RU2800798C2 - Enpp1 inhibitors and methods of modulating the immune response - Google Patents

Abstract

FIELD: pharmaceutical chemistry.

SUBSTANCE: group of inventions includes an ENPP1 inhibitor of formula (VI) and a pharmaceutical composition based on it. In formula (VI), X is selected from , , , , and ; L is selected from -CH₂ - and -(CH₂)₂-; each of Z¹ and Z² is independently selected from CR¹ and N; each of Z³ and Z⁴ is independently selected from CR and N, where R is H; each R¹ is independently selected from H and alkenyl having 2 to 6 carbon atoms substituted with pyridine; each of R² and R⁵ is independently selected from H, OH, alkoxy containing from 1 to 6 carbon atoms, and halogen; each of R³ and R⁴ is independently selected from H, OH, alkoxy containing from 1 to 6 carbon atoms, halogen and amino; or R³ and R⁴ together with the carbon atoms to which they are attached form dioxane.

(VI)

EFFECT: compound of formula (VI) with inhibitory activity against ENPP1.

13 cl, 11 dwg, 4 tbl, 16 ex

Description

cross reference

[0001] This application claims priority over U.S. Provisional Application No. 62/556,117, filed September 8, 2017, which is incorporated herein by reference in its entirety.

State rights

[0002] This invention was made with government support under NIH contracts CA190896 and CA228044 and Department of Defense contract W81XWH-18-1-0041. The government has certain rights to the present invention.

Introduction

[0003] Cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) activates the interferon gene stimulator (STING) pathway, which is an important antitumor innate immune pathway. The cGAS-cGAMP-STING pathway is activated in the presence of cytoplasmic DNA, either due to microbial infection or a pathophysiological condition, including cancer and autoimmune disease. Cyclic GMP-AMP synthase (cGAS) belongs to the family of nucleotidyl transferases and is a universal DNA sensor that is activated upon binding to cytosolic dsDNA to form a signal molecule (2'-5', 3'-5') of cyclic GMP-AMP (or 2 ', 3'-cGAMP or cyclic guanosine monophosphate-adenosine monophosphate, cGAMP). Acting as a second messenger during microbial infection, 2',3'-cGAMP binds and activates STING, resulting in the production of type I interferon (IFN) and other costimulatory molecules that trigger the immune response. In addition to its role in infectious diseases, the STING pathway has emerged as a promising new target for cancer immunotherapy and autoimmune diseases.

[0004] Ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) is a dominant cGAMP hydrolase that can cleave cGAMP. ENPP1 is a member of the ectonucleotide pyrophosphatase/phosphodiesterase (ENPP) family. The encoded protein is a type II transmembrane glycoprotein containing two identical subunits linked by a disulfide bond. The ENPP1 protein has broad specificity and can cleave various substrates, including phosphodiester bonds of nucleotides and nucleotide sugars and pyrophosphate bonds of nucleotides and nucleotide sugars. This protein can promote the hydrolysis of nucleoside 5'-triphosphates to their corresponding monophosphates and can also hydrolyze diadenosine polyphosphates.

The essence of the invention

[0005] Compounds, compositions, and methods for inhibiting ENPP1 are provided. Compounds that are inhibitors of ENPP1 can act extracellularly to block cGAMP degradation. Aspects of the methods of the present invention include contacting a sample with an ENPP1 inhibitor to inhibit the hydrolysis activity of cGAMP of ENPP1. In some cases, the ENPP1 inhibitor does not enter the cells. Compositions and methods for treating cancer are also provided. Aspects of the methods include administering to a subject a therapeutically effective amount of an ENPP1 inhibitor to treat the subject for cancer. In certain cases, the cancer is a solid tumor. Also provided are methods of administering radiation therapy to a subject before or after administration of an ENPP1 inhibitor. Radiation therapy may be administered at a dosage and/or frequency effective to reduce radiation injury to the subject's body while still eliciting an immune response.

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

Brief description of graphic materials

[0007] The invention may be better understood from the following detailed description when read in conjunction with the accompanying drawings. The patent or application file contains at least one figure in color. It is emphasized that, according to generally accepted practice, the various elements in the figures are presented without reference to any particular scale. On the contrary, the sizes of various elements are arbitrarily expanded or reduced for clarity. The following figures are included in the graphics. It is understood that the figures described below are for illustrative purposes only. The figures are not intended to limit the scope of the present invention in any way.

[0008] FIG. 1A-1C show data illustrating the ability of an exemplary ENPP1 inhibitor to increase the amount of extracellular cGAMP present in a cellular system.

[0009] FIG. 2A-2B show the ability of an exemplary ENPP1 inhibitor to enhance cGAMP-stimulated interferon transcription.

[0010] FIG. 3A-3B show data illustrating the ability of an exemplary ENPP1 inhibitor to increase tumor-associated dendritic cells in a mouse tumor model.

[0011] In FIG. 4A-4C show that ENPP1 inhibition is synergistic with ionizing radiation treatment and anti-CTLA-4 antibodies in producing antitumor effects.

[0012] FIG. 5 is a diagram illustrating the fact that ENPP1 is an innate checkpoint that regulates the cGAMP immunotransmitter.

Definitions

[0013] Before continuing the description of the embodiments of the present disclosure, it should be understood that this disclosure is not limited to the specific embodiments described, as these may, of course, vary. It should also be understood that the terminology used herein is used only to describe specific embodiments and is not limiting, since the scope of this disclosure is limited only by the appended claims.

[0014] Unless otherwise indicated, all technical and scientific terms used in this document have the generally accepted meanings understood by a person skilled in the art to which the present invention belongs. Any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of embodiments of this disclosure.

[0015] It should be noted that the use of the singular herein and in the appended claims includes reference to the plural unless the context clearly indicates otherwise. Thus, for example, reference to "compound" includes not only one compound but also a combination of two or more compounds, reference to "substituent" includes one substituent as well as two or more substituents, and the like.

[0016] In the description and claims of the present invention, the following terminology will be used in accordance with the definitions set forth below. It is understood that the definitions given in this document are not intended to be mutually exclusive. Accordingly, some chemical moieties may fall under the definition of more than one term.

[0017] As used herein, the expressions "for example", "for example", "such as" or "including" are intended to introduce examples that further clarify a more general subject. These examples are provided only as an aid to understanding the disclosure and are not intended to be limiting in any way.

[0018] The publications discussed herein are presented solely in relation to their content prior to the filing date of this application. Nothing herein should be interpreted as an admission that this invention is not entitled to predate such publication by virtue of an earlier invention. Moreover, the dates of the submitted publications may differ from the actual dates of the publications, which requires independent confirmation.

[0019] The terms “active agent,” “antagonist,” “inhibitor,” “drug,” and “pharmacologically active agent” are used interchangeably herein to refer to a chemical material or compound that, when administered to an organism (human or animal), induces the desired pharmacological and/or physiological effect through local and/or systemic action.

[0020] In the context of this document, the terms "treatment", "treat", etc. refer to obtaining the desired pharmacological and/or physiological effect, such as reducing tumor burden. The effect may be prophylactic in terms of completely or partially preventing a disease or its symptoms and/or may be therapeutic in terms of partially or completely curing a disease and/or an adverse event associated with the disease. As used herein, the term "treatment" encompasses any treatment of a disease in a mammal, in particular a human, and includes: (a) preventing the onset of a disease in a subject who may be predisposed to the disease but who has not yet been diagnosed with the disease (for example, including diseases that may be associated with or caused by the primary disease (as in liver fibrosis, which can occur with CHC), (b) inhibiting the disease, i.e. stopping its development, and (c) alleviating the disease, i.e. causing regression of the disease (eg, reduction of tumor burden).

[0021] The term "pharmaceutically acceptable salt" means a salt that is acceptable for administration to a patient, such as a mammal (counterion salts having acceptable mammalian safety for a given dosing regimen). Such salts may be derived from pharmaceutically acceptable inorganic or organic bases and from pharmaceutically acceptable inorganic or organic acids. A "pharmaceutically acceptable salt" refers to pharmaceutically acceptable salts of a compound that are derived from a variety of organic and inorganic counterions well known in the art and include, by way of example only, sodium, potassium, calcium, magnesium, ammonium, tetraalkyl ammonium, and the like. ; and when the molecule contains a basic functionality, salts of organic or inorganic acids such as hydrochloride, hydrobromide, formate, tartrate, besylate, mesylate, acetate, maleate, oxalate and the like.

[0022] The terms "individual", "host", "subject" and "patient" are used interchangeably herein and refer to an animal, including, but not limited to, humans and non-human primates, including monkeys and humans; rodents, including rats and mice; cattle; horse, sheep; feline; canine; etc. "Mammal" means a member or members of any species of mammal and includes, for example, canines; feline; horse; cattle, sheep; rodents, etc. and primates, such as humans and non-human primates. Non-human animal models, e.g. mammals, e.g. non-human primates, murines, lagomorphs, etc., may be used for experimental studies.

[0023] As used herein, the terms "definition", "measurement", "assessment" and "analysis" are used interchangeably and include both quantitative and qualitative definitions.

[0024] The terms "polypeptide" and "protein" are used interchangeably herein and refer to the polymeric form of amino acids of any length, which may include encoded and non-encoded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides with modified peptide backbones. This term includes fusion proteins, including, but not limited to, heterologous amino acid sequence fusion proteins, hybridizations with heterologous and native leader sequences, with or without N-terminal methionine residues; immunologically labeled proteins, fusion proteins with detectable hybridization partners, e.g. fusion proteins including a fluorescent protein, β-galactosidase, luciferase, and the like as a hybridization partner.

[0025] The terms "nucleic acid molecule" and "polynucleotide" are used interchangeably and refer to the polymeric form of nucleotides of any length, deoxyribonucleotides, or ribonucleotides, or analogs thereof. Polynucleotides can have any three-dimensional structure and can perform any function, known or unknown. Non-limiting examples of polynucleotides include gene, gene fragment, 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.

[0026] The term "therapeutically effective amount" or "effective amount" means an amount of a compound that, when administered to a mammal or other subject for the treatment of a disease, condition, or disorder, is sufficient to effect such treatment of the disease, condition, or disorder. The term "therapeutically effective amount" will vary depending on the compound, the disease and its severity, and the age, weight, etc. of the subject being treated.

[0027] As used herein, the term "unit dosage form" refers to physically discrete units suitable as unit dosages for humans and animals, with each unit containing a predetermined amount of a compound (i.e., an aminopyrimidine compound as used herein) , calculated in such an amount that will be sufficient to achieve the desired effect, in combination with a pharmaceutically acceptable diluent, carrier or liquid medium. Specifications for unit dosage forms may depend on the particular compound used and the effect to be achieved, as well as the pharmacodynamics associated with each compound in the host.

[0028] "A pharmaceutically acceptable excipient", a "pharmaceutically acceptable diluent", a "pharmaceutically acceptable carrier", and a "pharmaceutically acceptable adjuvant" means an excipient, diluent, carrier, and adjuvant that are used in the preparation of a pharmaceutical composition that is generally safe, non-toxic, and is neither biologically nor otherwise undesirable, and includes excipient, diluent, carrier and adjuvant that are acceptable for veterinary use as well as pharmaceutical use in humans. A "pharmaceutically acceptable excipient, diluent, carrier, and adjuvant" as used in the specification and claims includes one or more such excipient, diluent, carrier, and adjuvant.

[0029] As used herein, the term "pharmaceutical composition" means a composition suitable for administration to a subject such as a mammal, especially a human. In general, a "pharmaceutical composition" is sterile and preferably free of contaminants that are capable of causing an undesirable response in a subject (eg, the compound(s) in the pharmaceutical composition are pharmaceutically pure). Pharmaceutical compositions can be designed for administration to subjects or patients in need thereof via a number of different routes of administration, including oral, buccal, rectal, parenteral, intraperitoneal, intradermal, intratracheal, intramuscular, subcutaneous, and the like.

[0030] As used herein, the term "having a formula" or "having a structure" is not intended to be limiting and is used in the same way as the term "comprising" is commonly used. The term "independently selected from" is used herein to indicate that the listed elements, such as R groups or the like, may be the same or different.

[0031] As used herein, the terms "may," "optional," "optional," or "may optional" mean that the circumstance described subsequently may or may not occur, so that the description includes instances where the circumstance occurs and instances where when it doesn't. For example, the phrase "optionally substituted" means that a non-hydrogen substituent may or may not be present on a given atom, and thus the description includes structures in which a non-hydrogen substituent is present and structures in which a non-hydrogen substituent is absent.

[0032] "Acyl" refers to the groups H-C(O)-, alkyl-C(O)-, substituted alkyl-C(O)-, alkenyl-C(O)-, substituted alkenyl-C(O)-, alkynyl -C(O)-, substituted alkynyl-C(O)-, cycloalkyl-C(O)-, substituted cycloalkyl-C(O)-, cycloalkenyl-C(O)-, substituted cycloalkenyl-C(O)-, aryl-C(O)-, substituted aryl-C(O)-, heteroaryl-C(O)-, substituted heteroaryl-C(O)-, heterocyclyl-C(O)- and substituted heterocyclyl-C(O)- where alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclyl, and substituted heterocyclyl are as defined herein. For example, acyl includes the "acetyl" group CH3C(O)-

[0033] The term "alkyl" refers to a branched or straight chain saturated hydrocarbon group (i.e., a monoradical), usually, although not necessarily, containing from 1 to about 24 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n -butyl, isobutyl, t-butyl, octyl, decyl and the like, as well as cycloalkyl groups such as cyclopentyl, cyclohexyl and the like. Typically, though not necessarily, alkyl groups herein may contain from 1 to about 18 carbon atoms, and such groups may contain from 1 to about 12 carbon atoms. The term "lower alkyl" means an alkyl group with 1-6 carbon atoms. "Substituted alkyl" refers to alkyl substituted with one or more substituent groups, including cases in which two hydrogens from the same carbon atom in an alkyl substituent, such as a carbonyl group, are substituted (i.e., a substituted alkyl group may include a moiety -C(=O)-). The terms "heteroatom-containing alkyl" and "heteroalkyl" refer to an alkyl substituent in which at least one carbon atom is replaced by a heteroatom, as described in more detail below. Unless otherwise indicated, the terms "alkyl" and "lower alkyl" include linear, branched, cyclic, unsubstituted, substituted and/or heteroatom-containing alkyl or lower alkyl, respectively.

[0034] The term "substituted alkyl" is intended to include an alkyl group as defined herein, wherein one or more carbon atoms in the alkyl chain is optionally replaced by a heteroatom such as -O-, -N-, -S-, - S(O) n- (where n is 0-2), -NR- (where R is hydrogen or alkyl) and has 1 to 5 substituents selected from the group consisting of alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl , cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, aminoacyl, aminoacyloxy, hydroxyaminoacyl, 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-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-aryl, -SO2-heteroaryl and -NRaRb, where R' and R" may be the same or different and are selected from hydrogen, optionally substituted alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, heteroaryl and heterocyclic compounds.

[0035] The term "alkenyl" refers to a linear, branched or cyclic hydrocarbon group of 2 to about 24 carbon atoms containing at least one double bond, such as vinyl, n-propenyl, isopropenyl, n-butenyl, isobutenyl, octenyl , decenyl, tetradecenyl, hexadecenyl, eicosenyl, tetracosenyl and the like. Typically, though not necessarily, alkenyl groups herein may contain from 2 to about 18 carbon atoms, and, for example, may contain from 2 to 12 carbon atoms. The term "lower alkenyl" means an alkenyl group with 2-6 carbon atoms. The term "substituted alkenyl" refers to alkenyl substituted with one or more substituent groups, and the terms "heteroatom-containing alkenyl" and "heteroalkenyl" refer to alkenyl in which at least one carbon atom is replaced by a heteroatom. Unless otherwise indicated, the terms "alkenyl" and "lower alkenyl" include linear, branched, cyclic, unsubstituted, substituted and/or heteroatom-containing alkenyl and lower alkenyl, respectively.

[0036] The term "alkynyl" refers to a linear or branched hydrocarbon group of 2-24 carbon atoms containing at least one triple bond, such as ethynyl, n-propynyl, and the like. Typically, though not necessarily, alkynyl groups herein may contain from 2 to about 18 carbon atoms, and such groups may additionally contain from 2 to 12 carbon atoms. The term "lower alkynyl" means an alkynyl group with 2-6 carbon atoms. The term "substituted alkynyl" refers to alkynyl substituted with one or more substituent groups, and the terms "heteroatom-containing alkynyl" and "heteroalkynyl" refer to alkynyl in which at least one carbon atom is replaced by a heteroatom. Unless otherwise indicated, the terms "alkynyl" and "lower alkynyl" include linear, branched, unsubstituted, substituted, and/or heteroatom-containing alkynyl and lower alkynyl, respectively.

[0037] The term "alkoxy" refers to an alkyl group linked through one terminal ether bond; that is, an "alkoxy" group may be defined 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, etc. Substituents designated as "C1-C6 alkoxy" or "lower alkoxy" herein may, for example, contain from 1 to 3 carbon atoms, and as a further example, such substituents may contain 1 or 2 carbon atoms (ie, methoxy and ethoxy).

[0038] The term "substituted alkoxy" refers to the following groups: substituted alkyl-O-, substituted alkenyl-O-, substituted cycloalkyl-O-, substituted cycloalkenyl-O- and substituted alkynyl-O-, where substituted alkyl, substituted alkenyl, substituted cycloalkyl, substituted cycloalkenyl and substituted alkynyl are as defined herein.

[0039] The term "aryl", unless otherwise indicated, refers to an aromatic substituent, usually, although not necessarily, containing from 5 to 30 carbon atoms and containing one aromatic ring or more aromatic rings that are fused together, directly linked or indirectly linked (for example, different aromatic rings are linked to a common group such as a methylene or ethylene moiety). Aryl groups may, for example, contain from 5 to 20 carbon atoms, and, as another example, aryl groups may contain from 5 to 12 carbon atoms. For example, aryl groups may contain one aromatic ring or two or more fused or linked aromatic rings (ie, biaryl, aryl-substituted aryl, etc.). Examples include phenyl, naphthyl, biphenyl, diphenyl ether, diphenylamine, benzophenone and the like. "Substituted aryl" refers to an aryl moiety substituted with one or more substituent groups, and the terms "heteroatom-containing aryl" and "heteroaryl" refer to an aryl substituent in which at least one carbon atom has been replaced by a heteroatom, as will be described in more detail below. Aryl is intended to include stable cyclic, heterocyclic, polycyclic, and polyheterocyclic C3-C14 unsaturated moieties, examples of which include, but are not limited to, phenyl, biphenyl, naphthyl, pyridyl, furyl, thiophenyl, imidazoyl, pyrimidinyl, and oxazoyl; which may be further substituted with one to five members selected from the group consisting of hydroxy, C1-C8 alkoxy, C1-C8 branched or linear alkyl, acyloxy, carbamoyl, amino, N-acylamino, nitro, halogen, trifluoromethyl, cyano, and carboxyl (see, for example, Katritzky, Handbook of Heterocyclic Chemistry). Unless otherwise indicated, the term "aryl" includes unsubstituted, substituted and/or heteroaromatic aromatic substituents.

[0040] The term "aralkyl" refers to an alkyl group with an aryl substituent, and the term "alkaryl" refers to an aryl group with an alkyl substituent, where "alkyl" and "aryl" are as defined above. In general, aralkyl and alkaryl groups herein contain from 6 to 30 carbon atoms. Aralkyl and alkaryl groups may, for example, contain from 6 to 20 carbon atoms, and as another example, such groups may contain from 6 to 12 carbon atoms.

[0041] The term "alkylene" or refers to a diradical alkyl group. Unless otherwise indicated, such groups include saturated hydrocarbon chains containing from 1 to 24 carbon atoms, which may be substituted or unsubstituted, may contain one or more alicyclic groups, and may contain a heteroatom. "Lower alkylene" refers to alkylene bonds containing from 1 to 6 carbon atoms. Examples include methylene (--CH2--), ethylene (--CH2CH2--), propylene (--CH2 CH2CH2--), 2-methylpropylene (--CH2--CH)(CH3)--CH2--) , hexylene (--(CH2)6--) and the like.

[0042] Similarly, the terms "alkenylene", "alkynylene", "arylene", "aralkylene", and "alkarylene" refer to diradical alkenyl, alkynyl, aryl, aralkyl, and alkaryl groups, respectively.

[0043] The term "amino" refers to the group -NRR', where R and R' independently represent hydrogen or non-hydrogen substituents, where non-hydrogen substituents include, for example, alkyl, aryl, alkenyl, aralkyl and their substituted and/or heteroatom-containing variants.

[0044] The term "halogen" is used in the conventional sense to denote a substituent representing chlorine, bromine, fluorine or iodine.

[0045] "Carboxyl", "carboxy" or "carboxylate" refers to -CO2H or its salts.

[0046] "Cycloalkyl" refers to cyclic alkyl groups with 3-10 carbon atoms having one or more 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 chain structures such as cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl and the like, or multiple ring structures such as adamantanyl and the like.

[0047] The term "substituted cycloalkyl" refers to cycloalkyl groups having 1 to 5 substituents or 1 to 3 substituents selected from alkyl, substituted alkyl, alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, hydroxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxyalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioal coxy, 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.

[0048] The term "heteroatom-containing", as in "heteroatom-containing alkyl group" (also called "heteroalkyl" group) or "heteroatom-containing aryl group" (also called "heteroaryl" group), refers to a molecule, linkage or substituent in which one or several carbon atoms are replaced by an atom other than carbon, such as a nitrogen, oxygen, sulfur, phosphorus or silicon atom, usually nitrogen, oxygen or sulfur. Similarly, the term "heteroalkyl" refers to an alkyl substituent that contains a heteroatom, the term "heterocycloalkyl" refers to a cycloalkyl substituent that contains a heteroatom, the terms "heterocyclic" or "heterocycle" refers to a cyclic substituent that contains a heteroatom, the terms "heteroaryl" and "heteroaromatic" respectively refer to "aryl" and "aromatic" substituents that contain a heteroatom, and the like. Examples of heteroalkyl groups include alkoxyaryl, alkylsulfanyl-substituted alkyl, N-alkylated aminoalkyl, and the like. Examples of heteroaryl substituents include pyrrolyl, pyrrolidinyl, pyridinyl, quinolinyl, indolyl, furyl, pyrimidinyl, imidazolyl, 1,2,4-triazolyl, tetrazolyl, etc., and examples of heteroatom-containing alicyclic groups are pyrrolidino, morpholino, piperazino, piperidino, tetrahydrofuranyl and etc.

[0049] "Heteroaryl" refers to an aromatic group of 1-15 carbon atoms, such as 1 to 10 carbon atoms and 1 to 10 heteroatoms selected from the group consisting of oxygen, nitrogen and sulfur in the ring. Such heteroaryl groups may have a single ring (such as pyridinyl, imidazolyl, or furyl) or multiple fused rings in the ring system (e.g., in groups such as indolizinyl, quinolinyl, benzofuran, benzimidazolyl, or benzothienyl), where at least one ring within the ring system is aromatic, provided that the point of attachment is on an atom of the aromatic ring. In certain embodiments, the nitrogen and/or sulfur atoms in the ring of the heteroaryl group are optionally oxidized to form N-oxide (N→O), sulfinyl, or sulfonyl moieties. This term includes, for example, pyridinyl, pyrrolyl, indolyl, thiophenyl and furanyl. Unless otherwise limited by the definition of a heteroaryl substituent, such heteroaryl groups may be optionally substituted with 1-5 substituents or 1-3 substituents selected from acyloxy, hydroxy, thiol, acyl, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, substituted alkyl, substituted alkoxy, substituted alkenyl, substituted alkynyl, substituted cycloalkyl, substituted cycloalkenyl, amino, substituted amino, aminoacyl, acylamino, alkaryl, aryl, aryloxy, azido, carboxyl, carboxylalkyl, cyano, halogen, nitro, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, aminoacyloxy, hydroxyacylamino, thioalkoxy, substituted thioalkoxy, thioaryloxy, thioheteroaryloxy, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl, and -SO2-heteroaryl and trihalomethyl.

[0050] The terms "heterocycle", "heterocyclic", and "heterocyclyl" refer to a saturated or unsaturated group having one or more fused rings, including fused bridged and spiro ring systems, and having 3 to 15 ring atoms, including 1 to 4 heteroatoms. Such ring heteroatoms are selected from nitrogen, sulfur and oxygen, where in fused ring systems, one or more rings may be cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, provided that the point of attachment is on a non-aromatic ring. In certain embodiments, the nitrogen and/or sulfur atoms of the heterocyclic group are optionally oxidized to form -SO2, -S(O)-, or -N-oxide moieties.

