ENPP1 inhibitors and methods of modulating immune response

Abstract

To provide compounds, compositions and methods for the inhibition of ENPP1.SOLUTION: Aspects of the subject methods include contacting a sample with an ENPP1 inhibitor compound to inhibit the cGAMP hydrolysis activity of ENPP1. In some cases, the ENPP1 inhibitor compound is cell impermeable. ENPP1 inhibitor compounds can act extracellularly to block the degradation of cGAMP. There are also provided pharmaceutical compositions and methods for treating cancer. 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 cancer. There are also provided methods of administering radiation therapy to a subject in conjunction with administering an ENPP1 inhibitor to the subject. The radiation therapy can be administered in the subject methods at a dosage and / or frequency effective to reduce radiation damage to the subject, but still instigate an immune response.SELECTED DRAWING: None

Description

Technical Field

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

[0002] This invention was made with government support under Contract No. CA190896 and CA228044, awarded by the National Institutes of Health, and Contract No. W81XWH-18-1-0041, awarded by the Department of Defense. The government has certain rights in this invention.

Background Art

[0003] Introduction Cyclic guanosine monophosphate - adenosine monophosphate (cGAMP) activates the stimulator of interferon genes (STING) pathway, which is an important anti - cancer innate immune pathway. The cGAS - cGAMP - STING pathway is activated in the presence of cytoplasmic DNA, either due to microbial infection or pathophysiological states including cancer and innate immune disorders. Cyclic GMP - AMP synthase (cGAS) belongs to the nucleotidyltransferase family and is a universal DNA sensor that is activated upon binding to cytosolic dsDNA and produces the signaling molecule (2’ - 5’, 3’ - 5’) cyclic GMP - AMP (or 2’,3’ - cGAMP, or cGAMP which is cyclic guanosine monophosphate - adenosine monophosphate). When 2’,3’ - cGAMP acts as a second messenger during microbial infection, it binds to and activates STING, leading to the production of type I interferons (IFNs) and other co - stimulatory molecules that trigger an immune response. The STING pathway has emerged as a target for cancer immunotherapy and autoimmune diseases in addition to its role in infectious diseases.

[0004] Ectonucleotide pyrophosphatase / phosphodiesterase 1 (ENPP1) is the major hydrolase of cGAMP that can degrade cGAMP. ENPP1 is a member of the ecto-nucleotide pyrophosphatase / phosphodiesterase (ENPP) family and is a type II transmembrane glycoprotein containing two identical disulfide-bonded subunits. ENPP1 has a broad specificity for cleaving various substrates, including phosphodiester bonds of nucleotides and nucleotide sugars, as well as pyrophosphate bonds of nucleotides and nucleotide sugars. ENPP1 can have the function of hydrolyzing nucleoside 5'-triphosphate to its corresponding monophosphate and can also hydrolyze diadenosine polyphosphate.

Summary of the Invention

Means for Solving the Problems

[0005] Compounds, compositions and methods for inhibiting ENPP1 are provided. Aspects of the subject methods include contacting a sample with an ENPP1 inhibitor compound to inhibit the cGAMP hydrolysis activity of ENPP1. In some cases, the ENPP1 inhibitor compound is cell non-permeable. The ENPP1 inhibitor compound acts extracellularly and can block the degradation of cGAMP. Similarly, pharmaceutical compositions and methods for treating cancer are provided. Aspects of the method include administering to a subject a therapeutically effective amount of an ENPP1 inhibitor to treat the cancer of the subject. In certain cases, the cancer is a solid tumor cancer. Similarly, a method of administering radiotherapy to a subject in conjunction with the step of administering an ENPP1 inhibitor to the subject is provided. The radiotherapy can be administered in an amount and / or frequency effective to reduce radiation damage to the subject in the subject method but still cause an immune response.

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

[0007] The present invention will be best understood from the following detailed description when read in conjunction with the accompanying drawings. This patent or application document includes at least one drawing made in color. In accordance with common practice, it is emphasized that the various features of the drawings are not to scale. On the contrary, the dimensions of the various features are arbitrarily enlarged or reduced for clarity. The following figures are included in the drawings. It is understood that the drawings described below are for illustrative purposes only. The figures are not intended to limit the scope of the present teachings in any way.

Brief Description of the Drawings

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[0030] **Definitions** Before further describing embodiments of the present disclosure, it should be understood that the present disclosure is not limited to the specific embodiments described, and thus can naturally vary in various ways. Since the scope of the present disclosure is limited only by the appended claims, it should also be understood that the terms used herein are for the purpose of describing specific embodiments only and are not intended to be limiting.

[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of embodiments of the present disclosure.

[0032] As used herein and in the appended claims, the singular forms "a", "and", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a compound" includes not only a single compound, but also combinations of two or more compounds, reference to "a substituent" includes a single substituent as well as two or more substituents, and the like.

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

[0034] The phrases "for example", "for instance", "such as", or "including" are intended to introduce examples that further clarify a more general subject. These examples are presented only to assist in understanding the disclosure and are in no way intended to be limiting.

[0035] The publications discussed herein are presented only to show that they were available prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publications by virtue of prior invention. Further, the dates of the publications presented may be different from the actual publication dates, which may need to be independently verified.

[0036] The terms "agent", "antagonist", "inhibitor", "drug" and "pharmacological agent" are used interchangeably herein to refer to a chemical substance or compound that, when administered to a living organism (human or animal), elicits a desired pharmacological and / or physiological effect by local and / or systemic action.

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

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

[0039] The terms "individual", "host", "subject" and "patient" are used interchangeably herein and refer to animals including, but not limited to, humans and non-human primates including monkeys and humans; rodents including rats and mice; cattle; horses; sheep; cats; dogs, etc. "Mammal" means a member (singular or plural) of any mammalian species, and by way of example, dogs; cats; horses; cattle; sheep; rodents, etc., and primates, for example, including non-human primates and humans. Non-human animal models, for example, mammals such as non-human primates, murine, lagomorphs, etc. can be used for experimental studies.

[0040] The terms "determine", "measure", "evaluate" and "assay" are used interchangeably and include both quantitative and qualitative determinations.

[0041] As used interchangeably herein, the terms “polypeptide” and “protein” refer to a polymeric form of amino acids of any length, which can include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones. The term includes, but is not limited to, fusion proteins having non-homologous amino acid sequences that are fusions with heterologous and native leader sequences with or without an N-terminal methionine residue; immunologically tagged proteins; fusion proteins having a detectable fusion partner, e.g., fusion proteins including, as the fusion partner, a fluorescent protein, β-galactosidase, luciferase, etc.

[0042] The terms “nucleic acid molecule” and “polynucleotide” are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three-dimensional structure and may perform any function, known or unknown. Non-limiting examples of polynucleotides include genes, gene fragments, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, regulatory regions, isolated RNA of any sequence, nucleic acid probes, and primers. Nucleic acid molecules may be linear or circular.

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

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

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

[0046] The term "pharmaceutical composition" is intended to encompass compositions suitable for administration to a subject, such as a mammal, particularly a human. Generally, a "pharmaceutical composition" is sterile and preferably does not contain contaminants capable of inducing an undesirable response within the subject (e.g., the compound(s) in the pharmaceutical composition is / are of pharmaceutical grade). The pharmaceutical composition can be designed to be administered to a subject or patient in need thereof via several different routes of administration, including oral, buccal, rectal, parenteral, intraperitoneal, intradermal, intratracheal, intramuscular, subcutaneous, etc.

[0047] The phrase "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 "selected independently from" is used herein to indicate that the recited elements, such as R groups, etc., can be the same or different.

[0048] The terms "may", "optional", "optionally", or "optionally, may" mean that the situation described thereafter may or may not occur, and as a result, this description includes examples where the situation occurs and examples where the situation does not occur. For example, the phrase "optionally substituted" means that a non-hydrogen substituent may or may not be present on a given atom, and thus this description includes structures where the non-hydrogen substituent is present and structures where the non-hydrogen substituent is not present.

[0049] "Acyl" refers to the groups 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, heterocyclic and substituted heterocyclic are as defined herein. For example, acyl includes the "acetyl" group CH3C(O)-.

[0050] The term "alkyl", while not necessarily so, usually refers to a branched or unbranched saturated hydrocarbon group (i.e., a monoradical) containing from 1 to about 24 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, octyl, decyl, and cycloalkyl groups such as cyclopentyl, cyclohexyl. Generally, although not necessarily so, an alkyl group can contain from 1 to about 18 carbon atoms in this specification, and such a group can contain from 1 to about 12 carbon atoms. The term "lower alkyl" is intended to mean an alkyl group consisting of 1 to 6 carbon atoms. "Substituted alkyl" refers to an alkyl that is substituted with one or more substituents, which includes examples where two hydrogen atoms from the same carbon atom in the alkyl substituent are replaced, such as in a carbonyl group (i.e., a substituted alkyl group can contain a -C(=O)- moiety). The terms "heteroatom-containing alkyl" and "heteroalkyl" refer to an alkyl substituent in which at least one carbon atom is replaced by a heteroatom, as further detailed below. The terms "alkyl" and "lower alkyl" include, respectively, linear, branched, cyclic, unsubstituted, substituted, and / or heteroatom-containing alkyl or lower alkyl, unless otherwise indicated.

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

[0052] The term "alkenyl" refers to a straight-chain, branched, or cyclic hydrocarbon group consisting of 2 to about 24 carbon atoms containing at least one double bond, such as ethenyl, n-propenyl, isopropenyl, n-butenyl, isobutenyl, octenyl, decenyl, tetradecenyl, hexadecenyl, eicosenyl, tetracosenyl, etc. Generally, although not necessarily, an alkenyl group can contain 2 to about 18 carbon atoms in this specification, and for example, can contain 2 to 12 carbon atoms. The term "lower alkenyl" is intended to mean an alkenyl group consisting of 2 to 6 carbon atoms. The term "substituted alkenyl" refers to an alkenyl substituted by one or more substituents, and the terms "heteroatom-containing alkenyl" and "heteroalkenyl" refer to an alkenyl in which at least one carbon atom is replaced by a heteroatom. The terms "alkenyl" and "lower alkenyl" include, respectively, straight-chain, branched, cyclic, unsubstituted, substituted, and / or heteroatom-containing alkenyl and lower alkenyl, unless otherwise indicated.

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

[0054] The term "alkoxy" refers to an alkyl group bonded through a single terminal ether linkage group. That is, an "alkoxy" group can be represented as -O-alkyl, where alkyl is as defined above. A "lower alkoxy" group refers to an alkoxy group containing 1 to 6 carbon atoms, and includes, for example, methoxy, ethoxy, n-propoxy, isopropoxy, t-butyloxy, etc. Substituents specified as "C1-C6 alkoxy" or "lower alkoxy" may, in this specification, contain, for example, 1 to 3 carbon atoms, and as a further example, such substituents may contain 1 or 2 carbon atoms (i.e., methoxy and ethoxy). The notations "-OMe" and "MeO-" refer to the methoxy group.

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

[0056] The term "aryl", unless otherwise specified, generally, but not necessarily, contains 5 to 30 carbon atoms and contains a single aromatic ring or a plurality of aromatic rings that are fused together, directly linked, or indirectly linked (such that different aromatic rings are bonded to a common group such as a methylene or ethylene moiety), and refers to an aromatic substituent. An aryl group can contain, for example, 5 to 20 carbon atoms, and as a further example, an aryl group can contain 5 to 12 carbon atoms. For example, an aryl group can contain one aromatic ring or two or more fused aromatic rings or linked aromatic rings (i.e., biaryl, aryl-substituted aryl, etc.). Examples include phenyl, naphthyl, biphenyl, diphenyl ether, diphenylamine, benzophenone, etc. "Substituted aryl" refers to an aryl moiety substituted by one or more substituents, and the terms "heteroatom-containing aryl" and "heteroaryl" refer to aryl substituents in which at least one carbon atom is replaced by a heteroatom, as described in more detail below. Aryl is exemplified, but not limited to, phenyl, biphenyl, naphthyl, pyridyl, furyl, thiophenyl, imidazolyl, pyrimidinyl, and oxazolyl, and may further be substituted by 1 to 5 members selected from the group consisting of hydroxy, C1-C8 alkoxy, branched or straight-chain C1-C8 alkyl, acyloxy, carbamoyl, amino, N-acylamino, nitro, halogen, trifluoromethyl, cyano, and carboxyl (see, for example, Katritzky, Handbook of Heterocyclic Chemistry). Unless otherwise indicated, the term "aryl" includes unsubstituted, substituted, and / or heteroatom-containing aromatic substituents. 14 is intended to include moieties, and these may be further substituted by 1 to 5 members selected from the group consisting of hydroxy, C1-C8 alkoxy, branched or straight-chain C1-C8 alkyl, acyloxy, carbamoyl, amino, N-acylamino, nitro, halogen, trifluoromethyl, cyano, and carboxyl (for example, see Katritzky, Handbook of Heterocyclic Chemistry). Unless otherwise specifically indicated, the term "aryl" includes unsubstituted, substituted, and and / or heteroatom-containing aromatic substituents.

[0057] The term "aralkyl" refers to an alkyl group having an aryl substituent, the term "alkaryl" refers to an aryl group having an alkyl substituent, and "alkyl" and "aryl" are as defined above. Generally, aralkyl groups and alkaryl groups contain from 6 to 30 carbon atoms herein. Aralkyl groups and alkaryl groups can contain, for example, from 6 to 20 carbon atoms, and as a further example, such groups can contain from 6 to 12 carbon atoms.

[0058] The term "alkylene" refers to a diradical alkyl group. Unless otherwise indicated, such groups include a saturated hydrocarbon chain containing from 1 to 24 carbon atoms, which may be substituted or unsubstituted, may contain one or more alicyclic groups, and may contain heteroatoms. "Lower alkylene" refers to an alkylene linking group containing from 1 to 6 carbon atoms. Examples include methylene (--CH2--), ethylene (--CH2CH2--), propylene (--CH2CH2CH2--), 2-methylpropylene (--CH2--CH(CH3)--CH2--), hexylene (--(CH2)6--), and the like.

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

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

[0061] The terms "halo" and "halogen" are used in their conventional meanings and refer to chloro, bromo, fluoro or iodo substituents.

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

[0063] "Cycloalkyl" refers to a cyclic alkyl group consisting of 3 to 10 carbon atoms having a monocyclic or polycyclic ring, including fused ring systems, bridged ring systems 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, monocyclic structures such as cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, or polycyclic structures such as adamantanyl.

[0064] The term "substituted cycloalkyl" refers to an alkyl, substituted alkyl, alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azide, cyano, halogen, hydroxyl, oxo, thioketone, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl and -SO2-heteroaryl, or a cycloalkyl group having 1 to 3 substituents selected from 1 to 5 substituents.

[0065] The term "heteroatom-containing", as in "heteroatom-containing alkyl group" (also referred to as "heteroalkyl" group) or "heteroatom-containing aryl group" (also referred to as "heteroaryl" group), refers to a molecule, linking group or substituent in which one or more carbon atoms are replaced by atoms other than carbon, such as nitrogen, oxygen, sulfur, phosphorus or silicon, usually nitrogen, oxygen or sulfur. Similarly, the term "heteroalkyl" refers to an alkyl substituent containing a heteroatom, the term "heterocycloalkyl" refers to a cycloalkyl substituent containing a heteroatom, the term "heterocyclic" or "heterocycle" refers to a cyclic substituent containing a heteroatom, and the terms "heteroaryl" and "heteroaromatic" refer to "aryl" and "aromatic" substituents containing a heteroatom, respectively, and so on. 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, and the like, and examples of heteroatom-containing alicyclic groups are pyrrolidino, morpholino, piperazino, piperidino, tetrahydrofuranyl, and the like.

[0066] "Heteroaryl" refers to an aromatic group consisting of 1 to 15 carbon atoms such as 1 to 10 carbon atoms in the ring, and 1 to 10 heteroatoms selected from the group consisting of oxygen, nitrogen and sulfur. Such heteroaryl groups can be monocyclic (such as pyridinyl, imidazolyl or furyl), or have fused polycycles in the ring system (such as in groups such as indolizinyl, quinolinyl, benzofuran, benzimidazolyl or benzothienyl), in which case at least one ring in the ring system is aromatic, provided that the point of attachment is via an atom of the aromatic ring. In certain embodiments, the nitrogen and / or sulfur ring atom(s) of the heteroaryl group are optionally oxidized to provide N-oxide (N→O), sulfinyl or sulfonyl moieties. This term includes, by way of example, pyridinyl, pyrrolyl, indolyl, thiophenyl and furanyl. Unless particularly restricted by definition with respect to the heteroaryl substituent, such heteroaryl groups can be optionally substituted by 1 to 5 substituents or 1 to 3 substituents selected from acyloxy, hydroxy, thiol, acyl, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, substituted alkyl, substituted alkoxy, substituted alkenyl, substituted alkynyl, substituted cycloalkyl, substituted cycloalkenyl, amino, substituted amino, aminoacyl, acylamino, alkaryl, aryl, aryloxy, azide, carboxyl, carboxylalkyl, cyano, halogen, nitro, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, aminoacyloxy, oxyacylamino, thioalkoxy, substituted thioalkoxy, thioaryloxy, thioheteroaryloxy, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl and -SO2-heteroaryl and trihalomethyl.

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

[0068] Examples of heterocyclic rings and heteroaryls include, but are not limited to, azetidine, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, dihydroindole, indazole, purine, quinolidine, 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 referred to as thiamorpholinyl), 1,1-dioxothiomorpholinyl, piperidinyl, pyrrolidine, tetrahydrofuranyl, etc.

[0069] Unless otherwise particularly restricted by the definition of the complex cyclic substituent, such a complex cyclic group may be optionally substituted by 1 to 5 substituents or 1 to 3 substituents selected from alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azide, cyano, halogen, hydroxyl, oxo, thioketone, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl, -SO2-heteroaryl and fused complex rings.

[0070] "Hydrocarbyl" refers to a monovalent hydrocarbyl radical containing from 1 to about 30 carbon atoms, including from 1 to about 24 carbon atoms, further including from 1 to about 18 carbon atoms, and still further including from about 1 to 12 carbon atoms, including linear, branched, cyclic, saturated and unsaturated species, such as alkyl groups, alkenyl groups, aryl groups, etc. Hydrocarbyl may be substituted by one or more substituents. The term "heteroatom-containing hydrocarbyl" refers to a hydrocarbyl in which at least one carbon atom is replaced by a heteroatom. Unless otherwise indicated, the term "hydrocarbyl" should be interpreted to include substituted and / or heteroatom-containing hydrocarbyl moieties.

[0071] "Substituted", as in "substituted hydrocarbyl", "substituted alkyl", "substituted aryl", etc., is intended, as implied in part by the above definitions, that at least one hydrogen atom bonded to a carbon (or other) atom in a hydrocarbyl, alkyl, aryl or other moiety is replaced by one or more non-hydrogen substituents. Examples of such substituents include, without limitation, functional groups, and hydrocarbyl moieties C1-C24 alkyl (including C1-C18 alkyl, further including C1-C12 alkyl, further including C1-C6 alkyl), C2-C24 alkenyl (including C2-C18 alkenyl, further including C2-C12 alkenyl, further including C2-C6 alkenyl), C2-C24 alkynyl (including C2-C18 alkynyl, further including C2-C12 alkynyl, further including C2-C6 alkynyl), C5-C30 aryl (including C5-C20 aryl, further including C5-C12 aryl) and C6-C30 aralkyl (including C6-C20 aralkyl, further including C6-C12 aralkyl). The above hydrocarbyl moieties may be further substituted by one or more functional groups, such as those specifically described, or additional hydrocarbyl moieties. Unless otherwise indicated, any group described herein should be construed to include a substituted moiety and / or a heteroatom-containing moiety in addition to the unsubstituted group.

[0072] "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-heterocyclic and SO2-substituted heterocyclic, where alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic and substituted heterocyclic are as defined herein. Examples of sulfonyl include methyl-SO2-, phenyl-SO2- and 4-methylphenyl-SO2-.

[0073] The term "functional group" refers to halo, hydroxyl, sulfhydryl, C1-C24 alkoxy, C2-C24 alkenyloxy, C2-C24 alkynyloxy, C5-C20 aryloxy, acyl (including C2-C24 alkylcarbonyl (-CO-alkyl) and C6-C20 arylcarbonyl (-CO-aryl)), acyloxy (-O-acyl), C2-C24 alkoxycarbonyl (-(CO)-O-alkyl), C6-C20 aryloxycarbonyl (-(CO)-O-aryl), halocarbonyl (-(CO)-X where X is halo), C2-C24 alkyl carbonate (-(O-(CO)-O-alkyl)), C6-C20 aryl carbonate (-(O-(CO)-O-aryl)), carboxy (-COOH), carboxylato (-(COO-)), carbamoyl (-(CO)-NH2), monosubstituted C1-C24 alkylcarbamoyl (-(CO)-NH(C1-C24 alkyl)), disubstituted alkylcarbamoyl (-(CO)-N(C1-C24 alkyl)2), monosubstituted arylcarbamoyl (-(CO)-NH-aryl), thiocarbamoyl (-(CS)-NH2), carbamide (-(NH-(CO)-NH2)), cyano (-(C≡N)), isocyano (-(N+≡C-)), cyanato (-(O-C≡N)), isocyanato (-(O-N+≡C-)), isothiocyanato (-(S-C≡N)), azide (-(N=N+=N-)), formyl (-(CO)-H), thioformyl (-(CS)-H), amino (-(NH2)), mono- and di-(C1-C24 alkyl)-substituted amino, mono- and di-(C5-C20 aryl)-substituted amino, C2-C24 alkylamide (-(NH-(CO)-alkyl)), C5-C20 arylamide (-(NH-(CO)-aryl)), imino (-(CR=NH where R is hydrogen, C1-C24 alkyl, C5-C20 aryl, C6-C20 alkaryl, C6-C20 aralkyl, etc.)), alkylimino (-(CR=N(alkyl) where R is hydrogen, alkyl, aryl, alkaryl, etc.)), arylimino (-(CR=N(aryl) where R is hydrogen, alkyl, aryl, alkaryl, etc.)), nitro (-(NO2)), nitroso (-(NO)), sulfo (-(SO2-OH)), sulfonato (-(SO2-O-)), C1-C24 alkylsulfanyl (-(S-alkyl;Chemical groups such as alkylthio (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 are contemplated. Further, the above functional groups may be further substituted by one or more additional functional groups, such as those specifically described above, or one or more hydrocarbyl moieties, provided that the specific groups permit it.;

[0074] The "linking" or "linker" in "linking group", "linker part", etc. is intended to be a linking moiety that links two groups by a covalent bond. The linker can be linear, branched, cyclic or a single atom. Examples of such linking groups include alkyl, alkenylene, alkynylene, arylene, alkarylene, aralkylene, and, without limitation, amide (-NH-CO-), ureylene (-NH-CO-NH-), imide (-CO-NH-CO-), epoxy (-O-), episulfide (-S-), epidioxy (-O-O-), carbonyldioxy (-O-CO-O-), alkyldioxy (-O-(CH2)n-O-), epoxyimino (-O-NH-), epiimino (-NH-), carbonyl (-CO-), etc., including linking moieties containing functional groups. In certain cases, one, two, three, four or five, or more carbon atoms of the linker backbone may be optionally substituted by sulfur, nitrogen or oxygen heteroatoms. The bonds between the backbone atoms may be saturated or unsaturated, and usually, one, two or three or less unsaturated bonds are present in the linker backbone. The linker may include one or more substituents having, for example, an alkyl group, an aryl group or an alkenyl group. The linker may, without limitation, include poly(ethylene glycol) units (singular or plural) (e.g., -(CH2-CH2-O)-); ether, thioether, amine, alkyl (e.g., (C1~C 12 )alkyl), which may be linear or branched, for example, methyl, ethyl, n-propyl, 1-methylethyl (iso-propyl), n-butyl, n-pentyl, 1,1-dimethylethyl (t-butyl), etc. The linker backbone may include a cyclic group, for example, an aryl, heterocyclic or cycloalkyl group, in which case two or more atoms of the cyclic group, for example, two, three or four atoms, are included in the backbone. The linker may be cleavable or non-cleavable. The convenient orientation and / or linking of the linker to the linked groups may be used.

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

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

[0077] In addition to the groups disclosed herein for the individual terms, substituents that replace one or more hydrogens (any two hydrogens on a single carbon can be replaced by =O, =NR 70 , =N-OR 70 , =N2 or =S) on saturated carbon atoms in the specified group or radical are, unless otherwise specified, -R 60 , halo, =O, -OR 70 , -SR 70 , -NR 80 R 80 , trihalomethyl, -CN, -OCN, -SCN, -NO, -NO2, =N2, -N3, -SO2R 70 , -SO2O - M + , -SO2OR 70 , -OSO2R 70 , -OSO2O - M + , -OSO2OR 70 , -P(O)(O - )2(M + )2, -P(O)(OR 70 )O - M + , -P(O)(OR 70 )2, -C(O)R 70 , -C(S)R 70 , -C(NR 70 )R 70 , -C(O)O -M + 、 -C(O)OR 70 、 -C(S)OR 70 、 -C(O)NR 80 R 80 、 -C(NR 70 )NR 80 R 80 、 -OC(O)R 70 、 -OC(S)R 70 、 -OC(O)O - M + 、 -OC(O)OR 70 、 -OC(S)OR 70 、 -NR 70 C(O)R 70 、 -NR 70 C(S)R 70 、 -NR 70 CO2 - M + 、 -NR 70 CO2R 70 、 -NR 70 C(S)OR 70 、 -NR 70 C(O)NR 80 R 80 、 -NR 70 C(NR 70 )R 70 and -NR 70 C(NR 70 )NR 80 R 80 wherein R 60 is independently selected from the group consisting of optionally substituted alkyl, cycloalkyl, heteroalkyl, heterocycloalkylalkyl, cycloalkylalkyl, aryl, arylalkyl, heteroaryl and heteroarylalkyl, and each R 70 is independently hydrogen or R 60 wherein each R 80 is independently R 70 or alternatively, two R 80Together with the nitrogen atom to which they are attached, form a 5-, 6- or 7-membered heterocycloalkyl, which may optionally contain 1 to 4 identical or different additional heteroatoms selected from the group consisting of O, N and S, and N of the heteroatoms may have -H or C1-C3 alkyl substitution, M + Each is a counterion having a net single positive charge. M + Each is independently, for example, K + 、Na + 、Li + and other alkali ions; + Ammonium ions such as N(R 60 )4; or [Ca 2+ 0.5 、[Mg 2+ 0.5 or [Ba 2+ 0.5 (The subscript 0.5 means that one of the counterions for such divalent alkaline earth ions can be the ionized form of the compound of the present invention, and other typical counterions such as chloride ions or the two ionic compounds disclosed herein can act as counterions for such divalent alkaline earth ions, or the double-ionized compound of the present invention can act as a counterion for such divalent alkaline earth ions). It can be an alkaline earth ion such as. As a specific example, -NR 80 R 80 is intended to include -NH2, -NH-alkyl, N-pyrrolidinyl, N-piperazinyl, 4N-methyl-piperazin-1-yl and N-morpholinyl.

