Patent Application
20230087994
This disclosure contains one or more sequences in a computer readable format in an accompanying text file titled “047162-7250US1_sequence_listing,” which is 36.8 KB in size and was created on Nov. 23, 2021, the contents of which are incorporated herein by reference in their entirety.
Among the mechanisms that regulate transport of molecules into a cell is receptor-mediated endocytosis. In this process, a receptor on the cell surface binds to a specific ligand (or a molecule comprising such specific ligand) that is present outside the cell—this ligand may be a small molecule, metabolite, hormone, protein, or even a virus. The binding process triggers the inward budding of the plasma membrane (invagination), forming a vesicle containing the receptor-ligand complex. The vesicle becomes an endosome and subsequently fuses with lysosomes, and the receptor is degraded along with ligand cargo bound thereto or the receptor is recycled to the cell surface for further harvesting of the circulating ligand.
One such receptor is the asialoglycoprotein receptor (ASGPR). This receptor is a C-type lectin, and its major biological role is to bind, internalize, and subsequently clear from circulation glycoproteins that contain terminal galactose or N-acetylgalactosamine residues (asialoglycoproteins). ASGPRs remove the target glycoproteins from circulation through endocytosis and subsequent lysosomal degradation. ASGPRs are highly expressed on the surface of hepatocytes, several human carcinoma cell lines, and liver cancers, and also weakly expressed by glandular cells of the gallbladder and the stomach. These receptors are known to be involved in the clearance of IgG subtypes and other antibody isotypes from circulation, removal of apoptotic cells, clearance of low density lipoprotein (LDL) and chylomicron remnants, and disposal of cellular fibronectin.
Tumor necrosis factor (TNF, also known as tumor necrosis factor alpha or TNFα) is a cell signaling protein (cytokine) involved in the acute phase systemic inflammation reaction. It is produced primarily by activated macrophages, but can be produced by other cell types such as CD4+ lymphocytes, NK cells, neutrophils, mast cells, eosinophils, and neurons. The primary role of TNF is in the regulation of immune cells. TNF is an endogenous pyrogen and can induce fever, apoptotic cell death, cachexia, and inflammation, as well as inhibit tumorigenesis and viral replication and respond to sepsis via IL1- & IL6-producing cells. Dysregulation of TNF production plays a role in diseases such as, but not limited to, Alzheimer's disease, cancer, major depression, psoriasis, and inflammatory bowel disease (IBD).
An autoantibody is an antibody that is produced by the immune system and reacts with one or more of the subject's own proteins. At times, the immune system ceases to recognize one or more of the body's normal constituents as “self,” leading to production of pathological (or disease-associated) autoantibodies. These autoantibodies proceed to attack the body's own healthy cells, tissues, or organs, causing inflammation and damage. Many autoimmune diseases, such as lupus erythematosus, are caused by such autoantibodies.
Pathological autoantibodies may target a specific organ or be systemic in nature. Autoantibodies contribute to the development and perpetuation of many diseases, such as but not limited to Guillain-Barre Syndrome, Multiple Sclerosis, Myasthenia Gravis, Atypical Hemolytic Uremic Syndrome (HUS), Catastrophic Antiphospholipid Syndrome (CAPS), Systemic Lupus Erythematosus (SLE), Chronic Inflammatory Demyelinating Polyradiculoneuropathy (CIDP), Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcal Infections, and Sydenham's Chorea.
Removal of disease-associated autoantibodies has been shown to attenuate symptoms and lead to improvement in clinical outcomes. Currently, strategies for reducing antibody titers include: plasmapheresis, in which the patient's plasma is separated extracorporeally from the whole blood by centrifugation/filtration and replaced by plasma from healthy donors or albumin; and intravenous immunoglobulin (IVIG), in which antibodies are pooled from donor human plasma and injected intravenously into the patient. These approaches have their limitations and drawbacks. Challenges with plasmapheresis include high cost, inconvenience, and considerable health risks and complications (such as stroke, hypotension, infection, and hypocalcemia). Similarly, IVIG has a number of drawbacks including cost, lengthy response time, and side effects (such as allergies).
There is a need in the art for novel compounds and methods that allow for inhibition, removal, and/or degradation of TNF so as to treat, ameliorate, and/or prevent certain diseases and/or disorders in a subject. There is a need in the art for novel compounds and methods that allow for inhibition, removal, and/or degradation of certain extracellular proteins so as to treat, ameliorate, and/or prevent certain diseases and/or disorders in a subject. There is a need in the art for novel compounds and methods that allow for inhibition, removal, and/or degradation of certain autoantibodies that mediate a disease and/or disorder in a subject. The present disclosure addresses these needs.
The disclosure provides a compound comprising formula (I), or a salt, geometric isomer, stereoisomer, or solvate thereof:
[Protein binder]k′—[CON]h—[Linker]i—[CON]h′—[CRBM]j′ (I),
wherein Protein binder, CON, Linker, CRBM, k′, h, i, h′, and j′ are defined elsewhere herein.
The disclosure further provides a compound comprising formula (II), or a salt, geometric isomer, stereoisomer, or solvate thereof:
[TNF binder]k′—[CON]h—[Linker]i—[CON]h′—[CRBM]j′ (II),
wherein TNF binder, CON, Linker, CRBM, k′, h, i, h′, and j′ are defined elsewhere herein.
The disclosure further provides a compound comprising formula (III), or a salt, geometric isomer, stereoisomer, or solvate thereof:
[AATM]k′—[CON]h—[Linker]i—[CON]h′—[CRBM]j′ (III),
wherein AATM, CON, Linker, CRBM, k′, h, i, h′, and j′ are defined elsewhere herein.
The present disclosure further provides a pharmaceutical composition comprising at least one compound contemplated herein and at least one pharmaceutically acceptable excipient.
The present disclosure further provides a method of treating a disease or disorder in a subject, the method comprising administering a therapeutically effective amount of at least one compound contemplated herein.
The drawings illustrate generally, by way of example, but not by way of limitation, various embodiments of the present application.
As used herein, the term “REAG” refers to any reagent comprising -CON, -Linker, -CON-Linker, -Linker-CON, -CON-Linker-CON, -CRBM, -CON-CRBM, -Linker-CRBM, -CON-Linker-CRBM, -Linker-CON-CRBM, and/or -CON-Linker-CON-CRBM. In certain embodiments, the REAG reacts with a TNF binder group so as to incorporate the TNF binder in the compound of the disclosure, or a fragment thereof, derivative thereof, or intermediate thereto. In certain embodiments, the REAG reacts with a Protein Binder group so as to incorporate the Protein Binder in the compound of the disclosure, or a fragment thereof, derivative thereof, or intermediate thereto. In certain embodiments, the REAG reacts with an AATM group so as to incorporate the AATM in the compound of the disclosure, or a fragment thereof, derivative thereof, or intermediate thereto. In certain embodiments, the symbol indicates no-limiting positions to which the REAG and/or Protein Binder and/or AATM can be covalently attached.
The present disclosure provides, in one aspect, bifunctional compounds that can be used to promote and/or enhance degradation of an extracellular protein (or “Protein”, which may be, in a non-limiting example, a circulating protein and/or a cell surface protein, which can be attached or embedded in the cell membrane) in a subject. In certain embodiments, treatment or management of the disease and/or disorder contemplated in the disclosure requires degradation, removal, and/or reduction in concentration of the extracellular protein in the subject. Thus, in certain embodiments, administration of a compound of the disclosure to the subject removes the extracellular protein and/or reduces the circulation concentration of the extracellular protein, thus treating, ameliorating, and/or preventing the disease and/or disorder in the subject. In some embodiments, the extracellular protein comprises TNF. In some embodiments, the extracellular protein is TNF.
In certain embodiments, the compound of the disclosure comprises a group that binds to the extracellular protein. In other embodiments, the compound of the disclosure further comprises another group (such as but not limited to a small molecule) that binds to a cellular receptor, whereby the binding leads to endocytosis of the compound (and/or the extracellular protein-compound complex). The receptor binder and the extracellular protein binder can be linked via a linker such as a polyethylene glycol (PEG), any other linker as described herein with adjustable length, or other linker as described herein and containing contains one or more connector molecule(s), which are referred to herein as CON. Once the extracellular protein-compound complex undergoes endocytosis, the extracellular protein is eventually degraded, and the compound may be degraded or recycled to the outside of the cell. In some embodiments, the extracellular protein is TNF and the compound of the disclosure is a TNF binder.
The present disclosure provides, in another aspect, bifunctional compounds that can be used to promote or enhance degradation of certain autoantibodies of interest. In certain embodiments, the autoantibody mediates a disease and/or disorder in a subject, and treatment or management of the disease and/or disorder requires degradation, removal, or reduction in concentration of the autoantibody in the subject. Thus, in certain embodiments, administration of a compound of the disclosure to the subject removes the autoantibody and/or reduces the circulation concentration of the autoantibody, thus treating, ameliorating, or preventing the disease and/or disorder in the subject.
In certain embodiments, the compound of the disclosure comprises another group (such as but not limited to a small molecule) that binds to a cellular receptor, whereby the binding leads to endocytosis of the compound (and/or the extracellular protein-compound complex). Further, in certain embodiments, the compound of the disclosure comprises an autoantibody-targeting moiety (AATM), such as but not limited to a autoantibody ligand, such as but not limited to a small molecule, peptide, and/or nucleic acid aptamer, which can bind to the autoantibody of interest. The receptor binder and the AATM can be linked via a linker such as a polyethylene glycol (PEG), any other linker as described herein with adjustable length, or other linker as described herein and containing contains one or more connector molecule(s), which are referred to herein as CON. Once the autoantibody-compound complex undergoes endocytosis, the autoantibody is eventually degraded, and the compound may be degraded or recycled to the outside of the cell.
Without wishing to be limited by any theory, the bifunctional compounds of the disclosure that can be used to promote or enhance degradation of certain autoantibodies of interest have distinctive advantages over existing methods of eliminating autoantibodies from a subject. The AATM provides specificity to the bifunctional compounds. By using ATMs, one can target specific populations of autoantibodies. As shown elsewhere herein, a compound of the disclosure comprising anti-DNP IgG as the model autoantibody successfully induced degradation of anti-DNP IgG injected in mice.
The present disclosure provides a molecular approach to achieve similar goals as to plasmapheresis in diseases caused by autoantibodies. Unlike plasmapheresis, the present technology can be easily administered by various medical professionals (not just those specialized in transfusion medicine). Since the present approach is based on small molecules derived from synthetic approaches, the present disclosure circumvents the need for expensive equipment and materials and complex manufacturing practices. Compared to plasmapheresis and IVIG, the present approach is more cost-effective, safer, and accessible to patients.
Further, the present disclosure affords routes of administration that are less invasive and safer compared to extracorporeal procedures, which may introduce additional complications. The presently described compounds are modular and versatile. The targeting motifs on either ends of the linker (CRBM and AATM) can be modified to bind to various autoantibodies of interest with great specificity. Further, the defined composition of the present compounds enables simpler and consistent manufacturing practices—reducing batch to batch variability. The fact that the AATM predictably binds to the autoantibody allows for prediction of treatment outcome, drug-drug interactions, and possible side effects.
In certain embodiments, the receptor is a hepatocyte asialoglycoprotein receptor (ASGPR). In that case, the binding moiety is referred to herein as ASGPR binding moiety, or ASGPRBM. The disclosure is not limited to the receptor, but rather contemplates the use of other receptor described herein or any other endocytic receptor known in the art.
Further, the disclosure is not limited to degradation performed in hepatocytes. Rather, the disclosure contemplates that non-hepatic cells in the body display certain degradation receptors, and those receptors are contemplated within the present disclosure.
In one aspect, the compounds of the disclosure bind to an extracellular protein and/or an autoantibody and cause it to be removed from circulation in the body (and from the body) through the liver. In some embodiments, the extracellular protein is extracellular TNF. Thus, the compounds of the disclosure harness the body's own machinery for degrading proteins and/or autoantibodies. Without wishing to be limited by any theory, the compounds of the disclosure bind to certain receptors located in certain cells, such as but not limited to hepatocytes, such as but not limited to ASGPR. Such binding triggers degradation of protein targets via endolysosomal proteolysis. As a consequence of this mechanism, there is a lowering in the circulating levels of the extracellular protein target and/or extracellular autoantibody. In some embodiments, the extracellular protein target is TNF. As a result, the corresponding disease symptoms are attenuated and/or eliminated from the subject administered the present compounds.
The ASPGR has the function of clearing desialylated glycoproteins with exposed non-reducing D-galactose (Gal) or N-acetylgalactosamine (GalNac) as end groups. ASGPR is expressed at a level of about 500,000 per hepatocyte, and has minimal existence elsewhere in the body. Internalization of the target glycoproteins by the ASGPR has a half-life of about 3 min. The disclosed bifunctional compounds selectively bind to the extracellular protein through the compound's extracellular protein binder moiety, thus forming a protein complex. When this protein complex reaches the liver, the asialoglycoprotein receptor binding moiety (ASGPRBM) of the molecule engages the end-lysosomal pathway of hepatocytes through the ASGPR. Endosomal bound ASPGR releases the extracellular protein ligand at pH 5.4, and the ligand is eliminated from circulation by the hepatocytes. However, the ASPGR remains available for recycling; it is spared from lysosomal degradation and buds into recycling endosomes. Indeed, it can be recycled up to about 200 times with a recycling rate of about 15-20 minutes, depending on the cell line. ASPGR has a very promiscuous ligand size requirement, most likely reaching diameters of about 70 nm. For comparison, the IgM pentamer is approximately 20 nm in diameter, and thus meets the ASPGR's ligand size requirement.
The disclosures of the International Patent Applications No. PCT/US2019/026260, filed Apr. 8, 2019 (and published as WO 2019/199634 on Oct. 17, 2019), and No. PCT/US2019/026239, filed Apr. 8, 2019 (and published as WO 2019/199621 on Oct. 17, 2019), are incorporated herein in their entireties by reference.
In accordance with the present disclosure, conventional chemical synthetic and pharmaceutical formulation methods, as well as pharmacology, molecular biology, microbiology, and recombinant DNA techniques within the skill of the art may be employed. Such techniques are well-known and are otherwise explained fully in the literature.
Reference will now be made in detail to certain embodiments of the disclosed subject matter, examples of which are illustrated in part in the accompanying drawings. While the disclosed subject matter will be described in conjunction with the enumerated claims, it will be understood that the exemplified subject matter is not intended to limit the claims to the disclosed subject matter.
Throughout this document, values expressed in a range format should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. For example, a range of “about 0.1% to about 5%” or “about 0.1% to 5%” should be interpreted to include not just about 0.1% to about 5%, but also the individual values (e.g., 1%, 2%, 3%, and 4%) and the sub-ranges (e.g., 0.1% to 0.5%, 1.1% to 2.2%, 3.3% to 4.4%) within the indicated range. The statement “about X to Y” has the same meaning as “about X to about Y,” unless indicated otherwise. Likewise, the statement “about X, Y, or about Z” has the same meaning as “about X, about Y, or about Z,” unless indicated otherwise.
In the methods described herein, the acts can be carried out in any order, except when a temporal or operational sequence is explicitly recited. Furthermore, specified acts can be carried out concurrently unless explicit claim language recites that they be carried out separately. For example, a claimed act of doing X and a claimed act of doing Y can be conducted simultaneously within a single operation, and the resulting process will fall within the literal scope of the claimed process.
The term “about” as used herein can allow for a degree of variability in a value or range, for example, within 10%, within 5%, or within 1% of a stated value or of a stated limit of a range, and includes the exact stated value or range.
In this document, the terms “a,” “an,” or “the” are used to include one or more than one unless the context clearly dictates otherwise. The term “or” is used to refer to a nonexclusive “or” unless otherwise indicated. The statement “at least one of A and B” or “at least one of A or B” has the same meaning as “A, B, or A and B.” In addition, it is to be understood that the phraseology or terminology employed herein, and not otherwise defined, is for the purpose of description only and not of limitation. Any use of section headings is intended to aid reading of the document and is not to be interpreted as limiting; information that is relevant to a section heading may occur within or outside of that particular section. All publications, patents, and patent documents referred to in this document are incorporated by reference herein in their entirety, as though individually incorporated by reference.
The term “abnormal” when used in the context of organisms, tissues, cells or components thereof, refers to those organisms, tissues, cells or components thereof that differ in at least one observable or detectable characteristic (e.g., age, treatment, time of day, and so forth) from those organisms, tissues, cells or components thereof that display the “normal” (expected) respective characteristic. Characteristics that are normal or expected for one cell or tissue type, might be abnormal for a different cell or tissue type.
The term “acyl” as used herein refers to a group containing a carbonyl moiety wherein the group is bonded via the carbonyl carbon atom. The carbonyl carbon atom is bonded to a hydrogen forming a “formyl” group or is bonded to another carbon atom, which can be part of an alkyl, aryl, aralkyl cycloalkyl, cycloalkylalkyl, heterocyclyl, heterocyclylalkyl, heteroaryl, heteroarylalkyl group or the like. An acyl group can include 0 to about 12, 0 to about 20, or 0 to about 40 additional carbon atoms bonded to the carbonyl group. An acyl group can include double or triple bonds within the meaning herein. An acryloyl group is an example of an acyl group. An acyl group can also include heteroatoms within the meaning herein. A nicotinoyl group (pyridyl-3-carbonyl) is an example of an acyl group within the meaning herein. Other examples include acetyl, benzoyl, phenylacetyl, pyridylacetyl, cinnamoyl, and acryloyl groups and the like. When the group containing the carbon atom that is bonded to the carbonyl carbon atom contains a halogen, the group is termed a “haloacyl” group. An example is a trifluoroacetyl group.
The term “alkyl” as used herein refers to straight chain and branched alkyl groups and cycloalkyl groups having from 1 to 40 carbon atoms, 1 to about 20 carbon atoms, 1 to 12 carbons or, in some embodiments, from 1 to 8 carbon atoms. Examples of straight chain alkyl groups include those with from 1 to 8 carbon atoms such as methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl, and n-octyl groups. Examples of branched alkyl groups include, but are not limited to, isopropyl, iso-butyl, sec-butyl, t-butyl, neopentyl, isopentyl, and 2,2-dimethylpropyl groups. As used herein, the term “alkyl” encompasses n-alkyl, isoalkyl, and anteisoalkyl groups as well as other branched chain forms of alkyl. Representative substituted alkyl groups can be substituted one or more times with any of the groups listed herein, for example, amino, hydroxy, cyano, carboxy, nitro, thio, alkoxy, and halogen groups.
The term “alkenyl” as used herein refers to straight and branched chain and cyclic alkyl groups as defined herein, except that at least one double bond exists between two carbon atoms. Thus, alkenyl groups have from 2 to 40 carbon atoms, or 2 to about 20 carbon atoms, or 2 to 12 carbon atoms or, in some embodiments, from 2 to 8 carbon atoms. Examples include, but are not limited to vinyl, —CH═C═CCH2, —CH═CH(CH3), —CH═C(CH3)2, —C(CH3)═CH2, —C(CH3)═CH(CH3), —C(CH2CH3)═CH2, cyclohexenyl, cyclopentenyl, cyclohexadienyl, butadienyl, pentadienyl, and hexadienyl among others.
The term “alkoxy” as used herein refers to an oxygen atom connected to an alkyl group, including a cycloalkyl group, as are defined herein. Examples of linear alkoxy groups include but are not limited to methoxy, ethoxy, propoxy, butoxy, pentyloxy, hexyloxy, and the like. Examples of branched alkoxy include but are not limited to isopropoxy, sec-butoxy, tert-butoxy, isopentyloxy, isohexyloxy, and the like. Examples of cyclic alkoxy include but are not limited to cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, cyclohexyloxy, and the like. An alkoxy group can include about 1 to about 12, about 1 to about 20, or about 1 to about 40 carbon atoms bonded to the oxygen atom, and can further include double or triple bonds, and can also include heteroatoms. For example, an allyloxy group or a methoxyethoxy group is also an alkoxy group within the meaning herein, as is a methylenedioxy group in a context where two adjacent atoms of a structure are substituted therewith.
The term “alkynyl” as used herein refers to straight and branched chain alkyl groups, except that at least one triple bond exists between two carbon atoms. Thus, alkynyl groups have from 2 to 40 carbon atoms, 2 to about 20 carbon atoms, or from 2 to 12 carbons or, in some embodiments, from 2 to 8 carbon atoms. Examples include, but are not limited to —C≡CH, —C≡C(CH3), —C≡C(CH2CH3), —CH2C≡CH, —CH2C≡C(CH3), and —CH2C≡C(CH2CH3) among others.
The term “amine” as used herein refers to primary, secondary, and tertiary amines having, e.g., the formula N(group)3 wherein each group can independently be H or non-H, such as alkyl, aryl, and the like. Amines include but are not limited to R—NH2, for example, alkylamines, arylamines, alkylarylamines; R2NH wherein each R is independently selected, such as dialkylamines, diarylamines, aralkylamines, heterocyclylamines and the like; and R3N wherein each R is independently selected, such as trialkylamines, dialkylarylamines, alkyldiarylamines, triarylamines, and the like. The term “amine” also includes ammonium ions as used herein.
The term “amino acid sequence variant” refers to polypeptides having amino acid sequences that differ to some extent from a native sequence polypeptide. Ordinarily, amino acid sequence variants possess at least about 70% homology, at least about 80% homology, at least about 90% homology, or at least about 95% homology to the native polypeptide. The amino acid sequence variants possess substitutions, deletions, and/or insertions at certain positions within the amino acid sequence of the native amino acid sequence.
The term “amino group” as used herein refers to a substituent of the form —NH2, —NHR, —NR2, —NR3+, wherein each R is independently selected, and protonated forms of each, except for —NR3+, which cannot be protonated. Accordingly, any compound substituted with an amino group can be viewed as an amine. An “amino group” within the meaning herein can be a primary, secondary, tertiary, or quaternary amino group. An “alkylamino” group includes a monoalkylamino, dialkylamino, and trialkylamino group.
The term “aminoalkyl” as used herein refers to amine connected to an alkyl group, as defined herein. The amine group can appear at any suitable position in the alkyl chain, such as at the terminus of the alkyl chain or anywhere within the alkyl chain.
The term “aralkyl” as used herein refers to alkyl groups as defined herein in which a hydrogen or carbon bond of an alkyl group is replaced with a bond to an aryl group as defined herein. Representative aralkyl groups include benzyl and phenylethyl groups and fused (cycloalkylaryl)alkyl groups such as 4-ethyl-indanyl. Aralkenyl groups are alkenyl groups as defined herein in which a hydrogen or carbon bond of an alkyl group is replaced with a bond to an aryl group as defined herein.
The term “aryl” as used herein refers to cyclic aromatic hydrocarbon groups that do not contain heteroatoms in the ring. Thus aryl groups include, but are not limited to, phenyl, azulenyl, heptalenyl, biphenyl, indacenyl, fluorenyl, phenanthrenyl, triphenylenyl, pyrenyl, naphthacenyl, chrysenyl, biphenylenyl, anthracenyl, and naphthyl groups. In some embodiments, aryl groups contain about 6 to about 14 carbons in the ring portions of the groups. Aryl groups can be unsubstituted or substituted, as defined herein. Representative substituted aryl groups can be mono-substituted or substituted more than once, such as, but not limited to, a phenyl group substituted at any one or more of 2-, 3-, 4-, 5-, or 6-positions of the phenyl ring, or a naphthyl group substituted at any one or more of 2- to 8-positions thereof.
The term “antibody,” as used herein, refers to an immunoglobulin molecule that specifically binds with an antigen. Antibodies can be intact immunoglobulins derived from natural sources or from recombinant sources, and can be immunoreactive portions of intact immunoglobulins. Antibodies are typically tetramers of immunoglobulin molecules. The antibodies in the present disclosure may exist in a variety of forms including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab and F(ab)2, as well as single chain antibodies and humanized antibodies (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, N.Y.; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426).
The term “antibody fragment” refers to a portion of an intact antibody and refers to the antigenic determining variable regions of an intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab′, F(ab′)2, and Fv fragments, linear antibodies, scFv antibodies, single-domain antibodies such as sdAb (either VL or VH), such as camelid antibodies (Riechmann, 1999, J. Immunol. Meth. 231:25-38), camelid VHH domains, composed of either a VL or a VH domain that exhibit sufficient affinity for the target, and multispecific 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 complementarity-determining region (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 & Hudson, 2005, Nature Biotech. 23:1126-1136). Antigen binding fragments can also be grafted into scaffolds based on polypeptides such as a fibronectin type III (Fn3) (U.S. Pat. No. 6,703,199, which describes fibronectin polypeptide minibodies). The antibody fragment also includes a human antibody or a humanized antibody or a portion of a human antibody or a humanized antibody.
The term “antigen” or “Ag” as used herein is defined as 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. Furthermore, antigens can be derived from recombinant or genomic DNA. A skilled artisan will understand that any DNA, which comprises a nucleotide sequence or a partial nucleotide sequence encoding a protein that elicits an immune response therefore encodes an “antigen” as that term is used herein. Furthermore, one skilled in the art will understand that an antigen need not be encoded solely by a full-length nucleotide sequence of a gene. It is readily apparent that the present disclosure includes, but is not limited to, the use of partial nucleotide sequences of more than one gene and that these nucleotide sequences are arranged in various combinations to elicit the desired immune response. Moreover, a skilled artisan will understand that an antigen need not be encoded by a “gene” at all. It is readily apparent that an antigen can be generated synthesized or can be derived from a biological sample. Such a biological sample can include, but is not limited to a tissue sample, a tumor sample, a cell or a biological fluid.
As used herein, “aptamer” refers to a small molecule that can bind specifically to another molecule. Aptamers are typically either polynucleotide- or peptide-based molecules. A polynucleotidal aptamer is a DNA or RNA molecule, usually comprising several strands of nucleic acids, that adopt highly specific three-dimensional conformation designed to have appropriate binding affinities and specificities towards specific target molecules, such as peptides, proteins, drugs, vitamins, among other organic and inorganic molecules. Such polynucleotidal aptamers can be selected from a vast population of random sequences through the use of systematic evolution of ligands by exponential enrichment. A peptide aptamer is typically a loop of about 10 to about 20 amino acids attached to a protein scaffold that bind to specific ligands. Peptide aptamers may be identified and isolated from combinatorial libraries, using methods such as the yeast two-hybrid system.
As used herein, the term “asialoglycoprotein receptor binding moiety” or “ASGPRBM” refers to a group that is capable of binding to at least one hepatocyte asialoglycoprotein receptor on the surface of a cell, such as but not limited to hepatocytes. Once the ASGPRBM, and any additional moiety to which it is attached, binds to the receptor on the surface of hepatocyte, the molecule comprising the ASGPRBM is taken into the hepatocyte via a phagocytosis mechanism wherein the molecule is at least partially degraded through lysosomal degradation.
As used herein, the term “C6-10-C6-10 biaryl” means a C6-10 aryl moiety covalently bonded through a single bond to another C6-10 aryl moiety. The C6-10 aryl moiety can be any of the suitable aryl groups described herein. Non-limiting example of a C6-10-C6-10 biaryl include biphenyl and binaphthyl.
The term “coding sequence,” as used herein, means a sequence of a nucleic acid or its complement, or a part thereof, that can be transcribed and/or translated to produce the mRNA and/or the polypeptide or a fragment thereof. Coding sequences include exons in a genomic DNA or immature primary RNA transcripts, which are joined together by the cell's biochemical machinery to provide a mature mRNA. The anti-sense strand is the complement of such a nucleic acid, and the coding sequence can be deduced therefrom. In contrast, the term “non-coding sequence,” as used herein, means a sequence of a nucleic acid or its complement, or a part thereof, that is not translated into amino acid in vivo, or where tRNA does not interact to place or attempt to place an amino acid. Non-coding sequences include both intron sequences in genomic DNA or immature primary RNA transcripts, and gene-associated sequences such as promoters, enhancers, silencers, and the like.
As used herein, the terms “complementary” or “complementarity” are used in reference to polynucleotides (i.e., a sequence of nucleotides) related by the base-pairing rules. For example, the sequence “A-G-T,” is complementary to the sequence “T-C-A.” Complementarity may be “partial,” in which only some of the nucleic acids' bases are matched according to the base pairing rules. Or, there may be “complete” or “total” complementarity between the nucleic acids. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands. This is of particular importance in amplification reactions, as well as detection methods that depend upon binding between nucleic acids.
As used herein, the term “composition” or “pharmaceutical composition” refers to a mixture of at least one compound described herein with a pharmaceutically acceptable carrier. The pharmaceutical composition facilitates administration of the compound to a patient or subject. Multiple techniques of administering a compound exist in the art including, but not limited to, intravenous, oral, aerosol, parenteral, ophthalmic, pulmonary and topical administration.
As used herein, the terms “conservative variation” or “conservative substitution” as used herein refers to the replacement of an amino acid residue by another, biologically similar residue. Conservative variations or substitutions are not likely to change the shape of the peptide chain. Examples of conservative variations, or substitutions, include the replacement of one hydrophobic residue such as isoleucine, valine, leucine or methionine for another, or the substitution of one polar residue for another, such as the substitution of arginine for lysine, glutamic for aspartic acid, or glutamine for asparagine.
