an effective amount of a particular chemotherapeutic agent, the production of 2'3'-cGAMP can be induced in the subject. The induced cGAMP level can be maintained and / or improved, e.g., improved compared to the level achieved by either agent alone, when the subject ENPP1 inhibitory compound is combined to prevent the degradation of cGAMP. Any convenient chemotherapeutic agent that can cause DNA damage and induce cGAMP production by dying cells due to the overwhelm of repair or degradation mechanisms, e.g., 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 by binding to microtubules. In some cases, the cGAMP-inducing chemotherapeutic agent is an antitumor agent.

[0196] The cancers of interest that can be treated using the subject combination therapy include, but are not limited to, 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, glioma, glioblastoma, melanoma, and various head and neck tumors.In some cases, the cancer is breast cancer.In certain examples, the cancer is glioma or glioblastoma.

[0197] Chemotherapeutic agents of interest include uracil analogs, fluorouracil prodrugs, thymidylate synthase inhibitors, deoxycytidine analogs, DNA synthesis inhibitors (e.g., causing S-phase apoptosis), folate analogs, dehydrofolate reductase inhibitors, anthracyclines, intercalating agents (e.g., causing double-strand breaks), topoisomerase IIa inhibitors, taxanes, microtubule disassembly inhibitors (e.g., leading to G2 / M arrest / apoptosis), microtubule assembly inhibitors, microtubule function stabilizers (e.g., leading to G2 / M apoptosis), tubulin polymerization promoters, tubulin binding agents (e.g., leading to apoptosis via M-phase arrest) epothilone B analogs, vinca alkaloids, nitrogen mustards, nitrosoureas, DNA alkylating agents (e.g., interstrand crosslinks, leading to apoptosis via p53), VEGF inhibitors, anti-angiogenic antibodies, HER2 inhibitors, quinazoline HER2 inhibitors, EGFR inhibitors, tyrosine kinase inhibitors, sirolimus analogs, mTORC1 inhibitors (e.g., in breast cancer in combination with exemestane = aromatase inhibitor to inhibit estrogen production), triazenes, dacarbazine prodrugs, methylhydrazine.

[0198] Exemplary breast cancer chemotherapeutic 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 glioma / glioblastoma-related antitumor agents include, but are not limited to, carmustine, lomustine, temozolomide, procarbazine, vincristine, and bevacicumab. Exemplary DNA-damaging chemotherapeutic agents of interest include, but are not limited to, melphalan, cisplatin, and etoposide, fluorouracil, gemcitabine.

[0199] Concomitant radiation therapy Alternatively, in the case of a method for treating cancer, the ENPP1 inhibitory compound (or a pharmaceutical composition comprising such a compound) may be administered in combination with radiation therapy. In certain embodiments, the method includes administering radiation therapy to the subject. Again, the ENPP1 inhibitory compound can be administered before or after the administration of radiation therapy. Thus, the subject method may further include administering radiation therapy to the subject. The combination of radiation therapy and administration of the subject compound may provide a synergistic therapeutic effect. When a subject is exposed to radiation of an appropriate dose and / or frequency during radiation therapy (RT), the production of 2'3'-cGAMP may be induced in the subject. The induced cGAMP level may be maintained and / or enhanced, e.g., compared to the level achieved by RT alone, when the subject ENPP1 inhibitory compound is combined to prevent the degradation of cGAMP. For example, FIG. 4A shows 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 administration of a reduced dose and / or frequency / regimen of radiation therapy compared to a therapeutically effective dose and / or frequency / regimen of radiation therapy alone. In some cases, the radiation therapy is administered in combination with the subject compound at a dose and / or frequency effective to reduce the risk of radiation damage to the subject, e.g., radiation damage expected to occur under a therapeutically effective dose and / or frequency / regimen of radiation therapy alone.

[0200] In some cases, the method comprises administering an ENPP1 inhibitor to the subject before radiation therapy.In some cases, the method comprises administering an ENPP1 inhibitor to the subject after exposing the subject to radiation therapy.In some cases, the method comprises sequentially administering radiation therapy, followed by an ENPP1 inhibitor, followed by a checkpoint inhibitor to the subject in need thereof.

[0201] Practicality The compounds and methods of the present invention, such as those described herein, are used in a variety of applications. The intended applications include, but are not limited to, research applications and therapeutic applications. The methods of the present invention are used in a variety of different applications, including any convenient application in which inhibition of ENPP1 is desired.

[0202] 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.

[0203] The subject compounds and methods can be used in a variety of therapeutic applications. Therapeutic applications of interest include those in cancer treatment. Thus, the subject compounds can be used to treat a variety of different conditions in which it is desired to inhibit and / or treat cancer in a host. For example, the subject compounds and methods can be used to treat solid tumor cancers (e.g., those described herein).

[0204] Pharmaceutical Compositions The compounds discussed herein may be formulated using any convenient excipients, reagents and methods. The compositions are provided in formulations that include a pharma- ceutically acceptable excipient(s). A wide variety of pharma-ceutically acceptable excipients are known in the art and need not be discussed in detail herein. Pharmaceutically acceptable excipients are described, for example, in A. Gennaro (2000) “Remington: The Science and Practice of Pharmacy,” 20th edition, Lippincott, Williams, & Wilkins, Pharmaceutical Dosage Forms and Drug Delivery Systems(1999)HCAnsel et al., eds.,7 th ed., Lippincott, Williams, & Wilkins, and Handbook of Pharmaceutical Excipients (2000) AHKibbe et al., eds., 3 rdand are fully described in various publications, including ed. Amer. Pharmaceutical Assoc.

[0205] The pharma- ceutically acceptable excipients, for example, vehicles, adjuvants, carriers or diluents, are readily available to the public. Moreover, pharma- ceutically acceptable auxiliary substances, for example, pH adjusting and buffering agents, isotonicity adjusting agents, stabilizers, wetting agents, and the like, are readily available to the public.

[0206] 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, varying 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, sucrose, and the like. In some embodiments, the aqueous buffer further comprises a non-ionic surfactant, such as polysorbate 20 or 80. Optionally, the formulation may further comprise a preservative. Suitable preservatives include, but are not limited to, benzyl alcohol, phenol, chlorobutanol, benzalkonium chloride, and the like. In many cases, the formulation is stored at about 4° C. The formulation may also be lyophilized, in which case the formulation is generally protected from the cryoprotectant. Examples of suitable sugars include sucrose, trehalose, lactose, maltose, mannitol, etc. Lyophilized formulations can also be stored at ambient temperature for extended periods of time. In some embodiments, the subject compounds are formulated for sustained release.

[0207] 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 or separate formulations) that includes a pharmacologic 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.

[0208] Another aspect of the present invention provides pharmaceutical compositions comprising or consisting essentially of the compounds of the present invention, or pharma- ceutically acceptable salts, isomers, tautomers, or prodrugs thereof, and further comprising one or more additional active agents of interest. Any convenient active agent may be used in the subject method in conjunction with the subject compounds. In some cases, the additional agent is a checkpoint inhibitor. The subject compounds and checkpoint inhibitors, as well as the additional therapeutic agents described herein for combination therapy, may be administered orally, subcutaneously, intramuscularly, intranasally, parenterally, or by other routes. The subject compounds and the second active agent (if present) may be administered by the same or different routes of administration. The therapeutic agents may 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 administration, or injection into the affected organ. In certain cases, the therapeutic agents may be administered intranasally. In some cases, the therapeutic agent may be administered intratumorally.

[0209] In some embodiments, the subject compound and the chemotherapeutic agent are administered to an individual in a formulation (e.g., in the same or separate formulations) that includes a pharma- ceutically acceptable excipient(s). The chemotherapeutic agent includes, but is not limited to, alkylating agents, nitrosoureas, antimetabolites, antitumor antibiotics, plant (vinca) alkaloids, and steroid hormones. Peptide compounds can 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 analogues and derivatives (see, e.g., EP 1391213, and Liu et al (1996) Proc. Natl. Acad. Sci. USA 93:8618-8623), duocarmycins and their active analogues and derivatives (including, e.g., synthetic analogues, KW-2189 and CB1-TM1), and benzodiazepines and their active analogues and derivatives (e.g., pyrrolobenzodiazepines (PBDs)).

[0210] The subject compound and the second chemotherapeutic agent, as well as the additional therapeutic agent described herein for combination therapy, may be administered orally, subcutaneously, intramuscularly, parenterally, or by other routes. The subject compound and the second chemotherapeutic agent may be administered by the same or different routes of administration. The therapeutic agent may 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 administration, or injection into the affected organ.

[0211] The subject compounds may be administered in unit dosage form and by any method known in the art. The pharmaceutical compositions may be prepared in the form of a pharmaceutical composition comprising a pharmaceutical agent, such as a pharmaceutical composition for use in a pharmaceutical composition comprising a pharmaceutical agent, a pharmaceutical composition for use in ...

[0212] Examples of suitable solid carriers include lactose, sucrose, gelatin, agar and bulk powder. Examples of suitable liquid carriers include water, pharma- ceutically acceptable oils and fats, alcohol or other organic solvents, such as esters, emulsions, syrups or elixirs, suspensions, solutions and / or suspensions, and solutions and / or suspensions reconstituted from non-effervescent granules and effervescents reconstituted from effervescent agents. Such liquid carriers may contain, for example, suitable solvents, preservatives, emulsifiers, suspending agents, diluents, sweeteners, thickeners, and melting agents. A preferred carrier is edible oil, such as corn oil or canola oil. Polyethylene glycol, such as PEG, is also a good carrier.

[0213] Any drug delivery device or system that provides the dosing regimen of the present disclosure may be used. A wide variety of delivery devices and systems are known to those of skill in the art.

[0214] Further embodiments Further embodiments are described in the following clauses.

[0215] Article 1. ENPP1 inhibitors of formula (I): YALX (I) wherein Y is selected from the group consisting of aryl, substituted aryl, heteroaryl, substituted heteroaryl, carbocycle, substituted carbocycle, heterocycle, and substituted heterocycle; A is selected from the group consisting of carbocycle, substituted carbocycle, heterocycle, and substituted heterocycle; L is a covalent bond or a linker; X is a hydrophilic head group; Or a prodrug, pharma- ceutically acceptable salt or solvate thereof.

[0216] Clause 2. The ENPP1 inhibitor of clause 1, wherein the hydrophilic head group (X) is selected from phosphonic acid, phosphonate, phosphonate ester, phosphate, phosphate ester, thiophosphate, thiophosphate ester, phosphoramidate, thiophosphoramidate, sulfonic acid, sulfonate, sulfate, hydroxamic acid, and carboxylic acid.

[0217] Clause 3. The ENPP1 inhibitor of clause 2, wherein the hydrophilic head group (X) is selected from phosphonic acid, phosphonate, phosphonate ester, phosphate, phosphate ester, thiophosphate, thiophosphate ester, phosphoramidate, and thiophosphoramidate.