[0078] In addition to the disclosure herein, substituents for hydrogen on unsaturated carbon atoms in "substituted" alkene, alkyne, aryl and heteroaryl groups, unless otherwise specified, are -R 60 , halo, -O - M + , -OR 70 , -SR 70 , -S - M​​​+ 、 -NR 80 R 80 、 trihalomethyl, -CF3, -CN, -OCN, -SCN, -NO, -NO2, -N3, -SO2R 70 、 -SO3 - M + 、 -SO3R 70 、 -OSO2R 70 、 -OSO3 - M + 、 -OSO3R 70 、 -PO3 -2 (M + )2, -P(O)(OR 70 )O - M + 、 -P(O)(OR 70 )2, -C(O)R 70 、 -C(S)R 70 、 -C(NR 70 )R 70 、 -CO2 - M + 、 -CO2R 70 、 -C(S)OR 70 、 -C(O)NR 80 R 80 、 -C(NR 70 )NR 80 R 80 、 -OC(O)R 70 、 -OC(S)R 70 、 -OCO2 - M + 、 -OCO2R 70 、 -OC(S)OR 70 、 -NR 70 C(O)R 70 、 -NR 70 C(S)R 70 、 -NR 70 CO2 - M + 、 -NR 70 CO2R 70 、 -NR 70 C(S)OR 70 、 -NR 70 C(O)NR 80 R 80 、 -NR 70 C(NR 70 )R 70 and -NR 70 C(NR70 )NR 80 R 80 is, and R 60 , R 70 , R 80 and M + are as previously defined, provided that in the case of a substituted alkene or alkyne, the substituent is not -O - M + , -OR 70 , -SR 70 or -S - M + .

[0079] In addition to the groups disclosed for the individual terms herein, the substituents for the hydrogen on the nitrogen atom in "substituted" heteroalkyl and cycloheteroalkyl groups are, unless otherwise specified, -R 60 , -O - M + , -OR 70 , -SR 70 , -S - M + , -NR 80 R 80 , trihalomethyl, -CF3, -CN, -NO, -NO2, -S(O)2R 70 , -S(O)2O - M + , -S(O)2OR 70 , -OS(O)2R 70 , -OS(O)2O - M + , -OS(O)2OR 70 , -P(O)(O - )2(M + )2, -P(O)(OR 70 )O - M + , -P(O)(OR 70 )(OR 70 ), -C(O)R 70 , -C(S)R 70 , -C(NR 70 )R 70 , -C(O)OR 70 , -C(S)OR 70 , -C(O)NR 80 R 80 , -C(NR70 )NR 80 R 80 、 -OC(O)R 70 、 -OC(S)R 70 、 -OC(O)OR 70 、 -OC(S)OR 70 、 -NR 70 C(O)R 70 、 -NR 70 C(S)R 70 、 -NR 70 C(O)OR 70 、 -NR 70 C(S)OR 70 、 -NR 70 C(O)NR 80 R 80 、 -NR 70 C(NR 70 )R 70 and -NR 70 C(NR 70 )NR 80 R 80 wherein R 60 、 R 70 、 R 80 and M + are as defined above.

[0080] In addition to the disclosure herein, in certain embodiments, a substituted group has one, two, three or four substituents, one, two or three substituents, one or two substituents, or one substituent.

[0081] Unless otherwise indicated, the naming of substituents not explicitly defined herein is achieved by naming the terminal portion of the functional group, then the adjacent functional group, in the direction towards the point of attachment. For example, the substituent "arylalkyloxycarbonyl" refers to an (aryl)-(alkyl)-O-C(O)- group.

[0082] Regarding any of the groups disclosed herein that contain one or more substituents, it will of course be understood that such groups do not include any substitution or substitution patterns that are sterically difficult to achieve and / or not synthetically feasible. Further, the subject compounds include all of the stereochemical isomers resulting from the substitution of these compounds.

[0083] In certain embodiments, substituents may contribute to the optical and / or steric isomerism of the compound. Also of interest are the salts, solvates, hydrates and prodrug forms of the compounds. All such forms are encompassed by the present disclosure. Accordingly, the compounds described herein include their salts, solvates, hydrates, prodrugs and isomeric forms, including pharmaceutically acceptable salts, solvates, hydrates, prodrugs and isomers thereof. In certain embodiments, the compounds may be metabolized to pharmaceutically active derivatives.

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

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

[0086] Devices and methods are described, or are to be described, with functional descriptive flexibility, but unless otherwise expressly set forth under 35 U.S.C. § 112, claims are not to be construed as necessarily limited by reciting "means" or "steps," but rather are to be construed to cover the full scope of the meaning and equivalents of the definition presented by the claim under the doctrine of equivalents, and it should be clearly understood that when a claim is expressly set forth under 35 U.S.C. § 112, it is to cover full statutory equivalents thereunder.

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

[0088] As summarized above, aspects of the disclosure include compounds, compositions, and methods for inhibiting ENPP1. Aspects of the methods include contacting a sample with an ENPP1 inhibitor compound to inhibit the cGAMP hydrolysis activity of ENPP1. These compounds, compositions, and methods find use in a variety of applications where inhibition of ENPP1 is desirable.

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

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

[0091] The term "hydrophilic head group" refers to a group that is hydrophilic, is sufficiently solvated in an aqueous environment, such as physiological conditions, and is linked to a core aryl or heteroaryl ring system that has low permeability across cell membranes. In some cases, having low permeability across cell membranes means that the permeability coefficient, as measured by any convenient method of passive diffusion for an isolated hydrophilic head group across a membrane (e.g., a cell monolayer such as the colorectal Caco-2 or renal MDCK cell lines), is 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 lower such as 10 -4It is intended to be cm / s or less. For example, see Yang and Hinner, Methods Mol Biol. 2015; 1266: 29-53. The hydrophilic head group can provide improved water solubility and reduced cell permeability to the molecule to which the hydrophilic head group is attached. The hydrophilic head group can be any convenient hydrophilic group that is sufficiently solvated in an aqueous environment and has low permeability to the membrane. In certain examples, the hydrophilic group is a discrete functional group (e.g., as described herein), or a substituted form thereof. Generally, charged groups, or larger uncharged polar groups, have low permeability. In some cases, the hydrophilic head group is charged, e.g., positively or negatively charged. In some embodiments, the hydrophilic head group itself is not cell permeable, but confers cell impermeability to the target compound. It is understood that the hydrophilic head group or its prodrug form can be selected to provide the desired cell permeability of the target compound. In certain cases, the hydrophilic head group is a neutral hydrophilic group. In some cases, the hydrophilic head group is included in a prodrug form and thus contains a pro moiety that can be removed in vivo. In certain examples, the target compound is cell permeable.

[0092] The hydrophilic head group can be any convenient group, or a prodrug form thereof, that can bind to or chelate with zinc ions. In certain cases, the hydrophilic head group is a phosphorus-containing group. Exemplary phosphorus-containing groups that can be utilized in the target ENPP1 inhibitors include, but are not limited to, phosphonic acid or phosphonate, phosphonic acid ester, phosphate, phosphate ester, thiophosphate, thiophosphate ester, phosphoramidate and thiophosphoramidate or salts thereof, or prodrug forms thereof (e.g., as described herein).

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

[0094] In one case, the ENPP1 inhibitor compound of interest has the formula (I):

Chemical formula

[0095] In certain embodiments of formula (I), Z 3 is absent. In certain embodiments of formula (I), Z 3 is NR 22 , and R 22 is selected from H, C (1~6) alkyl and substituted C (1~6) alkyl. In certain cases, Z 3 is NH. In certain cases, Z 3 is NR 22 , and R 22 is C (1~6) alkyl, e.g., methyl, ethyl, propyl, pentyl or hexyl. In certain cases, Z 3 is NR 22 , and R 22 is substituted C (1~6) alkyl. In certain cases of formula (I), Z 3 is O. In certain cases of formula (I), Z 3 is S.

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

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

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

[0099] In certain embodiments of formula (I), L 1 and L 2 are each a covalent bond. In certain cases, L 1 and L 2 are each a linker. In certain cases, L 1 is a covalent bond and L 2 is a linker. In certain cases, L 1 is a linker and L 2 is a covalent bond. Any convenient linker can be utilized to link A to X and / or A to Z 3 (e.g., as described herein). In some cases, A is linked to X by a covalent bond. In certain cases, A is linked to X via a linear linker consisting of 1 to 12 atoms, such as 1 to 10 atoms, 1 to 8 atoms, or 1 to 6 atoms, for example, 1, 2, 3, 4, 5, or 6 atoms in length. The linker L 2 can be a (C 1~6 )alkyl linker, or a substituted (C 1~6 )alkyl linker optionally substituted with heteroatoms or linking functional groups such as esters (-CO2-), amides (CONH), carbamates (OCONH), ethers (-O-), thioethers (-S-), and / or amino groups (-NR-, where R is H or alkyl). In some cases, A is linked to Z 3is linked to. In certain cases, A is a linear linker consisting of 1 to 12 atoms, such as 1 to 10 atoms, 1 to 8 atoms, or 1 to 6 atoms in length, for example, 1, 2, 3, 4, 5, or 6 atoms in length, via a linker consisting of 1 to 12 atoms, and is linked to Z 3 is linked to. The linker L 1 can be a (C 1~6 ) alkyl linker, or a substituted (C 1~6 ) alkyl linker optionally substituted by a heteroatom or linking functional group such as keto (CO), ester (-CO2-), amide (CONH), carbamate (OCONH), ether (-O-), thioether (-S-), and / or amino group (-NR-, where R is H or alkyl). When Z 3 is NR 22 , the linker L 1 can include a terminal keto (C=O) group that, together with Z 3 , results in an amide group (NR 22 CO) linking group. When Z 31 is O or S, the linker L 1 can include a terminal keto (C=O) group that, together with Z 31 , results in an ester or thioester group linking group.

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

[0101] In certain examples of formula (I), Z 3 is selected from NR 22 , O, and S. Thus, the subject ENPP1 inhibitor compounds of formula (I) can be described by formula (II):

Chemical formula

[0102] In certain embodiments of formula (II), Z 31 is NR 22 , where R 22 is selected from H, C (1~6) alkyl and substituted C (1~6) alkyl. In certain cases, Z 31 is NH. In certain cases, Z 31 is NR 22 , where R 22 is C (1~6) alkyl, such as methyl, ethyl, propyl, pentyl or hexyl. In certain cases, Z 31 is NR 22 , where R 22 is substituted C (1~6) alkyl. In certain cases of formula (I), Z 31 is O. In certain cases of formula (I), Z 31 is S.

[0103] In some examples of formula (II), Z 1 is CR 11 , where R 11 is selected from hydrogen, cyano, trifluoromethyl, halogen, alkyl and substituted alkyl hydrogen. In some cases, the alkyl or substituted alkyl is C 1~5 alkyl. In some examples of formula (II), Z 1 is CR 11 , where R 11 is hydrogen. In some cases, R 11 is cyano. In some cases, R 11 is trifluoromethyl. In some cases, R 11 is halogen, such as Br, I, Cl or F. In some cases, R 11 is alkyl, such as C 1~5 alkyl. In some cases, R 11 is substituted alkyl, such as substituted C 1~5 alkyl.

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

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

[0106] In certain embodiments of formula (II), L 1 and L 2 are each a covalent bond. In certain cases, L 1 and L 2 are each a linker. In certain cases, L 1 is a covalent bond and L 2 is a linker. In certain cases, L 1is a linker, L 2 is a covalent bond. Any convenient linker can be used to link A to X and / or A to Z 3 (e.g., as described herein). In some cases, A is linked to X by a covalent bond. In certain cases, A is linked to X via a straight-chain linker consisting of 1 to 12 atoms, such as 1 to 10, 1 to 8, or 1 to 6 atoms, for example, 1, 2, 3, 4, 5, or 6 atoms in length. Linker L 2 can be a (C 1~6 )alkyl linker, or a substituted (C 1~6 )alkyl linker optionally substituted by a heteroatom or linking functional group such as keto (CO), ester (-CO2-), amide (CONH), carbamate (OCONH), ether (-O-), thioether (-S-), and / or amino group (-NR-, where R is H or alkyl). In some cases, A is linked to Z 3 by a covalent bond. In certain cases, A is linked to Z via a straight-chain linker consisting of 1 to 12 atoms, such as 1 to 10, 1 to 8, or 1 to 6 atoms, for example, 1, 2, 3, 4, 5, or 6 atoms in length. Linker L 3 can be a (C 1 )alkyl linker, or a substituted (C 1~6 )alkyl linker optionally substituted by a heteroatom or linking functional group such as keto (C=O), ester (-CO2-), amide (CONH), carbamate (OCONH), ether (-O-), thioether (-S-), and / or amino group (-NR-, where R is H or alkyl). When Z 1~6 is NR 31 , linker L 22 can include a terminal keto (C=O) group that, together with Z 1 , results in an amide group (NR 31 CO) linking group. When Z 22 is O or S, linker L 31 ​1 together with Z 31 can include a terminal keto (C=O) group that, together with Z, provides an ester or thioester group linking moiety.

[0107] In some cases of formula (II), the ENPP1 inhibitor compound of interest is of formula (III):

Chemical formula

[0108] In certain embodiments of formula (III), Z 31 is NR 22 where R 22 is selected from H, C (1~6) alkyl, and substituted C (1~6) alkyl. In certain cases, Z 31 is NH. In certain cases, Z 31 is NR 22 where R 22 is C (1~6) alkyl, such as methyl, ethyl, propyl, pentyl, or hexyl. In certain cases, Z 31 is NR 22 where R 22 is substituted C (1~6) alkyl. In certain cases of formula (III), Z 31 is O. In certain cases of formula (III), Z 31 is S.

[0109] In formula (II), when Z 31 is NR 22 , the linker L 1 can, together with Z 31 , form an amide group (NR 22 CO) linking group and contain a terminal keto (C=O) group. Thus, in some cases of formula (II), the ENPP1 inhibitor compound of interest is of formula (IIIa): [Chemical formula] (wherein Z 41 is -NR 22 C(=O)-, R 31 ~R 34 are each independently selected from H, halogen, alkyl and substituted alkyl, or R 31 and R 32 or R 33 and R 34 are cyclically linked and, together with the carbon atoms to which they are attached, form a cycloalkyl, substituted cycloalkyl, heterocyclyl or substituted heterocyclyl ring, n and m are each independently an integer from 0 to 6 (for example, 0 to 3)) .

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

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

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

[0113] In certain embodiments of formulas (III) to (IIIa), R 31 to R 34 are each hydrogen. In certain embodiments, at least one of R 31 to R 34 is halogen. In certain embodiments, at least one of R 31 to R 34 is alkyl. In certain embodiments, at least one of R 31 to R 34 is substituted alkyl. In certain cases, one of R 31 to R 34 is halogen and the rest are selected from hydrogen, halogen, alkyl, and substituted alkyl. In certain cases, one of R 31 to R 34 is alkyl and the rest are selected from hydrogen, halogen, alkyl, and substituted alkyl. In certain cases, one of R 31 to R 34 is substituted alkyl and the rest are selected from hydrogen, halogen, alkyl, and substituted alkyl. In certain cases, one of R 31 to R 34 is halogen and the rest are hydrogen. In certain cases, one of R 31 to R 34 is alkyl and the rest are hydrogen. In certain cases, one of R 31 to R 34 is substituted alkyl and the rest are hydrogen.

[0114] In certain embodiments of formulas (III) to (IIIa), n is an integer from 0 to 3. In certain cases, n is 0. In certain cases, n is 1. In certain cases, n is 2. In certain cases, n is 3. In certain embodiments of formulas (III) to (IIIa), m is an integer from 0 to 3. In certain cases, m is 0. In certain cases, m is 1. In certain cases, m is 2. In certain cases, m is 3. In certain cases, n is 0 and m is 1. In certain cases, n is 0 and m is 2. In certain cases, n is 0 and m is 3. In certain cases, n is 1 and m is 0. In certain cases, n is 1 and m is 1. In certain cases, n is 1 and m is 2. In certain cases, n is 1 and m is 3. In certain cases, n is 2 and m is 0. In certain cases, n is 2 and m is 1. In certain cases, n is 2 and m is 2. In certain cases, n is 2 and m is 3. In certain cases, n is 3 and m is 0. In certain cases, n is 3 and m is 1. In certain cases, n is 3 and m is 2. In certain cases, n is 3 and m is 3. In certain cases, n + m is an integer from 0 to 3. In certain cases, n + m is 0. In certain cases, n + m is 1. In certain cases, n + m is 2. In certain cases, n + m is 3.

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

[0116] In some embodiments, ring system A is of formula (A1):

Chemical formula

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

[0118] In some embodiments, the A1 ring is of formula (A1a):

Chemical formula

[0119] In some embodiments, ring system A is of formula (A2):

Chemical formula

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

[0121] In some embodiments, ring system A is of formula (A3):

Chemical formula

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

[0123] In some embodiments, ring system A is of formula (A4):

Chemical formula

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

[0125] In some cases of formulas (III) to (IIIa), the ENPP1 inhibitor compounds are of formulas (IV) to (IVa): [Chemical formula] (wherein Z 31 is selected from NR 22 , O and S, Z 41 is -NR 22 C(=O)-, Z 11 and Z 21 are independently selected from N and C(CN), R 31 ~R 34 are each independently selected from H, halogen, alkyl and substituted alkyl, or R 31 and R 32 or R 33 and R 34 are cyclically linked, and together with the carbon atoms to which they are attached, result in a cycloalkyl, substituted cycloalkyl, heterocyclyl or substituted heterocyclyl ring, R 6 are each independently selected from H, alkyl, substituted alkyl, hydroxy, alkoxy, substituted alkoxy, trifluoromethyl and halogen, p is an integer from 0 to 4, n and m are each independently integers from 0 to 6 (for example, 0 to 3). are those of.

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

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

[0128] In certain embodiments of formulas (IV) to (IVa), R 31 to R 34 are each hydrogen. In certain embodiments, at least one of R 31 to R 34 is halogen. In certain embodiments, at least one of R 31 to R 34 is alkyl. In certain embodiments, at least one of R 31 to R 34 is substituted alkyl. In certain cases, one of R 31 to R 34 is halogen, and the rest are selected from hydrogen, halogen, alkyl, and substituted alkyl. In certain cases, one of R 31 to R 34 is alkyl, and the rest are selected from hydrogen, halogen, alkyl, and substituted alkyl. In certain cases, one of R 31 to R 34One of them is a substituted alkyl, and the rest are selected from hydrogen, halogen, alkyl, and substituted alkyl. In certain cases, R 31 ~R 34 One of them is halogen, and the rest are hydrogen. In certain cases, R 31 ~R 34 One of them is alkyl, and the rest are hydrogen. In certain cases, R 31 ~R 34 One of them is a substituted alkyl, and the rest are hydrogen.

[0129] In certain embodiments of Formula (IV)-(IVa), n is an integer from 0 to 3. In certain cases, n is 0. In certain cases, n is 1. In certain cases, n is 2. In certain cases, n is 3. In certain embodiments of Formula (IV)-(IVa), m is an integer from 0 to 3. In certain cases, m is 0. In certain cases, m is 1. In certain cases, m is 2. In certain cases, m is 3. In certain cases, n is 0 and m is 1. In certain cases, n is 0 and m is 2. In certain cases, n is 0 and m is 3. In certain cases, n is 1 and m is 0. In certain cases, n is 1 and m is 1. In certain cases, n is 1 and m is 2. In certain cases, n is 1 and m is 3. In certain cases, n is 2 and m is 0. In certain cases, n is 2 and m is 1. In certain cases, n is 2 and m is 2. In certain cases, n is 2 and m is 3. In certain cases, n is 3 and m is 0. In certain cases, n is 3 and m is 1. In certain cases, n is 3 and m is 2. In certain cases, n is 3 and m is 3. In certain cases, n + m is an integer from 0 to 3. In certain cases, n + m is 0. In certain cases, n + m is 1. In certain cases, n + m is 2. In certain cases, n + m is 3.

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

[0131] In some cases of formula (IV) to (IVa), the ENPP1 inhibitor compound is of formula (V) to (Va):

Chemical formula

[0132] In certain embodiments of formula (V) to (Va), at least one of Z 11 and Z 21 is N. In certain embodiments of formula (V) to (Va), Z 11 is C(CN), and Z 21 is N. In certain cases of formula (V) to (Va), Z 11 is N, and Z 21 is C(CN). In certain examples of formula (V) to (Va), Z 11 is C(CN), and Z 21 is C(CN). In certain cases of formula (V) to (Va), Z 11 is N, and Z 21 is N.

[0133] In some cases of formula (V) to (Va), the ENPP1 inhibitor compound of interest is one of formula (VIa) to (VId):

Chemical formula

[0134] In certain embodiments of formula (VIa) to (VId), R 41 ~R 44 are each hydrogen. In certain embodiments, R 41 ~R 44At least one of them is alkyl or substituted alkyl. In certain embodiments, R 41 ~R 44 At least one of them is hydroxy. In certain embodiments, R 41 ~R 44 At least one of them is alkoxy or substituted alkoxy. In certain cases, R 41 ~R 44 At least one of them is trifluoromethyl. In certain cases, R 41 ~R 44 At least one of them is halogen. In certain cases, R 41 ~R 44 At least one of them is acyl or substituted acyl. In certain cases, R 41 ~R 44 At least one of them is carboxy. In certain cases, R 41 ~R 44 At least one of them is carboxamide or substituted carboxamide. In certain cases, R 41 ~R 44 At least one of them is sulfonyl or substituted sulfonyl. In certain cases, R 41 ~R 44 At least one of them is sulfonamide and substituted sulfonamide. In certain cases, R 31 ~R 34 One of them is hydrogen, and the rest are selected from hydrogen, alkyl, substituted alkyl, hydroxy, alkoxy, substituted alkoxy, trifluoromethyl, halogen, acyl, substituted acyl, carboxy, carboxamide, substituted carboxamide, sulfonyl, substituted sulfonyl, sulfonamide and substituted sulfonamide. In certain cases, R 31 ~R 34 Two of them are hydrogen, and the rest are selected from hydrogen, alkyl, substituted alkyl, hydroxy, alkoxy, substituted alkoxy, trifluoromethyl, halogen, acyl, substituted acyl, carboxy, carboxamide, substituted carboxamide, sulfonyl, substituted sulfonyl, sulfonamide and substituted sulfonamide. In certain cases, R31 ~R 34 Three of them are hydrogen, and the rest are selected from hydrogen, alkyl, substituted alkyl, hydroxy, alkoxy, substituted alkoxy, trifluoromethyl, halogen, acyl, substituted acyl, carboxy, carboxamide, substituted carboxamide, sulfonyl, substituted sulfonyl, sulfonamide and substituted sulfonamide.

[0135] In certain embodiments of Formulas (VIa)-(VId), n is an integer from 0 to 3. In certain cases, n is 0. In certain cases, n is 1. In certain cases, n is 2. In certain cases, n is 3. In certain embodiments of any of Formulas (VIa)-(VId), m is an integer from 0 to 3. In certain cases, m is 0. In certain cases, m is 1. In certain cases, m is 2. In certain cases, m is 3. In certain cases, n is 0 and m is 1. In certain cases, n is 0 and m is 2. In certain cases, n is 0 and m is 3. In certain cases, n is 1 and m is 0. In certain cases, n is 1 and m is 1. In certain cases, n is 1 and m is 2. In certain cases, n is 1 and m is 3. In certain cases, n is 2 and m is 0. In certain cases, n is 2 and m is 1. In certain cases, n is 2 and m is 2. In certain cases, n is 2 and m is 3. In certain cases, n is 3 and m is 0. In certain cases, n is 3 and m is 1. In certain cases, n is 3 and m is 2. In certain cases, n is 3 and m is 3. In certain cases, n + m is an integer from 0 to 3. In certain cases, n + m is 0. In certain cases, n + m is 1. In certain cases, n + m is 2. In certain cases, n + m is 3.

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

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

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

[0139] In some cases of formulas (VIa) - (VId), the ENPP1 inhibitor compound is one of formulas (VIIa) - (VIIb):

Chemical formula

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

[0141] In certain embodiments of any of formulas (I)-(VIIb), R 2 ~R 5 are hydrogen, OH, C (1~6) alkoxy, -OCF3, C (1~6) alkylamino, di-C (1~6) alkylamino, F, Cl, Br, and CN.

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

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

[0144] In some cases of formula (VIc)-(VId), R 41 ~R 44 are each independently H, halogen, C (1~6) alkyl or C (1~6) alkoxy. In some cases of formula (VIc)-(VId), m is 1 or 2. In some cases of formula (VIc)-(VId), R2 is H, and R 3 ~R 5 are independently selected from hydrogen, C (1~6) alkoxy, F, Cl and C (1~6) alkyl.

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

Chemical formula

[0146] In some cases of formula (VIa), the ENPP1 inhibitor compound is of formula (VIIm):

Chemical formula

[0147] In certain embodiments of formula (VIIm), R 2 ~R 5 are independently selected from H, OH, alkyl, substituted alkyl, alkoxy, substituted alkoxy, -OCF3, halogen, cyano, amine, substituted amine, amide, heterocycle and substituted heterocycle. In certain embodiments of formula (VIIm), R 2 ~R 5is hydrogen, OH, C (1~6) alkoxy, -OCF3, C (1~6) alkylamino, di-C (1~6) alkylamino, F, Cl, Br, and CN, independently selected. In certain embodiments of formula (VIIm), n + m = 1. In certain embodiments of formula (VIIm), n + m = 2. In certain embodiments of formula (VIIm), n is 1 and m is 0.

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

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

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

[0151] In certain examples of the ENPP1 inhibitor compound of formula (I), Z 3 is absent. In certain embodiments of formula (I), Z 3 is absent, and Z 2 is CR 12 , and R 12 is cyano, and the compound is of formula (X):

Chemical formula

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

[0153] In some embodiments, ring system A is any one of formulas (A1) to (A4):

Chemical formula

[0154] In some embodiments, ring A is of formula (A5):

Chemical formula

[0155] In certain cases, A5 is piperidine or substituted piperidine. In certain cases, A5 is piperazine or substituted piperazine. In certain cases, A5 is cyclohexyl or substituted cyclohexyl. In certain embodiments of A5, r is greater than 0, such as 1, 2, 3, 4, 5, 6, 7 or 8. In some cases, A5 contains one R 16 group. In some cases, A5 contains two R 16 groups. In some cases, A5 contains three R 16 groups. In some cases, A5 contains four R 16 groups. In certain embodiments, the substituents are selected from lower alkyl (e.g., methyl, ethyl, propyl, butyl, pentyl and hexyl), trifluoromethyl and halogen (e.g., F, Cl, I or Br).

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

Chemical formula

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

Chemical formula

[0158] In certain cases of formula (X), the ENPP1 inhibitor compound of interest is of formula (XI): [Chemical formula] (wherein Z 5 is independently selected from N and CR 16 respectively, and R 16 is independently selected from hydrogen, alkyl, substituted alkyl, hydroxy, alkoxy, substituted alkoxy, trifluoromethyl, halogen, acyl, substituted acyl, carboxy, carboxamide, substituted carboxamide, sulfonyl, substituted sulfonyl, sulfonamide and substituted sulfonamide respectively, and r is an integer from 0 to 8). It is of the following form.

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

[0160] In certain embodiments of any one of the compounds of formula (X) - (XI), L 11 and L 12 are each a covalent bond. In certain cases, L 11 and L 12 are each a linker. In certain cases, L 11 is a covalent bond and L 12 is a linker. In certain cases, L 11 is a linker and L 12 is a covalent bond. Any convenient linker can be used as L 11 and L 12 . In some cases, L 11 is a covalent bond. In certain cases, L 11is a linear linker consisting of 1 to 12 atoms, for example, 1 to 10 atoms, 1 to 8 atoms, or 1 to 6 atoms in length, such as 1, 2, 3, 4, 5, or 6 atoms in length. Linker L 11 is a (C 1~6 )alkyl linker, or a substituted (C 1~6 )alkyl linker optionally substituted by a heteroatom or a linking functional group such as an ester (-CO2-), an amide (CONH), a carbamate (OCONH), an ether (-O-), a thioether (-S-), and / or an amino group (-NR- where R is H or alkyl). In some cases, L 12 is a covalent bond. In certain cases, L 12 is a linker consisting of 1 to 12 atoms, for example, 1 to 10 atoms, 1 to 8 atoms, or 1 to 6 atoms in length, such as 1, 2, 3, 4, 5, or 6 atoms in length. Linker L 12 is a (C 1~6 )alkyl linker, or a substituted (C 1~6 )alkyl linker optionally substituted by a heteroatom or a linking functional group such as an ester (-CO2-), an amide (CONH), a carbamate (OCONH), an ether (-O-), a thioether (-S-), and / or an amino group (-NR- where R is H or alkyl).