The term “cycloalkyl” as used herein refers to cyclic alkyl groups such as, but not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl groups. In some embodiments, the cycloalkyl group can have 3 to about 8-12 ring members, whereas in other embodiments the number of ring carbon atoms range from 3 to 4, 5, 6, or 7. Cycloalkyl groups further include polycyclic cycloalkyl groups such as, but not limited to, norbornyl, adamantyl, bornyl, camphenyl, isocamphenyl, and carenyl groups, and fused rings such as, but not limited to, decalinyl, and the like. Cycloalkyl groups also include rings that are substituted with straight or branched chain alkyl groups as defined herein. Representative substituted cycloalkyl groups can be mono-substituted or substituted more than once, such as, but not limited to, 2,2-, 2,3-, 2,4-2,5- or 2,6-disubstituted cyclohexyl groups or mono-, di- or tri-substituted norbornyl or cycloheptyl groups, which can be substituted with, for example, amino, hydroxy, cyano, carboxy, nitro, thio, alkoxy, and halogen groups. The term “cycloalkenyl” alone or in combination denotes a cyclic alkenyl group.
A “disease” is a state of health of an animal wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal's health continues to deteriorate.
In contrast, a “disorder” in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal's state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the animal's state of health.
A disease or disorder is “alleviated” if the severity of a symptom of the disease or disorder, the frequency with which such a symptom is experienced by a patient, or both, is reduced.
As used herein, the terms “effective amount,” “pharmaceutically effective amount” and “therapeutically effective amount” refer to a nontoxic but sufficient amount of an agent to provide the desired biological result. That result may be reduction and/or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. An appropriate therapeutic amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation.
As used herein, the term “efficacy” refers to the maximal effect (Emax) achieved within an assay.
“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 (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene 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.
As used herein, the term “fragment,” as applied to a nucleic acid, refers to a subsequence of a larger nucleic acid. A “fragment” of a nucleic acid can be at least about 15 nucleotides in length; for example, at least about 50 nucleotides to about 100 nucleotides; at least about 100 to about 500 nucleotides, at least about 500 to about 1000 nucleotides; at least about 1000 nucleotides to about 1500 nucleotides; about 1500 nucleotides to about 2500 nucleotides; or about 2500 nucleotides (and any integer value in between). As used herein, the term “fragment,” as applied to a protein or peptide, refers to a subsequence of a larger protein or peptide. A “fragment” of a protein or peptide can be at least about 20 amino acids in length; for example, at least about 50 amino acids in length; at least about 100 amino acids in length; at least about 200 amino acids in length; at least about 300 amino acids in length; or at least about 400 amino acids in length (and any integer value in between).
As used herein, the term “GN3” refers to the group
The terms “halo,” “halogen,” or “halide” group, as used herein, by themselves or as part of another substituent, mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom.
The term “haloalkyl” group, as used herein, includes mono-halo alkyl groups, poly-halo alkyl groups wherein all halo atoms can be the same or different, and per-halo alkyl groups, wherein all hydrogen atoms are replaced by halogen atoms, such as fluoro. Examples of haloalkyl include trifluoromethyl, 1,1-dichloroethyl, 1,2-dichloroethyl, 1,3-dibromo-3,3-difluoropropyl, perfluorobutyl, and the like.
The term “heteroaryl” as used herein refers to aromatic ring compounds containing 5 or more ring members, of which, one or more is a heteroatom such as, but not limited to, N, O, and S; for instance, heteroaryl rings can have 5 to about 8-12 ring members. A heteroaryl group is a variety of a heterocyclyl group that possesses an aromatic electronic structure. A heteroaryl group designated as a C2-heteroaryl can be a 5-ring with two carbon atoms and three heteroatoms, a 6-ring with two carbon atoms and four heteroatoms and so forth. Likewise a C4-heteroaryl can be a 5-ring with one heteroatom, a 6-ring with two heteroatoms, and so forth. The number of carbon atoms plus the number of heteroatoms sums up to equal the total number of ring atoms. Heteroaryl groups include, but are not limited to, groups such as pyrrolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiazolyl, pyridinyl, thiophenyl, benzothiophenyl, benzofuranyl, indolyl, azaindolyl, indazolyl, benzimidazolyl, azabenzimidazolyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, imidazopyridinyl, isoxazolopyridinyl, thianaphthalenyl, purinyl, xanthinyl, adeninyl, guaninyl, quinolinyl, isoquinolinyl, tetrahydroquinolinyl, quinoxalinyl, and quinazolinyl groups. Heteroaryl groups can be unsubstituted, or can be substituted with groups as is discussed herein. Representative substituted heteroaryl groups can be substituted one or more times with groups such as those listed herein.
Additional examples of aryl and heteroaryl groups include but are not limited to phenyl, biphenyl, indenyl, naphthyl (1-naphthyl, 2-naphthyl), N-hydroxytetrazolyl, N-hydroxytriazolyl, N-hydroxyimidazolyl, anthracenyl (1-anthracenyl, 2-anthracenyl, 3-anthracenyl), thiophenyl (2-thienyl, 3-thienyl), furyl (2-furyl, 3-furyl) , indolyl, oxadiazolyl, isoxazolyl, quinazolinyl, fluorenyl, xanthenyl, isoindanyl, benzhydryl, acridinyl, thiazolyl, pyrrolyl (2-pyrrolyl), pyrazolyl (3-pyrazolyl), imidazolyl (1-imidazolyl, 2-imidazolyl, 4-imidazolyl, 5-imidazolyl), triazolyl (1,2,3-triazol-1-yl, 1,2,3-triazol-2-yl 1,2,3-triazol-4-yl, 1,2,4-triazol-3-yl), oxazolyl (2-oxazolyl, 4-oxazolyl, 5-oxazolyl), thiazolyl (2-thiazolyl, 4-thiazolyl, 5-thiazolyl), pyridyl (2-pyridyl, 3-pyridyl, 4-pyridyl), pyrimidinyl (2-pyrimidinyl, 4-pyrimidinyl, 5-pyrimidinyl, 6-pyrimidinyl), pyrazinyl, pyridazinyl (3-pyridazinyl, 4-pyridazinyl, 5-pyridazinyl), quinolyl (2-quinolyl, 3-quinolyl, 4-quinolyl, 5-quinolyl, 6-quinolyl, 7-quinolyl, 8-quinolyl), isoquinolyl (1-isoquinolyl, 3-isoquinolyl, 4-isoquinolyl, 5-isoquinolyl, 6-isoquinolyl, 7-isoquinolyl, 8-isoquinolyl), benzo[b]furanyl (2-benzo[b]furanyl, 3-benzo[b]furanyl, 4-benzo[b]furanyl, 5-benzo[b]furanyl, 6-benzo[b]furanyl, 7-benzo[b]furanyl), 2,3-dihydro-benzo[b]furanyl (2-(2,3-dihydro-benzo[b]furanyl), 3-(2,3-dihydro-benzo[b]furanyl), 4-(2,3-dihydro-benzo[b]furanyl), 5-(2,3-dihydro-benzo[b]furanyl), 6-(2,3-dihydro-benzo[b]furanyl), 7-(2,3-dihydro-benzo[b]furanyl), benzo[b]thiophenyl (2-benzo[b]thiophenyl, 3-benzo[b]thiophenyl, 4-benzo[b]thiophenyl, 5-benzo[b]thiophenyl, 6-benzo[b]thiophenyl, 7-benzo[b]thiophenyl), 2,3-dihydro-benzo[b]thiophenyl, (2-(2,3-dihydro-benzo[b]thiophenyl), 3-(2,3-dihydro-benzo[b]thiophenyl), 4-(2,3-dihydro-benzo[b]thiophenyl), 5-(2,3-dihydro-benzo[b]thiophenyl), 6-(2,3-dihydro-benzo[b]thiophenyl), 7-(2,3-dihydro-benzo[b]thiophenyl), indolyl (1-indolyl, 2-indolyl, 3-indolyl, 4-indolyl, 5-indolyl, 6-indolyl, 7-indolyl), indazole (1-indazolyl, 3-indazolyl, 4-indazolyl, 5-indazolyl, 6-indazolyl, 7-indazolyl), benzimidazolyl (1-benzimidazolyl, 2-benzimidazolyl, 4-benzimidazolyl, 5-benzimidazolyl, 6-benzimidazolyl, 7-benzimidazolyl, 8-benzimidazolyl), benzoxazolyl (1-benzoxazolyl, 2-benzoxazolyl), benzothiazolyl (1-benzothiazolyl, 2-benzothiazolyl, 4-benzothiazolyl, 5-benzothiazolyl, 6-benzothiazolyl, 7-benzothiazolyl), carbazolyl (1-carbazolyl, 2-carbazolyl, 3-carbazolyl, 4-carbazolyl), 5H-dibenz[b,f]azepine (5H-dibenz[b,f]azepin-1-yl, 5H-dibenz[b,f]azepine-2-yl, 5H-dibenz[b,f]azepine-3-yl, 5H-dibenz[b,f]azepine-4-yl, 5H-dibenz[b,f]azepine-5-yl), 10,11-dihydro-5H-dibenz[b,f]azepine (10,11-dihydro-5H-dibenz[b,f]azepine-1-yl, 10,11-dihydro-5H-dibenz[b,f]azepine-2-yl, 10,11-dihydro-5H-dibenz[b,f]azepine-3-yl, 10,11-dihydro-5H-dibenz[b,f]azepine-4-yl, 10,11-dihydro-5H-dibenz[b,f]azepine-5-yl), and the like.
The term “heteroarylalkyl” as used herein refers to alkyl groups as defined herein in which a hydrogen or carbon bond of an alkyl group is replaced with a bond to a heteroaryl group as defined herein.
As used herein, the term “C6-10-5-6 membered heterobiaryl” means a C6-10 aryl moiety covalently bonded through a single bond to a 5- or 6-membered heteroaryl moiety. The C6-10 aryl moiety and the 5-6-membered heteroaryl moiety can be any of the suitable aryl and heteroaryl groups described herein. Non-limiting examples of a C6-10-5-6 membered heterobiaryl include:
When the C6-10-5-6 membered heterobiaryl is listed as a substituent (e.g., as an “R” group), the C6-10-5-6 membered heterobiaryl is bonded to the rest of the molecule through the C6-10 moiety.
As used herein, the term “5-6 membered-C6-10 heterobiaryl ” is the same as a C6-10-5-6 membered heterobiaryl, except that when the 5-6 membered-C6-10 heterobiaryl is listed as a substituent (e.g., as an “R” group), the 5-6 membered-C6-10 heterobiaryl is bonded to the rest of the molecule through the 5-6-membered heteroaryl moiety.
The term “heterocyclyl” as used herein refers to aromatic and non-aromatic ring compounds containing three or more ring members, of which one or more is a heteroatom such as, but not limited to, N, O, and S. Thus, a heterocyclyl can be a cycloheteroalkyl, or a heteroaryl, or if polycyclic, any combination thereof. In some embodiments, heterocyclyl groups include 3 to about 20 ring members, whereas other such groups have 3 to about 15 ring members. A heterocyclyl group designated as a C2-heterocyclyl can be a 5-ring with two carbon atoms and three heteroatoms, a 6-ring with two carbon atoms and four heteroatoms and so forth. Likewise a C4-heterocyclyl can be a 5-ring with one heteroatom, a 6-ring with two heteroatoms, and so forth. The number of carbon atoms plus the number of heteroatoms equals the total number of ring atoms. A heterocyclyl ring can also include one or more double bonds. A heteroaryl ring is an embodiment of a heterocyclyl group. The phrase “heterocyclyl group” includes fused ring species including those that include fused aromatic and non-aromatic groups. For example, a dioxolanyl ring and a benzdioxolanyl ring system (methylenedioxyphenyl ring system) are both heterocyclyl groups within the meaning herein. The phrase also includes polycyclic ring systems containing a heteroatom such as, but not limited to, quinuclidyl. Heterocyclyl groups can be unsubstituted, or can be substituted as discussed herein. Heterocyclyl groups include, but are not limited to, pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, pyrrolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiazolyl, pyridinyl, thiophenyl, benzothiophenyl, benzofuranyl, dihydrobenzofuranyl, indolyl, dihydroindolyl, azaindolyl, indazolyl, benzimidazolyl, azabenzimidazolyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, imidazopyridinyl, isoxazolopyridinyl, thianaphthalenyl, purinyl, xanthinyl, adeninyl, guaninyl, quinolinyl, isoquinolinyl, tetrahydroquinolinyl, quinoxalinyl, and quinazolinyl groups. Representative substituted heterocyclyl groups can be mono-substituted or substituted more than once, such as, but not limited to, piperidinyl or quinolinyl groups, which are 2-, 3-, 4-, 5-, or 6-substituted, or disubstituted with groups such as those listed herein.
The term “heterocyclylalkyl” as used herein refers to alkyl groups as defined herein in which a hydrogen or carbon bond of an alkyl group as defined herein is replaced with a bond to a heterocyclyl group as defined herein. Representative heterocyclyl alkyl groups include, but are not limited to, furan-2-yl methyl, furan-3-yl methyl, pyridine-3-yl methyl, tetrahydrofuran-2-yl ethyl, and indol-2-yl propyl.
The term “independently selected from” as used herein refers to referenced groups being the same, different, or a mixture thereof, unless the context clearly indicates otherwise. Thus, under this definition, the phrase “X1, X2, and X3 are independently selected from noble gases” would include the scenario where, for example, X1, X2, and X3 are all the same, wherein X1, X2, and X3 are all different, wherein X1 and X2 are the same but X3 is different, and other analogous permutations.
The term “immunoglobulin” or “Ig” as used herein is defined as a class of proteins, which function as antibodies. Antibodies expressed by B cells are sometimes referred to as the BCR (B cell receptor) or antigen receptor. The five members included in this class of proteins are IgA, IgG, IgM, IgD, and IgE. IgA is the primary antibody that is present in body secretions, such as saliva, tears, breast milk, gastrointestinal secretions, and mucus secretions of the respiratory and genitourinary tracts. IgG is the most common circulating antibody. IgM is the main immunoglobulin produced in the primary immune response in most subjects. It is the most efficient immunoglobulin in agglutination, complement fixation, and other antibody responses, and is important in defense against bacteria and viruses. IgD is the immunoglobulin that has no known antibody function, but may serve as an antigen receptor. IgE is the immunoglobulin that mediates immediate hypersensitivity by causing release of mediators from mast cells and basophils upon exposure to allergen.
“Isolated” means altered or removed from the natural state. For example, a nucleic acid or a polypeptide naturally present in a living animal is not “isolated,” but the same nucleic acid or polypeptide partially or completely separated from the coexisting materials of its natural state is “isolated.” An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.
By the term “modulating,” as used herein, is meant mediating a detectable increase or decrease in the activity and/or level of a mRNA, polypeptide, or a response in a subject compared with the activity and/or level of a mRNA, polypeptide or a response in the subject in the absence of a treatment or compound, and/or compared with the activity and/or level of a mRNA, polypeptide, or a response in an otherwise identical but untreated subject. The term encompasses activating, inhibiting and/or otherwise affecting a native signal or response thereby mediating a beneficial therapeutic response in a subject, preferably, a human.
The term “monovalent” as used herein refers to a substituent connecting via a single bond to a substituted molecule. When a substituent is monovalent, such as, for example, F or Cl, it is bonded to the atom it is substituting by a single bond.
The term “organic group” as used herein refers to any carbon-containing functional group. Examples can include an oxygen-containing group such as an alkoxy group, aryloxy group, aralkyloxy group, oxo(carbonyl) group; a carboxyl group including a carboxylic acid, carboxylate, and a carboxylate ester; a sulfur-containing group such as an alkyl and aryl sulfide group; and other heteroatom-containing groups. Non-limiting examples of organic groups include OR, OOR, OC(O)N(R)2, CN, CF3, OCF3, R, C(O), methylenedioxy, ethylenedioxy, N(R)2, SR, SOR, SO2R, SO2N(R)2, SO3R, C(O)R, C(O)C(O)R, C(O)CH2C(O)R, C(S)R, C(O)OR, OC(O)R, C(O)N(R)2, OC(O)N(R)2, C(S)N(R)2, (CH2)0-2N(R)C(O)R, (CH2)0-2N(R)N(R)2, N(R)N(R)C(O)R, N(R)N(R)C(O)OR, N(R)N(R)CON(R)2, N(R)SO2R, N(R)SO2N(R)2, N(R)C(O)OR, N(R)C(O)R, N(R)C(S)R, N(R)C(O)N(R)2, N(R)C(S)N(R)2, N(COR)COR, N(OR)R, C(═NH)N(R)2, C(O)N(OR)R, C(═NOR)R, and substituted or unsubstituted (C1-C100)hydrocarbyl, wherein R can be hydrogen (in examples that include other carbon atoms) or a carbon-based moiety, and wherein the carbon-based moiety can be substituted or unsubstituted.
The terms “patient,” “subject,” or “individual” are used interchangeably herein, and refer to any animal, or cells thereof whether in vitro or in situ, amenable to the methods described herein. In a non-limiting embodiment, the patient, subject or individual is a human.
As used herein, the term “pharmaceutically acceptable” refers to a material, such as a carrier or diluent, which does not abrogate the biological activity or properties of the compound, and is relatively non-toxic, i.e., the material may be administered to an individual without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.
As used herein, the language “pharmaceutically acceptable salt” refers to a salt of the administered compounds prepared from pharmaceutically acceptable non-toxic acids or bases, including inorganic acids or bases, organic acids or bases, solvates, hydrates, or clathrates thereof.
Suitable pharmaceutically acceptable acid addition salts may be prepared from an inorganic acid or from an organic acid. Examples of inorganic acids include hydrochloric, hydrobromic, hydriodic, nitric, carbonic, sulfuric (including sulfate and hydrogen sulfate), and phosphoric acids (including hydrogen phosphate and dihydrogen phosphate). Appropriate organic acids may be selected from aliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic, carboxylic and sulfonic classes of organic acids, examples of which include formic, acetic, propionic, succinic, glycolic, gluconic, lactic, malic, tartaric, citric, ascorbic, glucuronic, maleic, malonic, saccharin, fumaric, pyruvic, aspartic, glutamic, benzoic, anthranilic, 4-hydroxybenzoic, phenylacetic, mandelic, embonic (pamoic), methanesulfonic, ethanesulfonic, benzenesulfonic, pantothenic, trifluoromethanesulfonic, 2-hydroxyethanesulfonic, p-toluenesulfonic, sulfanilic, cyclohexylaminosulfonic, stearic, alginic, β-hydroxybutyric, salicylic, galactaric and galacturonic acid.
Suitable pharmaceutically acceptable base addition salts of compounds described herein include, for example, ammonium salts, metallic salts including alkali metal, alkaline earth metal and transition metal salts such as, for example, calcium, magnesium, potassium, sodium and zinc salts. Pharmaceutically acceptable base addition salts also include organic salts made from basic amines such as, for example, N,N′-dibenzylethylene-diamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine) and procaine. All of these salts may be prepared from the corresponding compound by reacting, for example, the appropriate acid or base with the compound.
As used herein, the term “pharmaceutically acceptable carrier” or “pharmaceutically acceptable excipient” means a pharmaceutically acceptable material, composition or carrier, such as a liquid or solid filler, stabilizer, dispersing agent, suspending agent, diluent, excipient, thickening agent, solvent or encapsulating material, involved in carrying or transporting a compound described herein within or to the patient such that it may perform its intended function. Typically, such compounds are carried or transported from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation, including the compound(s) described herein, and not injurious to the patient. Some examples of materials that may serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; surface active agents; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer solutions; and other non-toxic compatible substances employed in pharmaceutical formulations. As used herein, “pharmaceutically acceptable carrier” also includes any and all coatings, antibacterial and antifungal agents, and absorption delaying agents, and the like that are compatible with the activity of the compound(s) described herein, and are physiologically acceptable to the patient. Supplementary active compounds may also be incorporated into the compositions. The “pharmaceutically acceptable carrier” may further include a pharmaceutically acceptable salt of the compound(s) described herein. Other additional ingredients that may be included in the pharmaceutical compositions used with the methods or compounds described herein are known in the art and described, for example in Remington's Pharmaceutical Sciences (Genaro, Ed., Mack Publishing Co., 1985, Easton, Pa.), which is incorporated herein by reference.
As used herein, the term “polypeptide” refers to a polymer composed of amino acid residues, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof linked via peptide bonds. Synthetic polypeptides may be synthesized, for example, using an automated polypeptide synthesizer. As used herein, the term “protein” typically refers to large polypeptides. As used herein, the term “peptide” typically refers to short polypeptides. Conventional notation is used herein to represent polypeptide sequences: the left-hand end of a polypeptide sequence is the amino-terminus, and the right-hand end of a polypeptide sequence is the carboxyl-terminus.
As used herein, the term “potency” refers to the dose needed to produce half the maximal response (ED50).
As used herein, the term “Protein” refers to an extracellular protein of interest.
As used herein, the term “REAG” refers to any reagent comprising -CON, -Linker, -CON-Linker, -Linker-CON, -CON-Linker-CON, -CRBM, -CON-CRBM, -Linker-CRBM, -CON-Linker-CRBM, -Linker-CON-CRBM, and/or -CON-Linker-CON-CRBM. In certain embodiments, the REAG reacts with a TNF binder group so as to incorporate the TNF binder in the compound of the disclosure, or a fragment thereof, derivative thereof, or intermediate thereto.
The term “room temperature” as used herein refers to a temperature of about 15° C. to 28° C.
By the term “specifically binds,” as used herein with respect to an antibody, is meant an antibody which recognizes a specific antigen, but does not substantially recognize or bind other molecules in a sample. For example, an antibody that specifically binds to an antigen from one species may also bind to that antigen from one or more species. But, such cross-species reactivity does not itself alter the classification of an antibody as specific. In another example, an antibody that specifically binds to an antigen may also bind to different allelic forms of the antigen. However, such cross reactivity does not itself alter the classification of an antibody as specific. In some instances, the terms “specific binding” or “specifically binding,” can be used in reference to the interaction of an antibody, a protein, or a peptide with a second chemical species, to mean that the interaction is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, an antibody recognizes and binds to a specific protein structure rather than to proteins generally. If an antibody is specific for epitope “A”, the presence of a molecule containing epitope A (or free, unlabeled A), in a reaction containing labeled “A” and the antibody, will reduce the amount of labeled A bound to the antibody.
The term “solvent” as used herein refers to a liquid that can dissolve a solid, liquid, or gas. Non-limiting examples of solvents are silicones, organic compounds, water, alcohols, ionic liquids, and supercritical fluids.
The term “standard temperature and pressure” as used herein refers to 20° C. and 101 kPa.
The term “substantially” as used herein refers to a majority of, or mostly, as in at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, 99.99%, or at least about 99.999% or more, or 100%. The term “substantially free of” as used herein can mean having none or having a trivial amount of, such that the amount of material present does not affect the material properties of the composition including the material, such that the composition is about 0 wt % to about 5 wt % of the material, or about 0 wt % to about 1 wt %, or about 5 wt % or less, or less than, equal to, or greater than about 4.5 wt %, 4, 3.5, 3, 2.5, 2, 1.5, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.01, or about 0.001 wt % or less. The term “substantially free of” can mean having a trivial amount of, such that a composition is about 0 wt % to about 5 wt % of the material, or about 0 wt % to about 1 wt %, or about 5 wt % or less, or less than, equal to, or greater than about 4.5 wt %, 4, 3.5, 3, 2.5, 2, 1.5, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.01, or about 0.001 wt % or less, or about 0 wt %.
The term “substituted” as used herein in conjunction with a molecule or an organic group as defined herein refers to the state in which one or more hydrogen atoms contained therein are replaced by one or more non-hydrogen atoms. The term “functional group” or “substituent” as used herein refers to a group that can be or is substituted onto a molecule or onto an organic group. Examples of substituents or functional groups include, but are not limited to, a halogen (e.g., F, Cl, Br, and I); an oxygen atom in groups such as hydroxy groups, alkoxy groups, aryloxy groups, aralkyloxy groups, oxo(carbonyl) groups, carboxyl groups including carboxylic acids, carboxylates, and carboxylate esters; a sulfur atom in groups such as thiol groups, alkyl and aryl sulfide groups, sulfoxide groups, sulfone groups, sulfonyl groups, and sulfonamide groups; a nitrogen atom in groups such as amines, hydroxyamines, nitriles, nitro groups, N-oxides, hydrazides, azides, and enamines; and other heteroatoms in various other groups. Non-limiting examples of substituents that can be bonded to a substituted carbon (or other) atom include F, Cl, Br, I, OR, OC(O)N(R)2, CN, NO, NO2, ONO2, azido, CF3, OCF3, R, 0 (oxo), S (thiono), C(O), S(O), methylenedioxy, ethylenedioxy, N(R)2, SR, SOR, SO2R, SO2N(R)2, SO3R, C(O)R, C(O)C(O)R, C(O)CH2C(O)R, C(S)R, C(O)OR, OC(O)R, C(O)N(R)2, OC(O)N(R)2, C(S)N(R)2, (CH2)0-2N(R)C(O)R, (CH2)0-2N(R)N(R)2, N(R)N(R)C(O)R, N(R)N(R)C(O)OR, N(R)N(R)CON(R)2, N(R)SO2R, N(R)SO2N(R)2, N(R)C(O)OR, N(R)C(O)R, N(R)C(S)R, N(R)C(O)N(R)2, N(R)C(S)N(R)2, N(COR)COR, N(OR)R, C(═NH)N(R)2, C(O)N(OR)R, and C(═NOR)R, wherein R can be hydrogen or a carbon-based moiety; for example, R can be hydrogen, (C1-C100)hydrocarbyl, alkyl, acyl, cycloalkyl, aryl, aralkyl, heterocyclyl, heteroaryl, or heteroarylalkyl; or wherein two R groups bonded to a nitrogen atom or to adjacent nitrogen atoms can together with the nitrogen atom or atoms form a heterocyclyl.
By the term “synthetic antibody” as used herein, is meant an antibody which is generated using recombinant DNA technology, such as, for example, an antibody expressed by a bacteriophage as described herein. 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 DNA molecule expresses an antibody protein, or an amino acid sequence specifying the antibody, wherein the DNA or amino acid sequence has been obtained using synthetic DNA or amino acid sequence technology which is available and well known in the art.
A “therapeutic” treatment is a treatment administered to a subject who exhibits signs of pathology, for the purpose of diminishing or eliminating those signs.
The term “thioalkyl” as used herein refers to a sulfur atom connected to an alkyl group, as defined herein. The alkyl group in the thioalkyl can be straight chained or branched. Examples of linear thioalkyl groups include but are not limited to thiomethyl, thioethyl, thiopropyl, thiobutyl, thiopentyl, thiohexyl, and the like. Examples of branched alkoxy include but are not limited to iso-thiopropyl, sec-thiobutyl, tert-thiobutyl, iso-thiopentyl, iso-thiohexyl, and the like. The sulfur atom can appear at any suitable position in the alkyl chain, such as at the terminus of the alkyl chain or anywhere within the alkyl chain.
The terms “treat,” “treating” and “treatment,” as used herein, means reducing the frequency or severity with which symptoms of a disease or condition are experienced by a subject by virtue of administering an agent or compound to the subject.
As used herein, the term “wild-type” refers to a gene or gene product isolated from a naturally occurring source. A wild-type gene is that which is most frequently observed in a population and is thus arbitrarily designed the “normal” or “wild-type” form of the gene. In contrast, the term “modified” or “mutant” refers to a gene or gene product that displays modifications in sequence and/or functional properties (i.e., altered characteristics) when compared to the wild-type gene or gene product. It is noted that naturally occurring mutants can be isolated; these are identified by the fact that they have altered characteristics (including altered nucleic acid sequences) when compared to the wild-type gene or gene product.
The term “autoimmune disease” refers to a disease or illness that occurs when the body tissues are attacked by its own immune system. Examples of autoimmune diseases include, for example, systemic lupus erythematosus, Sjogren syndrome, Hashimoto thyroiditis, rheumatoid arthritis, juvenile (type 1) diabetes, polymyositis, scleroderma, Addison's disease, vitiligo, pernicious anemia, glomerulonephritis, and pulmonary fibrosis, among numerous others.
