Patent Application
20240374724
The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML file, created on May 23, 2024, is named AGT-103WOC1_SL.xml, and is 5,491 bytes in size.
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 gets activated in presence of cytoplasmic DNA either due to microbial infection or patho-physiological condition, including cancer and autoimmune disorder. Cyclic GMP-AMP synthase (cGAS) belongs to the nucleotidyltransferase family and is a universal DNA sensor that is activated upon binding to cytosolic dsDNA to produce the signaling molecule (2′-5′, 3′-5′) cyclic GMP-AMP (or 2′, 3′-cGAMP or cyclic guanosine monophosphate-adenosine monophosphate, cGAMP). Acting as a second messenger during microbial infection, 2′, 3′-cGAMP binds and activates STING, leading to production of type I interferon (IFN) and other co-stimulatory molecules that trigger the immune response. Besides its role in infectious disease, the STING pathway has is under exploration as a target for cancer immunotherapy and autoimmune diseases.
Ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) is the dominant hydrolase of cGAMP that can degrade cGAMP. ENPP1 is a member of the ecto-nucleotide pyrophosphatase/phosphodiesterase (ENPP) family. The encoded protein is a type II transmembrane glycoprotein comprising two identical disulfide-bonded subunits. The ENPP1 protein has broad specificity and can cleave a variety of substrates, including phosphodiester bonds of nucleotides and nucleotide sugars and pyrophosphate bonds of nucleotides and nucleotide sugars. This protein may function to hydrolyze nucleoside 5′ triphosphates to their corresponding monophosphates and may also hydrolyze diadenosine polyphosphates.
CAR cell therapy has shown promising results in the clinic in treating some hematological cancers, such CD-19-expressing B cell malignancies. However, studies exploring CAR cell therapy for treating other cancers have demonstrated variable efficacy, in part due to the limited persistence and proliferation of the CAR-expressing cells in vivo. In particular, treatment of patients with solid tumors using CAR T cell therapy has been less successful. Potential barriers to CAR T cell efficacy against solid tumors include suboptimal migration and persistence of CAR T cells in the tumor microenvironment (TME), impaired function mediated by the immunosuppressive TME, and CAR T cell exhaustion.
Thus, there is need for developing therapies that enhance the efficacy of CAR cell therapy in certain tumors such as solid tumors.
Compounds, compositions, and methods are provided for the inhibition of ENPP1 in combination with immune cells expressing chimeric antigen receptors for treating a disease associated with expression of a tumor antigen, e.g., a cancer. ENPP1 inhibitor compounds can act extracellularly to block the degradation of cGAMP. Aspects of the subject methods include contacting a sample with a cell impermeable ENPP1 inhibitor to inhibit the cGAMP hydrolysis activity of ENPP1 and enhance the migration and persistence of CAR T cells in the TME.
Disclosed for herein are compounds for stimulating the STING pathway, particularly through regulating ENPP1, used in combination with CAR T cells and methods of use of same in a patient-specific combination therapy that can be used to treat cancers and other diseases and/or conditions.
Provided for herein is a composition comprising: an ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) inhibitor.
In some aspects, the ENPP1 inhibitor comprises the formula (VI):
wherein, X is a hydrophilic head group selected from phosphonic acid, phosphonate, phosphonate ester, phosphate, phosphate ester, thiophosphate, thiophosphate ester, phosphoramidate and thiophosphoramidate; L is a linker; Z1 and Z2 are each independently selected from CR1 and N; Z3 and Z4 are each independently selected from CR and N, wherein R is H, alkyl or substituted alkyl; each R1 is independently selected from H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, heterocycle and substituted heterocycle; R2 and R5 are each independently selected from H, OH, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, —OCF3, halogen, amine, substituted amine, amide, heterocycle and substituted heterocycle; R3 and R4 are each independently selected from H, OH, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, —OCF3, halogen, amine, substituted amine, amide, heterocycle and substituted heterocycle; or R3 and R4 together with the carbon atoms to which they are attached form a fused selected from heterocycle, substituted heterocycle, cycloalkyl, substituted cycloalkyl, aryl and substituted aryl; or a pro-drug, a pharmaceutically acceptable salt or a solvate thereof.
In some aspects, L is selected from —CH2—, —(CH2)2—, —(CH2)3—, —(CH2)4—, —(CH2)5— and —(CH2)6—; X is selected from:
wherein: Ra and Rb are each independently selected from aryl, alkyl, —CH2OC(O)Re, —CH2OC(O)ORe; and Re and Rd are each independently selected from —C(CH3)C(O)ORe, alkyl and wherein Re is alkyl.
In some aspects, the ENPP1 inhibitor is of the formula:
wherein, Z1 and Z2 are each N; Z3 is N; and Z4 is CH or N.
In some aspects, the ENPP1 inhibitor comprises a group selected from:
In some aspects, the inhibitor is a compound of Table 1, Table 2, Table 3, Table 4, Table 5, or Table 6.
In some aspects, the composition further comprises a cyclic GMP-AMP Synthase (cGAS)/Stimulator of Interferon Genes (STING) pathway agonist. In some aspects, the cGAS/STING pathway agonist is a cyclic-dinucleotide (CDN). In some aspects, the CDN is 2′3′-cyclic-GMP-AMP (2′3′-cGAMP).
In some aspects, the cGAS/STING pathway agonist is a cGAS ligand. In some aspects, the cGAS ligand is a virus-derived nucleic acid.
In some aspects, the composition further comprises an additional adjuvant, optionally wherein the additional adjuvant is selected from the group consisting of: alum, CpG oligonucleotides, Freund's adjuvant, 1018 ISS, aluminium salts, Amplivax, AS15, BCG, CP-870,893, CpG7909, CyaA, dSLIM, GM-CSF, IC30, IC31, Imiquimod, ImuFact IMP321, IS Patch, ISS, ISCOMATRIX, JuvImmune, LipoVac, MF59, monophosphoryl lipid A, lipopolyscharride, Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, Montanide ISA-51, OK-432, OM-174, OM-197-MP-EC, ONTAK, PepTel vector system, PLG microparticles, resiquimod, SRL172, Virosomes and other Virus-like particles, YF-17D, VEGF trap, R848, beta-glucan, Pam3Cys, Aquila's QS21 stimulon, mycobacterial extracts, synthetic bacterial cell wall mimics, and Ribi's Detox.
Also provided for herein is a method of treating a cancer in a subject in need thereof, comprising administering a therapeutically effective amount of a CAR expressing immune cell in combination with a therapeutically effective amount of a composition comprising an ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) inhibitor.
Also provided for herein is a method of treating a cancer in a subject in need thereof, comprising administering a therapeutically effective amount of a CAR expressing immune cell in combination with a therapeutically effective amount of a composition, wherein the composition is any one of the compositions provided herein.
Also provided for herein is a method of stimulating an immune response, treating a disease, or preventing a disease in a subject, the method comprising administering to the subject a therapeutically effective amount of a CAR expressing immune cell in combination with a therapeutically effective amount of a composition comprising an ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) inhibitor.
Also provided for herein is a method of stimulating an immune response, treating a disease, or preventing a disease in a subject, the method comprising administering to the subject a therapeutically effective amount of a CAR expressing immune cell in combination with a therapeutically effective amount of a composition, wherein the composition is any one of the compositions provided herein.
Also provided for herein is a method of treating or preventing a disease in a subject, optionally wherein the disease is a cancer, the method comprising administering to the subject a therapeutically effective amount of a CAR expressing immune cell in combination with a therapeutically effective amount of a composition comprising an ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) inhibitor.
Also provided for herein is a method of stimulating an immune response in a subject, the method comprising administering to the subject a therapeutically effective amount of a CAR expressing immune cell in combination with a therapeutically effective amount of a composition comprising an ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) inhibitor.
Also provided for herein is a method of stimulating an immune response in a subject, the method comprising administering to the subject a therapeutically effective amount of a CAR expressing immune cell in combination with a therapeutically effective amount of a composition comprising an ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) inhibitor, wherein the ENPP1 inhibitor is of the formula (VI):
wherein, X is a hydrophilic head group selected from phosphonic acid, phosphonate, phosphonate ester, phosphate, phosphate ester, thiophosphate, thiophosphate ester, phosphoramidate and thiophosphoramidate; L is a linker; Z1 and Z2 are each independently selected from CR1 and N; Z3 and Z4 are each independently selected from CR and N, wherein R is H, alkyl or substituted alkyl; each R1 is independently selected from H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, heterocycle and substituted heterocycle; R2 and R5 are each independently selected from H, OH, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, —OCF3, halogen, amine, substituted amine, amide, heterocycle and substituted heterocycle; R3 and R4 are each independently selected from H, OH, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, —OCF3, halogen, amine, substituted amine, amide, heterocycle and substituted heterocycle; or R3 and R4 together with the carbon atoms to which they are attached form a fused selected from heterocycle, substituted heterocycle, cycloalkyl, substituted cycloalkyl, aryl and substituted aryl; or a pro-drug, a pharmaceutically acceptable salt or a solvate thereof. In some embodiments, the composition further comprises a cyclic GMP-AMP Synthase (cGAS)/Stimulator of Interferon Genes (STING) pathway agonist, wherein the cGAS/STING pathway agonist comprises 2′3′-cGAMP.
In some aspects, the ENPP1 inhibitor and the cGAS/STING pathway agonist are co-formulated. In some aspects, the ENPP1 inhibitor and/or the cGAS/STING pathway agonist are administered by mucosal delivery. In some aspects, the mucosal delivery comprises buccal delivery, sublingual delivery, or intranasal delivery.
Also provided for herein is a pharmaceutical composition comprising: a) an ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) inhibitor; and b) a nanoparticle, wherein the pharmaceutical composition is formulated for mucosal delivery.
Also provided for herein is a pharmaceutical composition comprising: a) a cyclic GMP-AMP Synthase (cGAS)/Stimulator of Interferon Genes (STING) pathway agonist; and b) a nanoparticle, wherein the pharmaceutical composition is formulated for mucosal delivery, wherein the mucosal delivery comprises buccal delivery or sublingual delivery.
Also provided for herein is a pharmaceutical composition comprising: a) an ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) inhibitor; b) a cyclic GMP-AMP Synthase (cGAS)/Stimulator of Interferon Genes (STING) pathway agonist; and c) a nanoparticle, wherein the pharmaceutical composition is formulated for mucosal delivery.
In some aspects, the nanoparticle comprises a liposome. In some aspects, the nanoparticle comprises a hydrogel. In some aspects, the liposome comprises a pulmonary surfactant, a pulmonary surfactant membrane constituent, and/or a pulmonary surfactant biomimetic. In some aspects, the liposome, the pulmonary surfactant, the pulmonary surfactant membrane constituent, and/or the pulmonary surfactant biomimetic is negatively charged.
In some aspects, the mucosal delivery comprises buccal delivery, sublingual delivery, or intranasal delivery.
Also provided for herein is a method of stimulating an immune response, treating a disease, or preventing a disease in a subject, the method comprising administering to the subject a therapeutically effective amount of a CAR expressing immune cell in combination with a therapeutically effective amount of a composition, wherein the composition is any one of the compositions provided herein.
Also provided for herein is a method of treating or preventing a disease in a subject, optionally wherein the disease is cancer, the method comprising administering to the subject a therapeutically effective amount of a CAR expressing immune cell in combination with a therapeutically effective amount of a pharmaceutical composition comprising an ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) inhibitor and/or a cyclic GMP-AMP Synthase (cGAS)/Stimulator of Interferon Genes (STING) pathway agonist; and b) a nanoparticle, wherein the administering the pharmaceutical composition is administered by mucosal delivery.
Also provided for herein is a method of stimulating an immune response in a subject, the method comprising administering to the subject a therapeutically effective amount of a CAR expressing immune cell in combination with a therapeutically effective amount of a pharmaceutical composition comprising an ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) inhibitor and/or a cyclic GMP-AMP Synthase (cGAS)/Stimulator of Interferon Genes (STING) pathway agonist; and b) a nanoparticle, wherein the administering the pharmaceutical composition is administered by mucosal delivery.
In some aspects, the mucosal delivery comprises buccal delivery, sublingual delivery, or intranasal delivery. In some aspects, the ENPP1 inhibitor and the cGAS/STING pathway agonist are co-formulated.
Also provided for herein is a method of enhancing persistence of a CAR expressing immune cell in a tumor microenvironment, comprising administering a therapeutically effective amount of the CAR expressing immune cell in combination with a therapeutically effective amount of a composition comprising an ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) inhibitor.
Also provided for herein is a method of enhancing infiltration of a CAR expressing immune cell in a tumor microenvironment, comprising administering a therapeutically effective amount of the CAR expressing immune cell in combination with a therapeutically effective amount of a composition comprising an ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) inhibitor.
Also provided for herein is use of a pharmaceutical composition for enhancing persistence of a CAR expressing immune cell in a tumor microenvironment, wherein the composition comprises an ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) inhibitor, and wherein the composition is administered in combination with the CAR expressing immune cell.
Also provided for herein is use of a pharmaceutical composition for enhancing infiltration of a CAR expressing immune cell in a tumor microenvironment, wherein the composition comprises an ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) inhibitor, and wherein the composition is administered in combination with the CAR expressing immune cell.
In some aspects, the immune cell of the CAR expressing immune cell is an alpha/beta T cell, a gamma/delta T cell, a B cell, a natural killer (NK) cell, a natural killer T (NKT) cell, a mast cell, or a myeloid-derived phagocyte.
These and other advantages and features of the disclosure will become apparent to those persons skilled in the art upon reading the details of the compositions and methods of use, which are more fully described below.
The invention is best understood from the following detailed description when read in conjunction with the accompanying figures. The patent or application file contains at least one figure executed in color. It is emphasized that, according to common practice, the various features of the figures are not to-scale. On the contrary, the dimensions of the various features are arbitrarily expanded or reduced for clarity. Included in the drawings are the following figures. It is understood that the figures, described below, are for illustration purposes only. The figures are not intended to limit the scope of the present teachings in any way.
The disclosure is based, in part, upon the discovery that ENPP1 inhibitors can promote the trafficking and proliferation of immune cells expressing chimeric antigen receptors (CAR cells). As summarized above, aspects of the present disclosure include compounds, compositions, and methods for the inhibition of ENPP1. Aspects of the methods include contacting a sample with a cell impermeable ENPP1 inhibitor to inhibit cGAMP hydrolysis activity of ENPP1.
Also provided are compositions and methods for treating cancer. Aspects of the methods include administering to a subject an effective amount of an ENPP1 inhibitor to treat the subject for cancer. Aspects of the methods include administering to a subject an effective amount of a cell impermeable ENPP1 inhibitor to inhibit the hydrolysis of cGAMP and treat the subject for cancer.
These compounds and methods find use in a variety of applications in which inhibition of ENPP1 is desired.
As summarized above, aspects of the disclosure include ENPP1 inhibitor compounds. The subject compounds can include a core structure based on an aryl or heteroaryl ring system, e.g., a quinazoline, isoquinoline or pyrimidine group, which is linked to a hydrophilic head group. The linker between the aryl or heteroaryl ring system and the hydrophilic head group can include a monocyclic carbocycle or heterocycle and an acyclic linker. In some cases, the linker includes a 1,4-disubstituted 6-membered ring, such as cyclohexyl, piperidinyl or piperazinyl. The aryl or heteroaryl ring system is optionally further substituted. Exemplary ENPP1 inhibitor compounds of interest including quinazoline, isoquinoline and pyrimidine ring systems are set forth in formulae I IV, V, VI and VII and the following structures 1-106.
In some cases, the subject ENPP1 inhibitor compound is of formula (I):
Y-A-L-X (I)
The term “hydrophilic head group” refers to a linked group of the subject compounds that is hydrophilic and well solvated in aqueous environments e.g., under physiological conditions, and has low permeability to cell membranes. In some cases, by low permeability to cell membranes is meant a permeability coefficient of 10−4 cm/s or less, such as 10−5 cm/s or less, 10−6 cm/s or less, 10−7 cm/s or less, 10−8 cm/s or less, 10−9 cm/s or less, or even less, as measured via any convenient methods of passive diffusion for an isolated hydrophilic head group through a membrane (e.g., cell monolayers such as the colorectal Caco-2 or renal MDCK cell lines). See e.g., Yang and Hinner, Methods Mol Biol. 2015; 1266: 29-53.
The hydrophilic head group can impart improved water solubility and reduced cell permeability upon the molecule to which it is attached. The hydrophilic head group may be any convenient hydrophilic group that is well solvated in aqueous environments and which has low permeability to membranes. In certain instances, the hydrophilic group is a discrete functional group (e.g., as described herein) or a substituted version thereof. In general terms, larger, uncharged polar groups or charged groups have low permeability. In some cases, the hydrophilic head group is charged, e.g., positively or negatively charged. In some embodiments, the hydrophilic head group is not cell permeable and imparts cell impermeability upon the subject compound. It is understood that a hydrophilic headgroup, or a prodrug form thereof, can be selected to provide for a desired cell permeability of the subject compound. In certain cases, the hydrophilic head group is a neutral hydrophilic group. In some cases, the hydrophilic head group comprises a promoiety. In certain instances, the subject compound is cell permeable.
In some embodiments of formula (I), the hydrophilic head group (X) is selected from phosphonic acid or phosphonate, phosphonate ester, phosphate, phosphate ester, thiophosphate, thiophosphate ester, phosphoramidate, thiophosphoramidate, sulfonate, sulfonic acid, sulfate, hydroxamic acid, keto acid, amide and carboxylic acid. In some embodiments of formula (I), the hydrophilic head group is phosphonic acid, phosphonate, or a salt thereof. In some embodiments of formula (I), the hydrophilic head group is phosphate or a salt thereof. In some embodiments of formula (I), the hydrophilic head group is phosphonate ester or phosphate ester.
Particular examples of hydrophilic head groups of interest include, but are not limited to, a head group comprising a first molecule selected from phosphates (RPO4H−), phosphonates (RPO3H−), boric acid (RBO2H2), carboxylates (RCO2−), sulfates (RSO4−), sulfonates (RSO3−), amines (RNH3+), glycerols, sugars such as lactose or derived from hyaluronic acid, polar amino acids, polyethylene oxides and oligoethyleneglycols, that is optionally conjugated to a residue of a second molecule selected from choline, ethanolamine, glycerol, nucleic acid, sugar, inositol, and serine. The head group may contain various other modifications, for instance, in the case of the oligoethyleneglycols and polyethylene oxide (PEG) containing head groups, such PEG chain may be terminated with a methyl group or have a distal functional group for further modification. Examples of hydrophilic head groups also include, but are not limited to, thiophosphate, phosphocholine, phosphoglycerol, phosphoethanolamine, phosphoserine, phosphoinositol, ethylphosphosphorylcholine, polyethyleneglycol, polyglycerol, melamine, glucosamine, trimethylamine, spermine, spermidine, and conjugated carboxylates, sulfates, boric acid, sulfonates, sulfates and carbohydrates.
Any convenient linkers can be utilized to link A to X. In some cases, A is linked to X via a covalent bond. In certain cases, A is linked to X via a linear linker of 1-12 atoms in length, such as 1-10, 1-8 or 1-6 atoms in length, e.g., 1, 2, 3, 4, 5 or 6 atoms in length. The linker L can be a (C1-6)alkyl linker or a substituted (C1-6)alkyl linker, optionally substituted with a heteroatom or linking functional group, such as an ester (—CO2—), amido (CONH), carbamate (OCONH), ether (—O—), thioether (—S—) and/or amino group (—NR— where R is H or alkyl).
In some instances of formula (I), L is selected from alkyl, substituted alkyl, alkyloxy and substituted alkoxy; and X is selected from phosphonic acid, phosphonate, phosphate, thiophosphate, phosphoramidate and thiophosphoramidate. In some embodiments of formula (I), L-X comprises a group of the formula (XI):
In some embodiments of formula (XI), Z12, Z13 and Z14 are all oxygen atoms and Z15 is CH2. In other cases, Z12 is a sulfur atom, Z13 and Z14 are both oxygen atoms and Z15 is CH2. In other cases, Z12 is a sulfur atom, Z13, Z14, Z15 are all oxygen atoms. In some cases, Z12 is an oxygen atom, Z13 is NR′, Z14 is an oxygen atom and Z15 is a carbon atom. In other cases, Z12 is an oxygen atom, Z13 is a nitrogen atom, Z14 and Z15 are both oxygen atoms. In other cases, Z12 is an oxygen atom, Z13 and Z14 are each independently NR′ and Z15 is an oxygen atom. In yet other cases, Z12 is an oxygen atom, Z13 and Z14 are each independently NR′ and Z15 is CH2.
In some embodiments of formula (XI), R15 and R16 are both hydrogen atoms. In other cases, both R15 and R16 are substituents other than hydrogen. In some cases, R15 and R16 are each independently alkyl or substituted alkyl groups. In some other cases, R15 and R16 are each independently aryl groups. In some cases, R15 and R16 are each independently alkyl groups. In some cases, R15 and R16 are both alkyl groups substituted with an ester. In other cases, R15 and R16 are both alkyl groups substituted with an ester. In certain cases, both R15 and R16 are phenyl groups. In some cases, R15 and R16 are each the same substituent. In other cases, R15 and R16 are different substituents.
In some embodiments of formula (XI), Z15 is a carbon atom and q1 is 0. In other cases, Z15 is a carbon atom and q1 is greater than 0, such as 1, 2, 3, 4, 5 or 6. In some cases, Z15 is a carbon atom and q1 is 1. In other embodiments, Z15 is an oxygen atom and q1 is 1. In other cases, Z15 is an oxygen atom and q1 is greater than 1, such as 2, 3, 4, 5 or 6. In some cases, Z15 is an oxygen atom and q1 is 2.
In some embodiments of formula (XI), L-X is selected from one of the formula groups:
In some embodiments of formula (I), L-X comprises a group of the formula (XII):
In some embodiments of formula (XII), R17 and R18 are both hydrogen atoms. In other cases, both R17 and R18 are substituents other than hydrogen. In certain embodiments of formula (XII), q2 is 1. In certain cases, q2 is greater than 1, such as 2, 3, 4, 5 or 6. In some cases of formula (XII), q2 is 2. In certain embodiments of formula (XII), the hydrophilic head group is of the structure:
In some embodiments of formula (I), L-X comprises a group of the formula (XIII):
wherein q3 is an integer from 1 to 6. In certain embodiments, q3 is 1. In certain embodiments, q3 is greater than 1, such as 2, 3, 4, 5 or 6. In certain embodiments, q3 is 2. In certain embodiments of formula (XIII), the hydrophilic head group is of the structure:
In some embodiments of formula (I), L-X comprises a group of the formula (XIV):
In some embodiments of formula (XIV), Z16 is CH2 and q4 is 0. In other cases, Z16 is CH2 and q1 is greater than 0, such as 1, 2, 3, 4, 5 or 6. In some cases, Z16 is CH2 and q1 is 1. In other embodiments, Z16 is an oxygen atom and q1 is 1. In other cases, Z16 is an oxygen atom and q1 is greater than 1, such as 2, 3, 4, 5 or 6. In some cases, Z16 is an oxygen atom and q1 is 2.
In some embodiments of formula (XIV), the hydrophilic head group is selected from one of the following groups:
In some embodiments of formula (I), L-X comprises a group of the formula (XV):
wherein q5 is an integer from 1 to 6. In certain embodiments, q5 is 1. In certain embodiments, q5 is greater than 1, such as 2, 3, 4, 5 or 6. In certain embodiments, q5 is 2. In certain embodiments of formula (XV), the hydrophilic head group is of the structure:
In some embodiments of formula (I), L-X comprises a group of the formula (XVI):
In some embodiments of formula (XVI), R19 is hydrogen. In other cases, R19 is a substituent other than hydrogen. In certain embodiments, R19 is alkyl or substituted alkyl. In certain embodiments of formula (XVI), q6 is 1. In certain cases, q6 is greater than 1, such as 2, 3, 4, 5 or 6. In some cases of formula (XVI), q6 is 2. In certain embodiments of formula (XVI), the -L-X is of the structure:
In some embodiments of formula (I), L-X is of the formula (XVII):
wherein q7 is an integer from 1 to 6. In certain embodiments, q7 is 1. In certain embodiments, q7 is greater than 1, such as 2, 3, 4, 5 or 6. In certain embodiments, q7 is 2. In certain embodiments of formula (XVII), L-X is of the structure:
In some embodiments of formula (I), A is a heterocycle or substituted heterocycle. In some cases, A is a saturated heterocycle or substituted saturated heterocycle. The heterocycle can be a 5-, 6- or 7-membered monocyclic heterocycle. Heterocycles of interest include, but are not limited to, piperidine, piperazine, morpholine, tetrahydropyran, dioxane, imidazolidine, pyrazolidine, oxazolidine, isoxazolidine, and the like. In certain cases, the heterocycle is a 6-membered ring that is linked to Y and L via a 1, 4-configuration. In certain cases, the heterocycle is a 5- or 6-membered ring that is linked to Y and L via a 1, 3-configuration. In certain cases, the heterocycle is piperidine, substituted piperidine, piperazine or substituted piperazine. When the linking atom of the ring is C, the heterocycle can include a chiral center. In some cases, A is selected from one of the following heterocyclic groups:
In some embodiments of formula (I), A is a carbocycle. In some cases, A is a saturated carbocycle or substituted saturated carbocycle. The carbocycle can be a 5-, 6- or 7-membered monocyclic carbocycle, such as a cycloalkyl ring. Carbocycle of interest include, but are not limited to, cyclopentane, cyclohexane, cycloheptane, and the like. In certain cases, the carbocycle is a 6-membered ring that is linked to Y and L via a 1, 4-configuration. In certain cases, the carbocycle is a 5- or 6-membered ring that is linked to Y and L via a 1, 3-configuration. In certain cases, the carbocycle is cyclohexane or substituted cyclohexane. The cyclohexane can include a chiral center. In some cases, A is of the structure:
In certain other cases, A is an aromatic carbocycle, i.e., aryl. The aryl ring can be monocyclic. In certain cases, A is phenylene or substituted phenylene. In some cases, A is a 1,4-phenylene of the structure:
In certain other cases, A is an aromatic heterocycle, i.e., heteroaryl or substituted heteroaryl. The heteroaryl ring can be monocyclic. Heteroaryls of interest include, but are not limited to, pyridine, pyridazine, pyrimidine and pyrazine.