[0161] In some cases of formula (XI), the ENPP1 inhibitor compound of interest is of formula (XII):

Chemical formula

[0162] In certain embodiments of the compounds of formula (XII), Z 5 is CR 16 , and R 16are each independently selected from hydrogen, alkyl, substituted alkyl, hydroxy, alkoxy, substituted alkoxy, trifluoromethyl, halogen, acyl, substituted acyl, carboxy, carboxamide, substituted carboxamide, sulfonyl, substituted sulfonyl, sulfonamide and substituted sulfonamide. In certain cases of the compounds of formula (XII), Z 5 is N.

[0163] In certain embodiments of the compounds of formula (XII), L 12 is a covalent bond. In certain cases, L 12 is a linker. Any convenient linker can be used as L 12 . In certain cases, L 12 is a straight-chain linker consisting of 1 to 12 atoms, such as 1 to 10 atoms, 1 to 8 atoms or 1 to 6 atoms in length, for example 1, 2, 3, 4, 5 or 6 atoms in length. The linker L 12 can be a (C 1~6 )alkyl linker or a substituted (C 1~6 )alkyl linker optionally substituted by heteroatoms or linking functional groups such as ester (-CO2-), amide (CONH), carbamate (OCONH), ether (-O-), thioether (-S-) and / or amino group (-NR-, where R is H or alkyl).

[0164] In some cases of formula (XII), the ENPP1 inhibitor compound of interest has the formula (XIII):

Chemical formula

[0165] In certain embodiments of formula (XIII), R 35 and R 36 are each hydrogen. In certain embodiments, R 35 or R 36 at least one of which is halogen. In certain embodiments, R 35 or R 36 at least one of which is alkyl. In certain embodiments, R 35 or R 36 at least one of which is substituted alkyl. In certain cases, R 35 is halogen and R 36 is selected from hydrogen, halogen, alkyl and substituted alkyl. In certain cases, R 35 is alkyl and R 36 is selected from hydrogen, halogen, alkyl and substituted alkyl. In certain cases, R 35 is substituted alkyl and R 36 is selected from hydrogen, halogen, alkyl and substituted alkyl. In certain cases, R 35 is halogen and R 36 is hydrogen. In certain cases, R 35 is alkyl and R 36 is hydrogen. In certain cases, R 35 is substituted alkyl and R 36 is hydrogen.

[0166] In certain embodiments of formula (XIII), s is an integer from 0 to 3. In certain cases, s is 0. In certain cases, s is 1. In certain cases, s is 2. In certain cases, s is 3.

[0167] In some cases of formula (XIII), the ENPP1 inhibitor compound of interest is of formula (XIV):

Chemical formula

[0168] In certain embodiments of formula (XIII), s is an integer from 0 to 3. In certain cases, s is 0. In certain cases, s is 1. In certain cases, s is 2. In certain cases, s is 3.

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

[0170] In certain embodiments of any of formulas (X) to (XIV), R 2 ~R 5 are hydrogen, OH, C (1~6) alkoxy, -OCF3, C (1~6) alkylamino, di-C (1~6) alkylamino, F, Cl, Br, and CN, independently selected.

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

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

[0173] In some cases of formula (XIV), the ENPP1 inhibitor compound of interest is one of formulas (XIVa) - (XIVe):

Chemical formula

[0174] In some cases of formula (I), the ENPP1 inhibitor compound of interest is formula (XVa) or (XVb):

Chemical formula

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

[0176] In some examples of formulas (XV a) to (XV b), the ENPP1 inhibitor compound of interest is one of the following structures:

Chemical formula

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

[0178] In some embodiments of formulas (I) to (XV b), the hydrophilic head group (X 1) is selected from phosphonic acid or phosphonate, phosphonic acid 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 any one of formulas (I) to (XVb), the hydrophilic head group is phosphonic acid, phosphonate or a salt thereof. In some embodiments of any one of formulas (I) to (XVb), the hydrophilic head group is phosphate or a salt thereof. In some embodiments of any one of formulas (I) to (XVb), the hydrophilic head group is a phosphonic acid ester or a phosphate ester. In some embodiments of any one of formulas (I) to (XVb), the hydrophilic head group is thiophosphate. In some embodiments of any one of formulas (I) to (XVb), the hydrophilic head group is a thiophosphate ester. In some embodiments of any one of formulas (I) to (XVb), the hydrophilic head group is phosphoramidate. In some embodiments of any one of formulas (I) to (XVb), the hydrophilic head group is thiophosphoramidate.

[0179] Specific examples of the target hydrophilic head groups that can be incorporated into any one of the embodiments of formulas (I) to (XVb) described herein are not limited to the following, but include phosphate (RPO4H - ), phosphonate (RPO3H - ), borate (RBO2H2), carboxylate (RCO2 - ), sulfate (RSO4 - ), sulfonate (RSO3 - ), amine (RNH3 +) a head group comprising a first moiety selected from glycerol, sugars such as lactose or sugars derived from hyaluronic acid, polar amino acids, polyethylenoxide, and oligoethylene glycol, the head group being optionally conjugated to a residue of a second moiety selected from choline, ethanolamine, glycerol, nucleic acids, sugars, inositol, amino acids or amino acid esters (e.g., serine) and lipids (e.g., fatty acids, or hydrocarbon chains such as C8-C30 saturated or unsaturated hydrocarbons). The head group may contain various other modifications, for example, in the case of oligoethylene glycol and polyethylenoxide (PEG)-containing head groups, such PEG chains may be terminated by methyl groups and may have distal functional groups for further modification. Examples of hydrophilic head groups also include, but are not limited to, thiophosphate, phosphocholine, phosphoglycerol, phosphoethanolamine, phosphoserine, phosphoinositol, ethylphosphosphorylcholine, polyethylene glycol, polyglycerol, melamine, glucosamine, trimethylamine, spermine, spermidine and conjugated carboxylates, sulfates, borates, sulfonates, sulfates and carbohydrates.

[0180] In some examples of any one of formulas (I)-(XVb), the hydrophilic head group X 1 is of formula (XVI):

Chemical formula

[0181] In some embodiments of formula (XVI), Z 6 is absent. In other cases, Z 6 is CH2. In other cases, Z 6 is oxygen. In some embodiments of formula (XVI), Z 7 is oxygen and Z 9 is NR 10 In some cases, Z 7 is NR 10 and Z 9 is oxygen. In some cases, Z 7 and Z 9 are both oxygen. In other cases, Z 7 and Z 9 are both NR 10 In some cases, Z 8 is oxygen. In other cases, Z 8 is sulfur.

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

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

[0184] In some embodiments of formula (XVI), R 8 and R 9 are both hydrogen atoms. In some cases, at least one of R 8 and R 9 is a substituent other than hydrogen. In other cases, R 8 and R 9 are both substituents other than hydrogen. In some cases, at least one of R 8 and R 9 is alkyl or substituted alkyl. In some cases, at least one of R 8 and R 9 is alkenyl or substituted alkenyl. In some other cases, at least one of R 8 and R 9 is aryl or substituted aryl. In some cases, at least one of R 8 and R 9 is acyl or substituted acyl. In some cases, at least one of R 8 and R 9 is heteroaryl or substituted heteroaryl. In some cases, at least one of R 8 and R 9 is cycloalkyl or substituted cycloalkyl. In some cases, R 8 and R 9 are both alkyl groups (e.g., lower alkyl). In some cases, R 8 and R 9Both are substituted alkyl groups (e.g., C substituted by an alkoxy, substituted alkoxy, ester or carboxyl group) (1~6) alkyl). In some cases, R 8 and R 9 at least one of which contains a pro moiety. In certain cases, R 8 and R 9 are both phenyl groups. In some cases, R 8 and R 9 are the same. In other cases, R 8 and R 9 are different.

[0185] In some examples of any one of formulas (I) to (XVb), the hydrophilic head group X 1 is any one of formulas (XVIa) to (XVIf):

Chemical formula

[0186] In some embodiments of formulas (XVIa) to (XVIf), R 10 and R 11 are both hydrogen atoms. In some cases, at least one of R 10 and R 11 is a substituent other than hydrogen. In other cases, both R 10 and R 11 are substituents other than hydrogen. In some cases, R 10 and R 11 are the same. In other cases, R 10 and R 11 are different. In some cases, R 10 and R 11At least one of them is alkyl or substituted alkyl. In some cases, R 10 and R 11 At least one of them is aryl or substituted aryl. In some cases, R 10 and R 11 Both are alkyl or substituted alkyl. In some cases, R 10 and R 11 Both are aryl or substituted aryl. In some cases, R 10 and R 11 Both are acyl or substituted acyl. In some cases, R 10 and R 11 Both are lower alkyl groups. In some cases, R 10 and R 11 Both are substituted alkyl groups (e.g., C (1~6) alkyl substituted by an alkoxy, substituted alkoxy, ester or carboxyl group). In some cases, R 10 and R 11 At least one of them contains a pro moiety. In certain cases, R 10 and R 11 Both are phenyl groups.

[0187] In certain cases of formulae (XVIa) to (XVId), at least one of R 10 and R 11 contains a cleavable group or a self-destructive pro moiety. The self-destructive group can be a disulfide-linked pro moiety or a self-destructive ester-containing pro moiety. In some cases, R 10 and / or R 11 is a disulfide-linked pro moiety of the following formula: -CH2CH2-SS-R 12 (R 12 is alkyl or substituted alkyl) is included. In a specific example, R 12 is a C8-C30 saturated or unsaturated hydrocarbon chain. In some cases, R 10 and / or R 11 is a pro moiety of the following formula: -CH2OCOR 13 (R 13includes (wherein R is H, alkyl or substituted alkyl). In some cases, R 10 and / or R 11 is a pro moiety of the formula: -CH2C(R 14 )2CO2R 14 (wherein each of R 14 is independently H, alkyl or substituted alkyl).

[0188] In some examples of any one of formulas (I) to (XVb), the hydrophilic head group X 1 or its prodrug form is as follows:

Chemical formula

Chemical formula

[0189] In some examples of any one of formulas (I) to (XVb), the hydrophilic head group X 1 is of formula (XVI):

Chemical formula

[0190] In some embodiments of formula (XVI), R 81 and R 91 are both hydrogen atoms. In other cases, R 81 and R 91 are both substituents other than hydrogen.

[0191] In some examples of any one of formulas (I) to (XVb), the hydrophilic head group X 1 is of formula (XVII):

Chemical formula

[0192] In some examples of any one of formulas (I) to (XVb), the hydrophilic head group X 1 is of formula (XVIII):

Chemical formula

[0193] In some embodiments of formula (XVIII), the hydrophilic head group is one of the following groups:

Chemical formula

[0194] In some examples of any one of formulas (I) to (XVb), the hydrophilic head group X 1 is of formula (XIX):

Chemical formula

[0195] In some examples of any one of formulas (I) to (XVb), the hydrophilic head group X 1 is of formula (XX):

Chemical formula

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

Chemical formula

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

Chemical formula

[0198] For any group X in formulas (I)-(XVb) 1 it will be understood that any of the hydroxyl and amine groups may be further substituted, if desired, by any convenient group, such as an alkyl group, a substituted alkyl group, a phenyl group, a substituted phenyl group, an ester group, etc. It will be understood that any convenient alternative hydrophilic group may be utilized as the group X 1 in any of the compounds of formulas (I)-(XVb).

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

Table 1-1

Table 1-2

Table 1-3

Table 1-4

Table 1-5

Table 1-6

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

Table 2-1

Table 2-2

Table 2-3

Table 2-4

Table 2-5

[0201] In certain embodiments, the ENPP1 inhibitor compound is described by one of the structures in Table 3 or its prodrug (e.g., as described herein), or a pharmaceutically acceptable salt thereof.

Table 3-1

Table 3-2

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

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

[0204] In some embodiments, the ENPP1 inhibitor compound of interest or its prodrug form is provided in the form of a pharmaceutically acceptable salt. Compounds containing an amine or nitrogen-containing heteroaryl group are basic in nature and can therefore react with various inorganic and organic acids to form pharmaceutically acceptable acid addition salts. Acids commonly used to form such salts include inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid and phosphoric acid, and organic acids such as para-toluenesulfonic acid, methanesulfonic acid, oxalic acid, para-bromophenylsulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid and acetic acid, and related inorganic and organic acids. Accordingly, such pharmaceutically acceptable salts include sulfate, pyrosulfate, 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 salt, hexyne-1,6-dioate salt, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, terephthalate, sulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, β-hydroxybutyrate, glycolate, maleate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, mandelate, hippurate, gluconate, lactobionate, and similar salts. In certain embodiments, the pharmaceutically acceptable acid addition salts include those formed with inorganic acids such as hydrochloric acid and hydrobromic acid, and those formed with organic acids such as fumaric acid and maleic acid.

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

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

[0207] In some embodiments, the subject compound, its prodrug, stereoisomer or salt is provided in the form of a solvate (e.g., hydrate). As used herein, the term "solvate" refers to a complex or aggregate formed by one or more molecules of a solute, such as a prodrug or a pharmaceutically acceptable salt thereof, and one or more molecules of a solvent. Such solvates are typically crystalline solids having a substantially fixed molar ratio of solute to solvent. Representative solvents include, by way of example, water, methanol, ethanol, isopropanol, acetic acid, and the like. When the solvent is water, the solvate formed is a hydrate.

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

[0209] In some embodiments, the subject compound is metabolically stable (e.g., remains substantially intact in vivo during the half-life of the compound). In certain embodiments, the compound has a half-life (e.g., in vivo half-life) of 5 minutes or longer, such as 10 minutes or longer, 12 minutes or longer, 15 minutes or longer, 20 minutes or longer, 30 minutes or longer, 60 minutes or longer, 2 hours or longer, 6 hours or longer, 12 hours or longer, 24 hours or longer, or even longer. Method for inhibiting ENPP1

[0210] As summarized above, aspects of the present disclosure include ENPP1 inhibitors, and methods of using the same. ENPP1 is a member of the ecto-nucleotide pyrophosphatase / phosphodiesterase (ENPP) family. Accordingly, aspects of the subject methods include inhibiting the hydrolase activity of ENPP1 towards cGAMP. The inventors have discovered that cGAMP can have important extracellular biological functions, which can be enhanced by blocking the extracellular degradation of cGAMP, such as hydrolysis by its degrading enzyme ENPP1. In certain examples, the ENPP1 target of inhibition is extracellular, and the compound that inhibits the ENPP1 of interest is cell non-permeable and thus cannot diffuse into cells. Accordingly, the subject methods can achieve selective extracellular inhibition of the hydrolase activity of ENPP1 and increase the extracellular levels of cGAMP. Thus, in some cases, the ENPP1 inhibitory compound is a compound that inhibits the activity of ENPP1 extracellularly. Experiments conducted by the inventors have shown that inhibiting the activity of ENPP1 can increase extracellular cGAMP and, as a result, promote the STING pathway.

[0211] To inhibit ENPP1 is intended to reduce the activity of this enzyme by 10% or more, such as by more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, more than 95% (e.g., compared to a control in any convenient in vitro inhibition assay). In some cases, inhibition of ENPP1 means reducing the activity of the enzyme to less than one-half fold, such as less than one-third fold, less than one-fifth fold, less than one-tenth fold, less than one-hundredth fold, or less than one-thousandth fold, compared to its normal activity (e.g., compared to a control measured by any convenient assay).

[0212] In one case, the method is a method of inhibiting ENPP1 in a sample. As used herein, the term "sample" generally relates to a substance or mixture of substances in fluid form that contains one or more components of interest, although not necessarily.

[0213] In some embodiments, provided is a method of inhibiting ENPP1, the method comprising contacting a sample with a cell-impermeable ENPP1 inhibitor to inhibit the cGAMP hydrolysis activity of ENPP1. In some cases, the sample is a cell sample. In some cases, the sample contains cGAMP. In certain cases, the cGAMP level is elevated in the cell sample (e.g., compared to a control sample not contacted with the inhibitor). The subject method can result in an elevation of the level of cGAMP. "Elevation of the cGAMP level" is intended to mean a level of cGAMP in a cell sample contacted with a subject compound, wherein the cGAMP level in the sample is 20% or higher, 30% or higher, 40% or higher, 50% or higher, 60% or higher, 70% or higher, 80% or higher, 90% or higher, 100% or higher, or even higher, such as 10% or higher, compared to the cGAMP level in a control sample not contacted with the agent.

[0214] In certain embodiments, the ENPP1 inhibitor is an inhibitor as defined herein. In some embodiments, the ENPP1 inhibitor is an inhibitor according to any one of formulas (I)-(XVb) (e.g., as described herein). In some cases, the ENPP1 inhibitor is any one of the compounds of Tables 1-3 (e.g., as described herein). In some cases, the ENPP1 inhibitor is cell-impermeable.

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

[0216] In some embodiments, the subject compound has an ENPP1 inhibition profile that reflects activity against additional enzymes. In some embodiments, the subject compound specifically inhibits ENPP1 without unwanted inhibition of one or more other enzymes.

[0217] In some embodiments, the compounds of the disclosure interfere with the interaction of cGAMP and ENPP1. For example, the subject compound can act to increase extracellular cGAMP by inhibiting the hydrolase activity of ENPP1 against cGAMP. Without being bound to any particular theory, it is believed that the increase in extracellular cGAMP activates the STING pathway.

[0218] In some embodiments, the subject compound is determined by an inhibition assay, for example, an assay that determines the activity level of an enzyme in either a cell-free system or cells after treatment with the subject compound compared to a control, IC 50 or EC 50 values, respectively, inhibits ENPP1. In certain embodiments, the subject compound has an IC 50 value (or EC 50 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.

[0219] As summarized above, aspects of the present disclosure include methods of inhibiting ENPP1. The subject compounds (e.g., as described herein) can inhibit the activity of ENPP1 in the range of 10% to 100%, e.g., 10% or higher, 20% or higher, 30% or higher, 40% or higher, 50% or higher, 60% or higher, 70% or higher, 80% or higher, or 90% or higher. In certain assays, the subject compounds can inhibit their target at an IC -6 of 1×10 -6 M or less (e.g., 1×10 -7 M or less, 1×10 -8 M or less, 1×10 -9 M or less, 1×10 -10 M or less, or 1×10 -11 M or less). 50 There are numerous protocols that can be used to determine ENPP1 activity, including but not limited to cell-free assays, such as binding assays; assays using purified enzyme, cell assays where the cell phenotype is measured, such as gene expression assays; and in vivo assays involving specific animals (which in certain embodiments may be animal models of conditions associated with a target pathogen).

[0220] In some embodiments, the subject method is an in vitro method that includes contacting a sample with a subject compound that specifically inhibits ENPP1. In certain embodiments, the sample is suspected of containing ENPP1, and the subject method further includes the step of assessing whether the compound inhibits ENPP1.

[0221]

[0222] ​In certain embodiments, the subject compound is a modified compound that includes a label, such as a fluorescent label, and the subject method further includes the step of detecting, if present, the label in a sample, for example, using optical detection.

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

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

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

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

[0227] "STING-mediated response" refers to any response mediated by STING, including, but not limited to, immune responses against 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 is also involved in certain autoimmune diseases initiated by inappropriate recognition of self-DNA (e.g., Gall et al. Immunity (See, e.g., 36: 120-131 (2012)), and functions when inducing adaptive immunity in response to a DNA vaccine (see, e.g., Ishikawa et al. Nature 461: 788-792 (2009)). Increasing the STING-mediated response in a subject is intended to result in an increase in the STING-mediated response in the subject as compared to a control subject (e.g., a subject not receiving administration of the subject compound). In some cases, the subject is human and the subject compound and method result in activation of human STING. In some cases, the STING-mediated response includes modulation of the immune response. In some examples, the subject method is a method of modulating the immune response in a subject.

[0228] In some cases, the STING-mediated response includes an increase in the production of interferons (e.g., type I interferons (IFN), type III interferons (IFN)) in the subject. Interferons (IFN) are proteins having various biological activities, such as antiviral, immunomodulatory, and antiproliferative. IFN are relatively small, species-specific single-chain polypeptides produced by mammalian cells in response to exposure to various inducers such as viruses, polypeptides, mitogens, etc. Interferons are an important host defense mechanism that protects animal tissues and cells from viral attack. Interferons can be classified as type I, type II, and type III interferons. Type I interferons of the mammalian species of interest include IFN-α (alpha), IFN-β (beta), IFN-κ (kappa), IFN-δ (delta), IFN-ε (epsilon), IFN-τ (tau), IFN-ω (omega), and IFN-ζ (zeta, also known as limitin).

[0229] Interferons have been found to be used in the treatment of various cancers because these molecules have anti-cancer activity that acts at multiple levels. Interferon proteins can directly inhibit the growth of human tumor cells. In some cases, the anti-proliferative activity is also synergistic with various approved chemotherapeutic agents such as cisplatin, 5FU, and paclitaxel. The immunomodulatory activity of interferon proteins can also result in the induction of an anti-tumor immune response. This response includes the activation of NK cells, the stimulation of macrophage activity, and the induction of MHC class I surface expression, leading to the induction of anti-tumor cytotoxic T lymphocyte activity. Furthermore, interferon plays a role in the cross-presentation of antigens in the immune system. Additionally, some studies further show that IFN-β protein can have anti-angiogenic activity. Angiogenesis, the formation of new blood vessels, is important for the growth of solid tumors. IFN-β can inhibit angiogenesis by inhibiting the expression of angiogenesis-promoting factors such as bFGF and VEGF. Interferon proteins can also inhibit tumor invasiveness by modulating the expression of enzymes such as collagenase and elastase, which are important in tissue remodeling.

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

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

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

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

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

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

[0236] In some embodiments, an “effective amount” is the amount of a subject compound effective to inhibit ENPP1 by about 20% (20% inhibition), at least about 30% (30% inhibition), at least about 40% (40% inhibition), at least about 50% (50% inhibition), at least about 60% (60% inhibition), at least about 70% (70% inhibition), at least about 80% (80% inhibition), or at least about 90% (90% inhibition), compared to the ENPP1 activity in an individual in the absence of treatment with the compound, or alternatively compared to the ENPP1 activity in an individual before or after treatment with the compound, when one or more doses of the compound are administered to the individual in monotherapy or combination therapy.

[0237] In some embodiments, a “therapeutically effective amount” is the amount of a subject compound effective to reduce the tumor burden in a subject by about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%, compared to the tumor burden in an individual in the absence of treatment with the compound, or alternatively compared to the tumor burden in a subject before or after treatment with the compound, when one or more doses of the compound are administered to the individual in monotherapy or combination therapy. As used herein, the term “tumor burden” refers to the total mass of tumor tissue in a subject having cancer.

[0238] In some embodiments, a “therapeutically effective amount” is the amount of a subject compound effective to reduce the dose of radiation therapy required to observe tumor shrinkage in a subject by about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%, compared to the dose of radiation therapy required to observe tumor shrinkage in an individual in the absence of treatment with the compound, when one or more doses of the compound are administered to the individual in monotherapy or combination therapy.

[0239] In some embodiments, a "therapeutically effective amount" of a compound is an amount effective to achieve a tumor size reduction of 1.5 log, 2 log, 2.5 log, 3 log, 3.5 log, 4 log, 4.5 log, or 5 log when administered to an individual having cancer in one or more doses.

[0240] In some embodiments, an effective amount of the compound is an amount in the range of about 50 ng / ml to about 50 μg / ml (e.g., about 50 ng / ml to about 40 μg / ml, about 30 ng / ml to about 20 μg / ml, about 50 ng / ml to about 10 μg / ml, about 50 ng / ml to about 1 μg / ml, about 50 ng / ml to about 800 ng / ml, about 50 ng / ml to about 700 ng / ml, about 50 ng / ml to about 600 ng / ml, about 50 ng / ml to about 500 ng / ml, about 50 ng / ml to about 400 ng / ml, about 60 ng / ml to about 400 ng / ml, about 70 ng / ml to about 300 ng / ml, about 60 ng / ml to about 100 ng / ml, about 65 ng / ml to about 85 ng / ml, about 70 ng / ml to about 90 ng / ml, about 200 ng / ml to about 900 ng / ml, about 200 ng / ml to about 800 ng / ml, about 200 ng / ml to about 700 ng / ml, about 200 ng / ml to about 600 ng / ml, about 200 ng / ml to about 500 ng / ml, about 200 ng / ml to about 400 ng / ml, or about 200 ng / ml to about 300 ng / ml).

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

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

[0243] Administering a therapeutically effective amount of a subject compound to an individual having cancer results in one or more of the following: 1) a decrease in tumor burden, 2) a decrease in the dose of radiation therapy required to effect tumor shrinkage, 3) a reduction in the spread of cancer from one cell to another in the individual, 4) a decrease in morbidity or mortality in the clinical outcome, 5) a shortening of the total duration of treatment when combined with other anti-cancer agents, and 6) an improvement in the indicators of disease response (e.g., a decrease in one or more symptoms of cancer). Any of the various methods can be used to determine whether the treatment method is effective. For example, a biological sample obtained from an individual treated by the subject method can be assayed.

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

[0245] In some embodiments, the compound specifically inhibits ENPP1. In some embodiments, the compound modulates the activity of cGAMP. In some embodiments, the compound interferes with the interaction between ENPP1 and cGAMP. In some embodiments, the compound results in the activation of the STING pathway.

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

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

[0248] The subject compound can be administered to a subject alone or in addition to, i.e., in combination with, a second active agent. A combination therapy method in which an ENPP1 inhibitor compound of interest can be used in combination with a second active agent or an additional therapy, such as radiation therapy. The terms "agent", "compound" and "drug" are used interchangeably herein. For example, an ENPP1 inhibitor compound can be administered alone or in conjunction with one or more other drugs, such as drugs used in the treatment of a disease of interest, including but not limited to immunomodulatory diseases and conditions, and cancer. In some embodiments, the subject method further comprises the step of co-administering a second agent, such as a small molecule, chemotherapeutic agent, antibody, antibody fragment, antibody-drug conjugate, aptamer, protein or checkpoint inhibitor, either simultaneously or in sequence. In some embodiments, the method further comprises the step of performing radiation therapy on the subject.

[0249] The terms "co-administer" and "in combination with" include either simultaneous, concurrent or sequential administration of two or more therapeutic agents without a specific time limit. In one embodiment, the agents are present in the cell or the subject's body simultaneously or exert their biological or therapeutic effects simultaneously. In one embodiment, the therapeutic agents are present in the same composition or unit dosage form. In other embodiments, the therapeutic agents are present in separate compositions or unit dosage forms. In certain embodiments, the first agent can be administered before (e.g., minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks or 12 weeks before) the administration of the second therapeutic agent, simultaneously with 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 after) the second therapeutic agent.

[0250] "Concurrent administration" of a known therapeutic agent or additional therapy with the pharmaceutical composition of the present disclosure means administering the compound and the second agent or additional therapy at a time such that both the known drug and the composition of the invention have a therapeutic effect. Such concurrent administration can include co-administration (i.e., at the same time), pre-administration, or subsequent administration of the drug with respect to the administration of the subject compound. The routes of administration of the two agents can vary, and in this case, representative routes of administration are described in further detail below. It is not difficult for a person skilled in the art to determine the appropriate timing, sequence, and dosage of administration for a particular drug or therapy and the compounds of the present disclosure.