A more complete list of autoimmune diseases which may be treated by compounds and pharmaceutical compositions according to the present disclosure includes Addison's Disease, Autoimmune polyendodrine syndrome (APS) types 1, 2 and 3, autoimmune pancreatitis (AIP), diabetes mellitus type 1, autoimmune thyroiditis, Ord's thyroiditis, Grave's disease, autoimmune oophoritis, endometriosis, autoimmune orchitis, Sjogren's syndrome, autoimmune enteropathy, coeliac disease, Crohn's disease, microscopic colitis, ulcerative colitis, autophospholipid syndrome (APlS), aplastic anemia, autoimmune hemolytica anemia, autoimmune lymphoproliferative syndrome, autoimmune neutropenia, autoimmune thrombocytopenic purpura, cold agglutinin disease, essential mixed cryoglulinemia, Evans syndrome, pernicious anemia, pure red cell aplasia, thrombocytopenia, adiposis dolorosa, adult-onset Still's disease, ankylosing spondylitis, CREST syndrome, drug-induced lupus, enthesitis-related arthritis, esosiniphilic fasciitis, Felty syndrome, AgG4-related disease, juvenile arthritis, Lyme disease (chronic), mixed connective tissue disease (MCTD), palindromic rheumatism, Parry Romberg syndrome, Parsonage-Turner syndrome, psoriatic arthritis, reactive arthritis, relapsing polychondritis, retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis, sarcoidosis, Schnitzler syndrome, systemic lupus erythematosus, undifferentiated connective tissue disease (UCTD), dermatomyositis, fibromyalgia, myositis, inclusion body myositis, myasthenia gravis, neuromyotonia, paraneoplastic cerebellar degeneration, polymyositis, acute disseminated encephalomyelitis (ADEM), acute motor axonic neuropathy, anti-NMDA receptor encephalitis, Balo concentric sclerosis, Bickerstaff's encephalitis, chronic inflammatory demyelinating polyneuropathy, Guillain-Barré syndrome, Hashimoto's encephalopathy, idiopathic inflammatory demyelinating diseases, Lambert-Eaton myasthenic syndrome, multiple sclerosis, pattern II, Oshtoran Syndrome, Pediatric Autoimmune Neuropsychiatric Disorder Associated with Streptococcus (PANDAS), progressive inflammatory neuropathy, restless leg syndrome, stiff person syndrome, Syndenham chorea, transverse myelitis, autoimmune retinopathy, autoimmune uveitis, Cogan syndrome, Graves ophthalmopathy, intermediate uveitis, ligneous conjunctivitis, Mooren's ulcer, neuromyelitis optica, opsoclonus myoclonus syndrome, optic neuritis, scleritis, Susac's syndrome, sympathetic ophthalmia, Tolosa-Hunt syndrome, autoimmune inner ear disease (AIED), Méniére's disease, Behçet's disease, Eosinophilic granulomatosis with polyangiitis (EGPA), giant cell arteritis, granulomatosis with polyangiitis (GPA), IgA vasculitis (IgAV), IgA nephropathy, Kawasaki's disease, leukocytoclastic vasculitis, lupus vasculitis, rheumatoid vasculitis, microscopic polyangiitis (MPA), polyarteritis nodosa (PAN), polymyalgia rheumatica, urticarial vasculitis, vasculitis, primary immune deficiency, chronic fatigue syndrome, complex regional pain syndrome, eosinophilic esophagitis, gastritis, interstitial lung disease, POEMS syndrome, Raynaud's syndrome, primary immunodeficiency and pyoderma gangrenosum, among others.
The term “cancer” or “neoplasia” is used throughout the specification to refer to the pathological process that results in the formation and growth of a cancerous or malignant neoplasm, i.e., abnormal tissue that grows by cellular proliferation, often more rapidly than normal and continues to grow after the stimuli that initiated the new growth cease. Malignant neoplasms show partial or complete lack of structural organization and functional coordination with the normal tissue and most invade surrounding tissues, metastasize to several sites, and are likely to recur after attempted removal and to cause the death of the patient unless adequately treated. As used herein, the term neoplasia is used to describe all cancerous disease states and embraces or encompasses the pathological process associated with malignant hematogenous, ascitic and solid tumors. Neoplasms include, without limitation, morphological irregularities in cells in tissue of a subject or host, as well as pathologic proliferation of cells in tissue of a subject, as compared with normal proliferation in the same type of tissue. Additionally, neoplasms include benign tumors and malignant tumors (e.g., colon tumors) that are either invasive or noninvasive. Malignant neoplasms (cancer) are distinguished from benign neoplasms in that the former show a greater degree of anaplasia, or loss of differentiation and orientation of cells, and have the properties of invasion and metastasis. Examples of neoplasms or neoplasias from which the target cell of the present disclosure may be derived include, without limitation, carcinomas (e.g., squamous-cell carcinomas, adenocarcinomas, hepatocellular carcinomas, and renal cell carcinomas), particularly those of the bladder, bowel, breast, cervix, colon, esophagus, head, kidney, liver, lung, neck, ovary, pancreas, prostate, and stomach; leukemias; benign and malignant lymphomas, particularly Burkitt's lymphoma and Non-Hodgkin's lymphoma; benign and malignant melanomas; myeloproliferative diseases; sarcomas, particularly Ewing's sarcoma, hemangiosarcoma, Kaposi's sarcoma, liposarcoma, myosarcomas, peripheral neuroepithelioma, and synovial sarcoma; tumors of the central nervous system (e.g., gliomas, astrocytomas, oligodendrogliomas, ependymomas, gliobastomas, neuroblastomas, ganglioneuromas, gangliogliomas, medulloblastomas, pineal cell tumors, meningiomas, meningeal sarcomas, neurofibromas, and Schwannomas); germ-line tumors (e.g., bowel cancer, breast cancer, prostate cancer, cervical cancer, uterine cancer, lung cancer, ovarian cancer, testicular cancer, thyroid cancer, astrocytoma, esophageal cancer, pancreatic cancer, stomach cancer, liver cancer, colon cancer, and melanoma); mixed types of neoplasias, particularly carcinosarcoma and Hodgkin's disease; and tumors of mixed origin, such as Wilms' tumor and teratocarcinomas (Beers and Berkow (eds.), The Merck Manual of Diagnosis and Therapy, 17.sup.th ed. (Whitehouse Station, N.J.: Merck Research Laboratories, 1999) 973-74, 976, 986, 988, 991). All of these neoplasms may be treated using compounds according to the present disclosure.
Representative common cancers to be treated with compounds according to the present disclosure include, for example, prostate cancer, metastatic prostate cancer, stomach, colon, rectal, liver, pancreatic, lung, breast, cervix uteri, corpus uteri, ovary, testis, bladder, renal, brain/CNS, head and neck, throat, Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma, leukemia, melanoma, non-melanoma skin cancer, acute lymphocytic leukemia, acute myelogenous leukemia, Ewing's sarcoma, small cell lung cancer, choriocarcinoma, rhabdomyosarcoma, Wilms' tumor, neuroblastoma, hairy cell leukemia, mouth/pharynx, oesophagus, larynx, kidney cancer and lymphoma, among others, which may be treated by one or more compounds according to the present disclosure. Because of the activity of the present compounds, the present disclosure has general applicability treating virtually any cancer in any tissue, thus the compounds, compositions and methods of the present disclosure are generally applicable to the treatment of cancer and in reducing the likelihood of development of cancer and/or the metastasis of an existing cancer.
In certain particular aspects of the present disclosure, the cancer which is treated is metastatic cancer, a recurrent cancer or a drug resistant cancer, especially including a drug resistant cancer. Separately, metastatic cancer may be found in virtually all tissues of a cancer patient in late stages of the disease, typically metastatic cancer is found in lymph system/nodes (lymphoma), in bones, in lungs, in bladder tissue, in kidney tissue, liver tissue and in virtually any tissue, including brain (brain cancer/tumor). Thus, the present disclosure is generally applicable and may be used to treat any cancer in any tissue, regardless of etiology.
The term “anticancer agent” or “additional anticancer agent” refers to a compound other than the chimeric compounds according to the present disclosure which may be used in combination with a compound according to the present disclosure for the treatment of cancer. Exemplary anticancer agents which may be co-administered in combination with one or more chimeric compounds according to the present disclosure include, for example, antimetabolites, inhibitors of topoisomerase I and II, alkylating agents and microtubule inhibitors (e.g., taxol), among others. Exemplary anticancer compounds for use in the present disclosure may include everolimus, trabectedin, abraxane, TLK 286, AV-299, DN-101, pazopanib, GSK690693, RTA 744, ON 0910.Na, AZD 6244 (ARRY-142886), AMN-107, TKI-258, GSK461364, AZD 1152, enzastaurin, vandetanib, ARQ-197, MK-0457, MLN8054, PHA-739358, R-763, AT-9263, a FLT-3 inhibitor, a VEGFR inhibitor, an EGFR TK inhibitor, an aurora kinase inhibitor, a PIK-1 modulator, a Bcl-2 inhibitor, an HDAC inhbitor, a c-MET inhibitor, a PARP inhibitor, a Cdk inhibitor, an EGFR TK inhibitor, an IGFR-TK inhibitor, an anti-HGF antibody, a PI3 kinase inhibitors, an AKT inhibitor, a JAK/STAT inhibitor, a checkpoint-1 or 2 inhibitor, a focal adhesion kinase inhibitor, a Map kinase kinase (MEK) inhibitor, a VEGF trap antibody, pemetrexed, erlotinib, dasatanib, nilotinib, decatanib, panitumumab, amrubicin, oregovomab, Lep-etu, nolatrexed, azd2171, batabulin, ofatumumab (Arzerra), zanolimumab, edotecarin, tetrandrine, rubitecan, tesmilifene, oblimersen, ticilimumab, ipilimumab, gossypol, Bio 111, 131-I-TM-601, ALT-110, BIO 140, CC 8490, cilengitide, gimatecan, IL13-PE38QQR, INO 1001, IPdR1 KRX-0402, lucanthone, LY 317615, neuradiab, vitespan, Rta 744, Sdx 102, talampanel, atrasentan, Xr 311, romidepsin, ADS-100380, sunitinib, 5-fluorouracil, vorinostat, etoposide, gemcitabine, doxorubicin, irinotecan, liposomal doxorubicin, 5′-deoxy-5-fluorouridine, vincristine, temozolomide, ZK-304709, seliciclib; PD0325901, AZD-6244, capecitabine, L-Glutamic acid, N-[4-[2-(2-amino-4,7-dihydro-4-oxo-1 H-pyrrolo[2,3- d ]pyrimidin-5-yl)ethyl]benzoyl]-, disodium salt, heptahydrate, camptothecin, PEG-labeled irinotecan, tamoxifen, toremifene citrate, anastrazole, exemestane, letrozole, DES(diethylstilbestrol), estradiol, estrogen, conjugated estrogen, bevacizumab, IMC-1C11, CHIR-258, 3-[5-(methylsulfonylpiperadinemethyl)-indolylj-quinolone, vatalanib, AG-013736, AVE-0005, the acetate salt of [D-Ser(Bu t) 6,Azgly 10] (pyro-Glu-His-Trp-Ser-Tyr-D-Ser(Bu t)-Leu-Arg-Pro-Azgly-NH2 acetate [C59H84N18Oi4-(C2H4O2)X wherein x=1 to 2.4], goserelin acetate, leuprolide acetate, triptorelin pamoate, medroxyprogesterone acetate, hydroxyprogesterone caproate, megestrol acetate, raloxifene, bicalutamide, flutamide, nilutamide, megestrol acetate, CP-724714; TAK-165, HKI-272, erlotinib, lapatanib, canertinib, ABX-EGF antibody, erbitux, EKB-569, PKI-166, GW-572016, lonafarnib, BMS-214662, tipifarnib; amifostine, NVP-LAQ824, suberoyl analide hydroxamic acid, valproic acid, trichostatin A, FK-228, SU11248, sorafenib, KRN951, aminoglutethimide, arnsacrine, anagrelide, L-asparaginase, Bacillus Calmette-Guerin (BCG) vaccine, bleomycin, buserelin, busulfan, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, clodronate, cyproterone, cytarabine, dacarbazine, dactinomycin, daunorubicin, diethylstilbestrol, epirubicin, fludarabine, fludrocortisone, fluoxymesterone, flutamide, gemcitabine, gleevac, hydroxyurea, idarubicin, ifosfamide, imatinib, leuprolide, levamisole, lomustine, mechlorethamine, melphalan, 6-mercaptopurine, mesna, methotrexate, mitomycin, mitotane, mitoxantrone, nilutamide, octreotide, oxaliplatin, pamidronate, pentostatin, plicamycin, porfimer, procarbazine, raltitrexed, rituximab, streptozocin, teniposide, testosterone, thalidomide, thioguanine, thiotepa, tretinoin, vindesine, 13-cis-retinoic acid, phenylalanine mustard, uracil mustard, estramustine, altretamine, floxuridine, 5-deooxyuridine, cytosine arabinoside, 6-mecaptopurine, deoxycoformycin, calcitriol, valrubicin, mithramycin, vinblastine, vinorelbine, topotecan, razoxin, marimastat, COL-3, neovastat, BMS-275291, squalamine, endostatin, SU5416, SU6668, EMD121974, interleukin-12, IM862, angiostatin, vitaxin, droloxifene, idoxyfene, spironolactone, finasteride, cimitidine, trastuzumab, denileukin diftitox,gefitinib, bortezimib, paclitaxel, irinotecan, topotecan, doxorubicin, docetaxel, vinorelbine, bevacizumab (monoclonal antibody) and erbitux, cremophor-free paclitaxel, epithilone B, BMS-247550, BMS-310705, droloxifene, 4-hydroxytamoxifen, pipendoxifene, ERA-923, arzoxifene, fulvestrant, acolbifene, lasofoxifene, idoxifene, TSE-424, HMR-3339, ZK186619, PTK787/ZK 222584, VX-745, PD 184352, rapamycin, 40—O-(2-hydroxyethyl)-rapamycin, temsirolimus, AP-23573, RAD001, ABT-578, BC-210, LY294002, LY292223, LY292696, LY293684, LY293646, wortmannin, ZM336372, L-779,450, PEG-filgrastim, darbepoetin, erythropoietin, granulocyte colony-stimulating factor, zolendronate, prednisone, cetuximab, granulocyte macrophage colony-stimulating factor, histrelin, pegylated interferon alfa-2a, interferon alfa-2a, pegylated interferon alfa-2b, interferon alfa-2b, azacitidine, PEG-L-asparaginase, lenalidomide, gemtuzumab, hydrocortisone, interleukin-11, dexrazoxane, alemtuzumab, all-transretinoic acid, ketoconazole, interleukin-2, megestrol, immune globulin, nitrogen mustard, methylprednisolone, ibritgumomab tiuxetan, androgens, decitabine, hexamethylmelamine, bexarotene, tositumomab, arsenic trioxide, cortisone, editronate, mitotane, cyclosporine, liposomal daunorubicin, Edwina-asparaginase, strontium 89, casopitant, netupitant, an NK-1 receptor antagonists, palonosetron, aprepitant, diphenhydramine, hydroxyzine, metoclopramide, lorazepam, alprazolam, haloperidol, droperidol, dronabinol, dexamethasone, methylprednisolone, prochlorperazine, granisetron, ondansetron, dolasetron, tropisetron, pegfilgrastim, erythropoietin, epoetin alfa and darbepoetin alfa, vemurafenib among others, including immunotherapy agents such as IDO inhibitors (an inhibitor of indoleamine 2,3-dioxygenase (IDO) pathway) such as Indoximod (NLG-8187), Navoximod (GDC-0919) and NLG802, PDL1 inhibitors (an inhibitor of programmed death-ligand 1) including, for example, nivolumab, durvalumab and atezolizumab, PD1 inhibitors such as pembrolizumab (Merck) and CTLA-4 inhibitors (an inhibitor of cytotoxic T-lymphocyte associated protein 4/cluster of differentiation 152), including ipilimumab and tremelimumab, among others.
In addition to anticancer agents, a number of other agents may be co-administered with chimeric compounds according to the present disclosure in the treatment of cancer. These include active agents, minerals, vitamins and nutritional supplements which have shown some efficacy in inhibiting cancer tissue or its growth or are otherwise useful in the treatment of cancer. For example, one or more of dietary selenium, vitamin E, lycopene, soy foods, curcumin (turmeric), vitamin D, green tea, omega-3 fatty acids and phytoestrogens, including beta-sitosterol, may be utilized in combination with the present compounds to treat cancer.
The term “inflammatory disease” is used to describe a disease or illness with acute, but more often chronic inflammation as a principal manifestation of the disease or illness. Inflammatory diseases include diseases of neurodegeneration (including, for example, Alzheimer's disease, Parkinson's disease, Huntington's disease; other ataxias), diseases of compromised immune response causing inflammation (e.g., dysregulation of T cell maturation, B cell and T cell homeostasis, counters damaging inflammation), chronic inflammatory diseases including, for example, inflammatory bowel disease, including Crohn's disease, rheumatoid arthritis, lupus, multiple sclerosis, chronic obstructive pulmonary disease/COPD, pulmonary fibrosis, cystic fibrosis, Sjogren's disease; hyperglycemic disorders, diabetes (I and II), affecting lipid metabolism islet function and/or structure, pancreatic β-cell death and related hyperglycemic disorders, including severe insulin resistance, hyperinsulinemia, insulin-resistant diabetes (e.g. Mendenhall's Syndrome, Werner Syndrome, leprechaunism, and lipoatrophic diabetes) and dyslipidemia (e.g. hyperlipidemia as expressed by obese subjects, elevated low-density lipoprotein (LDL), depressed high-density lipoprotein (HDL), elevated triglycerides and metabolic syndrome, liver disease, renal disease (apoptosis in plaques, glomerular disease), cardiovascular disease (especially including infarction, ischemia, stroke, pressure overload and complications during reperfusion), muscle degeneration and atrophy, low grade inflammation, gout, silicosis, atherosclerosis and associated conditions such as cardiac and neurological (both central and peripheral) manifestations including stroke, age-associated dementia and sporadic form of Alzheimer's disease, and psychiatric conditions including depression), stroke and spinal cord injury, arteriosclerosis, among others. In these diseases, elevated MIF is very often observed, making these disease states and/or conditions response to therapy using compounds and/or pharmaceutical compositions according to the present disclosure. It is noted that there is some overlap between certain autoimmune diseases and inflammatory diseases as described herein.
Throughout this disclosure, various aspects of the disclosure can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.
In one aspect, the disclosure provides a compound comprising formula (I), or a salt, geometric isomer, stereoisomer, or solvate thereof:
[Protein binder]k′—[CON]h—[Linker]i—[CON]h′—[CRBM]j′ (I),
In certain embodiments, the compound comprises formula (Ia), or a salt, geometric isomer, stereoisomer, or solvate thereof:
[Protein binder]—[CON]0-1—[Linker]—[CON]0-1—[CRBM] (Ia).
In (I) and/or (Ia), the Protein binder is a molecule, such as but not limited to a small molecule and/or a peptide, that binds to an extracellular protein of interest (“Protein”). In certain embodiments, treatment or management of the disease and/or disorder requires degradation, removal, and/or reduction in concentration of the extracellular protein in the subject. In certain embodiments, the extracellular protein binder within (I) and/or (Ia) is capable of binding to the circulating extracellular protein in the plasma of the subject with identical affinity or substantially similar affinity as compared to the extracellular protein binder itself.
In (I) and/or (Ia), the CRBM is a cellular receptor binding moiety that binds to at least one receptor on the surface of hepatocytes or other degrading cells in the subject, whereby binding of (I) or (Ia) leads to endocytosis and degradation of (I) and/or (Ia) and/or extracellular protein. In certain embodiments, the CRBM is ASGPRBM, which is a cellular receptor binding moiety that binds to at least one asialoglycoprotein receptor on the surface of hepatocytes or other degrading cells in the subject.
In (I) and/or (Ia), each CON is independently a bond or a group that covalently links a Protein binder to a CRBM, a Protein binder to a Linker, and/or a Linker to a CRBM.
In (I) and/or (Ia), the Linker is a group having a valence ranging from 1 to 15. In certain embodiments, the valence of the Linker is 1 to 10. In certain embodiments, the valence of the Linker is 1 to 5. In certain embodiments, the valence of the Linker is 1, 2, or 3. In certain embodiments, the Linker covalently links one or more CRBM and/or Protein binder groups, optionally through a CON, wherein the Linker optionally itself contains one or more CON groups.
In certain embodiments, k′ is an integer ranging from 1 to 15. In certain embodiments, k′ is an integer ranging from 1 to 10. In certain embodiments, k′ is an integer ranging from 1 to 5. In certain embodiments, k′ is an integer ranging from 1 to 3. In certain embodiments, k′ is 1, 2 or 3.
In certain embodiments, j is an integer ranging from 1 to 15. In certain embodiments, j is an integer ranging from 1 to 10. In certain embodiments, j is an integer ranging from 1 to 5. In certain embodiments, j is an integer ranging from 1 to 3. In certain embodiments, j is 1, 2 or 3.
In certain embodiments, h is an integer ranging from 0 to 15. In certain embodiments, h is an integer ranging from 1 to 15. In certain embodiments, h is an integer ranging from 1 to 10. In certain embodiments, h is an integer ranging from 1 to 5. In certain embodiments, h is an integer ranging from 1 to 3. In certain embodiments, h is 1, 2, or 3.
In certain embodiments, h′ is an integer ranging from 0 to 15. In certain embodiments, h′ is an integer ranging from 1 to 15. In certain embodiments, h′ is an integer ranging from 1 to 10. In certain embodiments, h′ is an integer ranging from 1 to 5. In certain embodiments, h′ is an integer ranging from 1 to 3. In certain embodiments, h′ is 1, 2, or 3.
In certain embodiments, i is an integer ranging from 0 to 15. In certain embodiments, i is an integer ranging from 1 to 15. In certain embodiments, i is an integer ranging from 1 to 10. In certain embodiments, i is an integer ranging from 1 to 5. In certain embodiments, i is an integer ranging from 1 to 3. In certain embodiments, i is 1, 2, or 3.
In certain embodiments, at least one of h, h′, and i is at least 1.
In certain embodiments, k′, j′, h, h′, and i are each independently 1, 2, or 3.
In certain embodiments, k′ is 1, and j′ is 1, 2, or 3.
In another aspect, the disclosure provides a compound comprising formula (II), or a salt, geometric isomer, stereoisomer, or solvate thereof:
[TNF binder]k′—[CON]h—[Linker]i—[CON]h′—[CRBM]j′ (II).
In certain embodiments, the compound comprises formula (IIa), or a salt, geometric isomer, stereoisomer, or solvate thereof:
[TNF binder]—[CON]0-1—[Linker]—[CON]0-1—[CRBM]′ (IIa).
In (II) and/or (IIa), the TNF binder is a molecule, such as but not limited to a small molecule and/or a peptide, that binds to TNF. In certain embodiments, treatment or management of the disease and/or disorder requires degradation, removal, and/or reduction in concentration of TNF in the subject. In certain embodiments, the TNF binder within (II) and/or (IIa) is capable of binding to the circulating TNF in the plasma of the subject with identical affinity or substantially similar affinity as compared to the TNF binder itself.
In (II) and/or (IIa), the CRBM is a cellular receptor binding moiety that binds to at least one receptor on the surface of hepatocytes or other degrading cells in the subject, whereby binding of (II) or (IIa) leads to endocytosis and degradation of (II) and/or (IIa) and/or TNF. In certain embodiments, the CRBM is ASGPRBM, which is a cellular receptor binding moiety that binds to at least one asialoglycoprotein receptor on the surface of hepatocytes or other degrading cells in the subject.
In (II) and/or (IIa), each CON is independently a bond or a group that covalently links a TNF binder to a CRBM, a TNF binder to a Linker, and/or a Linker to a CRBM.
In (II) and/or (IIa), the Linker is a group having a valence ranging from 1 to 15. In certain embodiments, the valence of the Linker is 1 to 10. In certain embodiments, the valence of the Linker is 1 to 5. In certain embodiments, the valence of the Linker is 1, 2, or 3. In certain embodiments, the Linker covalently links one or more CRBM and/or TNF binder groups, optionally through a CON, wherein the Linker optionally itself contains one or more CON groups.
In certain embodiments, k′ is an integer ranging from 1 to 15. In certain embodiments, k′ is an integer ranging from 1 to 10. In certain embodiments, k′ is an integer ranging from 1 to 5. In certain embodiments, k′ is an integer ranging from 1 to 3. In certain embodiments, k′ is 1, 2 or 3.
In certain embodiments, j is an integer ranging from 1 to 15. In certain embodiments, j is an integer ranging from 1 to 10. In certain embodiments, j is an integer ranging from 1 to 5. In certain embodiments, j is an integer ranging from 1 to 3. In certain embodiments, j is 1, 2 or 3.
In certain embodiments, h is an integer ranging from 0 to 15. In certain embodiments, h is an integer ranging from 1 to 15. In certain embodiments, h is an integer ranging from 1 to 10. In certain embodiments, h is an integer ranging from 1 to 5. In certain embodiments, h is an integer ranging from 1 to 3. In certain embodiments, h is 1, 2, or 3.
In certain embodiments, h′ is an integer ranging from 0 to 15. In certain embodiments, h′ is an integer ranging from 1 to 15. In certain embodiments, h′ is an integer ranging from 1 to 10. In certain embodiments, h′ is an integer ranging from 1 to 5. In certain embodiments, h′ is an integer ranging from 1 to 3. In certain embodiments, h′ is 1, 2, or 3.
In certain embodiments, i is an integer ranging from 0 to 15. In certain embodiments, i is an integer ranging from 1 to 15. In certain embodiments, i is an integer ranging from 1 to 10. In certain embodiments, i is an integer ranging from 1 to 5. In certain embodiments, i is an integer ranging from 1 to 3. In certain embodiments, i is 1, 2, or 3.
In certain embodiments, at least one of h, h′, and i is at least 1.
In certain embodiments, k′, j′, h, h′, and i are each independently 1, 2, or 3.
In certain embodiments, k′ is 1, and j′ is 1, 2, or 3.
In yet another aspect, the disclosure provides a compound comprising formula (III), or a salt, geometric isomer, stereoisomer, or solvate thereof:
[AATM]k′—[CON]h—[Linker]i—[CON]h′—[CRBM]j′ (III).
In certain embodiments, the compound comprises formula (IIIa), or a salt, geometric isomer, stereoisomer, or solvate thereof:
[AATM]—[CON]0-1—[Linker]—[CON]0-1—[CRBM]′ (IIIa).
In (III) or (IIIa), the AATM is a ligand of an autoantibody. That ligand can be, for example, a small molecule, peptide, and/or nucleic acid aptamer. In certain embodiments, the autoantibody mediates a disease and/or disorder in a subject, and treatment or management of the disease and/or disorder requires degradation, removal, or reduction in concentration of the autoantibody in the subject. In certain embodiments, the AATM within (III) or (IIIa) is capable of binding to the autoantibody in the plasma of the subject with identical affinity or substantially similar affinity as compared to the AATM itself.
In (III) or (IIIa), the CRBM is a cellular receptor binding moiety that binds to at least one receptor on the surface of hepatocytes or other degrading cells in the subject, whereby binding leads to endocytosis and degradation of (III) and/or (IIIa) and/or autoantibody. In certain embodiments, the CRBM is ASGPRBM, which is a cellular receptor binding moiety that binds to at least one asialoglycoprotein receptor on the surface of hepatocytes or other degrading cells in the subject.
In (III) or (IIIa), each CON is independently a bond or a group that covalently links an AATM to an CRBM, an AATM to a Linker, and/or a Linker to a CRBM.
In (III) or (IIIa), the Linker is a group having a valence ranging from 1 to 15. In certain embodiments, the valence of the Linker is 1 to 10. In certain embodiments, the valence of the Linker is 1 to 5. In certain embodiments, the valence of the Linker is 1, 2, or 3. In certain embodiments, the Linker covalently links one or more CRBM and/or AATM groups, optionally through a CON, wherein the Linker optionally itself contains one or more CON groups.
In certain embodiments, k′ is an integer ranging from 1 to 15. In certain embodiments, k′ is an integer ranging from 1 to 10. In certain embodiments, k′ is an integer ranging from 1 to 5. In certain embodiments, k′ is an integer ranging from 1 to 3. In certain embodiments, k′ is 1, 2 or 3.
In certain embodiments, j is an integer ranging from 1 to 15. In certain embodiments, j is an integer ranging from 1 to 10. In certain embodiments, j is an integer ranging from 1 to 5. In certain embodiments, j is an integer ranging from 1 to 3. In certain embodiments, j is 1, 2 or 3. In certain embodiments, h is an integer ranging from 0 to 15. In certain embodiments, h is an integer ranging from 1 to 15. In certain embodiments, h is an integer ranging from 1 to 10. In certain embodiments, h is an integer ranging from 1 to 5. In certain embodiments, h is an integer ranging from 1 to 3. In certain embodiments, h is 1, 2, or 3.
In certain embodiments, h′ is an integer ranging from 0 to 15. In certain embodiments, h′ is an integer ranging from 1 to 15. In certain embodiments, h′ is an integer ranging from 1 to 10.
In certain embodiments, h′ is an integer ranging from 1 to 5. In certain embodiments, h′ is an integer ranging from 1 to 3. In certain embodiments, h′ is 1, 2, or 3.
In certain embodiments, i is an integer ranging from 0 to 15. In certain embodiments, i is an integer ranging from 1 to 15. In certain embodiments, i is an integer ranging from 1 to 10. In certain embodiments, i is an integer ranging from 1 to 5. In certain embodiments, i is an integer ranging from 1 to 3. In certain embodiments, i is 1, 2, or 3.
In certain embodiments, at least one of h, h′, and i is at least 1.
In certain embodiments, k′, j′, h, h′, and i are each independently 1, 2, or 3.
In certain embodiments, k′ is 1, and j′ is 1, 2, or 3.
Folic Acid (Folate) Receptor:
In certain embodiments, the CRBM is folic acid, or any fragment or derivative thereof that is capable of binding to the folic acid (folate) receptor. Folate receptors bind folate and reduced folic acid derivatives and mediates delivery to the interior of cells of tetrahydrofolate, which is then converted from monoglutamate to polyglutamate forms (such as 5-methyltetrahydrofolate) as only monoglutamate forms can be transported across cell membranes. Human proteins from this family include folate receptor 1 (adult), folate receptor 2 (fetal), and folate receptor gamma.
In certain embodiments, the folic acid CRBM comprises methotrexate or a biologically active fragment thereof:
In certain embodiments, the folic acid CRBM comprises premetrexed or a biologically active fragment thereof:
In certain embodiments, the folic acid CRBM can be incorporated into the compound of the disclosure through one of its carboxylic acid, as illustrated in
Mannose Receptor:
In certain embodiments, the CRBM is a group that binds to a mannose receptor. In certain embodiments, the CRBM comprises the group:
In certain embodiments, the mannose receptor CRBM can be attached to the compound of the disclosure (such as but not limited to the REAG) using one of the following reagents (which may be optionally protected with appropriately protecting groups):
wherein X is S or O, wherein R is selected from the group consisting of:
and wherein each occurrence of ‘n’ is independently 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.