[0218] Clause 4. The ENPP1 inhibitor according to any one of clauses 1 to 3, wherein LX comprises a group of formula (XI): [ka] During the ceremony, Z 12 is selected from O and S; Z 13 and Z 14 are each independently selected from O and NR', where R' is H, alkyl or substituted alkyl; Z 15 is selected from O and CH2; R 15 and R 16 are each independently selected from H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, aryl, substituted aryl, acyl, heterocycle, substituted heterocycle cycloalkyl, and substituted cycloalkyl; q 1is an integer from 0 to 6 (for example, 0 to 5).

[0219] Clause 5. The ENPP1 inhibitor according to clause 4, wherein LX is selected from: [ka] [ka] and [ka]

[0220] Clause 6. The ENPP1 inhibitor according to any one of clauses 1 to 5, wherein X is a phosphonic acid or a phosphonate ester.

[0221] Clause 7. The ENPP1 inhibitor according to clause 1, wherein LX comprises a group of formula (XII): [ka] During the ceremony, R 17 and R 18 are each independently selected from H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, aryl, substituted aryl, acyl, heterocycle, substituted heterocycle cycloalkyl, and substituted cycloalkyl; or R 17 and R 18 together with the atoms to which they are attached form a ring selected from heterocycles and substituted heterocycles; q 2 is an integer from 1 to 6.

[0222] Clause 8. The ENPP1 inhibitor according to clause 7, wherein LX is of the following structure: [ka]

[0223] Clause 9. The ENPP1 inhibitor according to clause 1, wherein LX comprises a group of formula (XIII): [ka] In the formula, q3 is an integer of 1 to 6.

[0224] Clause 10. The ENPP1 inhibitor according to clause 9, wherein LX is selected from: [ka] and [ka]

[0225] Clause 11. The ENPP1 inhibitor according to clause 1, wherein LX comprises a group of formula (XIV): [ka] In the formula, Z 16 is selected from O and CH2; q 4 is an integer from 0 to 6.

[0226] Clause 12. The ENPP1 inhibitor according to clause 11, wherein LX is selected from the following: [ka] and [ka]

[0227] Clause 13. The ENPP1 inhibitor according to clause 1, wherein LX comprises a group of formula (XV): [ka] In the formula, q 5 is an integer from 1 to 6.

[0228] Clause 14. The ENPP1 inhibitor according to clause 13, wherein LX is selected from the following: [ka] and [ka]

[0229] Clause 15. The ENPP1 inhibitor according to clause 1, wherein LX comprises a group of formula (XVI): [ka] During the ceremony, R 19 is selected from H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, aryl, substituted aryl, acyl group, heterocycle, substituted heterocycle cycloalkyl, and substituted cycloalkyl; q 6 is an integer from 1 to 6.

[0230] Clause 16. The ENPP1 inhibitor according to clause 15, wherein LX is of the following structure: [ka]

[0231] Clause 17. The ENPP1 inhibitor according to clause 1, wherein LX comprises a group of formula (XVII): [ka] In the formula, q7 is an integer of 1 to 6.

[0232] Clause 18. The ENPP1 inhibitor according to clause 17, wherein LX is of the following structure: [ka]

[0233] Clause 19. An ENPP1 inhibitor according to any one of clauses 1 to 18, wherein A is a heterocycle or a substituted heterocycle.

[0234] Clause 20. The ENPP1 inhibitor according to clause 19, wherein A is selected from piperidine, substituted piperidine, piperazine and substituted piperazine.

[0235] Clause 21.A: [ka] or [ka] The ENPP1 inhibitor according to any one of claims 19 to 20,

[0236] Clause 22. An ENPP1 inhibitor according to any one of clauses 1 to 18, wherein A is a carbocycle (e.g. a 5-, 6- or 7-membered monocyclic carbocycle).

[0237] The ENPP1 inhibitor of claim 22, wherein A is cycloalkyl or substituted cycloalkyl.

[0238] Clause 24.A: [ka] The ENPP1 inhibitor of claim 23,

[0239] The ENPP1 inhibitor of claim 22, wherein A is aryl or substituted aryl.

[0240] Clause 26. The ENPP1 inhibitor of claim 25, wherein A is phenylene or substituted phenylene.

[0241] Clause 27.A: [ka] The ENPP1 inhibitor of claim 26,

[0242] Clause 28. An ENPP1 inhibitor according to any one of clauses 1 to 27, wherein L is a linear linker having a backbone length of 1 to 12 atoms and containing one or more groups selected from alkylene, substituted alkylene, -CO-, -O-, -NR'-, -NR'CO-, -CO2- and -NR'CO2-, where R' is H, alkyl or substituted alkyl.

[0243] Clause 29. An ENPP1 inhibitor according to clause 28, wherein L is -(CH2)n-, where n is an integer from 1 to 6 (e.g., 1, 2, 3, 4, 5 or 6).

[0244] Clause 30. An ENPP1 inhibitor according to clause 29, wherein n is 1 or 2.

[0245] Clause 31. The ENPP1 inhibitor according to any one of clauses 1 to 30, wherein Y is selected from quinazoline, substituted quinazoline, quinoline, substituted quinoline, naphthalene, substituted naphthalene, isoquinoline, substituted isoquinoline, 7H-purine, substituted 7H-purine, pyrimidine, substituted pyrimidine.

[0246] Clause 32. The ENPP1 inhibitor according to any one of clauses 1 to 30, wherein Y is selected from 4-quinazolinyl, substituted 4-quinazolinyl, 4-quinolinyl, substituted 4-quinolinyl, 1-naphthalyl, substituted 1-naphthalyl, 4-isoquinolinyl, substituted 4-isoquinolinyl, 6-(7H-purinyl), substituted 6-(7H-purinyl), 4-pyrimidinyl, substituted 4-pyrimidinyl.

[0247] Article 33. The ENPP1 inhibitor according to any one of articles 31 to 32, wherein Y is a group of the following formula: [ka] During the ceremony, Z 1 and Z 2 are each independently1 and N, Each R 1 is independently selected from H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, heterocycle, and substituted heterocycle; R 2 and R 5 are each independently selected from the group consisting of H, OH, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, -OCF3, halogen, amine, substituted amine, amide, heterocycle, and substituted heterocycle; R 3 and R 4 are each independently selected from the group consisting of H, OH, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, -OCF3, halogen, amine, substituted amine, amide, heterocycle, and substituted heterocycle; Or, R 3 and R 4 together with the carbon atom to which they are attached form a fused ring selected from a heterocycle, a substituted heterocycle, a cycloalkyl, a substituted cycloalkyl, an aryl, and a substituted aryl.

[0248] Clause 34. An ENPP1 inhibitor according to clause 33 of the following formula: [ka] During the ceremony, L is selected from -CH2-, -(CH2)2-, -(CH2)3-, -(CH2)4-, -(CH2)5- and -(CH2)6-; X is [ka] and [ka] is selected from Here, R a and R b each independently represents an aryl, an alkyl, or -CHOC(O)Re , -CH2OC(O)OR e is selected from R c and R d are each independently -C(CH3)C(O)OR e , alkyl, where R e is alkyl, Z 3 and Z 4 are each independently selected from CR and N, where R is H, alkyl or substituted alkyl.

[0249] Clause 35. An ENPP1 inhibitor according to clause 34 of the following formula: [ka]

[0250] Clause 36. ENPP1 inhibitor according to clause 33 of the following formula: [ka] During the ceremony, L is selected from -CH2-, -(CH2)2-, -(CH2)3-, -(CH2)4-, -(CH2)5- and -(CH2)6-; X is [ka] [ka] and [ka] is selected from Here, R a and R b each independently represents an aryl, an alkyl, or -CHOC(O)R e , -CH2OC(O)OR e is selected from R c and Rd are each independently -C(CH3)C(O)OR e , alkyl, where R e is alkyl.

[0251] Article 37:R 1 But hydrogen, C 1-5 selected from alkyl and vinyl heterocycles; R 2 and R 5 are each independently hydrogen, C 1-5 selected from alkyl, amine, triazole, imidazole, amide, alkoxy, OCF3, halogen and hydroxy; R 3 and R 4 are each independently hydrogen, C 1-5 or R is selected from alkyl, triazole, imidazole, amine, amide, alkoxy, OCF3, halogen and hydroxy; 3 and R 4 may, together with the carbon atoms to which they are attached, form a fused heterocycle. 37. The ENPP1 inhibitor according to any one of clauses 33 to 36,

[0252] Clause 38. The ENPP1 inhibitor according to any one of clauses 33 to 36, wherein Y is a group of the following formula: [ka] During the ceremony, R 7 is selected from H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, heterocycle, and substituted heterocycle; R 8 is selected from the group consisting of OH, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, -OCF3, halogen, amine, substituted amine, amide, heterocycle, and substituted heterocycle.

[0253] Article 39:R 7 But hydrogen, C 1-5 Alkyl, substituted C1-5 selected from alkyl, vinyl-heterocycle and substituted vinyl-heterocycle; R 8 But hydrogen, C 1-5 39. The ENPP1 inhibitor according to clause 38, selected from alkyl, triazole, imidazole, amine, amide, alkoxy, halogen, OCF3 and hydroxy.

[0254] Clause 40. The ENPP1 inhibitor according to any one of clauses 33 to 36, wherein Y is a group of the following formula: [ka] During the ceremony, R 7 is selected from H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, heterocycle, and substituted heterocycle; R 8 and R 9 are each independently selected from OH, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, -OCF3, halogen, amine, substituted amine, amide, heterocycle, and substituted heterocycle; or R 8 and R 9 together with the carbon atom to which they are attached form a fused ring selected from heterocycle, substituted heterocycle, cycloalkyl, substituted cycloalkyl, aryl, and substituted aryl.

[0255] Article 41:R 7 But hydrogen, C 1-5 selected from alkyl and vinyl heterocycles; R 8 and R 9 are each independently hydrogen, C 1-5 or R is selected from alkyl, triazole, imidazole, amine, amide, alkoxy, halogen, OCF3, and hydroxy; 8 and R 9 and together with the carbon atom to which they are attached form a fused heterocycle or a fused substituted heterocycle.

[0256] Clause 42. The ENPP1 inhibitor according to any one of clauses 33 to 36, wherein Y is of the following formula: [ka] During the ceremony, R 7 is selected from H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, heterocycle, and substituted heterocycle; R 10 is selected from OH, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, -OCF3, halogen, amine, substituted amine, amide, heterocycle, and substituted heterocycle; R 8 and R 9 are each independently selected from OH, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, -OCF3, halogen, amine, substituted amine, amide, heterocycle, and substituted heterocycle; or R 8 and R 9 together with the carbon atom to which they are attached form a fused ring selected from heterocycle, substituted heterocycle, cycloalkyl, substituted cycloalkyl, aryl, and substituted aryl.