[0051] Examples of heterocycles and heteroaryls include, but are not limited to, azetidine, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, dihydroindole, indazole, purine, quinolysine, isoquinoline, quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, phenanthroline, isothiazole, phenazine, isoxazole, phenoxazine, phenothiazine, imidazolidine, imidazoline, piperidine, piperazine, indoline, phthalimide, 1,2,3,4- tetrahydroisoquinoline, 4,5,6,7-tetrahydrobenzo[b]thiophene, thiazole, thiazolidine, thiophene, benzo[b]thiophene, morpholinyl, thiomorpholinyl (also called thiamorpholinyl), 1,1-dioxothiomorpholinyl, piperidinyl, pyrrolidine, tetrahydrofuranyl, etc. similar.

[0052] Unless otherwise limited by the definition of a heterocyclic substituent, such heterocyclic groups may be optionally substituted with 1 to 5 or 1 to 3 substituents selected from alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, hydroxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxyalkyl, 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 and a fused heterocycle.

[0053] "Hydrocarbyl" refers to monovalent hydrocarbon radicals containing from 1 to about 30 carbon atoms, including from 1 to about 24 carbon atoms, further comprising from 1 to about 18 carbon atoms, and additionally containing from about 1 to 12 carbon atoms including linear, branched, cyclic, saturated and unsaturated compounds such as alkyl groups, alkenyl groups, aryl groups and the like. The hydrocarbyl may be substituted with one or more substituent groups. The term "heteroatom-containing hydrocarbyl" refers to hydrocarbyl in which at least one carbon atom is replaced by a heteroatom. Unless otherwise indicated, the term "hydrocarbyl" should be interpreted as including substituted and/or heteroatom-containing hydrocarbyl moieties.

[0054] By "substituted", as in "substituted hydrocarbyl", "substituted alkyl", "substituted aryl", etc., as mentioned in some of the above definitions, is meant that in a hydrocarbyl, alkyl, aryl or other moiety at least one hydrogen atom bonded to a carbon atom (or other atom) is replaced by one or more non-hydrogen substituents. Examples of such substituents include, without limitation, C1-C24 alkyl functional groups and hydrocarbyl moieties (including C1-C18 alkyl, further including C1-C12 alkyl and further including C1-C6 alkyl), C2-C24 alkenyl (including C2-C18 alkenyl further comprising C2-C12 alkenyl and further comprising C2-C6 alkenyl), C2-C24 alkynyl (including C2-C18 alkynyl further comprising C2-C12 alkynyl and further comprising C2-C6 alkynyl), C5-C30 aryl (including C5-C20 aryl, further including C5-C12 aryl) and C6-C30 aralkyl (including C6-C20 aralkyl, further comprising C6-C12 aralkyl). The above hydrocarbyl groups may be further substituted with one or more functional groups or additional hydrocarbyl groups such as those specifically listed. Unless otherwise indicated, any of the groups described herein should be interpreted as including substituted and/or heteroatom-containing moieties in addition to unsubstituted groups.

[0055] "Sulfonyl" refers to the groups SO2-alkyl, SO2-substituted alkyl, SO2-alkenyl, SO2-substituted alkenyl, SO2-cycloalkyl, SO2-substituted cycloalkyl, SO2-cycloalkenyl, SO2-substituted cycloalkenyl SO2-aryl, SO2- substituted aryl, SO2-heteroaryl, SO2-substituted heteroaryl, SO2-heterocyclyl and SO2-substituted heterocyclyl, where alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclyl and substituted heterocyclyl are as defined herein. Sulfonyl includes by way of example methyl-SO2-, phenyl-SO2- and 4-methylphenyl-SO2-.

[0056] The term "functional groups" refers to chemical groups such as halogen, hydroxyl, sulfhydryl, C1-C24 alkoxy, C2-C24 alkenyloxy, C2-C24 alkynyloxy, C5-C20 aryloxy, acyl (including C2-C24 alkylcarbonyl(- CO-alkyl) and C6-C20 arylcarbonyl(-CO-aryl)), acyloxy (-O-acyl), C2-C24 alkoxycarbonyl(-(CO)-O-alkyl), C6-C20 aryloxycarbonyl(-(CO)- O-aryl), halocarbonyl(-CO)-X where X is halogen), C2-C24 alkyl carbonato (-O-(CO)-O-alkyl), C6-C20 aryl carbonato (-O-(CO)-O- aryl), carboxy (-COOH), carboxylate (-COO- ), carbamoyl (-(CO)-NH2), monosubstituted C1-C24 alkylcarbamoyl (-(CO)-NH(C1-C24 alkyl)), disubstituted alkylcarbamoyl (-( CO)-N(C1-C24 alkyl)2), monosubstituted arylcarbamoyl (-(CO)-NH-aryl), thiocarbamoyl (-(CS)-NH2), urea (-NH-(CO)-NH2), cyano ( -C≡N), isocyano (-N+≡C-), cyanato (-O-C≡N), isocyanato (-O-N+≡C-), isothiocyanato (-S-C≡N), azido (-N=N+=N -), formyl (-(CO)-H), thioformyl (-(CS)-H), amino (-NH2), mono- and di-(C1-C24 alkyl)-substituted amino, mono- and di-( C5-C20 aryl)-substituted amino, C2-C24 alkylamido (-NH-(CO)-alkyl), C5-C20 arylamido (-NH-(CO)-aryl), imino (-CR=NH 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. 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 called "alkylthio"), arylsulfanyl (-S-aryl; also called "arylthio"), C1-C24 alkylsulfinyl (-(SO)-alkyl), C5-C20 arylsulfinyl (-(SO)-aryl), C1-C24 alkylsulfonyl (-SO2-alkyl), C5-C20 arylsulfonyl (-SO2-aryl), phosphono (-P(O)(OH)2), phosphonato (-P(O)(O-)2), phosphinato (-P (O)(O-)), phospho (-PO2) and phosphino (-PH2), mono- and di-(C1-C24 alkyl)-substituted phosphino, mono- and di-(C5-C20 aryl)-substituted phosphine . In addition, the aforementioned functional groups may, if the particular group permits, be further substituted with one or more additional functional groups or one or more hydrocarbyl groups such as those specifically listed above.

[0057] By "linking" or "linker", as in "linking group", "linker fragment", etc., is meant a linking fragment that connects two groups through covalent bonds. The linker may be linear, branched, cyclic, or a single atom. Examples of such linking groups include alkyl, alkenylene, alkynylene, arylene, alkarylene, aralkylene, and linking moieties containing functional groups including, but not limited to: amido (-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-), etc. In certain cases, one, two, three, four or five or more carbon atoms of the main chain of the linker may optionally be substituted with a sulfur, nitrogen or oxygen heteroatom. The bonds between the backbone atoms can be saturated or unsaturated, typically there will be no more than one, two or three unsaturated bonds in the linker backbone. The linker may include one or more substituent groups, for example with an alkyl, aryl or alkenyl group. The linker may include, without limitation, poly(ethylene glycol) elements (eg -(CH2-CH2-O)-); ethers, thioethers, amines, alkyls (e.g. (C1-C12) alkyl), which may be straight or branched, e.g. methyl, ethyl, n-propyl, 1-methylethyl (isopropyl), n-butyl, n-pentyl, 1,1-dimethylethyl (tert-butyl) and the like. The main chain of the linker may include a cyclic group, for example, aryl, heterocycle or cycloalkyl group, where 2 or more atoms, for example, 2, 3 or 4 atoms of the cyclic group, are included in the main chain. The linker may be cleavable or non-cleavable. Any convenient orientation and/or connection of linkers with linked groups can be used.

[0058] When the term "substituted" is in front of a list of possible substituted groups, it is understood that this term applies to each member of this group. For example, the phrase "substituted alkyl and aryl" should be interpreted as "substituted alkyl and substituted aryl".

[0059] In addition to the disclosure herein, the term "substituted" when used to modify a specified group or radical may also mean that one or more hydrogen atoms of a specified group or radical, each independently of one another, are replaced by the same or different groups of substituents in according to the definition below.

[0060] In addition to the groups disclosed with respect to individual terms herein, groups of substituents to replace one or more hydrogen atoms (any two hydrogen atoms on the same carbon atom can be replaced by =O, =NR70, =N-OR70, =N2 or =S) on saturated carbons in the indicated group or radical, unless otherwise indicated, are -R60, halogen, =O, -OR70, -SR70, -NR80R80, trihalomethyl, -CN, -OCN, -SCN , -NO, -NO2, =N2, -N3, -SO2R70, -SO2O-M+, -SO2OR70, -OSO2R70, -OSO2O-M+, -OSO2OR70, -P(O)(O-)2(M+)2, -P(O)(OR70)O-M+, -P(O)(OR70) 2, -C(O)R70, -C(S)R70, -C(NR70)R70, -C(O)O- M+, -C(O)OR70, -C(S)OR70, -C(O)NR80R80, -C(NR70)NR80R80, -OC(O)R70, -OC(S)R70, -OC(O)O -M+, -OC(O)OR70, -OC(S)OR70, -NR70C(O)R70, -NR70C(S)R70, -NR70CO2-M+, -NR70CO2R70, -NR70C(S)OR70, -NR70C(O )NR80R80, -NR70C(NR70)R70 and -NR70C(NR70)NR80R80, wherein R60 is selected from the group consisting of optionally substituted alkyl, cycloalkyl, heteroalkyl, heterocycloalkylalkyl, cycloalkylalkyl, aryl, arylalkyl, heteroaryl and heteroarylalkyl, each R70 is independently hydrogen or R60; each R80 is independently R70 or alternatively two R80' taken together with the nitrogen atom to which they are bonded form a 5-, 6- or 7-membered heterocycloalkyl which may optionally include from 1 to 4 of the same or different additional heteroatoms selected from the group consisting of O, N and S, of which N may have -H or C1-C3 alkyl substitution; and each M+ is a counterion with a net positive charge of unity. Each M+ may independently be, for example, an alkali metal ion such as K+, Na+, Li+; an ammonium ion such as +N (R60)4; or an alkaline earth metal ion such as [Ca2+]0.5, [Mg2+]0.5, or [Ba2+]0.5 (“the subscript 0.5 means that one of the counterions for such divalent alkaline earth metal ions may be an ionized form of the compound of the invention, and another exemplary counterion such as chloride, or the two ionized compounds disclosed herein may serve as counterions for such divalent alkaline earth ions, or the doubly ionized compound of the invention may serve as counterion for such divalent alkaline earth ions). As specific examples, -NR80R80 is intended to include -NH2, -NH-alkyl, N -pyrrolidinyl, N -piperazinyl, 4N -methylpiperazin-1-yl, and N -morpholinyl.

[0061] In addition to the disclosure herein, substituent groups for hydrogen atoms on unsaturated carbon atoms in "substituted" alkene, alkyne, aryl, and heteroaryl groups are, unless otherwise indicated, -R60, halogen, -O-M+, - OR70, -SR70, -S-M+, -NR80R80, trihalomethyl, -CF3, -CN, -OCN, -SCN, -NO, -NO2, -N3, -SO2R70, -SO3-M+, -SO3R70, -OSO2R70, -OSO3-M+, -OSO3R70, -PO3-2(M+)2, -P(O)(OR70)O-M+, -P(O)(OR70)2, -C(O)R70, -C(S )R70, -C(NR70)R70, -CO2-M+, -CO2R70, -C(S)OR70, -C(O)NR80R80, -C(NR70)NR80R80, -OC(O)R70, -OC(S )R70, -OCO2-M+, -OCO2R70, -OC(S)OR70, -NR70C(O)R70, -NR70C(S)R70, -NR70CO2-M+, -NR70CO2R70, -NR70C(S)OR70, -NR70C( O)NR80R80, -NR70C(NR70)R70 and -NR70C(NR70)NR80R80, where R60, R70, R80 and M+ are as previously defined, provided that in the case of a substituted alkene or alkyne, the substituents are not -O-M+, - OR70, -SR70, or -S-M+.

[0062] In addition to the groups disclosed in relation to individual terms in this specification, substituent groups for hydrogen atoms on nitrogen atoms in "substituted" heteroalkyl and cycloheteroalkyl groups are, unless otherwise indicated, -R60, -O-M+, -OR70, -SR70, -S-M+, -NR80R80, trihalomethyl, -CF3, -CN, -NO, -NO2, -S(O)2R70, -S(O)2O-M+, -S(O)2OR70 , -OS(O)2R70, -OS(O)2O-M+, -OS(O)2OR70, -P(O)(O-)2(M+)2, -P(O)(OR70)O-M+ , -P(O)(OR70)(OR70), -C(O)R70, -C(S)R70, -C(NR70)R70, -C(O)OR70, -C(S)OR70, -C (O)NR80R80, -C(NR70)NR80R80, -OC(O)R70, -OC(S)R70, -OC(O)OR70, -OC(S)OR70, -NR70C(O)R70, -NR70C( S)R70, -NR70C(O)OR70, -NR70C(S)OR70, -NR70C(O)NR80R80, -NR70C(NR70)R70 and -NR70C(NR70)NR80R80 where R60, R70, R80 and M+ are as above definitions.

[0063] In addition to the disclosure herein, in a specific embodiment, a group that is substituted has 1, 2, 3, or 4 substituents, 1, 2, or 3 substituents, 1 or 2 substituents, or 1 substituent.

[0064] Unless otherwise indicated, the nomenclature of substituents that are not explicitly defined herein is achieved by denoting a terminal piece of functionality followed by adjacent functionality towards the point of attachment. For example, the substituent "arylalkyloxycarbonyl" refers to the group (aryl)-(alkyl)-OC(O)-.

[0065] With regard to any of the groups disclosed herein that contain one or more substituents, it is understood that such groups do not contain any substituents or substitution structures that are sterically impractical and/or synthetically impracticable. In addition, the compounds of the present invention include all stereochemical isomers resulting from the substitution of these compounds.

[0066] In certain embodiments, the substituent may promote optical isomerism and/or stereoisomerism of the compound. Also of interest are salts, solvates, hydrates and prodrugs of the compound. All such forms are covered by this disclosure. Thus, the compounds described herein include their salts, solvates, hydrates, prodrugs and isomers, including their pharmaceutically acceptable salts, solvates, hydrates, prodrugs and isomers. In certain embodiments, the implementation of the connection can be metabolized into a pharmaceutically active derivative.

[0067] Unless otherwise indicated, reference to an atom is intended to include isotopes of that atom. For example, a reference to H means 1H, 2H (deuterium) and 3H (tritium), and a reference to C means 12C and all isotopes of carbon (such as 13C).

[0068] Definitions of other terms and concepts are given in the detailed description.

Detailed Description of the Invention

[0069] As summarized above, aspects of the present disclosure include compounds, compositions, and methods for inhibiting ENPP1. Aspects of the methods include contacting the sample with a non-cell-penetrating inhibitor of ENPP1 to inhibit the hydrolysis activity of cGAMP in ENPP1.

[0070] Compositions and methods for treating cancer are also provided. Aspects of the methods include administering to a subject a therapeutically effective amount of an ENPP1 inhibitor to treat the subject for cancer. Aspects of the methods include administering to a subject a therapeutically effective amount of a non-cell-penetrating ENPP1 inhibitor to inhibit cGAMP hydrolysis and treat the subject for cancer.

[0071] These compounds and methods can be used in a variety of applications in which ENPP1 inhibition is desired.

Compounds that are inhibitors of ENPP1

[0072] As summarized above, aspects of the disclosure include compounds that are inhibitors of ENPP1. The compounds of the present invention may include a nuclear structure based on an aryl or heteroaryl ring system, for example a quinazoline, isoquinoline or pyrimidine group, which is linked to a hydrophilic head group. The linker between the aryl or heteroaryl ring system and the hydrophilic head group may 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. Typical ENPP1 inhibitors of interest, including quinazoline, isoquinoline, and pyrimidine cyclic systems, are shown in formulas I IV, V, VI and VII and in the following structures 1-106.

[0073] In some cases, the considered compound, which is an inhibitor of ENPP1 has the formula (I):

Y-A-L-X

(I)

Where:

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 linker; And

X is a hydrophilic head group,

or a prodrug, pharmaceutically acceptable salt or solvate thereof.

[0074] The term "hydrophilic head group" refers to a related group of contemplated compounds that is hydrophilic and readily soluble in aqueous media, eg, under physiological conditions, and has low cell membrane permeability. In some cases, low cell membrane permeability refers to a permeability coefficient of 10-4 cm/s or less, such as 10-5 cm/s or less, 10-6 cm/s or less, 10-7 cm/s or less, 10-8 cm/s or less, 10-9 cm/s or less or even less as measured using any suitable passive diffusion methods for an isolated hydrophilic head group across the membrane (e.g., cell monolayers such as Caco colorectal cell lines -2 or renal MDCK). See, for example, Yang and Hinner, Methods Mol Biol. 2015; 1266:29-53,

[0075] A hydrophilic head group can confer improved water solubility and reduced cell permeability to the molecule to which it is attached. The hydrophilic head group can be any suitable hydrophilic group that is highly soluble in aqueous media and has low membrane permeability. In certain instances, the hydrophilic group is a discrete functional group (eg, as described herein) or a substituted version thereof. In general terms, large uncharged polar groups or charged groups have low permeability. In some cases, the hydrophilic head group has a charge, such as a positive or negative charge. In some embodiments, the hydrophilic head group is not cell-penetrating and imparts cell-impermeability to the compound in question. It is understood that the hydrophilic head group, or prodrug form thereof, may be selected to provide the desired cellular permeability of the compound in question. In certain instances, the hydrophilic head group is a neutral hydrophilic group. In some cases, the hydrophilic head group includes a temporary modifying protective group. In certain cases, the claimed compound penetrates the cells.

[0076] 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 a phosphonic acid, a phosphonate, or a salt thereof. In some embodiments of formula (I), the hydrophilic head group is a phosphate or a salt thereof. In some embodiments of Formula (I), the hydrophilic head group is a phosphonate ester or a phosphate ester.

[0077] Specific examples of hydrophilic head groups of interest include, but are not limited to, a head group comprising a first molecule selected from RPO4H- phosphates, RPO3H- phosphonates), boric acid (RBO2H2), carboxylates (RCO2-), sulfates ( RSO4-), sulfonates (RSO3-), amines (RNH3+), glycerol, sugars such as lactose or derived from hyaluronic acid, polar amino acids, polyethylene oxides and oligoethylene glycols, which are optionally conjugated to the remainder of a second molecule selected from choline, ethanolamine, glycerol , nucleic acid, sugar, inositol and serine. The head group may contain various other modifications, for example, in the case of oligoethylene glycols and polyethylene oxide (PEG) containing head groups, such a PEG chain may end in a methyl group or have a distal functional group for further modification. Examples of hydrophilic head groups also include, but are not limited to, thiophosphate, phosphocholine, phospholycerol, phosphoethanolamine, phosphoserine, phosphoinositols, ethylphosphorylcholine, polyethylene glycol, polyglycerol, melamine, glucosamine, trimethylamine, spermine, spermidine, and conjugated carboxylates, sulfates, boric acid, sulfonates, sulfates and carbohydrates.

[0078] Any convenient linkers can be used to link A to X. In some cases, A is linked to X via a covalent bond. In certain instances, A is linked to X via a linear linker 1-12 atoms long, eg 1-10, 1-8 or 1-6 atoms long, eg 1, 2, 3, 4, 5 or 6 atoms long. Linker L may be a (C1-6) alkyl linker or a substituted (C1-6) alkyl linker optionally substituted with a heteroatom or linker functional group such as ester (-CO2-), amido (CONH), carbamate (OCONH), an ether (-O-), a thioether (-S-) and/or an amino group (-NR-, where R is H or alkyl).

[0079] In some specific 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), L-X includes a group of formula (XI):

(XI),

Where:

Z12 is selected from O and S;

each of Z13 and Z14 is independently selected by O and NR';

Z15 is selected from O and CH2;

each of R15 and R16 is 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; And

q1 is an integer from 0 to 6.

[0080] In some embodiments of Formula (XI), Z12, Z13, and Z14 are all oxygen atoms and Z15 is CH2. In other cases, Z12 is a sulfur atom, both of Z13 and Z14 are oxygen atoms, and Z15 is CH2. In other cases, Z12 is a sulfur atom, all of Z13, Z14, Z15 are oxygen atoms. In some cases, Z12 is an oxygen atom, Z13 is NR', Z14 is an oxygen atom, and Z15 is a carbon atom. In other cases Z12 is an oxygen atom, Z13 is a nitrogen atom, both of Z14 and Z15 are oxygen atoms. In other cases, Z12 is an oxygen atom, Z13 and Z14 are each independently NR', and Z15 is an oxygen atom. In some other cases, Z12 is an oxygen atom, Z13 and Z14 are each independently NR', and Z15 is CH2. It is understood that the group of formula (XI) may include one or more tautomeric forms of the depicted structure, and that all such forms and their salts are intended to be included.

[0081] In some embodiments of formula (XI), both of R15 and R16 are hydrogen atoms. In other cases, both of R15 and R16 are substituents other than hydrogen. In some instances, R15 and R16 are each independently an alkyl or substituted alkyl group. In some other cases, each of R15 and R16 independently represents an aryl group. In some instances, R15 and R16 are each independently alkyl groups. In some instances, both of R15 and R16 are ester-substituted alkyl groups. In other cases, both of R15 and R16 are ester-substituted alkyl groups. In certain instances, both of R15 and R16 are phenyl groups. In some cases, R15 and R16 are the same substituent. In other cases, R15 and R16 are different substituents.

[0082] In some embodiments of formula (XI), Z15 is a carbon atom and q1 is 0. In other cases, Z15 is a carbon atom and q1 is greater than 0, such as 1, 2, 3, 4, 5, or 6. In some cases, Z15 is a carbon atom and q1 is 1. In other embodiments, Z15 is an oxygen atom and q1 is 1. In other cases, Z15 is an oxygen atom and q1 is greater than 1, such as 2, 3, 4, 5, or 6. B in some cases Z15 is an oxygen atom and q1 is 2.

[0083] In some embodiments of Formula (XI), LX is selected from one of the following groups:

, , , , ,

, , , .

[0084] In some embodiments of formula (I), L-X includes a group of formula (XII):

(XII)

Where:

each of R17 and R18 is 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 R17 and R18 together with the atoms to which they are attached form a group selected from heterocycle and substituted heterocycle; And

q2 is an integer from 1 to 6.

[0085] In some embodiments of formula (XII), both of R17 and R18 are hydrogen atoms. In other cases, both of R17 and R18 are substituents other than hydrogen. In certain embodiments of formula (XII), q2 is 1. In certain instances, q2 is greater than 1, such as 2, 3, 4, 5, or 6. In certain instances of formula (XII), q2 is 2. In certain embodiments of formula (XII), the hydrophilic the head group has the structure:

.

[0086] In some embodiments of formula (I), L-X includes a group of formula (XIII):

(XIII)

where q3 is an integer from 1 to 6. In certain embodiments, q3 is equal to 1. In certain embodiments, q3 is greater than 1, such as 2, 3, 4, 5, or 6. In certain embodiments, q3 is equal to 2. In certain embodiments, the implementation of the formula (XIII) the hydrophilic head group has the structure:

or .

[0087] In some embodiments of formula (I), L-X includes a group of formula (XIV):

(XIV)

where Z16 is selected from O and CH 2; And

q1 is an integer from 0 to 6 (eg 0-5).

In some embodiments of formula (XIV), Z16 is CH2 and q4 is 0. In other instances, Z16 is CH2 and q1 is greater than 0, such as 1, 2, 3, 4, 5, or 6. In some instances, Z16 is CH2 and q1 is 1. In other embodiments, Z16 is an oxygen atom and q1 is 1. In other cases, Z16 is an oxygen atom and q1 is greater than 1, such as 2, 3, 4, 5, or 6. In some cases, Z16 is an oxygen atom and q1 is equal to 2.

[0088] In some embodiments of formula (XIV), the hydrophilic head group is selected from one of the following groups:

or .

[0089] In some embodiments of formula (I), L-X includes a group of formula (XV):

(XV)

where q5 is an integer from 1 to 6. In certain embodiments, q5 is equal to 1. In certain embodiments, q5 is greater than 1, such as 2, 3, 4, 5, or 6. In certain embodiments, q5 is equal to 2. In certain embodiments, the implementation of the formula (XV) the hydrophilic head group has the structure:

or .

[0090] In some embodiments of formula (I), L-X includes a group of formula (XVI):

(XVI),

Where:

R19 is 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; And

q6 is an integer from 1 to 6.