In certain embodiments, the mannose receptor CRBM is part of a polymeric molecule. Such molecule can comprise one or more independently selected mannose receptor CRBMs as part of a polymeric chain. In certain embodiments, the CRBMs are incorporated into the polymeric molecule using CRBM reagents recited elsewhere herein.
Mannose-6-Phosphate (M6P) Receptor:
In certain embodiments, the CRBM is a group that binds to a mannose-6-phosphate (M6P) receptor. In certain embodiments, the CRBM comprises the group:
wherein X is O or S, and wherein R1 is selected from the group consisting of:
In certain embodiments, the CRBM can be attached to the compound of the disclosure (such as but not limited to the REAG) using one of the following reagents (which may be optionally protected with appropriately protecting groups):
wherein X and R1 are as defined elsewhere herein, wherein R2 is selected from the group consisting of:
and wherein each occurrence of ‘n’ is independently 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.
In certain embodiments, the M6P receptor CRBM is part of a polymeric molecule. Such molecule can comprise one or more independently selected M6P receptor CRBMs as part of a polymeric chain. In certain embodiments, the CRBMs are incorporated into the polymeric molecule using CRBM reagents recited elsewhere herein.
In certain embodiments, the M6P receptor CRBM is one of the following (Yamaguchi, et al., 2016, J. Am. Chem. Soc. 138(38):12472-12485):
In certain embodiments, the M6P receptor CRBM is one of the following (US 2011/0110960 to Platenburg):
Low Density Lipoprotein Receptor-Related Protein 1 (LRP1) Receptor:
In certain embodiments, the CRBM is a LRP1 [Low density lipoprotein receptor-related protein 1; also known as alpha-2-macroglobulin receptor (A2MR), apolipoprotein E receptor (APOER) or cluster of differentiation 91 (CD91)] binding group comprising one of the following amino acid sequences:
Low Density Lipoprotein Receptor (LDLR):
In certain embodiments, the CRBM is a LDLR (low density lipoprotein receptor) binding group comprising one of the following amino acid sequences:
FcγRI Receptor:
In certain embodiments, the CRBM is a FcγRI binding group comprising one of the following amino acid sequences:
Transferrin Receptor:
In certain embodiments, the CRBM is a transferrin receptor binding group comprising one of the following amino acid sequences:
Macrophage Scavenger Receptor:
In certain embodiments, the CRBM is a macrophage scavenger receptor binding moiety comprising one of the following amino acid sequences:
As used herein, Pen is Penicillamine, Thz is thiazolidine-4-carboxylic acid, Sar is sarcosine, Pip is pipecolic acid, Nleu is norleucine, and NMeLeu is N-methylleucine.
G-Protein Coupled Receptor:
In certain embodiments, the CRBM is a G-protein coupled receptor (GPCR) binding moiety. In certain embodiments, the binding moiety binds to the GPCR and induces receptor internalization. In certain embodiments, the receptor is CXCR7 (see, for example, Nalawansha, et al., 2019, ACS Cent. Sci. 5(6):1079-1084). In certain embodiments, the binding moiety comprises the following:
wherein each occurrence of R is independently H or C1-C6 alkyl. In certain embodiments, the CRBM can be attached to the compound of the disclosure (such as but not limited to the REAG) using one of the following reagents (which may be optionally protected with appropriately protecting groups):
wherein at least one occurrence of R is REAG, and wherein the remaining occurrences of R are independently H or C1-C6 alkyl.
Asialoglycoprotein Receptor (ASGPR):
The disclosure contemplates the use of a ASGPR binding moiety (ASGPRBM).
In certain embodiments, the ASGPRBM group is any such group recited in Huang, et al., 2017, Bioconjugate Chem. 28:283-295, which is incorporated herein in its entirety by reference.
In certain embodiments, the ASGPRBM group comprises the structure:
wherein X is a linker of 1-4 atoms in length and comprises O, S, N(RN1), or C(RN1)(RN1) groups, such that:
In certain embodiments, each occurrence of RN1 is independently H or C1-C3 alkyl optionally substituted with 1-3 independently selected halogens and/or 1-2 hydroxyl groups.
In certain embodiments, the X in ASGPRBM is —O—C(RN1)(RN1)—, —C(RN1)(RN1)—O—, —S—C(RN1)(RN1)—, —C(RN1)(RN1)—S—, —N(RN1)—C(RN1)(RN1)—, —C(RN1)(RN1)—N(RN1)—, or —C(RN1)(RN1)—C(RN1)(RN1)—, when X is 2 atoms in length.
In certain embodiments, the X in ASGPRBM is —O—C(RN1)(RN1)—C(RN1)(RN1)—, —C(RN1)(RN1)—O —C(RN1)(RN1)—, —O—C(RN1)(RN1)—O—, —O—C(RN1)(RN1)—S—, —O—C(RN1)(RN1)—N(RN1)—, —S—C(RN1)(RN1)—C(RN1)(RN1)—, —C(RN1)(RN1)—S—C(RN1)(RN1)—, —C(RN1)(RN1)—C(RN1)(RN1)—S, —S—C(RN1)(RN1)—S—, —S—C(RN1)(RN1)—O—, —S—C(RN1)(RN1)—N(RN1)—, —N(RN1)—C(RN1)(RN1)—C(RN1)(RN1)—, —C(RN1)(RN1)—N(RN1)—C(RN1)(RN1)—, —C(RN1)(RN1)—C(RN1)(RN1)—N(RN1)—, —N(RN1)—C(RN1)(RN1)—N(RN1)—, or —C(RN1)(RN1)—C(RN1)(RN1)—C(RN1)(RN1), when X is 3 atoms in length.
In certain embodiments, the X in ASGPRBM is —O—C(RN1)(RN1)—C(RN1)(RN1)—C(RN1)(RN1)—, —C(RN1)(RN1)—O—C(RN1)(RN1)—C(RN1)(RN1)—, —O—C(RN1)(RN1)—O—C(RN1)(RN1)—, —S—C(RN1)(RN1)—C(RN1)(RN1)—C(RN1)(RN1)—, —C(RN1)(RN1)—S—C(RN1)(RN1)—C(RN1)(RN1)—, —C(RN1)(RN1)—C(RN1)(RN1)—S—C(RN1)(RN1)—, —S—C(RN1)(RN1)—S—C(RN1)(RN1)—, —N(RN1)—C(RN1)(RN1)—C(RN1)(RN1)—C(RN1)(RN1)—, or —C(RN1)(RN1)—N(RN1)—C(RN1)(RN1)—C(RN1)(RN1)—, when X is 4 atoms in length.
In certain embodiments, X is OCH2 and RN1 is H.
In certain embodiments, X is CH2O and RN1 is H.
In certain embodiments, the ASGPRBM comprises the structure:
In certain embodiments, the ASGPRBM comprises the structure:
In certain embodiments, R1 is a group depicted in
In certain embodiments, R1 and R3 are each independently H, —(CH2)KOH, —(CH2)KO(C1-C4 alkyl) optionally substituted with 1-3 independently selected halogens, C1-C4 alkyl optionally substituted with 1-3 independently selected halogens, —(CH2)K(vinyl), —O(CH2)K(vinyl), —(CH2)K(alkynyl), —(CH2)KCOOH, —(CH2)KC(═O)O(C1-C4 alkyl) optionally substituted with 1-3 independently selected halogens, —OC(═O)(C1-C4 alkyl) optionally substituted with 1-3 independently selected halogens, or —C(═O)(C1-C4 alkyl) optionally substituted with 1-3 independently selected halogens.
In certain embodiments, R1 and R3 are each independently Ph(CH2)K—, which is optionally substituted with: 1-3 independently selected halogens; C1-C4 alkyl optionally substituted with 1-3 independently selected halogens and/or 1-2 hydroxyl groups; or C1-C4 alkoxy optionally substituted with 1-3 independently selected halogens and/or 1-2 hydroxyl groups.
In certain embodiments, R1 and R3 are each independently a group of structure
—O—(CH2)K—CH(OH)—(CH2)K′—R7,
wherein R7 is: C1-C4 alkoxy optionally substituted with 1-3 independently selected halogens and/or 1-2 hydroxy groups; —NRN3RN4; or —(CH2)K′—O—(CH2)K—CH2—CH═CH2.
In certain embodiments, K is 0. In certain embodiments, K is 1. In certain embodiments, K is 2. In certain embodiments, K is 3. In certain embodiments, K is 4.
In certain embodiments, K′ is 1. In certain embodiments, K′ is 2. In certain embodiments, K′ is 3. In certain embodiments, K′ is 4.
In certain embodiments, each occurrence of RN3 is independently H or C1-C3 alkyl. In certain embodiments, each occurrence of RN3 is independently H or C1-C3 alkyl optionally substituted with 1-3 independently selected halogens and/or 1-2 hydroxyl groups;
In certain embodiments, each occurrence of RN4 is independently H, C1-C3 alkyl, or Ph-(CH2)K—. In certain embodiments, each occurrence of RN4 is independently H, C1-C3 alkyl optionally substituted with 1-3 independently selected halogens and/or 1-2 hydroxyl groups, or Ph-(CH2)K—.
In certain embodiments, R1 and R3 are each independently selected from the group consisting of:
L1-≡-, L1-(CH2)K—, and CYC—(CH2)K—, wherein CYC is selected from the group consisting of:
wherein the bond marked with indicating the site on CYC whereto —(CH2)K is connected.
In certain embodiments, L1 is a bond, -Linker, —CON-Linker, or —CON-Linker-CON. In certain embodiments, L1 is a bond. In certain embodiments, L1 is -Linker. In certain embodiments, L1 is —CON-Linker. In certain embodiments, L1 is —CON-Linker-CON.
In certain embodiments, RC is absent, H, C1-C4 alkyl optionally substituted with 1-3 optionally substituted halogens and/or 1-2 hydroxyl groups, or a group of structure:
wherein R4, R5, and R6 are each independently H, F, Cl, Br, I, CN, NRN1RN2, —(CH2)KOH, —(CH2)KO(C1-C4 alkyl) optionally substituted with 1-3 independently selected halogens, C1-C3 alkyl optionally substituted with 1-3 independently selected halogens, C1-C3-alkoxy optionally substituted with 1-3 independently selected halogens, —(CH2)KCOOH, —(CH2)KC(═O)O—(C1-C4 alkyl) optionally substituted with 1-3 independently selected halogens, O—C(═O)—(C1-C4 alkyl) optionally substituted with 1-3 independently selected halogens, or —C(═O)—(C1-C4 alkyl) optionally substituted with 1-3 independently selected halogens.
In certain embodiments, each occurrence of RN2 is independently H or C1-C3 alkyl optionally substituted with 1-3 independently selected halogens and/or 1-2 hydroxyl groups.
In certain embodiments, RC is
In certain embodiments, R1 and R3 are each independently (C3-C8 saturated carbocyclic)-(CH2)K—, wherein the carbocyclic is further substituted with -L1 and —RC.
In certain embodiments, each occurrence of RN is independently H or C1-C3 alkyl optionally substituted with 1-3 independently selected halogens and/or 1-2 hydroxyl groups.
In certain embodiments, R2 is a group depicted in
In certain embodiments, R2 is —(CH2)K—N(RN1)—C(═O)RAM.
In certain embodiments, RAM is H, C1-C4 alkyl optionally substituted with 1-3 independently selected halogens and/or 1-2 hydroxyl groups, —(CH2)KCOOH, —(CH2)KC(═O)O(C1-C4 alkyl) optionally substituted with 1-3 independently selected halogens, —OC(═O)(C1-C4 alkyl) optionally substituted with 1-3 independently selected halogens, —C(═O)(C1-C4 alkyl) optionally substituted with 1-3 independently selected halogens, or —(CH2)K—NRN3RN4.
In certain embodiments, R2 is
wherein:
optionally substituted with 1-3 C1-C3 alkyl groups optionally substituted with 1-3 independently selected halogens,
where in each —(CH2)K group is optionally substituted with 1-4 C1-C3 alkyl groups optionally substituted with 1-3 fluoro groups or 1-2 hydroxyl groups.
In certain embodiments, the ASGPRBM group comprises the structure:
wherein:
In certain embodiments, RA is methyl or ethyl, either of which is optionally substituted with 1-3 fluorines.
In certain embodiments, ZA is a PEG group containing from 1 to 4 ethylene glycol residues.
In certain embodiments, the ASGPRBM group comprises one of the following (Mamidyala, et al., 2012, J. Am. Chem. Soc. 134:1978-1981):
In certain embodiments, the ASGPRBM group comprises one of the following (Sanhueza, et al., 2017, J. Am. Chem. Soc. 139:3528-3536):
In certain embodiments, the Linker is a polyethylene glycol containing linker having 1-12 ethylene glycol residues.
In certain embodiments, the Linker comprises the structure:
—CH2CH2(OCH2CH2)mOCH2—, —(CH2)mCH2—, —[N(Ra)—CH(Rb)(C═O)]m—, or a polypropylene glycol or polypropylene-co-polyethylene glycol group containing 1-100 alkylene glycol units;
In certain embodiments, the Linker comprises the structure —[N(N)R′—(CH2)1-15—C(═O)]—, wherein R′ is H or a C1-C3 alkyl optionally substituted with 1-2 hydroxyl groups, and m is an integer ranging from 1 to 100.
In certain embodiments, the Linker comprises the structure
—Z-D-Z′—,
wherein:
—(CH2)i—C(R2)═C(R2)— (cis or trans), —(CH2)i—≡—, or —Y—C(═O)—Y—;
In certain embodiments, the Linker comprises a structure:
—CH2—(OCH2CH2)n—CH2—, —(CH2CH2O)n—CH2CH2—, or —(CH2CH2CH2O)n—,
wherein each n and n′ is independently an integer ranging from 1 to 25; in certain embodiments 1 to 15; in certain embodiments 1 to 12; in certain embodiments 2 to 11; in certain embodiments 2 to 10; in certain embodiments 2 to 8; in certain embodiments 2 to 6; in certain embodiments 2 to 5; in certain embodiments 2 to 4; in certain embodiments 2 or 3; in certain embodiments 1, 2, 3, 4, 5, 6, 7, or 8.
In certain embodiments, the Linker comprises a structure:
-PEG-CON-PEG-
wherein each PEG is independently a polyethylene glycol group containing from 1-12 ethylene glycol residues and CON is a triazole group
In certain embodiments, the CON comprises a structure:
wherein R′ and R″ are each independently H, methyl, or a bond.
In certain embodiments, the CON comprises a diamide structure:
—C(═O)—N(R1)—(CH2)n″—N(R1)C(═O)—,
—N(R1)—C(═O)(CH2)n″—C(═O)N(R1)—, or
—N(R1)—C(═O)(CH2)n″—N(R1)C(═O)—;
wherein each R1 is independently H or C1-C3 alkyl, and n″ is independently an integer from 0 to 8, in certain embodiments 1 to 7, in certain embodiments 1, 2, 3, 4, 5 or 6.
In certain embodiments, the CON comprises a structure:
wherein:
In certain embodiments, the CON comprises a structure:
Any Protein binder that binds to a protein of interest (which in certain embodiments is a circulating protein) is useful within formula (I) and formula (Ia) of the present disclosure. In certain non-limiting embodiments, the binder is a small molecule. In certain non-limiting embodiments, the binder is a peptide and/or polypeptide.
The Protein binder can be incorporated within the compounds of formula (I) and/or formula (Ia) using any methods known in the art and/or any techniques described or illustrated herein. For example, the Protein binder can be attached to a Linker and/or CON using amide coupling, ester coupling, nucleophilic displacement, electrophilic displacement, radical coupling, or any other synthetic method known in the art. The attachment position of the Protein binder should be such that the attached Protein binder in formula (I) or formula (Ia) can still bind to the protein of interest. It is within the standard experimentation expected from, and known to, one skilled in the art to contemplate the mode of binding of the Protein binder to the protein of interest and identify potential sites of attachment on the Protein binder, and/or attach the Protein binder to a CON and/or linker and ascertain whether such attachment disturbs binding of the Protein binder to the protein of interest.
In certain embodiments, the Protein binder is an antibody, such as, but not limited to, a monoclonal antibody. The antibody of interest can be incorporated within the compounds of formula (I) or formula (Ia) using any methods known in the art and/or any techniques described or illustrated herein. For example, the antibody can be attached to a Linker and/or CON through a carboxylic acid group on the antibody's surface, using for example amide or ester formation chemistry. For example, the antibody can be attached to a Linker and/or CON through an amine group on the antibody's surface, using for example amide formation chemistry. For example, the antibody can be attached to a Linker and/or CON through a thiol group on the antibody's surface, using for example nucleophilic substitution chemistry. In that case, the surface cysteine residue can exist in the wild-type form of the antibody and/or can be introduced by mutation, using for example site-directed mutagenesis. The Linker and/or CON useful within the disclosure can be any linker known in the art, as long as the presence of the linker does not significantly disturb the antibody's ability to bind to the protein of interest.
In certain embodiments, the Protein binder is a polypeptide. The polypeptide of interest can be incorporated within the compounds of formula (I) or formula (Ia) using any methods known in the art and/or any techniques described or illustrated herein. For example, the polypeptide can be attached to a Linker and/or CON through its C-terminus and/or its N-terminus, using for example amide or ester formation chemistry. For example, the polypeptide can be attached to a Linker and/or CON through any intermediate residue using for example amide or ester formation chemistry and/or nucleophilic displacement chemistry (for example, if the polypeptide has a thiol residue). The polypeptide can be synthesized by standard Fmoc-SPPS. Introduction of a linker at either the N- or C-terminus followed by a functional handle (N3, alkyne, and so forth) allows simple ligation to a targeting domain.
The Protein binders that are protein-based, such as antibodies, polypeptides, and the like, can be synthesized by various methods well known in the field, such as expression in E. coli for those not requiring post-translational modification (PTM) or in mammalian culture for those that do require PTM. These binding proteins can be made into bifunctional proteins by introduction of an unnatural amino acid tag for ligation (N3, alkyne, and so forth) followed by reaction with the corresponding targeting domain, or by many other well-known bioorthogonal reactions for specific tagging of proteins.
As will be understood by one skilled in the art, any Protein binder that may recognize and specifically bind to the protein of interest is useful in the present disclosure. The disclosure should not be construed to be limited to any one type of Protein binder, either known or heretofore unknown, provided that the Protein binder can specifically bind to the protein of interest, and prevent or minimize biological activity of the protein of interest.
In certain embodiments, the protein of interest is CD40OL. In certain embodiments, the Protein binder that binds to CD40OL comprises the following (wherein the wavy lines indicate potential non-limiting points of attachment to REAG within contemplated compounds of the disclosure):
In certain embodiments, the protein of interest is PCSK9. In certain embodiments, the Protein binder that binds to PCSK9 comprises the following (wherein the peptide C-terminus is optionally amidated, and wherein the wavy lines indicate potential non-limiting points of attachment to REAG within contemplated compounds of the disclosure):
In certain embodiments, the protein of interest is PCSK9. In certain embodiments, the Protein binder that binds to PCSK9 comprises any binder recited in WO2018/057409. In certain embodiments, the Protein binder comprises any of the following (wherein the wavy lines indicate potential non-limiting points of attachment to REAG within contemplated compounds of the disclosure):
In certain embodiments, the protein of interest is VEGF. In certain embodiments, the Protein binder that binds to VEGF comprises the following (wherein the peptide C-terminus is optionally amidated, and wherein the wavy lines indicate potential non-limiting points of attachment to REAG within contemplated compounds of the disclosure):
In certain embodiments, the protein of interest is TGF-beta. In certain embodiments, the Protein binder that binds to TGF-beta comprises the following (wherein the peptide C-terminus is optionally amidated, and wherein the wavy lines indicate potential non-limiting points of attachment to REAG within contemplated compounds of the disclosure):
In certain embodiments, the protein of interest is TSP-1. In certain embodiments, the Protein binder that binds to TSP-1 comprises the following (wherein the peptide C-terminus is optionally amidated, and wherein the wavy lines indicate potential non-limiting points of attachment to REAG within contemplated compounds of the disclosure):
In certain embodiments, the protein of interest is soluble uPAR. In certain embodiments, the Protein binder that binds to uPAR comprises the following (wherein the wavy lines indicate potential non-limiting points of attachment to REAG within contemplated compounds of the disclosure):
In certain embodiments, the protein of interest is soluble PSMA. In certain embodiments, the Protein binder that binds to PSMA comprises the following (wherein the wavy lines indicate potential non-limiting points of attachment to REAG within contemplated compounds of the disclosure):
In certain embodiments, the protein of interest is IL-2. In certain embodiments, the Protein binder that binds to IL-2 comprises the following (wherein the wavy lines indicate potential non-limiting points of attachment to REAG within contemplated compounds of the disclosure):
In certain embodiments, the protein of interest is GP120. In certain embodiments, the Protein binder that binds to GP120 comprises the following (wherein the wavy lines indicate potential non-limiting points of attachment to REAG within contemplated compounds of the disclosure):
In certain embodiments, the protein of interest is MIF. In certain embodiments, the Protein binder that binds to MIF comprises the following (wherein the wavy lines indicate potential non-limiting points of attachment to REAG within contemplated compounds of the disclosure):
In certain embodiments, the protein of interest is IgA, as known in the art or described elsewhere herein. In certain embodiments, the Protein binder that binds to MIF comprises any peptide recited in Hatanaka, et al., 2012, J. Biol. Chem. 287:43126-43136, such as but not limited to:
These peptides can be acyclic (as free thiols) or cyclized as oxidized thiols (disulfide bonds). Further, the disclosure contemplates incorporating these peptides in the compounds of the disclosure through N- and/or C-terminus conjugation.
In certain embodiments, the Protein binder that binds to IgA is any Fc-alpha receptor peptide mimetic recited in Heineke, et al., 2017, Eur. J. Immunol. 47:1835-1845, such as but not limited to:
These peptides can be acyclic (as free thiols) or cyclized as oxidized thiols (disulfide bonds). Further, the disclosure contemplates incorporating these peptides in the compounds of the disclosure through N- and/or C-terminus conjugation.
CLIPS indicates cyclization of linear peptides via reaction of thiol-functionalities of the cysteines with a small rigid entity; this anchor reacts exclusively with thiols and attaches to the peptide via covalent bonds. Non-limiting examples of CLIPS cross-linkers contemplated in the present disclosure include:
Any TNF binder that binds to TNF is useful within formula (II) and formula (IIa) of the present disclosure. In certain non-limiting embodiments, the binder is a small molecule. In certain non-limiting embodiments, the binder is a peptide and/or polypeptide.
The TNF binder can be incorporated within the compounds of formula (II) and formula (IIa) using any methods known in the art and/or any techniques described or illustrated herein. For example, the TNF binder can be attached to a Linker and/or CON using amide coupling, ester coupling, nucleophilic displacement, electrophilic displacement, radical coupling, or any other synthetic method known in the art. The attachment position of the TNF binder should be such that the attached TNF binder in formula (II) or formula (IIa) can still bind to TNF. It is within the standard experimentation expected from, and known to, one skilled in the art to contemplate the mode of binding of the TNF binder to TNF and identify potential sites of attachment on the TNF binder, and/or attach the TNF binder to a CON and/or linker and ascertain whether such attachment disturbs binding of the TNF binder to TNF.
In certain embodiments, the TNF binder is an antibody, such as, but not limited to, a monoclonal antibody. The antibody of interest can be incorporated within the compounds of formula (II) and formula (IIa) using any methods known in the art and/or any techniques described or illustrated herein. For example, the antibody can be attached to a Linker and/or CON through a carboxylic acid group on the antibody's surface, using for example amide or ester formation chemistry. For example, the antibody can be attached to a Linker and/or CON through an amine group on the antibody's surface, using for example amide formation chemistry. For example, the antibody can be attached to a Linker and/or CON through a thiol group on the antibody's surface, using for example nucleophilic substitution chemistry. In that case, the surface cysteine residue can exist in the wild-type form of the antibody and/or can be introduced by mutation, using for example site-directed mutagenesis. The Linker and/or CON useful within the disclosure can be any linker known in the art, as long as the presence of the linker does not significantly disturb the antibody's ability to bind to TNF.
In certain embodiments, the TNF binder is a polypeptide. The polypeptide of interest can be incorporated within the compounds of formula (II) and formula (IIa) using any methods known in the art and/or any techniques described or illustrated herein. For example, the polypeptide can be attached to a Linker and/or CON through its C-terminus and/or its N-terminus, using for example amide or ester formation chemistry. For example, the polypeptide can be attached to a Linker and/or CON through any intermediate residue using for example amide or ester formation chemistry and/or nucleophilic displacement chemistry (for example, if the polypeptide has a thiol residue). The polypeptide can be synthesized by standard Fmoc-SPPS. In certain embodiments, the C-terminus of the peptide is amidated. Introduction of a linker at either the N- or C-terminus followed by a functional handle (N3, alkyne, and so forth) allows simple ligation to an ASGPR targeting domain. In a non-limiting example:
The TNF binders that are protein-based, such as antibodies, polypeptides, and the like, can be synthesized by various methods well known in the field, such as expression in E. coli for those not requiring post-translational modification or in mammalian culture for those that do require PTM. These binding proteins can be made into bifunctional proteins targeting TNF-ASGPR by introduction of an unnatural amino acid tag for ligation (N3, alkyne, and so forth) followed by reaction with the corresponding ASGPR targeting domain, or by many other well-known bioorthogonal reactions for specific tagging of proteins.
As will be understood by one skilled in the art, any TNF binder that may recognize and specifically bind to TNF is useful in the present disclosure. The disclosure should not be construed to be limited to any one type of TNF binder, either known or heretofore unknown, provided that the TNF binder can specifically bind to TNF, and prevent or minimize biological activity of TNF.
In certain embodiments, the TNF binder comprises the polypeptide STPTRYS (SEQ ID NO:120) (Guangdong Yixue 2008, 29(1):55-57).
In certain embodiments, the TNF binder comprises the polypeptide CALWHWWHC SEQ ID NO:121) or C(T/S)WLHWWAC (SEQ ID NO:122) (Diyi Daxue Xuebao 2002, 22(7):597-599).
In certain embodiments, the TNF binder comprises any Tbab protein described in Zhu, et al., 2016, Protein Sci. 25:2066-2075.
In certain embodiments, the TNF binder comprises the polypeptide (L/M)HEL(Y/F)(L/M)X(W/Y/F) (SEQ ID NO:123), as described in Zhang, et al., 2003, Biochem. Biophys. Res. Commun. 310:1181-1187.
In certain embodiments, the TNF binder comprises one of the polypeptides:
In certain embodiments, the TNF binder comprises TNFR1 or TNFR2 (Yang & Yang, 2013, Fenxi Huaxue/Chinese J. Anal. Chem. 41:664-669).
In certain embodiments, the TNF binder comprises anticachexin C1 and/or C2 (Lian, et al., 2013, J. Am. Chem. Soc. 135:11990-11995).
In certain embodiments, the TNF binder comprises adalimumab, infliximab, etanercept, golimumab, and/or certolizumab.
In certain embodiments, the TNF binder comprises the 29.2 kDa scFv identified in Safarpour, et al., 2018, Iran. J. Pharm. Res. 17:743-752.
In certain embodiments, the TNF binder comprises GACPPCLWQVLCGGSGSGSG (SEQ ID NO:126) (which can be, in a non-limiting example, tris-bromomethyl mesitylene core sulfur linked; Luzi, et al., 2015, Protein Eng. Des. Sel. 28:45-52).
In certain embodiments, the TNF binder comprises any affibodies (˜60 amino acids) identified in Löfdahl, et al., 2009, N. Biotechnol. 26:251-259.
In certain embodiments, the TNF binder comprises any affibodies identified in Kronqvist, et al., 2008, Protein Eng. Des. Sel. 21:247-255.
In certain embodiments, the TNF binder comprises any affibodies identified in Jonsson, et al., 2009, Biotechnol. Appl. Biochem. 54:93-103.
In certain embodiments, the TNF binder comprises the bispecific albumin/TNF binding polypeptide identified in Nilvebrant, et al., 2011, PLoS One 6.
In certain embodiments, the TNF binder comprises the ubiquitin-based artificial binding protein identified in Hoffmann, et al., 2012, PLoS One 7:2-11.
In certain embodiments, the TNF binder comprises HIHDDLLRYYGW linear (SEQ ID NO:127) or tetra branched peptide (SEQ ID NO:128) identified in Brunetti, et al., 2014, Molecules 19:7255-7268.
In certain embodiments, the TNF binder comprises any TNF-α binding peptides (P51 and P52) identified in Alizadeh, et al., 2017, Eur. J. Pharm. Sci. 96:490-498.
In certain embodiments, the TNF binder comprises the scFv antibody identified in Alizadeh, et al., 2015, Adv. Pharm. Bull. 5:661-666.
In certain embodiments, the TNF binder comprises any TNF binding peptide recited in WO 2006/053568 (such as but not limited to KRWSRYF (SEQ ID NO:129), which may in certain embodiments be polyvalent), which is incorporated herein in its entirety by reference.