[0257] Article 43:R 7 But hydrogen, C 1-5 selected from alkyl and vinyl heterocycles; R 10 But hydrogen, C 1-5 selected from alkyl, amine, triazole, imidazole, amide, alkoxy, OCF3 and hydroxy; R 8 and R 9 are each independently hydrogen, C 1-5 or R is selected from alkyl, triazole, imidazole, amine, amide, alkoxy, OCF3, and hydroxy; 8 and R 9and R 1 and R 2 together with the carbon atom to which they are attached form a fused heterocycle or a substituted fused heterocycle.

[0258] Clause 44. The ENPP1 inhibitor according to any one of clauses 33 to 36, wherein Y is of the following formula: [ka] During the ceremony, R 7 is selected from H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, heterocycle, and substituted heterocycle; R 11 and R 12 are each independently selected from H, OH, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, -OCF3, halogen, amine, substituted amine, amide, heterocycle, and substituted heterocycle; or R 11 and R 12 together with the carbon atom to which they are attached form a fused ring selected from heterocycle, substituted heterocycle, cycloalkyl, substituted cycloalkyl, aryl, and substituted aryl.

[0259] Article 45.R 7 But hydrogen, C 1-5 Alkyl, substituted C 1-5 is selected from alkyl, vinylheterocycle and substituted vinyl-heterocycle; R 11 and R 12 are each independently hydrogen, C 1-5 Selected from alkyl, triazole, imidazole, amine, amide, alkoxy, halogen, OCF3 and hydroxy. or R 11 and R 12 and R 1 and R 2 together with the carbon atom to which they are attached form a fused heterocycle or a substituted fused heterocycle.

[0260] Article 46. The ENPP1 inhibitor according to any one of articles 33 to 36, wherein Y is a group of the following formula: [ka] During the ceremony, R 7 is selected from the group consisting of H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, heterocycle, and substituted heterocycle; R 11 and R 12 are each independently selected from the group consisting of H, OH, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, -OCF3, halogen, amine, substituted amine, amide, heterocycle, and substituted heterocycle; or R 11 and R 12 together with the carbon atom to which they are attached form a fused ring selected from heterocycle, substituted heterocycle, cycloalkyl, substituted cycloalkyl, aryl, and substituted aryl.

[0261] Article 47.R 7 But hydrogen, C 1-5 Alkyl, substituted C 1-5 is selected from alkyl, vinylheterocycle and substituted vinyl-heterocycle; R 11 and R 12 are each independently hydrogen, C 1-5 or R is selected from alkyl, triazole, imidazole, amine, amide, alkoxy, halogen, OCF3, and hydroxy; 11 and R 12 and together with the carbon atom to which they are attached form a fused heterocycle or a substituted fused heterocycle.

[0262] Clause 48. The ENPP1 inhibitor according to any one of clauses 1 to 47, wherein Y is selected from the following: [ka] [ka] [ka] [ka] and [ka]

[0263] Clause 49. The ENPP1 inhibitor according to any one of clauses 1 to 47, wherein Y is selected from the following: [ka] and [ka]

[0264] Clause 50. The ENPP1 inhibitor according to any one of clauses 1 to 30, wherein Y is of the following formula: [ka] During the ceremony, Z 1 and Z 2 are each independently selected from CH and N; R 1 is selected from H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, heterocycle, and substituted heterocycle; R 6 is selected from heterocycle, substituted heterocycle, cycloalkyl, substituted cycloalkyl, aryl, and substituted aryl.

[0265] Article 51. An ENPP1 inhibitor according to article 50 of the following formula: [ka] In the formula, Z 3 and Z 4 are each independently selected from CR and N, where R is H, alkyl or substituted alkyl.

[0266] Article 52. The ENPP1 inhibitor according to article 50 or 51, wherein Y is selected from the following: [ka] During the ceremony, Z 5 , Z 6 , Z 7 and Z 8 are each independently 14 and N, R 13 is selected from H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, heterocycle, and substituted heterocycle; Each R 14 is independently selected from H, OH, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, -OCF3, amine, substituted amine, amide, heterocycle, and substituted heterocycle; m is 0 to 5.

[0267] Article 53. The ENPP1 inhibitor according to article 50 or 51, wherein Y is selected from the following: [ka] During the ceremony, Z 9 , Z 10 and Z 11 are each independently 14 and N, R 13 is selected from H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, heterocycle, and substituted heterocycle; Each R 14is independently selected from H, OH, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, -OCF3, amine, substituted amine, amide, heterocycle, and substituted heterocycle; p is 0 to 4.

[0268] Clause 54. The ENPP1 inhibitor according to any one of clauses 50 to 53, wherein Y is selected from the following: [ka] and [ka]

[0269] Clause 55. The ENPP1 inhibitor according to any one of clauses 1 to 30, wherein Y is a group of the following formula: [ka] [ka] During the ceremony, Z 1 , Z 2 , Z 17 , Z 18 and Z 19 are each independently 20 and N, Each R 20 is independently selected from H, OH, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, -OCF3, amine, substituted amine, amide, heterocycle, and substituted heterocycle; p 1 is an integer from 0 to 4.

[0270] Article 56. The ENPP1 inhibitor according to article 55, wherein Y is of the following structure: [ka]

[0271] Clause 57. An ENPP1 inhibitor according to any one of clauses 1 to 56, wherein said compound is a compound selected from the compounds of Tables 1 and 2.

[0272] Clause 58. A pharmaceutical composition comprising an ENPP1 inhibitor according to any one of clauses 1 to 57, and a pharma- ceutical acceptable excipient.

[0273] Clause 59. A pharmaceutical composition for use in the treatment of cancer, comprising an ENPP1 inhibitor according to any one of clauses 1 to 57, and a pharma- ceutically acceptable excipient.

[0274] Clause 60. A method for inhibiting ENPP1, comprising contacting a sample with an ENPP1 inhibitor to inhibit the cGAMP hydrolysis activity of ENPP1.

[0275] Clause 61. The method of clause 60, wherein the ENPP1 inhibitor is a non-cell-permeable ENPP1 inhibitor.

[0276] Clause 62. The method of clause 60 or 61, wherein the sample is a cell sample.

[0277] Clause 63. The method of any one of clauses 60 to 62, wherein the sample contains cGAMP.

[0278] Clause 64. The method of clause 63, wherein cGAMP levels are elevated in said cell sample (e.g., relative to a control sample not contacted with said inhibitor).

[0279] Clause 65. The method of any one of clauses 60 to 64, wherein the cell-non-permeable ENPP1 inhibitor is an inhibitor according to any one of clauses 1 to 57.

[0280] Clause 66. A method for treating cancer, comprising administering to a subject having cancer a therapeutically effective amount of an ENPP1 inhibitor, thereby treating the cancer in the subject.

[0281] Clause 67. The method of clause 66, wherein the cancer is a solid tumor cancer.

[0282] Clause 68. The method of clause 66 or 67, wherein the cancer is breast cancer.

[0283] Clause 69. The method of any one of clauses 66 to 68, wherein said cancer is selected from adrenal, liver, kidney, bladder, breast, colon, stomach, ovary, 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.

[0284] Clause 70. The method of claim 66, wherein the cancer is lymphoma.

[0285] Clause 71. The method of claim 69, wherein the cancer is glioblastoma.

[0286] Clause 72. The method of any one of claims 66 to 71, further comprising administering one or more additional active agents.

[0287] Clause 73. The method of claim 72, wherein said one or more additional active agents is a chemotherapeutic agent or an immunotherapeutic agent.

[0288] Clause 74. The method of claim 72 or 73, wherein the one or more additional active agents are a small molecule, an antibody, an antibody fragment, an antibody drug conjugate, an aptamer, or a protein.

[0289] Clause 75. The method of any one of clauses 72 to 74, wherein the one or more further active agents comprises a checkpoint inhibitor.

[0290] Clause 76. The method of clause 75, 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.

[0291] Clause 77. The method of any one of clauses 72 to 76, wherein the one or more further active agents comprises a chemotherapeutic agent.

[0292] Clause 78. The method of clause 77, wherein said chemotherapeutic agent is a cGAMP-inducing chemotherapeutic agent.

[0293] Clause 79. The method of clause 78, wherein the cGAMP-inducing chemotherapeutic agent is an antimitotic or antitumor agent administered in an amount effective to induce production of cGAMP in said subject.

[0294] Clause 80. The method of any one of clauses 66 to 79, further comprising administering radiation therapy to the subject.

[0295] Clause 81. The method of claim 80, wherein the inhibitor is administered to the subject prior to radiation therapy.

[0296] Clause 82. The method of clause 80, wherein said inhibitor is administered after exposure of said subject to radiation therapy.

[0297] Clause 83. The method of clause 81 or 82, wherein said radiation therapy induces the production of cGAMP in said subject.

[0298] Clause 84. The method of any one of clauses 80 to 83, wherein said radiation therapy is administered at a dose and / or frequency effective to reduce radiation damage to said subject.

[0299] Clause 85. The method according to any one of claims 66 to 84, wherein the ENPP1 inhibitor is an inhibitor according to any one of clauses 1 to 57.

[0300] Clause 86. The method of clause 85, wherein the ENPP1 inhibitor is non-cell permeable.

[0301] Clause 87. The method of clause 85, wherein the ENPP1 inhibitor is cell-permeable.

[0302] Clause 88. A method for modulating an immune response in a subject, comprising administering to the subject a therapeutically effective amount of administering an ENPP1 inhibitor of any one of the preceding claims to treat the inflammatory condition in the subject.

[0303] Clause 89. The method according to claim 88, wherein the ENPP1 inhibitor is an inhibitor according to any one of clauses 1 to 57. EXAMPLES

[0304] The following examples are presented to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the embodiments of the present disclosure, and are not intended to limit the scope of the invention as contemplated by the inventors, 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.

[0305] Although the present invention has been described with reference to specific embodiments thereof, it will 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. In addition, 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 claims appended hereto.

[0306] Example 1a: Synthesis of Compound 1

[0307] Synthesis scheme [ka]

[0308] Preparation of dimethyl(2-(piperidin-4-yl)ethyl)phosphonate [ka]

[0309] Sodium hydride (2.16 g, 54.11 mmol) was carefully added to a stirred solution of bis(dimethoxyphosphoryl)methane (11.42 g, 49.19 mmol) in toluene (100 mL) at room temperature. The reaction mixture was then placed under a nitrogen atmosphere and a solution of 1-benzylpiperidine-4-carbaldehyde (10 g, 49.19 mmol) in toluene (50 mL) was added slowly, 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 (MgSO4) and evaporated to dryness. Chromatography (120 g of SiO2, 5-100% EtOAc gradient in hexanes) gave dimethyl (E)-(2-(1-benzylpiperidin-4-yl)vinyl)phosphonate (6.2 g, 16%) as a colorless oil.

[0310] 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 h, filtered, and evaporated to dryness under reduced pressure to give dimethyl (2-(piperidin-4-yl)ethyl)phosphonate (2.7 g, 100%) as a colorless oil.