In some embodiments of formula (I), L is —(CH2)n-. In certain cases n is 1 to 8, such as 1 to 5. In some cases, n is 1 to 3, such as 2 or 3. In some cases, n is less than 8, such as 7, 6, 5, 4, 3, 2 or 1. In some cases, n is 1 to 6, such as 1 to 4 or 1 to 3. In some cases, n is 1. In some other cases, n is 2. In some cases, L is an ethylene or substituted ethylene group. In some other cases, L is a methylene or substituted methylene group. In certain other cases L is a covalent bond.
In some embodiments of formula (I), Y is selected from quinazoline, substituted quinazoline, quinoline, substituted quinoline, naphthalene, substituted naphthalene, isoquinoline and substituted isoquinoline. In certain instances, Y is selected from quinazoline and substituted quinazoline. In certain instances, Y is selected from quinoline and substituted quinoline. In certain instances, Y is selected from naphthalene and substituted naphthalene. In certain instances, Y is selected from isoquinoline and substituted isoquinoline. In some embodiments of formula (I), Y is a group of formula (II):
In certain embodiments of formula (II), at least of Z1 and Z2 is N. In certain embodiments of formula (II), Z1 is C and Z2 is N. In certain cases of formula (II), Z1 is N and Z2 is C. In certain instances of formula (IIa), Z1 is C and Z2 is C. In certain cases of formula (II), Z1 is N and Z2 is N. In some instances of formula (II), R1 and R4 are not hydrogen. In some instances of formula (II), R1, R3 and R4 are not hydrogen. In some instances of formula (II), R1, R3, R4 and R5 are not hydrogen.
In some instances of formula (II), R1 is selected from hydrogen, C1-5 alkyl, vinyl heterocycle (e.g., —CH═CH-heterocycle). In certain instances, the -vinyl heterocycle is vinyl pyridine (e.g., —CH═CH-pyridine). In some instances of formula (IIa), R1 is hydrogen. In some cases, R1 is C1-5 alkyl. In other cases, R1 is a vinyl heterocycle. In certain cases, R1 is vinyl pyridine. In some instances, R2 and R5 are both hydrogen. In some cases, R5 is selected from C1-5 alkyl, amine, triazole, imidazole, amide, alkoxy, OCF3 and hydroxy. In certain cases, R5 is alkoxy, e.g., methoxy. In some instances, R3 and R4 are each independently selected from hydrogen, C1-5 alkyl, triazole, imidazole, amine, amide, alkoxy, OCF3, hydroxy, or R3 and R4 together with the carbon to which they are attached from a heterocycle. In some cases, R3 and R4 are alkoxy, e.g., in some cases R3 and R4 are both methoxy. In some cases, R5 is methoxy and each of R1-R4 are hydrogen. In some cases, R5 is methoxy, R1 is —CH═CH-heterocycle and each of R2-R4 are hydrogen.
In some embodiments of formula (II), Y is a group of formula (IIA):
In some instances of formula (IIA), R7 is selected from hydrogen, C1-5 alkyl, substituted C1-5 alkyl, vinyl-heterocycle and substituted vinyl-heterocycle. In some instances of formula (IIA), R7 is selected from hydrogen, C1-5 alkyl, vinyl heterocycle (e.g., —CH═CH-heterocycle). In certain instances, the -vinyl heterocycle is vinyl pyridine (e.g., —CH═CH-pyridine). In some instances of formula (IIA), R7 is hydrogen. In some cases, R7 is C1-5 alkyl. In other cases, R7 is a vinyl heterocycle. In certain cases, R7 is vinyl pyridine. In some instances, R8 is selected from hydrogen, C1-5 alkyl, triazole, imidazole, amine, amide, alkoxy, OCF3 and hydroxyl. In some cases, R8 is alkoxy, e.g., methoxy. In some cases, R8 is methoxy and R7 is hydrogen. In some cases, R8 is methoxy and R7 is —CH═CH— heterocycle. In some embodiments of formula (II), Y is a group of formula (IIB):
In some instances of formula (IIB), R7 is selected from hydrogen, C1-5 alkyl, vinyl heterocycle (e.g., —CH═CH-heterocycle). In certain instances, the -vinyl heterocycle is vinyl pyridine (e.g., —CH═CH-pyridine). In some instances of formula (IIB), R7 is hydrogen. In some cases, R7 is C1-5 alkyl. In other cases, R7 is a vinyl heterocycle. In certain cases, R7 is vinyl pyridine. In some instances, R8 and R9 are each independently selected from hydrogen, C1-5 alkyl, triazole, imidazole, amine, amide, alkoxy, OCF3 and hydroxy, or R8 and R9 together with the carbon atoms to which they are attached from a fused heterocycle. In some cases, R8 and R9 are alkoxy, e.g., in some cases R8 and R9 are both methoxy. In some embodiments of formula (II), Y is a group of formula (IIC):
In some instances of formula (IIC), R7 is selected from hydrogen, C1-5 alkyl, vinyl heterocycle (e.g., —CH═CH-heterocycle). In certain instances, the -vinyl heterocycle is vinyl pyridine (e.g., —CH═CH-pyridine). In some instances of formula (IIC), R7 is hydrogen. In some cases, R7 is C1-5 alkyl. In some cases, R7 is a vinyl heterocycle. In certain cases, R7 is vinyl pyridine. In some cases, R10 is selected from hydrogen, C1-5 alkyl, amine, triazole, imidazole, amide, alkoxy, OCF3 and hydroxy. In some cases, R10 is hydrogen. In certain cases, R10 is alkoxy, e.g., methoxy. In some instances, R8 and R9 are each independently selected from hydrogen, C1-5 alkyl, triazole, imidazole, amine, amide, alkoxy, OCF3, hydroxy, or R8 and R9 together with the carbon atoms to which they are attached from a fused heterocycle. In some cases, R8 and R9 are alkoxy, e.g., in some cases R8 and R9 are both methoxy. In some cases, R10 is methoxy and each of R7-R9 are hydrogen. In some cases, R10 is methoxy, R7 is —CH═CH-heterocycle and each of R8 and R9 are hydrogen. In some embodiments of formula (II), Y is a group of formula (IID):
In some instances of formula (IID), R7 is selected from hydrogen, C1-5 alkyl, vinyl heterocycle (e.g., —CH═CH-heterocycle). In certain instances, the -vinyl heterocycle is vinyl pyridine (e.g., —CH═CH-pyridine). In some instances of formula (IID), R7 is hydrogen. In some cases, R7 is C1-5 alkyl. In some cases, R7 is a vinyl heterocycle. In certain cases, R7 is vinyl pyridine. In some instances, R11 and R12 are each independently selected from hydrogen, C1-5 alkyl, triazole, imidazole, amine, amide, alkoxy, OCF3 and hydroxy, or R11 and R12 together with the carbon atoms to which they are attached from a fused heterocycle. In some cases, R11 and R12 are alkoxy, e.g., in some cases R11 and R12 are both methoxy.
In some embodiments of formula (II), Y is a group of formula (IIE):
In some instances of formula (IIE), R7 is selected from hydrogen, C1-5 alkyl, vinyl heterocycle (e.g., —CH═CH-heterocycle). In certain instances, the -vinyl heterocycle is vinyl pyridine (e.g., —CH═CH-pyridine). In some instances of formula (IIE), R7 is hydrogen. In some cases, R7 is C1-5 alkyl. In other cases, R7 is a vinyl heterocycle. In certain cases, R7 is vinyl pyridine. In some instances, R11 and R12 are each independently selected from hydrogen, C1-5 alkyl, triazole, imidazole, amine, amide, alkoxy, OCF3 and hydroxy, or R11 and R12 together with the carbon to which they are attached from a heterocycle. In some cases, R11 and R12 are alkoxy, e.g., in some cases R11 and R12 are both methoxy.
In some embodiments of formula (II), Y is a group selected from:
In some embodiments of formula (II), any of R1 to R5 may be a halogen, e.g., F, Cl, Br or I. In some embodiments of formula (II), at least one of R1 to R5 is a halogen atom. In some embodiments of formula (II), at least one of R1 to R5 is fluoride. In other embodiments of formula (II), at least one of R1 to R5 is chloride. In other embodiments of formula (II), at least one of R1 to R5 is bromide. In yet other embodiments of formula (II), at least one of R1 to R5 is iodide.
In some embodiments of formula (II), Y is a group selected from:
In some embodiments of formula (I), Y is a group of formula (XI):
In some instances of formula (XI), R1 and R4 are not hydrogen. In some instances of formula (XI), R1, R3 and R4 are not hydrogen. In some instances of formula (XI), R1, R3, R4 and R5 are not hydrogen.
In some instances of formula (XI), Z21 is CR1 and R1 is selected from hydrogen, C1-5 alkyl, vinyl heterocycle (e.g., —CH═CH-heterocycle). In certain instances, the -vinyl heterocycle is vinyl pyridine (e.g., —CH═CH-pyridine). In some instances of formula (XI), Z21 is CR1 and R1 is hydrogen. In some cases, R1 is C1-5 alkyl. In other cases, Z21 is CR1 and R1 is a vinyl heterocycle. In certain cases, R1 is vinyl pyridine. In some instances, R2 and R5 are both hydrogen. In some cases, R5 is selected from C1-5 alkyl, amine, triazole, imidazole, amide, alkoxy, OCF3 and hydroxy. In certain cases, R5 is alkoxy, e.g., methoxy. In some instances, R3 and R4 are each independently selected from hydrogen, C1-5 alkyl, triazole, imidazole, amine, amide, alkoxy, OCF3, hydroxy, or R3 and R4 together with the carbon to which they are attached from a heterocycle. In some cases, R3 and R4 are alkoxy, e.g., in some cases R3 and R4 are both methoxy. In some cases, R5 is methoxy and each of R1-R4 are hydrogen. In some cases, R5 is methoxy, R1 is —CH═CH-heterocycle and each of R2-R4 are hydrogen.
In some embodiments of formula (I), Y is a group of the formula (III):
wherein:
In some embodiments of formula (III), Y is a group of the formula (IIIA):
In some instance of formula (IIIA), one and only one of Z5, Z6, Z7 and Z8 is N. In some instance of formula (IIIA), two and only two of Z5, Z6, Z7 and Z8 are N. In some instance of formula (IIIA), Z5 is N. In some instance of formula (IIIA), Z6 is N. In some instance of formula (IIIA), Z7 is N. In some instance of formula (IIIA), Z8 is N. In some instance of formula (IIIA), Z5 and Z7 are each N. In some instance of formula (IIIA), Z7 and Z8 are each N.
In some embodiments of formula (III), Y is a group of the formula (IIIB):
In some instance of formula (IIIB), one and only one of Z9, Z10 and Z11 is N. In some instance of formula (IIIB), two and only two of Z9, Z10 and Z11 are N. In some instance of formula (IIIB), Z9 is N. In some instance of formula (IIIA), Z10 is N. In some instance of formula (IIIB), Z11 is N. In some instances of formula (IIIB), R14 is selected form alkyl and substituted alkyl. In some instances of formula (IIIB), p is 0. In some instances of formula (IIIB), p is 1. In some instances of formula (IIIB), p is 2.
In some embodiments of formula (III), Y is a group selected from:
or a substituted version thereof.
In some embodiments of formula (I), Y is a group of formula (IIIC)
In some instances of formula (IIIC), Z1, Z2, Z17 and Z19 are each N and Z18 is CR20.
In some embodiments of formula (IIIC), Y is of the structure:
In some embodiments of formula (I), the structure has the formula (IV):
wherein,
In certain embodiments of formula (IV), at least one of Z1 and Z2 is N. In certain embodiments of formula (IV), Z1 is C and Z2 is N. In certain cases of formula (IV), Z1 is N and Z2 is C. In certain instances of formula (IV), Z1 is C and Z2 is C. In certain cases of formula (IV), Z1 is N and Z2 is N. In certain embodiments of formula (IV), at least one of Z3 and Z4 is N. In certain cases of formula (IV), Z3 is N and Z4 is N. In certain cases of formula (IV), Z3 is N and Z4 is CH. In certain cases of formula (IV), Z3 is CH and Z4 is N. In certain cases of formula (VI), Z3 is CH and Z4 is CH.
In some instances of formula (IV), R1 is selected from hydrogen, C1-5 alkyl, vinyl heterocycle (e.g., —CH═CH-heterocycle). In certain instances, the -vinyl heterocycle is vinyl pyridine (e.g., —CH═CH-pyridine). In some instances of formula (IV), R1 is hydrogen. In some cases, R1 is C1-5 alkyl. In other cases, R1 is a vinyl heterocycle. In certain cases, R1 is vinyl pyridine. In some instances, R2 and R5 are both hydrogen. In some cases, R5 is selected from C1-5 alkyl, amine, triazole, imidazole, amide, alkoxy, OCF3 and hydroxy. In certain cases, R5 is alkoxy, e.g., methoxy. In some instances, R3 and R4 are each independently selected from hydrogen, C1-5 alkyl, triazole, imidazole, amine, amide, alkoxy, OCF3, hydroxy, or R3 and R4 together with the carbon to which they are attached from a heterocycle. In some cases, R3 and R4 are alkoxy, e.g., in some cases R3 and R4 are both methoxy.
In some cases, R5 is methoxy and each of R1-R4 are hydrogen. In some cases, R5 is methoxy, R1 is —CH═CH-heterocycle and each of R2-R4 are hydrogen.
In some embodiments of formula (I), the structure has the formula (V)
wherein:
In certain embodiments of formula (V), at least one of Z1 and Z2 is N. In certain embodiments of formula (V), Z1 is CH and Z2 is N. In certain cases of formula (IV), Z1 is N and Z2 is CH. In certain instances of formula (V), Z1 is CH and Z2 is CH. In certain cases of formula (IV), Z1 is N and Z2 is N. In certain embodiments of formula (V), at least one of Z3 and Z4 is N. In certain cases of formula (V), Z3 is N and Z4 is N. In certain cases of formula (V), Z3 is N and Z4 is CH. In certain cases of formula (V), Z3 is CH and Z4 is N. In certain cases of formula (V), Z3 is CH and Z4 is CH.
In some embodiments of formula (I), the inhibitor has formula (VI):
wherein,
In some embodiments of formula (I), the structure has the formula (VI):
wherein,
In certain embodiments of formula (VI), at least one of Z1 and Z2 is N. In certain embodiments of formula (VI), Z1 is C and Z2 is N. In certain cases of formula (VI), Z1 is N and Z2 is C. In certain instances of formula (VI), Z1 is C and Z2 is C. In certain cases of formula (VI), Z1 is N and Z2 is N. In certain embodiments of formula (VI), at least one of Z3 and Z4 is N. In certain cases of formula (VI), Z3 is N and Z4 is N. In certain cases of formula (IVI Z3 is N and Z4 is C. In certain cases of formula (VI), Z3 is C and Z4 is N. In certain cases of formula (VI), Z3 is C and Z4 is C.
In some instances of formula (VI), R1 is selected from hydrogen, C1-5 alkyl, vinyl heterocycle (e.g., —CH═CH-heterocycle). In certain instances, the -vinyl heterocycle is vinyl pyridine (e.g., —CH═CH-pyridine). In some instances of formula (VI), R1 is hydrogen. In some cases, R1 is C1-5 alkyl. In other cases, R1 is a vinyl heterocycle. In certain cases, R1 is vinyl pyridine. In some instances, R2 and R5 are both hydrogen. In some cases, R5 is selected from C1-5 alkyl, amine, triazole, imidazole, amide, alkoxy, OCF3 and hydroxy. In certain cases, R5 is alkoxy, e.g., methoxy. In some instances, R3 and R4 are each independently selected from hydrogen, C1-5 alkyl, triazole, imidazole, amine, amide, alkoxy, OCF3, hydroxy, or R3 and R4 together with the carbon to which they are attached from a heterocycle. In some cases, R3 and R4 are alkoxy, e.g., in some cases R3 and R4 are both methoxy. In some cases, R5 is methoxy and each of R1-R4 are hydrogen. In some cases, R5 is methoxy, R1 is —CH═CH-heterocycle and each of R2-R4 are hydrogen.
In certain embodiments of formula (VI), L is —CH2—. In certain other cases of formula (VI), L is —(CH2)2—.
In certain embodiments of formula (VI), X is
In certain cases of formula (VI), X is
In certain other cases of formula (VI), X is
In certain cases of formula (VI), X is
In certain other cases of formula (VI), X is
In certain embodiments of formula (VI), X is
In certain cases of formula (VI), X is
In certain other cases of formula (VI), X is
In certain cases of formula (VI), X is
In certain other cases of formula (VI), X is
In certain cases of formula (VI), X is
In certain other cases of formula (VI), X is
In certain other cases of formula (VI), X is
wherein Ra and Rb are each independently selected from aryl, alkyl, —CH2OC(O)Re, —CH2OC(O)ORe, wherein Re is alkyl. In certain cases of formula (VI), X is
wherein Rc and Rd are each independently selected from —C(CH3)C(O)Ore and alkyl, wherein Re is alkyl. In certain other cases of formula (VI), X is
wherein Ra is selected from aryl, alkyl, —CH2OC(O)Re, —CH2OC(O)ORe and Re is selected from —C(CH3)C(O)Ore and alkyl, wherein Re is alkyl.
It will be understood that any of the hydroxyl and amine groups in group X in formula (VI) may be optionally further substituted with any convenient group, e.g., an alkyl group, a substituted alkyl group, a phenyl group, a substituted phenyl group, an ester group and the like. It will be understood that any convenient alternative hydrophilic group can be utilized as group X in a compound of formula (VI).
In some embodiments of formula (I), the structure has the formula (VII):
In certain embodiments of formula (VII), at least one of Z1 and Z2 is N. In certain embodiments of formula (VII), Z1 is C and Z2 is N. In certain cases of formula (VII), Z1 is N and Z2 is C. In certain instances of formula (VII), Z1 is C and Z2 is C. In certain cases of formula (VII), Z1 is N and Z2 is N.
In some instances of formula (VII), R1 is selected from hydrogen, C1-5 alkyl, vinyl heterocycle (e.g., —CH═CH-heterocycle). In certain instances, the -vinyl heterocycle is vinyl pyridine (e.g., —CH═CH-pyridine). In some instances of formula (VII), R1 is hydrogen. In some cases, R1 is C1-5 alkyl. In other cases R1 is a vinyl heterocycle. In certain cases, R1 is vinyl pyridine. In some instances, R2 and R5 are both hydrogen. In some cases, R5 is selected from C1-5 alkyl, amine, triazole, imidazole, amide, alkoxy, OCF3 and hydroxy. In certain cases, R5 is alkoxy, e.g., methoxy. In some instances, R3 and R4 are each independently selected from hydrogen, C1-5 alkyl, triazole, imidazole, amine, amide, alkoxy, OCF3, hydroxy, or R3 and R4 together with the carbon to which they are attached from a heterocycle. In some cases, R3 and R4 are alkoxy, e.g., in some cases R3 and R4 are both methoxy. In some cases, R5 is methoxy and each of R1-R4 are hydrogen. In some cases, R5 is methoxy, R1 is —CH═CH-heterocycle and each of R2-R4 are hydrogen.
In certain embodiments of formula (VII), L is —CH2—. In certain other cases of formula (VII), L is —(CH2)2—.
In certain embodiments of formula (VII), X is
In certain cases of formula (VII), X is
In certain other cases of formula (VII), X is
In certain cases of formula (VII), X is
In certain other cases of formula (VII), X is
In certain embodiments of formula (VII), X is
In certain cases of formula (VII), X is
In certain other cases of formula (VII), X is
In certain cases of formula (VII), X is
In certain other cases of formula (VII), X is
In certain cases of formula (VII), X is
In certain other cases of formula (VII), X is
In certain other cases of formula (VI), X is
wherein Ra and Rb are each independently selected from aryl, alkyl, —CH2OC(O)Re, —CH2OC(O)ORe, wherein Re is alkyl. In certain cases of formula (VI), X is
wherein Rc and Rd are each independently selected from —C(CH3)C(O)Ore and alkyl, wherein Re is alkyl. In certain other cases of formula (VI), X is
wherein Ra is selected from aryl, alkyl, —CH2OC(O)Re, —CH2OC(O)ORe and Rc is selected from —C(CH3)C(O)Ore and alkyl, wherein Re is alkyl.
It will be understood that any of the hydroxyl and amine groups in group X of formula (VII) may be optionally further substituted with any convenient group, e.g., an alkyl group, a substituted alkyl group, a phenyl group, a substituted phenyl group, an ester group and the like. It will be understood that any convenient alternative hydrophilic group can be utilized as group X in a compound of formula (VII).
In some cases, the subject ENPP1 inhibitor compound is of formula (I′):
In certain embodiments of formula (I′), Z3 is absent. In certain embodiments of formula (I′), Z3 is NR22, wherein R22 is selected from H, C(1-6)alkyl and substituted C(1-6)alkyl. In certain cases Z3 is NH. In certain cases, Z3 is NR22 and R22 is C(1-6)alkyl, e.g., methyl, ethyl, propyl, pentyl or hexyl. In certain cases, Z3 is NR22 and R22 is substituted C(1-6) alkyl. In certain cases of formula (I′), Z3 is O. In certain cases of formula (I′), Z3 is S.
In some instances of formula (I′), Z1 is CR11 and R11 is selected from hydrogen, cyano, trifluoromethyl, halogen, alkyl and substituted alkyl hydrogen. In some cases, the alkyl or substituted alky is C1-5 alkyl. In some instances of formula (I′), Z1 is CR11 and R11 is hydrogen. In some cases, R11 is cyano. In some cases, R11 is trifluoromethyl. In some cases, R11 is halogen, e.g., Br, I, Cl or F. In some cases, R11 is alkyl, e.g., C1-5 alkyl. In some cases, R11 is substituted alkyl, e.g., substituted C1-5 alkyl.
In some instances of formula (I′), Z2 is CR12 and R12 is selected from hydrogen, cyano, trifluoromethyl, halogen, alkyl and substituted alkyl hydrogen. In some cases, the alkyl or substituted alky is C1-5 alkyl. In some instances of formula (I′), Z2 is CR12 and R12 is hydrogen. In some cases, R12 is cyano. In some cases, R12 is trifluoromethyl. In some cases, R12 is halogen, e.g., Br, I, Cl or F.
In some cases, R12 is alkyl, e.g., C1-5 alkyl. In some cases, R12 is substituted alkyl, e.g., substituted C1-5 alkyl.
In certain embodiments of formula (I′), at least one of Z1 and Z2 is N. In certain embodiments of formula (I′), Z1 is CR11 and Z2 is N. In certain cases of formula (I′), Z1 is N and Z2 is CR12. In certain instances of formula (I′), Z1 is CR11 and Z2 is CR12. In certain cases of formula (I′), Z1 is N and Z2 is N.
In certain embodiments of formula (I′), L1 and L2 are each covalent bonds. In certain cases, L1 and L2 are each linkers. In certain cases, L1 is a covalent bond and L2 is a linker. In certain cases, L1 is a linker and L2 is a covalent bond. Any convenient linkers can be utilized to link A to X and/or A to Z3 (e.g., as described herein). In some cases, A is linked to X via a covalent bond. In certain cases, A is linked to X via a linear linker of 1-12 atoms in length, such as 1-10, 1-8 or 1-6 atoms in length, e.g., 1, 2, 3, 4, 5 or 6 atoms in length. The linker L2 can be a (C1-6)alkyl linker or a substituted (C1-6)alkyl linker, optionally substituted with a heteroatom or linking functional group, such as an ester (—CO2—), amido (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 Z3 via a covalent bond. In certain cases, A is linked to Z3 via a linear linker of 1-12 atoms in length, such as 1-10, 1-8 or 1-6 atoms in length, e.g., 1, 2, 3, 4, 5 or 6 atoms in length. The linker L1 can be a (C1-6)alkyl linker or a substituted (C1-6)alkyl linker, optionally substituted with a heteroatom or linking functional group, such as keto (CO), ester (—CO2—), amido (CONH), carbamate (OCONH), ether (—O—), thioether (—S—) and/or amino group (—NR— where R is H or alkyl). When Z3 is NR22, the linker L1 can include a terminal keto (C═O) group that together with Z3 provides an amido group (NR22CO) linkage. When Z31 is O or S, the linker L1 can include a terminal keto (C═O) group that together with Z31 provides an ester or thioester group linkage.
In certain embodiments of formula (I′), Z3 is phosphorus-containing group capable of binding zinc ion, or a prodrug form thereof.
In certain instances of formula (I′), Z3 is selected from NR22, O and S. As such, the subject ENPP1 inhibitor compound of formula (I′) can be described by formula (II′):
wherein Z31 is selected from NR22, O and S.
In certain embodiments of formula (II′), Z31 is NR22, wherein R22 is selected from H, C(1-6)alkyl and substituted C(1-6)alkyl. In certain cases Z31 is NH. In certain cases, Z31 is NR22 and R22 is C(1-6)alkyl, e.g., methyl, ethyl, propyl, pentyl or hexyl. In certain cases, Z31 is NR22 and R22 is substituted C(1-6)alkyl. In certain cases of formula (I′), Z31 is O. In certain cases of formula (I′), Z31 is S.
In some instances of formula (II′), Z1 is CR11 and R11 is selected from hydrogen, cyano, trifluoromethyl, halogen, alkyl and substituted alkyl hydrogen. In some cases, the alkyl or substituted alky is C1-5 alkyl. In some instances of formula (II′), Z1 is CR11 and R11 is hydrogen. In some cases, R11 is cyano. In some cases, R11 is trifluoromethyl. In some cases, R11 is halogen, e.g., Br, I, Cl or F. In some cases, R11 is alkyl, e.g., C1-5 alkyl. In some cases, R11 is substituted alkyl, e.g., substituted C1-5 alkyl.
In some instances of formula (II′), Z2 is CR12 and R12 is selected from hydrogen, cyano, trifluoromethyl, halogen, alkyl and substituted alkyl hydrogen. In some cases, the alkyl or substituted alky is C1-5 alkyl. In some instances of formula (II′), Z2 is CR12 and R12 is hydrogen. In some cases, R12 is cyano. In some cases, R12 is trifluoromethyl. In some cases, R12 is halogen, e.g., Br, I, Cl or F.