[0091] In some embodiments of Formula (XVI), R19 is hydrogen. In other cases, R19 is a substituent other than hydrogen. In certain embodiments, R19 is alkyl or substituted alkyl. In certain embodiments of formula (XVI), q6 is 1. In certain cases, q6 is greater than 1, such as 2, 3, 4, 5, or 6. In certain cases of formula (XVI), q6 is 2. In certain embodiments of formula (XVI), - L-X has the structure:

.

[0092] In some embodiments of formula (I), L-X correspond to formula (XVII):

(XVII),

where q7 is an integer from 1 to 6. In certain embodiments, q7 is equal to 1. In certain embodiments, q7 is greater than 1, such as 2, 3, 4, 5, or 6. In certain embodiments, q7 is equal to 2. In certain embodiments, the implementation of the formula (XVII)-L-X has the structure:

.

[0093] In some embodiments of Formula (I), A is a heterocycle or a substituted heterocycle. In some instances, A is a saturated heterocycle or a substituted saturated heterocycle. The heterocycle may 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 instances, a heterocycle is a 6-membered ring that is linked to Y and L via a 1,4-configuration. In certain instances, a heterocycle is a 5- or 6-membered ring that is linked to Y and L via a 1,3-configuration. In certain instances, the heterocycle is piperidine, substituted piperidine, piperazine, or substituted piperazine. When the linking ring atom is C, the heterocycle may include a chiral center. In some cases, A is selected from one of the following heterocyclic groups:

, or .

[0094] In some embodiments of Formula (I), A is a carbocycle. In some instances, A is a saturated carbocycle or a substituted saturated carbocycle. The carbocycle may 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 that is linked to Y and L through a 1,4 configuration. In certain cases, the carbocycle is a 5- or 6-membered ring that is linked to Y and L through a 1,3-configuration. In certain instances, the carbocycle is cyclohexane or a substituted cyclohexane. Cyclohexane may include a chiral center. In some cases, A has the structure:

.

[0095] In certain other instances, A is an aromatic carbocycle, ie aryl. The aryl ring may be monocyclic. In certain instances, A is phenylene or substituted phenylene. In some cases, A is a 1,4-phenylene of the structure:

.

[0096] In certain other instances, A is an aromatic heterocycle, ie heteroaryl or substituted heteroaryl. The heteroaryl ring may be monocyclic. Heteroaryls of interest include, but are not limited to, pyridine, pyridazine, pyrimidine, and pyrazine.

[0097] In some embodiments of Formula (I), L is -(CH2)n-. In certain cases, n is 1 to 8, such as 1 to 5. In some cases, n is 1 to 3, such as 2 or 3. In some cases, n is less than 8, such as 7, 6, 5, 4, 3, 2 or 1. In some instances, n is 1 to 6, such as 1 to 4 or 1 to 3. In some instances, n is 1. In some other embodiments, n is 2. In some instances, L is 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.

[0098] In some embodiments of formula (I), Y is selected from quinazoline, substituted quinoline, quinoline, substituted quinoline, naphthalene, substituted naphthalene, isoquinoline, and substituted isoquinoline. In certain instances, Y is selected from quinazoline and substituted quinazoline. In certain instances, Y is selected from quinoline and substituted quinoline. In certain instances, Y is selected from naphthalene and substituted naphthalene. In certain instances, Y is selected from isoquinoline and substituted isoquinoline. In some embodiments of formula (I), Y is a group of formula (II):

(II)

Where:

each of Z1 and Z2 is independently selected from CR1 and N;

each R1 is independently selected from H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, heterocycle, and substituted heterocycle;

each of R2 and R5 is independently selected from H, OH, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, -OCF3, amine, substituted amine, amide, heterocycle, and substituted heterocycle; And

each of R3 and R4 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 R3 and R4 together with the carbon atoms to which they are attached form a fused ring selected from heterocycle, substituted heterocycle, cycloalkyl, substituted cycloalkyl, aryl and substituted aryl.

[0099] In certain embodiments of formula (II), at least Z1 and Z2 are N. In certain embodiments of formula (II), Z1 is C and Z2 is N. In certain instances of formula (II), Z1 is N and Z2 is C. In certain cases of formula (IIa), Z1 is C and Z2 is C. In certain cases of formula (II), Z1 is N and Z2 is N. In certain cases of formula (II), R1 and R4 are not hydrogen. In some specific cases of formula (II), R1, R3 and R4 are not hydrogen. In some specific cases of formula (II), R1, R3, R4 and R5 are not hydrogen.

[00100] In some specific cases of formula (II), R1 is selected from hydrogen, C 1-5 alkyl, vinyl heterocycle (eg, -CH=CH-heterocycle). In certain instances, the α-vinylheterocycle is a vinylpyridine (eg, -CH=CH-pyridine). In some embodiments of formula (IIa), R1 is hydrogen. In some cases, R1 is C1-5 alkyl. In other cases, R1 is a vinyl heterocycle. In certain instances, R1 is vinylpyridine. In some specific instances, R2 and R5 are hydrogen. In some instances, R5 is selected from C1-5 alkyl, amine, triazole, imidazole, amide, alkoxy, OCF3 and hydroxy. In certain instances, R5 is alkoxy, eg methoxy. In some specific instances, R3 and R4 are each independently selected from hydrogen, C1-5 alkyl, triazole, imidazole, amine, amide, alkoxy, OCF3, hydroxy, or R3 and R4 together with the carbon atom to which they are attached. form a heterocycle. In some cases, R3 and R4 are alkoxy, for example, in some cases, both of R3 and R4 are methoxy. In some instances, R5 is methoxy and R1-R4 are each hydrogen. In some cases, R5 is methoxy, R1 is -CH=CH-heterocycle, and each of R2-R4 is hydrogen.

[00101] In some embodiments of formula (II), Y is a group of formula (IIA):

(IIA)

Where

R7 is selected from the group consisting of H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, heterocycle, and substituted heterocycle;

R8 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.

[00102] In some specific instances of formula (IIA), R7 is selected from hydrogen, C1-5 alkyl, substituted C1-5 alkyl, vinyl heterocycle, and substituted vinyl heterocycle. In some specific instances of formula (IIA), R7 is selected from hydrogen, C1-5 alkyl, vinyl heterocycle (eg -CH=CH-heterocycle). In certain instances, the α-vinylheterocycle is a vinylpyridine (eg, -CH=CH-pyridine). In some embodiments of formula (IIA), R7 is hydrogen. In some instances, R7 is C1-5 alkyl. In other cases, R7 is a vinyl heterocycle. In certain instances, R7 is vinylpyridine. In some specific instances, R8 is selected from hydrogen, C1-5 alkyl, triazole, imidazole, amine, amide, alkoxy, OCF3 and hydroxyl. In some instances, R8 is alkoxy, eg methoxy. In some cases R8 is methoxy and R7 is hydrogen. In some instances, R8 is methoxy and R7 is -CH=CH- heterocycle. In some cases of formula (II), Y is a group of formula (IIB):

(IIB)

Where

R7 is selected from the group consisting of H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, heterocycle, and substituted heterocycle;

each of R8 and R9 is 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 R8 and R9 together with the carbon atoms to which they are attached form a fused ring selected from heterocycle, substituted heterocycle, cycloalkyl, substituted cycloalkyl, aryl and substituted aryl.

[00103] In some specific instances of formula (IIB), R7 is selected from hydrogen, C1-5 alkyl, vinyl heterocycle (eg, -CH=CH-heterocycle). In certain instances, the α-vinylheterocycle is a vinylpyridine (eg, -CH=CH-pyridine). In some embodiments of formula (IIB), R7 is hydrogen. In some instances, R7 is C1-5 alkyl. In other cases, R7 is a vinyl heterocycle. In certain instances, R7 is vinylpyridine. In some specific cases, R8 and R9 are each independently selected from hydrogen, C1-5 alkyl, triazole, imidazole, amine, amide, alkoxy, OCF3 and hydroxy, or R8 and R9 together with the carbons to which they are attached form a fused heterocycle . In some cases, R8 and R9 are alkoxy, for example, in some cases, both of R8 and R9 are methoxy. In some embodiments of formula (II), Y is a group of formula (IIC):

(IIC)

Where

R7 is selected from the group consisting of H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, heterocycle, and substituted heterocycle;

R10 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;

each of R8 and R9 is 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 R8 and R9 together with the carbon atoms to which they are attached form a fused ring selected from heterocycle, substituted heterocycle, cycloalkyl, substituted cycloalkyl, aryl and substituted aryl.

[00104] In some specific instances of formula (IIC), R7 is selected from hydrogen, C1-5 alkyl, vinyl heterocycle (eg, -CH=CH-heterocycle). In certain instances, the α-vinylheterocycle is a vinylpyridine (eg, -CH=CH-pyridine). In some embodiments of Formula (IIC), R7 is hydrogen. In some instances, R7 is C1-5 alkyl. In some cases, R7 is a vinyl heterocycle. In certain instances, R7 is vinylpyridine. In some cases, R10 is selected from hydrogen, C 1-5 alkyl, amine, triazole, imidazole, amide, alkoxy, OCF3 and hydroxy. In some cases, R10 is hydrogen. In certain instances, R10 is alkoxy, eg methoxy. In some specific instances, R8 and R9 are each independently selected from hydrogen, C1-5 alkyl, triazole, imidazole, amine, amide, alkoxy, OCF3, hydroxy, or R8 and R9, together with the carbons to which they are attached, form a fused heterocycle . In some cases, R8 and R9 are alkoxy, for example, in some cases, both of R8 and R9 are methoxy. In some cases, R10 is methoxy and each of R7-R9 are hydrogen. In some cases, R10 is methoxy, R7 is -CH=CH-heterocycle, and each of R8 and R9 is hydrogen. In some embodiments of formula (II), Y is a group of formula (IID):

(IID)

Where

R7 is selected from the group consisting of H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, heterocycle, and substituted heterocycle;

each of R11 and R12 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; or R11 and R12 together with the carbon atoms to which they are attached form a fused ring selected from heterocycle, substituted heterocycle, cycloalkyl, substituted cycloalkyl, aryl and substituted aryl.

[00105] In some specific instances of formula (IID), R7 is selected from hydrogen, C1-5 alkyl, vinyl heterocycle (eg, -CH=CH-heterocycle). In certain instances, the α-vinylheterocycle is a vinylpyridine (eg, -CH=CH-pyridine). In some embodiments of Formula (IID), R7 is hydrogen. In some instances, R7 is C1-5 alkyl. In some cases, R7 is a vinyl heterocycle. In certain instances, R7 is vinylpyridine. In some specific cases, R11 and R12 are each independently selected from hydrogen, C1-5 alkyl, triazole, imidazole, amine, amide, alkoxy, OCF3 and hydroxy, or R11 and R12 together with the carbons to which they are attached form a fused heterocycle . In some cases, R11 and R12 are alkoxy, for example, in some cases, both of R11 and R12 are methoxy.

[00106] In some embodiments of formula (II), Y is a group of formula (IIE):

(IIE)

Where

R7 is selected from the group consisting of H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, heterocycle, and substituted heterocycle;

each of R11 and R12 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; or R11 and R12 together with the carbon atoms to which they are attached form a fused ring selected from heterocycle, substituted heterocycle, cycloalkyl, substituted cycloalkyl, aryl and substituted aryl.

[00107] In some specific instances of formula (IIE), R7 is selected from hydrogen, C1-5 alkyl, vinyl heterocycle (eg, -CH=CH-heterocycle). In certain instances, the α-vinylheterocycle is a vinylpyridine (eg, -CH=CH-pyridine). In some embodiments of formula (IIE), R7 is hydrogen. In some instances, R7 is C1-5 alkyl. In other cases, R7 is a vinyl heterocycle. In certain instances, R7 is vinylpyridine. In some specific instances, R11 and R12 are each independently selected from hydrogen, C1-5 alkyl, triazole, imidazole, amine, amide, alkoxy, OCF3 and hydroxy, or R11 and R12 together with the carbon to which they are attached form a heterocycle. In some cases, R11 and R12 are alkoxy, for example, in some cases, both of R11 and R12 are methoxy.

[00108] In some embodiments of formula (II), Y is a group selected from:

And .

[00109] In some embodiments of formula (II), any of R1-R5 may be halogen, such as F, Cl, Br, or I. In some embodiments of formula (II), at least one of R1-R5 is an atom halogen. In some embodiments of formula (II), at least one of R1-R5 is fluoride. In other embodiments of formula (II), at least one of R1-R5 is chloride. In other embodiments of formula (II), at least one of R1-R5 is bromide. In other embodiments of formula (II), at least one of R1-R5 is iodide.

[00110] In some embodiments of formula (II), Y is a group selected from:

, And .

[00111] In some embodiments of formula (I), Y is a group of formula (XI):

(XI),

Where:

Z21 is selected from CR1 and N;

R1, R21 and R22 are independently selected from H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, heterocycle, and substituted heterocycle;

R2 and R5 are independently selected from H, OH, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, -OCF3, amine, substituted amine, amide, heterocycle, and substituted heterocycle; And

R3 and R4 are independently selected from H, OH, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, -OCF3, amine, substituted amine, amide, heterocycle, and substituted heterocycle; or R3 and R4 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.

[00112] In some specific instances of Formula (XI), R1 and R4 are not hydrogen. In some specific cases of formula (XI), R1, R3 and R4 are not hydrogen. In some specific cases of formula (XI), R1, R3, R4 and R5 are not hydrogen.

[00113] In some specific instances of formula (XI), Z21 is CR1 and R1 is selected from hydrogen, C 1-5 alkyl, vinyl heterocycle (eg, -CH=CH-heterocycle). In certain instances, the α-vinylheterocycle is a vinylpyridine (eg, -CH=CH-pyridine). In some specific instances of formula (XI), Z21 is CR1 and R1 is hydrogen. In some cases, R1 is C1-5 alkyl. In other cases, Z21 is CR1 and R1 is a vinyl heterocycle. In certain instances, R1 is vinylpyridine. In some specific instances, R2 and R5 are hydrogen. In some instances, R5 is selected from C1-5 alkyl, amine, triazole, imidazole, amide, alkoxy, OCF3 and hydroxy. In certain instances, R5 is alkoxy, eg methoxy. In some specific instances, R3 and R4 are each independently selected from hydrogen, C1-5 alkyl, triazole, imidazole, amine, amide, alkoxy, OCF3, hydroxy, or R3 and R4 together with the carbon atom to which they are attached form a heterocycle. In some cases, R3 and R4 are alkoxy, for example, in some cases, both of R3 and R4 are methoxy. In some instances, R5 is methoxy and R1-R4 are each hydrogen. In some cases, R5 is methoxy, R1 is -CH=CH-heterocycle, and each of R2-R4 is hydrogen.

[00114] In some embodiments of formula (I), Y is a group of formula (III):

(III)

Where:

each of Z1 and Z2 is independently selected from CR1 and N;

each R1 is independently selected from the group consisting of H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, heterocycle, and substituted heterocycle; And

R6 is selected from the group consisting of heterocycle, substituted heterocycle, cycloalkyl, substituted cycloalkyl, aryl, and substituted aryl. In certain embodiments of formula (III), at least Z1 and Z2 are N. In certain embodiments of formula (III), Z1 is CH and Z2 is N. In certain instances of formula (III), Z1 is N and Z2 is a CH. In certain instances of formula (III), Z1 is CH and Z2 is CH. In certain instances of formula (III), Z1 is N and Z2 is N.

[00115] In some embodiments of formula (III), Y is a group of formula (IIIA):

(IIIA),

Where

each of Z5, Z6, Z7 and Z8 is independently selected from CR14 and N;

R13 is selected from the group consisting of H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, heterocycle, and substituted heterocycle;

R14 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; And

m is 0-5.

[00116] In some specific instances of formula (IIIA), one and only one of Z5, Z6, Z7, and Z8 is N. In some specific instances of formula (IIIA), two and only two of Z5, Z6, Z7, and Z8 are N In some specific cases of formula (IIIA), Z5 is N. In some specific cases of formula (IIIA), Z6 is N. In some specific cases of formula (IIIA), Z7 is N. In some specific cases of formula (IIIA), Z8 is is N. In some specific instances of formula (IIIA) each of Z5 and Z7 is N. In some instance of formula (IIIA) each of Z7 and Z8 is H.

[00117] In some embodiments of formula (III), Y is a group of formula (IIIB):

(IIIB)

Where

each of Z9, Z10 and Z11 is independently selected from CR14 and N;

R13 is selected from the group consisting of H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, heterocycle, and substituted heterocycle;

R14 is independently selected from the group consisting of and H, OH, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, -OCF3, amine, substituted amine, amide, heterocycle, and substituted heterocycle; And

p is 0-4.

[00118] In some specific instances of formula (IIIB), one and only one of Z9, Z10, and Z11 is N. In some specific instances of formula (IIIB), two and only two of Z9, Z10, and Z11 are N. In some specific instances, Z9, Z10, and Z11 are N. cases of formula (IIIB) Z9 is N. In some specific cases of formula (IIIA) Z10 is N. In some specific cases of formula (IIIB) Z11 is N. In some specific cases of formula (IIIB) R14 is selected from alkyl and substituted alkyl. In some specific instances of formula (IIIB), p is 0. In some specific instances of formula (IIIB), p is 1. In some specific instances of formula (IIIB), p is 2.

[00119] In some embodiments of formula (III), Y is a group selected from:

And or its replacement.

[00120] In some embodiments of formula (I), Y is a group of formula (IIIC)

(IIIC)

Where

each of Z1, Z2, Z17, Z18 and Z19 is independently selected from CR20 and N;

each R20 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; And

p1 is an integer 0-4.

In some specific instances of formula (IIIC), Z1, Z2, Z17 and Z19 are each N and Z18 is CR20.

In some embodiments of formula (IIIC), Y has the structure:

.

[00121] In some embodiments of formula (I), the structure has formula (IV):

(IV)

Where

each of Z1 and Z2 is independently selected from CR1 and N;

Z3 and Z4 are each independently selected from CR and N, where R is H, alkyl, or substituted alkyl;

R1 is selected from the group consisting of H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, heterocycle, and substituted heterocycle;

each of R2 and R5 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;

each of R3 and R4 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;

or R3 and R4 together with the carbon atom to which they are attached form a group selected from heterocycle, substituted heterocycle, cycloalkyl, substituted cycloalkyl, aryl and substituted aryl,

or a prodrug, pharmaceutically acceptable salt or solvate thereof.

[00122] In certain embodiments of formula (IV), at least one of Z1 and Z2 is N. In certain embodiments of formula (IV), Z1 is C and Z2 is N. In certain instances of formula (IV), Z1 is is N and Z2 is C. In certain instances of formula (IV), Z1 is C and Z2 is C. In certain instances of formula (IV), Z1 is N and Z2 is N. In certain embodiments of formula (IV) , at least one of Z3 and Z4 is N. In certain instances of formula (IV), Z3 is N and Z4 is N. In certain instances of formula (IV), Z3 is N and Z4 is CH. In certain instances of formula (IV), Z3 is CH and Z4 is N. In certain instances of formula (VI), Z3 is CH and Z4 is CH.

[00123] In some specific cases of formula (IV), R1 is selected from hydrogen, C1-5 alkyl, vinyl heterocycle (eg, -CH=CH-heterocycle). In certain instances, the α-vinylheterocycle is a vinylpyridine (eg, -CH=CH-pyridine). In some embodiments of formula (IV), R1 is hydrogen. In some cases, R1 is C1-5 alkyl. In other cases, R1 is a vinyl heterocycle. In certain instances, R1 is vinylpyridine. In some specific instances, R2 and R5 are hydrogen. In some instances, R5 is selected from C1-5 alkyl, amine, triazole, imidazole, amide, alkoxy, OCF3 and hydroxy. In certain instances, R5 is alkoxy, eg methoxy. In some specific instances, R3 and R4 are each independently selected from hydrogen, C1-5 alkyl, triazole, imidazole, amine, amide, alkoxy, OCF3, hydroxy, or R3 and R4 together with the carbon atom to which they are attached form a heterocycle. In some cases, R3 and R4 are alkoxy, for example, in some cases, both of R3 and R4 are methoxy. In some instances, R5 is methoxy and R1-R4 are each hydrogen. In some cases, R5 is methoxy, R1 is -CH=CH-heterocycle, and each of R2-R4 is hydrogen.

[00124] In some embodiments of formula (I), the structure has formula (V):

(V)

Where:

each of Z1 and Z2 is independently selected from CR1 and N;

Z3 and Z4 are each independently selected from CR and N, where R is H, alkyl, or substituted alkyl;

each R1 is independently selected from H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, heterocycle, and substituted heterocycle;

R6 is selected from heterocycle, substituted heterocycle, cycloalkyl, substituted cycloalkyl, aryl and substituted aryl,

or a prodrug, pharmaceutically acceptable salt or solvate thereof.

[00125] In certain embodiments of formula (V), at least one of Z1 and Z2 is N. In certain embodiments of formula (V), Z1 is CH and Z2 is N. In certain instances of formula (IV), Z1 is is N and Z2 is CH. In certain instances of formula (V), Z1 is CH and Z2 is CH. In certain instances of formula (IV), Z1 is N and Z2 is N. In certain embodiments of formula (V), at least one of Z3 and Z4 is N. In certain instances of formula (V), Z3 is N and Z4 is N. In certain instances of formula (V), Z3 is N and Z4 is CH. In certain instances of formula (V), Z3 is CH and Z4 is N. In certain instances of formula (V), Z3 is CH and Z4 is CH.

[00126] In some embodiments of formula (I), the inhibitor has formula (VI):

(VI)

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 a linker;

each of Z1 and Z2 is independently selected from CR1 and N;

Z3 and Z4 are each independently selected from CR and N, where R is H, alkyl, or substituted alkyl;

each R1 is independently selected from H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, heterocycle, and substituted heterocycle;

each of R2 and R5 is independently selected from H, OH, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, -OCF3, halogen, amine, substituted amine, amide, heterocycle, and substituted heterocycle;

each of R3 and R4 is 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 R3 and R4 together with the carbon atoms to which they are attached form a fused compound selected from heterocycle, substituted heterocycle, cycloalkyl, substituted cycloalkyl, aryl and substituted aryl;

or a prodrug, pharmaceutically acceptable salt or solvate thereof.

[00127] In some embodiments of formula (I), the structure has formula (VI):

(VI)

Where

L is selected from the group consisting of -CH2-, -(CH2)2-, -(CH2)3-, -(CH2)4-, -(CH2)5- and -(CH2)6-;

X is selected from the group consisting of , , , , , , , , , , , , And where Ra and Rb are each independently selected from aryl, alkyl, -CH2OC(O)Re, -CH2OC(O)ORe; Rc and Rd are each independently selected from -C(CH3)C(O)ORe, alkyl, and where Re is alkyl;

each of Z1, Z2, Z3 and Z4 is independently selected from CR1 and N;

R1 is selected from the group consisting of H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, heterocycle, and substituted heterocycle;

each of R2 and R5 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;

each of R3 and R4 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;

or R3 and R4 together with the carbon atom to which they are attached form a group selected from heterocycle, substituted heterocycle, cycloalkyl, substituted cycloalkyl, aryl and substituted aryl,

or a prodrug, pharmaceutically acceptable salt or solvate thereof.

[00128] In certain embodiments of formula (VI), at least one of Z1 and Z2 is N. In certain embodiments of formula (VI), Z1 is C and Z2 is N. In certain instances of formula (VI), Z1 is is N and Z2 is C. In certain instances of formula (VI), Z1 is C and Z2 is C. In certain instances of formula (VI), Z1 is N and Z2 is N. In certain embodiments of formula (VI) at least one of Z3 and Z4 is N. In certain instances of formula (VI), Z3 is N and Z4 is N. In certain instances of formula (IV), Z3 is N and Z4 is C. In certain instances of formula (VI) Z3 is C and Z4 is N. In certain instances of formula (VI), Z3 is C and Z4 is C.

[00129] In some specific instances of formula (VI), R1 is selected from hydrogen, C1-5 alkyl, vinyl heterocycle (eg, -CH=CH-heterocycle). In certain instances, the α-vinylheterocycle is a vinylpyridine (eg, -CH=CH-pyridine). In some embodiments of formula (VI), R1 is hydrogen. In some cases, R1 is C1-5 alkyl. In other cases, R1 is a vinyl heterocycle. In certain instances, R1 is vinylpyridine. In some specific cases, R2 and R5 are hydrogen. In some instances, R5 is selected from C1-5 alkyl, amine, triazole, imidazole, amide, alkoxy, OCF3 and hydroxy. In certain instances, R5 is alkoxy, eg methoxy. In some specific instances, R3 and R4 are each independently selected from hydrogen, C1-5 alkyl, triazole, imidazole, amine, amide, alkoxy, OCF3, hydroxy, or R3 and R4 together with the carbon atom to which they are attached form a heterocycle. In some cases, R3 and R4 are alkoxy, for example, in some cases, both of R3 and R4 are methoxy. In some instances, R5 is methoxy and R1-R4 are each hydrogen. In some cases, R5 is methoxy, R1 is -CH=CH-heterocycle, and each of R2-R4 is hydrogen.