[0311] Preparation of dimethyl(2-(1-(6,7-dimethoxyquinazolin-4-yl)piperidin-4-yl)ethyl)phosphonate [ka]

[0312] Diisopropylethylamine (0.6 g, 8.9 mmol) was added to a mixture of dimethyl (2-(piperidin-4-yl)ethyl)phosphonate (1.1 g, 4.9 mmol) and 4-chloro-6,7-dimethoxyquinazoline (1.0 g, 4.5 mmol) in isopropyl alcohol (20 mL). 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) gave 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).

[0313] Preparation of dimethyl(2-(1-(6,7-dimethoxyquinazolin-4-yl)piperidin-4yl)ethyl)phosphonic acid [ka]

[0314] Bromotrimethylsilane (3.67 g, 24 mmol) was added to a cold ice-bath 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). The reaction mixture was allowed to warm to room temperature and after 90 min was quenched 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 the volume, filtered to remove the precipitate and then evaporated to dryness. The residue was crystallized from dichloromethane, filtered and dried in 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).

[0315] Example 1b. General synthesis of substituted 2-(1-(quinazolin-4-yl)piperidin-4-yl)ethyl)phosphonic acid compounds. [ka]

[0316] (2-(1-(quinazolin-4-yl)piperidin-4-yl)ethyl)phosphonic acid was synthesized in a similar manner to compound 1, where dimethyl (2-(piperidin-4-yl)ethyl)phosphonate was reacted with a substituted 4-chloroquinazoline in the presence of a base such as diisopropylethylamine. The resulting adduct was deprotected using trimethylsilyl bromide in chloroform or neat trimethylsilyl iodide to give the desired phosphonate as shown in the table below. [Table 3-1] [Table 3-2] [Table 3-3]

[0317] Example 1c: Synthesis of isopropyl((2-(1-(6,7-dimethoxyquinazolin-4-yl)piperidin-4-yl)ethyl)(phenoxy)phosphoryl)-L-alaninate 77 and diphenyl(2-(1-(6,7-dimethoxyquinazolin-4-yl)piperidin-4-yl)ethyl)phosphonate 78. [ka]

[0318] A mixture of compound tert-butyl 4-(2-hydroxyethyl)piperidine-1-carboxylate (5.0 g, 21.8 mmol, 1.0 eq) and imidazole (2.23 g, 32.7 mmol, 1.5 eq) in DCM (50 mL) was stirred at room temperature for 5 min under nitrogen atmosphere. Then, a solution of I2 (8.3 g, 32.7 mmol, 1.5 eq) and PPh3 (8.6 g, 32.7 mmol, 1.5 eq) in DCM (20 mL) was added to the mixture. The mixture was stirred at room temperature for 10 min and filtered. The filtrate was diluted with DCM, washed with 5% Na2SO3 solution and brine, dried over Na2SO4, and concentrated in vacuum. The residue was purified by silica column chromatography (PE / EA, 6:1) to give compound tert-butyl 4-(2-iodoethyl)piperidine-1-carboxylate (6.1 g, 90%).

[0319] To a mixture of the compound diphenylphosphonate (15.6 g, 66.5 mmol, 5.0 equiv.) in CH3CN (45 mL), DBU (10.1 g, 66.5 mmol, 5.0 equiv.) was added. and the mixture was stirred at 0° C. for 10 minutes under nitrogen atmosphere. Then, tert-butyl 4-(2-iodoethyl)piperidine-1-carboxylate (4.5 g, 13.3 mmol, 1.0 equiv.) was added and the mixture was stirred at room temperature for another 2 hours. The mixture was concentrated to give a cloudy mixture, which was filtered to give the compound tert-butyl 4-(2-(diphenoxyphosphoryl)ethyl)piperidine-1-carboxylate (4.6 g, 75%).

[0320] To a solution of compound tert-butyl 4-(2-(diphenoxyphosphoryl)ethyl)piperidine-1-carboxylate (4.0 g, 8.97 mmol, 1.0 equiv) in methanol (40 mL) was added MeOH / HCl (5.0 M, 60 mL) and the mixture was stirred at room temperature for 3 h. The mixture was then evaporated to dryness. The residue was diluted with aqueous Na2CO3 and extracted with ethyl acetate. The organic layer was washed with brine, dried over Na2SO4, and concentrated to give compound diphenyl(2-(piperidin-4-yl)ethyl)phosphonate hydrochloride (3.4 g, 100%).

[0321] To a mixture of compound diphenyl(2-(piperidin-4-yl)ethyl)phosphonate hydrochloride (4.8 g, 13.8 mmol) in i-PrOH (100 mL) was added compound 5 (3.8 g, 16.8 mmol) and DIEA (5.4 g, 41.78 mmol). The mixture was stirred at 90° C. for 3 hours under nitrogen atmosphere. The mixture was concentrated. The residue was purified by column chromatography on silica gel (PE / EA, 1:1) to give compound 78 (2.2 g, 41%).

[0322] 1 H NMR (400 MHz, CDCl3): δ 8.64 (s, 1H), 7.33-7.06 (m, 12H), 4.20 (d, J = 8.0 Hz, 2H), 4.00 (s, 3H), 3.97 (s, 3H), 3.16 (t, J = 8.0 Hz, 2H), 2.18-2.10 (m, 2H), 1.79-1.71 (m, 4H), 1.57-1.51 (m, 1H), 1.45-1.39 (m, 2H).

[0323] To a mixture of compound 78 (1.59 g, 3 mmol, 1.0 equiv.) in THF (10 mL) and water (10 mL) was added sodium hydroxide (480 mg, 12 mmol, 4 equiv.) at room temperature. The mixture was stirred at room temperature for 12 h. The organic phase was removed under reduced pressure and the aqueous phase was adjusted to pH 1 with 1N HCl. The resulting solid was filtered and dried to give phenylhydrogen(2-(1-(6,7-dimethoxyquinazolin-4-yl)piperidin-4-yl)ethyl)phosphonate (1.3 g, 96%). A solution of the compound phenylhydrogen(2-(1-(6,7-dimethoxyquinazolin-4-yl)piperidin-4-yl)ethyl)phosphonate (1.4 g, 3.1 mmol, 1.0 equiv.), L-alanine isopropyl ester (1.04 g, 6.2 mmol) and TEA (620 mg, 6.2 mmol) in pyridine (20 mL) was heated to 60° C. for 5 min under N2. A solution of aldrithiol-2 (2.4 g, 10.9 mmol), PPh3 (2.9 g, 10.9 mmol) in pyridine (20 mL) was stirred at room temperature for 5 min and then added to the above solution at 60° C. under N2. The reaction was stirred for 12 h, concentrated and purified by FCC (CHCl:MeOH=20:1) to give 77 (200 mg, 11.4%). LCMS: [M+1] = 571.10

[0324] Example 1d: Synthesis of compound 72. Preparation of 2-(1-(6,7-dimethoxyquinazolin-4-yl)piperidin-4-yl)ethyl-N-diisopropylphosphanediamine 72. [ka]

[0325] A solution of compound 77 (700 mg, 1.8 mmol, 1.0 equiv), isopropylamine (319 mg, 5.4 mmol, 3 equiv) and triethylamine (364 mg, 3.6 mmol, 2 equiv) in pyridine (10 mL) was heated at 60° C. for 5 min under N2. A solution of aldrithiol-2 (1.4 g, 6.3 mmol, 3.5 equiv), PPh3 (1.7 g, 6.3 mmol, 3.5 equiv) in Py (10 mL) was stirred at room temperature for 5 min and then added to the above solution at 60° C. under N2. The mixture was stirred at 60° C. for 12 h. The mixture was then concentrated and purified by FCC (DCM:MeOH=20:1) to give 72 (200 mg, 24%). LCMS: [M+1] = 464.25. 1 H NMR (400 MHz, CDCl3) δ 8.63 (s, 1H), 7.38 (s, 1H), 7.07 (s, 1H), 4.32-4.29 (m, 2H), 4.03 (s, 3H), 3.98 (s, 3H), 3.42 (m, 2H), 3.10 (d, 2H), 2.00-1.42 (br m, 9H), 1.20 (s, 3H), 1.17 (s, 3H), 1.15 (s, 3H), 1.13 (s, 3H).

[0326] Example 1e: Preparation of O-((1-(6,7-dimethoxyquinazolin-4-yl)piperidin-4-yl)methyl) O,O-dihydrogen phosphorothioate 108. [ka]

[0327] A mixture of 4-chloro-6,7-dimethoxyquinazoline (900 mg, 4.018 mmol, 1.0 equiv) and piperidin-4-ylmethanol (508 mg, 4.420 mmol, 1.1 equiv) in i-PrOH (10 mL) was stirred at 100° C. for 16 h in a sealed tube. The progress of the reaction mixture was monitored by TLC. The reaction mixture was then concentrated under reduced pressure and the residue was purified by silica gel chromatography to give 4-(4-((λ 1 -Oxy To obtain 1 g (82%) of danyl)methyl)piperidin-1-yl)-6,7-dimethoxyquinazoline.

[0328] 4-(4-((λ 1 To a solution of (-oxidanyl)methyl)piperidin-1-yl)-6,7-dimethoxyquinazoline (100 mg, 0.330 mmol, 1.0 equiv) in dry pyridine (5 mL) was added phosphorothioyl trichloride (280 mg, 1.98 mmol, 6.0 equiv) at -15°C. After stirring at 0°C for 0.5 h, the mixture was poured into a solution of NaHCO3 (116 mg, 1.98 mmol, 6.0 equiv) in H2O (50 mL). The mixture was stirred at 0°C for 2 h. The progress of the reaction mixture was monitored by LCMS. The mixture was then concentrated under reduced pressure and the residue was purified by preparative HPLC to give compound 108 (10 mg, 86%) as a yellow solid. LCMS: [M+1] = 400.15. 1 H 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).

[0329] Example 1f: Preparation of O-(2-1-(6,7-dimethoxyquinazolin-4-yl)piperidin-4-yl)ethyl) O,O-dihydrogen phosphorothioate 109 [ka]

[0330] A mixture of 4-chloro-6,7-dimethoxyquinazoline (1 g, 4.46 mmol, 1.0 equiv) and piperidin-4-ylethanol (633 mg, 4.91 mmol, 1.1 equiv) in i-PrOH (10 mL) was stirred at 100° C. for 16 h in a sealed tube. The progress of the reaction mixture was monitored by TLC. The reaction mixture was then concentrated under reduced pressure and the residue was purified by silica gel chromatography to give 4-(4-(2-□ 1 4-(4-(2-Oxidaneyl)ethyl)piperidin-1-yl)-6,7-dimethoxyquinazoline was obtained (1.3 g, 91%). 1 To a solution of (-oxidanyl)ethyl)piperidin-1-yl)-6,7-dimethoxyquinazoline (150 mg, 0.473 mmol, 1.0 equiv) in dry pyridine (5 mL) was added 2-(piperidin-4-yl)ethan-1-ol (477 mg, 2.84 mmol, 6.0 equiv) at -15°C. After stirring at 0°C for 0.5 h, the mixture was poured into a solution of NaHCO3 (238 mg, 2.84 mmol, 6.0 equiv) in H2O (50 mL). The mixture was stirred at 0°C for 2 h. The progress of the reaction mixture was monitored by LCMS. The mixture was then concentrated under reduced pressure and the residue was purified by preparative HPLC to give compound 109 (16 mg, 8%) as a pale yellow solid. LCMS: [M +1] = 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).