In some cases, R12 is alkyl, e.g., C1-5 alkyl. In some cases, R12 is substituted alkyl, e.g., substituted C1-5 alkyl.
In certain embodiments of formula (II′), at least one of Z1 and Z2 is N. In certain embodiments of formula (I′), Z1 is CR11 and Z2 is N. In certain cases of formula (I′), Z1 is N and Z2 is CR12. In certain instances of formula (I′), Z1 is CR11 and Z2 is CR12. In certain cases of formula (I′), Z1 is N and Z2 is N.
In certain embodiments of formula (II′), L1 and L2 are each covalent bonds. In certain cases, L1 and L2 are each linkers. In certain cases, L1 is a covalent bond and L2 is a linker. In certain cases, L1 is a linker and L2 is a covalent bond. Any convenient linkers can be utilized to link A to X and/or A to Z3 (e.g., as described herein). In some cases, A is linked to X via a covalent bond. In certain cases, A is linked to X via a linear linker of 1-12 atoms in length, such as 1-10, 1-8 or 1-6 atoms in length, e.g., 1, 2, 3, 4, 5 or 6 atoms in length. The linker L2 can be a (C1-6)alkyl linker or a substituted (C1-6)alkyl linker, optionally substituted with a heteroatom or linking functional group, such as keto (CO), ester (—CO2—), amido (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 Z3 via a covalent bond. In certain cases, A is linked to Z3 via a linear linker of 1-12 atoms in length, such as 1-10, 1-8 or 1-6 atoms in length, e.g., 1, 2, 3, 4, 5 or 6 atoms in length. The linker L1 can be a (C1-6)alkyl linker or a substituted (C1-6)alkyl linker, optionally substituted with a heteroatom or linking functional group, such as keto (C═O), ester (—CO2—), amido (CONH), carbamate (OCONH), ether (—O—), thioether (—S—) and/or amino group (—NR— where R is H or alkyl). When Z31 is NR22, the linker L1 can include a terminal keto (C═O) group that together with Z31 provides an amido group (NR22CO) linkage. When Z31 is O or S, the linker L1 can include a terminal keto (C═O) group that together with Z31 provides an ester or thioester group linkage.
In some cases of formula (II′), the subject ENPP1 inhibitor compound is of formula (III′):
In certain embodiments of formula (III′), Z31 is NR22, wherein R22 is selected from H, C(1-6)alkyl and substituted C(1-6)alkyl. In certain cases Z31 is NH. In certain cases, Z31 is NR22 and R22 is C(1-6)alkyl, e.g., methyl, ethyl, propyl, pentyl or hexyl. In certain cases, Z31 is NR22 and R22 is substituted C(1-6)alkyl. In certain cases of formula (III′), Z31 is O. In certain cases of formula (III′), Z31 is S.
In formula (II′), when Z31 is NR22, the linker L1 can include a terminal keto (C═O) group that together with Z31 provides an amido group (NR22CO) linkage. As such, in some cases of formula (II′), the subject ENPP1 inhibitor compound is of formula (IIIa′):
wherein:
In some instances of formulae (III′)-(IIIa′), Z1 is CR11 and R11 is selected from hydrogen, cyano, trifluoromethyl, halogen, alkyl and substituted alkyl hydrogen. In some cases, the alkyl or substituted alky is C1-5 alkyl. In some instances of formulae (III′)-(IIIa′), Z1 is CR11 and R11 is hydrogen. In some cases, R11 is cyano. In some cases, R11 is trifluoromethyl. In some cases, R11 is halogen, e.g., Br, I, Cl or F. In some cases, R11 is alkyl, e.g., C1-5 alkyl. In some cases, R11 is substituted alkyl, e.g., substituted C1-5 alkyl.
In some instances of formulae (III′)-(IIIa′), Z2 is CR12 and R12 is selected from hydrogen, cyano, trifluoromethyl, halogen, alkyl and substituted alkyl hydrogen. In some cases, the alkyl or substituted alky is C1-5 alkyl. In some instances of formulae (III′)-(IIIa′), Z2 is CR12 and R12 is hydrogen. In some cases, R12 is cyano. In some cases, R12 is trifluoromethyl. In some cases, R12 is halogen, e.g., Br, I, Cl or F. In some cases, R12 is alkyl, e.g., C1-5 alkyl. In some cases, R12 is substituted alkyl, e.g., substituted C1-5 alkyl.
In certain embodiments of formulae (III′)-(IIIa′), at least one of Z1 and Z2 is N. In certain embodiments of formulae (III′)-(IIIa′), Z1 is CR11 and Z2 is N. In certain cases of formulae (III′)-(IIIa′), Z1 is N and Z2 is CR12. In certain instances of formulae (III′)-(IIIa′), Z1 is CR11 and Z2 is CR12. In certain cases of formulae (III′)-(IIIa′), Z1 is N and Z2 is N.
In certain embodiments of formulae (III′)-(IIIa′), R31 to R34 are each hydrogen. In certain embodiments, at least one of R31 to R34 is a halogen. In certain embodiments, at least one of R31 to R34 is alkyl. In certain embodiments, at least one of R31 to R34 is substituted alkyl. In certain cases, one of R31 to R34 is halogen and the remainder are selected from hydrogen, halogen, alkyl and substituted alkyl. In certain cases, one of R31 to R34 is alkyl and the remainder are selected from hydrogen, halogen, alkyl and substituted alkyl. In certain cases, one R31 to R34 is substituted alkyl and the remainder are selected from hydrogen, halogen, alkyl and substituted alkyl. In certain cases, one of R31 to R34 is halogen and the remainder are hydrogen. In certain cases, one of R31 to R34 is alkyl and the remainder are hydrogen. In certain cases, one R31 to R34 is substituted alkyl and the remainder are hydrogen.
In certain embodiments of formulae (III′)-(IIIa′), n is an integer from 0 to 3. In certain cases n is 0. In certain cases, n is 1. In certain cases, n is 2. In certain cases n is 3. In certain embodiments of formulae (III′)-(IIIa′), m is an integer from 0 to 3. In certain cases, m is 0. In certain cases, m is 1. In certain cases, m is 2. In certain cases, m is 3. In certain cases, n is 0 and m is 1. In certain cases, n is 0 and m is 2. In certain case, 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.
In some embodiments of any of formulae (I′) to (IIIa′), the ring system A is selected from phenyl, substituted phenyl, pyridyl, substituted pyridyl, pyrimidine, substituted pyrimidine, piperidine, substituted piperidine, piperazine, substituted piperazine, pyridazine, substituted pyridazine, cyclohexyl and substituted cyclohexyl. In certain cases, the ring system A is phenyl or substituted phenyl. In some cases, the ring system A is pyridyl or substituted pyridyl. In some cases, the ring system A is pyrimidine or substituted pyrimidine. In some cases, the ring system A is piperidine or substituted piperidine. In some cases, the ring system A is piperazine or substituted piperazine. In some cases, the ring system A is cyclohexyl or substituted cyclohexyl.
In some embodiments, the ring system A is described by the formula (A1′):
In certain cases, A1′ is phenylene. In certain cases, A1′ is a mono-substituted phenylene.
In certain cases, A1′ is a di-substituted phenylene. In certain cases, A1′ is a tri-substituted phenylene.
In certain cases, A1′ is a tetra-substituted phenylene. In certain cases, the substituents of the phenylene are selected from lower alkyl (e.g., methyl, ethyl, propyl, butyl, pentyl and hexyl) and halogen (e.g., F, Cl, I or Br).
In some embodiments, A1′ ring is described by the formula (A1a′):
In some embodiments the ring system A is described by the formula (A2′):
In certain cases, A2′ is pyridyl. In certain cases, A2′ is a substituted pyridyl. In some cases, the pyridyl is a mono-substituted pyridyl. In other cases, the pyridyl is a di-substituted pyridyl.
In other cases, the pyridyl is a tri-substituted pyridyl. In certain cases, Z5 is N, such that A2′ is a pyrimidyl. In some cases, A2′ is a substituted pyrimidyl. In some cases, the pyrimidyl is mono-substituted. In some cases, the pyrimidyl is di-substituted. 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).
In some embodiments, the ring system A is described by the formula (A3′):
In certain cases, A3′ is pyridyl. In certain cases, A3′ is a substituted pyridyl. In some cases, the pyridyl is a mono-substituted pyridyl. In other cases, the pyridyl is a di-substituted pyridyl. In other cases, the pyridyl is a tri-substituted pyridyl. In certain cases, Z5 is N, such that A3′ is a pyrimidyl. In some cases, A3′ is a substituted pyrimidyl. In some cases, the pyrimidyl is mono-substituted. In some cases, the pyrimidyl is di-substituted. 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).
In some embodiments, the ring system A is described by the formula (A4′):
In some cases, A4′ is a substituted pyrimidyl. In some cases, the pyrimidyl is mono-substituted. In some cases, the pyrimidyl is di-substituted. 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).
In some cases of formula (III′)-(IIIa′), the ENPP1 inhibitor compound is of formula (IV′)-(IVa′):
wherein:
In certain embodiments of formulae (IV′)-(IVa′), Z31 is NR22, wherein R22 is selected from H, C(1-6) alkyl and substituted C(1-6) alkyl. In certain cases Z31 is NH. In certain cases, Z31 is NR22 and R22 is C(1-6) alkyl, e.g., methyl, ethyl, propyl, pentyl or hexyl. In certain cases, Z31 is NR22 and R22 is substituted C(1-6) alkyl. In certain cases of formulae (IV′)-(IVa′), Z31 is O. In certain cases of formulae (IV′)-(IVa′), Z31 is S.
In certain embodiments of formulae (IV′)-(IVa′), at least one of Z11 and Z21 is N. In certain embodiments of formulae (IV′)-(IVa′), Z11 is C(CN) and Z21 is N. In certain cases of formulae (IV′)-(IVa′), Z11 is N and Z21 is C(CN). In certain instances of formulae (IV′)-(IVa′), Z11 is C(CN) and Z21 is C(CN). In certain cases of formulae (IV′)-(IVa′), Z11 is N and Z21 is N.
In certain embodiments of formulae (IV′)-(IVa′), R31 to R34 are each hydrogen. In certain embodiments, at least one of R31 to R34 is a halogen. In certain embodiments, at least one of R31 to R34 is alkyl. In certain embodiments, at least one of R31 to R34 is substituted alkyl. In certain cases, one of R31 to R34 is halogen and the remainder are selected from hydrogen, halogen, alkyl and substituted alkyl. In certain cases, one of R31 to R34 is alkyl and the remainder are selected from hydrogen, halogen, alkyl and substituted alkyl. In certain cases, one of R31 to R34 is substituted alkyl and the remainder are selected from hydrogen, halogen, alkyl and substituted alkyl. In certain cases, one of R31 to R34 is halogen and the remainder are hydrogen. In certain cases, one of R31 to R34 is alkyl and the remainder are hydrogen. In certain cases, one of R31 to R34 is substituted alkyl and the remainder are hydrogen.
In certain embodiments of formulae (IV′)-(IVa′), n is an integer from 0 to 3. In certain cases n is 0. In certain cases, n is 1. In certain cases, n is 2. In certain cases n is 3. In certain embodiments of formulae (IV′)-(IVa′), m is an integer from 0 to 3. In certain cases, m is 0. In certain cases, m is 1. In certain cases, m is 2. In certain cases, m is 3. In certain cases, n is 0 and m is 1. In certain cases, n is 0 and m is 2. In certain case, 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.
In some cases of formulae (IVa′), n is 0 and m is 0-2, such as m is 1 or 2.
In some cases of formulae (IV′)-(IVa′), the ENPP1 inhibitor compound is of formulae (V′)-(Va′):
In certain embodiments of formulae (V′)-(Va′), at least one of Z11 and Z21 is N. In certain embodiments of formulae (V′)-(Va′), Z11 is C(CN) and Z21 is N. In certain cases of formulae (V′)-(Va′), Z11 is N and Z21 is C(CN). In certain instances of formulae (V′)-(Va′), Z11 is C(CN) and Z21 is C(CN). In certain cases of formulae (V′)-(Va′), Z11 is N and Z21 is N.
In some cases of formulae (V′)-(Va′), the subject ENPP1 inhibitor compound is of one of formulae (VIa′)-(VId′):
In certain embodiments of formulae (VIa′)-(VId′), R41 to R44 are each hydrogen. In certain embodiments, at least one of R41 to R44 is alkyl or substituted alkyl. In certain embodiments, at least one of R41 to R44 is hydroxy. In certain embodiments, at least one of R41 to R44 is alkoxy or substituted alkoxy. In certain cases, at least one of R41 to R44 is trifluoromethyl. In certain cases, at least one of R41 to R44 is halogen. In certain cases, at least one of R41 to R44 is acyl or substituted acyl. In certain cases, at least one of R41 to R44 is carboxy. In certain cases, at least one of R41 to R44 is carboxyamide or substituted carboxyamide. In certain cases, at least one of R41 to R44 is sulfonyl or substituted sulfonyl. In certain cases, at least one of R41 to R44 is sulfonamide and substituted sulfonamide. In certain cases, one of R31 to R34 is hydrogen and the remainder are selected from hydrogen, alkyl, substituted alkyl, hydroxy, alkoxy, substituted alkoxy, trifluoromethyl, halogen, acyl, substituted acyl, carboxy, carboxyamide, substituted carboxyamide, sulfonyl, substituted sulfonyl, sulfonamide and substituted sulfonamide. In certain cases, two of R31 to R34 are hydrogen and the remainder are selected from hydrogen, alkyl, substituted alkyl, hydroxy, alkoxy, substituted alkoxy, trifluoromethyl, halogen, acyl, substituted acyl, carboxy, carboxyamide, substituted carboxyamide, sulfonyl, substituted sulfonyl, sulfonamide and substituted sulfonamide. In certain cases, three of R31 to R34 are hydrogen and the remainder are selected from hydrogen, alkyl, substituted alkyl, hydroxy, alkoxy, substituted alkoxy, trifluoromethyl, halogen, acyl, substituted acyl, carboxy, carboxyamide, substituted carboxyamide, sulfonyl, substituted sulfonyl, sulfonamide and substituted sulfonamide.
In certain embodiments of formulae (VIa′)-(VId′), n is an integer from 0 to 3. In certain cases n is 0. In certain cases, n is 1. In certain cases, n is 2. In certain cases n is 3. In certain embodiments of any of formulae (VIa′)-(VId′), m is an integer from 0 to 3. In certain cases, m is 0. In certain cases, m is 1. In certain cases, m is 2. In certain cases, m is 3. In certain cases, n is 0 and m is 1. In certain cases, n is 0 and m is 2. In certain case, 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.
In certain embodiments of any of formulae (VIa′)-(VId′), R22 is hydrogen. In certain cases, R22 is alkyl. In certain cases, R22 is substituted alkyl. In certain cases, the alkyl or substituted alkyl is C(1-6)alkyl.
In certain embodiments of any of formulae (I′)-(VId′), R1 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.
In certain cases of formulae (I′)-(VId′), R1 is hydrogen. In certain cases, R1 is aryl or substituted aryl. In certain cases, R1 is heteroaryl or substituted heteroaryl. In certain cases, R1 is alkylaryl or substituted alkylaryl. In certain cases, R1 is alkylheteroaryl or substituted alkylheteroaryl. In certain cases, R1 is alkenylaryl, or substituted alkenylaryl. In certain cases, R1 is ethenylaryl. In certain cases, R1 is substituted ethenylaryl. In some cases, R1 is ethenylheteroaryl. In certain cases, R1 is alkenylheteroaryl or substituted alkenylheteroaryl. In some cases, R1 is substituted ethenylheteroaryl.
In some cases of formula (VIa′)-(VId′), the ENPP1 inhibitor compound is of one of formulae (VIIa′)-(VIIb′):
In certain embodiments of any of formulae (I′)-(VIIb′), R2 to R5 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 any of formulae (I′)-(VIIb′), R2 to R5 are independently selected from hydrogen, OH, C(1-6)alkoxy, —OCF3, C(1-6)alkylamino, di-C(1-6)alkylamino, F, Cl, Br and CN. In certain cases, at least one of R2 to R5 is hydrogen. In certain cases, at least two of R2 to R5 are hydrogen. In certain cases, each of R2 to R5 is hydrogen. In certain cases, at least one of R2 to R5 is hydroxy. In certain cases, at least one of R2 to R5 is alkyl or substituted alkyl. In certain cases, at least one of R2 to R5 is alkoxy or substituted alkoxy. In certain cases, the alkoxy or substituted alkoxy is a C(1-6)alkoxy, e.g., methoxy, ethoxy, propoxy, butoxy, pentoxy, hexoxy. In certain cases, at least one of R2 to R5 is methoxy. In certain cases, at least one of R2 to R5 is —OCF3. In certain cases, at least one of R2 to R5 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 R2 to R5 is cyano. In certain cases, at least one of R2 to R5 is amine or substituted amine. In certain cases, at least one of R2 to R5 is C(1-6) alkylamino. In certain cases, at least one of R2 to R5 is di-C(1-6) alkylamino. In certain cases, at least one of R2 to R5 is amide. In certain cases, at least one of R2 to R5 is heterocycle or substituted heterocycle.
In some instances of formulae (I′)-(VIIb′), R3 and R4 are independently alkoxy; and R2 and R5 are both hydrogen. In certain cases, the alkoxy is methoxy. In some cases, R3 is alkoxy; and R2, R4 and R5 are hydrogen. In some cases, R4 is alkoxy; and R2, R3 and R5 are each hydrogen. In certain cases, R2, R3 and R4 are hydrogen and R5 is alkoxy. In certain cases, the alkoxy is a 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 pentoxy. In certain cases, the alkoxy is hexyloxy.
In some cases of formulae (VIc′)-(VId′), R41-R44 are each independently H, halogen, C(1 6)alkyl or C(1-6)alkoxy. In some cases of formulae (VIc′)-(VId′), m is 1 or 2. In some cases of formulae (VIc′)-(VId′), R2 is H, and R3 to R5 are independently selected from hydrogen, C(1-6) alkoxy, F, Cl and C(1-6)alkyl.
In some cases of formulae (VIIa′)-(VIIb′), the subject ENPP1 inhibitor compound is of one of formulae (VIIc′)-(VIII′):
In some cases of formula (VIa′), the ENPP1 inhibitor compound is of formula (VIIm′):
In certain embodiments of formula (VIIm′), R2 to R5 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′), R2 to R5 are independently selected from hydrogen, OH, C(1-6)alkoxy, —OCF3, C(1-6)alkylamino, di-C(1-6)alkylamino, F, Cl, Br and CN. 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.
In certain cases of formula (VIIm′), at least one of R3 to R5 is hydrogen. In certain cases, at least two of R3 to R5 are hydrogen. In certain cases, each of R3 to R5 is hydrogen. In certain cases, at least one of R3 to R5 is hydroxy. In certain cases, at least one of R3 to R5 is alkyl or substituted alkyl. In certain cases, at least one of R3 to R5 is alkoxy or substituted alkoxy. In certain cases of formula (VIIm′), the alkoxy or substituted alkoxy is a C(1-6) alkoxy, e.g., methoxy, ethoxy, propoxy, butoxy, pentoxy, hexoxy. In certain cases, at least one of R3 to R5 is methoxy. In certain cases of formula (VIIm′), at least one of R3 to R5 is —OCF3. In certain cases, at least one of R3 to R5 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 R3 to R5 is cyano. In certain cases, at least one of R3 to R5 is amine or substituted amine. In certain cases, at least one of R3 to R5 is C(1-6) alkylamino. In certain cases, at least one of R3 to R5 is di-C(1-6) alkylamino. In certain cases of formula (VIIm′), at least one of R3 to R5 is amide. In certain cases, at least one of R3 to R5 is heterocycle or substituted heterocycle.
In some instances of formula (VIIm′), R3 and R4 are independently alkoxy; and R2 and R5 are both hydrogen. In certain cases, the alkoxy is methoxy. In some cases, R3 is alkoxy; and R2, R4 and R5 are hydrogen. In some cases, R4 is alkoxy; and R2, R3 and R5 are each hydrogen. In certain cases of formula (VIIm′), R2, R3 and R4 are hydrogen and R5 is alkoxy. In certain cases, the alkoxy is a 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 pentoxy. In certain cases, the alkoxy is hexyloxy.
In certain embodiments of formula (VIIm′), n is 0-3 and m is 0-3. In some instances 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 case, 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.
In certain instances of the ENPP1 inhibitor compounds of formula (I′), Z3 is absent. In certain embodiments of formula (I′), Z3 is absent, Z2 is CR12, R12 is cyano, and the compound is described by formula (X′):
wherein L11 and L12 are independently covalent bond or linker. In some instances of formula (X′), L11 is covalent bond.
In some embodiments of formula (X′), the ring system A is selected from phenyl, substituted phenyl, pyridyl, substituted pyridyl, pyrimidine, substituted pyrimidine, piperidine, substituted piperidine, piperazine, substituted piperazine, pyridazine, substituted pyridazine, cyclohexyl and substituted cyclohexyl. In certain cases, the ring system A is phenyl or substituted phenyl. In some cases, the ring system A is pyridyl or substituted pyridyl. In some cases, the ring system A is pyrimidine or substituted pyrimidine. In some cases, the ring system A is piperidine or substituted piperidine. In some cases, the ring system A is piperazine or substituted piperazine. In some cases, the ring system A is cyclohexyl or substituted cyclohexyl.
In some embodiments, the ring system A is described by any one of formulae (A1′)-(A4′), (e.g., as described herein):
wherein:
In some embodiments the A ring is described by the formula (A5′):
wherein:
In certain cases, A5′ is piperidine or substituted piperidine. In certain cases, A5′ is piperazine or substituted piperazine. In certain cases, A5′ is a cyclohexyl or a substituted cyclohexyl. In certain embodiments of A5′, r is greater than 0, such as 1, 2, 3, 4, 5, 6, 7 or 8. In some cases, A5′ includes one R16 group. In some cases, A5′ includes two R16 groups. In some cases, A5′ includes three R16 groups. In some cases, A5′ includes four R16 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).
In certain embodiments, the A ring has any one of the formulae (A5a′)-(A5c′):
In certain embodiments, the A ring is a cyclohexyl having the relative configuration of formula (A5d′) or (A5e′):
In certain cases of formula (X′), the subject ENPP1 inhibitor compound is of the formula (XI′):
In certain embodiments of formula (XI′), at least one Z5 is N. In certain embodiments of formula (XI′), one Z5 is N and the other Z5 is CR16. In certain cases of formula (XI′), both Z5 groups are CR16. In certain cases of formula (XI′), both Z5 groups are N.
In certain embodiments of a compound of any one of formulae (X′)-(XI′), L1 and L12 are each covalent bonds. In certain cases, L11 and L12 are each linkers. In certain cases, L11 is a covalent bond and L12 is a linker. In certain cases, L11 is a linker and L12 is a covalent bond. Any convenient linkers can be utilized as L1 and L12. In some cases, L1 is a covalent bond. In certain cases, L11 is a linear linker of 1-12 atoms in length, such as 1-10, 1-8 or 1-6 atoms in length, e.g., 1, 2, 3, 4, 5 or 6 atoms in length. The linker L11 can be a (C1-6)alkyl linker or a substituted (C1-6)alkyl linker, optionally substituted with a heteroatom or linking functional group, such as an ester (—CO2—), amido (CONH), carbamate (OCONH), ether (—O—), thioether (—S—) and/or amino group (—NR— where R is H or alkyl). In some cases, L12 is a covalent bond. In certain cases, L12 is a linker of 1-12 atoms in length, such as 1-10, 1-8 or 1-6 atoms in length, e.g., 1, 2, 3, 4, 5 or 6 atoms in length. The linker L12 can be a (C1-6)alkyl linker or a substituted (C1-6)alkyl linker, optionally substituted with a heteroatom or linking functional group, such as an ester (—CO2—), amido (CONH), carbamate (OCONH), ether (—O—), thioether (—S—) and/or amino group (—NR— where R is H or alkyl).
In some cases of formula (XI′), the subject ENPP1 inhibitor compound is of the formula (XII′):
In certain embodiments of the compound of formula (XII′), Z5 is CR16, wherein R16 is selected from hydrogen, alkyl, substituted alkyl, hydroxy, alkoxy, substituted alkoxy, trifluoromethyl, halogen, acyl, substituted acyl, carboxy, carboxyamide, substituted carboxyamide, sulfonyl, substituted sulfonyl, sulfonamide and substituted sulfonamide. In certain cases of the compound of formula (XII′), Z5 is N.
In certain embodiments of a compound of formula (XII′), L12 is a covalent bond. In certain cases, L12 is a linker. Any convenient linkers can be utilized as L12. In certain cases, L12 is a linear linker of 1-12 atoms in length, such as 1-10, 1-8 or 1-6 atoms in length, e.g., 1, 2, 3, 4, 5 or 6 atoms in length. The linker L12 can be a (C1-6)alkyl linker or a substituted (C1-6)alkyl linker, optionally substituted with a heteroatom or linking functional group, such as an ester (—CO2—), amido (CONH), carbamate (OCONH), ether (—O—), thioether (—S—) and/or amino group (—NR— where R is H or alkyl). In some cases of formula (XII′), the subject ENPP1 inhibitor compound is of the formula (XIII′):
Wherein
In certain embodiments of formula (XIII′), R35 and R36 are each hydrogen. In certain embodiments, at least one of R35 or R36 is a halogen. In certain embodiments, at least one of R35 or R36 is alkyl. In certain embodiments, at least one of R35 or R36 is substituted alkyl. In certain cases, R35 is halogen and R36 is selected from hydrogen, halogen, alkyl and substituted alkyl. In certain cases, R35 is alkyl and R36 is selected from hydrogen, halogen, alkyl and substituted alkyl. In certain cases, R35 is substituted alkyl and R36 is selected from hydrogen, halogen, alkyl and substituted alkyl. In certain cases, R35 is halogen and R36 is hydrogen. In certain cases, R35 is alkyl and R36 is hydrogen. In certain cases, R35 is substituted alkyl and R36 is hydrogen.
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.
In some cases of formula (XIII′), the subject ENPP1 inhibitor compound is of the formula (XIV′):
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.