[0251] In some embodiments, the compounds (e.g., the subject compound and at least one additional compound or therapy) are administered to the subject within 24 hours of each other, within 12 hours of each other, within 6 hours of each other, within 3 hours of each other, or within 1 hour of each other. In certain embodiments, the compounds are administered within 1 hour of each other. In certain embodiments, the compounds are administered substantially simultaneously. Administered substantially simultaneously means that the compounds are intended to be administered to the subject within about 10 minutes or less of each other, such as within 5 minutes or less of each other, or within 1 minute or less of each other.

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

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

[0254] Regarding the treatment of cancer, the ENPP1 inhibitor compound can 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 replacement drugs, thymidylate targeting drugs, chimeric antigen receptor / T cell therapy, chimeric antigen receptor / NK cell therapy, apoptosis regulator inhibitors (e.g., B cell CLL / lymphoma 2 (BCL-2) BCL-2-like 1 (BCL-XL) inhibitors), CARP-1 / CCAR1 (cell division cycle and apoptosis regulator 1) inhibitors, colony stimulating factor 1 receptor (CSF1R) inhibitors, CD47 inhibitors, cancer vaccines (e.g., Th17-inducing dendritic cell vaccines or genetically modified tyrosinase (such as Oncept®)), and other cell therapies.

[0255] Specific chemotherapeutic agents for the purpose include, but are not limited to, gemcitabine, docetaxel, bleomycin, erlotinib, gefitinib, lapatinib, imatinib, dasatinib, nilotinib, bosutinib, crizotinib, ceritinib, trametinib, bevacizumab, sunitinib, sorafenib, trastuzumab, ado-trastuzumab emtansine, rituximab, ipilimumab, rapamycin, temsirolimus, everolimus, methotrexate, doxorubicin, abraxane, folfirinox, cisplatin, carboplatin, 5-fluorouracil, Teysumo, paclitaxel, prednisone, levothyroxine, pemetrexed, navitoclax, and ABT-199. Peptide compounds can also be used. Cancer chemotherapeutic agents for the purpose include, but are not limited to, dolastatin and its active analogs and derivatives; and auristatin and its active analogs and derivatives (e.g., monomethyl auristatin D (MMAD), monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), etc.). See, for example, WO96 / 33212, WO96 / 14856, and U.S. 6,323,315. Suitable cancer chemotherapeutic agents also include maytansinoid and its active analogs and derivatives (e.g., see EP1391213; and Liu et al (1996) Proc. Natl. Acad. Sci. USA 93:8618-8623); duocarmycin and its active analogs and derivatives (e.g., including synthetic analogs, KW-2189, and CB 1-TM1); and benzodiazepine and its active analogs and derivatives (e.g., pyrrolobenzodiazepine (PBD)). See also; duocarmycin and its active analogs and derivatives (e.g., including synthetic analogs, KW-2189, and CB 1-TM1); and benzodiazepine and its active analogs and derivatives (e.g., pyrrolobenzodiazepine (PBD)).

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

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

[0258] Any convenient cancer vaccination therapy and agent can be used in combination with the subject ENPP1 inhibitor compound, composition, and method. In the case of treating cancer, such as ovarian cancer, the ENPP1 inhibitor compound can be administered in combination with a vaccination therapy, such as a dendritic cell (DC) vaccine that promotes Th1 / Th17 immunity. Th17 cell infiltration is associated with a significant extension of overall survival among ovarian cancer patients. In some cases, the ENPP1 inhibitor compound has been found to be useful as an adjuvant therapy in combination with Th17-inducing vaccination.

[0259] Similarly, although not limited to the following, agents that are CARP-1 / CCAR1 (Cell Division Cycle and Apoptosis Regulator 1) inhibitors, including 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, are of interest.

[0260] In certain examples, the combination provides an enhanced effect compared to either component alone. In some cases, the combination achieves an effect greater than additive or a synergistic effect compared to the combined or additive effect of the components. Various combinations of the subject compounds and chemotherapeutic agents may be used, either sequentially or simultaneously. In the case of multiple doses, for example, the two drugs may be directly alternated in order, or multiple doses of one drug may be alternated in order with a single dose of the other drug. Co-administration of both drugs may also be alternated in order, or in other ways, the doses of the individual drugs may be dispersed. In some cases, the time between doses can range from about 1 to 6 hours, to about 6 to 12 hours, to about 12 to 24 hours, to about 1 to 2 days, to about 1 to 2 weeks, or longer after the start of treatment. Combination with cGAMP-inducing chemotherapeutic agents

[0261] Aspects of the present disclosure include methods of treating cancer, which include administering the ENPP1 inhibitor compound (or a pharmaceutical composition comprising such a compound) in combination with a chemotherapeutic agent capable of inducing the production of cGAMP in vivo. Exposure of a subject to an effective amount of a particular chemotherapeutic agent can induce the production of 2’3’-cGAMP in the subject. Co-administration of the ENPP1 inhibitor compound of interest to prevent the degradation of cGAMP can maintain and / or enhance the induced levels of cGAMP, for example, enhanced compared to the levels achieved using either agent alone. Cell death by overwhelming repair or degradation mechanisms can result in DNA damage, such as by alkylating agents, nucleic acid analogs, and intercalating agents, and any convenient chemotherapeutic agent capable of inducing cGAMP production can be used in the subject combination therapy. In some cases, the cGAMP-inducing chemotherapeutic agent is an anti-mitotic agent. Anti-mitotic agents are agents that act by damaging DNA or by binding to microtubules. In some cases, the cGAMP-inducing chemotherapeutic agent is an anti-tumor agent.

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

[0263] The chemotherapeutic agents for the purpose include, but are not limited to, uracil analogs, fluorouracil prodrugs, thymidylate synthase inhibitors, deoxycytidine analogs, DNA synthesis inhibitors (e.g., causing apoptosis in the S phase), folic acid analogs, dihydrofolate reductase inhibitors, anthracyclines, intercalating agents (e.g., causing double-strand breakage), topoisomerase IIa inhibitors, taxanes, microtubule depolymerization inhibitors (e.g., causing G2 / M phase arrest / apoptosis), microtubule assembly inhibitors, microtubule function stabilizers (e.g., causing G2 / M phase apoptosis), tubulin polymerization promoters, tubulin binders (e.g., causing apoptosis by M phase arrest), epothilone B analogs, vinca alkaloids, nitrogen mustard, nitrosourea, DNA alkylating agents (e.g., causing interstrand crosslinking, apoptosis by p53), VEGF inhibitors, anti-angiogenic antibodies, HER2 inhibitors, quinazoline HER2 inhibitors, EGFR inhibitors, tyrosine kinase inhibitors, sirolimus analogs, mTORC1 inhibitors (e.g., in breast cancer, a combination with an aromatase inhibitor that inhibits estrogen production, exemestane), triazenes, dacarbazine prodrugs, methylhydrazine.

[0264] Exemplary chemotherapeutic agents for breast cancer for the purpose include, but are not limited to, capecitabine, carmofur, fluorouracil, tegafur, gemcitabine, methotrexate, doxorubicin, epirubicin, docetaxel, ixabepilone, vindesine, vinorelbine, cyclophosphamide, bevacizumab, pertuzumab, trastuzumab, lap atinib and everolimus. Exemplary anti-neoplastic drugs related to glioma / glioblastoma include, but are not limited to, carmustine, lomustine, temozolomide, procarbazine, vincristine and bevacizumab. Exemplary chemotherapeutic agents for DNA damage for the purpose include, but are not limited to, melphalan, cisplatin and etoposide, fluorouracil, gemcitabine. Combined radiotherapy

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

[0266] In some cases, the method comprises the step of administering an ENPP1 inhibitor to a subject prior to radiation therapy. In some cases, the method comprises the step of administering an ENPP1 inhibitor to a subject after exposing the subject to radiation therapy. In certain cases, the method comprises sequentially administering radiation therapy, then an ENPP1 inhibitor, and then a checkpoint inhibitor to a subject in need thereof. Usefulness

[0267] For example, the compounds and methods of the present invention described herein have been found to have uses in a variety of applications. The intended applications include, but are not limited to, research applications and therapeutic applications. The methods of the present invention are found to have uses in a variety of different applications, including any convenient application where inhibition of ENPP1 is desirable.

[0268] The subject compounds and methods have been found to have uses in a variety of research applications. The subject compounds and methods can be used to optimize the bioavailability and metabolic stability of the compounds.

[0269] The subject compounds and methods have been found to have uses in a variety of therapeutic applications. The intended therapeutic applications include uses in cancer treatment. Thus, the subject compounds have been found to have uses in the treatment of a variety of different conditions where inhibition and / or treatment of cancer in a host is desirable. For example, the subject compounds and methods can be found to have uses in the treatment of solid tumor cancers (e.g., as described herein). Pharmaceutical composition

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

[0271] Pharmaceutically acceptable excipients such as vehicles, adjuvants, carriers or diluents are readily available publicly. Further, pharmaceutically acceptable auxiliary substances such as pH adjusters and buffers, isotonic agents, stabilizers, wetting agents, etc. are readily available publicly.

[0272] In some embodiments, the subject compound is formulated in an aqueous buffer. Suitable aqueous buffers include, but are not limited to, buffers of acetate, succinate, citrate and phosphate with strengths varying from 5 mM to 100 mM. In some embodiments, the aqueous buffer contains a reagent that provides an isotonic solution. Such reagents include, but are not limited to, sodium chloride; and sugars such as mannitol, dextrose, sucrose, etc. In some embodiments, the aqueous buffer further contains a nonionic surfactant such as polysorbate 20 or 80. Optionally, the formulation may further contain a preservative. Suitable preservatives include, but are not limited to, benzyl alcohol, phenol, chlorobutanol, benzalkonium chloride, etc. In many cases, the formulation is stored at about 4 °C. The formulations may also be lyophilized, in which case they generally contain cryoprotective substances such as sucrose, trehalose, lactose, maltose, mannitol, etc. Lyophilized formulations can be stored for a long time even at ambient temperature. In some embodiments, the subject compound is formulated for sustained release.

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

[0274] In another aspect of the invention, there is provided a pharmaceutical composition comprising or consisting essentially of a compound of the invention, or a pharmaceutically acceptable salt, isomer, tautomer or prodrug thereof, further comprising one or more additional active agents for additional purposes. Any convenient active agent can be utilized in the subject methods in conjunction with the subject compound. In some examples, the additive is a checkpoint inhibitor. The subject compound and the checkpoint inhibitor, as well as additional therapeutic agents described herein, for combination therapy can be administered by oral, subcutaneous, intramuscular, nasal, parenteral or other routes. The subject compound and the second active agent, if present, may be administered by the same route of administration or by different routes of administration. The therapeutic agent can be administered by any suitable means including, but not limited to, 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, the therapeutic agent can be administered nasally. In some cases, the therapeutic agent can be administered intratumorally.

[0275] In some embodiments, the subject compound and the chemotherapeutic agent are administered to a subject in a formulation (e.g., the same formulation or separate formulations) comprising a pharmaceutically acceptable excipient(s). Chemotherapeutic agents include, but are not limited to, alkylating agents, nitrosoureas, antimetabolites, antitumor antibiotics, plant (vinca) alkaloids, and steroid hormones. Peptide compounds may also be used. Suitable cancer chemotherapeutic agents include dolastatin and their active analogs and derivatives; and auristatin and their active analogs and derivatives (e.g., monomethyl auristatin D (MMAD), monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), etc.). See, for example, WO96 / 33212, WO96 / 14856, and U.S. 6,323,315. Suitable cancer chemotherapeutic agents also include maytansinoids and their active analogs and derivatives (e.g., see EP1391213; and Liu et al (1996) Proc. Natl. Acad. Sci. USA 93:8618-8623); duocarmycin and their active analogs and derivatives (including, for example, synthetic analogs, KW-2189, and CB 1-TM1); and benzodiazepines and their active analogs and derivatives (e.g., pyrrolobenzodiazepines (PBD)).

[0276] For combination therapy, the subject compound and the second chemotherapeutic agent, as well as the additional therapeutic agents described herein, can be administered orally, subcutaneously, intramuscularly, parenterally, or by other routes. The subject compound and the second chemotherapeutic agent may be administered by the same route of administration or by different routes of administration. The therapeutic agents can be administered by any suitable means, 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.

[0277] The subject compound may be provided in unit dosage form and may be prepared by any method well known in the art. Such methods include combining the subject compound with a pharmaceutically acceptable carrier or diluent that constitutes one or more accessory components. 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 injurious to the subject. This carrier can be either solid or liquid, and the type is generally selected based on the type of administration being used.

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

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

Examples

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

[0281] Although the present invention has been described with reference to specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made without departing from the true spirit and scope of the invention, and equivalents may be substituted. Furthermore, numerous modifications may be made to adapt a particular situation, material, composition of materials, process, process step(s) to the objectives, spirit and scope of the present disclosure. All such modifications are intended to be within the scope of the appended claims.

[0282] (Example 1) Synthesis of Compounds

[0283] The compounds may be synthesized using any convenient method. Methods that may be adapted for use in preparing the compounds of the present disclosure are the exemplary synthetic methods described in Examples 1a - 1c, and the methods described by Li et al. in PCT Application No. PCT / US2018 / 050018, filed September 7, 2018, the entire disclosure of which is incorporated herein by reference. Numerous general references presenting commonly known chemical synthetic schemes and conditions useful for synthesizing the disclosed compounds are also available (e.g., Smith and March, March’s Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, Fifth Edition, Wiley - Interscience, 2001; or Vogel, A Textbook of Practical Organic Chemistry, Including Qualitative Organic Analysis, Fourth Edition, New York: Longman, 1978. See). The reaction can be monitored by thin - layer chromatography (TLC), LC / MS, and the reaction products can be characterized by LC / MS and 1 1H NMR. The intermediates and final products can be purified by silica gel chromatography or by HPLC.

[0284] (Example 1a) Exemplary synthetic scheme - Compound 1

[0285] The synthesis of Compound 1, which may be adapted for use in the preparation of the compounds of this disclosure, is described below;

Chemical formula

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

[0287] To a stirred solution of bis(dimethoxyphosphoryl)methane (11.42 g, 49.19 mmol) in toluene (100 mL) was carefully added sodium hydride (2.16 g, 54.11 mmol) at room temperature. Next, the reaction mixture was placed under a nitrogen atmosphere and a solution of 1-benzylpiperidine-4-carbaldehyde (10 g, 49.19 mmol) in toluene (50 mL) was slowly added while maintaining the temperature below 40 °C. The resulting mixture was stirred at room temperature for 16 h and then quenched by adding saturated aqueous ammonium chloride solution. The organic phase was separated, washed with brine, dried (MgSO4), and evaporated to dryness. Chromatography (120 g SiO2; gradient of 5–100% EtOAc in hexane) gave dimethyl (E)-(2-(1-benzylpiperidin-4-yl)vinyl)phosphonate (6.2 g, 16%) as a colorless oil.

[0288] To a mixture of dimethyl (E)-(2-(1-benzylpiperidin-4-yl)vinyl)phosphonate (3.7 g, 12.0 mmol) in ethanol (40 mL) was added Pd / C (1.1 g, 10.3 mmol). The mixture was placed under a hydrogen atmosphere, stirred at room temperature for 12 h, filtered, and evaporated to dryness under reduced pressure to give dimethyl (2-(piperidin-4-yl)ethyl)phosphonate (2.7 g, 100%) as a colorless oil.

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

[0290] To a mixture of dimethyl(2-(piperidin-4-yl)ethyl)phosphonate (1.1 g, 4.9 mmol) and 4-chloro-6,7-dimethoxyquinazoline (1.0 g, 4.5 mmol) in isopropyl alcohol (20 mL) was added diisopropylethylamine (0.6 g, 8.9 mmol). After stirring at 90 °C for 3 h, the reaction mixture was cooled and evaporated to dryness. Purification by silica gel (5% MeOH in dichloromethane) gave dimethyl(2-(1-(6,7-dimethoxyquinazolin-4-yl)piperidin-4-yl)ethyl)phosphonate (755 mg, 37%) as an oil. LC-MS: m / z = 410.25 [M+H] + 1 H NMR (500 MHz, CDCl3) δ 8.65 (s, 1H), 7.23 (s, 1H), 7.09 (s, 1H), 4.19 (dq, J = 14.0,2.9, 2.4 Hz, 2H), 4.02 (s, 3H), 3.99 (s, 3H), 3.77 (s, 3H), 3.75 (s, 3H), 3.05 (td, J = 12.8,2.3 Hz, 2H), 1.93 - 1.77 (m, 4H), 1.67 (ddd, J = 14.1, 9.5, 5.9 Hz, 3H), 1.46 (qd, J =12.2, 3.7 Hz, 2H).

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

Chemical Structure

[0292] A cooled solution of dimethyl (2-(1-(6,7-dimethoxyquinazolin-4-yl)piperidin-4-yl)ethyl)phosphonate (3.25 g, 7.94 mmol) in chloroform (60 mL), cooled by an ice bath, was added bromotrimethylsilane (3.67 g, 24 mmol). The reaction mixture was warmed to room temperature and quenched after 90 minutes by the addition of methanol (20 mL). The mixture was evaporated to dryness under reduced pressure and then solvated in methanol (100 mL). The reaction mixture was concentrated to half its volume, filtered to remove the precipitate, and then evaporated to dryness. The residue was crystallized from dichloromethane, filtered and dried under vacuum to give dimethyl (2-(1-(6,7-dimethoxyquinazolin-4-yl)piperidin-4-yl)ethyl)phosphonic acid (2.1 g, 69%). LC-MS: m / z = 381.8 [M+H] + 1 H NMR (500 MHz, DMSO-d6) δ 8.77 (s, 1H), 7.34 (s, 1H), 7.23 (s, 1H), 4.71 (d, J = 13.1 Hz, 2H), 3.99 (s, 3H), 3.97 (s, 3H), 3.48 (t, J = 12.7 Hz, 2H), 3.18 (s, 1H), 1.97 - 1.90 (m, 2H), 1.62 - 1.43 (m, 4H), 1.40 - 1.27 (m, 2H).

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

[0294] Compound 5 was prepared using the synthetic scheme described below:

Chemical formula

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

[0296] Compound 6 was prepared using the synthetic scheme described below: [Chemical formula]

[0297] Chemical synthesis: Unless otherwise specified, reactions were carried out under ambient atmosphere. Qualitative TLC analysis was performed on 250 mm thickness, 60 Å, glass-backed, F254 silica (Silicycle, Quebec City, Canada). Visualization was done by exposure to UV light and p-anisaldehyde or KMnO4 staining solution followed by heating. All solvents used were ACS grade Sure-Seal products and all other reagents were used as received unless otherwise specified. The synthesis of commercially unavailable 4-chloroquinazoline and 4-chloro-3-quinoline nitrile is described in the supplementary information together with the amine building blocks. Flash chromatography was performed on a Teledyne Isco purification system using silica gel flash cartridges (SiliCycle® SiliaSep™ 40 - 63 μm, 60 Å). HPLC was performed on an Agilent 1260 Infinity preparative scale purification system using an Agilent PrepHT Zorbax Eclipse XDB-C18 reverse phase column (21.2×250 mm). Structure determination was performed using 1H spectra recorded on a Bruker AV-500 spectrometer and low-resolution mass spectra (ESI-MS) collected on a Shimadzu 20-20 ESI LCMS instrument. Structure determination was performed using 1H spectra recorded on either a Bruker AV-500 or AV-400 spectrometer and low-resolution mass spectra (ESI-MS) collected on a Shimadzu 20-20 ESI LCMS instrument. As determined by HPLC-MS, the purity of the final compound was >95%. The 1H spectra of the final compound 1 were all consistent with the expected structure. 1 were all consistent with the expected structure. 1 The 1H spectra of the final compound were all consistent with the expected structure.

[0298] Synthesis of Ureas 4 and 5.

[0299] [Chemical Formula]

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

[0301] To a solution of 1-(2-(piperidin-4-yl)ethyl)urea 64 (173 mg, 1.01 mmol) in isopropanol (5 mL) under a nitrogen atmosphere were added 4-chloro-6,7-dimethoxyquinazoline 63 (181 mg, 0.81 mmol) and N,N-diisopropylethylamine (391 mg, 3.03 mmol). The mixture was stirred at room temperature for 2 hours and then evaporated to dryness under reduced pressure. Purification (preparative HPLC) gave the title compound 4 (172 mg, 47%) as pale yellow crystals.

[0302] LCMS: [M+H] + m / z 360. 1 H NMR (400 MHz, DMSO-d6) δ 8.49 (s, 1H), 7.17 (s, 1H), 7.07 (s, 1H), 5.92 - 5.90 (m, 1H), 5.36 (br s, 2H), 4.13 - 4.09 (m, 2H), 3.90 (s, 3H), 3.88 (s, 3H), 3.04 - 2.94 (m, 4H), 1.81 - 1.78 (m, 2H), 1.62 - 1.56 (m, 1H) and 1.38 - 1.33 (m, 4H).

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

[0304] To a solution of 1-(piperidin-4-ylmethyl)urea 65 (155 mg, 0.97 mmol) in isopropanol (10 mL) was added 4-chloro-6,7-dimethoxyquinazoline 63 (174 mg, 0.78 mmol) and N,N-diisopropylethylamine (394 mg, 2.9 mmol) under a nitrogen atmosphere. The mixture was stirred at 10 °C for 3 hours and then evaporated to dryness under reduced pressure. The desired product 5 (150 mg, 44%) was obtained as a white solid by chromatography (SiO2: 0 - 6% MeOH in dichloromethane).

[0305] LCMS: [M+H] + m / z 346.0 1 H NMR (400 MHz, methanol-d4) δ 8.44 (s, 1H), 7.14 (s, 1H), 7.12 (s, 1H), 4.28 - 4.24 (m, 2H), 3.96 (s, 3H), 3.94 (s, 3H), 3.13 - 3.07 (m, 4H), 1.94 - 1.87 (m, 3H) and 1.50 - 1.41 (m, 2H).

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

[0307]

Chemical Structure

[0308] 4-Chloro-6,7-dimethoxy-quinazoline 63 (3.14 g, 13.98 mmol) and 3-(4-piperidyl)propanoic acid (2.0 g, 12.72 mmol) were suspended in isopropanol (100 mL) and stirred at 90 °C for 3 hours. Once cooled, the mixture was evaporated to dryness under reduced pressure. The residue was then triturated with CH2Cl2 (20 mL) to afford the title compound 6 (1.87 g, 42%) as a white solid.

[0309] 11H NMR (400 MHz, methanol-d4) δ

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

[0311] [Chemical formula]

[0312] A solution of tert-butyl 4-ethynylpiperidine-1-carboxylate 66 (2.92 g, 13.95 mmol), bis(cyclopentadienyl)zirconium chloride hydride (150 mg, 0.518 mmol) and 4,4,5,5-tetramethyl-1,3,2-dioxaborolane 67 (1.49 g, 11.63 mmol) was stirred at 60 °C for 16 h, then diluted with ether and evaporated to dryness under reduced pressure. 68 (4.2 g, 89%) was obtained by chromatography (SiO2; 2 - 5% ethyl acetate in petroleum ether). A mixture of tert-butyl (E)-4-(2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)vinyl)piperidine-1-carboxylate 68 (4.2 g, 12.46 mmol) and palladium on carbon (840 mg, 20% w / w) in MeOH (500 mL) was placed under a hydrogen atmosphere and stirred at room temperature for 16 h. The mixture was then filtered through a pad of Celite® and then evaporated to dryness under reduced pressure to give 69 (4.2 g, 92%). A solution of 1M aqueous HCl (4 mL) was added to a mixture of 73 (460 mg, 1.36 mmol) in cooled (0 °C) MeOH / hexane (5 mL / 5 mL). The mixture was warmed to room temperature, stirred for 3 h and then evaporated to dryness under reduced pressure to give (2-(piperidin-4-yl)ethyl)boronic acid 70 (180 mg, 68%) as the hydrochloride salt. To a solution of 70 (140 mg, 1.04 mmol) in THF (5 mL) was added 4-chloro-6,7-dimethoxyquinazoline 63 (180 mg, 0.935 mmol), then N,N-diisopropylethylamine (360 mg, 1.87 mmol). The mixture was stirred at 80 °C for 16 h and then evaporated to dryness under reduced pressure. Purification (preparative HPLC) gave the title compound as a pale yellow solid (105 mg; 37%).

[0313] LCMS: [M+H] + m / z 346.3. 1 H NMR (400 MHz, DMSO-d6) δ 8.67 (s, 1H), 7.26 (s, 1H), 7.25 (s, 1H), 4.62 - 4.59 (m, 2H), 3.92 (s, 3H), 3.90 (s, 3H), 3.42 - 3.36 (m, 4H), 2.46 (s, 1H), 1.88 - 1.86 (m, 2h), 1.29 - 1.14 (m, 3H) and 0.60 - 0.56 (m, 2H).

[0314] Preparation of hydroxamic acids 8 and 9.

[0315] [Chemical formula]

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

[0317] A mixture of 4-chloro-6,7-dimethoxyquinazoline 63 (600 mg, 2.68 mmol) and ethyl 2-(piperidin-4-yl)acetate 71 (504 mg, 2.95 mmol) in i-PrOH (6 mL) was stirred at 100 °C for 16 h in a sealed tube. Next, the reaction mixture was concentrated under reduced pressure and the residue was purified by silica gel chromatography to give ethyl 2-(1-(6,7-dimethoxyquinazolin-4-yl)piperidin-4-yl)acetate (750 mg, 77%). To a mixture of ethyl 2-(1-(6,7-dimethoxyquinazolin-4-yl)piperidin-4-yl)acetate (250 mg, 0.696 mmol) in THF (10 mL) was added 2M NaOH solution (1 mL) in H2O. The mixture was stirred at room temperature for 16 h and then quenched by adding 1M HCl solution. The organic phase was extracted with ethyl acetate, washed with brine, dried (Na2SO4), and evaporated to dryness under reduced pressure to give acid 72 (200 mg, 86%) as a white solid.

[0318] To a mixture of acid 72 (300 mg, 0.906 mmol) in THF (10 mL) was added NH₂OH·HCl (76 mg, 1.09 mmol), DIEA (468 mg, 3.63 mmol) and (benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP) (481 mg, 1.09 mmol). The mixture was stirred at room temperature for 16 h, then diluted with water and extracted with ethyl acetate, washed with brine solution, dried (Na₂SO₄) and evaporated to dryness under reduced pressure. The title product 8 (180 mg, 77%) was obtained as a white solid by chromatography (SiO₂, solvent). LCMS: [M+H] + m / z 347.10. 1 H NMR (400 MHz, D₂O) δ 8.42 (s, 1H), 7.13 (s, 1H), 7.00 (s, 1H), 4.68 - 4.62 (m, 2H), 3.95 (s, 3H), 3.91 (s, 3H), 3.51 - 3.45 (m, 2H), 2.21 - 2.15 (m, 3H), 1.93 - 1.0 (m, 2H) and 1.45 - 1.36 (m, 2H). Preparation of 1-(6,7-dimethoxyquinazolin-4-yl)-N-hydroxypiperidine-4-carboxamide 9 (in Table 3a)

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

[0320] LCMS: [M+H] + m / z 333.25. 1 H NMR (400 MHz, D₂O) δ 8.39 (s, 1H), 7.04 (s, 1H), 6.94 (s, 1H), 4.62 - 4.58 (m, 2H), 3.91 (s, 3H), 3.86 (s, 3H), 3.47 - 3.41 (m, 2H), 2.65 - 2.60 (m, 1H), 1.97 - 1.94 (m, 2H) and 1.82 - 1.77 (m, 2H).