[0331] [ka] A mixture of compound 4-chloro-6,7-dimethoxyquinazoline (600 mg, 2.68 mmol, 1.0 equiv.) and compound ethyl 2-(piperidin-4-yl)acetate (504 mg, 2.95 mmol, 1.1 equiv.) in i-PrOH (6 mL) was stirred at 100° C. for 16 hours in a sealed tube. The progress of the reaction mixture was monitored by TLC. 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%).

[0332] To a mixture of ethyl 2-(1-(6,7-dimethoxyquinazolin-4-yl)piperidin-4-yl)acetate (250 mg, 0.696 mmol, 1.0 equiv) in THF (10 mL / 5 mL) was added 2M NaOH (1 mL, 2.09 mmol, 3.0 equiv). The mixture was stirred at room temperature for 16 h. The progress of the reaction mixture was monitored by LCMS. The reaction mixture was then concentrated under reduced pressure to give the corresponding acid (200 mg, 86%).

[0333] To a mixture of the acid (300 mg, 0.906 mmol, 1.0 equiv) in THF (10 mL) was added NH2OH.HCl (76 mg, 1.09 mmol, 1.2 equiv), DIEA (468 mg, 3.63 mmol, 4.0 equiv) and BOP (481 mg, 1.09 mmol, 1.2 equiv). The mixture was stirred at room temperature for 16 h. The progress of the reaction mixture was monitored by TLC. The reaction mixture was then diluted with water and extracted with ethyl acetate. The organic layer was dried over anhydrous Na2SO4 and concentrated under reduced pressure. The residue was purified by silica gel chromatography to give 2-(1-(6,7-dimethoxyquinazolin-4-yl)piperidin-4-yl)-N-hydroxyacetamide 30 (180 mg, 77%) as a white solid. 1H NMR (400 MHz, D2O) δ 8.39 (s, 1H), 7.04 (s, 1H), 6.95 (s, 1H), 4.60 (d, J = 13.2 Hz, 2H), 3.89 (d, J = 16.7 Hz, 6H), 3.45 (t, J = 12.3 Hz, 2H), 2.63 (s, 1H), 1.96 (d, J = 11.7 Hz, 2H), 1.83-1.72 (m, 2H).

[0334] Example 1g: Preparation of (2-(1-(6,7-dimethoxyquinazolin-4-yl)piperidin-4-yl)ethyl)phosphonthio O,O-acid 83 [ka]

[0335] To a solution of compound diethylphosphonate (10 g, 72.46 mmol, 1.0 equiv.) in toluene (1000 mL), Lawesson's reagent (29.3 g, 72.46 mmol, 1.0 equiv.) was added under nitrogen atmosphere. The mixture was stirred at 110° C. for 16 h. The progress of the reaction mixture was monitored by TLC. The mixture was then filtered and concentrated under reduced pressure. The residue was purified by silica gel chromatography to obtain O,O-diethylphosphonothioate (3.4 g, 25%). To a solution of O,O-diethylphosphonothioate (1 g, 6.49 mmol, 1.5 equiv.) in MeCN (1 L), DBU (3.29 g, 21.65 mmol, 5.0 equiv.) was added. After stirring at 0° C. for 10 min, tert-butyl 4-(2-iodoethyl)piperidine-1-carboxylate (1.47 g, 4.33 mmol, 1.0 equiv) was added slowly. The mixture was allowed to warm to room temperature and stirred for 1 h. The progress of the reaction mixture was monitored by TLC. The mixture was then concentrated under reduced pressure and the residue was purified by silica gel chromatography to give tert-butyl 4-(2-(diethoxyphosphorothioyl)ethyl)piperidine-1-carboxylate (500 mg, 31%). A solution of tert-butyl 4-(2-(diethoxyphosphorothioyl)ethyl)piperidine-1-carboxylate (500 mg, 1.37 mmol, 1.0 equiv) in TFA / DCM (10 mL / 10 mL) was stirred at room temperature for 1 h. The progress of the reaction mixture was monitored by TLC. The mixture was then concentrated under reduced pressure to give crude O,O-diethyl(2-(piperidin-4-yl)ethyl)phosphonothioate (400 mg, 100%). To a stirred solution of O,O-diethyl(2-(piperidin-4-yl)ethyl)phosphonothioate (400 mg, 1.51 mmol, 0.84 equiv.) and DIEA (927 mg, 7.19 mmol, 4.0 equiv.) in DMSO (10 mL) was added compound 1-1 (403 mg, 1.80 mmol, 1.0 equiv.) under nitrogen atmosphere. The mixture was stirred at 80° C. for 16 h. The progress of the reaction mixture was monitored by TLC. The mixture was then diluted with water and extracted with ethyl acetate. The organic layer was dried over anhydrous Na2SO4 and concentrated under reduced pressure.The residue was purified by silica gel chromatography to give 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, 1.0 equiv) in TMSI (7 mL) was stirred at 60° C. for 16 h. The progress of the reaction mixture was monitored by TLC. The mixture was then diluted with water and extracted with ethyl acetate. The organic layer was dried over anhydrous Na2SO4 and concentrated under reduced pressure. The residue was purified by preparative HPLC to give (2-(1-(6,7-dimethoxyquinazolin-4-yl)piperidin-4-yl)ethyl)phosphonthio O,O-acid 83 (13 mg, 32%) as a white solid. LCMS: [M+1] = 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).

[0336] Example 1h: Preparation of (2-((1,4-cis)-4-(6,7-dimethoxyquinazolin-4-yl)cyclohexyl)ethyl)phosphonic acid 81 and (2-((1,4-trans)-4-(6,7-dimethoxyquinazolin-4-yl)cyclohexyl)ethyl)phosphonic acid 82 [ka]

[0337] To a solution of 60% NaH (5.54 g, 64.1 mmol, 1.0 equiv) in anhydrous THF (1000 mL) was added ethyl 2-(diethoxyphosphoryl)acetate (12.7 mL, 64.10 mmol, 1.0 equiv) dropwise at 0° C. under nitrogen atmosphere. After stirring for 0.5 h, 1,4-dioxaspiro[4.5]decan-8-one (10 g, 64.10 mmol, 1.0 equiv) was added dropwise. The mixture was allowed to warm to room temperature and stirred under nitrogen atmosphere for 2 h. The progress of the reaction mixture was monitored by TLC. The mixture was then diluted with saturated Et2O and extracted with water. The combined organic phase was washed with brine (3×500 mL), dried over anhydrous Na2SO4, and concentrated under reduced pressure to give the crude ethyl ester (13.9 g, 95%) as a colorless oil. To a stirred solution of the crude ethyl ester (13.9 g, 61.50 mmol, 1.0 equiv) in MeOH (200 mL) was added HCOONH4 (34.9 g, 0.554 mol, 9.0 equiv) and 10% Pd / C (2.09 g, 15% w / w). The mixture was stirred at reflux for 1.5 h. The progress of the reaction mixture was monitored by LCMS. After cooling, the reaction mixture was filtered through a pad of Celite and a sintered funnel and concentrated under reduced pressure. The residue was dissolved in DCM and washed with water. The organic layer was dried over anhydrous Na2SO4 and concentrated under reduced pressure to give the saturated ethyl ester (12.43 g, 88%) as a colorless oil.

[0338] To a mixture of ethyl ester (12.43 g, 54.52 mmol, 1.0 equiv) in dry THF (150 mL) was added LiAlH4 (2.5 M in THF, 17.4 mL, 43.61 mmol, 0.8 equiv) at -78 °C. The mixture was allowed to warm to room temperature and stirred under nitrogen for 2.5 h. The progress of the reaction mixture was monitored by LCMS. Na2SO4.H2O was then added portionwise at -20 °C until gas evolution ceased. The mixture was filtered through a pad of Celite and concentrated under reduced pressure to give the crude alcohol (10.27 g, 100%) as a white solid. A solution of PPh3 (21.7 g, 82.82 mmol, 1.5 equiv), imidazole (5.6 g, 82.82 mmol, 1.5 equiv) in DCM (150 mL) was stirred at room temperature for 5 min. I2 (21 g, 82.82 mmol, 1.5 equiv) was then added and stirred for 10 min, after which 2-(1,4-dioxaspiro[4.5]decan-8-yl)ethan-1-ol (10.27 g, 55.22 mmol, 1.0 equiv) was added. The mixture was stirred for 2 h. The progress of the reaction mixture was monitored by TLC. The mixture was concentrated under reduced pressure and the residue was purified by silica gel chromatography to give 8-(2-iodoethyl)-1,4-dioxaspiro[4.5]decane (12.35 g, 75%).

[0339] To a solution of dibenzylphosphonate (32.8 g, 0.125 mol, 3.0 equiv) in MeCN (200 mL) was added DBU (31.7 g, 0.209 mol, 5.0 equiv) at 0° C. After stirring for 30 min, I2 (21 g, 82.82 mmol, 1.5 equiv) was added and stirred for 10 min, followed by a solution of 8-(2-iodoethyl)-1,4-dioxaspiro[4.5]decane (12.35 g, 41.72 mmol, 1.0 equiv) in ACN (70 mL). The mixture was stirred for 16 h. The progress of the reaction mixture was monitored by TLC. The mixture was concentrated under reduced pressure and the residue was purified by silica gel chromatography to give the mixed phosphone intermediate (18.35 g, 100%).

[0340] To a solution of this phosphonate (18.35 g, 42.67 mmol, 3.0 equiv) in EtOH (200 mL) was added 2 M HCl (200 mL) at 0 °C. The mixture was then warmed to room temperature and stirred for 2 h. The progress of the reaction mixture was monitored by TLC. The mixture was neutralized with K2CO3 and extracted with ether. The organic layer was dried over anhydrous Na2SO4 and concentrated under reduced pressure. The residue was purified by silica gel chromatography to give dibenzyl (2-(4-oxocyclohexyl)ethyl)phosphonate (7.56 g, 45%). To a solution of the compound dibenzyl(2-(4-oxocyclohexyl)ethyl)phosphonate (3 g, 7.77 mmol, 1.0 equiv.) and 1,1,1-trifluoro-N-phenyl-N-((trifluoromethyl)sulfonyl)methanesulfonamide (3.6 g, 10.10 mmol, 1.3 equiv.) in THF (30 mL) was added LiHMDS (1 M in THF, 10.1 mL, 10.10 mmol, 1.3 equiv.) dropwise at -78°C. The mixture was stirred at -78°C for 4 h. The mixture was then allowed to warm to room temperature and stirred for 16 h. The progress of the reaction mixture was monitored by TLC. The mixture was quenched with NH4Cl and extracted with ethyl acetate. The organic layer was dried over anhydrous Na2SO4 and concentrated under reduced pressure. The residue was purified by silica gel chromatography (PE / EA, 3:1 to 1:1) to give 4-(2-(bis(benzyloxy)phosphoryl)ethyl)cyclohex-1-en-1-yl trifluoromethanesulfonate (2.15 g, 53%). To a mixture of 4-(2-(bis(benzyloxy)phosphoryl)ethyl)cyclohex-1-en-1-yl trifluoromethanesulfonate (2.15 g, 4.15 mmol, 1.0 equiv.) in dioxane (20 mL) was added B2Pin2 (1.37 g, 5.40 mmol, 1.3 equiv.), Pd(dppf)Cl2 (364 mg, 0.415 mmol, 0.1 equiv.) and KOAc (1.22 g, 12.45 mmol, 3.0 equiv.). The mixture was stirred at 90 °C for 16 h under nitrogen atmosphere. The progress of the reaction mixture was monitored by TLC. The mixture was then filtered under reduced pressure through a Celite pad and a sintered funnel to give crude dibenzyl(2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)cyclohexa-3,4-dioxaborolan-2-yl)cyclohexa-3. This afforded the 3-en-1-yl)ethyl)phosphonate which was used directly in the next step.