[00130] In certain embodiments of Formula (VI), L is -CH2-. In certain other instances of formula (VI), L is -(CH2)2-.

[00131] In certain embodiments of Formula (VI), X is . In certain cases of formula (VI), X is . In certain other instances of formula (VI), X is . In certain cases of formula (VI), X is . In certain other instances of formula (VI), X is . In certain embodiments of Formula (VI), X is . In certain cases of formula (VI), X is . In certain other instances of formula (VI), X is . In certain cases of formula (VI), X is . In certain other instances of formula (VI), X is . In certain cases of formula (VI), X is . In certain other instances of formula (VI), X is . In certain other instances of formula (VI), X is where Ra and Rb are each independently selected from aryl, alkyl, -CH2OC(O)Re, -CH2OC(O)ORe, where Re is alkyl. In certain cases of formula (VI), X is where Rc and Rd are each independently selected from -C(CH3)C(O)ORe and alkyl, where Re is alkyl. In certain other instances of formula (VI), X is where Ra is selected from aryl, alkyl, -CH2OC(O)Re, -CH2OC(O)ORe and Rc is selected from -C(CH3)C(O)ORe and alkyl, where Re is alkyl.

[00132] It is understood that any of the hydroxyl and amine groups in group X of formula (VI) may be further substituted with any suitable group, for example, an alkyl group, a substituted alkyl group, a phenyl group, a substituted phenyl group, an ester group, and the like. It is understood that any suitable alternative hydrophilic group may be used as the X group in the compound of formula (VI).

[00133] In some embodiments of formula (I), the structure has formula (VII):

(VII)

Where

L is selected from the group consisting of -CH2-, -(CH2)2-, -(CH2)3-, -(CH2)4-, -(CH2)5- and -(CH2)6-;

X is selected from the group consisting of , , , , , , , , , , , , And where Ra and Rb are each independently selected from aryl, alkyl, -CH2OC(O)Re, -CH2OC(O)ORe; Rc and Rd are each independently selected from -C(CH3)C(O)ORe, alkyl, and where Re is alkyl:

each of Z1 and Z2 is independently selected from C and N;

R1 is selected from the group consisting of H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, heterocycle, and substituted heterocycle;

each of R2 and R5 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;

each of R3 and R4 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;

or R3 and R4 together with the carbon atom to which they are attached form a group selected from heterocycle, substituted heterocycle, cycloalkyl, substituted cycloalkyl, aryl and substituted aryl,

or a prodrug, pharmaceutically acceptable salt or solvate thereof.

[00134] In certain embodiments of formula (VII), at least one of Z1 and Z2 is N. In certain embodiments of formula (VII), Z1 is C and Z2 is N. In certain instances of formula (VII) , Z1 is N and Z2 is C. In certain instances of formula (VII), Z1 is C and Z2 is C. In certain instances of formula (VII), Z1 is N and Z2 is N.

[00135] In some specific cases of formula (VII), R1 is selected from hydrogen, C1-5 alkyl, vinyl heterocycle (eg, -CH=CH-heterocycle). In certain instances, the α-vinylheterocycle is a vinylpyridine (eg, -CH=CH-pyridine). In some embodiments of formula (VII), R1 is hydrogen. In some cases, R1 is C1-5 alkyl. In other cases, R1 is a vinyl heterocycle. In certain instances, R1 is vinylpyridine. In some specific instances, R2 and R5 are hydrogen. In some instances, R5 is selected from C1-5 alkyl, amine, triazole, imidazole, amide, alkoxy, OCF3 and hydroxy. In certain instances, R5 is alkoxy, eg methoxy. In some specific instances, R3 and R4 are each independently selected from hydrogen, C1-5 alkyl, triazole, imidazole, amine, amide, alkoxy, OCF3, hydroxy, or R3 and R4 together with the carbon atom to which they are attached form a heterocycle. In some cases, R3 and R4 are alkoxy, for example, in some cases, both of R3 and R4 are methoxy. In some instances, R5 is methoxy and R1-R4 are each hydrogen. In some cases, R5 is methoxy, R1 is -CH=CH-heterocycle, and each of R2-R4 is hydrogen.

[00136] In certain embodiments of Formula (VII), L is -CH2-. In certain other instances of formula (VII), L is -(CH2)2-.

[00137] In certain embodiments of Formula (VII), X is . In certain cases of formula (VII), X is . In certain other instances of formula (VII), X is . In certain cases of formula (VII), X is . In certain other instances of formula (VII), X is . In certain embodiments of Formula (VII), X is . In certain cases of formula (VII), X is . In certain other instances of formula (VII), X is . In certain cases of formula (VII), X is . In certain other instances of formula (VII), X is . In certain cases of formula (VII), X is . In certain other instances of formula (VII), X is . In certain other instances of formula (VI), X is where Ra and Rb are each independently selected from aryl, alkyl, -CH2OC(O)Re, -CH2OC(O)ORe, where Re is alkyl. In certain cases of formula (VI), X is where Rc and Rd are each independently selected from -C(CH3)C(O)ORe and alkyl, where Re is alkyl. In certain other instances of formula (VI), X is where Ra is selected from aryl, alkyl, -CH2OC(O)Re, -CH2OC(O)ORe and Rc is selected from -C(CH3)C(O)ORe and alkyl, where Re is alkyl.

[00138] It is understood that any of the hydroxyl and amine groups in group X of formula (VII) may be further substituted with any suitable group, for example, an alkyl group, a substituted alkyl group, a phenyl group, a substituted phenyl group, an ester group, and the like. It is understood that any suitable alternative hydrophilic group may be used as the X group in the compound of formula (VII).

[00139] In certain embodiments, a compound is described by the structure of one of the compounds in Table 1 or Table 2.

Table 1. Connections

No. Structure No. Structure No. Structure 1 2 3 4 5 6 7 8 9 10 eleven 12

Table 1, continued

No. Structure No. Structure No. Structure 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27

Table 1, continued

No. Structure No. Structure No. Structure 28 29 thirty 31 32 33 34 35 36 37 38 39 40 41 42

Table 1, continued

No. Structure No. Structure No. Structure 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57

Table 1, continued

No. Structure No. Structure No. Structure 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72

Table 1, continued

No. Structure No. Structure No. Structure 73 74 75 76 77 78 79 80 81 82 83 84

Table 1, continued

No. Structure No. Structure No. Structure 85 86 87 88 89 90 91 92 107 108 109

Table 2. Connections

Comm. Structure Comm. Structure Comm. Structure 93 94 95 96 97 98 99 100 101 102 103 104

Table 2, continued

No. Structure No. Structure 105 106

[00140] In certain embodiments, a compound is described 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 may be present in salt form. In some instances, the salt form of a compound is a pharmaceutically acceptable salt. It is understood that any of the compounds shown in Table 1 or Table 2 may be present in the form of a prodrug.

[00141] Aspects of the present invention include compounds that are inhibitors of ENPP1 (eg, as described herein), their salts (eg, pharmaceutically acceptable salts), and/or their solvate, hydrate, and/or prodrug forms. Additionally, it should be understood that in any compound described herein that has one or more chiral centers, unless the absolute stereochemistry has been clearly stated, then each center may independently have the R-configuration, S-configuration, or a mixture of both. It is clear that all variations and combinations of salts, solvates, hydrates, prodrugs and stereoisomers should be covered by the scope of this disclosure.

[00142] In some embodiments, the ENPP1 inhibitor compounds or prodrugs thereof are in the form of pharmaceutically acceptable salts. Compounds containing an amino or nitrogen-containing heteroaryl group may be basic in nature and, accordingly, may be reacted with any amount of inorganic and organic acids to form pharmaceutically acceptable acid addition salts. Acids commonly used to form such salts include inorganic acids such as hydrochloric, hydrobromic, hydroiodic, sulfuric and phosphoric acids, as well as organic acids such as p-toluenesulfonic, methanesulfonic, oxalic, p-bromophenolsulfonic, carbonic, succinic, citric, benzoic and acetic acids, as well as related inorganic and organic acids. Such pharmaceutically acceptable salts thus include 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, hexine-1,6-dioate, benzoate, chlorobenzoate, methyl benzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, terephthalate , sulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, β-hydroxybutyrate, glycolate, maleate, tartrate, methanesulfonate, propanesulfonates, naphthalene-1-sulfonate, naphthalene-2-sulfonate, mandelate, hippurate, gluconate, lactobionate, etc. similar salts. In certain specific embodiments, pharmaceutically acceptable acid addition salts include salts formed with mineral acids such as hydrochloric acid and hydrobromic acid, and salts formed with organic acids such as fumaric acid and maleic acid.

[00143] In some embodiments, the compounds of the present invention are in the form of a prodrug. "Prodrug" refers to a derivative of the active agent that requires conversion in the body to release the active agent. In certain embodiments, the transformation is an enzymatic transformation. Prodrugs are often, though not necessarily, pharmacologically inactive until converted to an active agent. A "temporary modifying protecting group" refers to a form of a protecting group which, when used to modify a functional group in an active agent, converts the active agent into a prodrug. In some cases, a temporary modifying protecting group will be attached to the drug through bonds that are cleaved by enzymatic or non-enzymatic methods in vivo . Any convenient prodrug forms of the compounds of the present invention may be prepared, for example, in accordance with the strategies and methods described by Rautio et al. (“Prodrugs: design and clinical applications”, Nature Reviews Drug Discovery 7, 255-270 (February 2008)). In some cases, a temporary modifying protecting group is attached to the hydrophilic head group of the compounds in question. In some cases, a temporary modifying protecting group is attached to the hydroxy or carboxylic acid group of the compounds in question. In certain instances, the temporary modifying protecting group is an acyl or substituted acyl group. In certain cases, the temporary modifying group is an alkyl or substituted alkyl group, for example, forming an ester functional group when attached to a hydrophilic head group of the compounds in question, for example, a phosphonate ester, a phosphate ester, etc.

[00144] In some embodiments, the subject compound is a phosphonate ester or phosphate ester prodrug that can be transformed into a compound comprising a phosphonic acid or phosphonate, or a phosphate head group. In certain embodiments, the prodrug is one of compounds 74, 77, and 78 of Table 1.

[00145] In some embodiments, the compounds of the present invention, prodrugs, stereoisomers, or salts thereof are in the form of a solvate (eg, hydrate). As used herein, the term “solvate” refers to a complex or aggregate formed by one or more molecules of a solute, i.e., a prodrug or a pharmaceutically acceptable salt thereof, and one or more molecules of a solvent. Such solvates are typically solid crystals having a substantially fixed solute to solvent molar ratio. 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.

[00146] In some embodiments, the compounds of the present invention are administered orally and are absorbed into the bloodstream. In some embodiments, the oral bioavailability of the compounds of the present invention is 30% or more. Modifications may be made to the compounds of the invention or formulations thereof using any convenient means to increase intestinal lumen absorption or bioavailability.

[00147] In some embodiments, the compounds of the present invention are metabolically stable (eg, 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, e.g., 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.

ENPP1 inhibition methods

[00148] As summarized above, aspects of the present disclosure include ENPP1 inhibitors and methods of inhibition using them. ENPP1 is a member of the ectonucleotide pyrophosphatase/phosphodiesterase (ENPP) family. As such, aspects of the methods of the present invention include the inhibition of cGAMP hydrolase activity of ENPP1. The inventors have found that cGAMP can have significant extracellular biological functions that can be enhanced by blocking the extracellular degradation of cGAMP, such as hydrolysis by its degrading enzyme ENPP1. In certain instances, the target of ENPP1 inhibition is extracellular and ENPP1 inhibitory compounds do not enter cells and therefore cannot diffuse into cells. As such, the methods of the present invention can provide selective extracellular inhibition of ENPP1 hydrolase activity and increased extracellular levels of cGAMP. As such, in some instances, ENPP1 inhibitory compounds are compounds that inhibit ENPP1 activity extracellularly. Experiments by the inventors show that inhibition of ENPP1 activity increases extracellular cGAMP and therefore can enhance the STING pathway.

[00149] Inhibition of 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 (eg, relative to a control in any suitable in vitro inhibition assay). In some cases, inhibition of ENPP1 means a decrease in the activity of the enzyme by 2 or more times, for example, 3 or more, 5 or more, 10 or more, 100 or more, or 1000 or more times its normal activity (for example, relative to the control, measured by any suitable assay ).

[00150] In some cases, this method is a method of inhibiting ENPP1 in the sample. As used herein, the term "sample" refers to a material or mixture of materials, usually, although not necessarily, in liquid form, containing one or more components of interest.

[00151] In some embodiments, a method for inhibiting ENPP1 is provided, comprising contacting a sample with a non-cell-penetrating inhibitor of ENPP1 to inhibit cGAMP hydrolysis activity of ENPP1. In some cases, the sample is a cellular sample. In some cases, the sample contains cGAMP. In certain instances, cGAMP levels are elevated in a cell sample (eg, relative to a non-inhibitor-bound control sample). The methods of the present invention can provide increased levels of cGAMP. By "elevated cGAMP" is meant the level of cGAMP in a cell sample in contact with a test compound where the level of cGAMP in the sample is increased by 10% or more, such as 20% or more, 30% or more, 40% or more, 50% or more. 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 did not come into contact with the agent.

[00152] In certain embodiments, the non-cell-penetrating ENPP1 inhibitor is an inhibitor as defined herein. In some embodiments, non-cell-penetrating ENPP1 is an inhibitor according to any of the formulasI,IV V, VI orVII. IN in some cases, a non-cell-penetrating inhibitor of ENPP1 is one of the compounds1-106.

[00153] In some embodiments, the ENPP1 inhibitor enters the cells. In some embodiments, a method for inhibiting ENPP1 is provided, comprising contacting a sample with a cell-penetrating inhibitor of ENPP1 to inhibit ENPP1.

[00154] In some embodiments, the compounds of the present invention have an ENPP1 inhibition profile that reflects activity relative to additional enzymes. In some embodiments, the compounds of the present invention specifically inhibit ENPP1 without undesirably inhibiting one or more other enzymes.

[00155] In some embodiments, the compounds of the present invention interfere with the interaction of cGAMP and ENPP1. For example, the compounds of the present invention may serve to increase extracellular cGAMP by inhibiting cGAMP hydrolase activity of ENPP1. Without being bound by any particular theory, it is believed that an increase in extracellular cGAMP activates the STING pathway.

[00156] In some embodiments, subject compounds inhibit ENPP1 as determined by an inhibition assay, e.g., by an assay that measures the level of enzyme activity in a cell-free system or in a cell after treatment with a compound of the present invention relative to a control compound by measuring an IC50 value or EC50 respectively. In certain embodiments, the compounds of the present invention have an IC50 value (or an EC50 value) of 10 μM or less, such as 3 μM or less, 1 μM or less, 500 nM or less, 300 nM or less, 200 nM or less, 100 nM or less, 50 nM or less, 30 nM or less, 10 nM or less, 5 nM or less, 3 nM or less, 1 nM or less, or even lower.

[00157] As summarized above, aspects of the disclosure include methods for inhibiting ENPP1. The target compound (e.g., as described herein) can inhibit ENPP1 activity 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 compound in question may inhibit its target with an IC50 of 1x10-6M or less (e.g., 1x10-6M or less, 1x10-7M or less, 1x10-8M or less, 1x10-9M or less, 1×10-10M or less, or 1×10-11M or less).

[00158] The protocols that can be used in determining ENPP1 activity are numerous and include, but are not limited to, cell-free assays, eg, binding assays; assays using purified enzymes, cellular assays that measure cellular phenotype, such as gene expression assays; and in vivo assays that involve a specific animal (which, in certain embodiments, may be an animal model for a condition associated with a target pathogen).

[00159] In some embodiments, the method in question is an in vitro method that includes contacting a sample with a compound in question that specifically inhibits ENPP1. In certain embodiments, the sample is expected to contain ENPP1 and the method of the present invention further comprises assessing whether the compound inhibits ENPP1.

[00160] In certain embodiments, the compound of the present invention is a modified compound that includes a label, such as a fluorescent label, and the method of the present invention further comprises detecting the label, if present, in the sample, such as using optical detection.

[00161] In certain embodiments, the compound is modified with a carrier or affinity groups that bind to the carrier (eg, biotin) such that any sample that does not bind to the compound can be removed (eg, by washing). Specifically bound ENPP1, if present, can then be detected using any suitable means, such as, for example, binding a labeled target-specific probe or using a fluorescent protein-reactive reagent.

[00162] In another embodiment of the present method, the sample is known to contain ENPP1.

[00163] In some embodiments, the method is a method of reducing cancer cell proliferation, wherein the method comprises contacting a cell with an effective amount of an ENPP1 inhibitory compound (e.g., as described herein) to reduce cancer cell proliferation. In certain cases, the considered compounds, which are inhibitors of ENPP1, can act intracellularly. The method may be performed in combination with a chemotherapeutic agent (eg, as described herein). Cancer cells may be in vitro or in vivo . In certain instances, the method includes contacting a cell with an ENPP1 inhibitory compound (eg, as described herein) and contacting the cell with a chemotherapeutic agent. The target can be any suitable cancer cells.

Methods of treatment

[00164] Aspects of the present invention include methods for inhibiting cGAMP hydrolase activity of ENPP1, resulting in elevated levels of cGAMP and/or downstream modulation (eg, activation) of the STING pathway. The inventors found that cGAMP is present in the extracellular space and that ENPP1 can control extracellular levels of cGAMP. The inventors have also found that cGAMP can have significant extracellular biological functions in vivo (eg, see FIGS. 3A-4C). The results described and demonstrated herein indicate that the inhibition of ENPP1 according to the methods of the present invention may modulate the activity of STING in vivo and thus find application in the treatment of various diseases, for example as a target for cancer immunotherapy. Thus, the methods of the present invention can provide selective extracellular inhibition of ENPP1 activity (eg, cGAMP hydrolase activity) to increase extracellular cGAMP levels and activate the interferon gene stimulation (STING) pathway. In some specific cases, the present method is a method of increasing the STING-mediated response in a subject. In some specific instances, the method in question is a method of modulating an immune response in a subject.

[00165] "STING-mediated response" refers to any response that is mediated by STING, including, but not limited to, immune responses to, for example, bacterial pathogens, viral pathogens, and eukaryotic pathogens. See, for example, Ishikawa et al. Immunity 29: 538-550 (2008); Ishikawa et al. Nature 461: 788-792 (2009); and Sharma et al. Immunity 35: 194-207 (2011). STING also functions in certain autoimmune diseases caused by misrecognition 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 Nature 461: 788-792 (2009) An increase in the STING-mediated response in a subject refers to an increase in the STING-mediated response in the subject compared to a control subject (eg, a subject not receiving the compound of interest). is a human, and the compounds and according to the invention provide activation of human STING.In some cases, the STING-mediated response includes the modulation of the immune response.In some specific cases, the present method is a method of modulating the immune response in a subject.

[00166] In some cases, the STING-mediated response includes an increase in the production of interferon (eg, type I interferon (IFN), type III interferon (IFN)) in the subject. Interferons (IFN) are proteins with various types of biological activity, such as antiviral, immunomodulatory and antiproliferative. IFNs are relatively small species-specific single chain polypeptides produced by mammalian cells in response to various inducers such as viruses, polypeptides, mitogens, and the like. Interferons protect animal tissues and cells from viral attack and are an important host defense mechanism. Interferons can be classified as type I, type II and type III interferons. Type I mammalian interferons of interest include IFN-α (alpha), IFN-β (beta), IFN-κ (kappa), IFN-δ (delta), IFN-ε (epsilon), IFN-τ (tau), IFN- ω (omega) and IFN-ζ (zeta, also known as limitin).

[00167] Interferons find use in the treatment of various types of cancer because these molecules have anti-cancer activity that acts at several levels. Interferon proteins can directly inhibit the proliferation of human tumor cells. In some cases, the antiproliferative activity is also synergistic with various approved chemotherapeutic agents such as cisplatin, 5FU and paclitaxel. The immunomodulatory activity of interferon proteins can also lead to the induction of an antitumor immune response. This response includes activation of NK cells, stimulation of macrophage activity and induction of surface expression of MHC class I, which leads to the induction of antitumor cytotoxic activity of T-lymphocytes. In addition, interferons play a role in cross-presentation in the immune system. Moreover, some studies also show that the IFN-β protein may have anti-angiogenic activity. Angiogenesis, the formation of new blood vessels, is critical for the growth of solid tumors. IFN-β can inhibit angiogenesis by inhibiting the expression of pro-angiogenic factors such as bFGF and VEGF. Interferon proteins can also inhibit tumor invasiveness by modulating the expression of enzymes such as collagenase and elastase that are important for tissue remodeling.

[00168] Aspects of the methods include administering to a subject with cancer a therapeutically effective amount of an ENPP1 inhibitor to treat the subject's cancer. In some specific cases, the subject is a person diagnosed or suspected of having cancer. Any suitable inhibitors of ENPP1 may be used in the subject cancer treatments. In certain instances, an ENPP1 inhibitor compound is a compound as described herein. In certain instances, the ENPP1 inhibitor is a non-cell-penetrating compound. In certain cases, the ENPP1 inhibitor is a cell-penetrating compound. In certain cases, the cancer is a solid tumor. In certain embodiments, the cancer is selected from adrenal, liver, kidney, bladder, breast, colon, stomach, ovary, cervix, uterus, esophagus, colorectal, prostate, pancreatic, lung (small cell and non-small cell) cancers , thyroid, carcinomas, sarcomas, glioblastomas, melanomas and various tumors of the head and neck. In some cases, the cancer is breast cancer. In some embodiments, the cancer is a lymphoma.

[00169] Aspects of the methods include administering to a subject a therapeutically effective amount of a non-cell-penetrating ENPP1 inhibitor to inhibit cGAMP hydrolysis and treat the subject for cancer. In certain cases, the cancer is a solid tumor. In certain embodiments, the cancer is selected from adrenal, liver, kidney, bladder, breast, colon, stomach, ovary, cervix, uterus, esophagus, colorectal, prostate, pancreatic, lung (small cell and non-small cell) cancers , thyroid, carcinomas, sarcomas, glioblastomas, melanomas and various tumors of the head and neck. In certain embodiments, the cancer is breast cancer. In some cases, the cancer is a lymphoma.

[00170] In some embodiments of the methods disclosed herein, the non-cell-penetrating ENPP1 inhibitor is an inhibitor according to any of formulas I , IV , V , VI , or VII . In some cases, the non-cell-penetrating inhibitor of ENPP1 is any of compounds 1-106 .

[00171] In some embodiments of the methods disclosed herein, the ENPP1 inhibitor is cell penetrating.

[00172] Thus, aspects of the method include contacting the sample with the compound in question (eg, as described above) under conditions under which the compound inhibits ENPP1. Any suitable protocol may be used to contact the compound with the sample. The specific protocol used may vary, for example, depending on whether the sample is in vitro or in vivo . For in vitro protocols, contact of the sample with the compound can be achieved using any suitable protocol. In some specific cases, the sample includes cells that are maintained in a suitable culture medium and the complex is introduced into the culture medium. For in vivo protocols, any suitable administration protocol may be used. Depending on the activity of the compound, the cells of interest, the route of administration, the number of cells present, various protocols may be used.

[00173] In some embodiments, said method is a method of treating a subject for cancer. In some embodiments, the method of the present invention comprises administering to the subject an effective amount of a compound of the present invention (eg, as described herein) or a pharmaceutically acceptable salt thereof. The compound of the present invention can be administered as part of a pharmaceutical composition (eg, as described herein). In certain instances of the method, the compound administered is a compound of one of the formulas (I), (IV), (V), (VI) or (VII). In certain instances of the method, the compound administered is described by one of the compounds in Table 1 or 2.

[00174] In some embodiments, an "effective amount" is an amount of a compound of the invention that, when administered to an individual in single or multiple doses, alone or in combination therapy, is effective in inhibiting ENPP1 by about 20% (20% inhibition), at least 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) of ENPP1 activity in an individual in the absence of compound treatment, or , alternatively compared to ENPP1 activity in humans before or after compound treatment.