In certain embodiments, the TNF binder comprises any TNF binding peptide recited in WO 2015/055597 (such as but not limited to HIHDDLLRYYGW (SEQ ID NO:127), which may in certain embodiments be polyvalent), which is incorporated herein in its entirety by reference.
In certain embodiments, the TNF binder comprises YCWSQYLCY (SEQ ID NO:130) as identified in Arthritis & Rheumatism 2007,56(4):1164-74.
In certain embodiments, the TNF binder comprises DFLPHYKNTSLGHRP (SEQ ID NO:131) as identified in Chirinos-Rojas, et al., 1998, J. Immunol. 161:5621-5626.
In certain embodiments, the TNF binder comprises YCLYQSWCY (SEQ ID NO:132). In certain embodiments, the TNF binder is its reduced form (i.e., with an internal disulfide bond). In certain embodiments, the TNF binder is its oxidized form (i.e., without an internal disulfide bond). See
In certain embodiments, the TNF binder comprises one of the following:
(Zaka, et al., 2019, J. Biomol. Struct. Dyn. 37:2464-2476).
In certain embodiments, the TNF binder comprises one of the following:
(Shen, et al., 2014, Eur. J. Med. Chem. 85:119-126). See
In certain embodiments, the TNF binder comprises:
See
In certain embodiments, the TNF binder comprises:
See
In certain embodiments, the TNF binder comprises:
See
In certain embodiments, the TNF binder comprises one of the following (Saddala & Huang, 2019, J. Transl. Med. 17:1-16):
In certain embodiments, the TNF binder comprises SPD-304 and analogs thereof (He, et al., 2005, Science 310:1022-1025; Papaneophytou, et al., 2015, Medchemcomm 6:1196-1209):
Non-limiting chemical schemes to prepare and derivatize these compound is provided herein:
(Mettou, et al., 2018, SLAS Discov. 23:84-93). See
In certain embodiments, the TNF binder comprises a compound of formula (2a):
wherein:
For example, when A1 and A2 are 1-(3-(trifluoromethyl)phenyl)-1H-indole and 6,7-dimethyl-4H-chomen-4-one respectively, and X1 and X2 are both CH2, R3 and R4 form a heterocyclyl ring, such as but not limited to a piperazinyl ring.
In certain embodiments, the TNF binder comprises the small molecule IA-14069.
In certain embodiments, the TNF binder comprises
(Mouhsine, et al., 2017, Sci. Rep. 7:1-10 (2017). In certain embodiments, the Linker and/or Con can be attached the sulfonamido phenyl ring. See
In certain embodiments, the TNF binder comprises one of the following:
(Melagraki, et al., 2017, PLoS Comput. Biol. 13:1-27).
In certain embodiments, the TNF binder comprises one of the following:
(Melagraki, et al., 2018, Front. Pharmacol. 9:1-12).
In certain embodiments, the TNF binder comprises
(Ma, et al., 2014, J. Biol. Chem. 289:12457-12466). In certain embodiments, the Linker and/or CON can be attached to the phenyl group marked with an arrow. See
In certain embodiments, the TNF binder comprises one of the following:
wherein R indicates a non-limiting site of derivatization (Kumar, et al., 2011, Chem. Commun. 47:5010-5012). See
In certain embodiments, the TNF binder comprises
In certain embodiments, the TNF binder comprises any dihydro-benzo[cd]indole-6-sulfonamide or analogues depicted herein (non-limiting attachment points for REAG include R1 or the hydrophobic R group, including naphthyl, on the right hand side of the molecule):
In certain embodiments, the TNF binder comprises any of the following:
In certain embodiments, the TNF binder comprises any of the following:
In certain embodiments, the TNF binder comprises any of the following:
(Chen, et al., 2017, J. Chem. Inf. Model. 57:1101-1111).
In certain embodiments, the TNF binder comprises any compound disclosed in U.S. Pat. No. 10,266,532, which is incorporated herein in its entirety by reference.
In certain embodiments, the TNF binder comprises any compound disclosed in U.S. Pat. No. 9,879,016, which is incorporated herein in its entirety by reference.
In certain embodiments, the TNF binder comprises any compound disclosed in WO 2008/142623, which is incorporated herein in its entirety by reference.
In certain embodiments, the TNF binder comprises
In certain embodiments, the Linker and/or CON can be attached to the compound through the piperizinyl group (Blevitt, et al., 2017, J. Med. Chem. 60:3511-3517). See
In certain embodiments, the TNF binder comprises a compound of formula (2b):
or a pharmaceutically acceptable salt, tautomer, geometric isomer, or stereoisomer thereof, wherein:
In certain embodiments, the compound is not 1-(2-methylphenyl)-7-[2-(morpholin-4-yl)pyrimidin-5-yl]-2,3-dihydro-1H-pyrrolo[1,2-a]benzimidazole; 7-[2-(morpholin-4-yl)pyrimidin-5-yl]-1-phenyl-2,3-dihydro-1H-pyrrolo[1,2-a]benzimidazole; (1R or S)-7-(6-methylsulfonyl-3-pyridyl)-1-phenyl-2,3-dihydro-1H-pyrrolo[1,2-a]benzimidazole; [5-[(1R or S)-1-phenyl-2,3-dihydro-1H-pyrrolo[1,2-a]benzimidazol-7-yl]-2-pyridyl]methanol; tert-butyl 4-[5-[(1R or S)-1-phenyl-2,3-dihydro-1H-pyrrolo[1,2-a]benzimidazol-7-yl]-2-pyridyl]piperazine-1-carboxylate; (1R or S)-7-[6-chloromethyl)-3-pyridyl]-1-phenyl-2,3-dihydro-1H-pyrrolo[1,2-a]benzimidazole; (1R or S)-7-[(6-(methylsulfonylmethyl)-3-pyridyl]-1-phenyl-2,3-dihydro-1H-pyrrolo[1,2-a]benzimidazole; or (1R or S)-1-phenyl-7-[6-(piperazin-1-yl)pyridine-3-yl]-2,3-dihydro-1H-pyrrolo[1,2-a]benzimidazole.
In certain embodiments, the compound is 1-(2-methylphenyl)-7-[2-(morpholin-4-yl)pyrimidin-5-yl]-2,3-dihydro-1H-pyrrolo[1,2-a]benzimidazole; 7-[2-(morpholin-4-yl)pyrimidin-5-yl]-1-phenyl-2,3-dihydro-1H-pyrrolo[1,2-a]benzimidazole; (1R or S)-7-(6-methylsulfonyl-3-pyridyl)-1-phenyl-2,3-dihydro-1H-pyrrolo[1,2-a]benzimidazole; [5-[(1R or S)-1-phenyl-2,3-dihydro-1H-pyrrolo[1,2-a]benzimidazol-7-yl]-2-pyridyl]methanol; tert-butyl 4-[5-[(1R or S)-1-phenyl-2,3-dihydro-1H-pyrrolo[1,2-a]benzimidazol-7-yl]-2-pyridyl]piperazine-1-carboxylate; (1R or S)-7-[6-chloromethyl)-3-pyridyl]-1-phenyl-2,3-dihydro-1H-pyrrolo[1,2-a]benzimidazole; (1R or S)-7-[(6-(methylsulfonylmethyl)-3-pyridyl]-1-phenyl-2,3-dihydro-1H-pyrrolo[1,2-a]benzimidazole; or (1R or S)-1-phenyl-7-[6-(piperazin-1-yl)pyridine-3-yl]-2,3-dihydro-1H-pyrrolo[1,2-a]benzimidazole.
In certain embodiments, the compound of formula (2a) comprises one of the following:
wherein A2 is CH or N; A3 is CH or N; B1 is CH2 or O; B2 is CH2 or O; X is C or N; Y is C or N; Z1 is CH2 or O; and Z2 is CH2 or O.
In certain embodiments, R3a is selected from the group consisting of:
In certain embodiments, R3b is selected from the group consisting of:
In certain embodiments, R3a or R3b can be used to attach the TNF linker to the compound of the disclosure. This can be done, for example, using any hydroxyl, amino, amido, thiyl, or carboxylic acid group that is present in R3a or R3b as listed herein or that can be introduced therein. In any such cases, the hydroxyl group in R3a or R3b can be used for example to form an ester bond; the carboxylic group in R3a or R3b can be used for example to form an ester bond or an amide bond; the amino group in R3a or R3b can be used for example to form an amide group and an imine group, and so forth; the amino, amido, or thiyl group in R3a or R3b can be used for example to form a chemical linkage through alkylation or nucleophilic displacement, and so forth, as known to those skilled in the art.
In certain embodiments, R1 is selected from the group consisting of H, methyl, and hydroxyl.
In certain embodiments, R1 and R2 combine to form one of the following:
In certain embodiments, R4 is selected from the group consisting of:
In certain embodiments, the compound is selected from the group consisting of:
(4-(5-((S)-4-phenyl-3,4-dihydro-1H-benzo[4,5]imidazo[2,1-c][1,4]oxazin-7-yl)pyrimidin-2-yl)morpholin-2-yl)methanol;
In certain embodiments, the compound is selected from the group consisting of:
The disclosures of U.S. Pat. No. 10,266,532 B2 and No. 9,856,253 B2, and U.S. Patent Applications No. US20160304517A1 and US2018/0179198 A1, are incorporated herein in their entireties by reference.
or a pharmaceutically acceptable salt, tautomer, geometric isomer, or stereoisomer thereof, wherein:
In certain embodiments, the compound of formula (2c) comprises
wherein G is N or CH; and Z is CH or CF.
In certain embodiments of the compound of formula (2c), R1 is selected from the group consisting of:
In certain embodiments of the compound of formula (2c), R2 is selected from the group consisting of:
In certain embodiments, R2 can be used to attach the TNF linker to the compound of the disclosure. This can be done, for example, using any hydroxyl, amino, amido, thiyl, or carboxylic acid group that is present in R2 as listed herein or that can be introduced therein. In any such cases, the hydroxyl group in R2 can be used for example to form an ester bond; the carboxylic group in R2 can be used for example to form an ester bond or an amide bond; the amino group in R2 can be used for example to form an amide group and an imine group, and so forth; the amino, amido, or thiyl group in R2 can be used for example to form a chemical linkage through alkylation or nucleophilic displacement, and so forth, as known to those skilled in the art.
In certain embodiments, the compound is selected from the group consisting of:
Any autoantibody targeting moiety (AATM) that binds to an autoantibody is useful within the present disclosure. In certain non-limiting embodiments, the autoantibody is pathological. Any autoantibodies known in the art is contemplated within the present disclosure.
In certain embodiments, the AATM is any peptide and/or small molecule that binds to FcRn, as known in the art or described elsewhere herein.
In certain embodiments, the AATM comprises a FcRn antagonist, such as but not limited to rozanolixizumab (see, for example, Kiessling, et al., 2017, Sci. Transl. Med. 9:eaan1208).
In certain embodiments, the AATM comprises a FcRn antagonist, such as but not limited to efgartigimod (see, for example, Ulrichts, et al., 2018, J. Clin. Invest. 128(10):4372).
In certain embodiments, the AATM comprises 2,4-dinitrobenzene or any derivative or analogue thereof (wherein the phenyl ring is optionally substituted):
In certain embodiments, the AATM comprises the following cyclic peptide FcIII, or any reduced form thereof (e.g., any corresponding free thiol derivative thereof; see for example Science 2000, 287:1279-1283). The chemical group marked with * is a non-limiting position for attachment of Linker or CON in the compound of the disclosure.
also represented as
internal cystine form with C-terminus amidated).
In certain embodiments, the AATM comprises the following cyclic peptide FcIII-4C (amide), or any reduced form thereof (e.g., any corresponding free thiol derivative thereof; see Bioconjugate Chem. 2016, 27:1569). The chemical group marked with * is a non-limiting position for attachment of Linker or CON in the compound of the disclosure.
also represented as
internal cystine form with C-terminus amidated).
In certain embodiments, the AATM comprises a compound of formula (3a) or (3b):
wherein:
In certain embodiments, the AATM comprises one of the following compounds (see WO 2006/024175 A1). Each of the chemical groups marked with * illustrates a non-limiting position for attachment of Linker or CON in the compound of the disclosure.
In certain embodiments, the AATM comprises the following compound, wherein the chemical bond marked with illustrates a non-limiting position for attachment of Linker or CON in the compound of the disclosure (see Chemistry & Biology 18:1179-1188).
wherein:
The disclosures of U.S. Pat. No. 9,879,016 B2 and U.S. Patent Application No. US 2016/0304526 A1 are incorporated herein in their entireties by reference.
Additional Galactose- and Talose-Based ASGPR Binding Moieties
In certain embodiments, the present disclosure is directed to compounds which are useful for removing circulating proteins which are associated with a disease state or condition in a patient or subject according to the general chemical structure of Formula II:
The term “Extracellular Protein Targeting Ligand” as used herein is interchangeably used with the term CPBM (cellular protein binding moiety). The term “ASGPR Ligand” as used herein is interchangeably used with an asialoglycoprotein receptor (ASGPR) binding moiety as defined herein.
In the compound of Formula II, each [CON] is an optional connector chemical moiety which, when present, connects directly to [CPBM] or to [CRBM] or connects the [LINKER-2]to [CPBM] or to [CRBM].
In the compound of Formula II:
[LINKER-2]is a chemical moiety having a valency from 1 to 15 which covalently attaches to one or more [CRBM] and/or [CPBM] group, optionally through a [CON], including a [MULTICON] group, wherein said [LINKER-2]optionally itself contains one or more [CON] or [MULTICON] group(s);
A [MULTICON] group can connect one or more of a [CRBM] or [CPBM] to one or more of a [LINKER-2]. In various embodiments, [LINKER-2]has a valency of 1 to 10. In various embodiments, [LINKER-2]has a valency of 1 to 5. In various embodiments, [LINKER-2]has a valency of 1, 2 or 3. In various embodiments, in the compound of Formula II, the [LINKER-2]includes one or more of LinkerA, LinkerB, LinkerC, LinkerD, and/or combinations thereof as defined herein.
In the compound of Formula II, xx is independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, and 25.
In the compound of Formula II, yy is independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, and 25.
In the compound of Formula II, zz is independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, and 25.
In the compound of Formula II, X1 is 1 to 5 contiguous atoms independently selected from O, S, N(Rb), and C(R4)(R4), wherein if X1 is 1 atom then X1 is O, S, N(R6), or C(R4)(R4), if X1 is 2 atoms then no more than 1 atom of X1 is O, S, or N(R6), if X1 is 3, 4, or 5 atoms then no more than 2 atoms of X1 are O, S, or N(R6);
R3 at each occurrence is independently selected from hydrogen, alkyl, heteroalkyl, haloalkyl (including —CF3, —CHF2, —CH2F, —CH2CF3, —CH2CH2F, and —CF2CF3), arylalkyl, heteroarylalkyl, alkenyl, alkynyl, and, heteroaryl, heterocycle, —OR', and —NR8R9;
R4 is independently selected at each occurrence from hydrogen, heteroalkyl, alkyl, haloalkyl, arylalkyl, heteroarylalkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocycle, —OR6, —NR6R7,
R6 and R7 are independently selected at each occurrence from hydrogen, heteroalkyl, alkyl, arylalkyl, heteroaryl alkyl, alkenyl, alkynyl, and, haloalkyl, heteroaryl, heterocycle, -alkyl-OR8, -alkyl-NR8R9, C(O)R3, S(O)R3, C(S)R3, and S(O)2R3;
R8 and R9 are independently selected at each occurrence from hydrogen, heteroalkyl, alkyl, arylalkyl, heteroarylalkyl, alkenyl, alkynyl, aryl, heteroaryl, and heterocycle.
A. Galactose-Based ASGPR-Binding Cellular Receptor Binding Moieties of Formula II
In certain embodiments, the compound of Formula II is selected from:
In certain embodiments, the compound of Formula II has one of the following structures:
In various embodiments, the ASGPR ligand is linked at either the C1 or C5 (R1 or R5) position to form a degrading compound. In various embodiments, the ASGPR ligand is linked at C6 position to form a degrading compound. For example, when the ASGPR ligand is
then non-limiting examples of ASGPR binding compounds of Formula II include:
or the bi- or tri-substituted versions thereof or pharmaceutically acceptable salts thereof, where the bi- or tri-substitution refers to the number additional galactose derivatives attached to a linker moiety.
In any of the embodiments herein where an ASGPR ligand is drawn for use in a degrader the ASGPR ligand is typically linked through to the Extracellular Protein Targeting Ligand in the C5 position (e.g., which can refer to the adjacent C6 carbon hydroxyl or other functional moiety that can be used for linking purposes). When the linker and Extracellular Protein Targeting Ligand is connected through the C1 position, then that carbon is appropriately functionalized for linking, for example with a hydroxyl, amino, allyl, alkyne or hydroxyl-allyl group.
In various embodiments, the ASGPR ligand is not linked in the C3 or C4 position, because these positions chelate with the calcium for ASGPR binding in the liver. In certain embodiments, an ASGPR ligand useful for incorporation into a compound of Formula II is selected from:
In certain embodiments, the compound of Formula II is selected from:
B. Talose-Based ASGPR-Binding Cellular Receptor Binding Moieties of Formula II
In certain embodiments, the compound of Formula II is selected from:
In certain embodiments, the compound of Formula II is an Extracellular Protein degrading compound in which the ASGPR ligand is a ligand as described herein
In certain embodiments, in the compound of Formula II, the ASGPR ligand is linked at either the C1 or C5 (R1 or R5) position to form a degrading compound. In certain embodiments, in the compound of Formula II, the ASGPR ligand is linked at C6. In various embodiments, when the ASGPR ligand is
then non-limiting examples of ASGPR binding compounds of Formula II include:
or the bi- or tri-substituted versions thereof or pharmaceutically acceptable salts thereof, where the bi- or tri-substitution refers to the number additional galactose derivatives attached to a linker moiety. In certain embodiments the compound of Formula II is selected from:
wherein in certain embodiments R2 is selected from —NR6COR3, —NR6-(5-membered heteroaryl), and-NR6-(6-membered heteroaryl), each of which R2 groups is optionally substituted with 1, 2, 3, or 4 independent, substituents as described herein, for example 1, 2, 3, or 4 substituents independently selected from F, Cl, Br, haloalkyl, or alkyl.
In certain embodiments, the compound of Formula II is selected from:
wherein in certain embodiments R2 is selected from —NR6COR3, —NR6-(5-membered heteroaryl), and-NR6-(6-membered heteroaryl), each of which R2 groups is optionally substituted with 1, 2, 3, or 4 independent, substituents as described herein, for example 1, 2, 3, or 4 substituents independently selected from F, Cl, Br, haloalkyl, or alkyl.
In certain embodiments, the compound of Formula II is selected from:
wherein in certain embodiments R2 is selected from —NR6COR3, —NR6-(5-membered heteroaryl), and-NR6-(6-membered heteroaryl), each of which R2 groups is optionally substituted with 1, 2, 3, or 4 independent, substituents as described herein, for example 1, 2, 3, or 4 substituents independently selected from F, Cl, Br, haloalkyl, or alkyl.
In certain embodiments, the compound of Formula II is selected from:
wherein in certain embodiments R2 is selected from —NR6COR3, —NR6-(5-membered heteroaryl), and-NR6-(6-membered heteroaryl), each of which R2 groups is optionally substituted with 1, 2, 3, or 4 independent, substituents as described herein, for example 1, 2, 3, or 4 substituents independently selected from F, Cl, Br, haloalkyl, or alkyl.
In certain embodiments, the compound of Formula II is selected from:
wherein in certain embodiments R2 is selected from —NR6COR3, —NR6-(5-membered heteroaryl), and-NR6-(6-membered heteroaryl), each of which R2 groups is optionally substituted with 1, 2, 3, or 4 independent, substituents as described herein, for example 1, 2, 3, or 4 substituents independently selected from F, Cl, Br, haloalkyl, or alkyl.
In certain embodiments, the compound of Formula II is selected from:
wherein in certain embodiments R2 is selected from —NR6COR3, —NR6-(5-membered heteroaryl), and-NR6-(6-membered heteroaryl), each of which R2 groups is optionally substituted with 1, 2, 3, or 4 independent, substituents as described herein, for example 1, 2, 3, or 4 substituents independently selected from F, Cl, Br, haloalkyl, or alkyl.
In certain embodiments, the compound of Formula II is selected from:
wherein in certain embodiments R2 is selected from —NR6COR3, —NR6-(5-membered heteroaryl), and-NR6-(6-membered heteroaryl), each of which R2 groups is optionally substituted with 1, 2, 3, or 4 independent, substituents as described herein, for example 1, 2, 3, or 4 substituents independently selected from F, Cl, Br, haloalkyl, or alkyl.
In certain embodiments, the compound of Formula II is selected from:
wherein in certain embodiments R2 is selected from —NR6COR10, —NR6-(5-membered heteroaryl), and-NR6-(6-membered heteroaryl), each of which R2 groups is optionally substituted with 1, 2, 3, or 4 independent, substituents as described herein, for example 1, 2, 3, or 4 substituents independently selected from F, Cl, Br, haloalkyl, or alkyl.
In certain embodiments, the compound of Formula II is selected from:
wherein in certain embodiments R2 is selected from —NRbCOR10, —NR6-(5-membered heteroaryl), and-NR6-(6-membered heteroaryl), each of which R2 groups is optionally substituted with 1, 2, 3, or 4 independent, substituents as described herein, for example 1, 2, 3, or 4 substituents independently selected from F, Cl, Br, haloalkyl, or alkyl.
In certain embodiments, the compound of Formula II is selected from:
wherein in certain embodiments R2 is selected from —NR6COR10, —NR6-(5-membered heteroaryl), and-NR6-(6-membered heteroaryl), each of which R2 groups is optionally substituted with 1, 2, 3, or 4 independent, substituents as described herein, for example 1, 2, 3, or 4 substituents independently selected from F, Cl, Br, haloalkyl, or alkyl.
In certain embodiments, the compound of Formula II is selected from:
wherein in certain embodiments R2 is selected from —NR6COR10, —NR6-(5-membered heteroaryl), and-NR6-(6-membered heteroaryl), each of which R2 groups is optionally substituted with 1, 2, 3, or 4 independent, substituents as described herein, for example 1, 2, 3, or 4 substituents independently selected from F, Cl, Br, haloalkyl, or alkyl.
In certain embodiments, the compound of Formula II is selected from:
wherein in certain embodiments R2 is selected from —NR6COR10, —NR6-(5-membered heteroaryl), and-NR6-(6-membered heteroaryl), each of which R2 groups is optionally substituted with 1, 2, 3, or 4 independent, substituents as described herein, for example 1, 2, 3, or 4 substituents independently selected from F, Cl, Br, haloalkyl, or alkyl.
In certain embodiments, the compound of Formula II is selected from:
wherein in certain embodiments R2 is selected from —NR6COR10, —NR6-(5-membered heteroaryl), and-NR6-(6-membered heteroaryl), each of which R2 groups is optionally substituted with 1, 2, 3, or 4 independent, substituents as described herein, for example 1, 2, 3, or 4 substituents independently selected from F, Cl, Br, haloalkyl, or alkyl.
In certain embodiments, the compound of Formula II is selected from:
wherein in certain embodiments R2 is selected from —NR6COR10, —NR6-(5-membered heteroaryl), and-NR6-(6-membered heteroaryl), each of which R2 groups is optionally substituted with 1, 2, 3, or 4 independent, substituents as described herein, for example 1, 2, 3, or 4 substituents independently selected from F, Cl, Br, haloalkyl, or alkyl.
In certain embodiments, the compound of Formula II is selected from:
In certain embodiments, an ASGPR ligand useful for incorporation into a compound of Formula II is selected from:
C. The ASGPR Ligand/Binding Moiety in Compounds of Formula II
In certain embodiments, in the compound of Formula II, R1 is hydrogen.
In certain embodiments, in the compound of Formula II, R1 is
In certain embodiments, in the compound of Formula II, R1 is
In certain embodiments, in the compound of Formula II, R1 is
In certain embodiments, in the compound of Formula II, R1 is
In certain embodiments, in the compound of Formula II, R1 is
In certain embodiments, in the compound of Formula II, R1 is
In certain embodiments, in the compound of Formula II, R1 is C0-C6alkyl-cyano optionally substituted with 1, 2, 3, or 4 substituents.
In certain embodiments, in the compound of Formula II, R1 is alkyl optionally substituted with 1, 2, 3, or 4 substituents.
In certain embodiments, in the compound of Formula II, R1 is alkenyl optionally substituted with 1, 2, 3, or 4 substituents. In certain embodiments, in the compound of Formula II, R1 is alkynyl optionally substituted with 1, 2, 3, or 4 substituents. In certain embodiments, in the compound of Formula II, R1 is haloalkyl optionally substituted with 1, 2, 3, or 4 substituents. In certain embodiments, in the compound of Formula II, R1 is F.
In certain embodiments, in the compound of Formula II, R1 is Cl.
In certain embodiments, in the compound of Formula II, R1 is Br.
In certain embodiments, in the compound of Formula II, R1 is aryl optionally substituted with 1, 2, 3, or 4 substituents.
In certain embodiments, in the compound of Formula II, R1 is arylalkyl optionally substituted with 1, 2, 3, or 4 substituents.
In certain embodiments, in the compound of Formula II, R1 is heteroaryl optionally substituted with 1, 2, 3, or 4 substituents.
In certain embodiments, in the compound of Formula II, R1 is heteroaryl alkyl optionally substituted with 1, 2, 3, or 4 substituents.
In certain embodiments, in the compound of Formula II, R1 is heterocycle optionally substituted with 1, 2, 3, or 4 substituents.
In certain embodiments, in the compound of Formula II, R1 is heterocycloalkyl optionally substituted with 1, 2, 3, or 4 substituents.
In certain embodiments, in the compound of Formula II, R1 is haloalkoxy optionally substituted with 1, 2, 3, or 4 substituents.
In certain embodiments, in the compound of Formula II, R1 is —O-alkenyl, —O-alkynyl, C0-C6alkyl-OR6, C0-C6alkyl-SR6, C0-C6alkyl-NR6R7, C0-C6alkyl-C(O)R3, C0-C6alkyl-S(O)R3, C0-C6alkyl-C(S)R3, C0-C6alkyl-S(O)2R3, C0-C6alkyl-N(R8)—C(O)R3, C0-C6alkyl-N(R8)-S(O)R3, C0-C6alkyl-N(R8)—C(S)R3, C0-C6alkyl-N(R8)-S(O)2R3 C0-C6alkyl-O—C(O)R3, C0-C6alkyl-O—S(O)R3, C0-C6alkyl-O—C(S)R3, —N═S(O)(R3)2, C0-C6alkylN3, or C0-C6alkyl-O—S(O)2R3, each of which is optionally substituted with 1, 2, 3, or 4 substituents.
In certain embodiments, in the compound of Formula II, R2 is aryl optionally substituted with 1, 2, 3, or 4 substituents.
In certain embodiments, in the compound of Formula II, R2 is heterocycle optionally substituted with 1, 2, 3, or 4 substituents.
In certain embodiments, in the compound of Formula II, R2 is heteroaryl containing 1 or 2 heteroatoms independently selected from N, O, and S optionally substituted with 1, 2, 3, or 4 substituents.
In certain embodiments, in the compound of Formula II, R2 is selected from
In certain embodiments, in the compound of Formula II, R2 is heterocycle optionally substituted with 1, 2, 3, or 4 substituents.
In certain embodiments, in the compound of Formula II, R2 is —NR8—S(O)—R3 optionally substituted with 1, 2, 3, or 4 substituents.
In certain embodiments, in the compound of Formula II, R2 is —NR8—C(S)—R3 optionally substituted with 1, 2, 3, or 4 substituents.
In certain embodiments, in the compound of Formula II, R2 is —NR8—S(O)(NR6)—R3 optionally substituted with 1, 2, 3, or 4 substituents.
In certain embodiments, in the compound of Formula II, R2 is —N═S(O)(R3)2 optionally substituted with 1, 2, 3, or 4 substituents.
In certain embodiments, in the compound of Formula II, R2 is —NR8C(O)NR9S(O)2R3 optionally substituted with 1, 2, 3, or 4 substituents.
In certain embodiments, in the compound of Formula II, R2 is —NR8—S(O)2—R10 optionally substituted with 1, 2, 3, or 4 substituents.
In certain embodiments, in the compound of Formula II, R2 is —NR8—C(NR6)—R3 optionally substituted with 1, 2, 3, or 4 substituents.
In certain embodiments, in the compound of Formula II, R2 is hydrogen.
In certain embodiments, in the compound of Formula II, R2 is R10,
In certain embodiments, in the compound of Formula II, R2 is alkyl-C(O)—R3.
In certain embodiments, in the compound of Formula II, R2 is —C(O)—R3.
In certain embodiments, in the compound of Formula II, R2 is alkyl.
In certain embodiments, in the compound of Formula II, R2 is haloalkyl.
In certain embodiments, in the compound of Formula II, R2 is —OC(O)R3.
In certain embodiments, in the compound of Formula II, R2 is —NR8—C(O)R10.
In certain embodiments, in the compound of Formula II, R2 is alkenyl optionally substituted with 1, 2, 3, or 4 substituents.
In certain embodiments, in the compound of Formula II, R2 is allyl optionally substituted with 1, 2, 3, or 4 substituents.
In certain embodiments, in the compound of Formula II, R2 is alkynyl optionally substituted with 1, 2, 3, or 4 substituents.
In certain embodiments, in the compound of Formula II, R2 is —NR6-alkenyl optionally substituted with 1, 2, 3, or 4 substituents.