[0341] To a mixture of dibenzyl (2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)cyclohex-3-en-1-yl)ethyl)phosphonate from the previous step in dioxane (20 mL) was added 4-chloro-6,7-dimethoxyquinazoline (1.2 g, 5.40 mmol, 1.3 equiv), Pd(dppf)Cl2 (364 mg, 0.415 mmol, 0.1 equiv) and KOAc (1.22 g, 12.45 mmol, 3.0 equiv). The mixture was stirred at 90° C. for 16 h under nitrogen atmosphere. The progress of the reaction mixture was monitored by TLC. The mixture was then filtered under reduced pressure through a celite pad and a sintered funnel to give the crude dibenzylphosphonate (3.4 g, 100%) which was used directly in the next step.

[0342] To a solution of the crude dibenzylphosphonate (1.7 g, 3.05 mmol, 1.0 equiv) in MeOH (100 mL) was added Pd / C (340 mg, 20% w / w) under nitrogen atmosphere. The mixture was exchanged with hydrogen three times and stirred at 40° C. for 16 h. The progress of the reaction mixture was monitored by LCMS. The mixture was then filtered through a celite pad and a sintered funnel and Pd / C (340 mg, 20% w / w) was added under nitrogen atmosphere. The mixture was exchanged with hydrogen three times and stirred at 40° C. for 16 h. LCMS analysis of the reaction mixture showed complete conversion to the desired product. The mixture was filtered through a celite pad and a sintered funnel and the filtrate was concentrated under reduced pressure. The residue was purified by preparative HPLC to give (2-((1,4-cis)-4-(6,7-dimethoxyquinazolin-4-yl)cyclohexyl)ethyl)phosphonic acid 81 (78 mg, 6%, white solid) and (2-((1,4-trans)-4-(6,7-dimethoxyquinazolin-4-yl)cyclohexyl)ethyl)phosphonic acid 82 (185 mg, 16%, white solid).

[0343] Compound 81.LCMS: [M+1] = 381.25. 1H NMR (400 MHz, DMSO-d6) δ 8.94 (s, 1H), 7.46 (s, 1H), 7.29 (s, 1H), 3.94 (d, J = 6.1 Hz, 6H), 1.83 (d, J = 10.2 Hz, 4H), 1.67 (d, J = 12.1 Hz, 2H), 1.50 (dd, J = 33.3, 15.5 Hz, 4H), 1.22 (d, J = 18.6 Hz, 4H).

[0344] Compound 82.LCMS: [M+1] = 381.25. 1 H NMR (400 MHz, DMSO-d6) δ 8.98 (s, 1H), 7.47 (s, 1H), 7.30 (s, 1H), 3.94 (d, J = 4.5 Hz, 6H), 1.86 (d, J = 11.0 Hz, 2H), 1.76-1.56 (m, 10H), 1.52-1.42 (m, 2H).

[0345] Example 1i: Preparation of (4-(6,7-dimethoxyquinazolin-4-yl)piperidyl)methyl dihydrogen phosphate 60 [ka]

[0346] (4-(6,7-dimethoxyquinazolin-4-yl)piperidyl)methanol (100 mg, 0.33 mmol, 1.0 equiv) was dissolved in dry pyridine (3 mL), which was then cooled to -15 °C and stirred for 10 min. POCl3 (253 mg, 1.65 mmol, 5.0 equiv) was added dropwise under N2 atmosphere. The reaction temperature was slowly raised to 0 °C and then stirred for another 30 min. When LC-MS showed that compound 3 was completely consumed, the mixture was poured into NaHCO3 solution (160 mg in 50 mL water) at 0 °C. The desired compound was extracted with DCM (5 × 10 mL). The organic phase was concentrated to give a residue, which was purified by preparative HPLC to give (4-(6,7-dimethoxyquinazolin-4-yl)piperidyl)methyl dihydrogenphosphate 60 (70 mg, 55%) as a white powder after lyophilization. LC-MS: 384.20 [M+1] + . 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).

[0347] Example 1j: Preparation of 2-(4-(6,7-dimethoxyquinazolin-4-yl)piperidyl)ethyl dihydrogen phosphate 85 [ka]

[0348] 2-(4-(6,7-dimethoxyquinazolin-4-yl)piperidyl)ethan-1-ol (340 mg, 1.07 mmol, 1 equiv.) was dissolved in 10 mL of dry pyridine, which was then cooled to -15°C and stirred for 10 min. POCl3 (821 mg, 5.4 mmol, 5 equiv.) was added dropwise under N2 atmosphere. The reaction temperature was slowly raised to 0°C and then stirred for another 30 min. The mixture was poured into NaHCO3 solution (800 mg in 250 mL of water) at 0°C. The desired compound was extracted with DCM. The organic phase was concentrated and purified by preparative HPLC to give 2-(4-(6,7-dimethoxyquinazolin-4-yl)piperidyl)ethyl dihydrogen phosphate (52 mg, white powder, 12%). LC-MS: 398 [M+1] + . 1 H NMR (400 MHz, DMSO-d6) δ 8.54 (s, 1H), 7.16 (d, J = 25.4 Hz, 2H), 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), 1.44-1.32 (m, 2H).

[0349] Example 1k: Preparation of ((1-(6,7-dimethoxyquinazolin-4-yl)piperidin-4-yl)methyl)phosphonic acid 88 [ka]

[0350] A solution of PPh3 (3.39 g, 15 mmol, 1.5 eq.) and imidazole (1.02 g, 15 mmol, 1.5 eq.) in dry DCM (40 mL) was stirred in ice water for 10 min, then I2 (3.8 g, 15 mmol, 1.5 eq.) was added. It was stirred for 10 min under nitrogen atmosphere, after which (1-(6,7-dimethoxyquinazolin-4-yl)piperidin-4-yl)methanol (10 mmol) was added. The ice water was removed. The mixture was stirred for 10 min. It was then kept at room temperature overnight. After it was consumed, Na2S2O3 solution was added and stirred for 10 min. It was extracted with DCM, washed with water and brine, then dried over Na2SO4. 4-(4-(iodomethyl)piperidin-1-yl)-6,7-dimethoxyquinazoline (2.8 g, 56%) was obtained as a pale yellow solid after recrystallization with methanol. LC-MS: 414.3 [M+1] + . 1 H 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).

[0351] 4-(4-(iodomethyl)piperidin-1-yl)-6,7-dimethoxyquinazoline (9.5 g, 36.3 mmol, 3 eq.) was dissolved in 40 mL of dry MeCN, which was then cooled to 0° C. DBU (9.2 g, 60.5 mmol, 5 eq.) was added dropwise and then stirred for 10 min. Bis(benzyloxy)(oxo)-λ 4 The bis(benzyloxy)(oxo-λ)phosphane was dissolved in 20 mL of CAN. 4 -Phosphane solution The mixture was added dropwise at 0° C. The mixture was stirred overnight. The mixture was concentrated in vacuo. The residue was dissolved in EtOAc and then washed with water and brine. The dibenzyl ((1-(6,7-dimethoxyquinazolin-4-yl)piperidin-4-yl)methyl)phosphonate (1.1 g, colorless oil, 18%) was obtained by FCC eluting with DCM:MeOH (50:1). LC-MS: 548.20 [M+1] + . 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).

[0352] A mixture of dibenzyl((1-(6,7-dimethoxyquinazolin-4-yl)piperidin-4-yl)methyl)phosphonate (660 mg, 1.2 mmol, 1.0 equiv) and Pd / C (132 mg, 20% w / w) in CHOH (20 mL) was stirred at room temperature for 4 h under H2. Upon complete consumption of compound 4, the mixture was filtered through a pad of Celite. The filtrate was concentrated. The residue was purified by preparative HPLC to give ((1-(6,7-dimethoxyquinazolin-4-yl)piperidin-4-yl)methyl)phosphonic acid 88 (125 mg, 28%) as a pale yellow solid. LCMS: 368.10 [M+1] + . 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).

[0353] Example 2: Evaluation of Compound Activity

[0354] Selected compounds from Table 1, Table 2 and other derivatives were prepared and evaluated in ENPP1 activity assay using thymidine monophosphate paranitrophenol (TMP-pNP) as substrate. Enzyme reactions were prepared with TMP-pNP (2 μM), 5-fold dilutions of ENPP1 inhibitors, and purified recombinant mouse ENPP1 (0.5 nM) in 100 mM Tris, 150 mM NaCl, 2 mM CaCl2, 200 μM ZnCl2 at pH 7.5 at room temperature. Reaction progress was monitored by measuring the absorbance at 400 nm of paranitrophenolate formed over a 20-minute reaction. The IC was calculated by extracting, plotting, and fitting the slope of product formation using Graphpad Prism 7.03. 50 Got the value.

[0355] Compounds can also be used as substrates 32 ENPP1 enzyme activity was also assessed using P cGAMP. 32 PcGAMP 32 P-ATP doped unlabeled ATP (1 mM) and GTP (1 mM) were synthesized by incubation with 2 μM purified recombinant porcine cGAS in 20 mM Tris pH 7.5, 2 mM MgCl2, 100 μg / mL herring testis DNA overnight at room temperature, and the remaining nucleotide starting material was degraded with alkaline phosphatase at 37 °C for 4 h. 32P-cGAMP (5 μM) was incubated with purified recombinant mouse ENPP1 (20 nM) in 100 mM Tris, 150 mM NaCl, 2 mM CaCl2, 200 μM ZnCl2, pH 7.5 at room temperature for 5 h. Five-fold dilutions of ENPP1 inhibitors were included in the reactions to generate enzyme inhibition curves. Degradation was assessed by TLC (as described by Li et al. Nat. Chem. Biol. (2014) 10:1043-8). Plates were exposed to phosphorscreen (Molecular Dynamics) and imaged on a Typhoon 9400. 32 P signals were quantified using ImageJ. Inhibition curves were fitted using ad Prism 7.03, and IC 50 The IC values ​​of the compounds investigated were 50 The IC 50 The values ​​are in the range indicated by the letters A to D, where A is the IC 50 A represents an IC value less than 500 nM, B represents an IC 50 The values ​​represent 500 nM to 5 µM, and C is the IC 50 The values ​​represent 5 μM to 10 μM, and D is the IC 50 Values ​​above 10 μM (nd = undetermined). [Table 4-1] [Table 4-2]

[0356] Example 3: Demonstration of extracellular ENPP1 and inhibition of extracellular ENPP1

[0357] With reference to FIGS. 1A-1C, it was found that ENPP1 controls extracellular levels of cGAMP, and that cGAMP levels could be restored by treating cells with an exemplary ENPP1 inhibitor (compound 1).