In certain embodiments of any of formulae (X′)-(XIV′), R2 to R5 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 any of formulae (X′)-(XIV′), R2 to R5 are independently selected from hydrogen, OH, C(1-6) alkoxy, —OCF3, C(1-6) alkylamino, di-C(1-6) alkylamino, F, Cl, Br and CN.
In certain cases of any of formulae (X′)-(XIV′), at least one of R2 to R5 is hydrogen. In certain cases, at least two of R2 to R5 are hydrogen. In certain cases, at least three of R2 to R5 are hydrogen. In certain cases, each of R2 to R5 is hydrogen. In certain cases, at least one of R2 to R5 is hydroxy. In certain cases, at least one of R2 to R5 is alkyl or substituted alkyl. In certain cases, at least one of R2 to R5 is alkoxy or substituted alkoxy. In certain cases, the alkoxy or substituted alkoxy is a C(1-6) alkoxy, e.g., methoxy, ethoxy, propoxy, butoxy, pentoxy, hexoxy. In certain cases at least one of R2 to R5 is methoxy. In certain cases, at least one of R2 to R5 is —OCF3. In certain cases, at least one of R2 to R5 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 R2 to R5 is cyano. In certain cases, at least one of R2 to R5 is amine or substituted amine. In certain cases, at least one of R2 to R5 is C(1-6) alkylamino. In certain cases, at least one of R2 to R5 is di-C(1-6) alkylamino. In certain cases, at least one of R2 to R5 is amide. In certain cases, at least one of R2 to R5 is heterocycle or substituted heterocycle.
In some instances of any of formulae (X′)-(XIV′), R3 and R4 are independently alkoxy; and R2 and R5 are both hydrogen. In some cases, R3 is alkoxy; and R2, R4 and R5 are hydrogen. In some cases, R4 is alkoxy; and R2, R3 and R5 are each hydrogen. In certain cases, R2, R3 and R4 are hydrogen and R5 is alkoxy. In certain cases, the alkoxy is a 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 pentoxy. In certain cases, the alkoxy is hexyloxy.
In some cases of formula (XIV′), the subject ENPP1 inhibitor compound is of one of formulae (XIVa′)-(XIVe′):
In some cases of formula (I), the subject ENPP1 inhibitor compound is of the formula (XVa′) or (XVb′):
wherein:
In some cases of formula (XVa′)-(XVb′), R21 is selected from methyl, ethyl, n-propyl and isopropyl. In certain cases, R21 is methyl. In some cases of formula (XVa′)-(XVb′), R3 and R4 are Cl. In certain instances, R3 and R4 are F. In some cases of formula (XVa′)-(XVb′), s is 2. In certain instances, s is 1. In some embodiments of formulae (XVa′)-(XVb′), s is 2; R21 is methyl or isopropyl; and R3 and R4 are selected from Cl and F.
In some instances of formulae (XVa′)-(XVb′), the subject ENPP1 inhibitor compound is of one of the following structures, or a prodrug thereof (e.g., as described herein):
As described above, X1 is a hydrophilic head group or a prodrug version thereof. Any embodiments of a hydrophilic head group described herein can be incorporated into any one of the embodiments of formulae (I′)-(XVb′) described herein. In some embodiments of formulae (I′)-(XVb′), X1 is a hydrophilic head group comprising a charged group capable of binding zinc ion, or a prodrug form thereof. In certain cases, the hydrophilic head group capable of binding zinc ion is a phosphorus containing functional group (e.g., as described herein).
In some embodiments of formulae (I′)-(XVb′), the hydrophilic head group (X1) is selected from phosphonic acid or phosphonate, phosphonate ester, phosphate, phosphate ester, thiophosphate, thiophosphate ester, phosphoramidate, thiophosphoramidate, sulfonate, sulfonic acid, sulfate, hydroxamic acid, keto acid, amide and carboxylic acid. In some embodiments of any one of formulae (I′)-(XVb′), the hydrophilic head group is phosphonic acid, phosphonate, or a salt thereof. In some embodiments of any one of formulae (I′)-(XVb′), the hydrophilic head group is phosphate or a salt thereof. In some embodiments of any one of formulae (I′)-(XVb′), the hydrophilic head group is phosphonate ester or phosphate ester. In some embodiments of any one of formulae (I′)-(XVb′), the hydrophilic head group is a thiophosphate. In some embodiments of any one of formulae (I′)-(XVb′), the hydrophilic head group is a thiophosphate ester. In some embodiments of any one of formulae (I′)-(XVb′), the hydrophilic head group is a phosphoramidate. In some embodiments of any one of formulae (I′)-(XVb′), the hydrophilic head group is a thiophosphoramidate.
Particular examples of hydrophilic head groups of interest which can be incorporated into any one of the embodiments of formulae (I′)-(XVb′) described herein include, but are not limited to, a head group comprising a first moiety selected from phosphates (RPO4H−), phosphonates (RPO3H−), boric acid (RBO2H2), carboxylates (RCO2−), sulfates (RSO4−), sulfonates (RSO3−), amines (RNH3+), glycerols, sugars such as lactose or derived from hyaluronic acid, polar amino acids, polyethylene oxides and oligoethyleneglycols, that is optionally conjugated to a residue of a second moiety selected from choline, ethanolamine, glycerol, nucleic acid, sugar, inositol, amino acid or amino acid ester (e.g., serine) and lipid (e.g., fatty acid or hydrocarbon chain, such as a C8-C30 saturated or unsaturated hydrocarbon). The head group may contain various other modifications, for instance, in the case of the oligoethyleneglycols and polyethylene oxide (PEG) containing head groups, such PEG chain may be terminated with a methyl group or have a distal functional group for further modification. Examples of hydrophilic head groups also include, but are not limited to, thiophosphate, phosphocholine, phosphoglycerol, phosphoethanolamine, phosphoserine, phosphoinositol, ethylphosphosphorylcholine, polyethyleneglycol, polyglycerol, melamine, glucosamine, trimethylamine, spermine, spermidine, and conjugated carboxylates, sulfates, boric acid, sulfonates, sulfates and carbohydrates.
In some instances of any one of formulae (I′)-(XVb′), the hydrophilic head group X1 is of formula (XVI′):
In some embodiments of formula (XVI′), Z6 is absent. In other cases, Z6 is CH2. In other cases, Z6 is oxygen. In some embodiments of formula (XVI′), Z7 is oxygen and Z9 is NR10. In some cases, Z7 is NR10 and Z9 is oxygen. In some cases, both Z7 and Z9 are oxygen. In other cases, both Z7 and Z9 are NR10. In some cases, Z8 is oxygen. In other cases, Z8 is sulfur.
In some embodiments of formula (XVI′), Z7, Z8 and Z9 are all oxygen atoms and Z6 is absent or CH2. In other cases, Z8 is a sulfur atom, Z7 and Z9 are both oxygen atoms and Z6 is absent or CH2. In other cases, Z8 is a sulfur atom, Z6, Z7 and Z9 are all oxygen atoms. In some cases, Z8 is an oxygen atom, Z7 is NR10, Z9 is an oxygen atom and Z6 is absent or CH2. In other cases, Z8 is an oxygen atom, Z7 is NR10, Z6 and Z9 are both oxygen atoms. In other cases, Z8 is an oxygen atom, Z7 and Z9 are each independently NR10 and Z6 is an oxygen atom. In yet other cases, Z8 is an oxygen atom, Z7 and Z9 are each independently NR10 and Z6 is absent or CH2. In some cases, Z7 and Z9 are each the same. In other cases, Z7 and Z9 are different. It is understood that the group of formula (XVI′) may include one or more tautomeric forms of the structure depicted and that all such forms, and salts thereof, are meant to be included.
In some embodiments of formula (XVI′), at least one of Z7 and Z9 is NR10. In some cases, R10 is hydrogen. In some cases, R10 is alkyl. In some other cases, R10 is substituted alkyl. In some cases, both Z7 and Z9 are NR10. In some cases, both Z7 and Z9 are NR10 and each R10, R8 and R9 are independently hydrogen. In some cases, both Z7 and Z9 are NR10, each R10 is an alkyl group, and R8 and R9 are each hydrogen. In some cases, both Z7 and Z9 are NR10, each R10 is a substituted alkyl group (e.g., an alkyl group substituted with an ester or a carboxyl group), and R8 and R9 are each hydrogen.
In some embodiments of formula (XVI′), R8 and R9 are both hydrogen atoms. In some cases, at least one of R8 and R9 is a substituent other than hydrogen. In other cases, both R8 and R9 are substituents other than hydrogen. In some cases, at least one of R8 and R9 is an alkyl or substituted alkyl. In some cases, at least one of R8 and R9 is alkenyl or substituted alkenyl. In some other cases, at least one of R8 and R9 is aryl or substituted aryl. In some cases, at least one of R8 and R9 is acyl or substituted acyl. In some cases, at least one of R8 and R9 is heteroaryl or substituted heteroaryl. In some cases, at least one of R8 and R9 is cycloalkyl or substituted cycloalkyl. In some cases, R8 and R9 are both alkyl groups (e.g., lower alkyl). In some cases, R8 and R9 are both substituted alkyl groups (e.g., a C(1-6)alkyl, substituted with alkoxy, substituted alkoxy, ester or carboxyl group). In some cases, at least one of R8 and R9 includes a promoiety. In certain cases, both R8 and R9 are phenyl groups. In some cases, R8 and R9 are the same. In other cases, R8 and R9 are different.
In some instances of any one of formulae (I′)-(XVb′), the hydrophilic head group X1 is selected from any one of formulae (XVIa′) to (XVIf′):
In some embodiments of formulae (XVIa′) to (XVIf′), R10 and R11 are both hydrogen atoms. In some cases, at least one of R10 and R11 is a substituent other than hydrogen. In other cases, both R10 and R11 are substituents other than hydrogen. In some cases, R10 and R11 are the same. In other cases, R10 and R11 are different. In some cases, at least one of R10 and R11 is an alkyl or substituted alkyl. In some cases, at least one of R10 and R11 is aryl or substituted aryl. In some cases, both of R1 and R11 are alkyl or substituted alkyl. In some cases, both of R10 and R11 are aryl or substituted aryl. In some cases, both of R10 and R11 are acyl or substituted acyl. In some cases, R10 and R11 are both lower alkyl groups. In some cases, R10 and R11 are both substituted alkyl groups (e.g., a C(1-6) alkyl, substituted with alkoxy, substituted alkoxy, ester or carboxyl group). In some cases, at least one of R10 and R11 includes a promoiety. In certain cases, both R10 and R11 are phenyl groups.
In certain instances of formulae (XVIa′) to (XVId′), at least one of R10 and R11 includes a cleavable group or a self-immolative promoiety. A self-immolative group can be a disulfide linked promoiety or a self immolative ester containing promoiety. In some cases, R10 and/or R11 includes a disulfide linked promoiety of formula: —CH2CH2—SS—R12 where R12 is alkyl or substituted alkyl. In certain instances, R12 is a C8-C30 saturated or unsaturated hydrocarbon chain. In some cases, R11 and/or R11 includes a promoiety of formula: —CH2OCOR13 where R11 is H, alkyl or substituted alkyl. In some cases, R10 and/or R11 includes a promoiety of formula: —CH2C(R14)2CO2R14 where each R14 is independently H, alkyl or substituted alkyl.
In some instances of any one of formulae (I′)-(XVb′), the hydrophilic head group X1, or prodrug form thereof, is selected from:
or a pharmaceutically acceptable salt thereof.
In some instances of any one of formulae (I′)-(XVb′), the hydrophilic head group X1 is of the formula (XVI′):
In some embodiments of formula (XVI′), R81 and R91 are both hydrogen atoms. In other cases, both R81 and R91 are substituents other than hydrogen.
In some instances of any one of formulae (I′)-(XVb′), the hydrophilic head group X1 is of the formula
In some instances of any one of formulae (I′)-(XVb′), the hydrophilic head group X1 is of the formula (XVIII′):
wherein:
In some embodiments of formula (XVIII′), the hydrophilic head group is selected from one of the following groups:
In some instances of any one of formulae (I′)-(XVb′), the hydrophilic head group X1 is of the formula (XIX′):
In some instances of any one of formulae (I′)-(XVb′), the hydrophilic head group X1 is of the formula (XX′):
In some embodiments of formula (XX′), R92 is hydrogen. In other cases, R92 is a substituent other than hydrogen. In certain embodiments, R92 is alkyl or substituted alkyl. In certain embodiments of formula (XX′), the hydrophilic head group is of the structure:
In some instances of any one of formulae (I′)-(XVb′), the hydrophilic head group X1 is of the formula (XXI′):
It will be understood that any of the hydroxyl and amine groups in group X1 of any of formulae (I′)-(XVb′) may be optionally further substituted with any convenient group, e.g., an alkyl group, a substituted alkyl group, a phenyl group, a substituted phenyl group, an ester group and the like. It will be understood that any convenient alternative hydrophilic group can be utilized as group X1 in a compound of any of formulae (I′)-(XVb′).
In certain embodiments, the ENPP1 inhibitor compound is described by one of the structures of Table 1, Table 2, Table 3, Table 4, Table 5, or Table 6, or a prodrug thereof (e.g., as described herein), or a pharmaceutically acceptable salt thereof.
In certain embodiments, the compound is described by the structure of one of the compounds of Table 1, Table 2, Table 3, Table 4, Table 5, or Table 6. It is understood that any of the compounds shown in Table 1, Table 2, Table 3, Table 4, Table 5, or Table 6 may be present in a salt form. In some cases, the salt form of the compound is a pharmaceutically acceptable salt. It is understood that any of the compounds shown in Table 1 or Table 2 may be present in a prodrug form.
Aspects of the present disclosure include ENPP1 inhibitor compounds (e.g., as described herein), salts thereof (e.g., pharmaceutically acceptable salts), and/or solvate, hydrate and/or prodrug forms thereof. In addition, it is understood that, in any compound described herein having one or more chiral centers, if an absolute stereochemistry is not expressly indicated, then each center may independently be of R-configuration or S-configuration or a mixture thereof. It will be appreciated that all permutations of salts, solvates, hydrates, prodrugs and stereoisomers are meant to be encompassed by the present disclosure.
In some embodiments, the subject ENPP1 inhibitor compounds, or a prodrug form thereof, are provided in the form of pharmaceutically acceptable salts. Compounds containing an amine or nitrogen containing heteroaryl group may be basic in nature and accordingly may react with any number of inorganic and organic acids to form pharmaceutically acceptable acid addition salts. Acids commonly employed to form such salts include inorganic acids such as hydrochloric, hydrobromic, hydriodic, sulfuric and phosphoric acid, as well as organic acids such as para-toluenesulfonic, methanesulfonic, oxalic, para-bromophenylsulfonic, carbonic, succinic, citric, benzoic and acetic acid, and related inorganic and organic acids. Such pharmaceutically acceptable salts thus include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butyne-1,4-dioate, hexyne-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, terephathalate, sulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, β-hydroxybutyrate, glycollate, maleate, tartrate, methanesulfonate, propanesulfonates, naphthalene-1-sulfonate, naphthalene-2-sulfonate, mandelate, hippurate, gluconate, lactobionate, and the like salts. In certain specific embodiments, pharmaceutically acceptable acid addition salts include those formed with mineral acids such as hydrochloric acid and hydrobromic acid, and those formed with organic acids such as fumaric acid and maleic acid.
In some embodiments, the subject compounds are provided in a prodrug form. “Prodrug” refers to a derivative of an active agent that requires a transformation within the body to release the active agent. In certain embodiments, the transformation enzymatic transformation. Prodrugs are frequently, although not necessarily, pharmacologically inactive until converted to the active agent. “Promoiety” refers to a form of protecting group that, when used to mask a functional group within an active agent, converts the active agent into a prodrug. In some cases, the promoiety will be attached to the drug via bond(s) that are cleaved by enzymatic or non-enzymatic means in vivo. Any convenient prodrug forms of the subject compounds can be prepared, e.g., according to the strategies and methods described by Rautio et al. (“Prodrugs: design and clinical applications”, Nature Reviews Drug Discovery 7, 255-270 (February 2008)). In some cases, the promoiety is attached to a hydrophilic head group of the subject compounds. In some cases, the promoiety is attached to a hydroxy or carboxylic acid group of the subject compounds. In certain cases, the promoiety is an acyl or substituted acyl group. In certain cases, the promoiety is an alkyl or substituted alkyl group, e.g., that forms an ester functional group when attached to a hydrophilic head group of the subject compounds, e.g., a phosphonate ester, a phosphate ester, etc.
In some embodiments, the subject compounds, prodrugs, stereoisomers or salts thereof are provided in the form of a solvate (e.g., a hydrate). The term “solvate” as used herein refers to a complex or aggregate formed by one or more molecules of a solute, e.g., 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 and solvent. Representative solvents include by way of example, water, methanol, ethanol, isopropanol, acetic acid, and the like. When the solvent is water, the solvate formed is a hydrate.
In some embodiments, the subject compounds are provided by oral dosing and absorbed into the bloodstream. In some embodiments, the oral bioavailability of the subject compounds is 30% or more. Modifications may be made to the subject compounds or their formulations using any convenient methods to increase absorption across the gut lumen or their bioavailability.
In some embodiments, the subject compounds are metabolically stable (e.g., remain substantially intact in vivo during the half-life of the compound). In certain embodiments, the compounds have a half-life (e.g., an in vivo half-life) of 5 minutes or more, such as 10 minutes or more, 12 minutes or more, 15 minutes or more, 20 minutes or more, 30 minutes or more, 60 minutes or more, 2 hours or more, 6 hours or more, 12 hours or more, 24 hours or more, or even more.
In some embodiments, ENPP1 inhibitors include the formula:
or a pharmaceutically acceptable salt thereof, additional details of which are described in US Application Pub. No. US20190031655A1, herein incorporated by reference for all purposes.
In some embodiments, ENPP1 inhibitors include the formula:
or a pharmaceutically acceptable salt thereof, additional details of which are described in US Application Pub. No. US20200039979A1, herein incorporated by reference for all purposes.
In some embodiments, ENPP1 inhibitors include the formula:
or a pharmaceutically acceptable salt thereof, additional details of which are described in International Application Pub. No. WO2018119328A1, herein incorporated by reference for all purposes.
In some embodiments, ENPP1 inhibitors include the formula:
or pharmaceutically acceptable salts thereof, additional details of which are described in International Application Pub. No. WO2019046778A1 and US Application Pub. No. US20190282703A1, each herein incorporated by reference for all purposes.
In some embodiments, ENPP1 inhibitors include the formula:
or pharmaceutically acceptable salts thereof, additional details of which are described in International Application Pub. No. WO2019177971A1, herein incorporated by reference for all purposes.
In some embodiments, ENPP1 inhibitors include the formula:
or pharmaceutically acceptable salts thereof, additional details of which are described in International Application Pub. No. WO2019191504A1, herein incorporated by reference for all purposes.
As summarized above, aspects of the present disclosure include ENPP1 inhibitors, and methods of inhibition using the same. ENPP1 is a member of the ecto-nucleotide pyrophosphatase/phosphodiesterase (ENPP) family. As such, aspects of the subject methods include inhibition of the hydrolase activity of ENPP1 against cGAMP. The inventors discovered that cGAMP can have significant extracellular biological functions, which can be enhanced by blocking extracellular degradation of cGAMP, e.g., hydrolysis by its degradation enzyme ENPP1. In certain instances, the ENPP1 target of inhibition is extracellular, and the subject ENPP1 inhibiting compounds are cell-impermeable, and thus are not capable of diffusion into cells. As such, the subject methods can provide for selective extracellular inhibition of ENPP1's hydrolase activity and increased extracellular levels of cGAMP. As such, in some cases, the ENPP1 inhibiting compounds are compounds that inhibit the activity of ENPP1 extracellularly. Experiments conducted by the inventors indicate that inhibiting the activity of ENPP1 increases extracellular cGAMP and may consequently boost the STING pathway.
By inhibiting a ENPP1 it is meant that the activity of the enzyme is decreased by 10% or more, such as 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more (e.g., relative to a control in any convenient in vitro inhibition assay). In some cases, inhibiting a ENPP1 means decreasing the activity of the enzyme by a factor of 2 or more, such as 3 or more, 5 or more, 10 or more, 100 or more, or 1000 or more, relative to its normal activity (e.g., relative to a control as measured by any convenient assay).
In some cases, the method is a method of inhibiting ENPP1 in a sample. The term “sample” as used herein relates to a material or mixture of materials, typically, although not necessarily, in fluid form, containing one or more components of interest.
In some embodiments, there is provided a method of inhibiting ENPP1, the method comprising contacting a sample with a cell impermeable ENPP1 inhibitor to inhibit cGAMP hydrolysis activity of ENPP1. In some cases, the sample is a cellular sample. In some cases, the sample comprises cGAMP. In certain cases, the cGAMP levels are elevated in the cellular sample (e.g., relative to a control sample not contacted with the inhibitor). The subject methods can provide for increased levels of cGAMP. By “increased level of cGAMP” is meant a level of cGAMP in a cellular sample contacted with a subject compound, where the cGAMP level in the sample is increased by 10% or more, such as 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 100% or more, or even more, relative to a control sample that is not contacted with the agent.
In certain embodiments the cell impermeable ENPP1 inhibitor is an inhibitor as defined herein. In some embodiments, the cell impermeable ENPP1 inhibitor is an inhibitor according to any one of formulas I, IV V, VI or VII. In some cases, the cell impermeable ENPP1 inhibitor is any one of compounds 1-106.
In some embodiments the ENPP1 inhibitor is cell permeable. In some embodiments, there is provided a method of inhibiting ENPP1, the method comprising contacting a sample with a cell permeable ENPP1 inhibitor to inhibit ENPP1.
In some embodiments, the subject compounds have an ENPP1 inhibition profile that reflects activity against additional enzymes. In some embodiments, the subject compounds specifically inhibit ENPP1 without undesired inhibition of one or more other enzymes.
In some embodiments, the compounds of the disclosure interfere with the interaction of cGAMP and ENPP1. For example, the subject compounds may act to increase the extracellular cGAMP by inhibiting the hydrolase activity of ENPP1 against cGAMP. Without being bound to any particular theory, it is thought that increasing extracellular cGAMP activates the STING pathway.
In some embodiments, the subject compounds inhibit ENPP1, as determined by an inhibition assay, e.g., by an assay that determines the level of activity of the enzyme either in a cell-free system or in a cell after treatment with a subject compound, relative to a control, by measuring the IC50 or EC50 value, respectively. In certain embodiments, the subject compounds have an IC50 value (or EC50 value) of 10 μM or less, such as 3 μM or less, 1 μM or less, 500 nM or less, 300 nM or less, 200 nM or less, 100 nM or less, 50 nM or less, 30 nM or less, 10 nM or less, 5 nM or less, 3 nM or less, 1 nM or less, or even lower.
As summarized above, aspects of the disclosure include methods of inhibiting ENPP1. A subject compound (e.g., as described herein) may inhibit at activity of ENPP1 in the range of 10% to 100%, e.g., by 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more. In certain assays, a subject compound may inhibit its target with an IC50 of 1×10−6 M or less (e.g., 1×10−6 M or less, 1×10−7 M or less, 1×10−8 M or less, 1×10−19 M or less, 1×10−10 M or less, or 1×10−11 M or less).
The protocols that may be employed in determining ENPP1 activity are numerous, and include but are not limited to cell-free assays, e.g., binding assays; assays using purified enzymes, cellular assays in which a cellular phenotype is measured, e.g., gene expression assays; and in vivo assays that involve a particular animal (which, in certain embodiments may be an animal model for a condition related to the target pathogen).
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 comprises evaluating whether the compound inhibits ENPP1.
In certain embodiments, the subject compound is a modified compound that includes a label, e.g., a fluorescent label, and the subject method further includes detecting the label, if present, in the sample, e.g., using optical detection.
In certain embodiments, the compound is modified with a support or with affinity groups that bind to a support (e.g., biotin), such that any sample that does not bind to the compound may be removed (e.g., by washing). The specifically bound ENPP1, if present, may then be detected using any convenient means, such as, using the binding of a labeled target specific probe, or using a fluorescent protein reactive reagent.
In another embodiment of the subject method, the sample is known to contain ENPP1.
In some embodiments, the method is a method of reducing cancer cell proliferation, where the method includes contacting the cell with an effective amount of a subject ENPP1 inhibitor compound (e.g., as described herein) to reduce cancer cell proliferation. In certain cases, the subject ENPP1 inhibitor compounds can act intracellularly. The method can be performed in combination with a chemotherapeutic agent (e.g., as described herein). The cancer cells can be in vitro or in vivo. In certain instances, the method includes contacting the cell with an ENPP1 inhibitor compound (e.g., as described herein) and contacting the cell with a chemotherapeutic agent. Any convenient cancer cells can be targeted.
In some embodiments, the method is a method of enhancing tumor infiltration by immune effector cells (for example CAR expressing cells, CAR-T cells, T cells, or splenocytes) where the method includes contacting an ENPP1 expressing cell with an effective amount of a subject ENPP1 inhibitor compound (e.g., as described herein) to enhance tumor infiltration by immune effector cells.
In some embodiments, the method is a method of activating immune effector cells (for example CAR expressing cells, CAR-T cells, T cells, or splenocytes) where the method includes contacting an ENPP1 expressing cell with an effective amount of a subject ENPP1 inhibitor compound (e.g., as described herein) to activate immune effector cells.
Certain aspects of the present disclosure relate to chimeric receptors. In some embodiments, the chimeric receptor is a chimeric antigen receptor (CAR).
In some embodiments CARs are T cell receptors (TCRs). In certain embodiments the TCR comprises a TCR alpha chain and a TCR beta chain. In certain embodiments the TCR comprises a TCR gamma chain and a TCR delta chain. In some embodiments, the TCR further comprises additional signaling polypeptides such as CD3δ (delta), CD3γ (gamma), CD3ε (epsilon) and CD3ζ (zeta). In some embodiments, the TCR is an engineered TCR. In some embodiments, the TCR recognizes a specific epitope presented by an MHC.
In some embodiments, CARs are engineered receptors that graft or confer a specificity of interest onto an immune effector cell. In certain embodiments, CARs can be used to graft the specificity of an antibody onto an immune effector cell, such as a T cell. In some embodiments, CARs of the present disclosure comprise at least an extracellular antigen-binding domain, a transmembrane domain, and a cytoplasmic signaling domain (also referred to herein as “an intracellular signaling domain”) comprising a functional signaling domain derived from a stimulatory molecule as defined below.