In certain embodiments, in the compound of Formula II, R2 is —O-alkenyl optionally substituted with 1, 2, 3, or 4 substituents.
In certain embodiments, in the compound of Formula II, R2 is —NR6-alkynyl optionally substituted with 1, 2, 3, or 4 substituents.
In certain embodiments, in the compound of Formula II, R2 is —NR6-heteroaryl optionally substituted with 1, 2, 3, or 4 substituents.
In certain embodiments, in the compound of Formula II, R2 is —NR6-aryl optionally substituted with 1, 2, 3, or 4 substituents.
In certain embodiments, in the compound of Formula II, R2 is —O-heteroaryl optionally substituted with 1, 2, 3, or 4 substituents.
In certain embodiments, in the compound of Formula II, R2 is —O-aryl optionally substituted with 1, 2, 3, or 4 substituents.
In certain embodiments, in the compound of Formula II, R2 is —O-alkynyl optionally substituted with 1, 2, 3, or 4 substituents.
In certain embodiments, in the compound of Formula II, R2 is selected from and
In certain embodiments, in the compound of Formula II, R2 is selected from
In certain embodiments, in the compound of Formula II, R2 is selected from
wherein R is an optional substituent as defined herein.
In certain embodiments, in the compound of Formula II, R2 is selected from
In certain embodiments, in the compound of Formula II, R2A is selected from
wherein R is an optional substituent as defined herein.
In certain embodiments, in the compound of Formula II, R2A is selected from
In certain embodiments, in the compound of Formula II, R2 is selected from
In certain embodiments, in the compound of Formula II, R2 is selected from
In certain embodiments, in the compound of Formula II, R2 is selected from
In certain embodiments, in the compound of Formula II, R2 is selected from
In certain embodiments, in the compound of Formula II, R2 is selected from
In certain embodiments, in the compound of Formula II, R2 is selected from
In certain embodiments, in the compound of Formula II, R2 is selected from
In certain embodiments, in the compound of Formula II, R2 is selected from
In certain embodiments, in the compound of Formula II, R2 is selected from
In certain embodiments, in the compound of Formula II, R2 is selected from
In certain embodiments, in the compound of Formula II, R2 is selected from
In certain embodiments, in the compound of Formula II, R2 is selected from
In certain embodiments, in the compound of Formula II, R2 is selected from
In certain embodiments, in the compound of Formula II, R2 is selected from
In certain embodiments, in the compound of Formula II, R2 is selected from
In certain embodiments, in the compound of Formula II, R2 is selected from
In certain embodiments, in the compound of Formula II, R2 is selected from
In certain embodiments, in the compound of Formula II, R2 is selected from
In certain embodiments, in the compound of Formula II, R2 is selected from
In certain embodiments, in the compound of Formula II, R2 is selected from
In certain embodiments, in the compound of Formula II, R2 is selected from
In certain embodiments, in the compound of Formula II, R2 is selected from
In certain embodiments, in the compound of Formula II, R2 is selected from
In certain embodiments, in the compound of Formula II, R2 is selected from
In certain embodiments, in the compound of Formula II, R2 is selected from
In certain embodiments, in the compound of Formula II, R2 is selected from
In certain embodiments, in the compound of Formula II, R2 is selected from
In certain embodiments, in the compound of Formula II, R2 is selected from
In certain embodiments, in the compound of Formula II, R2 is selected from
In certain embodiments, in the compound of Formula II, R2 or R2A is selected from
In certain embodiments, in the compound of Formula II, R2 is selected from
In certain embodiments, in the compound of Formula II, R2 is selected from
In certain embodiments, in the compound of Formula II, R2 is selected from
In certain embodiments, in the compound of Formula II, R2 is selected from
In certain embodiments, in the compound of Formula II, R2 is selected from
In certain embodiments, in the compound of Formula II, R2 is a spirocyclic heterocycle, for example, and without limitation,
In certain embodiments, in the compound of Formula II, R2 is a silicon containing heterocycle, for example, and without limitation,
In certain embodiments, in the compound of Formula II, R2 is substituted with SF5, for example, and without limitation,
In certain embodiments, in the compound of Formula II, R2 is substituted with a sulfoxime, for example, and without limitation,
In certain embodiments, in the compound of Formula II, R10 is selected from bicyclic heterocycle.
In certain embodiments, in the compound of Formula II, R10 is selected from spirocyclic heterocycle.
In certain embodiments, in the compound of Formula II, R10 is selected from —NR6-heterocycle.
In certain embodiments, in the compound of Formula II, R10 is selected from
In certain embodiments, in the compound of Formula II, R10 is selected from
In certain embodiments, in the compound of Formula II, R10 is selected from
In certain embodiments, in the compound of Formula II, R10 is selected from
In certain embodiments, in the compound of Formula II, Cycle is selected from
In certain embodiments, in the compound of Formula II, R30 is selected from:
In certain embodiments, in the compound of Formula II, R200 is
In certain embodiments, in the compound of Formula II, R200 is
In certain embodiments, in the compound of Formula II, R200 is
In certain embodiments, in the compound of Formula II, R200 is
In certain embodiments, in the compound of Formula II, R200 is
In certain embodiments, in the compound of Formula II, R200 is
In certain embodiments, in the compound of Formula II, R200 is
In certain embodiments, in the compound of Formula II, R200 is
In certain embodiments, in the compound of Formula II, R200 is
In certain embodiments, in the compound of Formula II, R200 is
In certain embodiments, in the compound of Formula II, R200 is
In certain embodiments, in the compound of Formula II, R200 is
Linkers
In non-limiting embodiments, in the compound of Formula II, LinkerA and LinkerB are independently selected from:
wherein:
R11, R12, R13, R14, R15, R16, R17, R18, R19, and R20 are independently at each occurrence selected from the group consisting of a bond, alkyl, —C(O)—, —C(O)O—, —OC(O)—, —SO2—, —S(O)—, —C(S)—, —C(O)NR6—, —NR6C(O)—, —O—, —S—, —NR6—, —C(R21R21)—, —P(O)(R3)O—, —P(O)(R3)—, a divalent residue of a natural or unnatural amino acid, alkenyl, alkynyl, haloalkyl, alkoxy, and, heterocycle, heteroaryl, —CH2CH2—[O—(CH2)2]n—O—, CH2CH2—[O—(CH2)2]n—NR6—, —CH2CH2—[O—(CH2)2]n—, —[—(CH2)2—O]n—, —[O—(CH2)2]n—, —[O—CH(CH3)C(O)]n—, —[C(O)—CH(CH3)—O]n—,
—[O—CH2C(O)]n—, —[C(O)—CH2—O]n—, a divalent residue of a fatty acid, a divalent residue of an unsaturated or saturated mono- or di-carboxylic acid; each of which is optionally substituted with 1, 2, 3, or 4 substituents independently selected from R21;
n is independently selected at each instance from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;
R21 is independently at each occurrence selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, F, Cl, Br, I, hydroxyl, alkoxy, azide, amino, cyano, —NR6R7, —NR8SO2R3, —NR8S(O)R3, haloalkyl, heteroalkyl, and, heteroaryl, and heterocycle;
and the remaining variables are as defined herein.
In certain embodiments, in the compound of Formula II, LinkerA is bond and LinkerB is
In certain embodiments, in the compound of Formula II, LinkerB is bond and LinkerA is
In certain embodiments, in the compound of Formula II, a divalent residue of an amino acid is selected from
wherein the amino acid can be oriented in either direction and wherein the amino acid can be in the L- or D-form or a mixture thereof.
In certain embodiments, in the compound of Formula II, a divalent residue of a dicarboxylic acid is generated from a nucleophilic addition reaction:
Non-limiting embodiments of a divalent residue of a dicarboxylic acid generated from a nucleophilic addition reaction include:
In certain embodiments, in the compound of Formula II, a divalent residue of a dicarboxylic acid is generated from a condensation reaction:
Non-limiting embodiments of a divalent residue of a dicarboxylic acid generated from a condensation include:
Non-limiting embodiments of a divalent residue of a saturated dicarboxylic acid include:
Non-limiting embodiments of a divalent residue of a saturated dicarboxylic acid include:
Non-limiting embodiments of a divalent residue of a saturated monocarboxylic acid is selected from butyric acid (—OC(O)(CH2)2CH2—), caproic acid (—OC(O)(CH2)4CH2—), caprylic acid (—OC(O)(CH2)5CH2—), capric acid (—OC(O)(CH2)8CH2—), lauric acid (—OC(O)(CH2)10CH2—), myristic acid (—OC(O)(CH2)12CH2—), pentadecanoic acid (—OC(O)(CH2)13CH2—), palmitic acid (—OC(O)(CH2)14CH2—), stearic acid (—OC(O)(CH2)16CH2—), behenic acid (—OC(O)(CH2)20CH2—), and lignoceric acid (—OC(O)(CH2)22CH2—);
Non-limiting embodiments of a divalent residue of a fatty acid include residues selected from linoleic acid, palmitoleic acid, vaccenic acid, paullinic acid, oleic acid, elaidic acid, gondoic acid, gadoleic acid, nervonic acid, myristoleic acid, and erucic acid:
Non-limiting embodiments of a divalent residue of a fatty acid is selected from linoleic acid (—C(O)(CH2)7(CH)2CH2(CH)2(CH2)4CH2—), docosahexaenoic acid
(—C(O)(CH2)2(CHCHCH2)6CH2—), eicosapentaenoic acid (—C(O)(CH2)3(CHCHCH2)5CH2—), alpha-linolenic acid (—C(O)(CH2)7(CHCHCH2)3CH2—) stearidonic acid
(—C(O)(CH2)4(CHCHCH2)4CH2—), y-linolenic acid (—C(O)(CH2)4(CHCHCH2)3(CH2)3CH2—), arachidonic acid (—C(O)(CH2)3,(CHCHCH2)4(CH2)4CH2—), docosatetraenoic acid
(—C(O)(CH2)5(CHCHCH2)4(CH2)4CH2—), palmitoleic acid (—C(O)(CH2)7CHCH(CH2)5CH2—), vaccenic acid (—C(O)(CH2)9CHCH(CH2)5CH2—), paullinic acid
(—C(O)(CH2)11CHCH(CH2)5CH2—), oleic acid (—C(O)(CH2)7CHCH(CH2)7CH2—), elaidic acid
(—C(O)(CH2)7CHCH(CH2)7CH2—), gondoic acid (—C(O)(CH2)9CHCH(CH2)7CH2—), gadoleic acid (—C(O)(CH2)7CHCH(CH2)9CH2—), nervonic acid (—C(O)(CH2)13CHCH(CH2)3CH2—), mead acid (—C(O)(CH2)3(CHCHCH2)3(CH2)6CH2—), myristoleic acid (—C(O)(CH2)7CHCH(CH2)3CH2—), and erucic acid (—C(O)(CH2)11CHCH(CH2)7CH2—).
In certain embodiments, in the compound of Formula II, LinkerC is selected from:
wherein:
R22 is independently at each occurrence selected from the group consisting of alkyl, —C(O)N—, —NC(O)—, —N—, —C(R21)—, —P(O)O—, —P(O)—, —P(O)(NR6R7)N—, alkenyl, haloalkyl, aryl, heterocycle, and heteroaryl, each of which is optionally substituted with 1, 2, 3, or 4 substituents independently selected from R21;
and the remaining variables are as defined herein.
In certain embodiments, in the compound of Formula II, LinkerD is selected from:
wherein:
R32 is independently at each occurrence selected from the group consisting of alkyl, N+X—, —C—, alkenyl, haloalkyl, aryl, heterocycle, and heteroaryl, each of which is optionally substituted with 1, 2, 3, or 4 substituents independently selected from R21;
X— is an anionic group, for example Br— or Cl−; and
all other variables are as defined herein.
In certain embodiments, in the compound of Formula II, LinkerA is selected from:
wherein each heteroaryl, heterocycle, cycloalkyl, and aryl can optionally be substituted with 1, 2, 3, or 4 of any combination of halogen, alkyl, haloalkyl, and, heteroaryl, heterocycle, or cycloalkyl, as allowed by valence.
In certain embodiments, in the compound of Formula II, LinkerA is selected from:
wherein each heteroaryl, heterocycle, cycloalkyl, and and can optionally be substituted with 1, 2, 3, or 4 of any combination of halogen, alkyl, haloalkyl, aryl, heteroaryl, heterocycle, or cycloalkyl, as allowed by valence.
In certain embodiments, in the compound of Formula II, LinkerB is selected from:
In certain embodiments, in the compound of Formula II, LinkerB is selected from:
In certain embodiments, in the compound of Formula II, LinkerB, LinkerC, or LinkerD is selected from:
wherein tt is independently selected from 1, 2, or 3 and ss is 3 minus tt (3-tt).
In certain embodiments, in the compound of Formula II, LinkerB, LinkerC, or LinkerD is selected from:
wherein tt and ss are as defined herein.
In certain embodiments, in the compound of Formula II, LinkerB, LinkerC, or LinkerD is selected from:
wherein each heteroaryl, heterocycle, cycloalkyl, and aryl can optionally be substituted with 1, 2, 3, or 4 of any combination of halogen, alkyl, haloalkyl, aryl, heteroaryl, heterocycle, or cycloalkyl, as allowed by valence; and tt and ss are as defined herein.
In certain embodiments, in the compound of Formula II, LinkerB, LinkerC, or LinkerD is selected from:
wherein each heteroaryl, heterocycle, cycloalkyl, and aryl can optionally be substituted with 1, 2 3, or 4 of any combination of halogen, alkyl, haloalkyl, and, heteroaryl, heterocycle, or cycloalkyl, as allowed by valence: and tt and ss are as defined herein.
In certain embodiments, in the compound of Formula II, LinkerB, LinkerC, or LinkerD is selected from:
wherein each heteroaryl and aryl can optionally be substituted with 1, 2, 3, or 4 of any combination of halogen, alkyl, haloalkyl, aryl, heteroaryl, heterocycle, or cycloalkyl, as allowed by valence; and tt and ss are as defined herein.
In certain embodiments, in the compound of Formula II, LinkerA is selected from:
In certain embodiments, in the compound of Formula II, LinkerA is selected from:
In certain embodiments, in the compound of Formula II, LinkerA is selected from:
In certain embodiments, in the compound of Formula II, LinkerA is selected from:
In certain embodiments, in the compound of Formula II, LinkerB is selected from:
In certain embodiments, in the compound of Formula II, LinkerB is selected from:
In certain embodiments, in the compound of Formula II, LinkerB is selected from:
In certain embodiments, in the compound of Formula II, LinkerB is selected from:
In certain embodiments, in the compound of Formula II, LinkerC is selected from:
In certain embodiments, in the compound of Formula II, LinkerC is selected from:
In certain embodiments, in the compound of Formula II, LinkerC is selected from:
In certain embodiments, in the compound of Formula II, LinkerC is selected from:
In certain embodiments, in the compound of Formula II, LinkerC is selected from:
In certain embodiments, in the compound of Formula II, LinkerC is selected from:
In certain embodiments, in the compound of Formula II, LinkerC is selected from:
In certain embodiments, in the compound of Formula II, LinkerC is selected from:
In certain embodiments, in the compound of Formula II, LinkerD is selected from:
In certain embodiments, in the compound of Formula II, LinkerD is selected from:
In certain embodiments, in the compound of Formula II, LinkerD is selected from:
In certain embodiments, in the compound of Formula II, LinkerD is selected from:
In certain embodiments, in the compound of Formula II, LinkerD is selected from:
In certain embodiments, in the compound of Formula II, LinkerD is selected from:
In certain embodiments, in the compound of Formula II, LinkerD is selected from:
In certain embodiments, in the compound of Formula II, the LinkerA is selected from
In certain embodiments, in the compound of Formula II, the LinkerA is selected from
In certain embodiments, in the compound of Formula II, the LinkerA is selected from
In certain embodiments, the LinkerA is selected from
wherein each is optionally substituted with 1, 2, 3, or 4 substituents substituent selected from R21.
In certain embodiments, in the compound of Formula II, LinkerA is selected from:
In certain embodiments, in the compound of Formula II, the LinkerA is selected from
In certain embodiments, in the compound of Formula II, the LinkerA is selected from
In certain embodiments, in the compound of Formula II, the LinkerA is selected from
In certain embodiments, in the compound of Formula II, the LinkerA is selected from
In certain embodiments, in the compound of Formula II, the LinkerA is selected from
In certain embodiments, in the compound of Formula II, the LinkerA is selected from
In certain embodiments, in the compound of Formula II, the LinkerA is selected from
In certain embodiments, in the compound of Formula II, the LinkerA is selected from
In certain embodiments, in the compound of Formula II, the LinkerA is selected from
In certain embodiments, in the compound of Formula II, the LinkerA is selected from
In certain embodiments, in the compound of Formula II, the LinkerA is selected from
In certain embodiments, in the compound of Formula II, the LinkerA is selected from
In certain embodiments, in the compound of Formula II, the LinkerA is selected from
In certain embodiments, in the compound of Formula II, the LinkerB is selected from
In certain embodiments, in the compound of Formula II, the LinkerB is selected from
In certain embodiments, in the compound of Formula II, the LinkerB is selected from
In certain embodiments, in the compound of Formula II, the LinkerB is selected from wherein each is optionally substituted with 1, 2, 3, or 4 substituents substituent selected from R21.
In certain embodiments, in the compound of Formula II LinkerB is selected from:
In certain embodiments, in the compound of Formula II, the LinkerB is selected from:
In certain embodiments, in the compound of Formula II, the LinkerB is selected from:
In certain embodiments, in the compound of Formula II, the LinkerB is selected from:
In certain embodiments, in the compound of Formula II, the LinkerB is selected from:
In certain embodiments, in the compound of Formula II, the LinkerB is selected from:
In certain embodiments, in the compound of Formula II, the LinkerB is selected from:
In certain embodiments, in the compound of Formula II, the LinkerB is selected from:
In certain embodiments, in the compound of Formula II, LinkerB-LinkerA is selected from:
In certain embodiments, in the compound of Formula II, LinkerB-LinkerA is selected from:
In certain embodiments, in the compound of Formula II, the LinkerC is selected from:
In certain embodiments, in the compound of Formula II, the LinkerC is selected from:
In certain embodiments, in the compound of Formula II, the LinkerC is selected from:
In certain embodiments, in the compound of Formula II, the LinkerC is selected from:
In certain embodiments, in the compound of Formula II, the LinkerC is selected from:
In certain embodiments, in the compound of Formula II, the LinkerC is selected from:
In certain embodiments, in the compound of Formula II, the LinkerC is selected from:
In certain embodiments, in the compound of Formula II, the LinkerC is selected from: wherein each is optionally substituted with 1, 2, 3, or 4 substituents substituent selected from R21.
In certain embodiments, in the compound of Formula II, the LinkerC is selected from:
In certain embodiments, in the compound of Formula II, the LinkerC is selected from:
In certain embodiments, in the compound of Formula II, the LinkerC is selected from:
In certain embodiments, in the compound of Formula II, the LinkerC is selected from:
In certain embodiments, in the compound of Formula II, the LinkerC is selected from:
In certain embodiments, in the compound of Formula II, the LinkerC is selected from:
In certain embodiments, in the compound of Formula II, the LinkerC is selected from:
In certain embodiments, in the compound of Formula II, the LinkerC is selected from:
In certain embodiments, in the compound of Formula II, LinkerC-(LinkerA)2 is selected from:
In certain embodiments, in the compound of Formula II, LinkerC-(LinkerA)2 is selected from:
In certain embodiments, in the compound of Formula II, LinkerC-(LinkerA)2 is selected from:
In certain embodiments, in the compound of Formula II, LinkerC-(LinkerA)2 is selected from:
In certain embodiments, in the compound of Formula II, LinkerD is selected from:
In certain embodiments, in the compound of Formula II, LinkerD is selected from:
wherein each is optionally substituted with 1, 2, 3, or 4 substituents are selected from R21.
In certain embodiments, in the compound of Formula II, LinkerB-(LinkerA) is selected from
In certain embodiments, in the compound of Formula II, LinkerC-(LinkerA) is selected from
In certain embodiments, in the compound of Formula II, LinkerD-(LinkerA) is selected from
In various embodiments, R4 is independently selected at each occurrence from hydrogen, heteroalkyl, alkyl, haloalkyl, arylalkyl, heteroarylalkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocycle, —OR6, —NR6R7, C(O)R3, S(O)R3, C(S)R3, and S(O)2R3.
In various embodiments, in the compound of Formula II, R5 is independently selected from hydrogen, heteroalkyl,
C0-C6alkyl-cyano, alkyl, alkenyl, alkynyl, haloalkyl, F, Cl, Br, I, aryl, arylalkyl, heteroaryl, heteroarylalkyl, heterocycle, heterocycloalkyl, haloalkoxy, —O-alkenyl, —O-alkynyl, C0-C6alkyl-OR6, C0-C6alkyl-SR6, C0-C6alkyl-NR6R7, C0-C6alkyl-C(O)R3, C0-C6alkyl-S(O)R3, C0-C6alkyl-C(S)R3, C0-C6alkyl-S(O)2R3, C0-C6alkyl-N(R8)—C(O)R3, C0-C6alkyl-N(R8)—S(O)R3, C0-C6alkyl-N(R8)—C(S)R3, C0-C6alkyl-N(R8)—S(O)2R3 C0-C6alkyl-O—C(O)R3, C0-C6alkyl-O—S(O)R3, C0-C6alkyl-O—C(S)R3, —N═S(O)(R3)2, C0-C6alkylN3, and C0-C6alkyl-O—S(O)2R3, each of which is optionally substituted with 1, 2, 3, or 4 substituents.
In various embodiments, in the compound of Formula II, R6 and R7 are independently selected at each occurrence from hydrogen, heteroalkyl, alkyl, arylalkyl, heteroaryl alkyl, alkenyl, alkynyl, and, haloalkyl, heteroaryl, heterocycle, -alkyl-OR8, -alkyl-NR8R9, C(O)R3, S(O)R3, C(S)R3, and S(O)2R3.
In various embodiments, in the compound of Formula II, R8 and R9 are independently selected at each occurrence from hydrogen, heteroalkyl, alkyl, arylalkyl, heteroarylalkyl, alkenyl, alkynyl, aryl, heteroaryl, and heterocycle.
In various embodiments, the compound of Formula II has the structure of Formula II-A. In various embodiments, in the compound of Formula II, [Protein binder], [TNF binder] and [AATM] are as defined herein.
A compound of Formula II-A, having the structure:
Formula II-A
wherein:
[CPBM] is a cellular protein binding moiety selected from a [Protein binder], a [TNF binder], and a molecule that binds to an autoantibody [AATM];
[ASGPBM] is an asialoglycoprotein receptor binding moiety having the structure selected from
each [CON] is an optional connector chemical moiety which, when present, connects the [LIN] to [CPBM] or to [ASGPBM];
[LIN] is [LINKER] or [LINKER-2], each of which is a chemical moiety having a valency from 1 to 15, which covalently attaches to one or more [ASGPBM] or [CPBM] groups, optionally through a [CON], wherein the [LIN] optionally itself contains one or more [CON] groups;
R2 is
wherein RAM is H, C1-C4 alkyl optionally substituted with up to 3 halo groups and one or two hydroxyl groups, —(CH2)KCOOH, —(CH2)KC(O)O—(C1-C4 alkyl) optionally substituted with 1-3 halo groups, —O—C(O)—(C1-C4 alkyl) optionally substituted with 1-3 halo groups, —C(O)—(C1-C4 alkyl) optionally substituted with 1-3 halo groups, or —(CH2)K—NRN3RN4, or
R2 is
wherein
optionally substituted with up to three C1-C3 alkyl groups which are optionally substituted with up to three halo groups; or
RN, RN1, RN2, RN3, RN4 are each independently H or C1-C3 alkyl optionally substituted with one to three halo groups or one or two hydroxyl groups and each —(CH2)K group is optionally substituted with 1-4 C1-C3 alkyl groups which are optionally substituted with 1-3 fluoro groups or 1-2 hydroxyl groups;
IM is independently at each occurrence an integer from 0 to 6;
K is independently at each occurrence an integer from 0 to 4;
k′ is an integer ranging from 1 to 15;
j′ is an integer ranging from 1 to 15;
h and h′ are each independently an integer ranging from 0 to 15;
iL is 0 to 15;
with the proviso that at least one of h, h′, and iL is at least 1,
or a salt, stereoisomer, or solvate thereof.
In various embodiments, in the compound of Formula II-A, R2 is —NC(═O)CH3.
D. Other-Based ASGPR-Binding Moieties
In some embodiments, the ASGPR binding moieties can be any of the moieties described in: Reshitko, G. S., et al., “Synthesis and Evaluation of New Trivalent Ligands for Hepatocyte Targeting via the Asialoglycoprotein Receptor,” Bioconjugate Chem, doi: 10.1021/acs.bioconjchem.0c00202; Majouga, A. G., et al., “Identification of Novel Small-Molecule ASGP-R Ligands,” Current Drug Delivery, 2016,13, 1303-1312, doi: 10.2174/1567201813666160719144651; Olshanova, A. S., et al., “Synthesis of a new betulinic acid glycoconjugate with N-acetyl-D-galactosamine for the targeted delivery to hepatocellular carcinoma cells,” Russian Chemical Bulletin, International Edition, Vol. 69, No. 1, pp. 158-163, January 2020; Yamansarov, E. Yu., et al., “New ASGPR-targeted ligands based on glycoconjugated natural triterpenoids,” Russian Chemical Bulletin, International Edition, Vol. 68, No. 12, pp. 2331-2338, December 2019; Congdon, M. D., et al., “Enhanced Binding and Reduced Immunogenicity of Glycoconjugates Prepared via Solid-State Photoactivation of Aliphatic Diazirine Carbohydrates,” Bioconjugate Chem, doi: 10.1021/acs.bioconjchem.0c00555; and Dhawan, V., et al., “Polysaccharide conjugates surpass monosaccharide ligands in hepatospecific targeting—Synthesis and comparative in silico and in vitro assessment,” Carbohydrate Research 509 (2021) 108417, doi: 10.1016/j.carres.2021.108417. The following ASGPR binding moieties are illustrative and not intended to be limiting.
1. GalNAc-Tyrosine Based Moieties
In some embodiments, the ASGPR binding moiety can be a moiety having the structure of M1, M2, M3, or M4, or a combination thereof. In the structures of M1, M2, M3, and M4, X is independently at each occurrence O, NH, or S. In various embodiments, compounds of Formula I or Formula II can have one, two, or three ASGPR binding moieties with the structure of M1, M2, M3, or M4.
In various embodiments, ASGPR binding moieties M1 to M4 can be conjugated to any suitable [CON], [Linker], or [Linker-2]as described herein and in Congdon, M. D., et al., “Enhanced Binding and Reduced Immunogenicity of Glycoconjugates Prepared via Solid-State Photoactivation of Aliphatic Diazirine Carbohydrates,” Bioconjugate Chem, doi: 10.1021/acs.bioconjchem.0c00555.
2. Trivalent Triazole-Based Moieties
In some embodiments, the ASGPR binding moiety can be a moiety having the structure of M5:
In the structures M5, each R is independently at each occurrence R1 or R2,
In various embodiments, compounds of Formula I or Formula II contain an ASGPR binding moiety with the structure of M5. In various embodiments, each R in M5 is R1. In various embodiments, each R in M5 is R2.
In various embodiments, ASGPR binding moiety M5 can be conjugated/bonded to any suitable [CON], [Linker], or [Linker-2] as described herein and in Reshitko, G. S., et al., “Synthesis and Evaluation of New Trivalent Ligands for Hepatocyte Targeting via the Asialoglycoprotein Receptor,” Bioconjugate Chem, doi: 10.1021/acs.bioconjchem.0c00202.
3. Galactose- and Agarose-derived Behenic Acid Ester Moieties
In various embodiments, the ASGPR binding moiety can be the galactose behenic acid ester-derived moiety M7:
In the structure M7, Y is OH or NHAc.
In various embodiments, the ASGPR binding moiety can be the agarose behenic acid ester-derived moiety M8:
In various embodiments, ASGPR binding moieties M7 and M8 can be conjugated to any suitable [CON], [Linker], or [Linker-2]as described herein and in Dhawan, V., et al., “Polysaccharide conjugates surpass monosaccharide ligands in hepatospecific targeting—Synthesis and comparative in silico and in vitro assessment,” Carbohydrate Research 509 (2021) 108417, doi: 10.1016/j.carres.2021.108417.
4. Other Small Molecule ASGPR Binding Moieties
In various embodiments, the ASGPR binding moiety can be any of the compounds 2-18 below:
In various embodiments, in compounds 15 and 16, R is CH2OAc, COOH, or CH2OH. Compounds 2-18 can be conjugated/bonded to any suitable [CON], [Linker], or [Linker-2]as described herein and in Majouga, A. G., et al., “Identification of Novel Small-Molecule ASGP-R Ligands,” Current Drug Delivery, 2016, 13, 1303-1312, doi: 10.2174/1567201813666160719144651; Olshanova, A. S., et al., “Synthesis of a new betulinic acid glycoconjugate with N-acetyl-D-galactosamine for the targeted delivery to hepatocellular carcinoma cells,” Russian Chemical Bulletin, International Edition, Vol. 69, No. 1, pp. 158-163, January 2020; Yamansarov, E. Yu., et al., “New ASGPR-targeted ligands based on glycoconjugated natural triterpenoids,” Russian Chemical Bulletin, International Edition, Vol. 68, No. 12, pp. 2331-2338, December 2019. Compounds 2-18 can be attached through any suitable reactive group contained therein. Without limitation, compounds 2-13 can be attached to a CON], [Linker], or [Linker-2]through or by reaction with at least one OH, NH, vinyl, alkynyl, amide, acid, ester, ketone, or aromatic halogen contained in compounds 2-18. Suitable reaction modes for attaching compounds 2-18 to a [CON], [Linker], or [Linker-2]as described herein include, but are not limited to, substitution (e.g. alkylation of OH or NH groups), esterification (forming an ester), amidation (forming an amide), transesterification (exchanging one ester for another), transamidation (exchanging one amide for another), azide-alkyne cycloaddition, and other reactions capable of forming C—C, N—C, or O—C bonds with vinyl and alkynyl groups such as cycloadditions, aminations, oxidations, alkylations, rearrangement reactions (e.g. Claisen, Cope, etc.), and the like.