[00175] In some embodiments, a "therapeutically effective amount" is an amount of a subject compound that, when administered to an individual in single or multiple doses, alone or in combination therapy, is effective in reducing the tumor burden in a subject by about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90 % compared to the tumor burden in the individual in the absence of compound treatment, or alternatively compared to the tumor burden in the subject before or after treatment with the compound. As used herein, the term "tumor burden" refers to the total mass of tumor tissue in a patient with cancer.

[00176] In some embodiments, a "therapeutically effective amount" is an amount of a compound of the invention that, when administered to an individual in single or multiple doses, alone or in combination therapy, is effective in reducing the dose of radiation therapy required to observe 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 dose of radiation therapy required to observe tumor reduction in an individual in the absence of treatment with the compound.

[00177] In some embodiments, a "therapeutically effective amount" of a compound is an amount that, when administered in one or more doses to an individual suffering from cancer, is effective to achieve 1.5-log, 2-log, 2.5-log, 3-log, 3.5-log, 4-log, 4.5-log, or 5-log reduction in tumor size.

[00178] In some embodiments, an effective amount of a compound is an amount that is in the range of from about 50 ng/mL to about 50 µg/mL (e.g., from about 50 ng/mL to about 40 µg/mL, from about 30 ng /ml to about 20 μg/ml, from about 50 ng/ml to about 10 μg/ml, from about 50 ng/ml to about 1 μg/ml, from about 50 ng/ml to about 800 ng/ml, from about 50 ng/ml to about 700 ng/ml, from about 50 ng/ml to about 600 ng/ml, from about 50 ng/ml to about 500 ng/ml, from about 50 ng/ml to about 400 ng/ml, about 60 ng/mL to 400 ng/mL, 70 ng/mL to 300 ng/mL, 60 ng/mL to 100 ng/mL, 65 ng/mL to 85 ng/mL, from about 70 ng/mL ml to about 90 ng/ml, from about 200 ng/ml to about 900 ng/ml, from about 200 ng/ml to about 800 ng/ml, from about 200 ng/ml to about 700 ng/ml from about 200 ng/mL to about 600 ng/mL, from about 200 ng/mL to about 500 ng/mL, from about 200 ng/mL to about 400 ng/mL, or from about 200 ng/mL to about 300 ng/mL).

[00179] In some embodiments, an effective amount of a compound is an amount that is in the range of from about 10 pg to about 100 mg, for example, from about 10 pg to about 50 pg, from about 50 pg to about 150 pg, from about 150 pg to about 250 pg, about 250 pg to about 500 pg, about 500 pg to about 750 pg, about 750 pg to about 1 ng, about 1 ng to about 10 ng, about 10 ng to about 50 ng , 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 up to about 10 micrograms, from about 10 micrograms to about 50 micrograms, from about 50 micrograms to about 150 micrograms, from about 150 micrograms to about 250 micrograms, from about 250 micrograms to about 500 micrograms, from about 500 micrograms to about 750 micrograms, 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 amount or may be a total daily amount. The total daily amount may be from 10 pg to 100 mg, or from 100 mg to about 500 mg, or from 500 to about 1000 mg.

[00180] In some embodiments, a single dose of a compound is administered. In other embodiments, multiple doses are administered. If multiple doses are administered over a period of time, the compound can be administered twice a day (qid), daily (qd), every other day (qod), every third day, three times a week (tiw) or twice a week (biw ) over a specified period of time. For example, the compound is administered qid, qd, qod, tiw or biw over a period of one day to about 2 years or more. For example, the compound is administered at any of the above frequencies for one week, two weeks, one month, two months, six months, one year, or two years or more, depending on various factors.

[00181] Administration of a therapeutically effective amount of the subject compound to an individual with cancer may result in one or more of the following: 1) reduction in tumor burden; 2) reducing the dose of radiation therapy needed to cause shrinkage of the tumor; 3) reducing the spread of cancer from one cell to another cell in an individual; 4) reduction in morbidity or mortality in clinical outcomes; 5) reduction of the total duration of treatment in combination with other anticancer agents; and 6) improvement in disease response score (eg, reduction in one or more cancer symptoms). Any of a variety of methods can be used to determine the effectiveness of a treatment method. For example, a biological sample taken from an individual treated with the method in question can be analyzed.

[00182] Any of the compounds described herein can be used in the considered methods of treatment. In certain cases, the compound is one of the compounds of formulas I, IV, or V. In certain cases, the compound is one of the compounds of Table 1 or 2. In some cases, the compound used in the present methods does not enter cells. In some cases, the compound used in the present methods does not penetrate cells well.

[00183] In some embodiments, the implementation of the connection specifically inhibits ENPP1. In some embodiments, the compound modulates cGAMP activity. In some embodiments, the connection interferes with the interaction of ENPP1 and cGAMP. In some embodiments, the connection results in the activation of the STING pathway.

[00184] In some embodiments, the subject is a mammal. In certain cases, the subject is a person. Other subjects may include domestic animals (eg, dogs and cats), livestock (eg, cows, pigs, goats, horses, and the like), rodents (eg, mice, guinea pigs, and rats, eg, as in animal models of the disease) , as well as non-human primates (such as chimpanzees and lower apes). The subject may be in need of cancer treatment. In some specific cases, the methods of the present invention include the diagnosis of cancer, including any of the cancers described herein. In some embodiments, the compound is administered as a pharmaceutical formulation.

[00185] In certain embodiments, the ENPP1 inhibitory compound is a modified compound that includes a label, and the method further comprises detecting the label in the subject. The choice of label depends on the means of detection. Any convenient labeling and detection system can be used in the present methods, see, for example, Baker, “The whole picture,” Nature, 463, 2010, p977-980. In certain embodiments, the compound includes 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 connection includes a paramagnetic label suitable for tomographic detection. The compound of the present invention may be labeled as described above, although in some methods the compound is unlabeled and a secondary labeling agent is used for imaging.

Combination Therapies

[00186] The compounds in question may be administered to the subject alone or in combination with an additional, i.e. second, active agent. Combination therapeutic methods wherein the subject ENPP1 inhibitor compounds can be used in combination with a second active agent or adjunctive therapy such as radiation therapy. The terms "agent", "compound" and "drug" are used interchangeably herein. For example, ENPP1 inhibitor compounds can be administered alone or in combination with one or more other drugs, such as drugs used to treat diseases of interest, including, but not limited to, immunomodulatory diseases and conditions, and cancer. In some embodiments, the method of the present invention further comprises the simultaneous or sequential administration of a second agent, such as a small molecule, a chemotherapeutic agent, an antibody, an antibody fragment, an antibody-drug conjugate, an aptamer, a protein, or a checkpoint inhibitor. In some embodiments, the method further comprises administering radiation therapy to the subject.

[00187] The terms "co-administration" and "in combination with" include the administration of two or more therapeutic agents concomitantly, simultaneously, or sequentially, without any particular time limitation. In one embodiment, the agents are present in the cell or in the body of the subject at the same time or concurrently exert a biological or therapeutic effect. In one embodiment, the therapeutic agents are in the same formulation or unit dosage form. In another embodiment, the therapeutic agents are in various compositions or unit dosage forms. In certain embodiments, the first agent may be administered before (e.g., 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), at the same time, or after (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) administration of the second therapeutic agent.

[00188] "Concomitant administration" of a known therapeutic drug or adjunctive therapy with a pharmaceutical composition of the present disclosure means administering a compound and a second agent or adjunctive therapy at a time when the known drug and the composition of the present invention will have a therapeutic effect. Such concomitant administration may include simultaneous (ie, at the same time), prior or subsequent administration of the drug relative to the time of administration of the compound of the invention. Routes of administration of the two agents may differ, typical routes of administration are described in more detail below. The person skilled in the art will readily determine the appropriate timing, sequence, and doses of administration for particular drugs or treatments and the compounds of the present invention.

[00189] In some embodiments, the compounds (e.g., the compound in question and at least one additional compound or therapy) are administered to the subject within twenty-four hours of each other, e.g., within 12 hours of one another, within 6 hours of one after the other , within 3 hours of one after the other, or within 1 hour of one after the other. In certain embodiments, the compounds are administered 1 hour apart. In certain embodiments, the compounds are administered substantially simultaneously. By substantially simultaneous administration is meant that the compounds are administered to the subject within about 10 minutes or less, one after the other, for example, 5 minutes or less, or 1 minute or less, one after the other.

[00190] Pharmaceutical preparations of the compounds of the present invention and the second active agent are also provided. The compound in dosage forms may be administered in the form of their pharmaceutically acceptable salts, or the dosage forms may be used alone or in an appropriate combination, as well as in combination with other pharmaceutically active compounds.

[00191] In combination with any of the contemplated methods, compounds that are inhibitors of ENPP1 (for example, as described herein) (or pharmaceutical compositions containing such compounds) can be administered in combination with another drug designed to reduce or prevent inflammation , treatment or prevention of chronic inflammation or fibrosis or cancer treatment. In each case, the ENPP1 inhibitory compound may be administered before, simultaneously with, or after the administration of the other drug. In certain cases, the cancer is selected from adrenal, liver, kidney, bladder, breast, colon, stomach, ovary, cervix, uterus, esophagus, colorectal, prostate, pancreas, lung (small cell and non-small cell), thyroid, carcinomas, sarcomas, gliomas, glioblastomas, melanomas and various tumors of the head and neck.

[00192] For the treatment of cancer, ENPP1 inhibitory compounds 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 analogs, steroids, anthracyclines, thyroid hormone replacements, thymidylate-targeted drugs, chimeric T cell antigen receptor therapies, chimeric NK cell antigen receptor therapies, apoptosis regulator inhibitors (eg, B-cell CLL/lymphoma 2 (BCL) inhibitors) -2) BCL-2-like protein 1 (BCL-XL)), CARP-1/CCAR1 inhibitors (cell division and apoptosis cycle regulator 1), colony stimulating factor receptor-1 (CSF1R) inhibitors, CD47 inhibitors, cancer vaccine (e.g. , a Th17-inducing dendritic cell vaccine or a genetically engineered tyrosinase such as Oncept®) and other cell therapy options.

[00193] Specific chemotherapeutic agents of interest include, but are not limited to them, hemcitabin, docksel, bleomycin, eralotinib, gephicine, lapatinib, imatinib, dasatinib, nilotinib, barefoot, crisotinib, trametinib, bvasizumab, Sunini, Suninis, Suninis B, Sorafenib, Trustuzumab , adotrastuzumab, emtansine, rituximab, ipilimumab, rapamycin, temsirolimus, everolimus, methotrexate, doxorubicin, abraxane, folfirinox, cisplatin, carboplatin, 5-fluorouracil, teisumo, paclitaxel, prednisone, levothyroxine, pemetrexed, navitoclax, and ABT-199 . Peptide compounds may also be used. Cancer chemotherapeutic agents of interest include, but are not limited to, dolastatin and active analogs and derivatives thereof; and auristatin and its active analogs and derivatives (eg, monomethylauristatin D (MMAD), monomethylauristatin E (MMAE), monomethylauristatin F (MMAF) and the like). See, for example, WO 96/33212; WO 96/14856; and US Patent 6,323,315. Suitable anti-cancer chemotherapeutic agents also include maytansinoids and their active analogs and derivatives (see, for example, EP 1391213; and Liu et al. (1996) Proc. Natl. Acad. Sci. USA 93: 8618-8623); duocarmycins and their derivatives and active analogs (eg, including synthetic analogs, KW-2189 and CB 1-TM1); and benzodiazepines and their derivatives and active analogs (eg, pyrrolobenzodiazepine (PBD).

[00194] In some embodiments, compounds that are inhibitors of ENPP1 can be administered in combination with a chemotherapeutic agent for the treatment of cancer. In certain instances, the chemotherapeutic agent is gemcitabine. In some cases, the chemotherapeutic agent is docetaxel. In some cases, the chemotherapeutic agent is abraxane.

[00195] For the treatment of cancer (eg, solid cancer), an ENPP1 inhibitory compound may be administered in combination with an immunotherapeutic agent. An immunotherapeutic agent is any useful agent that finds use in the treatment of a disease by inducing, enhancing or suppressing an immune response. In some embodiments, the immunotherapeutic agent is an immune checkpoint inhibitor. For example, in FIG. fig. 4A-4C show that an exemplary ENPP1 inhibitor can act synergistically with an immune checkpoint inhibitor in a mouse model. Any suitable checkpoint inhibitors may be used, including, but not limited to, cytotoxic inhibitors of T-lymphocyte-associated antigen 4 (CTLA-4), programmed death inhibitor 1 (PD-1), and PD-L1 inhibitors. In certain instances, 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. Typical 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 disease, immunomodulatory polypeptides may be administered in combination with a colony stimulating factor receptor (CSF1R) inhibitor. CSF1R inhibitors of interest include, but are not limited to, emactuzumab.

[00196] Any suitable cancer vaccine therapy and agents may be used in combination with the compounds, compositions and methods of the ENPP1 inhibitor in question. For the treatment of cancer, eg ovarian cancer, ENPP1 inhibitory compounds can be administered in combination with vaccination, eg dendritic cell (DC) vaccination, to stimulate Th1/Th17 immunity. Th17 cell infiltration correlates with a markedly increased overall survival of patients with ovarian cancer. In some cases, a compound that is an inhibitor of ENPP1 finds use as an adjuvant therapy in combination with Th17-inducing vaccination.

[00197] Also of interest are agents that are inhibitors of CARP-1/CCAR1 (cell division cycle and apoptosis regulator 1), including but not limited to those described by Rishi et al., Journal of Biomedical Nanotechnology, Volume 11, Number 9, September 2015, pp. 1608-1627 (20) and CD47 inhibitors including, but not limited to, anti-CD47 antibody agents such as Hu5F9-G4.

[00198] In certain cases, the combination provides an enhanced effect compared to each of the components separately; in some cases, the combination provides a super-additive or synergistic effect relative to the combined or additive effects of the components. Various combinations of the subject compounds and the chemotherapeutic agent may be used, either sequentially or simultaneously. In the case of multiple doses, the two agents may be directly alternated, or two or more doses of one agent may be alternated, for example, with one dose of another agent. The simultaneous administration of both agents may also alternate or otherwise alternate with doses of the individual agents. In some cases, the time between dosages can be from about 1-6 to about 6-12 hours, from about 12-24 hours to about 1-2 days, up to about 1-2 weeks or more after the start of treatment.

Combination with cGAMP-inducing chemotherapy

[00199] Aspects of the present disclosure include methods of treating cancer, in which compounds that are inhibitors of ENPP1 (or pharmaceutical compositions containing such compounds) can be administered in combination with a chemotherapeutic agent that is capable of inducing cGAMP production in vivo. When a subject is exposed to an effective amount of a particular chemotherapeutic agent, the subject can be induced to produce 2'3'-cGAMP. Induced cGAMP levels can be maintained and/or increased when the subject ENPP1 inhibitor compounds are co-administered to prevent degradation of cGAMP, eg increased when compared to levels achieved with either agent. Any suitable chemotherapeutic agents that can cause DNA damage and can induce cGAMP production by dying cells due to overloaded repair or degradation mechanisms, such as alkylating agents, nucleic acid analogs, and intercalating agents, can be used in the contemplated combination therapeutic methods. In some instances, the cGAMP-inducing chemotherapeutic agent is an antimitotic agent. An antimitotic agent is an agent that acts by damaging DNA or binding to microtubules. In some cases, the cGAMP-inducing chemotherapeutic agent is an antineoplastic agent.

[00200] Cancers of interest that can be treated using the contemplated combination therapy include, but are not limited to, cancer of the adrenal gland, liver, kidney, bladder, breast, colon, stomach, ovary, cervix, uterus, esophagus , colorectal, prostate, pancreatic, lung (small and non-small cell), thyroid, carcinomas, sarcomas, gliomas, glioblastomas, melanomas, and various head and neck tumors. In some cases, the cancer is breast cancer. In certain cases, the cancer is a glioma or glioblastoma.

[00201] Chemotherapeutic agents of interest include, but are not limited to, uracil analogs, fluorouracil prodrug, thymidylate synthase inhibitors, deoxycytidine analog, DNA synthesis inhibitor (e.g. leading to S-phase apoptosis), folate analog, dehydrofolate reductase inhibitor, anthracycline, intercalating agent (eg causing double strand breaks), topoisomerase IIa inhibitor, taxane, microtubule disassembly inhibitor (eg causing G2/M phase arrest/apoptosis), microtubule assembly inhibitor, microtubule function stabilizers, (eg causing G2/M- apoptosis phases), tubulin polymerization promoters, tubulin binding agent (eg causing apoptosis upon M-phase arrest) epothilone B analog, vinca alkaloid, nitrogenous mustard, nitrosourea, DNA alkylater (eg causing interstrand crosslinks, apoptosis via p53) , VEGF inhibitor, anti-angiogenic antibody, HER2 inhibitor, quinazoline HER2 inhibitor, EGFR inhibitor, tyrosine kinase inhibitor, sirolimus analog, mTORC1 inhibitor (eg, in breast cancer in combination with exemestane=an aromatase inhibitor that inhibits estrogen production), triazene, dacarbazine prodrug, methylhydrazine.

[00202] Representative 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, bevacizumab, pertuzumab , trastuzumab, lapatinib, and everolimus. Typical anticancer drugs associated with glioma/glioblastoma include, but are not limited to, carmustine, lomustine, temozolomide, procarbazine, vincristine, and bevacizumab. Representative DNA damaging chemotherapeutic agents of interest include, but are not limited to, melphalan, cisplatin and etoposide, fluorouracil, gemcitabine.

Combined Radiation Therapy

[00203] Alternatively, for methods of treating cancer, compounds that are inhibitors of ENPP1 (or pharmaceutical compositions containing such compounds) can be administered in combination with radiation therapy. In certain embodiments, the methods further comprise administering radiation therapy to the subject. Again, an ENPP1 inhibitory compound may be administered before or after radiation therapy. Thus, the methods of the present invention may further include administering radiation therapy to the subject. The combination of radiation therapy and administration of the subject compounds may provide a synergistic therapeutic effect. When a subject is exposed to an appropriate dosage and/or frequency during radiotherapy (RT), the subject can be induced to produce 2'3'-cGAMP. Such induced cGAMP levels can be maintained and/or increased when the subject ENPP1 inhibitory compounds are co-administered to prevent cGAMP degradation, eg elevated when compared to levels achieved with RT alone. For example, in FIG. 4A shows that an exemplary ENPP1 inhibitor can act synergistically with radiation therapy (RT) to reduce tumor burden in a mouse model. As such, aspects of the methods of the present invention include the use of a reduced dose and/or frequency/schedule of radiation therapy compared to a therapeutically effective dose and/or frequency/schedule of radiation therapy alone. In some instances, radiation therapy is administered in combination with the compounds of the present invention at a dosage and/or frequency effective to reduce the risk of radiation damage in a subject, e.g., radiation damage expected to occur at a therapeutically effective dosage and/or frequency. / mode exclusively radiation therapy.

[00204] In some instances, the method includes administering an ENPP1 inhibitor to a subject prior to radiation therapy. In some instances, the method includes administering an ENPP1 inhibitor to a subject after the subject has been exposed to radiation therapy. In certain instances, the method comprises sequentially applying to a subject in need of radiation therapy followed by an ENPP1 inhibitor and then a checkpoint inhibitor.

Application

[00205] The compounds and methods of the invention, for example as described herein, can be used in a variety of applications. Applications of interest include, but are not limited to: research applications and therapeutic applications. The methods of the invention can be used in a variety of applications, including any convenient application where inhibition of ENPP1 is desired.

[00206] The compounds and methods of the present invention find use in a variety of research applications. The compounds and methods of the present invention can be used to optimize the bioavailability and metabolic stability of the compounds.

[00207] The compounds and methods of the present invention find use in a variety of therapeutic applications. Therapeutic applications of interest include these applications in the treatment of cancer. As such, the compounds of the present invention find use in the treatment of a variety of conditions in which inhibition and/or treatment of cancer in the host is desired. For example, the subject compounds and methods may find use in the treatment of cancer with a solid tumor (eg, as described herein).

Pharmaceutical compositions

[00208] The compounds discussed herein can be formulated using any suitable excipients, reagents, and methods. The compositions are supplied formulated with pharmaceutically acceptable excipients. A wide variety of pharmaceutically acceptable excipients are known in the art, so they do not need to be discussed in detail herein. Pharmaceutically acceptable excipients have been described in detail in various publications including, for example, A. Gennaro (2000) "Remington: The Science and Practice of Pharmacy," 20th edition, Lippincott, Williams, &Wilkins; Pharmaceutical Dosage Forms and Drug Delivery Systems (1999) H.C. Ansel et al., eds., 7th ed., Lippincott, Williams, &Wilkins; and Handbook of Pharmaceutical Excipients (2000) A.H. Kibbe et al., eds., 3rd ed. amer. Pharmaceutical Assoc.

[00209] Pharmaceutically acceptable excipients such as liquid media, adjuvants, carriers or diluents are commonly available. Moreover, pharmaceutically acceptable adjuvants such as pH adjusters and buffering agents, tonicity agents, stabilizers, wetting agents, and the like are commonly available.

[00210] In some embodiments, the subject compound is formulated in an aqueous buffer. Suitable aqueous buffers include, but are not limited to, acetate, succinate, citrate, and phosphate buffers ranging in strength from 5 to 100 mM. In some embodiments, the aqueous buffer includes reagents that provide an isotonic solution. Such reagents 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 formulations may further include a preservative. Suitable preservatives include, but are not limited to, benzyl alcohol, phenol, chlorobutanol, benzalkonium chloride, and the like. In many cases, the composition is stored at a temperature of about 4ºC. The compositions may also be lyophilized, in which case they typically include cryoprotectants such as sucrose, trehalose, lactose, maltose, mannitol, and the like. Freeze-dried formulations can be stored for extended periods of time, even at ambient temperature. In some embodiments, a compound of the present invention is formulated for sustained release.

[00211] In some embodiments, a compound of the present invention and a second active agent (e.g., as described herein), e.g., a small molecule, a chemotherapeutic agent, an antibody, an antibody fragment, an antibody-drug conjugate, an aptamer, or a protein, etc. are administered to individuals in a formulation (eg, in the same or separate formulations) with pharmaceutically acceptable excipients. 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.

[00212] In another aspect of the present invention, there is provided a pharmaceutical composition comprising or consisting essentially of a compound of the present invention, or a pharmaceutically acceptable salt, isomer, tautomer, or prodrug thereof, and further comprising one or more additional active agents of interest. Any suitable active agents may be used in the methods of the present invention in combination with the compounds of the present invention. In some specific cases, the additional agent is a checkpoint inhibitor. The compound of the present invention and the checkpoint inhibitor, as well as additional therapeutic agents for combination therapy as described herein, can be administered orally, subcutaneously, intramuscularly, intranasally, parenterally, or otherwise. The compound of the present invention and the second active agent (if present) may be administered by the same route of administration or by different routes of administration. Therapeutic agents may be administered by any suitable route, including, but not limited to, for example, oral, rectal, nasal, topical (including transdermal, aerosol, buccal, and sublingual), vaginal, parenteral (including subcutaneous, intramuscular, intravenous, and intradermal), intravesical, or injection into the affected organ. In certain cases, therapeutic agents can be administered intranasally. In some instances, therapeutic agents may be administered intratumorally.

[00213] In some embodiments, a compound of the present invention and a chemotherapeutic agent are administered to individuals in a formulation (eg, in the same or separate formulations) with pharmaceutically acceptable excipients. Chemotherapeutic agents include, but are not limited to, alkylating agents, nitrosoureas, antimetabolites, antineoplastic antibiotics, plant (vinca) alkaloids, and steroid hormones. Peptide compounds may also be used. Suitable anti-cancer chemotherapeutic agents include dolastatin and active analogs and derivatives thereof; and auristatin and its active analogs and derivatives (eg, monomethylauristatin D (MMAD), monomethylauristatin E (MMAE), monomethylauristatin F (MMAF) and the like). See, for example, WO 96/33212; WO 96/14856; and US Patent 6,323,315. Suitable anti-cancer chemotherapeutic agents also include maytansinoids and their active analogs and derivatives (see, for example, EP 1391213; and Liu et al. (1996) Proc. Natl. Acad. Sci. USA 93: 8618-8623); duocarmycins and their derivatives and active analogs (eg, including synthetic analogs, KW-2189 and CB 1-TM1); and benzodiazepines and their derivatives and active analogs (eg, pyrrolobenzodiazepine (PBD).