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

[0322]

Chem.

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

[0324] A mixture of 4-chloro-6,7-dimethoxyquinazoline 63 (1.0 g, 4.46 mmol) and piperidin-4-ylethanol 79 (633 mg, 4.91 mmol) in isopropanol (10 mL) was stirred at 100 °C for 16 h in a sealed tube. Upon cooling, the reaction mixture was concentrated under reduced pressure and the residue was purified by silica gel chromatography (SiO2; EtOAc in petroleum ether) to afford 2-(1-(6,7-dimethoxyquinolin-4-yl)piperidin-4-yl)ethan-1-ol 75 (1.3 g, 91%).

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

[0326] A mixture of 4-chloro-6,7-dimethoxyquinazoline 63 (900 mg, 4.02 mmol) and piperidin-4-ylmethanol 76 (508 mg, 4.42 mmol) in i-PrOH (10 mL) was stirred at 100 °C for 16 h in a sealed tube. Upon cooling, the reaction mixture was evaporated to dryness under reduced pressure. Purification by chromatography (SiO2; 10 - 80% ethyl acetate in petroleum ether) gave (1-(6,7-dimethoxyquinolin-4-yl)piperidin-4-yl)methanol 78 (1 g, 82%).

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

[0328] 2-(1-(6,7-Dimethoxyquinazolin-4-yl)piperidin-4-yl)ethan-1-ol 77 (340 mg, 1.07 mmol) was dissolved in 10 mL of dry pyridine, and then it was cooled to -15 °C and stirred for 10 minutes. POCl3 (821 mg, 5.4 mmol) was added dropwise under a N2 atmosphere. The reaction temperature was slowly raised to 0 °C and then stirred again for 30 minutes. The mixture was poured into a sodium bicarbonate solution (800 mg in 250 mL of water) at 0 °C. The desired compound was extracted with dichloromethane. The organic phase was concentrated and purified using preparative HPLC to give 2-(1-(6,7-Dimethoxyquinazolin-4-yl)piperidin-4-yl)ethyl dihydrogen phosphate 10 (52 mg, 12%) as a white solid.

[0329] LCMS: [M+H] + m / z 398. 1 H NMR (400 MHz, DMSO-d6) δ 8.54 (s, 1H), 7.17 (s, 1H), 7.15 (s, 1H), 4.28 - 4.16 (m, 2H), 3.93 (s, 8H), 3.13 - 3.04 (m, 2H), 1.90 - 1.80 (m, 2H), 1.75 (s, 1H), 1.59 (d, J = 6.4 Hz, 2H) and 1.44 - 1.32 (m, 2H).

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

[0331] (1-(6,7-Dimethoxyquinolin-4-yl)piperidin-4-yl)methanol 78 (100 mg, 0.33 mmol) was dissolved in dry pyridine (3 mL), then it was cooled to -15 °C and stirred for 10 minutes. POCl3 (253 mg, 1.65 mmol) was added dropwise under a nitrogen atmosphere. The reaction temperature was slowly raised to 0 °C and then stirred for an additional 30 minutes. The mixture was poured into an aqueous NaHCO3 solution (160 mg in 50 mL of water) at 0 °C. The desired compound was extracted with dichloromethane and then evaporated to dryness under reduced pressure. Purification by preparative HPLC gave (1-(6,7-dimethoxyquinolin-4-yl)piperidin-4-yl)methyl dihydrogen phosphate 11 (70 mg, 55%) as a white powder after lyophilization. LCMS: [M+H] + m / z 384.20. 1 H NMR (400 MHz, DMSO-d6) δ 8.74 (d, J = 1.7 Hz, 1H), 7.31 (s, 1H), 7.20 (s, 1H), 4.66 (d, J = 13.0 Hz, 1H), 3.97 (m, J = 12.6, 1.6 Hz, 8H), 3.76 (t, J = 6.6 Hz, 3H), 2.19 - 2.00 (m, 1H), 1.92 (d, J = 13.5 Hz, 2H), 1.45 (dd, J = 14.2, 10.7 Hz, 1H).

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

[0333] To a solution of 2-(1-(6,7-dimethoxyquinolin-4-yl)piperidin-4-yl)ethan-1-ol 77 (150 mg, 0.473 mmol) in dry pyridine (5 mL) was added P(S)Cl3 (477 mg, 2.84 mmol) at -15 °C. The mixture was stirred at 0 °C for 0.5 h and then poured into a solution of NaHCO3 (238 mg, 2.84 mmol) in H2O (50 mL). The mixture was stirred at 0 °C for 2 h. The progress of the reaction mixture was monitored by LCMS. Next, the mixture was concentrated under reduced pressure and the residue was purified by preparative HPLC to give compound 12 (16 mg, 8%) as a bright yellow solid. LCMS: [M+H] + m / z 414.05. 1 H NMR (400 MHz, DMSO-d6) δ 8.62 (s, 1H), 7.19 (d, J = 7.7 Hz, 2H), 4.45 (d, J = 12.3 Hz, 2H), 3.91 (d, J = 11.3 Hz, 10H), 1.86 (d, J = 12.2 Hz, 3H), 1.56 (d, J = 6.4 Hz, 2H), 1.34 (d, J = 10.7 Hz, 2H).

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

[0335] To a solution of (1-(6,7-dimethoxyquinolin-4-yl)piperidin-4-yl)methanol 78 (100 mg, 0.330 mmol) in dry pyridine (5 mL) was added P(S)Cl3 (280 mg, 1.98 mmol) at -15 °C. The mixture was stirred at 0 °C for 0.5 h and then poured into a solution of NaHCO3 (116 mg, 1.98 mmol) in H2O (50 mL). The mixture was stirred at 0 °C for 2 h. The mixture was evaporated to dryness under reduced pressure and the residue was purified by preparative HPLC to give compound 13 (10 mg, 7.6%) as a yellow solid. LCMS: [M+H] +m / z 400.15。 1 H NMR (400 MHz, DMSO-d6) δ 8.54 (s, 1H), 7.18 (s, 1H), 7.11 (s, 1H), 4.25 (d, J = 13.4 Hz, 2H), 3.89 (d, J = 9.1 Hz, 6H), 3.76 (s, 2H), 3.10 (d, J = 11.8 Hz, 3H), 1.94 (s, 1H), 1.81 (d, J = 12.7 Hz, 2H), 1.39 (d, J = 11.4 Hz, 1H).

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

[0337] PPh3 (3.39 g, 15 mmol) and imidazole (1.02 g, 15 mmol) in anhydrous CH2Cl2 (40 mL) were stirred at 0 °C for 10 minutes, then I2 (3.8 g, 15 mmol) was added. The crude reaction mixture was placed under a nitrogen atmosphere and stirred for an additional 10 minutes, then (1-(6,7-dimethoxyquinazolin-4-yl)piperidin-4-yl)methanol 78 (3.03 g, 10 mmol) was added. The reaction mixture was stirred at room temperature overnight. The reaction was quenched by the addition of aqueous Na2S2O3. The crude mixture was extracted with CH2Cl2, washed with water and brine, dried (Na2SO4), and evaporated to dryness under reduced pressure. Recrystallization from methanol gave 4-(4-(iodomethyl)piperidin-1-yl)-6,7-dimethoxyquinazoline (2.28 g, 56%) as a bright yellow solid. LCMS: [M+H] + m / z 414.3。 1 H NMR (400 MHz, CDCl3) δ 8.63 (d, J = 1.3 Hz, 1H), 7.28 (s, 1H), 7.07 (s, 1H), 4.23 (s, 2H), 4.00 (s, 6H), 3.19 (d, J = 6.5 Hz, 2H), 3.08 (s, 2H), 2.11 - 2.00 (m, 2H), 1.82 (s, 1H), 1.49 (s, 2H), 1.29 - 1.20 (m, 1H). In anhydrous MeCN (40 mL), a cooled (0 °C) solution of bis(benzyloxy)(oxo)-λ4 -phosphane (9.5 g, 36.3 mmol) was added 1,8-diazabicyclo(5.4.0)undec-7-ene (DBU) (9.2 g, 60.5 mmol). After 10 minutes, 4-(4-(iodomethyl)piperidin-1-yl)-6,7-dimethoxyquinazoline (5.0 g, 12.1 mmol) was added. The resulting mixture was stirred overnight and then evaporated to dryness under reduced pressure. The residue was dissolved in ethyl acetate, washed with water and brine, dried (MgSO4), and evaporated to dryness under reduced pressure. Purification by FCC [CH2Cl2:MeOH (50:1)] gave dibenzyl ((1-(6,7-dimethoxyquinazolin-4-yl)piperidin-4-yl)methyl)phosphonate (1.1 g, 18%) as a colorless viscous oil. LCMS: [M + H] + m / z 548.20. 1 H NMR (400 MHz, CDCl3) δ 8.57 (s, 1H), 7.89 (s, 1H), 7.39 - 7.33 (m, 10H), 6.99 (s, 1H), 5.08 (m, 3H), 4.96 (m, 2H), 4.64 (d, J = 13.5 Hz, 2H), 4.09 (s, 3H), 3.93 (s, 3H), 3.27 (d, J = 12.9 Hz, 2H), 2.05 (d, J = 13.9 Hz, 5H), 1.76 (m, 4H), 1.42 (d, J = 12.5 Hz, 2H).

[0338] A mixture containing dibenzyl ((1-(6,7-dimethoxyquinazolin-4-yl)piperidin-4-yl)methyl)phosphonate (660 mg, 1.2 mmol) and Pd / C (132 mg, 20% w / w) in MeOH (20 mL) was placed under an H2 atmosphere and stirred at room temperature. After 4 hours, the crude mixture was filtered through a Celite® pad and the filtrate was evaporated to dryness under reduced pressure. Purification by preparative HPLC gave ((1-(6,7-dimethoxyquinazolin-4-yl)piperidin-4-yl)methyl)phosphonic acid 16 (125 mg, 28%) as a pale yellow solid. LCMS: [M+H] + m / z 368.10. 1 H NMR (400 MHz, DMSO-d6) δ 8.72 (s, 1H), 7.29 (s, 2H), 4.60 (d, J = 12.8 Hz, 2H), 3.95 (d, J = 11.2 Hz, 6H), 3.46 (s, 2H), 2.09 (s, 3H), 1.61 (s, 2H), 1.42 (s, 2H).

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

[0340]

Chemical Structure

[0341] To a solution of PPh3 (1.4 g, 5.34 mmol) and imidazole (0.36 g, 5.34 mmol) in CH2Cl2 (20 mL) was added iodine (1.35 g, 5.34 mmol). The mixture was stirred at room temperature for 0.5 h, and then a solution of 79 (1.0 g, 4.11 mmol) in CH2Cl2 (5 mL) was added dropwise. The reaction mixture was stirred at room temperature for 4 h, then quenched with saturated Na2SO3 solution and extracted with CH2Cl2. The organic phase was washed with water and brine, dried (Na2SO4), and evaporated to dryness under reduced pressure. Chromatography (SiO2; 5% EtOAc in petroleum ether) gave tert-butyl 4-(3-iodopropyl)piperidine-1-carboxylate (1.0 g, 68% yield) as a pale yellow oil.

[0342] To a mixture of tert-butyl 4-(3-iodopropyl)piperidine-1-carboxylate (1.0 g, 2.83 mmol) in DMF (50 mL) were added diethyl phosphonate (0.58 g, 4.24 mmol) and Cs2CO3 (1.84 g, 5.66 mmol). The reaction mixture was stirred at room temperature overnight under a nitrogen atmosphere, and then quenched by the addition of water. The organic phase was washed with water and brine, dried (N a2SO4), and evaporated to dryness under reduced pressure. Chromatography (SiO2; 20% EtOAc in petroleum ether) gave 80 (0.78 g, 76%) as a pale yellow oil. LCMS: [M+H] + m / z 364.30.

[0343] To a solution of 80 (0.78 g, 2.14 mmol) in CH2Cl2 (8 mL) was added TFA (1.5 mL, 21.4 mmol). The mixture was stirred at room temperature for 4 h and then evaporated to dryness under reduced pressure to give crude 81 as an oil, which was used in the next step without further purification. LCMS: [M+H] + m / z 264.25.

[0344] To a solution of diethyl phosphonate (597 mg, 2.65 mmol) and crude 81 in CH2Cl2 (10 mL) was added DIPEA (1.37 g, 10.63 mmol). The mixture was stirred at room temperature overnight, then quenched with saturated aqueous NH4Cl and extracted with CH2Cl2. The organic phase was washed with water and brine, dried (Na2SO4), and evaporated to dryness under reduced pressure. The diethyl phosphonate (0.5 g, 39%) intermediate was obtained as a yellow oil by chromatography (SiO2, 5% MeOH in CH2Cl2). This was solvated in MeCN (10 mL) and TMSBr (1.46 mL, 11.07 mmol) was added. The resulting mixture was stirred at 60 °C for 6 h, then cooled to room temperature and evaporated to dryness under reduced pressure. By chromatography (preparative HPLC) 14 (220 mg, 50%) was obtained as a white solid. LCMS: [M+H] + m / z 396.20. 1 H NMR (400 MHz, methanol-d4) δ 8.51 (s, 1H), 7.33 (s, 1H), 7.14 (s, 1H), 4.02 (s, 3H), 3.97 (s, 3H), 3.49 (t, J = 12 Hz, 2H), 2.00 - 1.97 (m, 3H), 1.75 - 1.66 (m, 5H) and 1.45 - 1.37 (m, 5H).

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

[0346]

Chemical Structure

[0347] To a stirred solution of O,O-diethyl (2-(piperidin-4-yl)ethyl)phosphonothioate (400 mg, 1.51 mmol) and DIPEA (927 mg, 7.19 mmol) in DMSO (10 mL) was added 4-chloro-6,7-dimethoxy-quinazoline 66 (403 mg, 1.80 mmol). The reaction mixture was placed under a nitrogen atmosphere and then stirred at 80 °C for 16 h. The reaction mixture was cooled to room temperature, diluted with water and extracted with ethyl acetate. The organic phase was dried (Na2SO4) and evaporated to dryness under reduced pressure. Purification (SiO2, 0 - 100% EtOAc in hexane) gave O,O-diethyl (2-(1-(6,7-dimethoxyquinazolin-4-yl)piperidin-4-yl)ethyl)phosphonothioate (380 mg, 46%). A stirred solution of O,O-diethyl (2-(1-(6,7-dimethoxyquinazolin-4-yl)piperidin-4-yl)ethyl)phosphonothioate (45 mg, 0.099 mmol) in TMSI (7 mL) was stirred at 60 °C for 16 h and then cooled to room temperature. The mixture was diluted with water and extracted with ethyl acetate. The organic phase was dried (Na2SO4) and then evaporated to dryness under reduced pressure. Purification by preparative HPLC gave (2-(1-(6,7-dimethoxyquinazolin-4-yl)piperidin-4-yl)ethyl)phosphonothioic acid O,O-17 (13 mg, 32%) as a white solid. LCMS: [M+H] + m / z 396.25. 1 H NMR (400 MHz, DMSO-d6) δ 8.51 (s, 1H), 7.33 (s, 1H), 7.16 (s, 1H), 4.02 (s, 3H), 3.97 (s, 3H), 3.58 (d, J = 10.4 Hz, 3H), 3.48 (t, J = 12.0 Hz, 2H), 2.00 (d, J = 11.7 Hz, 2H), 1.81 (s, 1H), 1.64 (d, J = 17.9 Hz, 2H), 1.61 - 1.51 (m, 2H), 1.45 - 1.32 (m, 2H).

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

[0349]

Chem.

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

[0351]

Chem.

[0352] A mixture containing pyrazine 82 and compound 63 in propan-2-ol was heated to reflux for 30 min and then cooled to room temperature. The reaction mixture was quenched with water and extracted into chloroform. The organic phase was separated, washed with water, brine, dried (Na2SO4) and evaporated to dryness under reduced pressure. Crystallization from diethyl ether afforded 83 (1.65 g, 84%) as a white solid. Piperazine 83 (0.31 g, 1.1 mmol) was dissolved in water (20 mL) and vinyl phosphonate 84 (0.19 g, 1.2 mmol) was added. The resulting mixture was heated at 50° C. for 1 h and then cooled to room temperature. Extraction with chloroform, drying (Na2SO4) and evaporation to dryness under reduced pressure. Chromatography (12 g of SiO2; 15% MeOH in CH2Cl2) followed by crystallization from diethyl ether afforded the ethyl ester 85 (0.23 g; 49%) as a white solid. LCMS: [M+H] + m / z 410.10 1 H NMR (500 MHz, chloroform m-d) δ 8.67 (s, 1H), 7.25 (s, 1H), 7.09 (s, 1H), 4.01 (d, J = 18.8 Hz, 6H), 3.77 (d, J = 10.9 Hz, 6H), 3.68 (t, J = 4.9 Hz, 4H), 3.49 (s, 2H), 2.81-2.66 (m, 6H), 2.11-2.00 (m, 2H).To a solution of 4-[4-(2-diethoxyphosphorylethyl)piperazin-1-yl]-6,7-dimethoxy-quinazoline 85 (600 mg, 3.8 mmol) in chloroform (20 mL) and DMF (5 mL) was added trimethylsilyl bromide (198 mg, 1.3 mmol). The resulting solution was stirred at room temperature for 3 h and then quenched by adding methanol. The mixture was evaporated to dryness under reduced pressure and crystallized from methanol-diethyl ether to give the desired product 19 (0.23 g, 89%) as the HBr salt. LCMS: [M+H] + m / z 382.8. 1 H NMR (500 MHz, DMSO-d6) δ 8.97 (s, 1H), 7.96 (s, 1H), 7.42 (s, 1H), 7.35 (s, 1H), 4.02 (s, 3H), 4.00 (s, 3H), 3.34 (t, J = 8.6 Hz, 2H), 3.17 (s, 2H), 2.90 (s, 2H), 2.74 (s, 2H), 2.20 - 2.08 (m, 2H).

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

[0354] [Chemical formula]

[0355] To a solution of 2-(4-bromophenyl)ethan-1-ol 86 (5.0 g, 24.8 mmol) in anhydrous THF (100 mL) was added 2.5 M n-butyllithium (24 mL) at -78 °C under a nitrogen atmosphere. After stirring for 1 hour, triisopropyl borate (8.6 mL) was added to this mixture. The reaction mixture was stirred at room temperature for 1 hour and then quenched by adding 2 M HCl solution (100 mL) and stirred for 1 hour. The mixture was extracted with dichloromethane (3 × 100 mL), dried (Na2SO4), and evaporated to dryness under reduced pressure. Boronic acid 87 (1.34 g, 33%) was obtained as a bright yellow solid by chromatography (SiO2; dichloromethane:methanol, 1:0 - 20:0). Next, this substance was dissolved in a solution of THF (30 mL) and water (10 mL). To this solution were added 4-chloro-6,7-dimethoxyquinazoline 63 (2.24 g, 10.0 mmol), potassium carbonate (2.76 g, 20.0 mmol), and then tetrakis(triphenylphosphine)palladium (0.5 g, 0.43 mmol). The resulting mixture was stirred at 65 °C for 16 hours, then diluted with ethyl acetate, washed with brine, dried (Na2SO4), and evaporated to dryness under reduced pressure. 88 (1.43 g, 60%) was obtained as a bright yellow solid by chromatography (SiO2:methanol in dichloromethane 0 - 10%).

[0356] To a solution of triphenylphosphine (2.36 g, 9.0 mmol) in dichloromethane (24 mL) was added imidazole (700 mg, 10.28 mmol) at 0 °C. After stirring for 10 minutes, I2 (2.3 g, 9.0 mmol) was added. After further stirring for 10 minutes, compound 89 (1.5 g, 4.8 mmol) in dichloromethane (12 mL) was added. The mixture was warmed to room temperature and stirred for 5 hours. Next, the mixture was diluted with dichloromethane (36 mL), washed with brine and dried (Na2SO4), and evaporated to dryness under reduced pressure. 89 (4.0 g) was obtained as a colorless oil by chromatography (SiO2: petroleum ether: ethyl acetate 10:1).

[0357] To a mixture of crude 89 (930 mg, 2.2 mmol) and dibenzyl phosphonate (884 mg, 3.37 mmol) in DMF (20 mL) was added cesium carbonate (1.426 g, 4.4 mmol). The mixture was placed under a nitrogen atmosphere and stirred at room temperature for 3 hours. Once complete, the reaction mixture was filtered and evaporated to dryness under reduced pressure. Chromatography (C18 column: water: acetonitrile, 1:0 to 80:1), followed by lyophilization, gave the dibenzyl intermediate (750 mg, 79%) as an off-white solid. Dibenzyl (4-(6,7-dimethoxyquinazolin-4-yl)phenethyl)phosphonate (230 mg, 0.41 mmol) was dissolved in MeOH (20 mL). Pd / C (46 mg, 20% w / w) was added and the mixture was stirred under a hydrogen atmosphere at room temperature for 24 hours and then filtered through Celite (registered trademark). Compound 20 (55.4 mg, 36%) was obtained as a yellow solid by chromatography (preparative HPLC, under acidic conditions). LCMS: [M+H] + m / z 375.0。 1 H NMR (400 MHz, DMSO-d6)): δ 9.09 (s, 1H), 7.75 - 7.73 (d, J = 8.0 Hz, 2H), 7.45 - 7.43 (m, 2H), 7.41 (s, 1H), 7.32 (s, 1H), 4.08 (s, 1H), 3.98 (s, 3H), 3.91 (s, 1H), 3.81 (s, 3H), 2.89 - 2.87 (m, 3H) and 1.89 (m, 2H).

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

[0359]

Chemical formula

[0360] A mixture of 4 - chloro - 6,7 - dimethoxy - quinazoline 67 (0.67 g, 3.0 mmol) and diethyl (4 - aminophenyl)phosphonate (0.69 g, 3.0 mmol) in iPrOH (10 mL) was heated to reflux overnight. The solid precipitate was filtered, washed with EtOAc and dried to give diethyl (4 - ((6,7 - dimethoxyquinazolin - 4 - yl)amino)phenyl)phosphonate (0.92 g, 73% yield) as a white solid. This product was dissolved in MeCN (20 mL), to which trimethylsilyl bromide (2.8 mL, 22 mmol) was added. The resulting mixture was stirred at 60 °C for 6 h, cooled and then evaporated to dryness under reduced pressure. The crude residue was quenched by the addition of saturated aqueous NaHCO3 (adjusted to pH 8). The resulting mixture was purified by preparative HPLC (under neutral conditions) and then lyophilized to give the desired product 21 (300 mg, 35% yield) as an off - white solid. LCMS: [M + H] + m / z 362.10。 1 H NMR (400 MHz, D2O) δ 7.73 (s, 1H), 7.53 (t, J = 9.8 Hz, 2H), 7.22 (d, J = 7.4 Hz, 2H), 6.39 (s, 1H), 6.16 (s, 1H) and 3.39 (s, 6H).

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

[0362]

Chemical formula

[0363] A mixture of 4-chloro-6,7-dimethoxy-quinazoline 67 (0.34 g, 1.5 mmol) and diethyl (4-aminobenzyl)phosphonate (0.36 g, 3.0 mmol) in iPrOH (10 mL) was heated to reflux overnight. The precipitate was filtered, washed with EtOAc, evaporated to dryness under reduced pressure, and then dissolved in acetonitrile (20 mL). Trimethylsilyl bromide (0.58 mL, 4.6 mmol) was added thereto. The mixture was stirred at 60 °C for 6 hours. After concentration, the residue was treated with saturated aqueous NaHCO3 until the solution reached pH 8. Purification of this mixture by preparative HPLC (neutral) gave 22 (104 mg, 57%) as an off-white solid. LCMS: [M+H] + m / z 376.10。 1 H NMR (400 MHz, D2O) δ 7.77 (s, 1H), 7.15 (s, 4H), 6.49 (s, 1H), 6.21 (s, 1H), 3.47 (s, 6H) and 2.70 (d, J = 19.5 Hz, 2H).

[0364] (Sodium (4-(((6,7-dimethoxyquinolin-4-yl)amino)methyl)phenyl)phosphonate 23 (also referred to as 4 in Table 1)).

[0365]

Chemical formula

[0366] A mixture of 4-chloro-6,7-dimethoxyquinazoline 63 (0.93 g, 4.14 mmol) and (4-bromophenyl)methanamine 90 (0.77 g, 4.14 mmol) in iPrOH (10 mL) was heated to reflux overnight. The precipitated solid was filtered, washed with ethyl acetate, and evaporated to dryness under reduced pressure to give N-(4-bromobenzyl)-6,7-dimethoxyquinazolin-4-amine hydrochloride 91 (1.5 g, 88%) as a white solid. To a mixture of KOAc (11 mg, 0.112 mmol), Pd(OAc)2 (5.5 mg, 0.025 mmol), and dppf (27 mg, 0.049 mmol) in THF (10 mL) was added triethylamine (0.37 mL, 2.68 mmol), and the mixture was purged with nitrogen. Triethylamine (0.37 mL, 2.68 mmol) was added. After stirring at 70 °C for 15 minutes, a solution of N-(4-bromobenzyl)-6,7-dimethoxyquinazolin-4-amine hydrochloride (0.5 g, 1.22 mmol) and diethyl phosphonate (0.16 g, 1.22 mmol) in THF (10 mL) was added. The reaction mixture was refluxed and stirred for 6 hours and then partitioned between EtOAc (30 mL) and water (20 mL). The organic phase was separated, washed with water and brine, dried (Na2SO4), and evaporated to dryness under reduced pressure. Purification by column chromatography (SiO2; 50% petroleum ether in ethyl acetate) gave diethyl (4-(((6,7-dimethoxyquinazolin-4-yl)amino)methyl)phenyl)phosphonate (0.2 g, 38%) as a yellow solid.

[0367] To a solution of diethyl (4-(((6,7-dimethoxyquinazolin-4-yl)amino)methyl)phenyl)phosphonate (0.5 g, 1.16 mmol) in MeCN (20 mL) was added TMSBr (1.45 mL, 11.5 mmol). The mixture was stirred at 60 °C for 6 hours, cooled to room temperature, and then evaporated under reduced pressure. The residue was quenched with saturated aqueous NaHCO3 (pH 9), and the resulting mixture was purified by preparative HPLC (neutral) to give the title product 23 (102 mg, 22%) as an off-white solid. LCMS: [M-H]+ m / z: 374.00。 1 H NMR (400 MHz, D2O) δ 8.00 (s, 1H), 7.61 (s, 2H), 7.29 (d, J = 7.6 Hz, 2H), 6.70 (s, 1H), 6.55 (s, 1H), 4.62 (s, 2H), 3.75 (d, J = 18.2 Hz, 6H).

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

Chemical formula

[0369] To a stirred solution of bis(dimethoxyphosphoryl)methane 92 or bis(diethoxyphosphoryl)methane 93 (1 mol. equivalent) in toluene, sodium hydride (1.1 mol. equivalent) is carefully added at room temperature. Next, the reaction mixture is placed under a nitrogen atmosphere and a solution of 1-benzylpiperidine-4-carbaldehyde 94 (1 mol. equivalent) in toluene is slowly added while maintaining the temperature below 40 °C. The resulting mixture is stirred at room temperature for 16 h and then quenched by the addition of saturated aqueous NH4Cl. The organic phase is separated, washed with brine, dried (MgSO4), and evaporated to dryness. Dimethyl or diethyl (E)-(2-(1-benzylpiperidin-4-yl)vinyl)phosphonate is obtained as a colorless oil by chromatography (120 g SiO2; gradient of 5 - 100% EtOAc in hexane). To a mixture of dimethyl or diethyl (E)-(2-(1-benzylpiperidin-4-yl)vinyl)phosphonate (1 mol. equivalent) in ethanol, catalytic Pd / C is added. The mixture is placed under a hydrogen atmosphere, stirred at room temperature for 12 h, filtered, and evaporated to dryness under reduced pressure to obtain either dimethyl or diethyl (2-(piperidin-4-yl)ethyl)phosphonate 95 and 96 as colorless oils.