[0358] 293T cGAS ENPP1 - / -The cells were transfected with a human ENPP1 expression plasmid, and cGAMP hydrolase activity was confirmed in whole cell lysates (Fig. 1A). 293T cells were purchased from ATCC and transfected with a virus to stably express mouse cGAS. 293T mcGAS ENPP1 - / - ENPP1 was generated by viral transfection of CRISPR sgRNA targeting human ENPP1 (5'CACCGCTGGTTCTATGCACGTCTCC-3') (SEQ ID NO: 1). 293T mcGAS ENPP1 was transfected onto tissue culture-treated plates coated with DMEM (Corning Cellgro) containing PurCol (Advanced BioMatrix) and supplemented with 10% FBS (Atlanta Biologics) (v / v) and 100 U / mL penicillin-streptomycin (ThermoFisher). - / - Cells were seeded. 12–24 h after seeding, cells were transfected with Fugene6 (Promega) according to the manufacturer's instructions and the indicated concentrations of pcDNA3 plasmid DNA (empty or containing human ENPP1). 24 h after transfection Cells were lysed for analysis of ENPP1 expression by Western blotting (rabbit anti-ENPP1 (L520, 1:1000) and mouse anti-tubulin (DM1A, 1:2,000) antibodies, Cell Signaling Technologies). Total cell lysates were 1 × 10 6 Cells were lysed in 10 mM Tris, 150 mM NaCl, 1.5 mM MgCl2, 1% NP-40, pH 9.0. 32 P-cGAMP (5 μM) was incubated with whole cell lysates and degradation was monitored as described in Example 2 above (FIG. 1A).

[0359] In intact cells, expression of ENPP1 depletes extracellular cGAMP but does not affect intracellular cGAMP concentrations (Figure 1B). - / -24 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 cells were washed off the plate with cold PBS. Both medium and cells were centrifuged at 1000 rcf for 10 min at 4°C and prepared for cGAMP concentration measurement by liquid chromatography-tandem mass spectrometry (LC-MS / MS). Cells were incubated with 500 nM cyclic GMP- 13 C 10 , 15 The cells were dissolved 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 gel was supplemented with N5-AMP and 20% formic acid. Samples were analyzed for cGAMP, ATP, and GTP content on a Shimadzu HPLC (San Francisco, CA) connected to an AB Sciex 4000 QTRAP (Foster City, CA) with an autosampler set at 4°C. A volume of 10 μL 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 in acetonitrile (B). The initial condition was 90% B and held for 0.5 min. The mobile phase was ramped to 30% A from 0.5 to 2.0 min, held at 30% A from 2.0 to 3.5 min, ramped to 90% B from 3.5 to 3.6 min, and held 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 electrode spray positive ion mode with the ion source temperature set at 500 °C. Declustering and collision induced dissociation were performed using nitrogen gas. Declustering potential and collision energy were optimized by standard direct infusion. For each molecule, the MRM transition(s) (m / z), DP (V), and CE (V) were as follows: The following are the internal standard: ATP (508>136, 341, 55), GTP (524>152, 236, 43), cGAMP (675>136, 121, 97, 675>312, 121, 59, 675>152, 121, 73), 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).

[0360] Inhibiting ENPP1 blocks the degradation of extracellular cGAMP (Figure 1C). The same experiment as above was performed, but this time, an exemplary ENPP1 inhibitor (compound 1) was also included at 50 μM during the medium change. This inhibitor restored the extracellular cGAMP concentration in the medium to its previous level.

[0361] Figure 1A shows the results of transfection with the empty vector and the vector containing human ENPP1. After 24 hours, Western blot was used to measure ENPP1 protein expression (top) and thin layer chromatography (TLC) was used to measure ENPP1 expression. 32 P-cGAMP hydrolysis activity (bottom) 293T cGAS ENPP1 analyzed - / - Figure 1B shows intracellular and extracellular cGAMP concentrations using LC-MS / MS. BQL = below limit of quantification. Mean ± SEM (n = 2). ** p = 0.005 (Student's t test). Figure 1C shows the results of 293T cells transfected with an empty vector or a vector containing human ENPP1. cGAS ENPP1 - / - Figure 1 shows the intracellular and extracellular cGAMP concentrations of cells in the presence or absence of 50 μM compound 1. BQL = below limit of quantification. Mean ± SEM (n = 2). ** p=0.0013 (Student's t-test).

[0362] Example 4: ENPP1 inhibition increases cGAMP activation of primary CD14+ monocytes

[0363] Using an exemplary ENPP1 inhibitor (compound 1), 293T cGAS ENPP1 low cGAMP exported by the cell line is expressed by antigen-presenting cells (APCs), e.g., human CD14 + We investigated whether 293T cGAS ENPP1 could be detected by monocytes (Figure 2A). lowCells were transfected with pcDNA (empty or containing human ENPP1). Primary human peripheral blood mononuclear cells (PBMCs) were isolated by subjecting enriched buffy coats from whole blood to a density gradient of Percoll. CD14 + Monocytes are CD14 + Isolation was performed using MicroBeads (Miltenyi). CD14 + Monocytes were cultured in RMPI supplemented with 2% human serum and 100 U / mL penicillin-streptomycin. low Eight hours after transfection of the cells, the medium was replaced with RMPI supplemented with 2% human serum and 100 U / mL penicillin-streptomycin, with or without the exemplary ENPP1 inhibitor, Compound 1. 24 hours after medium replacement, 293T cGAS ENPP1 low The supernatant of cells was analyzed by CD14 + monocytes (Figure 2A). 24–26 h after supernatant transfer, total RNA was extracted using Trizol (Thermo Fisher Scientific) and reverse transcribed with Maxima H Minus Reverse Transcriptase (Thermo Fisher Scientific). Real-time RT-PCR was performed in duplicate with AccuPower 2X Greenstar qPCR Master Mix (Bioneer) on a 7900HT Fast Real-Time PCR System (Applied Biosystems). For each sample, data were normalized to CD14 expression. Fold induction was calculated using ΔΔCt. Primers for human IFNB1: fwd(5'-AAACTCATGAGCAGTCTGCA-3') (sequence number 2), rev(5'-AGGAGATCTTCAGTTTCGGAGG-3') (sequence number 3), human CD14: fwd(5'-GCCTTCCGTGTCCCCACTGC-3') (sequence number 4), rev(5'-TGAGGGGGCCCTCGACG-3') (sequence number 5).

[0364] cGAS-expressing 293T but not cGAS-null 293T cells cGAS ENPP1 low The cell supernatant induced CD14+IFNB1 expression, and extracellular cGAMP exported by cancer cells promoted CD14 as a signaling factor. + These results suggest that ENPP1 can be detected by 293T cGAS cells (Figure 2B). low Transient overexpression of ENPP1 in cells inhibits the degradation of extracellular cGAMP and the expression of CD14 + Addition of compound 1 caused a decrease in IFNB1 expression, but restored extracellular cGAMP levels and upregulated CD14 + IFNB1 expression was induced ( Fig. 2B ).

[0365] Referring to FIG. 1A, a schematic of the supernatant transfer experiment is shown. FIG. 2B shows the results of the analysis of cGAS null 293T cells or 293T cGAS ENPP1 cells transfected with DNA and incubated in the presence or absence of Compound 1. low The supernatants from these cells were cultured as primary CD14 + IFNB1 mRNA levels were normalized to CD14 and fold induction was calculated using the ratio of IFNB1 mRNA levels to CD14 levels in untreated human PBMCs. + Calculated for cells, mean ± SEM (n=2). * P < 0.05, *** P < 0.001( one-way analysis of variance).

[0366] Example 5: Combining ENPP1 inhibition and ionizing radiation (IR) treatment to increase tumor-associated dendritic cells.

[0367] We investigated whether cancer cell lines export cGAMP and whether ionizing radiation (IR) affects the levels of extracellular cGAMP produced. Ionizing radiation (IR) has been found to increase cytoplasmic 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). 24 hours after seeding, 4T1 cells were treated with 20 Gy of IR using a cesium source, and the medium was replaced and supplemented with 50 uM of an exemplary ENPP1 inhibitor, compound 1, to inhibit ENPP1 present in the cell culture. The medium was collected at the indicated time points, centrifuged at 1000 × g to remove residual cells, acidified with 0.5% acetic acid, and cyclically diluted with 100% acetic acid as an extraction standard. 13 C 10 , 15 5-GMP- 13 C 10 , 15 The medium was supplemented with N5-AMP (2 μM in 100 μL appropriate for final concentration). The medium was loaded onto a HyperSep Aminopropyl SPE column (ThermoFisher Scientific) and cGAMP was enriched as previously described (Gao et al., Proc. Natl. Acad. Sci. USA (2015) 112:E5699-705). The eluent was evaporated to dryness and reconstituted with 50:50 acetonitrile:water supplemented with 500 nM internal standard. The medium was subjected to mass spectrometry quantification of cGAMP.

[0368] Continuous cGAMP export was detected in 4T1 cells over a 48-h period, at which point cells treated with IR had significantly higher extracellular cGAMP levels than untreated ones.

[0369] We next examined the effect of IR in combination with compound 1, an exemplary ENPP1 inhibitor, on the number of tumor-associated dendritic cells in a murine 4T1 tumor model (Figure 3B). Seven- to nine-week-old female Balb / c mice (Jackson Laboratories) were inoculated with 1 × 10 64T1-luciferase tumor cells were inoculated via the mammary fat pad in 50 μL of PBS. Two days after injection, tumors were irradiated with 20 Gy using a 225 kVp X-ray irradiation cabinet (IC250, Kimtron Inc., CT) filtered with 0.5 mm Cu. Anesthetized animals were shielded with a 15×20 mm opening for tumor placement in a 3.2 mm lead shield. Mice were injected intratumorally with 100 μL of PBS containing 1 mM compound 1 or PBS alone. The next day, the tumors were excised and incubated with 20 μg / mL DNase I type IV (Sigma-Aldrich) and 1 mg / mL collagenase from Clostridium histolyticum (Sigma-Aldrich) in RPMI+10% FBS for 30 min at 37°C. Tumors were passed through a 100 μm cell strainer (Sigma-Aldrich) and red blood cells were lysed with red blood cell lysis buffer (155 mM NH4C1, 12 mM NaHCO3, 0.1 mM EDTA) for 5 min at room temperature. Cells were stained with Live / Dead fixable near-infrared dead cell staining kit (Thermo Fisher Scientific) and Fc blocked with TruStain fcX for 10 min, followed by antibody staining for CD11c, CD45, and IA / IE (all Biolegend). Cells were analyzed using a SH800S cell sorter (Sony) or an LSR II (BD Biosciences). Data were analyzed using FlowJo V10 software (Treestar) and Prism 7.04 software (Graphpad) for statistical analysis, and statistical significance was assessed using unpaired t-tests with Welch's correction.