[00214] The compound of the present invention and the second chemotherapeutic agent, as well as additional therapeutic agents as described herein for combination therapy, can be administered orally, subcutaneously, intramuscularly, parenterally, or otherwise. The compound of the present invention and the second chemotherapeutic agent may be administered by the same route of administration or by different routes of administration. Therapeutic agents may be administered by any suitable route, including, but not limited to, for example, oral, rectal, nasal, topical (including transdermal, aerosol, buccal, and sublingual), vaginal, parenteral (including subcutaneous, intramuscular, intravenous, and intradermal), intravesical, or injection into the affected organ.

[00215] The compositions in question may be administered in unit dosage form and may be prepared by any means well known in the art. Such methods include combining the subject compound with a pharmaceutically acceptable carrier or diluent which constitutes one or more additional ingredients. The pharmaceutically acceptable carrier is selected based on the chosen route of administration and standard pharmaceutical practice. Each carrier must be "pharmaceutically acceptable" in the sense of being compatible with the other ingredients of the formulation and not causing harm to the patient. Such a carrier may be solid or liquid, and the type is usually selected depending on the type of administration used.

[00216] Examples of suitable solid carriers include lactose, sucrose, gelatin, agar, and bulking powders. Examples of suitable liquid carriers include water, pharmaceutically acceptable fats and oils, alcohols or other organic solvents, including esters, emulsions, syrups or elixirs, suspensions, solutions and/or suspensions, and solution and/or suspensions reconstituted from effervescent granules; and effervescent preparations reconstituted from effervescent granules. Such liquid carriers may contain, for example, suitable solvents, preservatives, emulsifiers, suspending agents, diluents, sweeteners, thickeners, and melting agents. Preferred carriers are edible oils, such as corn or canola oil. Polyethylene glycols such as PEG are also good carriers.

[00217] Any drug delivery device or system that provides the dosing regimen of the present invention can be used. Those skilled in the art are aware of a wide variety of delivery devices and systems.

Additional Embodiments

[00218] Additional embodiments are set forth in the following paragraphs.

[00219] Item 1. ENPP1 inhibitor of formula (I):

Y-A-L-X

(I)

Where:

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 linker; And

X is a hydrophilic head group,

or a prodrug, pharmaceutically acceptable salt or solvate thereof.

[00220] Item 2. The ENPP1 inhibitor of claim 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.

[00221] Item 3. The ENPP1 inhibitor of claim 2, wherein the hydrophilic smut group (X) is selected from phosphonic acid, phosphonate, phosphonate ester, phosphate, phosphate ester, thiophosphate, thiophosphate ester, phosphoramidate, and thiophosphoramidate.

[00222] Item 4. The ENPP1 inhibitor according to any one of paragraphs. 1-3, in which L-X includes a group of formula (XI):

(XI),

Where:

Z12 is selected from O and S;

each of Z13 and Z14 is independently selected from O and NR', where R' is H, alkyl, or substituted alkyl;

Z15 is selected from O and CH 2;

each of R15 and R16 is independently selected from H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, aryl, substituted aryl, acyl, heterocycle, substituted heterocycle, cycloalkyl, and substituted cycloalkyl; And

q1 is an integer from 0 to 6 (eg 0-5).

[00223] Claim 5. The ENPP1 inhibitor of claim 4, wherein L-X is selected from

, , , . .

, , And .

[00224] Item 6. The ENPP1 inhibitor according to any one of paragraphs. 1-5, in which X is a phosphonic acid or an ester of a phosphonate.

[00225] Claim 7. The ENPP1 inhibitor of claim 1, wherein L-X contains a group of formula (XII):

(XII)

Where:

each of R17 and R18 is 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 R17 and R18. together with the atoms to which they are attached form a ring selected from a heterocycle and a substituted heterocycle; And

q2 is an integer from 1 to 6.

[00226] Claim 8. The ENPP1 inhibitor of claim 7, wherein L-X has the structure:

.

[00227] Claim 9. The ENPP1 inhibitor of claim 1, wherein L-X contains a group of formula (XIII):

(XIII)

where q3 is an integer from 1 to 6.

[00228] Claim 10. The ENPP1 inhibitor of claim 9, wherein L-X is selected from

And .

[00229] Claim 11. The ENPP1 inhibitor of claim 1, wherein L-X contains a group of formula (XIV):

(XIV)

where: Z16 is selected from O and CH2; and q4 is an integer from 0 to 6.

[00230] Claim 12. The ENPP1 inhibitor of claim 11, wherein L-X is selected from

And .

[00231] Claim 13. The ENPP1 inhibitor of claim 1, wherein L-X contains a group of formula (XV):

(XV)

where q5 is an integer from 1 to 6.

[00232] Claim 14. The ENPP1 inhibitor of claim 13, wherein L-X is selected from

And .

[00233] Claim 15. The ENPP1 inhibitor of claim 1, wherein L-X contains a group of formula (XVI):

(XVI),

Where:

R19 is selected from H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, aryl, substituted aryl, acyl, heterocycle, substituted heterocycle, cycloalkyl, and substituted cycloalkyl; And

q6 is an integer from 1 to 6.

[00234] Claim 16. The ENPP1 inhibitor of claim 15, wherein L-X has the structure:

.

[00235] Claim 17. The ENPP1 inhibitor of claim 1, wherein L-X contains a group of formula (XVII):

(XVII),

where q7 is an integer from 1 to 6.

[00236] Claim 18. The ENPP1 inhibitor of claim 17, wherein L-X has the structure:

.

[00237] Item 19. The ENPP1 inhibitor according to any one of paragraphs. 1-18, in which A is a heterocycle or a substituted heterocycle.

[00238] Claim 20. The ENPP1 inhibitor of claim 19, wherein A is selected from piperidine, substituted piperidine, piperazine, and substituted piperazine.

[00239] Item 21. The ENPP1 inhibitor according to any one of paragraphs. 19-20, in which A is:

or .

[00240] Item 22. The ENPP1 inhibitor according to any one of paragraphs. 1-18, wherein A is a carbocycle (eg, a 5-, 6-, or 7-membered monocyclic carbocycle).

[00241] Item 23. The ENPP1 inhibitor of claim 22, wherein A is cycloalkyl or substituted cycloalkyl.

[00242] Item 24. The ENPP1 inhibitor of claim 23, wherein A is:

.

[00243] Item 25. The ENPP1 inhibitor of claim 22, wherein A is aryl or substituted aryl.

[00244] Claim 26. The ENPP1 inhibitor of claim 25, wherein A is phenylene or substituted phenylene.

[00245] Item 27. The ENPP1 inhibitor of claim 26, wherein A is:

.

[00246] Item 28. The ENPP1 inhibitor according to any one of paragraphs. 1-27, in which L is a linear linker having a main chain from 1 to 12 atoms in length and including 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.

[00247] Item 29. The ENPP1 inhibitor of claim 28, wherein L is -(CH2)n- and n is an integer from 1 to 6 (e.g., 1, 2, 3, 4, 5, or 6) .

[00248] Claim 30. The ENPP1 inhibitor of claim 29, wherein n is 1 or 2.

[00249] Item 31. The ENPP1 inhibitor according to any one of paragraphs. 1-30, 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.

[00250] Item 32. The ENPP1 inhibitor according to any one of paragraphs. 1-30, 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.

[00251] Item 33. The ENPP1 inhibitor according to any one of paragraphs. 31-32, in which Y is a group of the formula:

Where:

each of Z1 and Z2 is independently selected from CR1 and N;

each R1 is independently selected from H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, heterocycle, and substituted heterocycle;

each of R2 and R5 is 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;

each of R3 and R4 is 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 R3 and R4 together with the carbon atoms to which they are attached form a fused group selected from heterocycle, substituted heterocycle, cycloalkyl, substituted cycloalkyl, aryl and substituted aryl.

[00252] Item 34. ENPP1 inhibitor according to item 33 of the formula:

(VI)

Where

L is selected from -CH2-, -(CH2)2-, -(CH2)3-, -(CH2)4-, -(CH2)5- and -(CH2)6-;

X is selected from:

, , , , , , , , , , , , And ;

Where:

Ra and Rb are each independently selected from aryl, alkyl, -CH2OC(O)Re, -CH2OC(O)ORe;

Rc and Rd are each independently selected from -C(CH3)C(O)ORe, alkyl, and where Re is alkyl; And

each of Z3 and Z4 is independently selected from CR and N, where R is H, alkyl or substituted alkyl.

[00253] Item 35. ENPP1 inhibitor according to item 34 of the formula:

.

[00254] Item 36. ENPP1 inhibitor according to item 33 of the formula:

(VII)

Where

L is selected from -CH2-, -(CH2)2-, -(CH2)3-, -(CH2)4-, -(CH2)5- and -(CH2)6-;

X is selected from:

, , , , , , , , , , , , And ;

Where:

Ra and Rb are each independently selected from aryl, alkyl, -CH2OC(O)Re, -CH2OC(O)ORe; And

each of Rc and Rd is independently selected from -C(CH3)C(O)ORe, alkyl, and wherein Re is alkyl.

[00255] Item 37. The ENPP1 inhibitor according to any one of paragraphs. 33-36, in which

R1 is selected from hydrogen, C 1-5 alkyl and vinyl heterocycle;

each of R2 and R5 is independently selected from hydrogen, C1-5 alkyl, amine, triazole, imidazole, amide, alkoxy, OCF3, halogen and hydroxy; And

each of R3 and R4 is independently selected from hydrogen, C1-5 alkyl, triazole, imidazole, amine, amide, alkoxy, OCF3, halogen and hydroxy, or R3 and R4 together with the carbon atoms to which they are attached form a fused heterocycle.

[00256] Item 38. The ENPP1 inhibitor according to any one of paragraphs. 33-36, in which Y is a group of the formula:

Where:

R7 is selected from H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, heterocycle, and substituted heterocycle;

R8 is selected from OH, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, -OCF3, halogen, amine, substituted amine, amide, heterocycle, and substituted heterocycle.

[00257] Item 39. The ENPP1 inhibitor of claim 38, wherein:

R7 is selected from hydrogen, C1-5 alkyl, substituted C1-5 alkyl, vinyl heterocycle, and substituted vinyl heterocycle; And

R8 is selected from hydrogen, C1-5 alkyl, triazole, imidazole, amine, amide, alkoxy, halogen, OCF3 and hydroxy.

[00258] Item 40. The ENPP1 inhibitor according to any one of paragraphs. 33-36, in which Y is a group of the formula:

Where

R7 is selected from H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, heterocycle, and substituted heterocycle;

each of R8 and R9 is independently selected from OH, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, -OCF3, halogen, amine, substituted amine, amide, heterocycle, and substituted heterocycle; or R8 and R9 together with the carbon atoms to which they are attached form a fused ring selected from heterocycle, substituted heterocycle, cycloalkyl, substituted cycloalkyl, aryl and substituted aryl.

[00259] Item 41. The ENPP1 inhibitor of claim 40, wherein

R7 is selected from hydrogen, C 1-5 alkyl and vinyl heterocycle;

each of R8 and R9 is independently selected from hydrogen, C1-5 alkyl, triazole, imidazole, amine, amide, alkoxy, halogen, OCF3 and hydroxy, or R8 and R9 together with the carbon atoms to which they are attached form a fused heterocycle or a fused substituted heterocycle .

[00260] Item 42. The ENPP1 inhibitor according to any one of paragraphs. 33-36, where Y corresponds to the formula:

Where

R7 is selected from H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, heterocycle, and substituted heterocycle;

R10 is selected from OH, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, -OCF3, halogen, amine, substituted amine, amide, heterocycle, and substituted heterocycle;

each of R8 and R9 is independently selected from OH, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, -OCF3, halogen, amine, substituted amine, amide, heterocycle, and substituted heterocycle; or R8 and R9 together with the carbon atoms to which they are attached form a fused ring selected from heterocycle, substituted heterocycle, cycloalkyl, substituted cycloalkyl, aryl and substituted aryl.

[00261] Item 43. The ENPP1 inhibitor of claim 42, wherein

R7 is selected from hydrogen, C 1-5 alkyl and vinyl heterocycle;

R10 is selected from hydrogen, C 1-5 alkyl, amine, triazole, imidazole, amide, alkoxy, OCF3 and hydroxy; And

each of R8 and R9 is independently selected from hydrogen, C1-5 alkyl, triazole, imidazole, amine, amide, alkoxy, OCF3 and hydroxy, or R8 and R9 together with the carbon atoms to which they are attached form a fused heterocycle or a substituted fused heterocycle.

[00262] Item 44. The ENPP1 inhibitor according to any one of paragraphs. 33-36, where Y corresponds to the formula:

Where

R7 is selected from H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, heterocycle, and substituted heterocycle;

each of R11 and R12 is 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 R11 and R12 together with the carbon atoms to which they are attached form a fused ring selected from heterocycle, substituted heterocycle, cycloalkyl, substituted cycloalkyl, aryl and substituted aryl.

[00263] Item 45. The ENPP1 inhibitor of claim 44, wherein

R7 is selected from hydrogen, C1-5 alkyl, substituted C1-5 alkyl, vinyl heterocycle, and substituted vinyl heterocycle; And

each of R11 and R12 is independently selected from hydrogen, C1-5 alkyl, triazole, imidazole, amine, amide, alkoxy, halogen, OCF3 and hydroxy, or R11 and R12 together with the carbon atoms to which they are attached form a fused heterocycle or a substituted fused heterocycle .

[00264] Item 46. The ENPP1 inhibitor according to any one of paragraphs. 33-36, in which Y is a group of the formula:

(IIE)

Where

R7 is selected from the group consisting of H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, heterocycle, and substituted heterocycle;

each of R11 and R12 is 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 R11 and R12 together with the carbon atoms to which they are attached form a fused ring selected from heterocycle, substituted heterocycle, cycloalkyl, substituted cycloalkyl, aryl and substituted aryl.

[00265] Item 47. The ENPP1 inhibitor of claim 46, wherein

R7 is selected from hydrogen, C1-5 alkyl, substituted C1-5 alkyl, vinyl heterocycle, and substituted vinyl heterocycle; And

each of R11 and R12 is independently selected from hydrogen, C1-5 alkyl, triazole, imidazole, amine, amide, alkoxy, halogen, OCF3 and hydroxy, or R11 and R12 together with the carbon atoms to which they are attached form a fused heterocycle or a substituted fused heterocycle .

[00266] Item 48. The ENPP1 inhibitor according to any one of paragraphs. 1-47, characterized in that Y is selected from:

And .

[00267] Item 49. The ENPP1 inhibitor according to any one of paragraphs. 1-47, characterized in that Y is selected from:

, And .

[00268] Item 50. The ENPP1 inhibitor according to any one of paragraphs. 1-30, where Y corresponds to the formula:

Where:

each of Z1 and Z2 is independently selected from CH and N;

R1 is selected from H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, heterocycle, and substituted heterocycle;

R6 is selected from heterocycle, substituted heterocycle, cycloalkyl, substituted cycloalkyl, aryl and substituted aryl.

[00269] Item 51. ENPP1 inhibitor according to item 50 of the formula:

where: Z3 and Z4 are each independently selected from CR and N, where R is H, alkyl or substituted alkyl.

[00270] Item 52. The ENPP1 inhibitor of claim 50 or 51, wherein Y is selected from:

Where

each of Z5, Z6, Z7 and Z8 is independently selected from CR14 and N;

R13 is selected from H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, heterocycle and substituted heterocycle;

each R14 is independently selected from H,OH, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, -OCF3, amine, substituted amine, amide, heterocycle, and substituted heterocycle; And

m is 0-5.

[00271] Item 53. The ENPP1 inhibitor of claim 50 or 51, wherein Y is selected from:

Where

each of Z9, Z10 and Z11 is independently selected from CR14 and N;

R13 is selected from H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, heterocycle and substituted heterocycle;

each R14 is independently selected from H,OH, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, -OCF3, amine, substituted amine, amide, heterocycle, and substituted heterocycle; And

p is 0-4.

[00272] Item 54. The ENPP1 inhibitor according to any one of paragraphs. 50-53, characterized in that Y is selected from:

And

[00273] Item 55. The ENPP1 inhibitor according to any one of paragraphs. 1-30, in which Y is a group of the formula:

(IIIC)

Where

each of Z1, Z2, Z17, Z18 and Z19 is independently selected from CR20 and N;

each R20 is independently selected from H, OH, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, -OCF3, amine, substituted amine, amide, heterocycle, and substituted heterocycle; And

p1 is an integer 0-4.

[00274] Item 56. The ENPP1 inhibitor of claim 55, wherein Y has the structure:

.

[00275] Item 57. The ENPP1 inhibitor according to any one of paragraphs. 1-56, wherein the compound is a compound selected from the compounds of Table 1 and Table 2.

[00276] Item 58. A pharmaceutical composition comprising:

an ENPP1 inhibitor according to any one of paragraphs. 1-57; And

pharmaceutically acceptable excipient.

[00277] Item 59. A pharmaceutical composition for the treatment of cancer, comprising:

an ENPP1 inhibitor according to any one of paragraphs. 1-57; And

pharmaceutically acceptable excipient.

[00278] Item 60. Method for inhibiting ENPP1, the method includes:

contacting the sample with an ENPP1 inhibitor to inhibit the hydrolysis activity of cGAMP in ENPP1.

[00279] Item 61. The method of claim 60, wherein the ENPP1 inhibitor is a non-cell penetrating ENPP1 inhibitor.

[00280] Clause 62. The method according to claims 60-61, wherein the sample is a cellular sample.

[00281] Item 63. The method according to any one of paragraphs. 60-62, in which the sample contains cGAMP.

[00282] Item 64. The method of claim 63, wherein cGAMP levels are elevated in the cell sample (eg, relative to a control sample not exposed to the inhibitor).

[00283] Item 65. The method according to any one of paragraphs. 60-64, in which the non-permeating ENPP1 inhibitor is an inhibitor according to any one of paragraphs. 1-57.

[00284] Item 66. A method of treating cancer, comprising administering to a subject with cancer a therapeutically effective amount of an ENPP1 inhibitor to treat the subject's cancer.

[00285] Item 67. The method of claim 66 wherein the cancer is a solid tumor.

[00286] Clause 68. The method according to claims 66 or 67, wherein the cancer is breast cancer.

[00287] Item 69. The method according to any one of paragraphs. 66-68, wherein the cancer is selected from adrenal, liver, kidney, bladder, breast, colon, stomach, ovary, cervix, uterus, esophagus, colorectal, prostate, pancreatic, lung (small cell and non-small cell), thyroid, carcinomas, sarcomas, glioblastomas, melanomas, and various head and neck tumors.

[00288] Item 70. The method of claim 66 wherein the cancer is lymphoma.

[00289] Item 71. The method of claim 69 wherein the cancer is a glioblastoma.

[00290] Item 72. The method according to any one of paragraphs. 66-71 further comprising administering one or more additional active agents.

[00291] Item 73. The method of claim 72, wherein the one or more additional active agents is a chemotherapeutic agent or an immunotherapeutic agent.

[00292] Item 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.

[00293] Item 75. The method according to any one of paragraphs. 72-74, wherein one or more additional active agents comprise a checkpoint inhibitor.

[00294] Item 76. The method of claim 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.

[00295] Item 77. The method according to any one of paragraphs. 72-76, in which one or more additional active agents include a chemotherapeutic agent.

[00296] Item 78. The method of claim 77, wherein the chemotherapeutic agent is a cGAMP-inducing chemotherapeutic agent.

[00297] Item 79. The method of claim 78, wherein the cGAMP-inducing chemotherapeutic agent is an antimitotic or antitumor agent administered in an amount effective to induce cGAMP production in the subject.

[00298] Item 80. The method according to any one of paragraphs. 66-79, further comprising administering radiation therapy to the patient.

[00299] Item 81. The method of claim 80, wherein the inhibitor is administered to the subject prior to the administration of radiation therapy.

[00300] Item 82. The method of claim 80, wherein the inhibitor is administered to the subject after the subject has been exposed to radiation therapy.

[00301] Item 83. The method of claim 81 or 82, wherein the radiation therapy induces cGAMP production in the subject.

[00302] Item 84. The method according to any one of paragraphs. 80-83, wherein radiation therapy is administered at a dosage and/or frequency effective to reduce radiation damage in a subject.

[00303] Item 85. The method according to any one of paragraphs. 66-84, in which the ENPP1 inhibitor is an inhibitor according to any one of paragraphs. 1-57.

[00304] Item 86. The method of claim 85, wherein the ENPP1 inhibitor is unable to enter cells.

[00305] Item 87. The method of claim 85, wherein the ENPP1 inhibitor is able to enter cells.

[00306] Item 88. A method of modulating an immune response in a subject, comprising administering to the subject a therapeutically effective amount of an ENPP1 inhibitor to treat the subject's inflammatory condition.

[00307] Item 89. The method according to item 88, in which the ENPP1 inhibitor is an inhibitor according to any one of paragraphs. 1-57.

Examples

[00308] The following examples are provided to provide those of skill in the art with a complete disclosure and description of how to make and use embodiments of the present disclosure, however, they are not intended to limit the scope of what the authors consider their invention to be, nor that the experiments below represent all or the only experiments performed. Care has been taken to maintain accuracy with respect to the numbers used (eg quantities, temperatures, etc.), however, some experimental errors and deviations should be taken into account. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric.

[00309] Although the present invention has been described with reference to specific embodiments thereof, however, experts in the art should understand that various changes can be made and replacement of equivalents can be made without departing from the essence and scope of this invention. In addition, numerous modifications may be made to adapt a particular situation, material, chemical composition, process, process step or process steps to the purpose, spirit, and scope of this disclosure. All such modifications are intended to be within the scope of the appended claims.

[00310] Example 1a. Synthesis of compound 1

[00311] Synthesis scheme

[00312] Preparation of dimethyl (2-(piperidin-4-yl)ethyl)phosphonate

[00313] 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, keeping the temperature below 40°C. The resulting mixture was allowed to stir at room temperature for 16 hours and then quenched by adding an aqueous saturated ammonium chloride solution. The organic phase was separated, washed with brine, dried (MgSO4) and evaporated to dryness. Chromatography (120 g SiO2; gradient EtOAc 5 to 100% in hexane) afforded dimethyl ( E )-(2-(1-benzylpiperidin-4-yl)vinyl)phosphonate (6.2 g, 16%) as colorless oily liquid.

[00314] To a mixture of dimethyl( E )-(2-(1-benzylpiperidin-4-yl)vinyl)phosphonate (3.7 g, 12.0 mmol) in ethanol (40 mL) was added Pd/C (1.1 g, 10.3 mmol). The mixture was placed under a hydrogen atmosphere and stirred at room temperature for 12 hours, filtered and evaporated to dryness under reduced pressure to give dimethyl (2-(piperidin-4-yl)ethyl)phosphonate (2.7 g, 100%) as a colorless oily liquid.

[00315] Preparation of dimethyl (2-(1-(6,7-dimethoxyquinazolin-4-yl)piperidin-4-yl)ethyl)phosphonate

[00316] 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 in 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 oily liquids.

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

1H NMR (500 MHz, CDCl3) δ 8.65 (s, 1H), 7.23 (s, 1H), 7.09 (s, 1H), 4.19 (dc, 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 (cd , J =

12.2, 3.7 Hz, 2H).

[00317] Preparation of dimethyl (2-(1-(6,7-dimethoxyquinazolin-4-yl)piperidin-4-yl)ethyl)phosphonic acid.

[00318] Bromotrimethylsilane (3.67 g, 24 mmol) was added to a chilled solution of dimethyl (2-(1-(6,7-dimethoxyquinazolin-4-yl)piperidin-4-yl)ethyl)phosphonate (3.25 g, 7.94 mmol) in chloroform (60 ml) which was cooled in an ice bath. The reaction mixture was allowed to warm to room temperature and quenched after 90 minutes by adding 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 volume, filtered to remove the precipitate and then evaporated to dryness. The residue was crystallized with dichloromethane, filtered and dried in vacuo to give dimethyl (2-(1-(6,7-dimethoxyquinazolin-4-yl)piperidin-4-yl)ethyl)phosphonic acid (2.1 g, 69%) .

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

1H NMR (500 MHz, DMSO- d 6) 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).

[00319] Example 1b. General synthesis of compounds of substituted (2-(1-(quinazolin-4-yl)piperidin-4-yl)ethyl)phosphonic acids.

[00320] (2-(1-(quinazolin-4-yl)piperidin-4-yl)ethyl)phosphonic acids were synthesized in a manner analogous to compound 1 . In this case, dimethyl (2-(piperidin-4-yl)ethyl)phosphonate was reacted with substituted 4-chloroquinazoline in the presence of a base such as diisopropylethylamine. The resulting adduct is deprotected using trimethylsilyl bromide in chloroform or pure trimethylsilyl iodide to give the desired phosphonates as shown in the table below.