In some embodiments, the stimulatory molecule is the zeta chain associated with the T cell receptor complex. In one aspect, the cytoplasmic signaling domain comprises a primary signaling domain (e.g., a primary signaling domain of CD3-zeta). In one aspect, the cytoplasmic signaling domain further comprises one or more functional signaling domains derived from at least one costimulatory molecule as defined below. In one aspect, the costimulatory molecule is chosen from 4-1BB (i.e., CD 137), OX40 (CD134), CD27, ICOS, and/or CD28.
As used herein, the term “intracellular signaling domain” refers to the functional portion of a protein which acts by transmitting information within the cell to regulate cellular activity via defined signaling pathways by generating second messengers or functioning as effectors by responding to such messengers. In some embodiments, the signaling domain of a chimeric receptor of the present disclosure is derived from a stimulatory molecule or co-stimulatory molecule described herein, or is a synthesized or engineered signaling domain.
In some embodiments, the CAR comprises one or more intracellular signaling domains, and the one or more intracellular signaling domains are selected from the group consisting of a CD3zeta-chain intracellular signaling domain, a CD97 intracellular signaling domain, a CD11a-CD18 intracellular signaling domain, a CD2 intracellular signaling domain, an ICOS intracellular signaling domain, a CD27 intracellular signaling domain, a CD154 intracellular signaling domain, a CD8 intracellular signaling domain, an OX40 intracellular signaling domain, a 4-1BB intracellular signaling domain, a CD28 intracellular signaling domain, a ZAP40 intracellular signaling domain, a CD30 intracellular signaling domain, a GITR intracellular signaling domain, an HVEM intracellular signaling domain, a DAP10 intracellular signaling domain, a DAP12 intracellular signaling domain, and a MyD88 intracellular signaling domain.
In some embodiments, the CAR comprises a transmembrane domain, and the transmembrane domain is selected from the group consisting of a CD8 transmembrane domain, a CD28 transmembrane domain a CD3zeta-chain transmembrane domain, a CD4 transmembrane domain, a 4-1BB transmembrane domain, an OX40 transmembrane domain, an ICOS transmembrane domain, a CTLA-4 transmembrane domain, a PD-1 transmembrane domain, a LAG-3 transmembrane domain, a 2B4 transmembrane domain, and a BTLA transmembrane domain.
In some embodiments, the antigen binding domain in the CAR is an antibody or an antigen-binding fragment thereof. In some embodiments, the antigen-binding domain comprises an antibody, an antigen-binding fragment of an antibody, a F(ab) fragment, a F(ab′) fragment, a single chain variable fragment (scFv), or a single-domain antibody (sdAb). In some embodiments, the antigen-binding domain comprises a single chain variable fragment (scFv). In some embodiments, the scFv comprises a heavy chain variable domain (VH) and a light chain variable domain (VL). In some embodiments, the VH and VL are separated by a peptide linker. In some embodiments, the antigen binding domain in the CAR binds to a tumor antigen. In some embodiments, the tumor antigen is associated with a solid tumor. Exemplary antigens and respective antibodies or antigen binding fragments are listed in Table 7.
In some embodiments the antigen binding domain in the CAR is a ligand or a functional fragment of a ligand of the antigen. Exemplary antigens and respective ligands are listed in Table 7.
In some embodiments, the CAR comprises a means to specifically bind an antigen. In some embodiments, the means to bind an antigen comprise a TCR, a fragment of a TCR, an engineered TCR, an antibody or antibody fragment, or an antigen ligand. In some embodiments, the means for binding an antigen comprise a scFv.
The term “antibody,” as used herein, refers to a protein, or polypeptide sequence derived from an immunoglobulin molecule which specifically binds with an antigen. Antibodies can be polyclonal or monoclonal, multiple or single chain, or intact immunoglobulins, and may be derived from natural sources or from recombinant sources. Antibodies can be tetramers of immunoglobulin molecules.
As used herein, the term “antibody fragment” refers to at least one portion of an intact antibody, or recombinant variants thereof, and refers to the antigen-binding domain, e.g., an antigenic determining variable region of an intact antibody, that is sufficient to confer recognition and specific binding of the antibody fragment to a target, such as an antigen. Examples of antibody fragments include, but are not limited to, Fab, Fab′, F(ab′)2, and Fv fragments, scFv antibody fragments, linear antibodies, single domain antibodies such as sdAb (either VL or VH), camelid VHH domains, and multi-specific antibodies formed from antibody fragments such as a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region, and an isolated CDR or other epitope binding fragments of an antibody. An antigen binding fragment can also be incorporated into single domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR and bis-scFv (see, e.g., Hollinger and Hudson, Nature Biotechnology 23: 1126-1 136, 2005). Antigen binding fragments can also be grafted into scaffolds based on polypeptides such as a fibronectin type III (Fn3)(see U.S. Pat. No. 6,703,199, which describes fibronectin polypeptide minibodies).
As used herein, the term “single-chain variable fragment” or “scFv” refers to a fusion protein comprising at least one antibody fragment comprising a variable region of a light chain and at least one antibody fragment comprising a variable region of a heavy chain, wherein the light and heavy chain variable regions are contiguously linked via a short flexible polypeptide linker, and capable of being expressed as a single chain poly peptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived. Unless specified, as used herein an scFv may have the VL and VH variable regions in either order, e.g., with respect to the N-terminal and C-terminal ends of the polypeptide, the scFv may comprise VL-linker-VH or may comprise VH-linker-VL.
The term “complementarity determining region” or “CDR,” as used herein, refers to the sequences of ammo acids within antibody variable regions which confer antigen specificity and binding affinity. For example, in general, there are three CDRs in each heavy chain variable region (e.g., HCDR1, HCDR2, and HCDR3) and three CDRs in each light chain variable region (LCDR1, LCDR2, and LCDR3). The precise ammo acid sequence boundaries of a given CDR can be determined using any of a number of well-known schemes, including those described by Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (“Kabat” numbering scheme), Al-Lazikani et al, (1997) JMB 273,927-948 (“Chothia” numbering scheme), or a combination thereof. Under the Kabat numbering scheme, in some embodiments, the CDR amino acid residues in the heavy chain variable domain (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3); and the CDR amino acid residues in the light chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3). Under the Chothia numbering scheme, in some embodiments, the CDR amino acids in the VH are numbered 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3); and the CDR amino acid residues in the VL are numbered 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3). In a combined Kabat and Chothia numbering scheme, in some embodiments, the CDRs correspond to the amino acid residues that are part of a Kabat CDR, a Chothia CDR, or both. For instance, in some embodiments, the CDRs correspond to amino acid residues 26-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3) in a VH, e.g., a mammalian VH, e.g., a human VH; and ammo acid residues 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) in a VL, e.g., a mammalian VL, e.g., a human VL.
The portion of the chimeric receptor of the present disclosure comprising an antibody or antibody fragment thereof may exist in a variety of forms where the antigen-binding domain is expressed as part of a contiguous polypeptide chain including, for example, scFv antibody fragments, linear antibodies, single domain antibodies such as sdAb (either VL or VH), camelid VHH domains, a humanized antibody, a bispecific antibody, an antibody conjugate (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al, 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al, 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al, 1988, Science 242:423-426). In one aspect, the antigen-binding domain of a chimeric receptor of the present disclosure comprises an antibody fragment. In a further aspect, the chimeric receptor comprises an antibody fragment that comprises an scFv.
As used herein, the term “antibody heavy chain” refers to the larger of the two types of polypeptide chains present in antibody molecules in their naturally occurring conformations, and which normally determines the class to which the antibody belongs.
As used herein, the term “antibody light chain” refers to the smaller of the two types of polypeptide chains present in antibody molecules in their naturally occurring conformations. Kappa (κ) and lambda (λ) light chains refer to the two major antibody light chain isotypes.
As used herein, the term “recombinant antibody” refers to an antibody which is generated using recombinant DNA technology, such as, for example, an antibody expressed by a bacteriophage or yeast expression system. The term should also be construed to mean an antibody which has been generated by the synthesis of a DNA molecule encoding the antibody and which DN A molecule expresses an antibody protein, or an amino acid sequence specifying the antibody, wherein the DNA or amino acid sequence has been obtained using recombinant DNA or amino acid sequence technology which is available and well known in the art.
As used herein, the term “antigen” or “Ag” refers to a molecule that provokes an immune response. This immune response may involve either antibody production, or the activation of specific immunologically-competent cells, or both. The skilled artisan will understand that any macromolecule, including virtually all proteins or peptides, can serve as an antigen.
A CAR of the present disclosure can include a first, second, or third generation CAR. “First generation” CARs comprise a single intracellular signaling domain, generally derived from a T cell receptor chain. “First generation” CARs generally have the intracellular signaling domain from the CD3-zeta (CD3z) chain, which is the primary transmitter of signals from endogenous TCRs. “First generation” CARs can provide de novo antigen recognition and cause activation of both CD4+ and CD8+ T cells through their CD3z chain signaling domain in a single fusion molecule, independent of HLA-mediated antigen presentation. “Second generation” CARs add a second intracellular signaling domain from one of various co-stimulatory molecules (e.g., CD28, 4-1BB, ICOS, OX40) to the cytoplasmic tail of the CAR to provide additional signals to the T cell. “Second generation” CARs provide both co-stimulation (e.g., CD28 or 4-1BB) and activation (CD3z). Preclinical studies have indicated that “Second Generation” CARs can improve the anti-tumor activity of immune effector cell, such as a T cell. “Third generation” CARs have multiple intracellular co-stimulation signaling domains (e.g., CD28 and 4-1BB) and an intracellular activation signaling domain (CD3z).
CARs can be incorporated into immune effector cells such as T cells, TILs, NK cells, macrophages, dendritic cells, induced pluripotent stem cells (iPSCs), or TCRs resulting in CAR-T, CAR-TILs, CAR-NK cells, CAR-M, CAR-DC, and TCR engineered CAR-T cells, respectively. For descriptions of CAR-T cells, methods of making CAR-T cells, and uses thereof, see, e.g., Brudno et al., (Nature Rev. Clin. Oncol., 2018, 15:31-46); Maude et al., (N. Engl. J. Med., 2014, 371:1507-1517); and Sadelain et al., (Cancer Disc., 2013, 3:388-398). For example, nucleic acids encoding CARs can be introduced into an immune effector cell using viral or non viral methods. CARs can be expressed in an immune effector cell for example from an introduced mRNA, an engineered genomic DNA, or a viral vector.
An immune effector cell or a progenitor, or progeny thereof is a cell that functions in an immune response (e.g., an immune effector response). Examples of immune effector cells include, without limitation, alpha/beta T cells (e.g., CD8+ T cells, CD4+ T cells, or CTLs), gamma/delta T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and myeloid-derived phagocytes. Immune effector cells can also include splenocytes and engineered splenocytes (for example TCR engineered splenocytes, or CAR engineered splenocytes). Immune effector cells can be collected, for example, from blood, lymph node, tumor, spleen, bone marrow, cord blood, or skin sample of a patient or a donor. Immune effector cells can be cultured and/or activated and/or expanded ex vivo (for example cultured, activated, and expanded). The immune effector cells can then be used fresh or stored frozen and thawed before use.
As used herein, the term “immune effector response” or “immune effector function” refers to a function or response, e.g., of an immune effector cell, that enhances or promotes an immune attack of a target cell. For example, an immune effector function or response may refer to a property of a T cell or NK cell that promotes killing or the inhibition of growth or proliferation, of a target cell. In the case of a T cell, primary stimulation and co-stimulation are examples of immune effector function or response.
In some embodiments, the CAR cell is an autologous CAR cell for example a CAR-T cell or a TIL cell. In some embodiments, the CAR cell is an allogeneic CAR-T cell for example a CAR-T cell or a TCR engineered CAR-T cell.
Aspects of the present disclosure include methods for inhibiting the hydrolase activity of ENPP1 against cGAMP provides for increased levels of cGAMP and/or downstream modulation (e.g., activation) of the STING pathway. The inventors have discovered that cGAMP is present in the extracellular space and that ENPP1 can control extracellular levels of cGAMP. The inventors have also discovered that cGAMP can have significant extracellular biological functions in vivo (e.g., see
A “STING mediated response” refers to any response that is mediated by STING, including, but not limited to, immune responses, e.g., to bacterial pathogens, viral pathogens, and eukaryotic pathogens. See, e.g., Ishikawa et al. Immunity 29: 538-550 (2008); Ishikawa et al. Nature 461: 788-792 (2009); and Sharma et al. Immunity 35: 194-207 (2011). STING also functions in certain autoimmune diseases initiated by inappropriate recognition of self DNA (see, e.g., Gall et al. Immunity 36: 120-131 (2012), as well as for the induction of adaptive immunity in response to DNA vaccines (see, e.g., Ishikawa et al. Nature 461: 788-792 (2009). By increasing a STING mediated response in a subject is meant an increase in a STING mediated response in a subject as compared to a control subject (e.g., a subject who is not administered a subject compound). In some cases, the subject is human and the subject compounds and methods provide for activation of human STING. In some cases, the STING mediated response includes modulation of an immune response. In some instances, the subject method is a method of modulating an immune response in a subject.
In some cases, the STING mediated response includes increasing the production of an interferon (e.g., a type I interferon (IFN), type III interferon (IFN)) in a subject. Interferons (IFNs) are proteins having a variety of biological activities, e.g., antiviral, immunomodulating and antiproliferative. IFNs are relatively small, species-specific, single chain polypeptides, produced by mammalian cells in response to exposure to a variety of inducers such as viruses, polypeptides, mitogens and the like. Interferons protect animal tissues and cells against viral attack and are an important host defense mechanism. Interferons may be classified as Type-I, Type-II and Type-III interferons. Mammalian Type-I interferons of interest include IFN-α (alpha), IFN-β (beta), IFN-κ (kappa), IFN-δ (delta), IFN-ε (epsilon), IFN-τ (tau), IFN-ω (omega), and IFN-ζ (zeta, also known as limiting).
Interferons find use in the treatment of a variety of cancers since these molecules have anti-cancer activity that acts at multiple levels. Interferon proteins can directly inhibit the proliferation of human tumor cells. In some cases, the anti-proliferative activity is also synergistic with a variety of approved chemotherapeutic agents such as cisplatin, 5FU and paclitaxel. The immunomodulatory activity of interferon proteins can also lead to the induction of an anti-tumor immune response. This response includes activation of NK cells, stimulation of macrophage activity and induction of MHC class I surface expression, leading to the induction of anti-tumor cytotoxic T lymphocyte activity. In addition, interferons play a role in cross-presentation of antigens in the immune system. Moreover, some studies further indicate that IFN-β protein may have anti-angiogenic activity. Angiogenesis, new blood vessel formation, is critical for the growth of solid tumors. IFN-β may inhibit angiogenesis by inhibiting the expression of pro-angiogenic factors such as bFGF and VEGF. Interferon proteins may also inhibit tumor invasiveness by modulating the expression of enzymes, such as collagenase and elastase, which are important in tissue remodeling.
Aspects of the methods include administering to a subject an effective amount of an ENPP1 inhibitor to treat the subject for cancer. Any convenient ENPP1 inhibitors can be used in the subject methods of treating cancer. In certain cases, the ENPP1 inhibitor compound is a compound as 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 cancer e.g., a lymphoma. In certain embodiments, the cancer is selected from adrenal, liver, kidney, bladder, breast, colon, gastric, ovarian, cervical, uterine, esophageal, colorectal, prostate, pancreatic, lung (both small cell and non-small cell), thyroid, carcinomas, sarcomas, glioblastomas, melanoma and various head and neck tumors.
Aspects of the methods include administering to a subject an effective amount of a cell impermeable ENPP1 inhibitor to inhibit the hydrolysis of cGAMP and treat the subject for cancer. In certain cases the cancer is a solid cancer e.g., a lymphoma. In certain embodiments, the cancer is selected from adrenal, liver, kidney, bladder, breast, colon, gastric, ovarian, cervical, uterine, esophageal, colorectal, prostate, pancreatic, lung (both small cell and non-small cell), thyroid, carcinomas, sarcomas, glioblastomas, melanoma and various head and neck tumors.
In some embodiments of the methods disclosed herein, the cell impermeable ENPP1 inhibitor is an inhibitor of any one of formulas I, IV, V, VI or VII. In some cases, the cell impermeable ENPP1 inhibitor is any one of compounds 1-106.
In some embodiments of the methods disclosed herein, the ENPP1 inhibitor is cell permeable.
As such, aspects of the method include contacting a sample with a subject compound (e.g., as described above) under conditions by which the compound inhibits ENPP1. Any convenient protocol for contacting the compound with the sample may be employed. The particular protocol that is employed may vary, e.g., depending on whether the sample is in vitro or in vivo. For in vitro protocols, contact of the sample with the compound may be achieved using any convenient protocol. In some instances, the sample includes cells that are maintained in a suitable culture medium, and the complex is introduced into the culture medium. For in vivo protocols, any convenient administration protocol may be employed. Depending upon the potency of the compound, the cells of interest, the manner of administration, the number of cells present, various protocols may be employed.
In some embodiments, the subject method is a method of treating a subject for cancer. In some embodiments, the subject method includes administering to the subject an effective amount of a subject compound (e.g., as described herein) or a pharmaceutically acceptable salt thereof. The subject compound may be administered as part of a pharmaceutical composition (e.g., as described herein). In certain instances of the method, the compound that is administered is a compound of one of formulae (I), (IV), (V), (VI) or (VII). In certain instances of the method, the compound that is administered is described by one of the compounds of Table 1, Table 2, Table 3, Table 4, Table 5, or Table 6.
In some embodiments, an “effective amount” is an amount of a subject compound that, when administered to an individual in one or more doses, in monotherapy or in combination therapy, is effective to inhibit ENPP1 by about 20% (20% inhibition), at least about 30% (30% inhibition), at least about 40% (40% inhibition), at least about 50% (50% inhibition), at least about 60% (60% inhibition), at least about 70% (70% inhibition), at least about 80% (80% inhibition), or at least about 90% (90% inhibition), compared to the ENPP1 activity in the individual in the absence of treatment with the compound, or alternatively, compared to the ENPP1 activity in the individual before or after treatment with the compound.
In some embodiments, an “effective amount” is an amount of a subject compound that, when administered to an individual in one or more doses, in monotherapy or in combination therapy, is effective to decrease tumor burden in the subject by about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%, compared to tumor burden in the individual in the absence of treatment with the compound, or alternatively, compared to the tumor burden in the subject before or after treatment with the compound. As used herein the term “tumor burden” refers to the total mass of tumor tissue carried by a subject with cancer.
In some embodiments, an “effective amount” is an amount of a subject compound that, when administered to an individual in one or more doses, in monotherapy or in combination therapy, is effective to reduce the dose of radiotherapy required to observe tumor shrinkage in the subject by about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%, compared to the dose of radiotherapy required to observe tumor shrinkage in the individual in the absence of treatment with the compound.
In some embodiments, an “effective amount” of a compound is an amount that, when administered in one or more doses to an individual having cancer, is effective to achieve a 1.5-log, a 2-log, a 2.5-log, a 3-log, a 3.5-log, a 4-log, a 4.5-log, or a 5-log reduction in tumor size.
In some embodiments, an effective amount of a compound is an amount that ranges from about 50 ng/ml to about 50 μg/ml (e.g., from about 50 ng/ml to about 40 μg/ml, from about 30 ng/ml to about 20 μg/ml, from about 50 ng/ml to about 10 μg/ml, from about 50 ng/ml to about 1 μg/ml, from about 50 ng/ml to about 800 ng/ml, from about 50 ng/ml to about 700 ng/ml, from about 50 ng/ml to about 600 ng/ml, from about 50 ng/ml to about 500 ng/ml, from about 50 ng/ml to about 400 ng/ml, from about 60 ng/ml to about 400 ng/ml, from about 70 ng/ml to about 300 ng/ml, from about 60 ng/ml to about 100 ng/ml, from about 65 ng/ml to about 85 ng/ml, from about 70 ng/ml to about 90 ng/ml, from about 200 ng/ml to about 900 ng/ml, from about 200 ng/ml to about 800 ng/ml, from about 200 ng/ml to about 700 ng/ml, from about 200 ng/ml to about 600 ng/ml, from about 200 ng/ml to about 500 ng/ml, from about 200 ng/ml to about 400 ng/ml, or from about 200 ng/ml to about 300 ng/ml).
In some embodiments, an effective amount of a compound is an amount that ranges from about 10 pg to about 100 mg, e.g., from about 10 pg to about 50 pg, from about 50 pg to about 150 pg, from about 150 pg to about 250 pg, from about 250 pg to about 500 pg, from about 500 pg to about 750 pg, from about 750 pg to about 1 ng, from about 1 ng to about 10 ng, from about 10 ng to about 50 ng, from about 50 ng to about 150 ng, from about 150 ng to about 250 ng, from about 250 ng to about 500 ng, from about 500 ng to about 750 ng, from about 750 ng to about 1 μg, from about 1 g to about 10 μg, from about 10 μg to about 50 μg, from about 50 μg to about 150 μg, from about 150 μg to about 250 μg, from about 250 μg to about 500 μg, from about 500 μg to about 750 μg, from about 750 μg to about 1 mg, from about 1 mg to about 50 mg, from about 1 mg to about 100 mg, or from about 50 mg to about 100 mg. The amount can be a single dose amount or can be a total daily amount. The total daily amount can range from 10 pg to 100 mg, or can range from 100 mg to about 500 mg, or can range from 500 mg to about 1000 mg.
In some embodiments, a single dose of a compound is administered. In other embodiments, multiple doses are administered. Where multiple doses are administered over a period of time, the compound can be administered twice daily (qid), daily (qd), every other day (qod), every third day, three times per week (tiw), or twice per week (biw) over a period of time. For example, a compound is administered qid, qd, qod, tiw, or biw over a period of from one day to about 2 years or more. For example, a compound is administered at any of the aforementioned frequencies for one week, two weeks, one month, two months, six months, one year, or two years, or more, depending on various factors.
Administration of an effective amount of a subject compound to an individual with cancer can result in one or more of: 1) a reduction in tumor burden; 2) a reduction in the dose of radiotherapy required to effect tumor shrinkage; 3) a reduction in the spread of a cancer from one cell to another cell in an individual; 4) a reduction of morbidity or mortality in clinical outcomes; 5) shortening the total length of treatment when combined with other anti-cancer agents; and 6) an improvement in an indicator of disease response (e.g., a reduction in one or more symptoms of cancer). Any of a variety of methods can be used to determine whether a treatment method is effective. For example, a biological sample obtained from an individual who has been treated with a subject method can be assayed.
Any of the compounds described herein can be utilized in the subject methods of treatment. In certain instances, the compound is of one of formulae I, IV or V. In certain cases, the compound is one of the compounds of Table 1, Table 2, Table 3, Table 4, Table 5, or Table 6. In some cases, the compound that is utilized in the subject methods is not cell permeable. In some cases, the compound that is utilized in the subject methods has poor cell permeability.
In some embodiments, the compound specifically inhibits ENPP1. In some embodiments, the compound modulates the activity of cGAMP. In some embodiments, the compound interferes with the interaction of ENPP1 and cGAMP. In some embodiments, the compound results in activation of the STING pathway.
In some embodiments, the subject is mammalian. In certain instances, the subject is human. Other subjects can include domestic pets (e.g., dogs and cats), livestock (e.g., cows, pigs, goats, horses, and the like), rodents (e.g., mice, guinea pigs, and rats, e.g., as in animal models of disease), as well as non-human primates (e.g., chimpanzees, and monkeys). The subject may be in need of treatment for cancer. In some instances, the subject methods include diagnosing cancer, including any one of the cancers described herein. In some embodiments, the compound is administered as a pharmaceutical preparation.
In certain embodiments, the ENPP1 inhibitor compound is a modified compound that includes a label, and the method further includes detecting the label in the subject. The selection of the label depends on the means of detection. Any convenient labeling and detection systems may be used in the subject methods, see e.g., Baker, “The whole picture,” Nature, 463, 2010, p 977-980. In certain embodiments, the compound includes a fluorescent label suitable for optical detection. In certain embodiments, the compound includes a radiolabel for detection using positron emission tomography (PET) or single photon emission computed tomography (SPECT). In some cases, the compound includes a paramagnetic label suitable for tomographic detection. The subject compound may be labeled, as described above, although in some methods, the compound is unlabeled and a secondary labeling agent is used for imaging.
The subject compounds can be administered to a subject alone or in combination with an additional, i.e., second, active agent. Combination therapeutic methods where the subject ENPP1 inhibitor compounds may be used in combination with a second active agent or an additional therapy, e.g., radiation therapy. The terms “agent,” “compound,” and “drug” are used interchangeably herein. For example, ENPP1 inhibitor compounds can be administered alone or in conjunction with one or more other drugs, such as drugs employed in the treatment of diseases of interest, including but not limited to, immunomodulatory diseases and conditions and cancer. In some embodiments, the subject method further includes coadministering concomitantly or in sequence a second agent, e.g., a small molecule, a chemotherapeutic, an antibody, an antibody fragment, an antibody-drug conjugate, an aptamer, a protein, or a checkpoint inhibitor. In some embodiments, the method further includes performing radiation therapy on the subject.
In some embodiments, the method further includes performing adoptive cell therapy on the subject. In certain aspects, use of adoptive immunotherapy in a subject in need of treatment in combination with an ENPP1 inhibitor compound, which latter treatment may be administered prior to, concurrently with, or subsequent to adoptive immunotherapy.
The terms “co-administration” and “in combination with” include the administration of two or more therapeutic agents either simultaneously, concurrently or sequentially within no specific time limits. In one embodiment, the agents are present in the cell or in the subject's body at the same time or exert their biological or therapeutic effect at the same time. In one embodiment, the therapeutic agents are in the same composition or unit dosage form. In other embodiments, the therapeutic agents are in separate compositions or unit dosage forms. In certain embodiments, a first agent can be administered prior to (e.g., minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concomitantly with, or subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of a second therapeutic agent.