The compounds described herein can possess one or more stereocenters, and each stereocenter can exist independently in either the (R) or (S) configuration. In certain embodiments, compounds described herein are present in optically active or racemic forms. It is to be understood that the compounds described herein encompass racemic, optically-active, regioisomeric and stereoisomeric forms, or combinations thereof that possess the therapeutically useful properties described herein. Preparation of optically active forms is achieved in any suitable manner, including by way of non-limiting example, by resolution of the racemic form with recrystallization techniques, synthesis from optically-active starting materials, chiral synthesis, or chromatographic separation using a chiral stationary phase. In certain embodiments, a mixture of one or more isomer is utilized as the therapeutic compound described herein. In other embodiments, compounds described herein contain one or more chiral centers. These compounds are prepared by any means, including stereoselective synthesis, enantioselective synthesis and/or separation of a mixture of enantiomers and/ or diastereomers. Resolution of compounds and isomers thereof is achieved by any means including, by way of non-limiting example, chemical processes, enzymatic processes, fractional crystallization, distillation, and chromatography.
The methods and formulations described herein include the use of N-oxides (if appropriate), crystalline forms (also known as polymorphs), solvates, amorphous phases, and/or pharmaceutically acceptable salts of compounds having the structure of any compound(s) described herein, as well as metabolites and active metabolites of these compounds having the same type of activity. Solvates include water, ether (e.g., tetrahydrofuran, methyl tert-butyl ether) or alcohol (e.g., ethanol) solvates, acetates and the like. In certain embodiments, the compounds described herein exist in solvated forms with pharmaceutically acceptable solvents such as water, and ethanol. In other embodiments, the compounds described herein exist in unsolvated form.
In certain embodiments, the compound(s) described herein can exist as tautomers. All tautomers are included within the scope of the compounds presented herein.
In certain embodiments, compounds described herein are prepared as prodrugs. A “prodrug” refers to an agent that is converted into the parent drug in vivo. In certain embodiments, upon in vivo administration, a prodrug is chemically converted to the biologically, pharmaceutically or therapeutically active form of the compound. In other embodiments, a prodrug is enzymatically metabolized by one or more steps or processes to the biologically, pharmaceutically or therapeutically active form of the compound.
In certain embodiments, sites on, for example, the aromatic ring portion of compound(s) described herein are susceptible to various metabolic reactions. Incorporation of appropriate substituents on the aromatic ring structures may reduce, minimize or eliminate this metabolic pathway. In certain embodiments, the appropriate substituent to decrease or eliminate the susceptibility of the aromatic ring to metabolic reactions is, by way of example only, a deuterium, a halogen, or an alkyl group.
Compounds described herein also include isotopically-labeled compounds wherein one or more atoms is replaced by an atom having the same atomic number, but an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes suitable for inclusion in the compounds described herein include and are not limited to 2H, 3H, 11C, 13C, 14C, 36Cl, 18F 123I, 125I, 13N, 15N, 15O, 17O, 18O, 32P, and 35S. In certain embodiments, isotopically-labeled compounds are useful in drug and/or substrate tissue distribution studies. In other embodiments, substitution with heavier isotopes such as deuterium affords greater metabolic stability (for example, increased in vivo half-life or reduced dosage requirements). In yet other embodiments, substitution with positron emitting isotopes, such as 11C, 18F, 15O and 13N, is useful in Positron Emission Topography (PET) studies for examining substrate receptor occupancy. Isotopically-labeled compounds are prepared by any suitable method or by processes using an appropriate isotopically-labeled reagent in place of the non-labeled reagent otherwise employed.
In certain embodiments, the compounds described herein are labeled by other means, including, but not limited to, the use of chromophores or fluorescent moieties, bioluminescent labels, or chemiluminescent labels.
The compounds described herein, and other related compounds having different substituents are synthesized using techniques and materials described herein and as described, for example, in Fieser & Fieser's Reagents for Organic Synthesis, Volumes 1-17 (John Wiley and Sons, 1991); Rodd's Chemistry of Carbon Compounds, Volumes 1-5 and Supplementals (Elsevier Science Publishers, 1989); Organic Reactions, Volumes 1-40 (John Wiley and Sons, 1991), Larock's Comprehensive Organic Transformations (VCH Publishers Inc., 1989), March, Advanced Organic Chemistry 4th Ed., (Wiley 1992); Carey & Sundberg, Advanced Organic Chemistry 4th Ed., Vols. A and B (Plenum 2000,2001), and Green & Wuts, Protective Groups in Organic Synthesis 3rd Ed., (Wiley 1999) (all of which are incorporated by reference for such disclosure). General methods for the preparation of compound as described herein are modified by the use of appropriate reagents and conditions, for the introduction of the various moieties found in the formula as provided herein.
Compounds described herein are synthesized using any suitable procedures starting from compounds that are available from commercial sources, or are prepared using procedures described herein.
In certain embodiments, reactive functional groups, such as hydroxyl, amino, imino, thio or carboxy groups, are protected in order to avoid their unwanted participation in reactions. Protecting groups are used to block some or all of the reactive moieties and prevent such groups from participating in chemical reactions until the protective group is removed. In other embodiments, each protective group is removable by a different means. Protective groups that are cleaved under totally disparate reaction conditions fulfill the requirement of differential removal.
In certain embodiments, protective groups are removed by acid, base, reducing conditions (such as, for example, hydrogenolysis), and/or oxidative conditions. Groups such as trityl, dimethoxytrityl, acetal and t-butyldimethylsilyl are acid labile and are used to protect carboxy and hydroxy reactive moieties in the presence of amino groups protected with Cbz groups, which are removable by hydrogenolysis, and Fmoc groups, which are base labile. Carboxylic acid and hydroxy reactive moieties are blocked with base labile groups such as, but not limited to, methyl, ethyl, and acetyl, in the presence of amines that are blocked with acid labile groups, such as t-butyl carbamate, or with carbamates that are both acid and base stable but hydrolytically removable.
In certain embodiments, carboxylic acid and hydroxy reactive moieties are blocked with hydrolytically removable protective groups such as the benzyl group, while amine groups capable of hydrogen bonding with acids are blocked with base labile groups such as Fmoc. Carboxylic acid reactive moieties are protected by conversion to simple ester compounds as exemplified herein, which include conversion to alkyl esters, or are blocked with oxidatively-removable protective groups such as 2,4-dimethoxybenzyl, while co-existing amino groups are blocked with fluoride labile silyl carbamates.
Allyl blocking groups are useful in the presence of acid- and base-protecting groups since the former are stable and are subsequently removed by metal or pi-acid catalysts. For example, an allyl-blocked carboxylic acid is deprotected with a palladium-catalyzed reaction in the presence of acid labile t-butyl carbamate or base-labile acetate amine protecting groups. Yet another form of protecting group is a resin to which a compound or intermediate is attached. As long as the residue is attached to the resin, that functional group is blocked and does not react. Once released from the resin, the functional group is available to react.
Typically blocking/protecting groups may be selected from:
Other protecting groups, plus a detailed description of techniques applicable to the creation of protecting groups and their removal are described in Greene & Wuts, Protective Groups in Organic Synthesis, 3rd Ed., John Wiley & Sons, New York, N.Y., 1999, and Kocienski, Protective Groups, Thieme Verlag, New York, N.Y., 1994, which are incorporated herein by reference for such disclosure.
The compositions containing the compound(s) described herein include a pharmaceutical composition comprising at least one compound as described herein and at least one pharmaceutically acceptable carrier. In certain embodiments, the composition is formulated for an administration route such as oral or parenteral, for example, transdermal, transmucosal (e.g., sublingual, lingual, (trans)buccal, (trans)urethral, vaginal (e.g., trans- and perivaginally), (intra)nasal and (trans)rectal, intravesical, intrapulmonary, intraduodenal, intragastrical, intrathecal, subcutaneous, intramuscular, intradermal, intra-arterial, intravenous, intrabronchial, inhalation, and topical administration.
The compounds of the disclosure can be used to treat certain diseases and/or disorders, such as, but not limited to, autoimmune diseases (such as but not limited to IgA nephropathy), cancer, inflammation, and any other disease or disorder contemplated herein.
The methods described herein include administering to the subject a therapeutically effective amount of at least one compound described herein, which is optionally formulated in a pharmaceutical composition. In various embodiments, a therapeutically effective amount of at least one compound described herein present in a pharmaceutical composition is the only therapeutically active compound in a pharmaceutical composition. In certain embodiments, the method further comprises administering to the subject an additional therapeutic agent that treats the disease or disorder.
In certain embodiments, administering the compound(s) described herein to the subject allows for administering a lower dose of the additional therapeutic agent as compared to the dose of the additional therapeutic agent alone that is required to achieve similar results in treating the disease or disorder in the subject. For example, in certain embodiments, the compound(s) described herein enhance(s) the activity of the additional therapeutic compound, thereby allowing for a lower dose of the additional therapeutic compound to provide the same effect.
In certain embodiments, the compound(s) described herein and the therapeutic agent are co-administered to the subject. In other embodiments, the compound(s) described herein and the therapeutic agent are coformulated and co-administered to the subject.
In certain embodiments, the subject is a mammal. In other embodiments, the mammal is a human.
The compounds useful within the methods described herein can be used in combination with one or more additional therapeutic agents useful for treating the disease or disorder, and/or with an additional therapeutic agents that reduce or ameliorate the symptoms and/or side-effects of therapeutic agent used in the treatment of the disease or disorder. These additional therapeutic agents may comprise compounds that are commercially available or synthetically accessible to those skilled in the art. When the additional therapeutic agents useful for treating the disease or disorder are used, these additional therapeutic agents are known to treat, or reduce the symptoms of the disease or disorder.
In various embodiments, a synergistic effect is observed when a compound as described herein is administered with one or more additional therapeutic agents or compounds. A synergistic effect may be calculated, for example, using suitable methods such as, for example, the Sigmoid-E. equation (Holford & Scheiner, 1981, Clin. Pharmacokinet. 6:429-453), the equation of Loewe additivity (Loewe & Muischnek, 1926, Arch. Exp. Pathol Pharmacol. 114:313-326) and the median-effect equation (Chou & Talalay, 1984, Adv. Enzyme Regul. 22:27-55). Each equation referred to above may be applied to experimental data to generate a corresponding graph to aid in assessing the effects of the drug combination. The corresponding graphs associated with the equations referred to above are the concentration-effect curve, isobologram curve and combination index curve, respectively.
The regimen of administration may affect what constitutes an effective amount. The therapeutic formulations may be administered to the subject either prior to or after the onset of the disease or disorder. Further, several divided dosages, as well as staggered dosages may be administered daily or sequentially, or the dose may be continuously infused, or may be a bolus injection. Further, the dosages of the therapeutic formulations may be proportionally increased or decreased as indicated by the exigencies of the therapeutic or prophylactic situation.
Administration of the compositions described herein to a patient, preferably a mammal, more preferably a human, may be carried out using known procedures, at dosages and for periods of time effective to treat the disease or disorder in the patient. An effective amount of the therapeutic compound necessary to achieve a therapeutic effect may vary according to factors such as the state of the disease or disorder in the patient; the age, sex, and weight of the patient;
and the ability of the therapeutic compound to treat the disease or disorder in the patient. Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation. A non-limiting example of an effective dose range for a therapeutic compound described herein is from about 1 and 5,000 mg/kg of body weight/per day. One of ordinary skill in the art would be able to study the relevant factors and make the determination regarding the effective amount of the therapeutic compound without undue experimentation.
Actual dosage levels of the active ingredients in the pharmaceutical compositions described herein may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.
In particular, the selected dosage level depends upon a variety of factors including the activity of the particular compound employed, the time of administration, the rate of excretion of the compound, the duration of the treatment, other drugs, compounds or materials used in combination with the compound, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well, known in the medical arts.
A medical doctor, e.g., physician or veterinarian, having ordinary skill in the art may readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the compounds described herein employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.
In particular embodiments, it is especially advantageous to formulate the compound in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the patients to be treated; each unit containing a predetermined quantity of therapeutic compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical vehicle. The dosage unit forms of the compound(s) described herein are dictated by and directly dependent on (a) the unique characteristics of the therapeutic compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding/formulating such a therapeutic compound.
In certain embodiments, the compositions described herein are formulated using one or more pharmaceutically acceptable excipients or carriers. In certain embodiments, the pharmaceutical compositions described herein comprise a therapeutically effective amount of a compound described herein and a pharmaceutically acceptable carrier.
The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms may be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it is preferable to include isotonic agents, for example, sugars, sodium chloride, or polyalcohols such as mannitol and sorbitol, in the composition. Prolonged absorption of the injectable compositions may be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate or gelatin.
In certain embodiments, the compositions described herein are administered to the patient in dosages that range from one to five times per day or more. In other embodiments, the compositions described herein are administered to the patient in range of dosages that include, but are not limited to, once every day, every two, days, every three days to once a week, and once every two weeks. It is readily apparent to one skilled in the art that the frequency of administration of the various combination compositions described herein varies from individual to individual depending on many factors including, but not limited to, age, disease or disorder to be treated, gender, overall health, and other factors. Thus, administration of the compounds and compositions described herein should not be construed to be limited to any particular dosage regime and the precise dosage and composition to be administered to any patient is determined by the attending physician taking all other factors about the patient into account.
The compound(s) described herein for administration may be in the range of from about 1 μg to about 10,000 mg, about 20 μg to about 9,500 mg, about 40 μg to about 9,000 mg, about 75 μg to about 8,500 mg, about 150 μg to about 7,500 mg, about 200 μg to about 7,000 mg, about 350 μg to about 6,000 mg, about 500 μg to about 5,000 mg, about 750 μg to about 4,000 mg, about 1 mg to about 3,000 mg, about 10 mg to about 2,500 mg, about 20 mg to about 2,000 mg, about 25 mg to about 1,500 mg, about 30 mg to about 1,000 mg, about 40 mg to about 900 mg, about 50 mg to about 800 mg, about 60 mg to about 750 mg, about 70 mg to about 600 mg, about 80 mg to about 500 mg, and any and all whole or partial increments therebetween.
In some embodiments, the dose of a compound described herein is from about 1 mg and about 2,500 mg. In some embodiments, a dose of a compound described herein used in compositions described herein is less than about 10,000 mg, or less than about 8,000 mg, or less than about 6,000 mg, or less than about 5,000 mg, or less than about 3,000 mg, or less than about 2,000 mg, or less than about 1,000 mg, or less than about 500 mg, or less than about 200 mg, or less than about 50 mg. Similarly, in some embodiments, a dose of a second compound as described herein is less than about 1,000 mg, or less than about 800 mg, or less than about 600 mg, or less than about 500 mg, or less than about 400 mg, or less than about 300 mg, or less than about 200 mg, or less than about 100 mg, or less than about 50 mg, or less than about 40 mg, or less than about 30 mg, or less than about 25 mg, or less than about 20 mg, or less than about 15 mg, or less than about 10 mg, or less than about 5 mg, or less than about 2 mg, or less than about 1 mg, or less than about 0.5 mg, and any and all whole or partial increments thereof.
In certain embodiments, a composition as described herein is a packaged pharmaceutical composition comprising a container holding a therapeutically effective amount of a compound described herein, alone or in combination with a second pharmaceutical agent; and instructions for using the compound to treat, or reduce one or more symptoms of a disease or disorder in a patient.
Formulations may be employed in admixtures with conventional excipients, i.e., pharmaceutically acceptable organic or inorganic carrier substances suitable for oral, parenteral, nasal, intravenous, subcutaneous, enteral, or any other suitable mode of administration, known to the art. The pharmaceutical preparations may be sterilized and if desired mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure buffers, coloring, flavoring and/or aromatic substances and the like. They may also be combined where desired with other active agents, e.g., other analgesic agents.
Routes of administration of any of the compositions described herein include oral, nasal, rectal, intravaginal, parenteral, buccal, sublingual or topical. The compounds for use in the compositions described herein can be formulated for administration by any suitable route, such as for oral or parenteral, for example, transdermal, transmucosal (e.g., sublingual, lingual, (trans)buccal, (trans)urethral, vaginal (e.g., trans- and perivaginally), (intra)nasal and (trans)rectal), intravesical, intrapulmonary, intraduodenal, intragastrical, intrathecal, subcutaneous, intramuscular, intradermal, intra-arterial, intravenous, intrabronchial, inhalation, and topical administration.
Suitable compositions and dosage forms include, for example, tablets, capsules, caplets, pills, gel caps, troches, dispersions, suspensions, solutions, syrups, granules, beads, transdermal patches, gels, powders, pellets, magmas, lozenges, creams, pastes, plasters, lotions, discs, suppositories, liquid sprays for nasal or oral administration, dry powder or aerosolized formulations for inhalation, compositions and formulations for intravesical administration and the like. It should be understood that the formulations and compositions described herein are not limited to the particular formulations and compositions that are described herein.
Oral Administration
For oral application, particularly suitable are tablets, dragees, liquids, drops, suppositories, or capsules, caplets and gelcaps. The compositions intended for oral use may be prepared according to any method known in the art and such compositions may contain one or more agents selected from the group consisting of inert, non-toxic pharmaceutically excipients that are suitable for the manufacture of tablets. Such excipients include, for example an inert diluent such as lactose; granulating and disintegrating agents such as cornstarch; binding agents such as starch; and lubricating agents such as magnesium stearate. The tablets may be uncoated or they may be coated by known techniques for elegance or to delay the release of the active ingredients. Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert diluent.
For oral administration, the compound(s) described herein can be in the form of tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., polyvinylpyrrolidone, hydroxypropylcellulose or hydroxypropyl methylcellulose); fillers (e.g., cornstarch, lactose, microcrystalline cellulose or calcium phosphate); lubricants (e.g., magnesium stearate, talc, or silica); disintegrates (e.g., sodium starch glycollate); or wetting agents (e.g., sodium lauryl sulphate). If desired, the tablets may be coated using suitable methods and coating materials such as OPADRY™ film coating systems available from Colorcon, West Point, Pa. (e.g., OPADRY™ OY Type, OYC Type, Organic Enteric OY-P Type, Aqueous Enteric OY-A Type, OY-PM Type and OPADRY™ White, 32K18400). Liquid preparation for oral administration may be in the form of solutions, syrups or suspensions. The liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, methyl cellulose or hydrogenated edible fats); emulsifying agent (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters or ethyl alcohol); and preservatives (e.g., methyl or propyl p-hydroxy benzoates or sorbic acid).
Parenteral Administration
For parenteral administration, the compounds as described herein may be formulated for injection or infusion, for example, intravenous, intramuscular or subcutaneous injection or infusion, or for administration in a bolus dose and/or continuous infusion. Suspensions, solutions or emulsions in an oily or aqueous vehicle, optionally containing other formulatory agents such as suspending, stabilizing and/or dispersing agents may be used.
Sterile injectable forms of the compositions described herein may be aqueous or oleaginous suspension. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. Sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono- or di-glycerides. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant, such as Ph. Hely or similar alcohol.
Additional Administration Forms
Additional dosage forms suitable for use with the compound(s) and compositions described herein include dosage forms as described in U.S. Pat. Nos. 6,340,475; 6,488,962; 6,451,808; 5,972,389; 5,582,837; and 5,007,790. Additional dosage forms suitable for use with the compound(s) and compositions described herein also include dosage forms as described in U.S. Patent Applications Nos. 20030147952; 20030104062; 20030104053; 20030044466; 20030039688; and 20020051820. Additional dosage forms suitable for use with the compound(s) and compositions described herein also include dosage forms as described in PCT Applications Nos. WO 03/35041; WO 03/35040; WO 03/35029; WO 03/35177; WO 03/35039; WO 02/96404; WO 02/32416; WO 01/97783; WO 01/56544; WO 01/32217; WO 98/55107; WO 98/11879; WO 97/47285; WO 93/18755; and WO 90/11757.
In certain embodiments, the formulations described herein can be, but are not limited to, short-term, rapid-offset, as well as controlled, for example, sustained release, delayed release and pulsatile release formulations.
The term sustained release is used in its conventional sense to refer to a drug formulation that provides for gradual release of a drug over an extended period of time, and that may, although not necessarily, result in substantially constant blood levels of a drug over an extended time period. The period of time may be as long as a month or more and should be a release which is longer that the same amount of agent administered in bolus form.
For sustained release, the compounds may be formulated with a suitable polymer or hydrophobic material which provides sustained release properties to the compounds. As such, the compounds for use with the method(s) described herein may be administered in the form of microparticles, for example, by injection or in the form of wafers or discs by implantation.
In some cases, the dosage forms to be used can be provided as slow or controlled-release of one or more active ingredients therein using, for example, hydropropylmethyl cellulose, other polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, liposomes, or microspheres or a combination thereof to provide the desired release profile in varying proportions. Suitable controlled-release formulations known to those of ordinary skill in the art, including those described herein, can be readily selected for use with the pharmaceutical compositions described herein. Thus, single unit dosage forms suitable for oral administration, such as tablets, capsules, gelcaps, and caplets, that are adapted for controlled-release are encompassed by the compositions and dosage forms described herein.
Most controlled-release pharmaceutical products have a common goal of improving drug therapy over that achieved by their non-controlled counterparts. Ideally, the use of an optimally designed controlled-release preparation in medical treatment is characterized by a minimum of drug substance being employed to cure or control the condition in a minimum amount of time. Advantages of controlled-release formulations include extended activity of the drug, reduced dosage frequency, and increased patient compliance. In addition, controlled-release formulations can be used to affect the time of onset of action or other characteristics, such as blood level of the drug, and thus can affect the occurrence of side effects.
Most controlled-release formulations are designed to initially release an amount of drug that promptly produces the desired therapeutic effect, and gradually and continually release of other amounts of drug to maintain this level of therapeutic effect over an extended period of time. In order to maintain this constant level of drug in the body, the drug must be released from the dosage form at a rate that will replace the amount of drug being metabolized and excreted from the body.
Controlled-release of an active ingredient can be stimulated by various inducers, for example pH, temperature, enzymes, water, or other physiological conditions or compounds. The term “controlled-release component” is defined herein as a compound or compounds, including, but not limited to, polymers, polymer matrices, gels, permeable membranes, liposomes, or microspheres or a combination thereof that facilitates the controlled-release of the active ingredient. In certain embodiments, the compound(s) described herein are administered to a patient, alone or in combination with another pharmaceutical agent, using a sustained release formulation. In certain embodiments, the compound(s) described herein are administered to a patient, alone or in combination with another pharmaceutical agent, using a sustained release formulation.
The term delayed release is used herein in its conventional sense to refer to a drug formulation that provides for an initial release of the drug after some delay following drug administration and that mat, although not necessarily, includes a delay of from about 10 minutes up to about 12 hours.
The term pulsatile release is used herein in its conventional sense to refer to a drug formulation that provides release of the drug in such a way as to produce pulsed plasma profiles of the drug after drug administration.
The term immediate release is used in its conventional sense to refer to a drug formulation that provides for release of the drug immediately after drug administration. As used herein, short-term refers to any period of time up to and including about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, or about 10 minutes and any or all whole or partial increments thereof after drug administration after drug administration.
As used herein, rapid-offset refers to any period of time up to and including about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, or about 10 minutes, and any and all whole or partial increments thereof after drug administration.
The therapeutically effective amount or dose of a compound described herein depends on the age, sex and weight of the patient, the current medical condition of the patient and the progression of the disease or disorder in the patient being treated. The skilled artisan is able to determine appropriate dosages depending on these and other factors.
A suitable dose of a compound described herein can be in the range of from about 0.01 mg to about 5,000 mg per day, such as from about 0.1 mg to about 1,000 mg, for example, from about 1 mg to about 500 mg, such as about 5 mg to about 250 mg per day. The dose may be administered in a single dosage or in multiple dosages, for example from 1 to 4 or more times per day. When multiple dosages are used, the amount of each dosage may be the same or different. For example, a dose of 1 mg per day may be administered as two 0.5 mg doses, with about a 12-hour interval between doses.
It is understood that the amount of compound dosed per day may be administered, in non-limiting examples, every day, every other day, every 2 days, every 3 days, every 4 days, or every 5 days. For example, with every other day administration, a 5 mg per day dose may be initiated on Monday with a first subsequent 5 mg per day dose administered on Wednesday, a second subsequent 5 mg per day dose administered on Friday, and so on.
In the case wherein the patient's status does improve, upon the doctor's discretion the administration of the compound(s) described herein is optionally given continuously; alternatively, the dose of drug being administered is temporarily reduced or temporarily suspended for a certain length of time (i.e., a “drug holiday”). The length of the drug holiday optionally varies between 2 days and 1 year, including by way of example only, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 15 days, 20 days, 28 days, 35 days, 50 days, 70 days, 100 days, 120 days, 150 days, 180 days, 200 days, 250 days, 280 days, 300 days, 320 days, 350 days, or 365 days. The dose reduction during a drug holiday includes from 10%-100%, including, by way of example only, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.
Once improvement of the patient's conditions has occurred, a maintenance dose is administered if necessary. Subsequently, the dosage or the frequency of administration, or both, is reduced to a level at which the improved disease is retained. In certain embodiments, patients require intermittent treatment on a long-term basis upon any recurrence of symptoms and/or infection.
The compounds described herein can be formulated in unit dosage form. The term “unit dosage form” refers to physically discrete units suitable as unitary dosage for patients undergoing treatment, with each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, optionally in association with a suitable pharmaceutical carrier. The unit dosage form may be for a single daily dose or one of multiple daily doses (e.g., about 1 to 4 or more times per day). When multiple daily doses are used, the unit dosage form may be the same or different for each dose.
Toxicity and therapeutic efficacy of such therapeutic regimens are optionally determined in cell cultures or experimental animals, including, but not limited to, the determination of the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between the toxic and therapeutic effects is the therapeutic index, which is expressed as the ratio between LD50 and ED50. The data obtained from cell culture assays and animal studies are optionally used in formulating a range of dosage for use in human. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with minimal toxicity. The dosage optionally varies within this range depending upon the dosage form employed and the route of administration utilized.
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures, embodiments, claims, and examples described herein. Such equivalents are considered to be within the scope of this disclosure and covered by the claims appended hereto. For example, it should be understood, that modifications in reaction conditions, including but not limited to reaction times, reaction size/volume, and experimental reagents, such as solvents, catalysts, pressures, atmospheric conditions, e.g., nitrogen atmosphere, and reducing/oxidizing agents, with art-recognized alternatives and using no more than routine experimentation, are within the scope of the present application.
It is to be understood that wherever values and ranges are provided herein, all values and ranges encompassed by these values and ranges, are meant to be encompassed within the scope of the present disclosure. Moreover, all values that fall within these ranges, as well as the upper or lower limits of a range of values, are also contemplated by the present application.
The following examples further illustrate aspects of the present disclosure. However, they are in no way a limitation of the teachings or disclosure of the present disclosure as set forth herein.
The disclosure is now described with reference to the following Examples. These Examples are provided for the purpose of illustration only and the disclosure should in no way be construed as being limited to these Examples, but rather should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.
Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the compounds of the present disclosure and practice the disclosed methods. The following working examples therefore, point out specific embodiments of the present disclosure, and are not to be construed as limiting in any way the remainder of the disclosure.
The materials and methods used in the experiments presented in this Experimental Example are now described.
N-Cbz and O-Bz protected phenylglycine was treated with carbon monoxide in the presence of hydrochloric acid and aluminum trichloride to give an intermediate aryl aldehyde, which was then reduced with hydrogen gas over ruthenium to give the cyclohexyl derivative. Any reduced aldehyde was reoxidized using Dess-Martin periodinane. The amine and carboxylic acid were reprotected using CbzCl and Obz respectively. The aldehyde was then oxidized with Jones reagent and reacted with oxaylyl chloride give an acyl chloride, which was reacted with an amine carboxylic acid terminating in a carboxylic acid. The amine and carboxylic acid protecting groups were removed with Pd/C under a hydrogen atmosphere and the carboxylic acids protected with OtBu groups using acid in t-butanol to give intermediate A.
Separately, 4-bromopyridine-2,6-dicarboxylic acid was reacted with oxalyl chloride to give the di-acyl chloride, which was then reacted with intermediate A and (S)-1-(pyrrolidin-2-ylmethyl)pyrrolidine in the presence of triethylamine to give compound B.
Separately, 4-bromopyridine-2,6-dicarboxylic acid was treated with oxalyl chloride and reacted with [1,1′-biphenyl]-3-ylmethanamine and (S)-1-(pyrrolidin-2-ylmethyl)pyrrolidine in the presence of triethylamine to give compound C.