[0370] Intratumoral injection of compound 1 did not alter tumor-associated leukocyte composition compared to the PBS control (Figure 1). 3B), suggesting that ENPP1 does not play a substantial role in removing basal levels of extracellular cGAMP in this tumor model. However, when tumors were pretreated with IR, compound 1 downregulated tumor-associated CD11c + It was found that the number of

[0371] These results are shown in Figures 3A and 3B. Figure 3A shows the extracellular cGAMP produced by 4T1 cells over a 48-hour period. At time 0, cells were left untreated or treated with 20 Gy of IR and filled with medium supplemented with 50 μM of Compound 1. Mean ± SEM (n=2). ** P = 0.004 (Student's t test). Figure 3B shows the effect of 4T1 cells (1 × 10 6 ) are shown. On day 2, tumors were left untreated or treated with 20 Gy of 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).

[0372] Example 6: ENPP1 inhibition synergizes with IR therapy and anti-CTLA-4 to exert antitumor effects

[0373] We investigated whether immune detection and tumor elimination could be enhanced by further increasing extracellular cGAMP in vivo using ionizing radiation (IR) and an exemplary ENPP1 inhibitor, e.g., Compound 1.

[0374] Female Balb / c mice (Jackson Laboratories), aged 7 to 9 weeks, were injected with 5 × 10 4 4T1-luciferase cells were suspended in 50 μL of PBS and inoculated into the mammary fat pad. Tumor volume (length x width x height) was 2 × width / 2) is 80mm 3 ~120mm 3When the tumors reached IR, they were irradiated with 20 Gy using a 225 kVp X-ray irradiation cabinet (IC250, Kimtron Inc., CT) filtered with 0.5 mm Cu. Anesthetized animals were shielded with a 15 x 20 mm opening for tumor placement in a 3.2 mm lead shield. On days 2, 4, and 7 after IR, 100 μL of 100 μM compound 1 and / or 10 μg cGAMP in PBS or PBS alone was injected intratumorally. Alternatively, 1 mM compound 1 in PBS or PBS alone was injected intratumorally and 200 μg anti-CTLA-4 antibody or Syrian hamster IgG antibody (both BioXCell) was injected intraperitoneally on days 2, 5, and 7 after IR. Mice from different treatment groups were placed together in each cage to eliminate cage effects. Experimenters were blinded throughout the study. Tumor volumes were recorded every other day. Tumor volumes were analyzed with generalized estimating equations to account for intra-mouse correlation. Pairwise comparisons of treatment groups at each time point were performed using post-hoc tests with Tukey adjustment for multiple comparisons. Animal deaths were plotted in 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 Committee on Laboratory Animal Care.

[0375] Administration of compound 1 improved the tumor shrinkage effect of IR treatment, but not significantly (Figure 4A). Intratumoral injection of cGAMP had no effect on IR treatment, but injection of compound 1 in addition to cGAMP synergistically shrank tumors, extended survival, and achieved a cure rate of 10% (Figures 4A and 4B).

[0376] We also investigated synergistic effects with anti-CTLA-4, an adaptive immune checkpoint blocker. Without IR, treatment with anti-CTLA-4 and compound 1 did not affect survival extension (Figure 4C). However, combining IR pretreatment with compound 1 and anti-CTLA-4 exerted significant synergistic effects, achieving a 10% cure rate. Taken together, these results suggest that combining IR treatment with ENPP1 inhibition can reduce extracellular cGAMP production. This demonstrates that the increase enhances tumor immunogenicity and exerts an antitumor effect.

[0377] These results are shown in Figure 4A, 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 of PBS or treatments on days 2, 4, and 7 after IR (n=9 per treatment group). Mice from different treatment groups were housed together and experimenters were blinded. Tumor volumes were analyzed with generalized estimating equations to account for intramouse correlation. Pairwise comparisons of treatment groups at each time point were performed using post hoc tests with Tukey adjustment for multiple comparisons. Figure 4B shows Kaplan-Meier curves for Figure 4A, and P values ​​were determined by log-rank Mantel-Cox test. Figure 4C shows the same treatments as in Figure 4B, plus anti-CTLA4 or IgG isotype control antibodies injected intraperitoneally on days 2, 5, and 7 after IR (n=8 for IR(0)+Compound 1+CTLA-4 treatment group, n=17–19 for all other treatment groups). Statistical analysis was performed as in Figure 4B.

[0378] In summary, these results indicate that cGAMP exists extracellularly and that the subject ENPP1 inhibitors act extracellularly, and thus that extracellular ENPP1 inhibition is sufficient for therapeutic efficacy. ENPP1 is considered a natural immune checkpoint. These experiments show that extracellular inhibition of ENPP1 allows cGAMP to enhance antitumor immunity and allows synergistic combination of cGAMP with immune checkpoint blockade already available as a treatment (Figure 5).

[0379] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those skilled in the art, in light of the present disclosure, that certain changes and modifications can be made thereto without departing from the spirit and scope of the appended claims.

[0380] Thus, the foregoing merely illustrates the principles of the invention. It should be understood that those skilled in the art can devise various configurations that embody the principles of the invention and fall within its spirit and scope, although not explicitly described or shown herein. Furthermore, all examples and conditional statements recited herein are intended primarily to aid the reader in understanding the principles of the invention and the concepts that the inventors contribute to furthering the art, and should not be construed as being limited to such specifically recited examples and conditions. Furthermore, all statements herein reciting principles, aspects, and embodiments of the invention, as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Moreover, such equivalents are also intended to include currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. The scope of the invention is therefore not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of the invention are embodied by the following:

[0381] According to a preferred embodiment of the present invention, for example, the following is provided: (Section 1) formula: [ka] ENPP1 inhibitors of the formula: X is a hydrophilic head group selected from phosphonic acid, phosphonate, phosphonate ester, phosphate, phosphate ester, thiophosphate, thiophosphate ester, phosphoramidate, and thiophosphoramidate; L is a linker, Z 1 and Z 2 are each independently 1 and N, Z 3 and Z 4 are each independently selected from CR and N, where R is H, alkyl or substituted alkyl; Each R 1 is independently selected from H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, heterocycle, and substituted heterocycle; R 2 and R 5 are each independently H, OH, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, -OCF 3 , halogen, amine, substituted amine, amide, heterocycle, and substituted heterocycle; R 3 and R 4 are each independently H, OH, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, -OCF 3 , halogen, amine, substituted amine, amide, heterocycle and substituted heterocycle; Or R 3 and R 4 taken together with the carbon atom to which they are attached form a fusion selected from heterocycle, substituted heterocycle, cycloalkyl, substituted cycloalkyl, aryl and substituted aryl, Or a prodrug, pharma- ceutically acceptable salt or solvate thereof. (Section 2) L is -CH 2 -, -(CH 2 ) 2 -, -(CH 2 ) 3 -, -(CH 2 ) 4 -, -(CH 2 ) 5 - and -(CH 2 ) 6 - selected from X,

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Claims

1. A pharmaceutical composition for use in the treatment of cancer, comprising a compound of the formula: 【Chemistry 1】 or a pharma- ceutically acceptable salt or solvate thereof, Where: X is a hydrophilic head group selected from phosphonic acid, phosphonate, phosphonate ester, phosphate, phosphate ester, thiophosphate, thiophosphate ester, phosphoramidate, and thiophosphoramidate; L is selected from -CH2-, -(CH2)2-, -(CH2)3-, -(CH2)4-, -(CH2)5- and -(CH2)6-; Z 1 and Z 2 are each independently selected from CR 1 and N; Z 3 and Z 4 are each independently selected from CR and N, where R is H, alkyl or substituted alkyl; each R 1 is independently selected from H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, heterocycle, and substituted heterocycle; R 2 and R 5 are each independently selected from H, OH, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, -OCF 3 , halogen, amine, substituted amine, heterocycle, and substituted heterocycle; R 3 and R 4 are each independently selected from H, OH, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, -OCF 3 , halogen, amine, substituted amine, heterocycle, and substituted heterocycle, or R 3 and R 4 together with the carbon atom to which they are attached form a fused ring selected from heterocycle, substituted heterocycle, cycloalkyl, substituted cycloalkyl, aryl, and substituted aryl; Pharmaceutical compositions.

2. X is 【Chemistry 2】 is selected from Where: R a and R b are each independently selected from aryl, alkyl, —CH 2 OC(O)R e , and —CH 2 OC(O)OR e ; and The method of claim 1 , wherein R e is alkyl.

3. The pharmaceutical composition of claim 2, wherein the compound has the formula: 【Chemistry 3】 The Where: Z 1 and Z 2 are each N; Z 3 is N, and Z 4 is CH or N; Pharmaceutical compositions.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein 【Chemistry 4-1】 The portion of the molecule represented by: 【Chemistry 4-2】 【Chemistry 4-3】 【Chemistry 4-4】 The pharmaceutical composition is selected from the group consisting of

5. X is 【Chemistry 5】 The pharmaceutical composition according to claim 1 or 2, 6. The pharmaceutical composition of claim 1, wherein L is -(CH 2 ) 2 -.

7. The pharmaceutical composition according to claim 1, wherein Z 1 and Z 2 are each N.

8. The pharmaceutical composition according to any one of claims 1 to 3, wherein R 1 is hydrogen.

9. The pharmaceutical composition according to claim 1, wherein R 5 is alkoxy.

10. The pharmaceutical composition of claim 9, wherein R 5 is methoxy.

11. The compound of claim 1 【Chemistry 11-1】 【Chemistry 11-2】 【Chemistry 11-3】 【Chemistry 11-4】 【Chemistry 11-5】 【Chemistry 11-6】 【Chemistry 11-7】 or a pharma- ceutically acceptable salt or solvate thereof.

12. The compound comprising: 【Chemistry 12】 or a pharma- ceutically acceptable salt or solvate thereof.

13. The compound of claim 12, 【Chemistry 13】 The pharmaceutical composition of claim 1 ,

14. The pharmaceutical composition described in any one of claims 1 to 3 and 11 to 13, wherein the cancer is a solid tumor cancer.

15. The pharmaceutical composition described in any one of claims 1 to 3 and 11 to 13, wherein the cancer is breast cancer.

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