“Concomitant administration” of a known therapeutic drug or additional therapy with a pharmaceutical composition of the present disclosure means administration of the compound and second agent or additional therapy at such time that both the known drug and the composition of the present invention will have a therapeutic effect. Such concomitant administration may involve concurrent (i.e., at the same time), prior, or subsequent administration of the drug with respect to the administration of a subject compound. Routes of administration of the two agents may vary, where representative routes of administration are described in greater detail below. A person of ordinary skill in the art would have no difficulty determining the appropriate timing, sequence and dosages of administration for particular drugs or therapies and compounds of the present disclosure.
In some embodiments, the compounds (e.g., a subject compound and the at least one additional compound or therapy) are administered to the subject within twenty-four hours of each other, such as within 12 hours of each other, within 6 hours of each other, within 3 hours of each other, or within 1 hour of each other. In certain embodiments, the compounds are administered within 1 hour of each other. In certain embodiments, the compounds are administered substantially simultaneously. By administered substantially simultaneously is meant that the compounds are administered to the subject within about 10 minutes or less of each other, such as 5 minutes or less, or 1 minute or less of each other.
Also provided are pharmaceutical preparations of the subject compounds and the second active agent. In pharmaceutical dosage forms, the compounds may be administered in the form of their pharmaceutically acceptable salts, or they may also be used alone or in appropriate association, as well as in combination, with other pharmaceutically active compounds.
In conjunction with any of the subject methods, the ENPP1 inhibitor compounds (e.g., as described herein) (or pharmaceutical compositions comprising such compounds) can be administered in combination with another drug designed to reduce or prevent inflammation, treat or prevent chronic inflammation or fibrosis, or treat cancer. In each case, the ENPP1 inhibitor compound can be administered prior to, at the same time as, or after the administration of the other drug. In certain cases, the cancer is selected from adrenal, liver, kidney, bladder, breast, colon, gastric, ovarian, cervical, uterine, esophageal, colorectal, prostate, pancreatic, lung (both small cell and non-small cell), thyroid, carcinomas, sarcomas, glioma, glioblastomas, melanoma and various head and neck tumors.
For the treatment of cancer, the ENPP1 inhibitor compounds 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-targeted drugs, Chimeric Antigen Receptor/T cell therapies, Chimeric Antigen Receptor/NK cell therapies, 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 vaccine (e.g., a Th17-inducing dendritic cell vaccine, or a genetically modified tyrosinase such as Oncept®) and other cell therapies.
Specific chemotherapeutic agents of interest include, but are not limited to, Gemcitabine, Docetaxel, Bleomycin, Erlotinib, Gefitinib, Lapatinib, Imatinib, Dasatinib, Nilotinib, Bosutinib, Crizotinib, Ceritinib, Trametinib, Bevacizumab, Sunitinib, Sorafenib, Trastuzumab, Ado-trastuzumab emtansine, Rituximab, Ipilimumab, Rapamycin, Temsirolimus, Everolimus, Methotrexate, Doxorubicin, Abraxane, Folfirinox, Cisplatin, Carboplatin, 5-fluorouracil, Teysumo, Paclitaxel, Prednisone, Levothyroxine, Pemetrexed, navitoclax, and ABT-199. Peptidic compounds can also be used. Cancer chemotherapeutic agents of interest include, but are not limited to, dolastatin and active analogs and derivatives thereof; and auristatin and active analogs and derivatives thereof (e.g., Monomethyl auristatin D (MMAD), monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), and the like). See, e.g., WO 96/33212, WO 96/14856, and U.S. Pat. No. 6,323,315. Suitable cancer chemotherapeutic agents also include maytansinoids and active analogs and derivatives thereof (see, e.g., EP 1391213; and Liu et al (1996) Proc. Natl. Acad. Sci. USA 93:8618-8623); duocarmycins and active analogs and derivatives thereof (e.g., including the synthetic analogues, KW-2189 and CB 1-TM1); and benzodiazepines and active analogs and derivatives thereof (e.g., pyrrolobenzodiazepine (PBD).
In some embodiments, the ENPP1 inhibitor compounds can be administered in combination with a chemotherapeutic agent to treat 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.
For the treatment of cancer (e.g., solid tumor cancer or lymphoma), the ENPP1 inhibitor compound can be administered in combination an immunotherapeutic agent. An immunotherapeutic agent is any convenient agent that finds use in the treatment of disease by inducing, enhancing, or suppressing an immune response. In some cases, the immunotherapeutic agent is an immune checkpoint inhibitor. For example,
Also of interest are agents that are CARP-1/CCAR1 (Cell division cycle and apoptosis regulator 1) inhibitors, including but not limited to those described by Rishi et al., Journal of Biomedical Nanotechnology, Volume 11, Number 9, September 2015, pp. 1608-1627(20), and CD47 inhibitors, including, but not limited to, anti-CD47 antibody agents such as Hu5F9-G4.
In certain instances, the combination provides an enhanced effect relative to either component alone; in some cases, the combination provides a supra-additive or synergistic effect relative to the combined or additive effects of the components. A variety of combinations of the subject compounds and the chemotherapeutic agent may be employed, used either sequentially or simultaneously. For multiple dosages, the two agents may directly alternate, or two or more doses of one agent may be alternated with a single dose of the other agent, for example. Simultaneous administration of both agents may also be alternated or otherwise interspersed with dosages of the individual agents. In some cases, the time between dosages may be for a period from about 1-6 hours, to about 6-12 hours, to about 12-24 hours, to about 1-2 days, to about 1-2 week or longer following the initiation of treatment.
Combination with cGAMP-Inducing Chemotherapeutics
Aspects of the present disclosure include methods of treating cancer, where the ENPP1 inhibitor compounds (or pharmaceutical compositions comprising such compounds) can be administered in combination with a chemotherapeutic that is capable of inducing production of cGAMP in vivo. When a subject is exposed to an effective amount of a particular chemotherapeutic, the production of 2′3′-cGAMP can be induced in the subject. The induced levels of cGAMP can be maintained and/or enhanced when the subject ENPP1 inhibitor compounds are co-administered to prevent the degradation of the cGAMP, e.g., enhanced by comparison to levels achieved with either agent alone. Any convenient chemotherapeutic agents which can lead to DNA damage and can induce cGAMP production by the dying cells due to overwhelmed repair or degradation mechanisms can be used in the subject combination therapeutic methods, such as alkylating agents, nucleic acid analogues, and intercalating agents. In some cases, the cGAMP-inducing chemotherapeutic is an anti-mitotic agent. An anti-mitotic agent is an agent that acts by damaging DNA or binding to microtubules. In some cases, the cGAMP-inducing chemotherapeutic is an antineoplastic agent.
Cancers of interest which may be treated using the subject combination therapies include, but are not limited to, adrenal, liver, kidney, bladder, breast, colon, gastric, ovarian, cervical, uterine, esophageal, colorectal, prostate, pancreatic, lung (both small cell and non-small cell), thyroid, carcinomas, sarcomas, glioma, glioblastomas, melanoma and various head and neck tumors. In some cases, the cancer is breast cancer. In certain instances, the cancer is glioma or glioblastoma.
Chemotherapeutic of interest include, but are not limited to, Uracil analogues, Fluorouracil prodrug, Thymidylate Synthase inhibitors, Deoxycytidine analogue, DNA synthesis inhibitor (e.g. leading to S-phase apoptosis), Folate analogue, Dehydrofolate Reductase inhibitor, Anthracycline, intercalating agent, (e.g., leading to double strand breaks), Topoisomerase IIa inhibitor, Taxane, microtubule disassembly inhibitor (e.g. leading to G2/M phase arrest/apoptosis), microtubule assembly inhibitor, microtubule function stabilizers (e.g. leading to G2/M-phase apoptosis), tubulin polymerization promoters, tubulin binding agent (e.g. leading to apoptosis by M-phase arrest) Epothilone B analogue, Vinka alkaloid, Nitrogen mustard, Nitrosourea, DNA alkylater (e.g., leading to interstrand crosslinks, apoptosis via p53), VEGF inhibitor, anti-angiogenic antibody, HER2 inhibitor, Quinazoline HER2 inhibitor, EGFR inhibitor, tyrosine kinase inhibitor, Sirolimus analogue, mTORC1 inhibitor (e.g., in breast cancer combination with Exemestane=Aromastase inhibitor inhibiting Estrogen production), Triazene, Dacarbazine prodrug, Methylhydrazine.
Exemplary breast cancer chemotherapeutic of interest include, but are not limited to, Capecitabine, Carmofur, Fluorouracil, Tegafur, Gemcitabine, Methotrexate, Doxorubicin, Epirubicin, Docetaxel, Ixabepilone, Vindesine, Vinorelbine, Cyclophosphamide, Bevacicumab, Pertuzumab, Trastuzumab, Lapatinib and Everolimus. Exemplary Glioma/Glioblastoma related antineoplastic drugs: include, but are not limited to, Carmustine, Lomustine, Temozolomide, Procarbazine, Vincristine and Bevacicumab. Exemplary DNA damaging chemotherapeutic agents of interest include, but are not limited to, Melphalan, Cisplatin, and Etoposide, Fluorouracil, Gemcitabine.
Alternatively, for the methods of treating cancer, the ENPP1 inhibitor compounds (or pharmaceutical compositions comprising such compounds) can be administered in combination with radiation therapy. In certain embodiments, the methods include administering radiation therapy to the subject. Again, the ENPP1 inhibitor compound can be administered prior to, or after the administration of the radiation therapy. As such, the subject methods can further include administering radiation therapy to the subject. The combination of radiation therapy and administration of the subject compounds can provide a synergistic therapeutic effect. When a subject is exposed to radiation of a suitable dosage and/or frequency during radiation therapy (RT), the production of 2′3′-cGAMP can be induced in the subject. These induced levels of cGAMP can be maintained and/or enhanced when the subject ENPP1 inhibitor compounds are co-administered to prevent the degradation of the cGAMP, e.g., enhanced by comparison to levels achieved with RT alone. For example,
In some cases, the method includes administering an ENPP1 inhibitor to the subject before radiation therapy. In some cases, the method includes administering an ENPP1 inhibitor to the subject following exposure of the subject to radiation therapy. In certain cases, the method includes sequential administration of radiation therapy, followed by an ENPP1 inhibitor, followed by a checkpoint inhibitor to a subject in need thereof.
For the methods of treating cancer, the ENPP1 inhibitor compounds (or pharmaceutical compositions comprising such compounds) can be administered in combination with adoptive cell therapy for example a TIL or engineered TIL therapy, Chimeric Antigen Receptor/T cell therapy, or Chimeric Antigen Receptor/NK cell therapy. For example, KYMRIAH® (tisagenlecleucel), is an autologous T cell therapy that includes T cells recombinantly expressing a chimeric antigen receptor (CAR) that binds CD19, for treatment of B-cell precursor acute lymphoblastic leukemia. Other CAR-T cell therapies include but are not limited to ciltacabtagene autoleucel (Carvykti™), idecabtagene vicleucel (Abecma®), lisocabtagene maraleucel (BREYANZI®), brexucabtagene autoleucel (TECARTUS™), and axicabtagene ciloleucel (YESCARTA™). In certain embodiments, the methods include administering adoptive cell therapy to the subject. Again, the ENPP1 inhibitor compound can be administered prior to, or after the administration of the adoptive cell therapy. As such, the subject methods can further include administering adoptive cell therapy to the subject. The combination of adoptive cell therapy and administration of the subject compounds can provide a synergistic therapeutic effect.
In some cases, the method includes administering an ENPP1 inhibitor to the subject before adoptive cell therapy. In some cases, the method includes administering an ENPP1 inhibitor to the subject following exposure of the subject to adoptive cell therapy. In certain cases, the method includes sequential administration of adoptive cell therapy, followed by an ENPP1 inhibitor, followed by a checkpoint inhibitor to a subject in need thereof.
In some embodiments, an effective amount of a compound is an amount that ranges from about 5 mg/kg to about 150 mg/kg. Non-limiting doses include about 5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg, 45 mg/kg, 50 mg/kg, 55 mg/kg, 60 mg/kg, 65 mg/kg, 70 mg/kg, 75 mg/kg, 80 mg/kg, 90 mg/kg, 95 mg/kg, 100 mg/kg, 105 mg/kg, 110 mg/kg, 115 mg/kg, 120 mg/kg, 125 mg/kg, 130 mg/kg, 135 mg/kg, 140 mg/kg, 145 mg/kg, or 150 mg/kg. In some embodiments, an effective amount of a compound is administered on days 1, 2, 3, 4, 5, 6, 7, 8, 9, and/or 10. In some embodiments, adoptive T cell therapy is administered on day 1, 2, 3, 4, 5, 6, 7, 8, 9, and/or 10.
Disclosed for herein are compounds for stimulating the STING pathway, particularly through regulating ENPP1, used in combination with CAR expressing immune effector cell and methods of use of same in a patient-specific combination therapy that can be used to treat cancers and other diseases and/or conditions.
The compounds and methods of the invention, e.g., as described herein, find use in a variety of applications. Applications of interest include, but are not limited to: research applications and therapeutic applications. Methods of the invention find use in a variety of different applications including any convenient application where inhibition of ENPP1 is desired.
The subject compounds and methods find use in a variety of research applications. The subject compounds and methods may be used in the optimization of the bioavailability and metabolic stability of compounds.
The subject compounds and methods find use in a variety of therapeutic applications. Therapeutic applications of interest include those applications in cancer treatment. As such, the subject compounds find use in the treatment of a variety of different conditions in which the inhibition and/or treatment of cancer in the host is desired. For example, the subject compounds and methods may find use in treating a solid tumor cancer (e.g., as described herein), such as a lymphoma.
The herein-discussed compounds can be formulated using any convenient excipients, reagents and methods. Compositions are provided in formulation with a 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 have been amply described in a variety of publications, including, for example, A. Gennaro (2000) “Remington: The Science and Practice of Pharmacy,” 20th edition, Lippincott, Williams, & Wilkins; Pharmaceutical Dosage Forms and Drug Delivery Systems (1999) H. C. Ansel et al., eds., 7th ed., Lippincott, Williams, & Wilkins; and Handbook of Pharmaceutical Excipients (2000) A. H. Kibbe et al., eds., 3rd ed. Amer. Pharmaceutical Assoc.
The pharmaceutically acceptable excipients, such as vehicles, adjuvants, carriers or diluents, are readily available to the public. Moreover, pharmaceutically acceptable auxiliary substances, such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents and the like, are readily available to the public.
In some embodiments, the subject compound is formulated in an aqueous buffer. Suitable aqueous buffers include, but are not limited to, acetate, succinate, citrate, and phosphate buffers varying in strengths from 5 mM to 100 mM. In some embodiments, the aqueous buffer includes reagents that provide for an isotonic solution. Such reagents include, but are not limited to, sodium chloride; and sugars e.g., mannitol, dextrose, sucrose, and the like. In some embodiments, the aqueous buffer further includes a non-ionic surfactant such as polysorbate 20 or 80. Optionally the formulations may further include a preservative. Suitable preservatives include, but are not limited to, a benzyl alcohol, phenol, chlorobutanol, benzalkonium chloride, and the like. In many cases, the formulation is stored at about 4° C. Formulations may also be lyophilized, in which case they generally include cryoprotectants such as sucrose, trehalose, lactose, maltose, mannitol, and the like. Lyophilized formulations can be stored over extended periods of time, even at ambient temperatures. In some embodiments, the subject compound is formulated for sustained release.
In some embodiments, the subject compound and a second active agent (e.g., as described herein), e.g., a small molecule, a chemotherapeutic, an antibody, an antibody fragment, an antibody-drug conjugate, an aptamer, or a protein, etc. are administered to individuals in a formulation (e.g., in the same or in separate formulations) with a pharmaceutically acceptable excipient(s). In some embodiments, the second active agent is a checkpoint inhibitor, e.g., a cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) inhibitor, a programmed death 1 (PD-1) inhibitor, or a PD-L1 inhibitor.
In certain cases, two or more therapeutic agents (e.g., cGAS ligands, STING ligands, and/or ENPP1 inhibitors) can be co-formulated. In certain cases, all of the therapeutic agents (e.g., cGAS ligands, STING ligands, and/or ENPP1 inhibitors) are co-formulated. In certain cases, two or more therapeutic agents can be administered as separate formulations.
In another aspect of the present invention, a pharmaceutical composition is provided, comprising, or consisting essentially of, a compound of the present invention, or a pharmaceutically acceptable salt, isomer, tautomer or prodrug thereof, and further comprising one or more additional active agents of interest. Any convenient active agents can be utilized in the subject methods in conjunction with the subject compounds. In some instances, the additional agent is a checkpoint inhibitor. The subject compound and checkpoint inhibitor, as well as additional therapeutic agents as described herein for combination therapies, can be administered orally, subcutaneously, intramuscularly, intranasally, parenterally, or other route. The subject compound and second active agent (if present) 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 an affected organ. In some cases, the therapeutic agents can be administered intratumorally.
In certain cases, the therapeutic agents can be administered as a pharmaceutical composition formulated for mucosal delivery. Examples of mucosal delivery of cGAS/STING pathway agonists are described in more detail in Martin et al. (Vaccine. 2017 Apr. 25; 35(18): 2511-2519) and Dubensky et al. (Ther Adv Vaccines (2013) 1(4) 131-143), each herein incorporated by reference for all purposes. Mucosal delivery can include, but is not limited to, buccal delivery, sublingual delivery, or intranasal delivery. In certain cases, the therapeutic agents can be administered buccally. In certain cases, the therapeutic agents can be administered sublingually. In certain cases, the therapeutic agents can be administered intranasally. Pharmaceutical compositions formulated for mucosal delivery can include formulation in a nanoparticle, such as liposomes. Liposomes useful for mucosal delivery are known to those skilled in the art. For example, liposomes useful for mucosal delivery can contain a pulmonary surfactant, a pulmonary surfactant membrane constituent, and/or a pulmonary surfactant biomimetic are described in more detail in Wang et al. [Science 367, 869 (2020)], herein incorporated by reference for all purposes. In certain cases, two or more therapeutic agents (e.g., cGAS ligands, STING ligands, and/or ENPP1 inhibitors) can be co-formulated for mucosal delivery. In certain cases, all of the therapeutic agents (e.g., cGAS ligands, STING ligands, and/or ENPP1 inhibitors) are co-formulated for mucosal delivery. In certain cases, two or more therapeutic agents can be administered as separate formulations for mucosal delivery.
In some embodiments, the subject compound and a chemotherapeutic agent are administered to individuals in a formulation (e.g., in the same or in separate formulations) with a pharmaceutically acceptable excipient(s). The chemotherapeutic agents include, but are not limited to alkylating agents, nitrosoureas, antimetabolites, antitumor antibiotics, plant (vinca) alkaloids, and steroid hormones. Peptidic compounds can also be used. Suitable cancer chemotherapeutic agents include dolastatin and active analogs and derivatives thereof; and auristatin and active analogs and derivatives thereof (e.g., Monomethyl auristatin D (MMAD), monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), and the like). See, e.g., WO 96/33212, WO 96/14856, and U.S. Pat. No. 6,323,315. Suitable cancer chemotherapeutic agents also include maytansinoids and active analogs and derivatives thereof (see, e.g., EP 1391213; and Liu et al (1996) Proc. Natl. Acad. Sci. USA 93:8618-8623); duocarmycins and active analogs and derivatives thereof (e.g., including the synthetic analogues, KW-2189 and CB 1-TM1); and benzodiazepines and active analogs and derivatives thereof (e.g., pyrrolobenzodiazepine (PBD).
The subject compound and second chemotherapeutic agent, as well as additional therapeutic agents as described herein for combination therapies, can be administered orally, subcutaneously, intramuscularly, parenterally, or other route. The subject compound and 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 an affected organ.
The subject compounds may be administered in a unit dosage form and may be prepared by any methods well known in the art. Such methods include combining the subject compound with a pharmaceutically acceptable carrier or diluent which constitutes one or more accessory ingredients. A pharmaceutically acceptable carrier is selected on the basis of 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 a solid or liquid and the type is generally chosen based on the type of administration being used.
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, emulsions, syrups or elixirs, suspensions, solutions and/or suspensions, and solution and or suspensions reconstituted from non-effervescent granules and effervescent preparations reconstituted from effervescent granules. Such liquid carriers may contain, for example, suitable solvents, preservatives, emulsifying agents, suspending agents, diluents, sweeteners, thickeners, and melting agents. Preferred carriers are edible oils, for example, corn or canola oils. Polyethylene glycols, e.g., PEG, are also good carriers.
Any drug delivery device or system that provides for the dosing regimen of the instant disclosure can be used. A wide variety of delivery devices and systems are known to those skilled in the art.
Before embodiments of the present disclosure are further described, it is to be understood that this disclosure is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.
Unless defined otherwise, 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.
It must be noted that 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 a combination of two or more compounds, reference to “a substituent” includes a single substituent as well as two or more substituents, and the like.
In describing and claiming the present invention, certain terminology will be used in accordance with the definitions set out below. It will be appreciated that the definitions provided herein are not intended to be mutually exclusive. Accordingly, some chemical moieties may fall within the definition of more than one term.
As used herein, the phrases “for example,” “for instance,” “such as,” or “including” are meant to introduce examples that further clarify more general subject matter. These examples are provided only as an aid for understanding the disclosure, and are not meant to be limiting in any fashion.
The publications discussed herein are provided solely for their disclosure 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 publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.
The terms “active agent,” “antagonist”, “inhibitor”, “drug” and “pharmacologically active agent” are used interchangeably herein to refer to a chemical material or compound which, when administered to an organism (human or animal) induces a desired pharmacologic and/or physiologic effect by local and/or systemic action.
As used herein, the terms “treatment,” “treating,” and the like, refer to obtaining a desired pharmacologic and/or physiologic effect, such as reduction of tumor burden. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disease and/or adverse effect attributable to the disease. “Treatment,” as used herein, covers any treatment of a disease in a mammal, particularly in a human, and includes: (a) preventing the disease or a symptom of a disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it (e.g., including diseases that may be associated with or caused by a primary disease; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., causing regression of the disease (e.g., reduction in of tumor burden).
The term “pharmaceutically acceptable salt” means a salt which is acceptable for administration to a patient, such as a mammal (salts with counterions having acceptable mammalian safety for a given dosage regime). Such salts can be derived from pharmaceutically acceptable inorganic or organic bases and from pharmaceutically acceptable inorganic or organic acids. “Pharmaceutically acceptable salt” refers to pharmaceutically acceptable salts of a compound, which salts are derived from a variety of organic and inorganic counter ions well known in the art and include, by way of example only, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, and the like; and when the molecule contains a basic functionality, salts of organic or inorganic acids, such as hydrochloride, hydrobromide, formate, tartrate, besylate, mesylate, acetate, maleate, oxalate, and the like.
The terms “individual,” “host,” “subject,” and “patient” are used interchangeably herein, and refer to an animal, including, but not limited to, human and non-human primates, including simians and humans; rodents, including rats and mice; bovines; equines; ovines; felines; canines; and the like. “Mammal” means a member or members of any mammalian species, and includes, by way of example, canines; felines; equines; bovines; ovines; rodentia, etc. and primates, e.g., non-human primates, and humans. Non-human animal models, e.g., mammals, e.g., non-human primates, murines, lagomorpha, etc. may be used for experimental investigations.
As used herein, the terms “determining,” “measuring,” “assessing,” and “assaying” are used interchangeably and include both quantitative and qualitative determinations.
The terms “polypeptide” and “protein”, used interchangeably herein, 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 fusion proteins, including, but not limited to, fusion proteins with a heterologous amino acid sequence, fusions with heterologous and native leader sequences, with or without N-terminal methionine residues; immunologically tagged proteins; fusion proteins with detectable fusion partners, e.g., fusion proteins including as a fusion partner a fluorescent protein, β-galactosidase, luciferase, etc.; and the like.
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 a gene, a gene fragment, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, control regions, isolated RNA of any sequence, nucleic acid probes, and primers. The nucleic acid molecule may be linear or circular.
A “therapeutically effective amount” or “efficacious amount” means the amount of a compound that, when administered to a mammal or other subject for treating a disease, condition, or disorder, is sufficient to effect such treatment for the disease, condition, or disorder. The “therapeutically effective amount” will vary depending on the compound, the disease and its severity and the age, weight, etc., of the subject to be treated.
The term “unit dosage form,” as used herein, refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of a compound (e.g., an aminopyrimidine compound, as described herein) calculated in an amount sufficient to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier or vehicle. The specifications for unit dosage forms depend on the particular compound employed and the effect to be achieved, and the pharmacodynamics associated with each compound in the host.
A “pharmaceutically acceptable excipient,” “pharmaceutically acceptable diluent,” “pharmaceutically acceptable carrier,” and “pharmaceutically acceptable adjuvant” means an excipient, diluent, carrier, and adjuvant that are useful in preparing a pharmaceutical composition that are generally safe, non-toxic and neither biologically nor otherwise undesirable, and include an excipient, diluent, carrier, and adjuvant that are acceptable for veterinary use as well as human pharmaceutical use. “A pharmaceutically acceptable excipient, diluent, carrier and adjuvant” as used in the specification and claims includes both one and more than one such excipient, diluent, carrier, and adjuvant.
As used herein, a “pharmaceutical composition” is meant to encompass a composition suitable for administration to a subject, such as a mammal, especially a human. In general a “pharmaceutical composition” is sterile, and preferably free of contaminants that are capable of eliciting an undesirable response within the subject (e.g., the compound(s) in the pharmaceutical composition is pharmaceutical grade). Pharmaceutical compositions can be designed for administration to subjects or patients in need thereof via a number of different routes of administration including oral, buccal, rectal, parenteral, intraperitoneal, intradermal, intracheal, intramuscular, subcutaneous, and the like.
As used herein, the phrase “having the formula” or “having the structure” is not intended to be limiting and is used in the same way that the term “comprising” is commonly used. The term “independently selected from” is used herein to indicate that the recited elements, e.g., R groups or the like, can be identical or different.
As used herein, the terms “may,” “optional,” “optionally,” or “may optionally” mean that the subsequently described circumstance may or may not occur, so that the description includes instances where the circumstance occurs and instances where it does not. For example, the phrase “optionally substituted” means that a non-hydrogen substituent may or may not be present on a given atom, and, thus, the description includes structures wherein a non-hydrogen substituent is present and structures wherein a non-hydrogen substituent is not present.