Table 3. Analytical data of (2-(1-(quinazolin-4-yl)piperidin-4-yl)ethyl)phosphonic acids.

Compound 1H NMR data LCMS data 5 (400 MHz, DMSO- d6 ) δ8.64 (s, 1H), 7.96 (d, J =9.3 Hz, 1H), 7.09 (dd, J =9.2, 2.2 Hz, 1H), 7.00 (d, J =2.2 Hz, 1H), 4.62 (s, 2H), 3.38 (s, 2H), 1.87 (d, J =12.7 Hz, 2H), 1.72 (s, 1H), 1.58-1.38 (m, 4H), 1.26 (d, J =11.3 Hz, 2H). [M +1]= 338.25 7 (400 MHz, CD3OD) δ8.47 (s, 1H), 7.20 (br.s. 1H), 7.00 (d, J =2.2 Hz, 1H), 5.00-4.82 ( m, 2H), 4.09 (s, 3H), 3.97 (s, 3H). 3.52 (m, 2H), 2.06-1.36 (m, 9H) [M+1]= 382.0 10 [M+1]= 352.1 eleven [M+1]= 380.1 12 (400 MHz, DMSO- d6 ) δ 8.03 (d, J = 9.2 Hz, 1H), 7.20 (d, J = 9.2 Hz, 1H), 7.04 (s, 1H), 4.70-4.55 (m, 2H), 3.93 (s, 3H), 2.48 (s, 3H). 2.48-1.26 (m, 9H). [M+1]= 366.15 13 (400 MHz, DMSO- d6 ) δ 8.63 (s, 1H), 7.48 (s 1H), 7.19 (s, 1H), 4.57-4.54 (m, 2H), 4, 45-4.37(m, 4H), 3.38-3.32(m, 2H), 1.88-1.85(m, 2H, 1.75-1.24(m, 7H). [M+1]= 380.15 16 [M+1]= 382.15 18 (400 MHz, CD3OD): δ 9.16 (br.s., 1H), 8.79 (m, 2H), 8.27 (d, J =15.6 Hz, 1H), 7.97 (m , 1H), 7.79 (d, J =9.2 Hz, 1H), 7.62 (d, J =9.2 Hz, 1H), 7.52 (d, J =15.6 Hz, 1H ), 7.42 (s, 1H), 5.23 (m, 2H), 5.02-4.99 (m, 2H), 3.96 (s, 3H), 3.54 (br. t, 2H), 2.07-1.26 (m, 9H). [M+1]= 455.35 19 (500 MHz, CD3OD): δ 8.57 (s, 1H), 7.73 (d, J =8.5 Hz, 1H), 7.56 (t, J =8.2 Hz, 1H), 7 .46 (br. s., 1H), 3.98 (s, 3H), 3.5 (br. s, 2H), 2.65 (m, 2H), 2.07-2.04 (m, 2H), 1.81-1.75 (m, 3H), 1.66-1.63 (m, 2H), 1.46-1.44 (m, 2H) [M+1]= 352.10 22 (400 MHz, DMSO- d6 ) δ 8.60 (s, 1H), 7.90 (d, J =9.2 Hz, 1H), 7.48 (d, J =9.2 Hz, 1H), 4.69 (m, 2H), 4.02 (s, 3H), 3.88 (s, 3H), 3.45 (m, 2H), 1.91-1.88 (m, 2H), 1 .70 (m, 1H), 1.53-1.27 (m, 6H). [M+1]= 382.15 23 (400 MHz, DMSO- d6 ) δ 8.48 (s, 1H), 7.65 (d, J = 8.4 Hz, 1H), 7.32 (d, J = 8.8 Hz, 1H), 7.18 (s, 1H), 4.19 (d, J =12.0 Hz, 2H), 2.97 (m, 2H), 1.82 (d, J =12.0 Hz, 2H), 1.53-1.49 (m, 5H), 1.30-1.19 (m, 2H). [M+1]= 338.15 thirty (400 MHz, DMSO- d6 ) δ 8.35 (s, 1H), 7.62 (d, J = 8.8 Hz, 1H), 6.81 (d, J = 9.2 Hz, 1H), 6.62 (s, 1H), 6.30 (s, 1H), 4.23 (d, J =11.6 Hz, 2H), 3.02 (m, 2H), 1.79-1.76 (m, 2H), 1.45 (m, 4H), 1.23 (d, J =11.6 Hz, 2H). [M+1]= 337.10 38 [M+1]= 381.10 70 [M+1]= 340.10 76 (400 MHz, DMSO- d6 ) δ 8.62 (s, 1H), 7.62-7.52 (m, 3H), 4.64 (d, J =12 Hz, 2H), 4.01 (s , 3H), 3.39-3.35 (m, 2H), 1.57-1.53 (m, 2H), 1.49 (b.s., 1H), 1.46-1.29 ( m, 6H). [M+1]= 352.10 79 (400 MHz, DMSO- d6 ) δ 8.55 (s, 1H), 7.41 (dd, J =17.5, 7.2 Hz, 2H), 4.25 (s, 2H), 3.95 (s, 3H), 3.23 (t, J =12.1 Hz, 2H), 1.81 (d, J =11.7 Hz, 3H), 1.63 (s, 1H), 1.53 -1.33 (m, 5H), 1.2 -1.09 (m, 2H). [M+1]= 370.10 84 [M+1]= 356.10 93 (400 MHz, DMSO- d6 ) δ 8.43 (s, 1H), 7.62 (s, 1H), 6.89 (s, 1H), 6.84 (s, 1H), 4.56-4 .48 (m, 2H), 2.91-2.89 (m, 2H), 2.53 (s, 3H), 1.76-1.02 (9H). [M+1]= 352.15 107 (400 MHz, CD3OD): δ8.48 (s, 1H), 7.58 (d, J =8.5 Hz, 1H), 7.51 (t, J =8.2 Hz, 1H), 7, 36-7.29 (m, 1H), 4.95 (d, J =12.9 Hz, 2H), 3.50 (t, J =12.8 Hz, 2H), 2.03 (d, J =10.7Hz, 2H), 1.86(s, 1H), 1.67(dd, J =30.3, 13.2Hz, 4H), 1.47-1.35(m, 2H) . [M+1]= 338.15

[00321] 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.

[00322] A mixture of tert -butyl 4-(2-hydroxyethyl)piperidine-1-carboxylate compound (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 RT for 5 min under nitrogen. 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) were then added to the mixture. The mixture was stirred at rt for 10 min. and filtered. The filtrate was diluted with DCM, washed with 5% Na2SO3 and brine, dried over Na2SO4 and concentrated in vacuo. The residue was purified by silica gel column chromatography (PE/EA, 6:1) to give tert- butyl-4-(2-iodoethyl)piperidine-1-carboxylate (6.1 g, 90%).

[00323] To a mixture of diphenylphosphonate compound (15.6 g, 66.5 mmol, 5.0 eq.) in CH3CN (45 ml) was added DBU (10.1 g, 66.5 mmol, 5.0 eq.) and the mixture was stirred at 0°C for 10 min under a nitrogen atmosphere. Then tert- butyl 4-(2-iodoethyl)piperidine-1-carboxylate (4.5 g, 13.3 mmol, 1.0 eq) 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 tert- butyl 4-(2-(diphenoxyphosphoryl)ethyl)piperidine-1-carboxylate (4.6g, 75%).

[00324] MeOH was added to a solution of tert -butyl 4-(2-(diphenoxyphosphoryl)ethyl)piperidine-1-carboxylate (4.0 g, 8.97 mmol, 1.0 eq.) in methanol (40 mL). /HCl (5.0 M, 60 ml) and the mixture was stirred at room temperature for 3 hours. The mixture was then evaporated to dryness. The residue was diluted with aq. Na2CO3 solution, extracted with ethyl acetate. The organic layer was washed with brine, dried over Na2SO4, concentrated to give diphenyl(2-(piperidin-4-yl)ethyl)phosphonate hydrochloride compound (3.4g, 100%).

[00325] 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. The mixture was concentrated. The residue was purified by silica gel column chromatography (PE/EA, 1:1) to give compound 78 (2.2 g, 41%).

[00326] 1H 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).

[00327] To a mixture of compounds 78 (1.59 g, 3 mmol, 1.0 eq.) in THF (10 ml) and water (10 ml) was added sodium hydroxide (480 mg, 12 mmol, 4 eq.) at rt . The mixture was stirred at room temperature for 12 hours. The organic phase was removed under reduced pressure and the pH of the aqueous phase was adjusted to 1 with 1N HCl. HCl. The resulting solid was filtered off and dried to give phenylhydrogen (2-(1-(6,7-dimethoxyquinazolin-4-yl)piperidin-4-yl)ethyl)phosphonate (1.3 g, 96%).

Phenylhydrogen compound solution (2-(1-(6,7-dimethoxyquinazolin-4-yl)piperidin-4-yl)ethyl)phosphonate (1.4 g, 3.1 mmol, 1.0 eq.), L-alanine -isopropyl ether (1.04 g, 6.2 mmol) and TEA (620 mg, 6.2 mmol) in pyridine (20 ml) were heated to 60°C for 5 min under N2. 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 minutes, then added to the above solution at 60° C in an N2 atmosphere. The reaction mixture was stirred for 12 hours, concentrated and purified with FCC (CH2Cl2:MeOH=20:1) to give 77 (200 mg, 11.4%).

LCMS: [M+1]= 571.10

[00328] Example 1d. Synthesis of compound 72. Preparation of 2-(1-(6,7-dimethoxyquinazolin-4-yl)piperidin-4-yl)ethyl- N -diisopropylphosphandiamine 72.

[00329] Compound solution77 (700 mg, 1.8 mmol, 1.0 eq.), isopropylamine (319 mg, 5.4 mmol, 3 equiv.) and triethylamine (364 mg, 3.6 mmol, 2 equiv.) in pyridine (10 ml) were heated to 60°C for 5 min under N2 atmosphere. Aldrithiol-2 (1.4 g, 6.3 mmol, 3.5 eq.), PPh 3 (1.7 g, 6.3 mmol, 3.5 eq.) in Py (10 ml) was stirred at room temperature for 5 minutes, then added to the above solution at 60°C under N2 atmosphere. The mixture was stirred at 60°C for 12 hours. The mixture was then concentrated and purified with FCC (DCM:MeOH=20:1) to give72 (200 mg, 24%).HCMS: [M+1]= 464.25.1H 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).

[00330] Example 1e. Preparation of O -((1-(6,7-dimethoxyquinazolin-4-yl)piperidin-4-yl)methyl) O, O- dihydrophosphorothioate 108.

[00331] A mixture of 4-chloro-6,7-dimethoxyquinazoline (900 mg, 4.018 mmol, 1.0 eq.) and piperidin-4-ylmethanol (508 mg, 4.420 mmol, 1.1 eq.) in i-PrOH ( 10 ml) were stirred at 100°C for 16 h in a closed 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-((λ1-oxidanyl)methyl)piperidin-1-yl)-6,7-dimethoxyquinazoline (1 g, 82%).

[00332] To a solution of 4-(4-((λ1-oxidenyl)methyl)piperidin-1-yl)-6,7-dimethoxyquinazoline (100 mg, 0.330 mmol, 1.0 eq.) in dry pyridine (5 ml) phosphorothioyl trichloride (280 mg, 1.98 mmol, 6.0 eq.) was added 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 eq.) 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. 1H NMR (400 MHz, DMSO- d6 ) δ 8.54 (s, 1H), 7.18 (s, 1H), 7.11 (s, 1H), 4.25 (d, J =13.4 Hz , 2H), 3.89 (d, J = 9.1 Hz, 6H), 3.76 (s, 2H), 3.10 (d, J = 11.8 Hz, 3H), 1.94 (s , 1H), 1.81 (d, J =12.7 Hz, 2H), 1.39 (d, J =11.4 Hz, 1H).

[00333] Example 1f. Preparation of O-( 2-(1-(6,7-dimethoxyquinazolin-4-yl)piperidin-4-yl)ethyl) O,O- dihydrophosphorothioate 10

[00334] A mixture of 4-chloro-6,7-dimethoxyquinazoline (1 g, 4.46 mmol, 1.0 eq.) and piperidin-4-ylethanol (633 mg, 4.91 mmol, 1.1 eq.) in i-PrOH (10 ml) was stirred at 100°C for 16 h in a closed 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-(λ1-oxidaneyl)ethyl)piperidin-1-yl)-6,7-dimethoxyquinazoline (1.3 g, 91% ).

To a solution of 4-(4-(2-(λ1-oxidaneyl)ethyl)piperidin-1-yl)-6,7-dimethoxyquinazoline (150 mg, 0.473 mmol, 1.0 eq.) in dry pyridine (5 ml) was added 2-(piperidin-4-yl)ethan-1-ol (477 mg, 2.84 mmol, 6.0 eq) 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 eq.) 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 light yellow solid. LCMS: [M+1]=414.05. 1H 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).

[00335] A mixture of 4-chloro-6,7-dimethoxyquinazoline compound (600 mg, 2.68 mmol, 1.0 eq) 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 h in a closed 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%).

[00336] To a mixture of ethyl 2-(1-(6,7-dimethoxyquinazolin-4-yl)piperidin-4-yl)acetate (250 mg, 0.696 mmol, 1.0 eq.) in THF (10 ml/5 ml ) 2 M NaOH (1 ml, 2.09 mmol, 3.0 eq.). The mixture was stirred at room temperature for 16 hours. The progress of the reaction mixture was monitored by LC-MS. The reaction mixture was then concentrated under reduced pressure to give the corresponding acid (200 mg, 86%).

[00337] To a mixture of acid (300 mg, 0.906 mmol, 1.0 eq.) in THF (10 mL) was added NH2OH HCl (76 mg, 1.09 mmol, 1.2 eq.), DIEA (468 mg, 3.63 mmol, 4.0 eq.) and BOP (481 mg, 1.09 mmol, 1.2 eq.). The mixture was stirred at room temperature for 16 hours. The progress of the reaction mixture was monitored by TLC. Thereafter, the reaction mixture was 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).

[00338] Example 1g. Preparation of (2-(1-(6,7-dimethoxyquinazolin-4-yl)piperidin-4-yl)ethyl)phosphonothioic O, O- acid 83

[00339] To a compound solution diethylphosphonate (10 g, 72.46 mmol, 1.0 eq.) in toluene (1000 ml) was added Lawesson's reagent (29.3 g, 72.46 mmol, 1.0 eq.) 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 evaporated under reduced pressure. The residue was purified by silica gel chromatography to giveO, O-diethylphosphonothioate (3.4 g, 25%). To solutionO, O- diethylphosphonothioate (1 g, 6.49 mmol, 1.5 eq.) in MeCN (1 L) was added DBU (3.29 g, 21.65 mmol, 5.0 eq.). After stirring at 0°C for 10 minutes, slowly addtert-butyl 4-(2-iodoethyl)piperidine-1-carboxylate (1.47 g, 4.33 mmol, 1.0 eq.). The mixture was left 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 obtaintert-butyl 4-(2-(diethoxyphosphorothioyl)ethyl)piperidine-1-carboxylate (500mg, 31%). Solutiontert-butyl 4-(2-(diethoxyphosphorothioyl)ethyl)piperidine-1-carboxylate (500 mg, 1.37 mmol, 1.0 eq.) in TFA/DCM (10 ml/10 ml) was stirred at room temperature for 1 hours. The progress of the reaction mixture was monitored by TLC. The mixture was then concentrated under reduced pressure to give crudeO, O-diethyl (2-(piperidin-4-yl)ethyl)phosphonothioate (400 mg, 100%). To the stirred solutionO, O-diethyl (2-(piperidin-4-yl)ethyl)phosphonothioate (400 mg, 1.51 mmol, 0.84 eq.) and DIEA (927 mg, 7.19 mmol, 4.0 eq.) in DMSO (10 ml) compound was added1-1 (403 mg, 1.80 mmol, 1.0 eq.) under nitrogen. 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 preparative HPLC to give (2-(1-(6,7-dimethoxyquinazolin-4-yl)piperidin-4-yl)ethyl)phosphonothioicO, O-acids83 (13 mg, 32%) as a white solid.HCMS: [M+1]= 396.25.1H 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).

[00340] 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

[00341] Ethyl 2-(diethoxyphosphoryl)acetate (12.7 ml, 64, 10 mmol, 1.0 eq.) at 0 °C under nitrogen. After stirring for 0.5 h, 1,4-dioxaspiro[4,5]decan-8-one (10 g, 64.10 mmol, 1.0 eq.) was added dropwise. The mixture was allowed to warm to room temperature and stirred for 2 hours under nitrogen. 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 phases were washed with brine (3×500 mL), dried over anhydrous Na2SO4 and concentrated under reduced pressure to give crude ethyl ester (13.9 g, 95%) as a colorless oil. To a stirred solution of crude ethyl ether( 13.9 g, 61.50 mmol, 1.0 eq.) in MeOH (200 ml) were added HCOONH4 (34.9 g, 0.554 mol, 9.0 eq.) and 10% Pd/C (2.09 g , 15% w/w). The mixture was stirred at reflux for 1.5 hours. The progress of the reaction mixture was monitored by LCMS. After cooling, the reaction mixture was filtered through a pad of celite and a glass-ceramic 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 saturated ethyl ester (12.43 g, 88%) as a colorless oil.

[00342] To a mixture of ethyl ether (12.43 g, 54.52 mmol, 1.0 eq.) in dry THF (150 ml) was added LiAlH 4 (2.5 M in THF, 17.4 ml, 43.61 mmol, 0.8 equiv.) at -7 8 °C. The mixture was allowed to warm to room temperature and stirred for 2.5 hours under nitrogen. The progress of the reaction mixture was monitored by LCMS. Then Na2SO4 H2O was added in portions at –20°C until the evolution of gas 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 eq.), imidazole (5.6 g, 82.82 mmol, 1.5 eq.) in DCM (150 ml) was stirred at room temperature for 5 minutes. Then I2 (21 g, 82.82 mmol, 1.5 eq.) was added and stirred for 10 minutes, and then 2-(1,4-dioxaspiro[4,5]decan-8-yl)ethan-1 was added -ol (10.27 g, 55.22 mmol, 1.0 eq.). The mixture was stirred for 2 hours. 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%).

[00343] To a solution of dibenzylphosphonate (32.8 g, 0.125 mol, 3.0 eq.) in MeCN (200 ml) was added DBU (31.7 g, 0.209 mol, 5.0 eq.) at 0°C. After stirring for 30 minutes, I2 (21 g, 82.82 mmol, 1.5 eq) was added and stirred for 10 minutes, and then a solution of 8-(2-iodoethyl)-1,4-dioxaspiro[4, 5] dean. (12.35 g, 41.72 mmol, 1.0 eq.) in ACN (70 ml). The mixture was stirred for 16 hours. 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 crude phosphonic intermediate (18.35 g, 100%).

[00344] To a solution of the phosphonate (18.35 g, 42.67 mmol, 3.0 eq.) in EtOH (200 ml) was added 2 M HCl (200 ml) at 0°C. The mixture was then allowed to warm to room temperature and stirred for 2 hours. 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 eq.) and 1,1,1-trifluoro -N- phenyl- N -((trifluoromethyl)sulfonyl )methanesulfonamide (3.6 g, 10.10 mmol, 1.3 eq.) in THF (30 mL) and LiHMDS was added. (1 M in THF, 10.1 ml, 10.10 mmol, 1.3 eq.) dropwise at -78°C. The mixture was stirred at -78°C for 4 hours. The mixture was then allowed to warm to room temperature and stirred for 16 hours. 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 column chromatography (PE/EA=3/1 to 1/1) to obtain 4-(2-(bis(benzyloxy)phosphoryl)ethyl)cyclohex-1-en-1-yltrifluoromethanesulfonate (2.15 g , 53%). B2Pin2 (1.37 g, 5.40 mmol, 1.3 eq.), Pd(dppf)Cl 2 (364 mg, 0.415 mmol, 0.1 eq.) and KOAc (1.22 g, 12.45 mmol, 3.0 equiv.). The mixture was stirred at 90°C for 16 hours under nitrogen. The progress of the reaction mixture was monitored by TLC. The mixture was then filtered through a pad of celite and a glass ceramic funnel and under reduced pressure to obtain crude dibenzyl(2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)cyclohex-3- en-1-yl)ethyl)phosphonate, which was directly used for the next step.

[00345] 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 with the previous stage 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 eq.) and KOAc (1.22 g, 12.45 mmol, 3.0 eq.). The mixture was stirred at 90°C for 16 hours under nitrogen. The progress of the reaction mixture was monitored by TLC. The mixture was then filtered through a pad of celite and a glass ceramic funnel and under reduced pressure to obtain crude dibenzylphosphonate (3.4 g, 100%), which was used directly for the next step.

[00346] To a solution of crude dibenzylphosphonate (1.7 g, 3.05 mmol, 1.0 eq.) in MeOH (100 ml) was added Pd/C (340 mg, 20% w/w) under nitrogen atmosphere. The mixture was treated with hydrogen 3 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 pad of celite and a glass-ceramic funnel and Pd/C (340 mg, 20% w/w) was added under nitrogen. The mixture was treated with hydrogen 3 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 pad of celite and a glass-ceramic 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 acid81 (78 mg, 6%, white solid) and (2-((1,4-trance)-4-(6,7-dimethoxyquinazolin-4-yl)cyclohexyl)ethyl)phosphonic acid82(185 mg, 16%, white solid).

[00347] 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).

[00348] Compound 82. LCMS: [M+1] = 381.25. 1H 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).

[00349] Example 1i. Preparation of (4-(6,7-dimethoxyquinazolin-4-yl)cyclohexyl)methyl dihydrogen phosphate 60

[00350] (4-(6,7-Dimethoxyquinazolin-4-yl)cyclohexyl)methanol (100mg, 0.33mmol, 1.0eq) was dissolved in dry pyridine (3mL) then cooled to -15° C and stirred for 10 min., POCl3 (253 mg, 1.65 mmol, 5.0 eq.) was added dropwise under N2 atmosphere. The reaction temperature was slowly raised to 0°C, then stirred for another 30 min. Once LCMS indicated that compound 3 was completely consumed. The mixture was poured into a solution of NaHCO3 (160 mg in 50 ml of 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)cyclohexyl)methyl dihydrogen phosphate 60 (70 mg, 55%) as a white powder after lyophilization. LC-MS: 384.20 [M+1]+. 1H 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).

[00351] Example 1j. Preparation of 2-(4-(6,7-dimethoxyquinazolin-4-yl)cyclohexyl)ethyldihydrophosphate 85

[00352] 2-(4-(6,7-Dimethoxyquinazolin-4-yl)cyclohexyl)ethan-1-ol (340 mg, 1.07 mmol, 1 eq.) was dissolved in 10 ml dry pyridine, 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, then stirred again for another 30 minutes. The mixture was poured into a solution of NaHCO3 (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)cyclohexyl)ethyl dihydrogen phosphate (52 mg, white powder, 12%). LC-MS: 398 [M+1]+. 1H 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).

[00353] Example 1k. Preparation of ((1-(6,7-dimethoxyquinazolin-4-yl)piperidin-4-yl)methyl)phosphonic acid 88

[00354] PPh 3 (3.39 g, 15 mmol, 1.5 eq.) and imidazole (1.02 g, 15 mmol, 1.5 eq.) in dry DCM (40 ml) were stirred in ice water for 10 minutes, then I2 (3.8 g, 15 mmol, 1.5 eq.) was added. It was stirred under nitrogen atmosphere for 10 minutes, after which (1-(6,7-dimethoxyquinazolin-4-yl)piperidin-4-yl)methanol (10 mmol) was added. Ice water was removed. The mixture was stirred for 10 minutes, then kept at rt overnight. After its consumption, Na2S2O3 solution was added and stirred for 10 minutes. It was extracted with DCM, washed with water and brine, then dried with Na2SO4. Received 4-(4-(iodomethyl)piperidin-1-yl)-6,7-dimethoxyquinazoline (2.28 g, 56%) as a light yellow solid after recrystallization from methanol. LC-MS: 414.3 [M+1]+. 1H NMR (400 MHz, CDCl3) δ 8.63 (d, J =1.3 Hz, 1H), 7.28 (s, 1H), 7.07 (s, 1H), 4.23 (s, 2H ), 4.00 (s, 6H), 3.19 (d, J =6.5 Hz, 2H), 3.08 (s, 2H), 2.11-2.00 (m, 2H), 1 .82 (s, 1H), 1.49 (s, 2H), 1.29-1.20 (m, 1H).