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

[0371]

Chemical formula

[0372] Iodine (1.5 mol equivalent) was added to a CH2Cl2 solution of PPh3 (1.5 mol equivalent) and imidazole (1.5 mol equivalent). After stirring for 10 minutes, a CH2Cl2 solution of 97 (1.0 mol equivalent) was added dropwise. The mixture was stirred at room temperature for 2 hours, filtered through a Celite® pad, and treated with a 5% sodium thiosulfate solution. The mixture was extracted with ethyl acetate, washed with brine, dried (Na2SO4), and evaporated to dryness under reduced pressure. 98 was obtained as an oil by chromatography.

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

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

[0375] (2-(1-(quinazolin-4-yl)piperidin-4-yl)ethyl)phosphonic acid, (2-(1-(quinolin-4-yl)piperidin-4-yl)ethyl)phosphonic acid and (2-(1-(isoquinolin-1-yl)piperidin-4-yl)ethyl)phosphonic acid general method of synthesis.

[0376] Method A

[0377] A mixture (reaction concentration 0.1 M) of dimethyl(2-(piperidin-4-yl)ethyl)phosphonate 95 or diethyl(2-(piperidin-4-yl)ethyl)phosphonate 96 (1.1 mol. equivalent) and either 4-chloroquinazoline, 4-chloroquinoline or 1-chloroisoquinoline (1 mol. equivalent) was added to diisopropylethylamine (2 mol. equivalent). After stirring at 90 °C for 3 h, the reaction mixture was cooled and evaporated to dryness. Purification on silica gel (5% MeOH in dichloromethane) gave dimethylphosphonate or diethylphosphonate. To a cooled (0 °C) solution of phosphonate (1 mol. equivalent) in chloroform or dichloromethane (reaction concentration 0.5 M) was added trimethylsilyl bromide (3 mol. equivalent). The reaction mixture was warmed to room temperature and quenched after 90 min by addition of methanol. The mixture was evaporated to dryness under reduced pressure and then solvated in methanol. The reaction mixture was concentrated to half volume, filtered to remove precipitate and then evaporated to dryness. The residue was crystallized from dichloromethane, filtered and dried under reduced pressure to give the desired phosphonic acid as the bromide salt.

[0378] Method B:

[0379] A mixture (reaction concentration 0.1 M) of dimethyl(2-(piperidin-4-yl)ethyl)phosphonate 95 or diethyl(2-(piperidin-4-yl)ethyl)phosphonate 96 (1.1 mol. equivalent) and any one of 4-chloroquinazoline, 4-chloroquinoline or 1-chloroisoquinoline (1 mol. equivalent) was added with diisopropylethylamine (3 mol. equivalent). After stirring overnight at room temperature, the reaction mixture was quenched by the addition of saturated aqueous NH4Cl solution. The organic phase was separated, washed with water and brine, dried (Na2SO4) and evaporated to dryness under reduced pressure. Purification by silica gel (5% MeOH in dichloromethane) gave dimethylphosphonate or diethylphosphonate. To a cooled (0 °C) solution (reaction concentration 0.1 M) of dimethylphosphonate or diethylphosphonate (7 mol. equivalent) in acetonitrile was added trimethylsilyl bromide (3 mol. equivalent). The reaction mixture was stirred at 60 °C for 6 hours, cooled and evaporated to dryness under reduced pressure, and the crude residue was quenched by the addition of saturated aqueous NaHCO3 solution (until pH 8 - 9 was observed). Purification of this crude residue by preparative HPLC (neutral) gave the phosphonic acid as the sodium salt.

[0380] Method C:

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

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

Chemical Structure

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

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

[0385] Prepared according to Method B. The product was isolated as an off - white solid (9% yield) by preparative HPLC. LCMS: [M + H] + m / z 390.15. 1 H NMR (400 MHz, D2O) δ 8.12 (s, 1H), 7.22 (s, 4H), 7.11 (s, 1H), 6.91 (s, 1H), 4.79 (s, 2H), 4.76 (s, 2H), 3.98 (s, 3H), 3.91 (s, 3H), 2.79 (s, 1H), 2.74 (s, 1H)

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

[0387] [Chemical formula]

[0388] Prepared according to Method A, and 25 (50% yield) was obtained as an off - white solid. LCMS: [M + H] + m / z 352.10. 1 H NMR (400 MHz, methanol - d4) δ 8.57 (s, 1H), 7.74 - 7.73 (m, 1H), 7.68 - 7.66 (m, 1H), 7.46 (d, 1H), 4.96 (br s, 2H), 3.98, (s, 3H), 3.57 (br s, 2H), 2.65 (s, 2H), 2.07 - 2.04 (m, 2H), 1.81 (m, 1H), 1.79 - 1.75 (m, 2H), 1.66 - 1.63 (m, 2H) and 1.46 - 1.44 (m, 2H).

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

[0390]

Chem.

[0391] Prepared according to Method C, 26 (7% yield) was obtained as an off-white solid. LCMS: [M+H] + m / z 338.15. 1 H NMR (400 MHz, DMSO-d6) δ 8.48 (s, 1H), 7.65 (d, J = 8.8 Hz, 1H), 7.32 (d, J = 8.8 Hz, 1H), 7.18 (s, 1H), 4.21 - 4.17 (m, 2H), 2.99 - 2.95 (m, 2H), 1.82 - 1.79 (m, 2H), 1.53 - 1.49 (m, 5H) and 1.30 - 1.19 (m, 2H). (Preparation of (2-(1-(7-methoxyquinazolin-4-yl)piperidin-4-yl)ethyl)phosphonic acid 27

[0392]

Chem.

[0393] Prepared according to Method A, 27 (95% yield) was obtained as an off-white solid. LCMS: [M+H] + m / z 352.0 1 H NMR (500 MHz, methanol-d4) δ 8.55 (s, 1H), 8.10 (d, J = 10 Hz, 1H), 7.30 (dd, J = 10 Hz and 5 Hz, 1H), 7.10 (d, J = 5 Hz, 1H), 4.01 (s, 3H), 3.57 - 3.48 (m, 2H), 2.65 (s, 1H), 2.05 - 2.02 (m, 2H), 1.94 - 1.90 (m, 1H), 1.80 - 1.74 (m, 2H), 1.65 - 1.60 (m, 2H) and 1.46 - 1.41 (m, 2H).

[0394] (2-(1-(7-Ethoxyquinazolin-4-yl)piperidin-4-yl)ethyl)phosphonic acid 28 was prepared. [Chemical formula]

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

[0396] (2-(1-(7-Hydroxyquinazolin-4-yl)piperidin-4-yl)ethyl)phosphonic acid 29 was prepared

[0397] [Chemical formula]

[0398] Prepared according to Method B, 29 (4% yield) was obtained as a pale yellow solid. LCMS: [M+H] + m / z 338.25. 11H NMR (400 MHz, DMSO-d6) δ 11.49 (s, 1H), 8.64 (s, 1H), 7.96 (d, J = 9.2 Hz, 1H), 7.10 (dd, J = 9.2 and 2.2 Hz, 1H), 7.00 (d, J = 2.2 Hz, 1H), 4.62 (br s, 2H), 3.38 (br s, 2H), 1.87 (d, J = 12.7 Hz, 2H), 1.72 (br s, 1H), 1.58 - 1.38 (m, 4H) and 1.28 - 1.22 (m, 2H). (2-(1-(7-Aminoquinazolin-4-yl)piperidin-4-yl)ethyl)phosphonic acid 30 was prepared

[0399]

Chemical Structure

[0400] Prepared according to Method C and 30 was obtained as a pale yellow solid. LCMS: [M + H] + m / z 337.10 1 1H NMR (400 MHz, DMSO-d6) δ 8.35 (s, 1H), 7.62 (d, J = 8.8 Hz, 1H), 6.81 (d, J = 8.8 Hz, 1H), 6.61 (br s, 1H), 6.30 (br s, 2H), 4.26 - 4.20 (m, 2H), 3.10 - 2.90 (m, 2H), 1.79 - 1.76 (m, 2H), 1.60 - 1.30 (m, 5H) and 1.25 - 1.20 (m, 2H).

[0401] (2-(1-(7-Isopropoxyquinazolin-4-yl)piperidin-4-yl)ethyl)phosphonic acid 31 was prepared

[0402]

Chemical Structure

[0403] Prepared according to Method B and obtained 31 as a bright yellow solid. LCMS: [M+H] + m / z 337.10 1 H NMR (400 MHz, DMSO-d6) δ 8.35 (s, 1H), 7.62 (d, J = 8.8 Hz, 1H), 6.81 (d, J = 9.2 Hz, 1H), 6.66 (s, 1H), 6.30 (br s, 2H), 4.25 - 4.21 (m, 2H), 3.08 - 2.96 (m, 2H), 1.81 - 1.75 (m, 2H), 1.65 - 1.31 (m, 5H) and 1.27 - 1.18 (m, 2H).

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

[0405]

Chemical Structure

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

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

[0408] [Chem.]

[0409] Prepared according to Method B and obtained 33 as a white solid. LCMS: [M+H] + m / z 366.20. 1 H NMR (400 MHz, DMSO-d6) δ 1 H NMR (400 MHz, D2O) δ 8.17 (s, 1H), 7.21 - 7.09 (m, 2H), 4.13 - 3.98 (m, 4H), 2.97 (t, J = 12.4 Hz, 2H), 1.82 (d, J = 13.0 Hz, 2H), 1.56 - 1.35 (m, 8H), 1.26 (q, J = 11.4 Hz, 2H). Preparation of (2-(1-(8-Isopropoxyquinazolin-4-yl)piperidin-4-yl)ethyl)phosphonic acid 34 (in Table 3a)

[0410] [Chem.]

[0411] Prepared according to Method B and obtained 34 (43% yield) as an off-white solid. LCMS: [M+H] + m / z 1 H NMR (400 MHz, DMSO-d6) δ 1 H NMR (400 MHz, D2O) δ 8.10 (s, 1H), 7.11 (d, J = 7.5 Hz, 2H), 7.03 (d, J = 7.1 Hz, 1H), 3.94 (d, J = 13.0 Hz, 2H), 2.86 (t, J = 12.6 Hz, 2H), 1.67 (d, J = 13.1 Hz, 2H), 1.40 - 1.07 (m, 13H). (Preparation of (2-(1-(8-hydroxyquinazolin-4-yl)piperidin-4-yl)ethyl)phosphonic acid 36 (in Table 3a))

[0412] [Chemical formula] Prepared according to Method B, 35 was obtained as an off-white solid.

[0413] LCMS: [M+H] + m / z 1 H NMR (400 MHz, DMSO-d6) δ 1 H NMR (400 MHz, D2O) (Preparation of (2-(1-(5,8-dimethoxyquinazolin-4-yl)piperidin-4-yl)ethyl)phosphonic acid 36)

[0414] [Chemical formula]

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

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

[0417] [Chemical formula]

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

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

[0420]

Chemical Structure

[0421] Prepared according to Method C and obtained 38 (16% yield) as an off-white solid. LCMS: [M+H] + m / z 382.15 1 H NMR (400 MHz, DMSO-d6) δ8.60 (s, 1H), 7.90 (d, J = 9.6 Hz, 1H), 7.49 (d, J = 9.2 Hz, 1H), 4.69 (m, 2H), 4.02 (s, 3H), 3.89 (s, 3H), 3.46 (m, 2H), 1.90 (d, J = 12.8 Hz, 2H), 1.75 (m, 1H), 1.53 - 1.49 (m, 4 H) and 1.31 - 1.28 (m, 2H). (Preparation of (2-(1-(7,8-dihydro-[1,4]dioxino[2,3-g]quinazolin-4-yl)piperidin-4-yl)ethyl)phosphonic acid 39 (in Table 3a)

[0422] [Chem.]

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

[0424] [Chem.]

[0425] Prepared according to Method C to give 40 (7% yield) as a bright yellow solid. LCMS: [M+H] + m / z 370.10. 1 H NMR (400 MHz, DMSO-d6) δ8.55 (s, 1H), 7.44 - 7.38 (m, 2H), 4.28 - 4.23 (m, 2H), 3.94 (s, 3H), 3.28 - 3.18 (m, 2H), 1.82 - 1.78 (m, 2H), 1.70 - 1.66 (m, 1H), 1.49 - 1.23 (m, 4H) and 1.26 - 1.09 (m, 2H).

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

[0427] [Chemical formula]

[0428] Prepared according to Method B, 41 (44% yield) was obtained as a white solid. LCMS: [M+H] + m / z 366.15. 1 H NMR (400 MHz, DMSO-d6) δ8.02 (d, J = 9.2 Hz, 1H), 7.21 (d, J = 9.2 Hz, 1H), 7.04 (s, 1H), 3.93 (s, 3H), 2.49 (s, 3H), 1.91-1.88 (m, 2 H), 1.74(m, 1 H), 1.53-1.49(m, 5 H), and 1.29-1.27 (m, 3 H).

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

[0430] [Chemical formula]

[0431] Prepared according to Method B, 42 (42% yield) was obtained as a bright yellow solid. LCMS: [M+H] + m / z 386.10. 1 H NMR (400 MHz, D2O) δ8.03 (s, 1H), 6.91-6.88 (m, 2H), 3.92-3.89 (m, 2H), 3.77 (s, 3H), 2.93-2.87 (m, 2H), 1.70-1.67 (m, 2H) and 1.41-1.12 (m, 7H).

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

[0433]

Chem.

[0434] Prepared according to Method B, 43 (50% yield) was obtained as a white solid. LCMS: [M+H] + m / z 386.05. 1 H NMR (400 MHz, D2O) δ8.07 (s, 1H), 7.10 - 7.06 (m, 2H), 3.95 - 3.91 (m, 2H), 3.71 (s, 3H), 2.96 - 2.90 (m, 2H), 1.71 - 1.68 (m, 2H) and 1.42 - 1.01 (m, 7H).

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

[0436]

Chem.

[0437] Prepared according to Method B, 44 (49% yield) was obtained as a bright yellow solid. LCMS: [M+H] + m / z 485.25. 1 H NMR (400 MHz, D2O) δ8.09 (s, 1H), 7.94 (s, 1H), 7.36 (d, J = 8 Hz, 1H), 7.06 (s, 1H), 6.74 (d, J = 16.8 Hz 1H), 6.58 (d, J = 3.2 Hz, 1H), 6.40 (d, J = 3.2 Hz, 1H), 6.24 (d, J = 16.8 Hz, 1H), 3.94-3.91 (m, 2H), 3.84 (s, 3H), 3.67 (s, 3H), 2.96-2.90 (m, 2H), 1.96-1.93 (m, 2H) and 1.56-1.32 (m, 7H). (E)-(2-(1-(8-Methoxy-2-(2-(pyridin-3-yl)vinyl)quinazolin-4-yl)piperidin-4-yl)ethyl)phosphonic acid 45 was prepared

[0438]

Chemical formula

[0439] Prepared according to Method B, 45 was obtained as a yellow solid in 79% yield. LCMS: [M+H] + m / z 455.20. 1 H NMR (400 MHz, methanol-d4) δ9.07 (br s, 1H), 8.70 (br s, 1H), 8.62-8.60 (m, 1H), 8.31 (d, 1H), 7.89-7.88 (m, 1H), 7.70-7.54 (m, 4H), 5.20-5.00 (m, 2H) 4.13 (s, 3H), 3.58-3.50 (m, 2H), 2.07-2.02 (m, 2H), 1.88-1.82 (m, 1H), 1.78-1.64 (m, 4H) and 1.51-1.46 (m, 2H).

[0440] (2-(1-(6,7-Dimethoxyquinolin-4-yl)piperidin-4-yl)ethyl)phosphonic acid 46 was prepared

[0441]

Chemical formula

[0442] Prepared according to Method C and obtained 46 (22% yield) as a white solid. LCMS: [M+H] + m / z 381.30. 1 H NMR (400 MHz, methanol-d4) δ8.35 (d, J = 6.8 Hz, 1H), 7.29 - 7.27 (m, 2H), 7.11 (d, J = 6.8 Hz, 1H), 4.27 - 4.23 (m, 2H), 4.03 (s, 3H), 4.02 (s, 3H), 3.40 - 3.32 (m, 2H), 2.06 - 2.03 (m, 4H) 1.82 - 1.79 (m, 3H) and 1.62 - 1.48 (m, 2H).

[0443] (2-(1-(3-Cyano-6,7-dimethoxyquinolin-4-yl)piperidin-4-yl)ethyl)phosphonic acid 47 (also referred to as 42 in Table 2) Preparation

[0444]

Chemical Structure

[0445] Prepared according to Method B and obtained 47 (47% yield) as an off-white solid. LCMS: [M+H] + m / z 406.20. 1 H NMR (400 MHz, D2O) δ8.00 (s, 1H), 6.62 (s, 1H), 6.40 (s, 1H), 3.75 (s, 3H), 3.66 (s, 3H), 3.18 (d, J = 12.3 Hz, 2H), 2.94 (t, J = 12.2 Hz, 2H), 1.72 (d, J = 12.7 Hz, 2H), 1.43 - 1.30 (m, 6H), 1.17 - 1.04 (m, 2H).

[0446] (2-(1-(3-Cyano-6-methoxyquinolin-4-yl)piperidin-4-yl)ethyl)phosphonic acid 48 (also referred to as 44 in Table 2) Preparation

[0447] [Chem.]

[0448] Prepared according to Method B and obtained 48 (16% yield) as an off-white solid. LCMS: [M+H] + m / z 376.20. 1 H NMR (400 MHz, D2O) δ8.15 (s, 1H), 7.51 (s, 1H), 7.18 (s, 1H), 6.88 (s, 1H), 3.71 (s, 3H), 3.60 - 3.51 (m, 2H), 3.15 - 3.08 (m, 2H), 1.81 - 1.74 (m, 2H) and 1.41 - 1.15 (m, 7H).

[0449] Preparation of (2-(1-(3-cyano-7-methoxyquinolin-4-yl)piperidin-4-yl)ethyl)phosphonic acid 49 (also referred to as 43 in Table 2)

[0450] [Chem.]

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

[0452] (2-(1-(3-Cyano-8-methoxyquinolin-4-yl)piperidin-4-yl)ethyl)phosphonic acid 50 (also referred to as 45 in Table 1) was prepared

[0453] [Chemical formula]

[0454] Prepared according to Method B, 50 was obtained as an off-white solid. LCMS: [M+H] + m / z 376.20. 1 H NMR (400 MHz, D2O) δ8.03 (s, 1H), 7.27 - 7.23 (m, 1H), 7.11 - 7.09 (m, 1H), 7.04 - 7.02 (m, 1H), 3.90 (s, 3H), 3.43 (br d, J = 12.4 Hz, 2H), 3.06 (br t, J = 12 Hz, 2H), 1.80 (br d, J = 12.8 Hz, 2H), 1.50 - 1.47 (m, 5H) and 1.31 - 1.24 (m, 2H). (2-(1-(6,7-Dimethoxyisoquinolin-1-yl)piperidin-4-yl)ethyl)phosphonic acid 51 was prepared

[0455] [Chemical formula]

[0456] Prepared according to Method B, 51 (30% yield) was obtained as an off-white solid. LCMS: [M+H] + m / z 381.10. 1 H NMR (400 MHz, D2O) δ 1 H NMR (400 MHz, DMSO-d6): δ 7.79 (d, J = 6.4 Hz, 1H), 7.51 - 7.44 (m, 2H), 7.31 (s, 1H), 3.96 (d, J = 4.8 Hz, 6H), 3.23 (m, 4H), 1.88 (m, 2H), 1.55 (m, 2H), 1.48 (m, 1H) and 1.45 (m, 4H).

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

[0458]

Chem.

[0459] Prepared according to Method B, 52 (50% yield) was obtained as an off - white solid. LCMS: [M + H] + m / z 1 1H NMR (400 MHz, D2O) δ

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

[0461]

Chem.

[0462] ((1-(8-Methoxyquinazolin-4-yl)piperidin-4-yl)methanol (prepared using the same method as Compound 80) (500 mg, 1.83 mmol)) in pyridine (5 mL) was added dropwise with phosphorus thiochloride (1.6 g, 9.45 mmol) at -15 °C. The reaction mixture was stirred at 0 °C for 1 hour and then added to an aqueous solution of sodium hydrogen carbonate (923 mg, 10.98 mmol) (20 mL) at 0 °C. The resulting mixture was stirred at 0 °C for 2 hours. Next, it was evaporated to dryness under reduced pressure. By purification (preparative HPLC), 53 (83 mg, 12%) was obtained as a white solid. LCMS: [M+H] + m / z 354.10 1 H NMR (400 MHz,, DMSO-d6) δ8.59 (s, 1H), 7.59-7.50 (m, 2H), 7.45 (dd, J = 6.5, 2.4 Hz, 1H), 4.53 (d, J = 12.7 Hz, 2H), 3.97 (s, 3H), 3.80-3.74 (m, 4H), 2.03 (s, 1H), 1.86 (d, J = 13.5 Hz, 2H), 1.41 (q, J = 11.8 Hz, 2H). Preparation of (((1-(8-Methoxyquinazolin-4-yl)piperidin-4-yl)oxy)methyl)phosphonic acid 54 (in Table 3a)

[0463]

Chemical Structure

[0464] Prepared using the same method as Compounds 10 and 11. When this mixture was purified (preparative HPLC, 0.1% TFA), 54 was obtained as an off-white solid.

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

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

[0467]

Chem.

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

[0469] LCMS: [M + H] + m / z 345.10. 1 H NMR (400 MHz, D2O) δ

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

[0471]

Chem.

[0472] Prepared according to the same method as for Compound 23. LCMS: [M + H] + m / z 346.10. 1 H NMR (400 MHz, D2O) δ8.20 (s, 1H), 7.62 - 7.57 (m, 2H), 7.43 - 7.41 (m, 2H), 7.31 - 7.28 (m, 2H), 7.23 - 7.21 (m, 1H) and 2.93 (s, 3H). (Preparation of (4-(((3-cyano-8-methoxyquinolin-4-yl)amino)methyl)phenyl)phosphonic acid 57 (also referred to as 52 in Table 1))

[0473]

Chem.

[0474] Prepared according to the same method as compound 23, 57 was obtained as an off-white solid. LCMS: [M+H] + m / z 370.10 1 H NMR (400 MHz, D2O) δ8.02 (s, 1H), 7.48 - 7.43 (m, 2H, 7.36 - 7.30 (m, 2H), 7.15 - 7.09 (m, 3H), 4.77 (s, 2H) and 3.81 (s, 3H).

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

[0476]

Chem.

[0477] Prepared according to the same method as compound 23, 58 was obtained as a white solid. LCMS: [M+H] + m / z 384.15 1 H NMR (400 MHz, methanol - d4) δ8.32 (s, 1H), 7.77 (d, J = 8.4 Hz, 1H), 7.50 (t, J = 8.4 Hz, 1H), 7.35 - 7.33 (m, 2H), 7.26 (d, J = 7.6 Hz, 1H), 7.20 (d, J = 8.4 Hz, 2H), 5.02 (s, 2H), 3.99 (s, 3H) and 2.85 (d, J = 20 Hz, 2H).

[0478] Preparation of (3 - (1 - (3 - cyano - 8 - methoxyquinolin - 4 - yl)piperidin - 4 - yl)propyl)phosphonic acid 59 (also referred to as 210 in Table 2)

[0479]

Chem.

[0480] Prepared according to the same method as Compound 14, and 59 was obtained as a white solid. LCMS: [M+H] + m / z 390.20. 1 H NMR (400 MHz, D2O) δ8.30 (s, 1H), 7.39 (br s, 2H), 7.19 (br s, 1H), 3.98 (s, 3H), 3.73 - 3.70 (m, 2H), 3.30 (t, J = 12 Hz, 2H), 1.92 - 1.88 (m, 2H), 1.70 - 1.45 (m, 3H) and 1.40 - 1.27 (m, 6H).

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

[0482]

Chemical Structure

[0483] To a solution of compound 101 (0.97 g, 5.0 mmol) in 2-methoxyethanol (10 mL) were added (4-bromophenyl)methanamine 90 (1.74 g, 10.0 mmol) and Et3N (1.51 g, 15 mmol). The mixture was heated at 100 °C overnight, cooled to room temperature, and then evaporated to dryness under reduced pressure. 102 (1.5 g, 88%) was obtained as a white solid by chromatography (35% EtoAc in petroleum ether). To a solution of compound 102 (69 mg, 0.2 mmol) in DMSO (3 mL) were added bis(pinacolato)diboron (61.0 mg, 0.24 mmol), potassium acetate (58.8 mg, 3.0 mmol), and Pd(dppf)Cl2 (7.4 mg, 0.05 mmol). The reaction was degassed by purging with nitrogen and then heated at 80 °C for 48 h. The mixture was cooled to room temperature, diluted with ethyl acetate, and then filtered through a Celite® pad. The filtrate was evaporated to dryness under reduced pressure. The residue was dissolved in EtOAc (10 mL), and a solution of HCl (4 M, 0.2 mL, 4.0 mmol) in EtOAc was added. The mixture was stirred at room temperature overnight and then evaporated to dryness under reduced pressure. 60 (40.5 mg, 65% over two steps) was obtained as a white solid by chromatography [preparative HPLC (TFA)]. LCMS: [M+H] + m / z 334.15. 1 H NMR (400 MHz, methanol-d4) δ 8.69 (s, 1H), 7.90 (d, J = 8.4 Hz, 1H), 7.73 (t, J = 8.3 Hz, 2H), 7.61 (d, J = 8.1 Hz, 2H), 7.41 (dd, J = 17.3, 7.8 Hz, 2H), 5.05 (s, 2H), 4.13 (s, 3H).

[0484] Preparation of (2-(1-(3-cyano-8-methoxyquinolin-4-yl)piperidin-4-yl)ethyl)boronic acid 61 (also referred to as 216 in Table 2)

[0485]

Chemical Structure

[0486] It was prepared according to the same procedure as for Compound 7. Compound 61 was isolated as a yellow solid.

[0487] LCMS: [M+H] + m / z 340.20. 1 H NMR (400 MHz, methanol-d4) δ8.81 (s, 1H), 7.83 (d, J = 12 Hz, 1H), 7.72 (t, J = 8 Hz, 1H), 7.60 (d, J = 8 Hz, 1H), 4.51 (d, J = 12 Hz, 2H), 3.81 (t, J = 12 Hz, 2H), 3.31 (s, 3H), 2.66 (s, 1H). 2.05 (br d, J = 12 Hz, 2H), 1.72 - 1.30 (m, 4H) and 0.91 - 0.85 (m, 2H).

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

[0489]

Chemical Structure

[0490] A solution of 101 (2.0 g, 8.9 mmol) and 103 (1.5 g, 8.9 mmol) in 2 - methoxyethanol (40 mL) was heated to reflux overnight and then cooled to room temperature. The reaction mixture was evaporated to dryness under reduced pressure and then triturated with EtOAc, filtered, and dried to give crude compound 104 (1.7 g) as a bright yellow solid.