“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)—, wherein 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)—.
The term “alkyl” refers to a branched or unbranched saturated hydrocarbon group (i.e., a mono-radical) typically although not necessarily containing 1 to about 24 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, octyl, decyl, and the like, as well as cycloalkyl groups such as cyclopentyl, cyclohexyl and the like. Generally, although not necessarily, alkyl groups herein may contain 1 to about 18 carbon atoms, and such groups may contain 1 to about 12 carbon atoms. The term “lower alkyl” intends an alkyl group of 1 to 6 carbon atoms. “Substituted alkyl” refers to alkyl substituted with one or more substituent groups, and this includes instances wherein two hydrogen atoms from the same carbon atom in an alkyl substituent are replaced, such as in a carbonyl group (i.e., a substituted alkyl group may include a —C(═O)— moiety). The terms “heteroatom-containing alkyl” and “heteroalkyl” refer to an alkyl substituent in which at least one carbon atom is replaced with a heteroatom, as described in further detail infra. If not otherwise indicated, the terms “alkyl” and “lower alkyl” include linear, branched, cyclic, unsubstituted, substituted, and/or heteroatom-containing alkyl or lower alkyl, respectively.
The term “substituted alkyl” is meant to include an alkyl group as defined herein wherein one or more carbon atoms in the alkyl chain have been optionally replaced with a heteroatom such as —O—, —N—, —S—, —S(O)n— (where n is 0 to 2), —NR— (where R is hydrogen or alkyl) and having from 1 to 5 substituents selected from the group consisting of alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, —SO-alkyl, —SO-aryl, —SO-heteroaryl, —SO2-alkyl, —SO2-aryl, —SO2-heteroaryl, and —NRaRb, wherein R′ and R″ may be the same or different and are chosen from hydrogen, optionally substituted alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, heteroaryl and heterocyclic.
The term “alkenyl” refers to a linear, branched or cyclic hydrocarbon group of 2 to about 24 carbon atoms containing at least one double bond, such as ethenyl, n-propenyl, isopropenyl, n-butenyl, isobutenyl, octenyl, decenyl, tetradecenyl, hexadecenyl, eicosenyl, tetracosenyl, and the like. Generally, although again not necessarily, alkenyl groups herein may contain 2 to about 18 carbon atoms, and for example may contain 2 to 12 carbon atoms. The term “lower alkenyl” intends an alkenyl group of 2 to 6 carbon atoms. The term “substituted alkenyl” refers to alkenyl substituted with one or more substituent groups, and the terms “heteroatom-containing alkenyl” and “heteroalkenyl” refer to alkenyl in which at least one carbon atom is replaced with a heteroatom. If not otherwise indicated, the terms “alkenyl” and “lower alkenyl” include linear, branched, cyclic, unsubstituted, substituted, and/or heteroatom-containing alkenyl and lower alkenyl, respectively.
The term “alkynyl” refers to a linear or branched hydrocarbon group of 2 to 24 carbon atoms containing at least one triple bond, such as ethynyl, n-propynyl, and the like. Generally, although again not necessarily, alkynyl groups herein may contain 2 to about 18 carbon atoms, and such groups may further contain 2 to 12 carbon atoms. The term “lower alkynyl” intends an alkynyl group of 2 to 6 carbon atoms. The term “substituted alkynyl” refers to alkynyl substituted with one or more substituent groups, and the terms “heteroatom-containing alkynyl” and “heteroalkynyl” refer to alkynyl in which at least one carbon atom is replaced with a heteroatom. If not otherwise indicated, the terms “alkynyl” and “lower alkynyl” include linear, branched, unsubstituted, substituted, and/or heteroatom-containing alkynyl and lower alkynyl, respectively.
The term “alkoxy” refers to an alkyl group bound through a single, terminal ether linkage; that is, an “alkoxy” group may be represented as —O-alkyl where alkyl is as defined above. A “lower alkoxy” group refers to an alkoxy group containing 1 to 6 carbon atoms, and includes, for example, methoxy, ethoxy, n-propoxy, isopropoxy, t-butyloxy, etc. Substituents identified as “C1-C6 alkoxy” or “lower alkoxy” herein may, for example, may contain 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 term “substituted alkoxy” refers to the groups substituted alkyl-O—, substituted alkenyl-O-substituted cycloalkyl-O—, substituted cycloalkenyl-O—, and substituted alkynyl-O— where substituted alkyl, substituted alkenyl, substituted cycloalkyl, substituted cycloalkenyl and substituted alkynyl are as defined herein.
The term “aryl”, unless otherwise specified, refers to an aromatic substituent generally, although not necessarily, containing 5 to 30 carbon atoms and containing a single aromatic ring or multiple aromatic rings that are fused together, directly linked, or indirectly linked (such that the different aromatic rings are bound to a common group such as a methylene or ethylene moiety). Aryl groups may, for example, contain 5 to 20 carbon atoms, and as a further example, aryl groups may contain 5 to 12 carbon atoms. For example, aryl groups may contain one aromatic ring or two or more fused or linked aromatic rings (i.e., biaryl, aryl-substituted aryl, etc.). Examples include phenyl, naphthyl, biphenyl, diphenylether, diphenylamine, benzophenone, and the like. “Substituted aryl” refers to an aryl moiety substituted with one or more substituent groups, and the terms “heteroatom-containing aryl” and “heteroaryl” refer to aryl substituent, in which at least one carbon atom is replaced with a heteroatom, as will be described in further detail infra. Aryl is intended to include stable cyclic, heterocyclic, polycyclic, and polyheterocyclic unsaturated C3-C14 moieties, exemplified but not limited to phenyl, biphenyl, naphthyl, pyridyl, furyl, thiophenyl, imidazoyl, pyrimidinyl, and oxazoyl; which may further be substituted with one to five members selected from the group consisting of hydroxy, C1-C8 alkoxy, C1-C8 branched or straight-chain alkyl, acyloxy, carbamoyl, amino, N-acylamino, nitro, halogen, trifluoromethyl, cyano, and carboxyl (see e.g. Katritzky, Handbook of Heterocyclic Chemistry). If not otherwise indicated, the term “aryl” includes unsubstituted, substituted, and/or heteroatom-containing aromatic substituents.
The term “aralkyl” refers to an alkyl group with an aryl substituent, and the term “alkaryl” refers to an aryl group with an alkyl substituent, wherein “alkyl” and “aryl” are as defined above. In general, aralkyl and alkaryl groups herein contain 6 to 30 carbon atoms. Aralkyl and alkaryl groups may, for example, contain 6 to 20 carbon atoms, and as a further example, such groups may contain 6 to 12 carbon atoms.
The term “alkylene” refers to a di-radical alkyl group. Unless otherwise indicated, such groups include saturated hydrocarbon chains containing from 1 to 24 carbon atoms, which may be substituted or unsubstituted, may contain one or more alicyclic groups, and may be heteroatom-containing. “Lower alkylene” refers to alkylene linkages 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.
Similarly, the terms “alkenylene,” “alkynylene,” “arylene,” “aralkylene,” and “alkarylene” refer to di-radical alkenyl, alkynyl, aryl, aralkyl, and alkaryl groups, respectively.
The term “amino” refers to the group —NRR′ wherein R and R′ are independently hydrogen or nonhydrogen substituents, with nonhydrogen substituents including, for example, alkyl, aryl, alkenyl, aralkyl, and substituted and/or heteroatom-containing variants thereof.
The terms “halo” and “halogen” are used in the conventional sense to refer to a chloro, bromo, fluoro, or iodo substituent.
“Carboxyl,” “carboxy” or “carboxylate” refers to —CO2H or salts thereof.
“Cycloalkyl” refers to cyclic alkyl groups of from 3 to 10 carbon atoms having single or multiple cyclic rings including fused, bridged, and spiro ring systems. Examples of suitable cycloalkyl groups include, for instance, adamantyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl and the like. Such cycloalkyl groups include, by way of example, single ring structures such as cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, and the like, or multiple ring structures such as adamantanyl, and the like.
The term “substituted cycloalkyl” refers to cycloalkyl groups having from 1 to 5 substituents, or from 1 to 3 substituents, selected from alkyl, substituted alkyl, alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, —SO-alkyl, —SO-substituted alkyl, —SO-aryl, —SO— heteroaryl, —SO2-alkyl, —SO2-substituted alkyl, —SO2-aryl and —SO2-heteroaryl.
The term “heteroatom-containing” as in a “heteroatom-containing alkyl group” (also termed a “heteroalkyl” group) or a “heteroatom-containing aryl group” (also termed a “heteroaryl” group) refers to a molecule, linkage or substituent in which one or more carbon atoms are replaced with an atom other than carbon, e.g., nitrogen, oxygen, sulfur, phosphorus or silicon, typically nitrogen, oxygen or sulfur. Similarly, the term “heteroalkyl” refers to an alkyl substituent that is heteroatom-containing, the term “heterocycloalkyl” refers to a cycloalkyl substituent that is heteroatom-containing, the terms “heterocyclic” or “heterocycle” refer to a cyclic substituent that is heteroatom-containing, the terms “heteroaryl” and “heteroaromatic” respectively refer to “aryl” and “aromatic” substituents that are heteroatom-containing, and the like. Examples of heteroalkyl groups include alkoxyaryl, alkylsulfanyl-substituted alkyl, N-alkylated amino alkyl, and the like. Examples of heteroaryl substituents include pyrrolyl, pyrrolidinyl, pyridinyl, quinolinyl, indolyl, furyl, pyrimidinyl, imidazolyl, 1,2,4-triazolyl, tetrazolyl, etc., and examples of heteroatom-containing alicyclic groups are pyrrolidino, morpholino, piperazino, piperidino, tetrahydrofuranyl, etc.
“Heteroaryl” refers to an aromatic group of from 1 to 15 carbon atoms, such as from 1 to 10 carbon atoms and 1 to 10 heteroatoms selected from the group consisting of oxygen, nitrogen, and sulfur within the ring. Such heteroaryl groups can have a single ring (such as, pyridinyl, imidazolyl or furyl) or multiple condensed rings in a ring system (for example as in groups such as, indolizinyl, quinolinyl, benzofuran, benzimidazolyl or benzothienyl), wherein at least one ring within the ring system is aromatic, provided that the point of attachment is through an atom of an aromatic ring. In certain embodiments, the nitrogen and/or sulfur ring atom(s) of the heteroaryl group are optionally oxidized to provide for the N-oxide (N→O), sulfinyl, or sulfonyl moieties. This term includes, by way of example, pyridinyl, pyrrolyl, indolyl, thiophenyl, and furanyl. Unless otherwise constrained by the definition for the heteroaryl substituent, such heteroaryl groups can be optionally substituted with 1 to 5 substituents, or from 1 to 3 substituents, selected from acyloxy, hydroxy, thiol, acyl, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, substituted alkyl, substituted alkoxy, substituted alkenyl, substituted alkynyl, substituted cycloalkyl, substituted cycloalkenyl, amino, substituted amino, aminoacyl, acylamino, alkaryl, aryl, aryloxy, azido, carboxyl, carboxylalkyl, cyano, halogen, nitro, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, aminoacyloxy, oxyacylamino, thioalkoxy, substituted thioalkoxy, thioaryloxy, thioheteroaryloxy, —SO-alkyl, —SO-substituted alkyl, —SO-aryl, —SO-heteroaryl, —SO2-alkyl, —SO2-substituted alkyl, —SO2-aryl and —SO2-heteroaryl, and trihalomethyl.
The terms “heterocycle,” “heterocyclic” and “heterocyclyl” refer to a saturated or unsaturated group having a single ring or multiple condensed rings, including fused bridged and spiro ring systems, and having from 3 to 15 ring atoms, including 1 to 4 hetero atoms. These ring heteroatoms are selected from nitrogen, sulfur and oxygen, wherein, in fused ring systems, one or more of the rings can be cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, provided that the point of attachment is through the non-aromatic ring. In certain embodiments, the nitrogen and/or sulfur atom(s) of the heterocyclic group are optionally oxidized to provide for the N-oxide, —S(O)—, or —SO2— moieties.
Examples of heterocycles and heteroaryls include, but are not limited to, azetidine, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, dihydroindole, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, phenanthroline, isothiazole, phenazine, 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, and the like.
Unless otherwise constrained by the definition for the heterocyclic substituent, such heterocyclic groups can be optionally substituted with 1 to 5, or from 1 to 3 substituents, selected from alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, —SO— alkyl, —SO-substituted alkyl, —SO-aryl, —SO-heteroaryl, —SO2-alkyl, —SO2-substituted alkyl, —SO2— aryl, —SO2-heteroaryl, and fused heterocycle.
“Hydrocarbyl” refers to univalent hydrocarbyl radicals containing 1 to about 30 carbon atoms, including 1 to about 24 carbon atoms, further including 1 to about 18 carbon atoms, and further including about 1 to 12 carbon atoms, including linear, branched, cyclic, saturated and unsaturated species, such as alkyl groups, alkenyl groups, aryl groups, and the like. A hydrocarbyl may be substituted with one or more substituent groups. The term “heteroatom-containing hydrocarbyl” refers to hydrocarbyl in which at least one carbon atom is replaced with a heteroatom. Unless otherwise indicated, the term “hydrocarbyl” is to be interpreted as including substituted and/or heteroatom-containing hydrocarbyl moieties.
By “substituted” as in “substituted hydrocarbyl,” “substituted alkyl,” “substituted aryl,” and the like, as alluded to in some of the aforementioned definitions, is meant that in the hydrocarbyl, alkyl, aryl, or other moiety, at least one hydrogen atom bound to a carbon (or other) atom is replaced with one or more non-hydrogen substituents. Examples of such substituents include, without limitation, functional groups, and the hydrocarbyl moieties C1-C24 alkyl (including C1-C18 alkyl, further including C1-C12 alkyl, and further including C1-C6 alkyl), C2-C24 alkenyl (including C2-C18 alkenyl, further including C2-C12 alkenyl, and further including C2-C6 alkenyl), C2-C24 alkynyl (including C2-C18 alkynyl, further including C2-C12 alkynyl, and further including C2-C6 alkynyl), C5-C30 aryl (including C5-C20 aryl, and further including C5-C12 aryl), and C6-C30 aralkyl (including C6-C20 aralkyl, and further including C6-C12 aralkyl). The above-mentioned hydrocarbyl moieties may be further substituted with one or more functional groups or additional hydrocarbyl moieties such as those specifically enumerated. Unless otherwise indicated, any of the groups described herein are to be interpreted as including substituted and/or heteroatom-containing moieties, in addition to unsubstituted groups.
“Sulfonyl” refers to the group SO2-alkyl, SO2-substituted alkyl, SO2-alkenyl, SO2-substituted alkenyl, SO2-cycloalkyl, SO2-substituted cycloalkyl, SO2-cycloalkenyl, SO2-substituted cylcoalkenyl, SO2-aryl, SO2-substituted aryl, SO2-heteroaryl, SO2-substituted heteroaryl, SO2-heterocyclic, and SO2— substituted heterocyclic, wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein. Sulfonyl includes, by way of example, methyl-SO2—, phenyl-SO2—, and 4-methylphenyl-SO2—.
By the term “functional groups” is meant chemical groups such as 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 alkylcarbonato (—O—(CO)—O-alkyl), C6-C20 arylcarbonato (—O—(CO)—O-aryl), carboxy (—COOH), carboxylato (—COO—), carbamoyl (—(CO)—NH2), mono-substituted C1-C24 alkylcarbamoyl (—(CO)—NH(C1-C24 alkyl)), di-substituted alkylcarbamoyl (—(CO)—N(C1-C24 alkyl)2), mono-substituted arylcarbamoyl (—(CO)—NH-aryl), thiocarbamoyl (—(CS)—NH2), carbamido (—NH—(CO)—NH2), cyano (—C≡N), isocyano (—N+—C—), cyanato (—O—C≡N), isocyanato (—O—N+≡C—), isothiocyanato (—S—C≡N), azido (—N═N+β2 N—), formyl (—(CO)—H), thioformyl (—(CS)—H), amino (—NH2), mono- and di-(C1-C24 alkyl)-substituted amino, mono- and di-(C5-C20 aryl)-substituted amino, C2-C24 alkylamido (—NH—(CO)-alkyl), C5-C20 arylamido (—NH—(CO)-aryl), imino (—CR═NH where R=hydrogen, C1-C24 alkyl, C5-C20 aryl, C6-C20 alkaryl, C6-C20 aralkyl, etc.), alkylimino (—CR═N(alkyl), where R=hydrogen, alkyl, aryl, alkaryl, etc.), arylimino (—CR═N(aryl), where R=hydrogen, alkyl, aryl, alkaryl, etc.), nitro (—NO2), nitroso (—NO), sulfo (—SO2—OH), sulfonato (—SO2—O—), C1-C24 alkylsulfanyl (—S-alkyl; also termed “alkylthio”), arylsulfanyl (—S-aryl; also termed “arylthio”), C1-C24 alkylsulfinyl (—(SO)-alkyl), C5-C20 arylsulfinyl (—(SO)-aryl), C1-C24 alkylsulfonyl (—SO2-alkyl), C5-C20 arylsulfonyl (—SO2-aryl), phosphono (—P(O)(OH)2), phosphonato (—P(O)(O—)2), phosphinato (—P(O)(O—)), phospho (—PO2), and phosphino (—PH2), mono- and di-(C1-C24 alkyl)-substituted phosphino, mono- and di-(C5-C20 aryl)-substituted phosphine. In addition, the aforementioned functional groups may, if a particular group permits, be further substituted with one or more additional functional groups or with one or more hydrocarbyl moieties such as those specifically enumerated above.
By “linking” or “linker” as in “linking group,” “linker moiety,” etc., is meant a linking moiety that connects two groups via covalent bonds. The linker may be linear, branched, cyclic or a single atom. Examples of such linking groups include alkyl, alkenylene, alkynylene, arylene, alkarylene, aralkylene, and linking moieties containing functional groups including, without limitation: amido (—NH—CO—), ureylene (—NH—CO—NH—), imide (—CO—NH—CO—), epoxy (—O—), epithio (—S—), epidioxy (—O—O—), carbonyldioxy (—O—CO—O—), alkyldioxy (—O—(CH2)n-O—), epoxyimino (—O—NH—), epimino (—NH—), carbonyl (—CO—), etc. In certain cases, one, two, three, four or five or more carbon atoms of a linker backbone may be optionally substituted with a sulfur, nitrogen or oxygen heteroatom. The bonds between backbone atoms may be saturated or unsaturated, usually not more than one, two, or three unsaturated bonds will be present in a linker backbone. The linker may include one or more substituent groups, for example with an alkyl, aryl or alkenyl group. A linker may include, without limitations, poly(ethylene glycol) unit(s) (e.g., —(CH2—CH2—O)—); ethers, thioethers, amines, alkyls (e.g., (C1-C12)alkyl), which may be straight or branched, e.g., methyl, ethyl, n-propyl, 1-methylethyl (iso-propyl), n-butyl, n-pentyl, 1,1-dimethylethyl (t-butyl), and the like. The linker backbone may include a cyclic group, for example, an aryl, a heterocycle or a cycloalkyl group, where 2 or more atoms, e.g., 2, 3, or 4 atoms, of the cyclic group are included in the backbone. A linker may be cleavable or non-cleavable. Any convenient orientation and/or connections of the linkers to the linked groups may be used.
When the term “substituted” appears prior to a list of possible substituted groups, it is intended that the term apply to every member of that group. For example, the phrase “substituted alkyl and aryl” is to be interpreted as “substituted alkyl and substituted aryl.”
In addition to the disclosure herein, the term “substituted,” when used to modify a specified group or radical, can also mean that one or more hydrogen atoms of the specified group or radical are each, independently of one another, replaced with the same or different substituent groups as defined below.
In addition to the groups disclosed with respect to the individual terms herein, substituent groups for substituting for one or more hydrogens (any two hydrogens on a single carbon can be replaced with ═O, ═NR70, ═N—OR70, ═N2 or ═S) on saturated carbon atoms in the specified group or radical are, unless otherwise specified, —R60, halo, ═O, —OR70, —SR70, —NR80R80, trihalomethyl, —CN, —OCN, —SCN, —NO, —NO2, ═N2, —N3, —SO2R70, —SO2O−
In addition to the disclosure herein, substituent groups for hydrogens on unsaturated carbon atoms in “substituted” alkene, alkyne, aryl and heteroaryl groups are, unless otherwise specified, —R60, halo, —O−M+, —OR70, —SR70, —S−M+, —NR80R80,
In addition to the groups disclosed with respect to the individual terms herein, substituent groups for hydrogens on nitrogen atoms in “substituted” heteroalkyl and cycloheteroalkyl groups are, unless otherwise specified, —R60, —O−M+, —OR70, —SR70, —S−M+, —NR80R80, trihalomethyl, —CF3, —CN, —NO, —NO2, —S(O)2R70, —S(O)2O−M+, —S(O)2OR70, —OS(O)2R70, —OS(O)2O−M+, —OS(O)2OR70, —P(O)(O−)2(M+)2, —P(O)(OR70)O−M+, —P(O)(OR70)(OR70), —C(O)R70, —C(S)R70, —C(NR70) R70, —C(O)OR70, —C(S)OR70, —C(O)NR80R80, —C(NR70)NR80R80, —OC(O)R70, —OC(S)R70, —OC(O)OR70, —OC(S)OR70, —NR70C(O)R70, —NR70C(S)R70, —NR70C(O)OR70, —NR70C(S)OR70, —NR70C(O)NR80R80, —N R70C(NR70)R70 and —NR70C(NR70)NR80R80, where R60, R70, R80 and M+ are as previously defined.
In addition to the disclosure herein, in a certain embodiment, a group that is substituted has 1, 2, 3, or 4 substituents, 1, 2, or 3 substituents, 1 or 2 substituents, or 1 substituent.
Unless indicated otherwise, the nomenclature of substituents that are not explicitly defined herein are arrived at by naming the terminal portion of the functionality followed by the adjacent functionality toward the point of attachment. For example, the substituent “arylalkyloxycarbonyl” refers to the group (aryl)-(alkyl)-O—C(O)—.
As to any of the groups disclosed herein which contain one or more substituents, it is understood, of course, that such groups do not contain any substitution or substitution patterns which are sterically impractical and/or synthetically non-feasible. In addition, the subject compounds include all stereochemical isomers arising from the substitution of these compounds.
In certain embodiments, a substituent may contribute to optical isomerism and/or stereo isomerism of a compound. Salts, solvates, hydrates, and prodrug forms of a compound are also of interest. All such forms are embraced by the present disclosure. Thus, the compounds described herein include salts, solvates, hydrates, prodrug and isomer forms thereof, including the pharmaceutically acceptable salts, solvates, hydrates, prodrugs, and isomers thereof. In certain embodiments, a compound may be a metabolized into a pharmaceutically active derivative.
Unless otherwise specified, reference to an atom is meant to include isotopes of that atom. For example, reference to H is meant to include 1H, 2H (i.e., D) and 3H (i.e., T), and reference to C is meant to include 12C and all isotopes of carbon (such as 3C).
As used herein, the term “anti-tumor effect” or “anti-tumor activity” refers to a biological effect which can be manifested by various means, including but not limited to, e.g., a decrease in tumor volume, a decrease in the number of tumor cells, a decrease in the number of metastases, an increase in life expectancy, decrease in tumor cell proliferation, decrease in tumor ceil survival, or amelioration of various physiological symptoms associated with the cancerous condition. An “anti-tumor effect” can also be manifested by the ability of the peptides, polynucleotides, cells and antibodies of the present disclosure in prevention of the occurrence of tumor in the first place.
As used herein, the term “affinity” refers to a measure of binding strength. Without being bound to theory, affinity depends on the closeness of stereochemical fit between antibody combining sites and antigen determinants, on the size of the area of contact between them, and on the distribution of charged and hydrophobic groups. Affinity also includes the term “avidity,” which refers to the strength of the antigen-antibody bond after formation of reversible complexes. Methods for calculating the affinity of an antibody for an antigen are known in the art, including use of binding experiments to calculate affinity. Antibody activity in functional assays (e.g., flow cytometry assay) is also reflective of antibody affinity. Antibodies and affinities can be phenotypically characterized and compared using functional assays (e.g., flow cytometry assay).
As used herein, the term “immunosuppressive activity” refers to the induction of signal transduction or changes in protein expression in a cell, such as an activated immune effector cell, that results in a decrease in an immune response. Non-limiting examples of polypeptides known to suppress or decrease an immune response via their binding include CD47, PD-1, CTLA-4, and their corresponding ligands, including SIRPa, PD-L1, PD-L2, B7-1, and B7-2. Such polypeptides may be present in the tumor microenvironment and can inhibit immune responses to neoplastic cells. In various embodiments, inhibiting, blocking, or otherwise antagonizing the interaction of immunosuppressive polypeptides and/or their ligands may enhance the immune response of the immune effector cell.
As used herein, the term “enzymatic inhibitory domain” refers to a protein domain that inhibits an intracellular signal transduction cascade, for example a native T cell activation cascade. In some embodiments, the enzymatic inhibitory domain of a chimeric inhibitory receptor of the present disclosure comprises at least a portion of an extracellular domain, a transmembrane domain, and/or an intracellular domain. In some embodiments, the enzymatic inhibitory domain comprises at least a portion of an enzyme. In some embodiments, the enzyme is selected from CSK, SHP-1, PTEN, CD45, CD148, PTP-MEG1, PTP-PEST, c-CBL, CBL-b, PTPN22, LAR, PTPH1, SHIP-1, and RasGAP (see e.g., Stanford et al., Regulation of TCR signaling by tyrosine phosphatases: from immune homeostasis to autoimmunity, Immunology, 2012 September; 137(1): 1-19). In some embodiments, the portion of the enzyme comprises an enzyme domain(s), an enzyme fragment(s), or a mutant(s) thereof. In some embodiments, the portion of the enzyme is a catalytic domain of the enzyme. In some embodiments, the enzyme domain(s), enzyme fragment(s), or mutants(s) thereof are selected to maximize efficacy and minimize basal inhibition.