[00355] 4-(4-(iodomethyl)piperidin-1-yl)-6,7-dimethoxyquinazoline (9.5g, 36.3mmol, 3eq) was dissolved in 40ml dry MeCN then cooled to 0° WITH. DBU (9.2 g, 60.5 mmol, 5 eq.) was added dropwise, then stirred for 10 minutes. Bis(benzyloxy)(oxo)-λ4-phosphane was dissolved in 20 ml of CAN. A solution of bis(benzyloxy)(oxo)-λ4-phosphane was added dropwise to the mixture at 0°C. The mixture was stirred overnight. The mixture was concentrated in vacuo. The residue was taken up in EtOAc and then washed with water and brine. 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]+. 1H 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).

[00356] A mixture containing dibenzyl ((1-(6,7-dimethoxyquinazolin-4-yl)piperidin-4-yl)methyl)phosphonate (660 mg, 1.2 mmol, 1.0 eq.) and Pd/C (132 mg, 20% w/w) in CH3OH (20 ml) under H2 was stirred at room temperature for 4 hours. After complete reaction 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 light yellow solid. LC-MS : 368.10 [M+1]+. 1H NMR (400 MHz, DMSO- d 6) δ 8.72 (s, 1H), 7.29 (s, 2H), 4.60 (d, J =12.8 Hz, 2H), 3.95 ( e, J = 11.2 Hz, 6H), 3.46 (s, 2H), 2.09 (s, 3H), 1.61 (s, 2H), 1.42 (s, 2H).

[00357] Example 2 Evaluation of Compound Activity

[00358] Selected compounds from Table 1, Table 2, and other derivatives were generated and evaluated in an ENPP1 activity assay using paranitrophenol thymidine monophosphate (TMP-pNP) as a substrate. Enzymatic reactions were prepared with TMP-pNP (2 μM), 5-fold dilution of ENPP1 inhibitor and purified recombinant mouse ENPP1 (0.5 nM) in 100 mM Tris, 150 mM NaCl, 2 mM CaCl2, 200 μM ZnCl2, pH 7.5 at room temperature. The progress of the reaction was monitored by measuring the absorbance at 400 nm of the resulting paranitrophenolate reaction for 20 minutes. Product formation slopes were obtained, plotted and fitted to obtain IC50 values using Graphpad Prism 7.03.

[00359] Compounds were also evaluated in an ENPP1 enzyme activity assay using 32P cGAMP as a substrate. Radiolabeled 32P cGAMP was synthesized by incubating unlabeled ATP (1 mM) and GTP (1 mM), supplemented with 32P-ATP, with 2 μM purified recombinant porcine cGAS in 20 mM Tris pH 7.5, 2 mM MgCl2, 100 μg/ ml of herring milt DNA overnight at room temperature, and the remaining nucleotide starting materials were digested with alkaline phosphatase for 4 h at 37°C. The 32P-cGAMP probe (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 hours. To obtain enzyme inhibition curves, 5-fold dilutions of the ENPP1 inhibitor were included in the reaction. Degradation was assessed by TLC (as described in Li et al. Nat. Chem. Biol . (2014) 10:1043-8). The plates were exposed to a phosphor screen (Molecular Dynamics) and displayed on a Typhoon 9400 and the 32P signal was quantified using ImageJ. Inhibition curves were fitted to obtain IC50 values using Graphpad Prism 7.03. The IC50's of the compounds tested are shown in Table 4. IC50 values fall within the range labeled AD where A represents an IC50 value of less than 500 nM, B represents an IC50 value between 500 nM and 5 µM, C represents an IC50 value between 5 µM and 10 µM, D is an IC50 value greater than 10 µM (N.O. = not determined).

Table 4. A (<500 nM); B (500 nM-5 µM); C (5 µM - 10 µM); D (> 10µM)

Compound IC50 values
(TMP-pNP; μM) IC50 values
(cGAMP; µM) 1 A A 5 A BUT. 7 A BUT. 10 A A eleven D BUT. 12 A B 13 B BUT. 16 B BUT. 18 A A 19 A BUT. 22 A BUT. 23 B BUT. 25 A BUT. thirty A BUT. 38 A BUT. 42 D BUT. 60 A BUT. 61 B BUT. 67 A BUT. 68 B BUT. 70 A BUT. 71 A BUT. 73 D BUT. 75 A B 76 A A 81 A BUT. 82 A BUT. 83 A BUT. 84 A BUT. 86 A BUT. 87 A BUT. 88 A BUT. 93 A BUT. 100 A BUT.

[00360] Example 3 Demonstration of extracellular ENPP1 and inhibition of extracellular ENPP1

[00361] As illustrated in FIG. 1A-1C, it was noted that ENPP1 controls extracellular cGAMP levels and that cGAMP levels can be restored by treating cells with a typical inhibitor of ENPP1 (compound 1).

[00362] 293T CGAS ENPP1 -/- cells were transfected with 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 viruses to stably express mouse cGAS. 293T mcGAS ENPP1 -/- were generated by viral transfection of CRISPR sgRNA targeting human ENPP1 (5'CACCGCTGGTTCTATGCACGTCTCC-3') (SEQ ID NO:1). 293T mcGAS ENPP1 -/- cells were seeded on tissue culture-treated plates coated with PurCol (Advanced BioMatrix) in DMEM (Corning Cellgro) supplemented with 10% PBS (Atlanta Biologics) (v/v) and 100 U/ml penicillin- streptomycin (ThermoFisher). 12-24 hours after seeding, cells were transfected with Fugene 6 (Promega) according to the manufacturer's instructions, supplemented with the indicated concentrations of pcDNA3 plasmid DNA (blank or containing human ENPP1 ). Cells were lysed 24 hours after transfection to analyze ENPP1 expression by Western blotting (using rabbit anti-ENPP1 antibody (L520, 1:1000) and mouse anti-tubulin antibody (DM1A, 1:2000), Cell Signaling Technologies). Whole cell lysates were prepared by lysing 1 x 106 cells in 10 mM Tris, 150 mM NaCl, 1.5 mM MgCl2, 1% NP-40, pH 9.0. 32P-cGAMP (5 μM) was incubated with whole cell lysates and degradation was monitored as described above in Example 2 (FIG. 1A).

[00363] In intact cells, ENPP1 expression depletes extracellular cGAMP but does not affect intracellular cGAMP concentration (FIG. 1B). 24 hours after transfection of 293T mcGAS ENPP1-/- with pcDNA3 (blank or containing human ENPP1 ), 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 changing the medium, it was removed and the cells were washed from the plate with cold PBS. Both medium and cells were centrifuged at 1000 rpm for 10 minutes at 4°C and prepared for cGAMP concentration measurement by liquid chromatography with tandem mass spectrometry (LC-MS/MS). Cells were lysed in 30-100 µl of 50:50 acetonitrile:water supplemented with 500 nM cyclic GMP-13C10, 15N5-AMP as an internal standard and centrifuged at 15,000 rpm for 20 minutes at 4°C to remove insoluble factions. The medium was removed, 500 nM cyclic GMP-13C10, 15N5-AMP as an internal standard, and 20% formic acid were added. Samples were analyzed for cGAMP, ATP, and GTP on a Shimadzu HPLC (San Francisco, CA) with an autosampler set at 4°C and connected to an AB Sciex 4000 QTRAP (Foster City, CA). A volume of 10 μl was injected into a Biobasic AX LC column, 5 μm, 50 x 3 mm (Thermo Scientific). The mobile phase consisted of 100 mM ammonium carbonate (A) and 0.1% formic acid in acetonitrile (B). The initial state was 90% B, held for 0.5 min. The mobile phase was increased to 30% A from 0.5 to 2.0 min, maintained at 30% A from 2.0 to 3.5 min, increased to 90% B from 3.5 to 3.6 min and maintained at 90% B 3.6 to 5 min. The flow rate was set to 0.6 ml/min. The mass spectrometer was operated in the electrospray mode of positively charged ions with the source temperature set to 500 °C. Declustering and collision-induced dissociation were achieved using nitrogen gas. The declustering potential and collision energy were optimized by direct infusion of standards. For each molecule, the MRM ( m/z ), DP (V), and CE (V) transitions are as follows: ATP (508 > 136, 341, 55), GMP (524 > 152, 236, 43), cGAMP (675 > 136, 121, 97; 675 > 312, 121, 59; 675 > 152, 121, 73), intracellular standard cyclic GMP-13C10,15N5-AMP(690 > 146, 111, 101; 690 > 152, 111, 45; 690 > 327, 111, 47), extractable standard cyclic 13C10.15N5-GMP-13C10.15N5-AMP (705 > 156, 66, 93; 705 > 162, 66, 73).

[00364] Inhibition of ENPP1 blocks the degradation of extracellular cGAMP (Fig. 1C). The same experiment as above was performed , this time also including an exemplary inhibitor of ENPP1 (compound 1) at a concentration of 50 μM with a change of medium. When using an inhibitor, the concentrations of extracellular cGAMP in the medium returned to the previous levels.

[00365] FIG. 1A shows 293T cGAS ENPP1-/- cells that were transfected with an empty vector and a vector containing human ENPP1, and analyzed after 24 hours to determine the expression of the ENPP1 protein using Western blotting (top) and the hydrolysis activity of ENPP1 32P-cGAMP using thin layer chromatography (TLC) (bottom). In FIG. 1B shows intracellular and extracellular cGAMP concentrations using LC-MS/MS.BQL=below the limit of quantitation. Mean ± S.E.M. (n=2). **P=0.005 (t-Student's criterion). In FIG. 1C shows intracellular and extracellular concentrations of cGAMP for 293T cGAS ENPP1-/- cells transfected with an empty vector or a vector containing human ENPP1. in the presence or absence of 50 μM compound 1.BQL=below the limit of quantitation. Mean ± S.E.M. (n=2). **P=0.0013 (t-Student's criterion).

[00366] Example 4 Inhibition of ENPP1 increases cGAMP activation of primary CD14+ monocytes

[00367] Using an exemplary ENPP1 inhibitor (compound 1), the ability of cGAMP exported by the 293T cGAS ENPP1low cell line to be detected by antigen presenting cells (APCs) such as human CD14+ monocytes was tested (FIG. 2A). 293T CGAS ENPP1low cells were transfected with pcDNA (blank or containing human ENPP1). Primary human peripheral blood mononuclear cells (PBMCs) were isolated by treating an enriched buffy coat of whole blood with a Percoll density gradient. CD14+ monocytes were isolated using CD14+ microbeads (Miltenyi). CD14+ monocytes were cultured in RMPI supplemented with 2% human serum and 100 U/ml penicillin-streptomycin. 8 hours after transfection of 293T cGAS ENPP1low cells, medium was changed to RMPI supplemented with 2% human serum and 100 U/ml penicillin-streptomycin with or without exemplary ENPP1 inhibitor compound-1. 24 hours after medium change, supernatant from 293T cGAS ENPP1low cells was transferred into CD14+ monocytes (FIG. 2A). 24-26 hours after supernatant transfer, total RNA was extracted using Trizol (Thermo Fisher Scientific) and reverse transcribed using Maxima H Minus reverse transcriptase (Thermo Fisher Scientific). Real-time RT-PCR was performed in duplicate using an AccuPower 2X Greenstar qPCRMaster Mix (Bioneer) on a 7900HT fast PCR system (Applied Biosystems). Data were normalized to CD14 expression for each sample. The induction fold was calculated using ΔΔCt. Primers for human IFNB1 : forward (5'-AAACTCATGAGCAGTCTGCA-3') (SEQ ID NO: 2), reverse (5'-AGGAGATCTCAGTTTCGGAGG-3') (SEQ ID NO: 3); Human CD14 : forward (5'-GCCTTCCGTGTCCCCACTGC-3') (SEQ ID NO: 4), reverse (5'-TGAGGGGGCCCTCGACG-3') (SEQ ID NO: 5).

[00368] The supernatant of cGAS-expressing 293T cGAS ENPP1low cells, but not cGAS-null 293T cells, induced CD14+ IFNB1 expression, suggesting that extracellular cGAMP, which is exported by cancer cells, can be detected using CD14+ cells as a signaling factor (Fig. 2B). Transient overexpression of ENPP1 in 293T cGAS ENPP1low cells caused extracellular cGAMP degradation and decreased IFNB1 CD14+ expression, but addition of Compound 1 restored extracellular cGAMP levels and induced IFNB1 CD14+ expression (Fig. 2B).

[00369] See FIG. 1A, which shows a schematic of a supernatant transfer experiment. In FIG. 2B shows cGAS null 293T cells or 293T cGAS ENPP1low cells that were transfected with DNA and incubated in the presence or absence of Compound 1. The supernatant from these cells was transferred into human CD14+ primary PBMCs. IFNB1 mRNA levels were normalized to CD14 and fold induction was calculated relative to untreated CD14+ cells. Mean ± S.E.M. ( n =2). * P <0.05, *** P <0.001 (one-way ANOVA).

[00370] Example 5 Inhibition of ENPP1 in combination with ionizing radiation (IR) treatment to increase tumor associated dendritic cells.

[00371] The test was performed to determine whether cancer cell lines export cGAMP and whether ionizing radiation (IR) affects the levels of extracellular cGAMP produced. Ionizing radiation (IR) has been shown to increase cytosolic DNA and activate cGAS-dependent IFN-β production in tumor cells (Bakhoum et al. Nat. Commun . (2015) 6:1-10; and Vanpouille Nat. Commun . (2017 ) 8:15618). 24 hours after seeding, 4T1 cells were treated with IR at a dose of 20 Gy using a cesium source, and the medium was changed by adding 50 μM of an exemplary inhibitor of ENPP1 (compound 1) to inhibit ENPP1 present in cell culture. The medium was collected at the indicated time, centrifuged at 1000 xg to remove residual cells, acidified with 0.5% acetic acid, and cyclic-13C10, 155-GMP-13C10, 15N5-AMP was added as an extraction standard (corresponding amount for a final concentration of 2 μM in 100 µl). The medium was applied to HyperSep Aminopropyl SPE columns (ThermoFisher Scientific) for cGAMP enrichment as previously described (Gao et al., Proc. Natl. Acad. Sci. USA (2015) 112:E5699-705). The eluents were evaporated to dryness and reconstituted in a 50:50 mixture of acetonitrile: water with the addition of 500 nM internal standard. The medium was subjected to mass spectrometric quantification of cGAMP.

[00372] Continuous export of cGAMP was detected in 4T1 cells for 48 hours. After 48 hours, IR-treated cells had significantly higher levels of extracellular cGAMP than untreated cells.

[00373] The effect of IR in combination with an exemplary inhibitor of ENPP1 (Compound 1) on tumor dendritic cell numbers was then examined in a 4T1 mouse tumor model (FIG. 3B). Seven to nine week old female Balb/c mice (Jackson Laboratories) were inoculated into the mammary fat pad with 1 x 106 4T1-luciferase tumor cells suspended in 50 µl of PBS. Two days after injection, the tumors were irradiated with a dose of 20 Gy using a 225 kVp cabinet X-ray irradiator filtered with 0.5 mm Cu (IC250, Kimtron Inc., CT). Anesthetized animals were shielded with a lead shield 3.2 mm in diameter with a hole 15 × 20 mm, into which the tumor was placed. Mice were injected intramuscularly with 100 μl of 1 mM compound 1 in PBS or PBS alone. The next day, the tumor was extracted and incubated in RPMI+10% PBS with 20 µg/ml DNase type IV (Sigma-Aldrich) and 1 mg/ml Clostridium histolyticum collagenase (Sigma-Aldrich) at 37°C for 30 minutes. Tumors were passed through a 100 μm sieve (Sigma-Aldrich) and erythrocytes were lysed using erythrocyte lysis buffer (155 mM NH4Cl, 12 mM NaHCO3, 0.1 mM EDTA) for 5 min at room temperature. Cells were stained with the LIVE/DEAD Near IR Fixed Dead Cell Stain Kit (Thermo Fisher Scientific), Fc-blocked for 10 minutes with TruStain fcX, and then stained with CD11c, CD45, and I-A/I-E antibodies (all from Biolegend). Cells were analyzed using a SH800S (Sony) or LSRII (BD Biosciences) cell sorter. Statistical analysis of data was performed using FlowJo V10 software (Treestar) and Prism 7.04 software (Graphpad) and statistical significance was assessed using Welch's unpaired t-test (corrected).

[00374] Compound 1 intratumoral injection did not alter tumor-associated leukocyte compositions compared to PBS control (Figure 3B), suggesting that ENPP1 does not play a significant role in clearing extracellular basal cGAMP in this tumor model. However, upon pre-treatment of tumors with IR, Compound 1 was observed to increase the tumor-associated CD11c+ population (FIG. 3B).

[00375] The results are shown in FIG. 3A and FIG. 3b. In FIG. 3A shows extracellular cGAMP produced by 4T1 cells over 48 hours. At time 0, cells were left untreated or treated with 20 Gy IR and refreshed with medium supplemented with 50 μM Compound 1. Mean±SEM ( n =2). ** P = 0.004 (Student's t -test). In FIG. 3B shows 4T1 cells (1 x 106) orthotopically injected into BALB/cJ mice on day 0. Tumors were left untreated or treated with 20 Gy IR and injected with PBS intravenously ( n = 5 for IR (0 Gy); n = 4 for IR (20 Gy)) or compound 1 ( n =5) on day 2. Tumors were collected and analyzed by FACS on day 3. * P = 0.047 (Welch's t -test).

[00376] Example 6 Inhibition of ENPP1 is synergistic with ionizing radiation treatment and anti-CTLA-4 antibodies in producing antitumor effects.

[00377] A study was conducted as to whether it is possible to increase the immunological detection and clearance of tumors by further increasing extracellular cGAMP in vivo using ionizing radiation (IR) and an exemplary inhibitor of ENPP1, for example, compound 1.

[00378] Seven to nine week old female Balb/c mice (Jackson Laboratories) were inoculated into the mammary fat pad with 5 x 104 4T1-luciferase cells suspended in 50 μl of PBS. When the tumor volume (length 2 x width/2) reached 80 mm3 to 120 mm3, the tumors were irradiated with a dose of 20 Gy using a shielded 225 kVp X-ray emitter with 0.5 mm Cu filter (IC250, Kimtron Inc., CT). Anesthetized animals were shielded with a lead shield 3.2 mm in diameter with a hole 15 × 20 mm, into which the tumor was placed. On days 2, 4, and 7 after IR, 100 μl of compound 1 at a concentration of 100 μM and/or 10 μg of cGAMP in PBS or PBS alone were administered intravenously. Alternatively, 1 mM compound 1 in PBS or PBS alone was administered intratumorally, and 200 μg of anti-CTLA-4 antibody or Syrian hamster IgG antibody (both BioXCell) were administered intraperitoneally on days 2, 5 and 7 after IR. Mice from different treatment groups were housed in the same cage together to eliminate cage interference. The experimenter was in a "blind evaluation" state throughout the study. Tumor volumes were recorded every other day. Tumor volumes were analyzed in a generalized scoring equation to account for intra-individual correlation in mice. Pairwise comparisons of treatment groups at each time point were performed using post hoc analysis with Tukey's test for multiple comparisons. Animal mortality was plotted on a Kaplan-Meier survival curve using Graphpad Prism 7.03 and statistical significance was assessed using the Mantel-Cox log-rank test. All animal procedures have been approved by the Administrative Committee for Laboratory Animal Care.

[00379] Compound 1 administration enhanced the tumor shrinking effect of IR treatment, although not significantly (FIG. 4A). Although intratumoral cGAMP injection had no effect on IR treatment, compound 1 injection, in addition to synergistically reduced cGAMP tumors, increased survival and achieved a 10% cure rate (FIG. 4A and FIG. 4B).

[00380] A potential synergistic effect was also tested with an anti-CTLA-4 adaptive immune checkpoint blocker. Without IR, treatment with anti-CTLA-4 and Compound 1 had no effect on prolonging survival (Fig. 4C). However, the combination of IR pretreatment with compound 1 and anti-CTLA-4 showed a significant synergistic effect and achieved a 10% cure rate. Together, these results demonstrate that enhancement of extracellular cGAMP by combining IR treatment with ENPP1 inhibition enhances tumor immunogenicity and exerts antitumor effects.

[00381] The results are shown in FIG. 4A showing tumor reduction induced by Compound 1 in combination with IR. Established tumors (100 ± 20 mm3) were treated once with a dose of 20 Gy IR followed by three intratumoral injections of PBS or the drug on days 2, 4, and 7 after IR ( n = 9 per treatment group). Mice from different treatment groups were housed in shared cages and the experimenter was in a “blind evaluation” state. Tumor volumes were analyzed in a generalized scoring equation to account for intra-individual correlation in mice. Pairwise comparisons of treatment groups at each time point were performed using post hoc analysis with Tukey's test for multiple comparisons. In FIG. 4B shows the Kaplan-Meier curves for FIG. 4A, P values determined using the Mantel-Cox log rank test. In FIG. 4C show, in addition to the same procedure as in FIG. 4B, IgG isotype control antibodies or anti-CTLA 4 administered ip 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 in the same way as for Fig. 4b.

[00382] Thus, these results indicate that cGAMP can be found extracellularly and the ENPP1 inhibitors in question act extracellularly; therefore, indicate that extracellular inhibition of ENPP1 is sufficient for a therapeutic effect. ENPP1 falls under the definition of an innate immunity checkpoint. These experiments show that extracellular inhibition of ENPP1 allows cGAMP to enhance anti-cancer immunity and to be combined synergistically with immune checkpoint blocking drugs already available as a therapy (Fig. 5).

[00383] 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 apparent to those of ordinary skill in the art in light of the teachings of this description that certain changes and modifications may be made to this invention without depart from the spirit or scope of the appended claims.

[00384] Accordingly, the foregoing merely illustrates the principles of the invention. It is understood that those skilled in the art will be able to develop various devices which, although not explicitly described or shown here, embody the principles of the invention and are included within its spirit and scope. In addition, all examples and conventions given herein are primarily intended to help the reader understand the principles of the invention and the concepts introduced by the inventors in the development of the art, and should be considered as not limited to such specifically listed examples and conditions. In addition, all statements that give principles, aspects and embodiments of the invention, as well as its specific examples, are intended to cover its structural and functional equivalents. Further, such equivalents are intended to include both currently known equivalents and future developed equivalents, that is, any elements developed that perform the same function, regardless of structure. Therefore, the scope of the present invention is not intended to be limited by the exemplary embodiments shown and described herein. Rather, the scope and essence of the present invention are embodied in the following.

Claims (31)

1. ENPP1 inhibitor formula (VI) where X is selected from , , , , And ; L is selected from -CH ₂ - and -(CH ₂ ) ₂ -; each of Z ¹ and Z ² is independently selected from CR ¹ and N; each of Z ³ and Z ⁴ is independently selected from CR and N, where R is H; each R ¹ is independently selected from H and alkenyl having 2 to 6 carbon atoms substituted with pyridine; each of R ² and R ⁵ is independently selected from H, OH, alkoxy containing from 1 to 6 carbon atoms, and halogen; each of R ³ and R ⁴ is independently selected from H, OH, alkoxy containing from 1 to 6 carbon atoms, halogen and amino; or R ³ and R ⁴ together with the carbon atoms to which they are attached form dioxane. 2. The ENPP1 inhibitor according to claim 1, wherein X is selected from: , , And . 3. The ENPP1 inhibitor according to claim 2, wherein the inhibitor has the formula where each of Z ¹ and Z ² represents N; Z3 is N; And Z4 is CH or N. 4. The ENPP1 inhibitor according to any one of paragraphs. 1-3, in which the part of the molecule represented by , selected from: , , , , , , , , , , , , , , , , , And . 5. The ENPP1 inhibitor according to claim 1 or 2, wherein X is . 6. An ENPP1 inhibitor according to claim 1 or 2, wherein L is -(CH ₂ ) ₂ -. 7. The ENPP1 inhibitor according to claim 1 or 2, wherein each of Z ¹ and Z ² is N. 8. The ENPP1 inhibitor according to claims 1-3, in which R ¹ represents hydrogen. 9. The ENPP1 inhibitor according to claims 1-3, in which R ⁵ is alkoxy containing from 1 to 6 carbon atoms. 10. The ENPP1 inhibitor of claim 9 wherein R ⁵ is methoxy. 11. The ENPP1 inhibitor according to claim 1, selected from: , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , And . 12. The ENPP1 inhibitor according to claim 1, which is . 13. Pharmaceutical composition having inhibitory activity against ENPP1, containing an effective amount of an ENPP1 inhibitor according to any one of paragraphs. 1-12 and a pharmaceutically acceptable excipient.

Images