[0491] To a solution of compound 104 (0.5 g, 1.55 mmol) in THF (20 mL) was added NaOH (0.17 g, 4.65 mmol, dissolved in 2 mL of water). The mixture was heated to 45 °C overnight. The cooled solution was concentrated under reduced pressure, and the residue was treated with aqueous HCl (2N) until pH 5.5 was achieved. The resulting precipitate was filtered and dried to give the crude acid intermediate (0.3 g, 62% yield) as a light yellow solid. The crude acid was dissolved in DMF (10 mL), then cooled to 0 °C and placed under nitrogen. BOP (0.48 g, 1.06 mmol) and DIPEA (0.50 g, 3.88 mmol) were added, followed by HONH 2- HCl (0.09 g, 1.26 mmol). The mixture was stirred at room temperature overnight, quenched with water (50 mL), and extracted with EtOAc. The organic phase was washed with water and brine, dried (Na2SO3), and evaporated to dryness under reduced pressure. Chromatography (5% MeOH in CH2Cl2), then preparative HPLC (H + , 0.1% TFA) gave 62 (34 mg, 10%) as an off-white solid. LCMS: [M+H] + 1 H NMR (400 MHz, DMSO-d6) δ 11.19 (s, 1H), 9.06 (s, 1H), 8.55 (s, 1H), 8.45 (d, J = 8.7 Hz, 1H), 7.72 (d, J = 7.4 Hz, 2H), 7.39 (dd, J = 4.6, 2.9 Hz, 2H), 7.29 (dd, J = 12.3, 5.0 Hz, 2H), 5.11 (s, 2H), 3.93 (s, 3H).

[0492] (Example 2) Evaluation of Compound Activity

[0493] Prepare the selected compounds of Tables 1-3 and other derivatives and evaluate them in an ENPP1 activity assay using thymidine monophosphate paranitrophenol (TMP-pNP) as the substrate. Prepare the enzyme reaction at room temperature in 100 mM Tris, 150 mM NaCl, 2 mM CaCl2, 200 μM ZnCl2 (pH 7.5) using TMP-pNP (2 μM), a 5-fold dilution of the ENPP1 inhibitor, and purified recombinant mouse ENPP1 (0.5 nM). Monitor the progress of the reaction by measuring the absorbance of the paranitrophenolate produced by this reaction at 400 nm for 20 minutes. Extract the slope of product formation, plot it, and fit it using Graphpad Prism 7.03 to obtain the IC 50 value.

[0494] The compounds are also evaluated in an ENPP1 enzyme activity assay using cGAMP as the substrate. Methods that can be used to evaluate the compounds of interest include those described by Li et al. in PCT Application No. PCT / US2018 / 050018 filed on September 7, 2018. An exemplary method is described below.

[0495] Substances:

[0496] Mouse ENPP1: Expressed and purified according to Kato et al., PNAS (2012) 109(42):16876 - 8. cGAMP: Synthesized and purified according to Li et al., Nat. Chem. Biol. (2014) 10:1043 - 8. Polyphosphate:AMP phosphotransferase (PAP): The PAP gene (GenBank: AB092983.1) was synthesized (Integrated DNA Technologies) and cloned into the pTB146 vector with a His - SUMO C - terminal tag. BL21(DE3) cells transformed with the plasmid were grown and induced with 0.75 mM IPTG at OD600 = 1 overnight at 16°C. Cells were resuspended in a buffer containing 50 mM Tris (pH 7.5), 400 mM NaCl, 10 mM imidazole, 2 mM DTT, and protease inhibitor (Roche) and lysed by two freeze - thaw cycles and sonication. All subsequent steps were performed at 4°C. The lysate was clarified by centrifugation at 40,000 rcf for 1 hour, and the supernatant was incubated with HisPur cobalt resin (Thermo Fisher Scientific) for 2 hours. The resin was washed twice with 30 mL of buffer containing 50 mM Tris (pH 7.5), 150 mM NaCl, and the protein was eluted with 50 mM Tris (pH 7.5), 150 mM NaCl, 600 mM imidazole. Anion - exchange chromatography (HiTrap Q HP) was performed. Myokinase (MilliporeSigma). CellTiterGlo (Promega)

[0497] Exemplary procedure for ENPP1 enzyme activity assay:

[0498] In a buffer containing 50 mM Tris (pH 7.6), 250 nM NaCl, 500 μM CaCl2 and 1 μM ZnCl2 (total reaction volume = 10 μL), 5 μM cGAMP and serial 5-fold dilutions of the compound were incubated with 3 nM mouse ENPP1 for 3 hours at room temperature. Subsequently, the reaction was heat-inactivated at 95 °C for 10 minutes. The AMP degradation product was converted to ATP, which was detected using luciferase. To achieve this, an enzyme mixture consisting of polyphosphate:AMP phosphotransferase (PAP) and myokinase was prepared according to Goueli et al.'s EP2771480. Briefly, PAP was diluted to 2 mg / mL in a buffer containing 50 mM Tris (pH 7.5) and 0.1% NP-40. Myokinase was diluted to 2 KU / mL in a buffer containing 3.2 mM ammonium sulfate (pH 6.0), 1 mM EDTA and 4 mM polyphosphate. The heat-inactivated ENPP1 reaction was incubated with PAP (0.01 μg / μL) and myokinase (0.0075 U / μL) for 3 hours (total reaction volume = 20 μL) in a buffer containing 40 mM Tris (pH 7.5), 0.05 mg / mL Prionex, 5 mM MgCl2, 20 μM polyphosphate and 0.15 g / L phenol red (to facilitate pipetting). According to the manufacturer's protocol, CellTiterGlo (20 μL) was added to this reaction and the luminescence was measured. After normalizing the data to 100% enzyme activity (without compound) and 0% enzyme activity (without enzyme), it was fitted to the function 100 / (1 + ([compound] / IC50)).

[0499] The IC50 values fall within the ranges indicated by letters A - C, where A represents that the IC50 value is less than 50 nM, B represents that the IC50 value is between 50 nM and 100 nM, and C represents that the IC50 value is higher than 100 nM.

[0500]

Table 4-1

Table 4-2

Table 4-3

Table 4-4

Table 4-5

Table 4-6

Table 4-7

Table 4-8

Table 4-9

Table 4-10

Table 4-11

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

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

[0503] 293T cGAS ENPP1 - / - Cells were transfected with a human ENPP1 expression plasmid, and cGAMP hydrolase activity was confirmed in the whole cell lysate (FIG. 18A). 293T cells were purchased from ATCC and transfected with a virus to stably express mouse cGAS. 293T mcGAS ENPP1 - / -was generated by viral transfection of CRISPR sgRNA targeting human ENPP1 (5’CACCGCTGGTTCTATGCACGTCTCC-3’) (SEQ ID NO: 1). In DMEM (Corning Cellgro) supplemented with 10% FBS (Atlanta Biologics) (v / v) and 100 U / mL penicillin-streptomycin (ThermoFisher), 293T mcGAS ENPP1 cells were plated in tissue culture-treated plates coated with PurCol (Advanced BioMatrix). - / - After plating for 12 - 24 hours, pcDNA3 plasmid DNA (empty or containing human ENPP1) at the indicated concentration was added according to the manufacturer's instructions, and the cells were transfected with Fugene6 (Promega). Twenty-four hours after transfection, the cells were lysed to analyze ENPP1 expression by Western blotting (using rabbit anti-ENPP1 (L520, 1:1000) and mouse anti-tubulin (DM1A, 1:2,000), Cell Signaling Technologies). Whole cell lysates were generated by lysing 1×10 6 cells in 10 mM Tris, 150 mM NaCl, 1.5 mM MgCl2, 1% NP-40 (pH 9.0). 32 P-cGAMP (5 μM) was incubated with the whole cell lysates and degradation was monitored as described in Example 2 above (Figure 18A).

[0504] In naive cells, ENPP1 expression depletes extracellular cGAMP but does not affect intracellular cGAMP concentration (Figure 18B). 293T mcGAS ENPP1 - / -pcDNA3 (empty or containing human ENPP1) was transfected, and after 24 hours, the medium was removed and replaced with serum-free DMEM supplemented with 1% insulin-transferrin-selenium-sodium pyruvate (ThermoFisher) and 100 U / mL penicillin-streptomycin. 12 - 24 hours after medium replacement, the medium was removed and the cells were washed off the plate with cold PBS. Both the medium and the cells were centrifuged at 1000 rcf for 10 minutes at 4 °C to prepare for cGAMP concentration measurement by liquid chromatography-tandem mass spectrometry (LC-MS / MS). As an internal standard, 500 nM cyclic GMP- 13 C 10 , 15 N5-AMP was added to 30 - 100 μL of 50:50 acetonitrile:water, the cells were lysed, and centrifuged at 15,000 rcf for 20 minutes at 4 °C to remove the insoluble fraction. The medium was removed and 500 nM cyclic GMP- 13 C 10 , 15 N5-AMP and 20% formic acid were added. The cGAMP, ATP, and GTP contents of the samples were analyzed with a Shimadzu HPLC (San Francisco, CA) connected to an AB Sciex 4000 QTRAP (Foster City, CA) equipped with an autosampler set at 4 °C. 10 μL aliquots were injected onto a Biobasic AX LC column, 5 μm, 50×3 mm (Thermo Scientific). The mobile phase consisted of 100 mM ammonium carbonate (A) and 0.1% formic acid in acetonitrile (B). The initial conditions were B 90% and were maintained for 0.5 minutes. This mobile phase was improved to A 30% from 0.5 to 2.0 minutes, A 30% was maintained from 2.0 to 3.5 minutes, increased to B 90% from 3.5 to 3.6 minutes, and B 90% was maintained from 3.6 to 5 minutes. The flow rate was set at 0.6 mL / min. The mass spectrometer was operated in electrospray positive ion mode with the source temperature set at 500 °C. The decl Stallings and collision-induced dissociation were performed using nitrogen gas. The declustering potential and collision energy were optimized by direct injection of the standards. For each molecule, the MRM transitions (m / z), DP (V), and CE (V) are as follows: ATP (508>136, 341, 55), GTP (524>152, 236, 43), cGAMP (675>136, 121, 97; 675>312, 121, 59; 675>152, 121, 73), internal standard cyclic GMP- 13 C 10 , 15 N5-AMP (690>146, 111, 101; 690>152, 111, 45; 690>327, 111, 47), extraction standard cyclic 13 C 10 , 15 N5-GMP- 13 C 10 , 15 N5-AMP (705>156, 66, 93; 705>162, 66, 73).

[0505] Inhibition of ENPP1 blocks the degradation of extracellular cGAMP (Figure 18C). The same experiment was performed as above, this time including 50 μM of the ENPP1 inhibitor (Compound 1) when the medium was changed. When the inhibitor was used, the extracellular cGAMP concentration in the medium returned to previous levels.

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

[0507] (Example 4) Inhibition of ENPP1 improves cGAMP activation in primary CD14+ monocytes.

[0508] Using an ENPP1 inhibitor (compound 1), it was tested whether cGAMP excreted by the 293T cGAS ENPP1 low cell line could be detected by antigen-presenting cells (APCs) such as human CD14 + monocytes (Figure 19A). 293T cGAS ENPP1 low cells were transfected with pcDNA (empty or containing human ENPP1). Primary human peripheral blood mononuclear cells (PBMCs) were isolated by applying a Percoll density gradient to the concentrated buffy coat from whole blood. CD14 + monocytes were isolated using CD14 + microbeads (Miltenyi). CD14 monocytes were cultured in RPMI supplemented with 2% human serum and 100 U / mL penicillin-streptomycin. + 293T cGAS ENPP1 low After 8 hours of transfection of the cells, the medium was changed to RPMI supplemented with 2% human serum and 100 U / mL penicillin-streptomycin with or without the exemplary ENPP1 inhibitor compound 1. After 24 hours of changing the medium, the supernatant derived from the 293T cGAS ENPP1 low cells was added to CD14 +Transferred to single cells (Figure 19A). After 24 - 26 hours of transferring the supernatant, total RNA was extracted using Trizol (Thermo Fisher Scientific) and reverse transcribed using Maxima H Minus reverse transcriptase (Thermo Fisher Scientific). Real-time RT-PCR was performed in duplicate using AccuPower 2X Greenstar qPCR Master Mix (Bioneer) on a 7900HT Fast Real-Time PCR System (Applied Biosystems). Data were normalized to the CD14 expression level for each sample. The induction fold was calculated using ΔΔCt. Primers for human IFNB1: forward (5’-AAACTCATGAGCAGTCTGCA-3’) (SEQ ID NO: 2), reverse (5’-AGGAGATCTTCAGTTTCGGAGG-3’) (SEQ ID NO: 3); for human CD14: forward (5’-GCCTTCCGTGTCCCCACTGC-3’) (SEQ ID NO: 4), reverse (5’-TGAGGGGGCCCTCGACG-3’) (SEQ ID NO: 5).

[0509] 293T cGAS ENPP1 expressing cGAS low The supernatant derived from the cells induced CD14+ IFNB1 expression, while the supernatant derived from cGAS-null 293T cells did not. This suggests that extracellular cGAMP excreted by cancer cells can be detected by CD14 + cells as a signaling factor (Figure 19B). 293T cGAS ENPP1 low Transient overexpression of ENPP1 on the cell surface caused the degradation of extracellular cGAMP and a decrease in the CD14 + IFNB1 expression level. However, the addition of compound 1 rescued the extracellular cGAMP level and induced CD14 + IFNB1 expression (Figure 19B).

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

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

[0512] It was tested whether cancer cell lines excrete cGAMP and whether ionizing radiation (IR) affects the level of extracellular cGAMP produced. Ionizing radiation (IR) has been shown to increase cytosolic DNA in tumor cells and activate cGAS-dependent IFN-β production (Bakhoum et al. Nat. Commun. (2015) 6:1-10; and Vanpouille Nat. Commun. (2017) 8:15618). After 24 hours of plate culture, 4T1 cells were treated with 20 Gy IR using a cesium source and changed to a medium supplemented with 50 μM of the ENPP1 inhibitor (Compound 1) to inhibit ENPP1 present in the cell culture. The medium was collected at the indicated times, centrifuged at 1000 × g to remove residual cells, acidified with 0.5% acetic acid, and supplemented with cyclic- 13 C 10 , 15 5-GMP- 13 C 10 , 15 N5-AMP (in an appropriate amount to a final concentration of 2 μM in 100 μL). As previously described, the medium was applied to a HyperSep aminopropyl SPE column (ThermoFisher Scientific) to enrich cGAMP (Gao et al., Proc. Natl. Acad. Sci. U.S.A. (2015) 112:E5699-705). The eluate was evaporated to dryness and reconstituted in 50:50 acetonitrile:water supplemented with 500 nM internal standard. The medium was subjected to mass spectrometric quantification of cGAMP.

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

[0514] Next, the effect of IR combined with exemplary ENPP1 inhibitor compound 1 on tumor-associated dendritic cell numbers in a mouse 4T1 tumor model was examined (Figure 20B). 1 × 10 6 cells of 4T1-luciferase tumor cells suspended in 50 μL of PBS were inoculated into the mammary fat pad of 7- to 9-week-old female Balb / c mice (Jackson Laboratories). Two days after injection, the tumors were irradiated with 20 Gy of radiation using a 225 kVp cabinet-type X-ray irradiator (IC250, Kimtron Inc., CT) with a 0.5 mm Cu filter. Anesthetized animals were shielded using a 3.2 mm lead shield with a 15 × 20 mm gap where the tumor was located. 100 μL of 1 mM compound 1 in PBS or PBS alone was injected intratumorally into the mice. The next day, the tumors were excised and incubated at 37°C for 30 minutes in RPMI + 10% FBS containing 20 μg / mL DNase I type IV (Sigma-Aldrich) and 1 m g / mL collagenase from Clostridium histolyticum (Sigma-Aldrich). The tumors were passed through a 100 μm cell strainer (Sigma-Aldrich), and the red blood cell lysis buffer (155 mM NH4Cl, 12 mM Red blood cells were lysed at room temperature for 5 minutes using lysis buffer (150 mM NH4Cl, 10 mM KHCO3, 0.1 mM EDTA). Cells were stained using the Live / Dead Fixable Near-IR Dead Cell Stain Kit (Thermo Fisher Scientific), Fc-blocked for 10 minutes using TruStain fcX, and subsequently antibody stained using CD11c, CD45, and I-A / I-E (all from Biolegend). Cells were analyzed using an SH800S cell sorter (Sony) or an LSR II (BD Biosciences). For statistical analysis, data were analyzed using FlowJo V10 software (Treestar) and Prism 7.04 software (Graphpad), and statistical significance was evaluated using an unpaired t-test with Welch's correction.

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

[0516] These results are illustrated in Figures 20A and 20B. Figure 20A 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 in medium supplemented with 50 μM of compound 1. Mean ± standard error (n = 2). ** P = 0.004 (Student's t-test). Figure 20B shows 4T1 cells (1 × 10 6 ) syngeneically injected into BALB / cJ mice on day 0. On day 2, tumors were left untreated or treated with 20 Gy IR and injected intratumorally with PBS (n = 5 for IR(0 Gy); n = 4 for IR(20 Gy)) or compound 1 (n = 5). On day 3, tumors were harvested and analyzed by FACS. * P = 0.047 (Welch's t-test).

[0517] (Example 6) ENPP1 inhibition synergizes with IR treatment and anti-CTLA-4 to exert an antitumor effect

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

[0519] 5 × 10 4 cells of 4T1-luciferase suspended in 50 μL of PBS were inoculated into the mammary fat pads of 7- to 9-week-old female Balb / c mice (Jackson Laboratories). The tumor volume (determined by length 2 × width / 2) was 80 mm 3 ~120 mm 3Upon reaching, 225 kVp cabinet-type X-ray irradiation device (IC250, Kimtron Inc., CT) with a 0.5 mm Cu filter was used to irradiate the tumor with 20 Gy of radiation. Anesthetized animals were shielded using a 3.2 mm lead shield with a 15×20 mm gap where the tumor was present. On days 2, 4, and 7 after IR, 100 μL of 100 μM compound 1 and / or 10 μg of cGAMP in PBS or PBS alone were injected intratumorally. Alternatively, after IR, on days 2, 5, and 7, 1 mM compound 1 in PBS or PBS alone was injected intratumorally, and 200 μg of anti-CTLA-4 antibody or Syrian hamster IgG antibody (both from BioXCell) were injected intraperitoneally. Mice from different treatment groups were co-housed in each cage to eliminate cage effects. The experimenters were blinded throughout the study. Tumor volume was recorded every other day. To account for the correlation within mice, tumor volume was analyzed using a generalized estimating equation. Pairwise comparisons of treatment groups at each time point were performed using a post hoc test with Tukey's adjustment for multiple comparisons. Using Graphpad Prism 7.03, animal deaths were plotted on a Kaplan-Meier curve, and statistical significance was evaluated using a log-rank Mantel-Cox test. All animal procedures were approved by the Institutional Animal Care and Use Committee.

[0520] Administration of compound 1 enhanced the tumor shrinkage effect of the IR treatment, but it was not significant (Figure 21A). Intratumoral injection of cGAMP did not have as much effect as the IR treatment, but when compound 1 was injected in addition to cGAMP, it synergistically regressed the tumor, extended survival, and achieved a 10% cure rate (Figure 21A and Figure 21B).

[0521] The synergistic effect with anti-CTLA-4, an adaptive immune checkpoint inhibitor, was also tested. In the absence of IR, treatment with anti-CTLA-4 and Compound 1 had no effect on the extension of survival (Figure 21C). However, when IR pretreatment was combined with Compound 1 and anti-CTLA-4, a significant synergistic effect was exerted, and a cure rate of 10% was achieved. In summary, these results demonstrate that increasing extracellular cGAMP by combining IR treatment and ENPP1 inhibition increases tumor immunogenicity and exerts an antitumor effect.

[0522] These results are illustrated in Figure 21A, which shows the tumor shrinkage effect of Compound 1 in combination with IR. Established tumors (100±20 mm 3 ) were once treated with 20 Gy IR, and then PBS was intra-tumorally injected or treated three times on the 2nd, 4th, and 7th days after IR (n = 9 per treatment group). Mice from different treatment groups were co-housed and the experimenters were blinded. To account for the correlation within mice, tumor volume was analyzed with a generalized estimating equation. Pairwise comparisons of treatment groups at each time point were performed using a post hoc test with Tukey's adjustment for multiple comparisons. Figure 21B shows the Kaplan-Meier curve for the case of Figure 21A, and the P value was determined by the log-rank Mantel-Cox test. Figure 21C shows anti-CTLA4 or IgG isotype control antibody intraperitoneally injected on the 2nd, 5th, and 7th days after IR, in addition to the same procedure as in the case of Figure 21B (n = 8 for the IR(0)+Compound 1+CTLA-4 treatment group; n = 17 - 19 for all other treatment groups). Statistical analysis was performed in the same manner as in the case of Figure 21B.

[0523] In summary, these results indicate that cGAMP is present extracellularly and that the ENPP1 inhibitor of interest can act extracellularly, and thus extracellular inhibition of ENPP1 is sufficient for a therapeutic effect. ENPP1 is suitable as a natural immune checkpoint. These experiments show that by inhibiting ENPP1 extracellularly, cGAMP can activate anti-cancer immunity and can be synergistically combined with immune checkpoint blockers already available as therapeutic agents (Figure 22). (Example 7) 2’3’-cGAMP is an immunotransmitter produced by cancer cells and regulated by ENPP1 Introduction

[0524] 2’3’-Cyclic GMP-AMP (cGAMP) is synthesized in response to cytosolic dsDNA and is characterized as an intracellular second messenger that activates the innate immune STING pathway. Its extracellular hydrolase ENPP1 implies the presence of extracellular cGAMP. Using mass spectrometry, it was detected that cGAMP is continuously secreted as a soluble factor by engineered cell lines and then efficiently cleared by ENPP1. By developing a potent, specific, and cell-impermeable ENPP1 inhibitor, cGAMP secretion was detected in cancer cell lines commonly used in mouse tumor models. In tumors, depletion of extracellular cGAMP using a neutralizing protein led to a decrease in tumor-associated dendritic cells. Increased extracellular cGAMP due to genetic knockout and pharmacological inhibition of ENPP1 led to an increase in tumor-associated dendritic cells, tumor shrinkage, and synergism with ionizing radiation and anti-CTLA-4 to cure tumors. In conclusion, cGAMP is an anti-cancer immunotransmitter released by tumors and detected by the host's innate immunity.

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

[0526] In addition to activating STING in its original cell, cGAMP can diffuse to bystander cells via gap junctions in epithelial cells. This cell-cell communication mechanism alerts neighboring cells of damaged cells and, unfortunately, may also be a major cause of the spread of drug-induced liver toxicity and brain metastasis. Furthermore, cytosolic cGAMP can be encapsulated in budding viral particles and transmitted during the next stage of infection. In either mode of transmission, cGAMP is never exposed to the extracellular space.

[0527] The only enzyme responsible for detectable cGAMP hydrolase activity is ectonucleotide pyrophosphatase phosphodiesterase 1 (ENPP1) (e.g., Li, L. et al. See Nat. Chem. Biol. 10, 1043-8 (2014). This is surprising because ENPP1 is annotated as an extracellular enzyme in both a membrane-bound form anchored by a single transmembrane domain and as a cleaved soluble protein in serum. cGAMP, which has two negative charges and presumably cannot passively cross the cell membrane, can enter cells and activate STING (e.g., Gao, P. et al. Structure-function analysis of STING activation by c[G(2’,5’) pA(3’,5’)p] and targeting by antiviral DMXAA. Cell 154, 748-762 (2013); and Corrales, L. et al. Direct Activation of STING in the Tumor Microenvironment Leads to Potent and Systemic Tumor Regression and Immunity. See Cell Rep. 11, 1018-1030 (2015), suggesting the existence of a transport channel for cGAMP. Since cGAMP can enter cells, cGAMP analogs are currently being tested in clinical trials for treating metastatic solid tumors by intratumoral injection. Recognizing that extracellular cGAMP influx has an anti-cancer effect and that the major cGAMP hydrolase is extracellular, it was hypothesized that cGAMP is excreted into the extracellular space to signal to other cells and is regulated by extracellular degradation.

[0528] The role of extracellular cGAMP in cancer-induced cGAMP excretion and anti-cancer immunity detection has been demonstrated herein. Using genetic knockout and pharmacological inhibition, the role of ENPP1 in controlling extracellular cGAMP concentration, immune infiltration, and tumor progression was also investigated. Collectively, cGAMP was characterized as an immunotransmitter regulated by ENPP1. Substances and Methods

[0529] Reagents, Antibodies, and Cell Lines

[0530] [α- 32 P]ATP (800 Ci / mmol, 10 mCi / mL, 250 μCi) and 35 S]ATPαS (1250 Ci / mmol, 12.5 mCi / mL, 250 μCi) were purchased from Perkin Elmer. Adenosine triphosphate, guanosine triphosphate, adenosine- 13 C 10 , 15 N5,5’-triphosphate, guanosine- 13 C 10 , 15 N5-triphosphate, 4-nitrophenyl phosphate, and bis(4-nitrophenyl) phosphate were purchased from Sigma-Aldrich and were >98% atomically pure. 2’3’-cGAMP was purchased from Invivogen. The Caco-2 assay was purchased from Cyprotex. The kinome screening was performed by Eurofins. The PAMPA and MDCK permeability assays were performed by Quintara Discovery. Total protein content was quantified using the BCA assay (ThermoFisher). Cell viability was quantified using the CellTiterGlo assay (Promega). Full-length human ENPP1 was cloned into the pcDNA3 vector. A series of 4ON-TARGETplus ENPP1 siRNA (LQ-003809-00-0002) was purchased from Dharmacon. QS1 was synthesized as previously described 25The following monoclonal antibodies were used for Western blotting: rabbit anti-cGAS (D1D3G Cell Signaling, 1:1,000), rabbit anti-mouse cGAS (D2O8O Cell Signaling, 1:1,000), mouse anti-tubulin (DM1A Cell Signaling, 1:2,000), and rabbit anti-STING (D2P2F Cell Signaling, 1:1,000), IRDye 800CW goat anti-rabbit (LI-COR, 1:15,000), and IRDye 680RD goat anti-mouse (LI-COR, 1:15,000).

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

[0532] Cell cultures

[0533] Cell lines were maintained in DMEM (Corning Cellgro) (293T, MC38) supplemented with 10% FBS (Atlanta Biologics) (v / v) and 100 U / mL penicillin-streptomycin (ThermoFisher) or RPMI (Corning Cellgro) (4T1-Luc, E0771, MDA-MD-231). Primary human peripheral blood mononuclear cells (PBMCs) were isolated by applying a Percoll density gradient to buffy coat concentrates from whole blood. CD14 + PBMCs were + isolated using CD14 + microbeads (Miltenyi). CD14

[0534] Expression and purification of recombinant proteins

[0535] Using sscGAS: primer pair forward: (5’-CTGGAAGTTCTGTTCCAGGGGCCCCATATGGGCGCCTGGAAGCTCCAGAC-3’) and reverse: (5’-GATCTCAGTGGTGGTGGTGGTGGTGCTCGAGCCAAAAAACTGGAAATCCATTGT-3’), the DNA sequence encoding porcine cGAS (residues 135-497) was amplified from a porcine cDNA library. The PCR product was inserted into pDB-His-MBP by Gibson assembly and expressed in Rosetta cells. Cells were grown in 2×YT medium containing kanamycin (100 μg / ml), and when the OD 600 reached 1, they were induced with 0.5 mM IPTG and grown overnight at 16 °C. All of the following procedures involving proteins and cell lysates were performed at 4 °C. Cells were pelleted and lysed in 20 mM HEPES (pH 7.5), 400 mM NaCl, 10% glycerol, 10 mM imidazole, 1 mM DTT, and protease inhibitor cocktail (cOmplete EDTA-free tablets, Roche). The cell extract was clarified by ultracentrifugation at 50,000×g for 1 hour. The clarified supernatant was incubated with HisPur cobalt resin (ThermoFisher Scientific; 1 mL of resin per liter of bacterial culture). The cobalt resin was washed with 20 mM HEPES (pH 7.5), 1 M NaCl, 10% glycerol, 10 mM imidazole, 1 mM DTT. The protein was eluted from the resin with 300 mM imidazole in 20 mM HEPES (pH 7.5), 1 M NaCl, 10% glycerol, and 1 mM DTT. Fractions containing His-MBP-sscGAS were pooled and concentrated, and dialyzed against 20 mM HEPES (pH 7.5), 400 mM NaCl, 1 mM DTT. The protein was snap-frozen in aliquots for future use.