As used herein, the term “immunostimulatory activity” refers to induction of signal transduction or changes in protein expression in a cell, such as an activated immune effector cell, that results in an increase in an immune response. Immunostimulatory activity may include pro-inflammatory activity. Non-limiting examples of polypeptides known to stimulate or increase an immune response via their binding include CD28, OX-40, 4-1BB, and their corresponding ligands, including B7-1, B7-2, OX-40L, and 4-1BBL. Such polypeptides may be present in the tumor microenvironment and can activate immune responses to neoplastic cells. In various embodiments, promoting, stimulating, or otherwise agonizing pro-inflammatory polypeptides and/or their ligands may enhance the immune response of the immune effector cell.
Isolated nucleic acid molecules of the present disclosure include any nucleic acid molecule that encodes a polypeptide of the present disclosure, or fragment thereof. Such nucleic acid molecules need not be 100% homologous or identical with an endogenous nucleic acid sequence, but will typically exhibit substantial identity. Nucleic acids having “substantial identity” or “substantial homology” to an endogenous sequence are typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. As used herein, “hybridize” refers to pairing to form a double-stranded molecule between complementary polynucleotide sequences (e.g., a gene described herein), or portions thereof, under various conditions of stringency. For example, stringent salt concentration may be less than about 750 mM NaCl and 75 mM trisodium citrate, less than about 500 mM NaCl and 50 mM trisodium citrate, or less than about 250 mM NaCl and 25 mM trisodium citrate. Low stringency hybridization can be obtained in the absence of organic solvent, e.g., formamide, while high stringency hybridization can be obtained in the presence of at least about 35% formamide or at least about 50% formamide. Stringent temperature conditions will ordinarily include temperatures of at least about 30° C., at least about 37° C., or at least about 42° C. Varying additional parameters, such as hybridization time, the concentration of detergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art. Various levels of stringency may be accomplished by combining these various conditions as needed.
By “substantially identical” or “substantially homologous” is meant a polypeptide or nucleic acid molecule exhibiting at least about 50% homologous or identical to a reference amino acid sequence (for example, any one of the amino acid sequences described herein) or nucleic acid sequence (for example, any one of the nucleic acid sequences described herein). Preferably, such a sequence is at least about 60%, about 80%, about 85%, about 90%, about 95%, about 99%, or about 100% homologous or identical at the amino acid level or nucleic acid to the sequence used for comparison. Sequence identity is typically measured using sequence analysis software (for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP/PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and/or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary approach to determining the degree of identity, a BLAST program may be used, with a probability score between e-3 and e-100 indicating a closely related sequence.
As used herein, the term “encoding” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (e.g., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene, cDNA, or RNA, encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA. Unless otherwise specified, a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase nucleotide sequence that encodes a protein or an RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some version contain an intron(s).
As used herein, the term “ligand” refers to a molecule that binds to a receptor. In particular, the ligand binds a receptor on another cell, allowing for cell-to-cell recognition and/or interaction.
Definitions of other terms and concepts appear throughout the detailed description.
All references, issued patents, and patent applications cited within the body of the specification are hereby incorporated by reference in their entirety, for all purposes.
The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use embodiments of the present disclosure, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.
While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the present disclosure. All such modifications are intended to be within the scope of the claims appended hereto.
Sodium hydride (2.16 g, 54.11 mmol) was carefully added to a stirred solution of bis(dimethoxyphosphoryl)methane (11.42 g, 49.19 mmol) in toluene (100 mL) at room temperature. The reaction mixture was then placed under an atmosphere of nitrogen and a solution of 1-benzylpiperidine-4-carbaldehyde (10 g, 49.19 mmol) in toluene (50 mL) was slowly added keeping the temperature below 40° C. The resulting mixture was left to stir at room temperature for 16 h and then quenched by the addition of aqueous saturated ammonium chloride solution. The organic phase was separated, washed with brine, dried (MgSO4) and evaporated to dryness. Chromatography (120 g SiO2; 5 to 100% gradient of EtOAc in hexanes) provided dimethyl (E)-(2-(1-benzylpiperidin-4-yl)vinyl)phosphonate (6.2 g, 16%) as a colorless oil.
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 an atmosphere of hydrogen and stirred at room temperature for 12 h, filtered and evaporated to dryness under reduced pressure to give dimethyl (2-(piperidin-4 yl)ethyl)phosphonate (2.7 g, 100%) as colorless oil.
Diisopropylethylamine (0.6 g, 8.9 mmol) was added to a mixture of dimethyl (2-(piperidin-4-yl)ethyl)phosphonate (1.1 g, 4.9 mmol) and 4-chloro-6,7-dimethoxyquinazoline (1.0 g, 4.5 mmol) in isopropyl alcohol (20 mL). After stirring at 90° C. for 3 h, the reaction mixture was cooled and evaporated to dryness. Purification of silica gel (5% MeOH in dichloromethane) provided dimethyl (2-(1-(6,7-dimethoxyquinazolin-4-yl)piperidin-4-yl)ethyl)phosphonate (755 mg, 37%) as oil.
LC-MS: m/z=410.25 [M+H]+
1H NMR (500 MHz, CDCl3) δ 8.65 (s, 1H), 7.23 (s, 1H), 7.09 (s, 1H), 4.19 (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).
Bromotrimethylsilane (3.67 g, 24 mmol) was added to a cooled solution of dimethyl (2-(1-(6,7-dimethoxyquinazolin-4-yl)piperidin-4-yl)ethyl)phosphonate (3.25 g, 7.94 mmol) in chloroform (60 mL) that was cooled by an ice bath. The reaction mixture was allowed to warm to room temperature and after 90 minutes was quenched by the addition of methanol (20 mL). The mixture was evaporated to dryness under reduced pressure and then solvated in methanol (100 mL). The reaction mixture was concentrated to half volume, filtered to remove precipitate, and then evaporated to dryness. The residue was crystalized with 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]+
1H 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).
Selected compounds of Table 1, Table 2, Table 3, Table 4, Table 5, or Table 6 and other derivatives were prepared and assessed in an ENPP1 activity assay using thymidine monophosphate paranitrophenol (TMP-pNP) as a substrate. Enzyme reactions were prepared with TMP-pNP (2 μM), 5-fold dilutions of ENPP1 inhibitor, and purified recombinant mouse ENPP1 (0.5 nM) in 100 mM Tris, 150 mM NaCl, 2 mM CaCl2, 200 μM ZnCl2, pH 7.5 at room temperature. Reaction progress was monitored by measuring absorbance at 400 nm of paranitrophenolate produced by the reaction for 20 minutes. Slopes of product formation were extracted, plotted, and fit to obtain IC50 values with Graphpad Prism 7.03.
Compounds were also assessed in an ENPP1 enzyme activity assay using 32P cGAMP as a substrate. Radiolabeled 32P cGAMP was synthesized by incubating unlabeled ATP (1 mM) and GTP (1 mM) doped with 32P-ATP with 2 μM purified recombinant porcine cGAS in 20 mM Tris pH 7.5, 2 mM MgCl2, 100 μg/mL herring testes DNA) overnight at room temperature, and the remaining nucleotide starting materials were degraded with alkaline phosphatase for 4 h at 37° C. The probe 32P-cGAMP (5 μM) was incubated with purified recombinant mouse ENPP1 (20 nM) or whole cell lysates in 100 mM Tris, 150 mM NaCl, 2 mM CaCl2, 200 μM ZnCl2, pH 7.5 at room temperature for 5 hours. To generate enzyme inhibition curves, 5-fold dilutions of ENPP1 inhibitor were included in the reaction. Degradation was evaluated by TLC (as described by Li et al. Nat. Chem. Biol. (2014) 10:1043-8). Plates were exposed on a phosphor screen (Molecular Dynamics) and imaged on a Typhoon 9400 and the 32P signal was quantified using ImageJ. Inhibition curves were fit to obtain IC50 values using Graphpad Prism 7.03. The IC50 of the compounds tested is provided in Table 8. IC50 values fall in the range indicated by letters A-D, where A represents an IC50 value less than 0.005 μM, B represents an IC50 value between 0.005 μM and 0.05 μM, and C represents an IC50 value between 0.05 μM and 0.5 μM, D represents an IC50 value between 0.5 μM and 5 μM, and E represents an IC50 value greater than 5 μM (n.d.=not determined).
With reference to
In intact cells, ENPP1 expression depletes extracellular cGAMP, but does not affect the intracellular cGAMP concentration (
Inhibiting ENPP1 blocks degradation of extracellular cGAMP (
Using an exemplary ENPP1 inhibitor (compound 1), it was tested whether cGAMP exported by the 293T cGAS ENPP1low cell line could be detected by antigen presenting cells (APCs) such as human CD14+ monocytes (
Supernatant from the cGAS-expressing 293T cGAS ENPP1low cells, but not cGAS-null 293T cells, induced CD14+ IFNB1 expression, suggesting that extracellular cGAMP exported by cancer cells could be detected by CD14+ cells as a signaling factor (
With reference to
It was tested whether cancer cell lines export cGAMP and if ionizing radiation (IR) affects the levels of extracellular cGAMP produced. Ionizing radiation (IR) has been shown to increase cytosolic DNA and activate cGAS-dependent IFN-β production in tumor cells (Bakhoum et al. Nat. Commun. (2015) 6:1-10; and Vanpouille Nat. Commun. (2017) 8:15618). 24 hours after plating, 4T1 cells were treated with 20 Gy IR using a cesium source and the media was changed, supplemented with 50 uM of the exemplary ENPP1 inhibitor compound 1 to inhibit ENPP1 present in cell culture. Media was collected at indicated times, centrifuged at 1000×g to remove residual cells, acidified with 0.5% acetic acid, and supplemented with cyclic-13C10,155-GMP-13C10,15N5-AMP as an extraction standard extraction standard (the appropriate amount for a final concentration of 2 μM in 100 μL). Media was applied to HyperSep Aminopropyl SPE columns (ThermoFisher Scientific) to enrich for cGAMP as described previously (Gao et al., Proc. Natl. Acad. Sci. U.S.A. (2015) 112:E5699-705). Eluents were evaporated to dryness and reconstituted in 50:50 acetonitrile: water supplemented with 500 nM internal standard. The media was submitted for mass spectrometry quantification of cGAMP.
Continuous cGAMP export was detected in the 4T1 cells over 48 hours. At 48 hours, cells treated with IR had significantly higher extracellular cGAMP levels than untreated.
Next, the effect of IR combined with exemplary ENPP1 inhibitor compound 1 on the number of tumor-associated dendritic cells in a mouse 4T1 tumor model was investigated (
Intratumoral injection of compound 1 did not change tumor-associated leukocyte compositions compared to the PBS control (
The results are illustrated in
It was investigated whether immune detection and clearance of tumors could be increased by further increasing extracellular cGAMP in vivo using ionizing radiation (IR) and an exemplary ENPP1 inhibitor, e.g., compound 1.
Seven- to nine-week-old female Balb/c mice (Jackson Laboratories) were inoculated with 5×104 4T1-luciferase cells suspended in 50 μL of PBS into the mammary fat pad. When tumor volume (determine length2×width/2) reached 80 mm3 to 120 mm3, tumors were irradiated with 20 Gy using a 225 kVp cabinet X-ray irradiator filtered with 0.5 mm Cu (IC 250, Kimtron Inc., CT). Anaesthetized animals were shielded with a 3.2 mm lead shield with a 15×20 mm aperture where the tumor was placed. On day 2, 4 and 7 after IR, 100 μL of 100 μM compound 1 and/or 10 μg cGAMP in PBS or PBS alone were injected intratumorally. Alternatively, 1 mM compound 1 in PBS or PBS alone were injected intratumorally and 200 μg of anti-CTLA-4 antibody or Syrian hamster IgG antibody (both BioXCell) were injected intraperitoneally on day 2, 5, and 7 after IR. Mice from different treatment groups were co-housed in each cage to eliminate cage effects. The experimenter was blinded throughout the entire study. Tumor volumes were recorded every other day. Tumor volumes were analyzed in a generalized estimation equation in order to account for the within mouse correlation. Pair-wise comparisons of the treatment groups at each time point were done using post hoc tests with a Tukey adjustment for multiple comparisons. Animal death was plotted in a Kaplan Meier curve using Graphpad Prism 7.03 and statistical significance was assessed using the Logrank Mantel-Cox test. All animal procedures were approved by the administrative panel on laboratory animal care.
Administration of compound 1 enhanced tumor shrinkage effects of IR treatment, although not significantly (
The synergistic effect with the adaptive immune checkpoint blocker anti-CTLA-4 was also tested. Without IR, treatment with anti-CTLA-4 and compound 1 had no effect on prolonging survival (
The results are illustrated in
In summary, these results indicate that the cGAMP exists extracellulary and subject ENPP1 inhibitors act extracellularly; therefore, indicating that the extracellular inhibition of ENPP1 is sufficient for therapeutic effect. ENPP1 qualifies as an innate immune checkpoint. These experiments indicate that inhibiting ENPP1 extracellularly allows cGAMP to potentiate anti-cancer immunity and combine synergistically with immune checkpoint blocking drugs already available as therapies (
Inhibition of ENPP1 with small molecule inhibitor is assessed following adoptive T cell therapy in EG7-OVA, C57BL/6 OT-I female transgenic mice.
EG7-Ova tumors are implanted on flanks of C57BL/6 female mice and randomized at 120 mm3 into 6 groups and n=12 per group. In parallel, spleens are harvested from 18 OT-I female C57BL/6 mice and enriched CD3+ cells isolated (total of 180 million enriched CD3+ T cells).
The treatments for each group are: (1) control untreated; (2) untreated and adoptive T cell therapy on day 4; (3) an inhibitor of ENPP1 as described herein at 50 mg/kg on days 1, 2, 3, 8, 9, and 10; (4) an inhibitor of ENPP1 as described herein at 50 mg/kg on days 1, 2, 3, 8, 9, 10 and adoptive T cell therapy on day 4; (5) an inhibitor of ENPP1 as described herein at 50 mg/kg on days 1, 2, 3, 4, 5, 6, and 7; and (6) an inhibitor of ENPP1 as described herein at 50 mg/kg on days 1, 2, 3, 4, 5, 6, and 7 and adoptive T cell therapy on day 4.
The route of ENPP1 inhibitor administration is chosen taking into consideration antigen characteristics (e.g., antigen size) and desired localization of the immune response.
On day 4, for adoptive therapy, CD3+ splenocytes from OT-I C57BL/6 transgenic mice are labeled with CFSE and transferred (5×106 cells per mouse).
On day of harvest (study day 4), isolated OT-I T cells (labeled with CFSE), are transferred by intraperitoneal injection (5×10e6 cells/mouse) into EG7-Ova tumor-bearing mice.
An immunoprofile panel of CD45+ TILs is analyzed by FACS from animals (n=6 per group) on day 7 and day 12. On day 7, blood and half of the tumors are harvested from 6 mice per group and cells isolated for flow cytometry analysis. If tumor size permits, tumors are cut in ½ with one half used for flow cytometry analysis and the other for IHC. If tumor is not large enough preference is given to flow cytometry analysis. On day 12, blood and the other half of the tumors are harvested from 6 mice per group and cells isolated for flow cytometry analysis. If tumor size permits, tumors are cut in ½ with one half used for flow cytometry analysis and the other for IHC. If tumor is not large enough preference is given to flow cytometry analysis. Flow cytometry panels may include, but are not limited to: live/dead, CD45, CD3, CD4, CD8, CD11 b, F4/80, CFSE, siinfekl pentamer (H-2Kd), Foxp3, CD335, or Gr-1.
ENPP1 inhibition promotes increased T cell trafficking to the tumor following adoptive T cell therapy compared to adoptive T cell therapy alone.
This example demonstrates enhanced donor T cell trafficking to a B16 melanoma site in C57BL/6 mice after dosing with an ENPP1 inhibitor (Compound 76).
For the study, 0.2×106 B16F10 melanoma cells/mouse were implanted on day −8 on flanks of C57BL/6 female mice. On day 0, when tumor size had reached about 80 mm3, mice were randomized into 3 groups with an n=12/group. All mice in the three groups were subjected to whole body irradiation with 5.0 Gy for lymphodepletion. The three mouse groups were dosed as follows:
gp100 specific donor splenocytes were harvested from C57BL/6 pmel-1 donor mice (The Jackson Laboratory, world wide web.jax.org/strain/005023). Briefly, splenocytes comprising immune cells such as T cells from spleens of 18 pmel-1 female C57BL/6 mice (Jackson labs) were harvested yielding˜90 Million splenocytes. The pmel-1 splenocytes were isolated, the red blood cells lysed, and the remaining splenocytes expanded and activated with IL-2 and gp100 antigen for 4 days in vitro, and administered to the B16 melanoma bearing mice by i.v.
Pmel-1 mice are a transgenic mouse strain that carries a rearranged T cell receptor transgene specific for the mouse homologue (pmelSi or pmel-17) of human premelanosome protein (referred to as PMEL, SILV, or gp100), and the T lymphocyte specific Thy1a (Thy1.1) allele. gp100 is an enzyme involved in pigment synthesis that is expressed by the majority of malignant melanoma cells including the B16 melanoma model. Transgenic constructs containing the mouse alpha-chain and beta-chain of a T-cell receptor were used to create transgenic animals on a C57BL/6 background (termed C57BL/6 pmel-1). The mice were then crossed to B6.PL-Thy1a/CyJ (Stock No. 000406). The strain is maintained homozygous for the transgenic insert and homozygous for the Thy1a (Thy1.1) allele.
Mouse Thy1.2 also known as CD90.2 is expressed by thymocytes and mature T lymphocytes as well as hematopoietic stem cells, neurons, epithelial cells, and fibroblasts. Thy1.2 is expressed only by certain mouse strains including C57BL/6, BALB/c, CBA, C3H, C58/, SJL, DBA, and NZB/. Thy1.2 is a 25-35 kDa GPI-anchored membrane glycoprotein and a member of the immunoglobulin superfamily. Donor mouse cells and host mouse cells can be distinguished by their respective Thy1.1 or Thy1.2 expression.
On day 7, mice were sacrificed, and tumor, spleen, and blood samples were collected. Tumor size of all three treatment groups were measured on days 0, 2, 5 and 7 (
After mice were sacrificed, tumor samples were tested for the fraction of several live immune cells (CD45+ cells) in the tumor. Briefly, tumors were harvested, and samples digested with tumor digestion mix and cells collected and washed in FACS buffer. A sample of cells was stained with a live/dead stain (ef780) to test for live cells and enumerated by flow cytometry. Results show that the percentage of live cells in tumors from mice treated with Compound 76 (50 mg/kg) plus donor T cells and Compound 76 (5 mg/kg) plus donor T cells had a higher percentage of live cells compared to mice treated with PBS in combination with donor T cells (
Additional samples of harvested cells were blocked with Fc-Block (Mouse, BD) and stained for cell further cell surface markers (CD45, CD3, CD4, CD8, CD11B, Thy-1.1, and/or Thy-1.2). When staining for intracellular markers, cells were fixed and permeabilized before staining with markers (ki67). For example, cells were stained with markers for MDSC and F4/80 macrophages and enumerated by flow cytometry. Results show that the percentage of MDSC macrophages in tumors from mice treated with Compound 76 (50 mg/kg) plus donor T cells and Compound 76 (5 mg/kg) plus donor T cells is lower compared to mice treated with PBS in combination with donor T cells and the percentage of F4/80+ macrophages is higher (
Samples were also analyzed for the percentage of gp100+, CD8+ T cells actively proliferating (ki67+ cells) that were present in the tumor. Results show that the percentage of gp100+, CD8+, ki67+ T cells in tumors from mice treated with Compound 76 (50 mg/kg) plus donor splenocytes and Compound 76 (5 mg/kg) plus donor splenocytes is higher compared to mice treated with PBS in combination with donor splenocytes (
Samples were also analyzed for the percentage of gp100+, CD8+ T cell that came either from the C57BL/6 pmel-1 donor mice (Thy1.1+) or were present in the C57BL/6 host mice (Thy1.2+). Results show that the percentage of Thy1.1+(pmel-1 derived), gp100+, CD8+ donor T cells in tumors from mice treated with Compound 76 (50 mg/kg) plus donor splenocytes and Compound 76 (5 mg/kg) plus donor splenocytes, is higher compared to mice treated with PBS in combination with donor splenocytes (
This example describes the in vitro activation and expansion of donor C57BL/6 pmel-1 splenocytes by INF-γ ELISA and INF-γ Elispot analysis of splenocytes derived from B16F10 melanoma syngeneic tumor model inoculated with Pmel-1 donor splenocytes (T-cells).
Briefly, spleens and splenocytes from C57BL-B16F10 tumor bearing mice treated with or without an ENPP1 inhibitor and donor T cells are harvested and purified as described in Example 8. To test for cytokine secretion, the isolated splenocytes are then plated and co-cultured with gp100 peptide on a plate of an ELISpot kit and IFN-γ secretion is detected. The number of spots is counted for each well and summarized for each group.
Additionally, or alternatively, IFN-γ secretion is detected with a IFN-γ ELISA test. The result will show that IFN-γ secretion is elevated in the mice treated with Compound 76 (50 mg/kg) plus donor T cells and Compound 76 (5 mg/kg) plus donor T cells compared to mice treated with PBS in combination with donor T cells.
Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it is readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.
Accordingly, the preceding merely illustrates the principles of the invention. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. The scope of the present invention, therefore, is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of present invention is embodied by the following.
Notwithstanding the appended claims, the disclosure set forth herein is also described by the following clauses.
Clause 1. An ENPP1 inhibitor of the formula (I):
Y-A-L-X (I)
Clause 6. The ENPP1 inhibitor of any one of clauses 1-5, wherein X is phosphonic acid or phosphonate ester.
Clause 7. The ENPP1 inhibitor of clause 1, wherein L-X comprises a group of the formula (XII):
Clause 9. The ENPP1 inhibitor of clause 1, wherein L-X comprises a group of formula (XIII):
Clause 11. The ENPP1 inhibitor of clause 1, wherein L-X comprises a group of formula (XIV):
Clause 13. The ENPP1 inhibitor of clause 1, wherein L-X comprises a group of formula (XV):
wherein q5 is an integer from 1 to 6.
Clause 14. The ENPP1 inhibitor of clause 13, wherein L-X is selected from:
Clause 15. The ENPP1 inhibitor of clause 1, wherein L-X comprises a group of formula (XVI):
Clause 17. The ENPP1 inhibitor of clause 1, wherein L-X comprises a group of formula (XVII):
Clause 19. The ENPP1 inhibitor of any one of clauses 1-18, wherein A is a heterocycle or substituted heterocycle.
Clause 20. The ENPP1 inhibitor of clause 19, wherein A is selected from piperidine, substituted piperidine, piperazine and substituted piperazine.
Clause 21. The ENPP1 inhibitor of any one of clauses 19-20, wherein A is:
Clause 22. The ENPP1 inhibitor of any one of clauses 1-18, wherein A is a carbocycle (e.g., a 5-, 6- or 7-membered monocyclic carbocycle).
Clause 23. The ENPP1 inhibitor of clause 22, wherein A is a cycloalkyl or substituted cycloalkyl.
Clause 24. The ENPP1 inhibitor of clause 23, wherein A is:
Clause 25. The ENPP1 inhibitor of clause 22, wherein A is aryl or substituted aryl.
Clause 26. The ENPP1 inhibitor of clause 25, wherein A is phenylene or substituted phenylene.
Clause 27. The ENPP1 inhibitor of clause 26, wherein A is:
Clause 28. The ENPP1 inhibitor of any one of clauses 1 to 27, wherein L is a linear linker having a backbone of 1 to 12 atoms in length and comprising one or more groups selected from alkylene, substituted alkylene, —CO—, —O—, —NR′— —NR′CO—, —CO2— and —NR′CO2— wherein R′ is H, alkyl or substituted alkyl.
Clause 29. The ENPP1 inhibitor of clause 28, wherein L is —(CH2)n-, and n is an integer from 1 to 6 (e.g., 1, 2, 3, 4, 5 or 6).
Clause 30. The ENPP1 inhibitor of clause 29, wherein n is 1 or 2.
Clause 31. The ENPP1 inhibitor of any one of clauses 1 to 30, wherein Y is selected from quinazoline, substituted quinazoline, quinoline, substituted quinoline, naphthalene, substituted naphthalene, isoquinoline, substituted isoquinoline, 7H-purine, substituted 7H-purine, pyrimidine, substituted pyrimidine.
Clause 32. The ENPP1 inhibitor of any one of clauses 1 to 30, wherein Y is selected from 4-quinazolinyl, substituted 4-quinazolinyl, 4-quinolinyl, substituted 4-quinolinyl, 1-naphthalyl, substituted 1-naphthalyl, 4-isoquinolinyl, substituted 4-isoquinolinyl, 6-(7H-purinyl), substituted 6-(7H-purinyl), 4-pyrimidinyl, substituted 4-pyrimidinyl.
Clause 33. The ENPP1 inhibitor of any one of clauses 31 to 32, wherein Y is a group of the formula:
wherein,
wherein:
Clause 36. The ENPP1 inhibitor of clause 33, of the formula:
wherein,
wherein:
wherein:
wherein,
wherein,
wherein,
wherein,
Clause 49. The ENPP1 inhibitor of any one of any one of clauses 1-47, wherein Y is selected from:
Clause 50. The ENPP1 inhibitor of any one of clauses 1 to 30, wherein Y is of the formula:
wherein:
wherein: Z3 and Z4 are each independently selected from CR and N, wherein R is H, alkyl or substituted alkyl.
Clause 52. The ENPP1 inhibitor of clause 50 or 51, wherein Y is selected from:
wherein,
wherein,
Clause 55. The ENPP1 inhibitor of any one of clauses 1 to 30, wherein Y is a group of the formula:
wherein,
Clause 57. The ENPP1 inhibitor of any one of clauses 1 to 56, wherein the compound is a compound selected from the compounds of Table 1, Table 2, Table 3, Table 4, Table 5 and Table 6.
Clause 58. A pharmaceutical composition, comprising:
This application is a bypass continuation of PCT/US2022/075955, filed on Sep. 2, 2022, which claims the benefit of and U.S. priority to Provisional Patent Application No. 63/240,723, filed on Sep. 3, 2021, the disclosure of which is incorporated herein by reference in its entirety for all purposes.
| Number | Date | Country | |
|---|---|---|---|
| 63240723 | Sep 2021 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/US22/75955 | Sep 2022 | WO |
| Child | 18592874 | US |