[0536] STING: Mouse STING (residues 139-378) was inserted into the pTB146 His-SUMO vector and expressed in Rosetta cells. Cells were grown in 2×YT medium containing 100 μg / mL ampicillin until OD 600When it reached 1, it was induced with 0.75 mM IPTG at 16 °C overnight. All of the following procedures using the protein and cell lysates were performed at 4 °C. The cells were pelleted and lysed in 50 mM Tris (pH 7.5), 400 mM NaCl, 10 mM imidazole, 2 mM DTT and protease inhibitor (cOmplete, EDTA-free protease inhibitor cocktail, Roche). The cells were lysed by sonication and the lysate was clarified by ultracentrifugation at 50,000 rcf for 1 hour. The clear supernatant was incubated with HisPur cobalt resin (ThermoFisher Scientific; 1 mL of resin per 1 L of bacterial culture) for 30 minutes. The resin-bound protein was washed with 50 column volumes of 50 mM Tris (pH 7.5), 150 mM NaCl, 2% triton® X-114, 50 CV of 50 mM Tris (pH 7.5), 1 M NaCl (each wash was set at a drip rate of 1 drop / 2 - 3 seconds and took 2 - 3 hours) and 20 CV of 50 mM Tris (pH 7.5), 150 mM NaCl. The protein was eluted from the resin with 600 mM imidazole in 50 mM Tris (pH 7.5), 150 mM NaCl. The fractions containing His-SUMO-STING were pooled and concentrated, dialyzed against 50 mM Tris (pH 7.5), 150 mM NaCl while incubating with the SUMO protease enzyme His-ULP1, and the His-SUMO tag was removed overnight. This solution was incubated again with HisPur cobalt resin to remove the His-SUMO tag and STING was collected from the flow-through. The protein was dialyzed against 50 mM Tris (pH 7.5), loaded onto a HitrapQ anion exchange column (GE Healthcare) using an Aekta FPLC (GE Healthcare), and eluted with an NaCl gradient. The fractions containing STING were pooled, buffer-exchanged into PBS, and stored at 4 °C until use.

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

[0538] Liquid chromatography-tandem mass spectrometry

[0539] Cyclic GMP- 13 C 10 , 15 using N5-AMP as an internal standard, cyclic 13 C 10 , 15 5-GMP- 13 C 10 , 15N5-AMP was used as the extraction standard. The isotope-labeled cGAMP standard was synthesized by incubating 1 mM ATP (isotope-labeled), 1 mM GTP (isotope-labeled), 20 mM MgCl2, 0.1 mg / mL salmon testis DNA (Sigma), and 2 μM sscGAS in 100 mM Tris (pH 7.5) overnight. The reaction was heated at 95 °C and filtered through a 3 kDa centrifugal filter. Water was removed by rotary evaporator. cGAMP was purified from the crude reaction mixture using a preparative HPLC (1260 Infinity LC system; Agilent Technologies) with a PLRP-S polymer reverse-phase preparative column (100 Å, 8 μm, 300 × 25 mm; Agilent Technologies) connected to a UV-visible detector (ProStar; Agilent Technologies) and a fraction collector (440-LC; Agilent Technologies). The flow rate was set at 25 mL / min. The mobile phase consisted of 10 mM triethylammonium acetate in water and acetonitrile. The mobile phase was started as 2% acetonitrile for the first 5 minutes. Next, acetonitrile was increased to 30% from 5 - 20 minutes, to 90% from 20 - 22 minutes, maintained at 90% from 22 - 25 minutes, and then decreased to 2% from 25 - 28 minutes. The fractions containing cGAMP were lyophilized and resuspended in water. The concentration was determined by measuring the absorbance at 280 nm. The cGAMP, ATP, and GTP contents of the samples were analyzed by a Shimadzu HPLC (San Francisco, CA) connected to an AB Sciex 4000 QTRAP (Foster City, CA) equipped with an autosampler set at 4 °C. A 10 μL aliquot was injected onto a Biobasic AX LC column, 5 μm, 50 × 3 mm (Thermo Scientific). The mobile phase consisted of 100 mM ammonium carbonate (A) and 0.1% formic acid in acetonitrile (B). The initial conditions were B 90% and maintained for 0.5 minutes.The mobile phase was increased to 30% A over 0.5 to 2.0 minutes, maintained at 30% A from 2.0 to 3.5 minutes, increased to 90% B from 3.5 to 3.6 minutes, and maintained at 90% B from 3.6 to 5 minutes. The flow rate was set at 0.6 mL / min. The mass spectrometer was operated in electrospray positive ion mode with the source temperature set at 500 °C. Declustering and collision-induced dissociation were performed using nitrogen gas. The declustering potential and collision energy were optimized by direct injection of the standard. For each molecule, the MRM transitions (m / z), DP (V), and CE (V) were as follows: ATP (508>136, 341, 55), GTP (524>152, 236, 43), cGAMP (675>136, 121, 97; 675>312, 121, 59; 675>152, 121, 73), internal standard cyclic GMP-. 13 C 10 , 15 N5-AMP (690>146, 111, 101; 690>152, 111, 45; 690>327, 111, 47), extraction standard cyclic 13 C 10 , 15 N5-GMP- 13 C 10 , 15 N5-AMP (705>156, 66, 93; 705>162, 66, 73).

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

[0541] 293T cGAS ENPP1 - / -Cells were plated in tissue culture-treated plates (Advanced BioMatrix) coated with PurCol. After 24 hours, the medium was gently removed and replaced with serum-free DMEM supplemented with 1% insulin-transferrin-selenium-sodium pyruvate (ThermoFisher) and 100 U / mL penicillin-streptomycin. At the indicated times, the medium was removed and the cells were washed off the plate with cold PBS. Both the medium and cells were centrifuged at 1000 rcf for 10 minutes at 4 °C. Cells were lysed in 30 - 100 μL of 50:50 acetonitrile:water supplemented with 500 nM internal standard and centrifuged at 15,000 rcf for 20 minutes at 4 °C to remove the insoluble fraction. If concentration was not required, an aliquot of the medium was taken and supplemented with 500 nM internal standard and 20% formic acid. If concentration was required, the medium was acidified with 0.5% acetic acid and supplemented with extraction standard (an appropriate amount to a final concentration of 2 μM in 100 μL). As described by Gao, D. et al. (Activation of cyclic GMP-AMP synthase by self-DNA causes autoimmune diseases. Proc. Natl. Acad. Sci. U. S. A. 112, E5699-705 (2015)), the medium was applied to a HyperSep aminopropyl SPE column (ThermoFisher Scientific) to enrich for cGAMP. The eluate was evaporated to dryness and reconstituted in 50:50 acetonitrile:water supplemented with 500 nM internal standard. Medium and cell extracts were subjected to mass spectrometric quantification of cGAMP, ATP, and GTP.

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

[0543] In accordance with the manufacturer's instructions, pcDNA3 plasmid DNA (empty or containing human ENPP1) at the indicated concentrations was added to 293T cGAS ENPP1 - / -The cells were transfected with Fugene6 (Promega). Twenty-four hours after transfection, the efflux assay was performed as described above.

[0544] Transfer of conditioned medium

[0545] 293T cGAS ENPP1 low The cells were plated and transfected with plasmid DNA as described above. Twenty-four hours after transfection, the medium was changed to RPMI + 2% human serum + 1% penicillin-streptomycin, + / - 2 μM cGAMP, + / - 20 nM recombinant mENPP1 or + / - 50 μM compound 1. Twenty-four hours after changing the medium, 293T cGAS ENPP1 low The conditioned medium was removed from the cells and incubated with freshly isolated CD14 + PBMCs. The gene expression levels of CD14 + PBMCs were analyzed 14 - 16 hours later.

[0546] RT-PCR analysis

[0547] Total RNA was extracted using Trizol (Thermo Fisher Scientific), and Maxima H Minus reverse transcriptase (Thermo Fisher It was reverse-transcribed using (Scientific). Real-time RT-PCR was performed in duplicate using the 7900HT Fast Real-Time PCR System (Applied Biosystems) with AccuPower 2X Greenstar qPCR Master Mix (Bioneer). The data were normalized to the expression levels of CD14, ACTB, or GAPDH for each sample. The induction fold was calculated using ΔΔCt. Primers for human IFNB1: forward (5’-AAACTCATGAGCAGTCTGCA-3’), reverse (5’-AGGAGATCTTCAGTTTCGGAGG-3’); human CD14: forward (5’-GCCTTCCGTGTCCCCACTGC-3’), reverse (5’-TGAGGGGGCCCTCGACG-3’); human ACTB: forward (5’-GGCATCCTCACCCTGAAGTA-3’), reverse (5’-AGAGGCGTACAGGGATAGCA-3’); human GAPDH: forward (5’-CCAAGGTCATCCATGACAAC-3’); reverse (5’-CAGTGAGCTTCCCGTTCAG-3’).

[0548] 32 P-cGAMP degradation TLC assay

[0549] Radioactive labeling 32 P cGAMP contains unlabeled ATP (1 mM) and 2 μM of purified recombinant porcine cGAS in 20 mM Tris (pH 7.5), 2 mM MgCl2, 100 μg / mL salmon testis DNA 32 P-ATP-doped GTP (1 mM) was synthesized by incubating overnight at room temperature, and the remaining nucleotide starting materials were degraded using alkaline phosphatase for 4 hours at 37°C. Cell lysates were in 100 μL of 10 mM Tris, 150 mM NaCl, 1.5 mM MgCl2, 1% NP-40 (pH 9.0) containing 1×10 6 cells (293T) or 10×10 6Generated by lysing cells (4T1-Luc, E0771, and MDA-MB-231). For 4T1-Luc, E0771, and MDA-MB-231, the total protein concentration of the lysate was measured using a BCA assay (Pierce, Thermo Fisher), and the samples were normalized such that the same amount of protein was used for each lysate reaction. 100 mM In Tris, 150 mM NaCl, 2 mM CaCl2, 200 μM ZnCl2 (pH 7.5 or pH 9.0), the probe 32 P-cGAMP (5 μM) was incubated with mENPP1 (20 nM) or whole cell lysates for the indicated time periods. To generate inhibition curves, 5-fold dilutions of ENPP1 inhibitors were included in this reaction. Degradation was evaluated by TLC (see, for example, Li, L. et al. Hydrolysis of 2’3’-cGAMP by ENPP1 and design of nonhydrolyzable analogs. Nat. Chem. Biol. 10, 1043-8 (2014)). Plates were exposed to a fluorescence screen (Molecular Dynamics) and imaged on a Typhoon 9400, 32 and P signals were quantified using ImageJ. Inhibition curves were fitted, and IC 50 values were obtained using Graphpad Prism 7.03. The IC 50 values were converted to K i,app values using the Cheng-Prusoff equation K 50 =IC m / (1 + [S] / K i,app ).

[0550] ALPL and ENPP2 inhibition assays

[0551] In the 96-well plate format, the inhibition assay for other ectonucleotidases was performed by incubating the reaction components at room temperature and monitoring the generation of 4-nitrophenolate by measuring the absorbance at 400 nM in a plate reader (Tecan). ALPL: 0.1 nM ALPL, 2 μM 4-nitrophenyl phosphate, and various concentrations of inhibitor in a buffer (pH 9.0) containing 50 mM Tris, 20 μM ZnCl2, 1 mM MgCl2 at room temperature. ENPP2: 2 nM ENPP2, 500 μM bis(4-nitrophenyl) phosphate, and various concentrations of inhibitor in a buffer (pH 9.0) containing 100 mM Tris, 150 mM NaCl, 200 μM ZnCl2, 2 mM CaCl2.

[0552] Efflux assay in cancer cell lines

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

[0554] 4T1-Luc tumor mouse model

[0555] 5×10 4 or 5×10 5 4T1-Luc-luciferase cells suspended in 50 μL of PBS were inoculated into the mammary fat pads of 7- to 9-week-old female BALB / c mice (Jackson Laboratories). Tumor volume (determined as length 2 × width / 2) was 80 mm 3 ~120 mm 3Upon reaching, 20 Gy of radiation was delivered to the tumor using a 225 kVp cabinet-type X-ray irradiation device (IC250, Kimtron Inc., CT) with a 0.5 mm Cu filter. Anesthetized animals were shielded using a 3.2 mm lead shield with a 15 × 20 mm gap at the location of the tumor. On days 2, 4, and 7 after IR, 100 μL of 100 μM compound 1 and / or 10 μg of cGAMP in PBS or PBS alone were injected intratumorally. Alternatively, after IR, on days 2, 5, and 7, 1 mM compound 1 in PBS or PBS alone was injected intratumorally, and 200 μg of anti-CTLA-4 antibody or Syrian hamster IgG antibody (both from BioXCell) were injected intraperitoneally. Mice from different treatment groups were co-housed in each cage to eliminate cage effects. The experimenter was blinded throughout the study. Tumor volume was recorded every other day. To account for the correlation within mice, tumor volume was analyzed using a generalized estimating equation. Pairwise comparisons of treatment groups at each time point were performed using a post hoc test with Tukey's adjustment for multiple comparisons. Using Graphpad Prism 7.03, animal deaths were plotted on a Kaplan-Meier curve, and statistical significance was evaluated using a log-rank Mantel-Cox test. All mice were maintained at Stanford University in accordance with the regulations of the Stanford University Institutional Animal Care and Use Committee, and the procedures were approved by the Stanford University Administrative Panel on Laboratory Animal Care.

[0556] FACS analysis of tumors

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

[0558] in vivo imaging

[0559] 3 mg of XenoLight D-luciferin (Perkin-Elmer) in 200 μl of water was injected ip into mice and imaged using a Lago X in vivo imaging system (Spectral Instruments Imaging). The objective height was set to 1.5 cm, binning to 4, f-stop to 1.2, and the exposure time to 120 seconds. Images were analyzed using aura2.0.1 software (Spectral Instruments Imaging). Results

[0560] cGAMP is secreted as a soluble factor from 293T cGAS ENPP1 - / - cells. To test the hypothesis that cGAMP exists extracellularly, the inventors first developed a liquid chromatography-tandem mass spectrometry (LC-MS / MS) method for detecting cGAMP from complex mixtures. The inventors were able to quantify cGAMP concentrations down to 0.5 nM in both basal cell culture medium and serum-containing medium using two isotope-labeled cGAMP standards (panel A of FIG. 1), and the inventors were able to quantify intracellular cGAMP concentrations from cell extracts in the same experiment (panel B of FIG. 1, as well as panels A and B of FIG. 8). The inventors chose to use 293T cells that do not express either cGAS or STING. The inventors generated a 293T cGAS ENPP1 low cell line by stably expressing murine cGAS and knocking out ENPP1 using CRISPR (panel C of FIG. 8). Next, the inventors isolated single clones and generated a 293T cGAS ENPP1 - / - cell line (panel C of FIG. 8). Since serum contains a soluble form cleaved by proteolysis of ENPP1, the inventors also used serum-free medium. The inventors used this ENPP1-free cell culture system to culture 293T cGAS ENPP1 without any stimulation - / -A basal intracellular cGAMP concentration in the low micromolar range was detected in the cells (Panel C of FIG. 1). This is not surprising as cytosolic dsDNA is present in large amounts in cancer cells as a result of incorrect DNA segregation (see, for example, Mackenzie, K. J. et al. cGAS surveillance of micronuclei links genome instability to innate immunity. Nature 548, 461-465 (2017); Harding, S. M. Mitotic progression following DNA damage enables pattern recognition within micronuclei. Nature 548, 466-470 (2017); and Bakhoum, S. F. et al. Chromosomal instability drives metastasis through a cytosolic DNA response. Nature 553, 467-472 (2018)). After the inventors replenished the cells with fresh medium, a linear increase up to 100 nM in the extracellular cGAMP concentration was measured after 30 hours (Panel D of FIG. 1). At 30 hours, the number of cGAMP molecules outside the cells was equal to the number inside (Panel E of FIG. 1). The inventors detected a negligible amount of cell death based on extracellular lactate dehydrogenase (LDH) activity, suggesting that cGA MP is excreted by live cells (Panel E of FIG. 1). The inventors calculated that the excretion rate (v export ) is 220 molecules cell -1 s -1 (Panel F of FIG. 1). Finally, cGAMP in the medium was able to pass through a 10 kDa filter without any retention, suggesting that extracellular vesicles and proteins should have been retained and that cGAMP is excreted as a freely soluble molecule (Panel H of FIG. 1).

[0561] To further confirm that extracellular cGAMP secreted by 293T cells is mainly in a soluble form and not present in extracellular vesicles, the inventors used CD14 as a reporter + human peripheral blood mononuclear cells (PBMCs). These cells have previously been shown to absorb soluble cGAMP, which results in IFN-β production 17 . The inventors + observed that PBMCs respond to concentrations of soluble cGAMP below micromolar concentrations by upregulating IFNB1 (Figure 9). Conditioned medium from 293T cGAS ENPP1 low cells expressing transfected DNA cGAS induced IFNB1 expression in CD14 + cells, while conditioned medium from DNA-transfected cGAS-null 293T cells did not. This suggests that this activity is the result of extracellular cGAMP produced by 293T cells (panels H and I of Figure 1). Addition of purified soluble recombinant mouse ENPP1 (mENPP1) (panel D of Figure 8) depleted detectable cGAMP in the conditioned medium and also, again, lost this activity (panels H and J of Figure 1). Since soluble ENPP1 (MW = approximately 100 kDa) cannot penetrate the membrane and thus can only access soluble extracellular cGAMP, the inventors conclude that 293T cells secrete soluble cGAMP. In summary, the inventors' data demonstrate that this artificial cancer cell line maintains its intracellular cGAMP at a steady state by excreting intracellular cGAMP into the extracellular medium as a soluble factor

[0562] Panels A-J of Figure 1: cGAMP is present in 293T cGAS ENPP1 - / -It is secreted from cells as a soluble factor. a, Chemical structures of cGAMP and singly isotope-labeled cGAMP, b, cGAMP is detected by LC-MS / MS. Lower limit of quantification = 4 nM. (Left) Liquid chromatography traces of cGAMP at 0, 4, and 10 nM, and singly isotope-labeled cGAMP (15 Da heavier) at 500 nM as an internal standard; (Right) External standard curve of cGAMP, R 2 = 0.996. Data are representative of more than 10 independent experiments. c–d, Intracellular and extracellular concentrations of cGAMP from 293T cGAS ENPP1 cells measured using LC-MS / MS in the absence of exogenous stimulation - / - Intracellular and extracellular concentrations of cGAMP from cells. At 0 h, cells were supplemented with serum-free medium. Mean ± standard error with some error bars too small to visualize (n = 2). Data are representative of 3 independent experiments. e, Ratio of extracellular / total lactate dehydrogenase (LDH) activity (right y-axis) compared to the ratio of extracellular / total cGAMP molecules calculated from the data in (c) and (d) (left y-axis). f, Amount of cGAMP excreted per cell over time calculated from the data in (d). The excretion rate was found using linear regression. g, Intracellular and extracellular cGAMP concentrations produced by 293T cGAS ENPP1 cells measured before and after passing the medium through a 10 kDa filter. Mean ± standard error (n = 2). Data are representative of 2 independent experiments. h, Schematic of conditioned medium transfer experiments for (i) and (j). cGAS null 293T or 293T cGAS ENPP1 cells were transfected with an empty pcDNA vector and treated with + / - 20 nM recombinant mouse ENPP1 (mENPP1). Conditioned medium from these cells was transferred to primary CD14 - / - human PBMCs. i, IFNB1 mRNA levels were normalized to CD14, and the induction fold was calculated compared to untreated CD14 cells. Mean ± standard error (n = 4). low cells. j, cGAMP concentrations were measured in the conditioned medium. Mean ± standard error (n = 2). + P = 0.0003 (one-way ANOVA). cGAMP concentrations were measured in the conditioned medium. Mean ± standard error (n = 2). + Mean ± standard error (n = 4). *** P = 0.0003 (one-way ANOVA). cGAMP concentrations were measured in the conditioned medium. Mean ± standard error (n = 2). ***P = 0.0002 (one-way ANOVA). Data are representative of two independent experiments. j, IFNB1 mRNA levels were normalized to CD14, and fold induction was calculated relative to untreated CD14 + cells. Mean ± standard error (n = 2). * P = 0.04 (one-way ANOVA). cGAMP concentrations were measured in conditioned media. Mean ± standard error (n = 2). ** P = 0.002 (one-way ANOVA). Data are representative of two independent experiments.

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

[0564] Panel A of Figure 9: CD14 + ​​PBMC responds to extracellular cGAMP. a, Schematic diagram of the stimulation of CD14+ PBMC by extracellular cGAMP. b, Human CD14 stimulated by increasing the concentration of extracellular cGAMP for 16 hours + IFNB1 induction measured by RT-qPCR on PMBC. Mean ± standard error (n = 2, technical qPCR replicates).

[0565] ENPP1 regulates only extracellular cGAMP

[0566] The inventors knocked out ENPP1 derived from 293T cells and cultured these cells in ENPP1-free medium, and for the first time, extracellular cGAMP could be observed. Therefore, the inventors then examined whether extracellular cGAMP is regulated only by ENPP1. Despite its extracellular annotation, ENPP1 can invert its membrane surface orientation as in the case of the enzyme CD38 (see, for example, Zhao, Y. J., Lam, C. M. C. & Lee, H. C. The membrane-bound enzyme CD38 exists in two opposing orientations. Sci. Signal. 5, ra67 (2012)), or it is conceivable that ENPP1 can be activated when synthesized in the ER lumen and that cGAMP can pass through the ER membrane (Panel A of Figure 2). To examine the localization of ENPP1 activity, the inventors transfected 293T cGAS ENPP1 - / - cells with a human ENPP1 expression plasmid and confirmed its activity in whole cell lysates (Panel B of Figure 2). In intact cells, ENPP1 expression depletes extracellular cGAMP but does not affect intracellular cGAMP concentration (Panel C of Figure 2). Therefore, in these cells, extracellular cGAMP is regulated by ENPP1, but intracellular cGAMP is not.

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

[0568] Development of cell - impermeable ENPP1 inhibitors

[0569] To investigate the physiological relevance of extracellular cGAMP and why cGAMP needs to be regulated by specific hydrolases, the inventors attempted to manipulate its concentration by pharmacologically inhibiting ENPP1. The inventors first tested QS1, a non - specific ENPP1 inhibitor (Panel A of FIG. 10) (Patel, S. D. et al. Quinazolin - 4 - piperidin - 4 - methyl sulfamide PC - 1 inhibitors: Alleviating hERG interactions through structure based design. Bioorganic Med. Chem. Lett. 19, 3339 - 3343 (2009); and Shayhidin, E. E. et al. Quinazoline - 4 - piperidine sulfamides are specific inhibitors of human NPP1 and prevent pathological mineralization of valve interstitial cells. Br. J. Pharmacol. 172, 4189 - 4199 (2015)). QS1 can inhibit extracellular cGAMP degradation in cells overexpressing ENPP1, but this also partially blocked the excretion of cGAMP in ENPP1 knockout cells (Panel B of Figure 10). Treatment with QS1 improved intracellular cGAMP, also demonstrating that excretion is an important mechanism for maintaining cGAMP homeostasis in cancer cells. Due to the excretion blocking activity, QS1 is excluded as a means for examining extracellular cGAMP in our excretion studies. Compound 1, a phosphonate analog, was designed to chelate with Zn 2+ at the ENPP1 catalytic site, minimize cell permeability, and avoid intracellular off-targets (Panel A of Figure 3). Compound 1 has a K i,app of 110 ± 10 nM (Panel B of Figure 3), which is approximately 60-fold more potent than QS1 (Panel A of Figure 10).

[0570] We confirmed that Compound 1 is cell-impermeable by performing three independent permeability assays: parallel artificial membrane permeability assay (PAMPA) (Panel A of Figure 11); intestinal cell Caco-2 permeability assay (Panel B of Figure 11); and epithelial cell MDCK permeability assay (Panel C of Figure 11). Compared to control compounds with high and low cell permeabilities, Compound 1 falls into the classification of non-permeable compounds in all three assays. Furthermore, Compound 1 has a K i,app > 100 μM) for alkaline phosphatase, an ectonucleotidase closely related to it, and K i,appThe activity against (=5.5 μM) is low (Panel D of Figure 11). Although the inventors did not expect Compound 1 to have intracellular off-targets due to its low cell permeability, the inventors tested its binding to a panel of 468 kinases to further determine its specificity. Despite having a structural similarity to AMP, Compound 1 binds to only two kinases at 1 μM (Panel E of Figure 11). Compound 1 also shows high stability (t 1 / 2 > 159 minutes) in both human and mouse liver microsomes. In summary, the inventors demonstrated that Compound 1 is a potent, cell-impermeable, specific, and stable ENPP1 inhibitor.

[0571] Next, the inventors measured the efficacy of Compound 1 in maintaining the extracellular cGAMP concentration in 293T cGAS cells overexpressing ENPP1 and obtained an IC 50 value of 340 ± 160 nM (Panel C of Figure 3), and 10 μM was sufficient to completely block extracellular cGAMP degradation (Panel D of Figure 3). Unlike QS1, Compound 1 did not affect intracellular cGAMP, and it was demonstrated that Compound 1 did not affect the efflux of cGAMP (Panel D of Figure 3). Thus, Compound 1 is an excellent means compound of an ENPP1 inhibitor that specifically increases extracellular cGAMP concentration.

[0572] Finally, the inventors tested the efficacy of Compound 1 in enhancing the detectable extracellular cGAMP signal against CD14 + PBMC. The inventors first confirmed that Compound 1 was not toxic to PBMC at the concentrations used (Panel F of Figure 11). The conditioned medium derived from 293T cGAS cells overexpressing ENPP1 could not induce IFNB1 expression in CD14 + cells (Panels E and F of Figure 3). However, Compound 1 rescued the extracellular cGAMP levels in the medium and the induction of IFNB1 expression in CD14 + cells (Panel F of Figure 3). These results indicate that the enzymatic activity of ENPP1, without the potential scaffolding effect as a transmembrane protein, in CD14+ It has been demonstrated to suppress the response to extracellular cGAMP by PBMCs. In summary, our data suggest that extracellular cGAMP levels can be decreased by ENPP1 expression and can be enhanced by ENPP1 inhibition, and these affect the in vitro activation of CD14 + PBMCs.

[0573] Panels A–F of FIG. 3: Activity of cell-impermeable ENPP1 inhibitor. a, Chemical structure of compound 1. b, Inhibition activity of compound 1 against purified mouse ENPP1 with P-cGAMP as substrate at pH 7.5 (K 32 = 110 ± 10 nM). Mean ± standard error with some error bars too small to visualize (n = 3 independent experiments). c, Inhibition activity of compound 1 against human ENPP1 transiently expressed in 293T cGAS ENPP1 i,app cells (IC - / - = 340 ± 160 nM). Mean ± standard error (n = 2). d, Intracellular and extracellular cGAMP concentrations in 293T cGAS ENPP1 50 cells transfected with empty pcDNA vector or vector containing hu...

Claims

[Claim 1] The invention described in this specification.

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