B and C were cross-coupled using a palladium catalyst in DMF at elevated temperature, followed by treatment with aqueous sodium bicarbonate and treatment with TFA in DCM to give a carboxylic acid intermediate. This was then treated with HBTU, DIPEA, and H2N-GN3 in the presence of DMF. Deprotection with sodium methoxide afforded the final compound.
4-nitro-1H-pyrazole was treated with ethyl 2-bromo-2-methylpropanoate in the presence of organic base in DMF to give the alkylated product. The methyl ester was then deprotected with sodium hydroxide, and reacted with furan-2-ylmethanamine under standard amide coupling conditions to give the amide nitro product which was then reduced to afford an amine. This was then reacted with 6-(methoxycarbonyl)nicotinic acid under standard amide coupling conditions to afford the diamide methyl ester, which was deprotected to give the carboxylic acid. This was activated using HBTU and DIPEA in DMF and reacted with H2N-GN3. Deprotection of the GN3 acyl groups afforded the final compound.
1-(4-hydroxy-2-methylphenyl)ethan-1-one was treated with sodium hydride and reacted with 1-(bromomethyl)-4-ethynylbenzene to give the intermediate ketone, which was reacted with sodium hydride and treated with dimethyl oxalate to give the enol product. This was then condensed with 3-morpholinopropan-1-amine and isonicotinaldehyde to form a 5-membered ring. The resulting alkyne was reacted with N3—(CH2CH2O)3CH2C(═O)NH-GN3 (acyl-deprotected) under standard click coupling conditions to afford the product.
Peptide VWDLYEEWSTFVT (SEQ ID NO:135) was synthesized according to standard Fmoc protocols. The peptide was treated with 5-hexynoic acid on resin to afford the alkyne, which was cleaved from resin using Reagent L. The alkyne was reacted with N3—(CH2CH2O)3CH2C(═O)NH-GN3 (deprotected OAc groups with sodium methoxide) under standard copper-mediated conditions to give the final bifunctional molecule.
Peptide LREFCEWEWMVHIDCNPEV (SEQ ID NO:136) was synthesized according to standard Fmoc protocols. The peptide was treated with 5-hexynoic acid on resin to afford the alkyne, which was cleaved from resin using Reagent L, then cyclized overnight in pH 8 buffer. The alkyne was reacted with N3—(CH2CH2O)3CH2C(═O)NH-GN3 (deprotected OAc groups with sodium methoxide) under standard copper-mediated conditions to give the final bifunctional molecule.
Benzoquinone was reacted with two equivalents of 3-mercaptopropanoic acid to give the dicarboxylic acid. This was then reacted with 1 equivalent of HBTU in the presence of organic base in DMF and treated with H2N-GN3 (acyl deprotected with sodium methoxide) to give the bifunctional molecule.
Peptide CGGDQKFRK (SEQ ID NO:137) was synthesized on resin following standard solid phase protocols and treated with 5-hexynoic acid in the presence of HATU, NMM, and DMF. The alkynyl peptide was cleaved from resin using Reagent L and reacted with N3—(CH2CH2O)3CH2C(═O)NH-GN3 (acyl deprotected with sodium methoxide) under standard copper-mediated conditions to give the bifunctional molecule.
3-(4,5,6-trihydroxy-3-oxo-3H-xanthen-9-yl)propanoic acid was treated with HBTU in the presence of organic base in DMF and treated with H2N-GN3 (acetyl deprotected with sodium methoxide) to give the final compound.
Peptide LRLKSLIQGR (SEQ ID NO:138) was synthesized on resin following standard solid phase protocols and treated with 5-hexynoic acid in the presence of HATU, NMM, and DMF. The alkynyl peptide was cleaved from resin using Reagent L and reacted with N3—(CH2CH2O)3CH2C(═O)NH-GN3(acyl deprotected with sodium methoxide) under standard copper-mediated conditions to give the bifunctional molecule.
Benzyl (3-(2-(2-(3-aminopropoxy)ethoxy)ethoxy)propyl)carbamate was treated with (S)-1-(tert-butoxycarbonyl)piperidine-3-carboxylic acid under standard amide coupling conditions. This compound was then treated with TFA in DCM to give the monoprotected diamine A.
Separately, naphthalene-1,4-dione was treated with 1-trimethylsiloxy-1,3-butadine in DCM for 24 hours, then treated with triethylamine to afford the aromatic compound (
Treatment with A in the presence of organic base in DMF at elevated temperature gave the Cbz-protected amine, which was deprotected under a hydrogen atmosphere using Pd/C and reacted with succinic anhydride to give a carboxylic acid. This was then treated with HBTU in the presence of organic base in DMF and reacted with H2N-GN3 (acetyl deprotected with sodium methoxide) to afford the final compound (
O-(2-Azidoethyl)heptaethylene glycol was treated with sodium hydride and subsequently propargyl bromide to give the intermediate alkyne, which was then reduced using triphenyl phosphine in water/THF, followed by protection of the amine with Boc anhydride in the presence of organic base in methanol, affording A (
Separately, di-tert-butyl L-glutamate was treated with triphosgene followed by H-Lys(cbz)-Ot-Bu to give the urea intermediate. This was then reduced with hydrogen gas atmosphere over Pd/C to give the intermediate amine, which was converted to an azide by treatment with triflic azide and copper in the presence of base. This azide was then reacted with intermediate A under standard copper mediated cyclization conditions, then treated with TFA to provide amine B.
Separately, H2N-GN3 (acetyl deprotected using sodium methoxide in methanol) was treated with succinic anhydride in the presence of organic base in DMF to give a carboxylic acid, which was then activated with HBTU in the presence of DIPEA in DMF to react with amine B to give the final molecule (
Tert-butyl 4-formylpiperidine-1-carboxylate was treated with dimethyl-1-diazo-2-oxopropylphosphonate in the presence of potassium carbonate in methanol to afford the resulting alkyne amine, which was deprotected using acid in dioxane to afford the resulting amine. This was reacted under standard coupling conditions with N-Boc glycine to afford the Boc-protected amine, which was deprotected as above and coupled to N-Boc-D-leucine using standard coupling conditions. The resulting alkyne is intermediate A.
Separately, 2,3-dichlorophenol was treated with bromine in DCM for 2 hours to afford the mono-brominated product. This was then reacted with TIPS chloride to afford the silyl ether, which was treated with n-BuLi in THF at low temperature with bubbled carbon dioxide to afford the carboxylic acid. This carboxylic acid was converted to acyl chloride B upon treatment with oxyalyl chloride in DMF/DCM.
Separately, 5-formylfuran-2-carboxylic acid was reacted with trimethylsilyldiazomethane in benzene/methanol, followed by reduction with sodium borohydride to afford alcohol C.
A and B were condensed using copper (I) iodide in the presence of palladium catalyst and organic base. The resulting compound was treated with methylhydrazine in ethanol, followed by deprotection with fluoride at decreased temperature in THF to afford the intermediate phenol. This was treated with intermediate C in the presence of diethyl azodicarboxylate and triphenylphosphine to afford the ether product. The methyl ester was then deprotected with lithium hydroxide in THF, followed by Boc deprotection with acid in dioxane. This was then treated with N,N′-bis-Boc-1-guanylpyrazole in the presence of organic base. The remaining Boc groups were removed with TFA/DCM. The carboxylic acid was activated using HBTU for coupling with H2N-GN3 (acetyl groups removed with sodium methoxide in methanol) in the presence of organic base in DMF. Amide formation resulted in the final compound.
Hexapropylene glycol was treated with tosyl chloride in the presence of organic base, followed by treatment with sodium azide at elevated temperature to give the mono-azido alcohol A (
Separately, 1H-pyrrolo[2,3-b]pyridine was treated with methyl magnesium iodide followed by zinc (II) chloride and subsequently ClCOCOOMe to give the methyl ester derivative. Treatment with potassium bicarbonate deprotected the methyl ester to give the carboxylic acid, which was subjected to amide coupling with N-benzoyl-3-(R)-methylpiperazine in the presence of DEPBT and DIPEA in DMF. Treatment with mCPBA in acetone gave a zwitterionic intermediate, which upon treatment with nitric acid and TFA gave a mononitrated product. Treatment with intermediate A followed by reaction with PCl3 gave the azido derivative (
7-Bromo-4-methoxy-1H-indole was treated with oxalyl chloride in THF to give the acyl chloride intermediate. Following treatment with tert-butyl piperazine-1-carboxylate in the presence of organic base, the molecule was treated with TFA in DCM to give the secondary amine intermediate. This was coupled with benzoic acid using EDC and HOBT in the presence of organic base. The bromide was condensed with (5-(((tert-butoxycarbonyl)amino)methyl)furan-2-yl)boronic acid in the presence of sodium bicarbonate in water and DMF under microwave irradiation to give the coupled intermediate, which was then deprotected (TFA in DCM) and reacted with succinic anhydride to give a carboxylic acid. The carboxylic acid was activated as above for amide coupling with H2N-GN3 to give the final compound.
4-(((tert-butoxycarbonyl)amino)methyl)benzoic acid was treated with diimidazolyl ketone and magnesium ethyl malonate in THF to afford the intermediate ester, which was then treated with thiosemicarbazide in the presence of HCl, followed by stirring in ethyl acetate/ethanol, to give the intermediate bicyclic compound.
The amine was reprotected as the Fmoc using Fmoc chloride in the presence of organic base. The intermediate was then treated with sodium nitrite in the presence of strong acid, followed by treatment with 4-chloro-3-nitroaniline to give the diazo compound. Treatment of the intermediate with 2-bromo-1-phenylethan-1-one at elevated temperature in the presence of molecular sieves gave the cyclic product. Isomerization was then induced using UV light, followed by deprotection of the amine and reaction with succinic anhydride, to give the carboxylic acid product. This was then coupled with H2N—(CH2CH2O)3CH2C(═O)NH-GN3 under standard amide coupling conditions, which upon treatment with sodium methoxide in methanol gave the bifunctional molecule.
Tert-butyl 5-amino-1H-indole-1-carboxylate was treated with NBS at low temperature in acetonitrile to give the brominated intermediate, which was then reacted with (4-(methoxycarbonyl)phenyl)boronic acid under cross coupling conditions (Pd catalyst, potassium carbonate, DMF, elevated temperature) to give compound A.
Separately, benzo[cd]indol-2(1H)-one was treated with chlorosulfuric acid to give the mono-substituted product, which then was reacted with A in the presence of pyridine in THF to give the intermediate methyl ester. This was deprotected by treatment with sodium hydroxide in MeOH/water, followed by treatment with TFA in DCM. The carboxylic acid was then reacted with NHS under standard coupling conditions to give the NHS ester, which was reacted with GN3 and treated with sodium methoxide to give the bifunctional molecule.
Piperazine was treated with benzyl bromide at elevated temperature in THF to give the monoalkylated intermediate A. Separately, methyl 2-(chlorosulfonyl)benzoate and 2,3-dimethylaniline were treated with pyridine in THF to afford the sulfonamide intermediate. This was then treated with sodium hydride in DMF, followed by tert-butyl 2-bromoacetate to give the alkylated diester product. The t-butyl ester was deprotected with TFA in DCM, then reacted under standard amide coupling conditions with intermediate A to give the monoester intermediate. This intermediate was then deprotected using sodium hydroxide in water/methanol, then reacted using EDCI, HOBt, and DIPEA in DMF with H2N-GN3. Subsequent deprotection of the O-acyl groups with sodium methoxide in methanol gave the final compound.
1H-indole was reacted with 1-bromo-3-(trifluoromethyl)benzene in the presence of copper (I) iodide and cesium carbonate in DMF at elevated temperature to give the condensed product. This was then reacted with POCl3 in DMF and DCE at increasing temperature to give the aldehyde. N1-(3-azidopropyl)-N2-methylethane-1,2-diamine was then reacted with this aldehyde and 6,7-dimethyl-4-oxo-4H-chromene-3-carbaldehyde to give the dimine, which was reduced using sodium triacetoxyborohydride. The azide was then reduced using triphenylphosphine in water and THF, and reacted with succinic anhydride in the presence of organic base to give the resulting carboxylic acid. This was then treated with HBTU and DIPEA in DMF, followed by the addition of H2N—(CH2CH2O)3CH2C(═O)NH-GN3. Deprotecting of the OAc groups with sodium methoxide gave the final compound.
Peptide YCWSQYLCY (SEQ ID NO:132) was synthesized on rink amide resin following general solid phase synthesis protocols, then treated with 5-hexynoic acid in the presence of HATU and NMM in DMF to give the alkyne product, which was cleaved from resin using reagent L and cyclized overnight in pH8 buffer. This alkyne was reacted with N3—(CH2CH2O)3CH2C(═O)NH-GN3 described previously under click chemistry conditions to afford the final compound.
The synthesis of DNP-OH3 begins with an HBTU-mediated amide bond formation between the tri-carboxylic acid and three equivalents of the commercially available hydroxy amine 2-[2-(2-aminoethoxy)ethoxy]ethanol, affording the Cbz-protected intermediate. The intermediate is reduced to afford an amine which undergoes an HBTU-mediated cross coupling with a carboxylic acid, forming the final compound DNP-OH3.
It was investigated whether DNP-GN3 could mediate the formation of a ternary complex between a fluorescently labeled α-DNP antibody and ASGPR on the surface of immortalized human hepatocyte HepG2 cells in suspension. The extent of fluorescently-labeled antibody association with cells was found to be dependent on the concentration of DNP-GN3, with concentrations of 7.4 nM and 0.12 μM eliciting half-maximal fluorescence association (
Cell-associated fluorescence was found to be inhibited by reagents that bind competitively to either ASGPR or α-DNP antibody. Cellular fluorescence was decreased by increasing concentrations of both DNP-OH3 (IC50=36 nM) and monomeric GalNAc (GN) sugar (IC50=0.20 mM) (
It was next explored whether DNP-GN3 mediates the endocytosis of fluorescently labeled α-DNP antibody. It was observed that the intracellular fluorescence of adherent HepG2 cells was dependent on the concentration of both DNP-GN3 and α-DNP antibody (
The observed increase in intracellular fluorescence mediated by DNP-GN3 was inhibitable by reagents that were previously shown to interfere with ternary complex formation (
To explore the cellular mechanism of antibody uptake, cells were treated with chemical inhibitors of several endocytic pathways (
Having demonstrated that DNP-GN3 mediates the endocytosis of α-DNP antibodies, the subcellular localization of endocytosed fluorescently labeled α-DNP antibody was investigated. Accumulation of intracellular antibody-derived fluorescence was found to depend on the presence of both DNP-GN3 and α-DNP antibody (
No colocalization of endocytosed α-DNP antibody with the early endosome marker EEA1 was observed in cells after 12 hours (
In addition, western blotting with an antibody directed to Alexa Fluor 488 was used to determine if endocytosed fluorescently labeled α-DNP antibodies are degraded in vitro. Lysates from HepG2 cells treated with both DNP-GN3 and α-DNP antibody were found to accumulate fluorophore in a time- and DNP-GN3-dependent manner (
Having demonstrated that DNP-GN3 mediates the degradation of α-DNP antibodies in vitro, the viability of the MoDE-A (Molecular Degraders of Extracellular proteins through the Asialoglycoprotein receptor (ASGPR)) technology in vivo was evaluated. A dose of 1 mpk DNP-GN3 was found to be bioavailable via IP dosing in nude mice, with the maximal serum concentration reached after 1 h and a measured half-life in serum of 0.67 h. DNP-GN3 was well-tolerated up to doses of 100 mpk, with no significant differences in body weight or serum liver enzyme levels between control and treatment groups (
Treatment with DNP-GN3 was found to accelerate the depletion of monoclonal mouse IgG2 α-DNP antibodies from serum in nude mice in vivo (
No accelerated antibody depletion from serum was observed following treatment with the negative control compound DNP-OH3, which binds to the α-DNP antibody but not ASGPR (
Single doses of DNP-GN3 were also found to be efficacious at mediating α-DNP antibody depletion, albeit less effectively than daily dosing (
DNP-GN3 was also found to be efficacious in depleting polyclonal α-DNP antibody from serum collected from mice immunized with DNP-keyhole limpet haemocyanin (KLH) (
After demonstrating that DNP-GN3, which utilizes a trivalent GalNAc motif to bind to ASGPR, is effective at mediating target protein endocytosis and degradation both in vitro and in vivo, experiments were conducted with DNP-AF3. DNP-AF3 utilizes trivalent display of a higher affinity ASGPR ligand to engage the receptor. First, the effect of DNP-AF3 concentration on α-DNP antibody association with cells was determined (
Maximal ternary complex formation was observed at DNP-AF3 concentrations of 19.5 and 39.1 nM. These observations with DNP-AF3 showed similar trends to treatment with DNP-GN3, which exhibited maximal ternary complex formation at concentrations of 20 and 40 nM. A decrease in cellular fluorescence was also observed at high concentrations of DNP-AF3, which is consistent with ternary complex formation. Based on these data, it was concluded that DNP-AF3 mediates the association of α-DNP antibody with HepG2 cells.
Next, whether increasing concentrations of a competitive binder of the α-DNP antibody inhibits ternary complex formation was investigated. At a concentration of 40 nM DNP-AF3, a stable ternary complex formation with minimal variability was observed between experimental replicates within the same experimental group. However, different preparations of the HepG2 cells and/or compound gave varying mean fluorescence intensity for the cell population between different independent experimental replicates. Therefore, for competition studies, inhibition of ternary complex formation was reported rather than mean fluorescence intensity of the cell population. 100% ternary complex formation is corrected to the fluorescence of a cell population treated with both 40 nM DNP-AF3 and 100 nM α-DNP antibody, while 0% ternary complex formation is corrected to a mixture of cells and α-DNP antibody without DNP-AF3.
The competitive α-DNP antibody binding molecule DNP-OH3 inhibited ternary complex formation in a concentration-dependent manner (
It was then determined if competitive binders of the ASGPR protein impacted ternary complex formation. The small molecule AF is a synthetic sugar mimetic that binds to ASGPR more strongly than GalNAc. DNP-AF3 links together three AF sugars to bind strongly to ASGPR. The monomeric sugar AF was able to inhibit antibody association with cells at high concentrations, with an observed IC50 of 1.45 μM (
It was also investigated whether proteins which have been reported to bind selectively to ASGPR impact ternary complex formation mediated by DNP-AF3 (
In contrast to the observations with DNP-GN3, the asialoglycoprotein ASF did not inhibit ternary complex formation mediated by DNP-AF3 (
After establishing that DNP-AF3 is able to mediate the formation of a ternary complex between ASGPR present on the HepG2 cell surface and fluorescently labeled α-DNP antibody, next whether formation of this ternary complex resulted in α-DNP antibody endocytosis was investigated. The intracellular fluorescence of cells that were incubated at 37° C. with both fluorescently labeled α-DNP antibody and DNP-AF3 for a given amount of time was examined. Cells were then washed, removed from the plate with trypsin, and subjected to flow cytometry. Trypsin treatment is expected to cleave ASGPR and surface-bound α-DNP antibody from cells, and therefore the cellular fluorescence observed in these assays is expected to arise only from internalized antibodies.
Intracellular HepG2 cell fluorescence was dependent on the concentration of both α-DNP antibody and DNP-AF3 (
Alternatively, these data can be plotted to demonstrate the time-dependence of DNP-AF3-mediated α-DNP antibody endocytosis (
It was then determined if reagents which inhibit ternary complex formation inhibit endocytosis mediated by DNP-AF3. In order to account for non-specific antibody association with cells, the mean fluorescence intensity of an aliquot of cells which had been treated with α-DNP antibody but not DNP-AF3 was subtracted from each reading. In the absence of competitive inhibitor of endocytosis, the cell population had a mean fluorescence of approximately 1.31e6. In the presence of 2.38 μM (0.1 mg/mL) ASOR, the cell population fluorescence was decreased to 4.66e5 (
As discussed above, the asialoglycoprotein ASF was not effective at inhibiting ternary complex formation at any concentration of the protein tested. Although the protein was not effective at decreasing ternary complex formation, a significant decrease in endocytosis was observed after treatment of cells with ASF at a concentration of 2.07 μM. The serum proteins ORM and fetuin did not significantly impact fluorescent antibody endocytosis. Based on these data, which demonstrate that known proteins that bind to ASGPR inhibit DNP-AF3-mediated α-DNP antibody endocytosis, it was concluded that DNP-AF3 mediates endocytosis via ASGPR.
Cellular fluorescence was decreased to near-background levels by DNP-OH3, a competitive binder of the α-DNP antibody. DNP-OH3 was used at a concentration 15.6-fold greater than the IC50 observed in ternary complex experiments. Monomeric sugars also decreased cellular fluorescence. When present at 1.25 mM—approximately three orders of magnitude greater than its observed IC50 in ternary complex experiments—the monomeric AF sugar decreased cellular fluorescence to background levels. The monomeric GalNAc sugar, which has a lower affinity for ASGPR, also significantly decreased cellular fluorescence at a concentration of 1.25 mM. Based on these data, it was concluded that ternary complex formation is necessary for antibody endocytosis, and reagents which inhibit formation of a ternary complex also inhibit antibody endocytosis.
Next, whether inhibitors of specific endocytic pathways inhibited α-DNP antibody endocytosis was determined (
Antibody endocytosis was significantly inhibited by some inhibitors of macropinocytosis and phagocytosis. Treatment with cytochalisin D (CytD) gave a non-significant decrease in the fluorescence intensity of the cell population. One possible explanation for this is that CytD inhibits processes responsible for trafficking of endosomes throughout the cell, and disruption of those networks could decrease ASGPR recycling. A decrease in cellular fluorescence with the macropinocytosis inhibitor amiloride was seen, but not with the closely related compound EIPA. It is also possible that some of the inhibitors tested in these assays are not specific for their prescribed pathway, but rather have effects on several different endocytic pathways. Treatment with the caveolin-dependent endocytosis inhibitors nystatin, indomethacin, and genistein did not significantly decrease fluorescence of the cell population. In contrast, all tested inhibitors of clathrin-mediated endocytosis significantly decreased antibody endocytosis compared to uninhibited cells. These inhibitors are either weak bases that neutralize endosomes and lysosomes or inhibit proton pumps which acidify endolysosomal compartments. Based on these data, it was concluded that DNP-AF3 mediates α-DNP antibody endocytosis consistent with ASGPR targeting. A fraction of the endocytosis mediated by DNP-AF3 may occur through non-clathrin-dependent pathways.
In order to investigate the intracellular localization endocytosed of α-DNP antibody, colocalization experiments were undertaken using antibodies directed to makers of different cytoplasmic compartments. Punctae containing endocytosed Alexa 568-labeled α-DNP antibody were found to accumulate in cells over time, with distinct punctae present following one hour of incubation with α-DNP antibody and DNP-AF3 (
Then, whether fluorescence derived from endocytosed α-DNP antibody colocalized with early or late endosomes in cells was investigated. It was found that endocytosed α-DNP antibody did not colocalize strongly with an antibody that recognizes the early endosome protein EEA1, indicating that endocytosed antibody does not accumulate in early endosomes (
First, reducing conditions were used to visualize antibody degradation via PAGE gel. Under these conditions, strong bands in cell culture supernatant with molecular weights of 50 and 25 kDa were observed (
Antibody accumulation in cell lysates showed a dependence both on time and on the presence of DNP-AF3 (
In cell lysates, two fluorophore-associated protein fragments were observed that were not observed in cell supernatants. The first was found in the well of the gel, which is hypothesized to represent higher molecular weight aggregates of the α-DNP antibody in cell lysates. In addition, a fluorescent band below 10 kDa was observed that was found in the lysates of cells treated with DNP-AF3 after a six hour incubation. Because this band is strongly observed only in cell lysates and only in cells treated with DNP-AF3, it was hypothesized that this is a lower molecular weight protein fragment derived from lysosomal proteolysis of the α-DNP antibody.
In addition to observing increasing amounts of α-DNP antibody in cell lysates, a change in the abundance of each protein fragment was observed relative to the other protein fragments. In the cell supernatant samples, the brightness of the heavy chain was approximately 3-fold brighter than the light chain under all conditions (
The change in the intensity of individual protein bands was observed across many cell lysates. The intensity of fluorescence associated with proteins of 50 kDa molecular weight increased at time points up to six hours, after which a gradual degrease in their fluorescence was observed (
It was hypothesized that these observations are due to the proteolysis of endocytosed antibody by lysosomal proteases. For example, it was hypothesized that the buildup in protein fragments with a weight of 25 kDa is due to proteolysis of the heavy chain antibody protein. It was also hypothesized that the fragments observed at <10 kDa are proteolysis products of both the 50 and 25 kDa bands. A ratiometric representation of the intensity of the fluorescent signal observed associated with proteins of molecular weight 50 kDa divided by proteins of molecular weight 25 kDa is presented in
The accumulation of fluorescently labeled protein fragments at both 25 kDa and <10 kDa was inhibited by the addition of several protease inhibitors. The ratiometric comparison of band intensity at 50 kDa divided by the intensity of the 25 kDa was used as a measure of α-DNP antibody degradation. As endocytosed α-DNP antibody is degraded in cells, this ratio decreases. By analyzing data in this way, the endocytosis of α-DNP antibody did not need to be controlled for in order to determine whether protease inhibitors were effective. Each ratiometric measurement is produced using only the protein fragments present in the cell lysate.
In the absence of protease inhibitors, a general decrease was observed in the ratio of 50 kDa- and 25 kDa-chain derived signal, with ratios of 1.28, 1.02, and 0.74 observed at six, 12, and 24 hours respectively (
The protease inhibitor leupeptin was effective at inhibiting α-DNP antibody degradation at both 20 and 80 Leupeptin is an aldehyde-containing tripeptide that forms covalent bonds with active site residues of both serine and cysteine proteases, and has previously been used successfully in HepG2 cells to inhibit lysosomal degradation of proteins. E64 is a covalent inhibitor of cysteine proteases contains a trans-epoxysuccinyl group that has been effectively used in HepG2 cells to inhibit protein degradation. Unlike leupeptin and antipain, E64 is specific for cysteine proteases such as papain, actinidase, and cathepsins B, H, and L. E64 at concentrations of both 50 and 10 μM was effective at decreasing anti-DNP antibody degradation. Pepstatin is an inhibitor of aspartic proteases that has previously been shown to inhibit protein degradation in HepG2 cells. Pepstatin was not effective at decreasing antibody degradation at any time point. After 24 hours, a concentration of 5 μM pepstatin was toxic to cells. Antipain is an oligopeptide which inhibits both cysteine and serine proteases by forming a covalent bond with protease active site nucleophilic residues. Antipain was effective at inhibiting α-DNP antibody degradation at both 50 and 100 μM. Aprotinin is a 58-mer protein which inhibits serine proteases, and has been used successful to inhibit protein degradation in HepG2 cells. Aprotinin was not effective at inhibiting α-DNP antibody degradation at a concentration of either 400 or 800 nM. While this protein is reported to be cell-permeable, its proteinaceous character may mean that it is degraded by other proteases in the lysosome, or that aprotinin localizes to the cytosol rather than the lysosome. Bestatin is an inhibitor of the amino proteases, such as leucine aminopeptidase and aminopeptidase N. These proteases are responsible for cleaving single N-terminal amino acids from protein chains. Bestatin was not effective at inhibiting α-DNP degradation at either concentration tested. Because bestatin inhibits proteases that catalyze the removal of only a single amino acid from proteins, any degradation inhibition may not be significant enough to change the migration of fluorescently labeled protein fragments. Phenylmethylsulfonyl fluoride (PMSF) is a small molecule which covalently inhibits serine proteases, and has been used effectively in HepG2 cells. PMSF was not effective at inhibiting α-DNP antibody degradation at a concentration of either 100 or 500 μM. PMSF has been reported to be very unstable in aqueous solutions and to have poor solubility, so it is possible that this inhibitor was either inactivated in solution or at a very low concentration in cell culture supernatant. 4-(2-aminoethyl)benzeneulfonyl fluoride hydrochloride (AEBSF) is similar in structure to PMSF, but is reported to be more stable at lower pH values. The sulfonyl chloride group reacts with active site nucleophiles of proteases. AEBSF was not effective at decreasing α-DNP antibody degradation at 10 μM. At 100 μM, AEBSF was toxic to cells. Calpain Inhibitor I (Ac-LLnL-CHO, ALLN) covalently inhibits both serine and cysteine proteases. ALLN is similar to leupeptin, but may be more cell permeable because it is more hydrophobic. At 10 μM, ALLN was not active at inhibiting anti-DNP antibody degradation. At 100 μM, however, ALLN showed strong inhibition at the 12 hour time point. By 24 hours, 100 μM ALLN was observed to be toxic to cells.
After carrying out the initial screening for protease inhibitors which decrease α-DNP antibody degradation, a single time point (12 hours) was elected for future studies. The 24 hour time point was not chosen because at that time, most cell lysates showed a more drastic increase in low molecular weight protein fragments even in the presence of effective protease inhibitors. This is perhaps due to the protease inhibitors becoming hydrolyzed or otherwise inactivated in solution. This observation may also be due to the production of more proteases to compensate for the covalently inhibited proteases present in cells.
Next it was studied whether the inhibition of α-DNP antibody degradation by protease inhibitors was reproducible. Each protease inhibitor concentration was tested in triplicate in HepG2 cells (
Based on these data, a further repeat of this experiment was undertaken using only 5 conditions which showed the most robust inhibition of α-DNP antibody degradation. Pepstatin at a concentration 15 μM was included as a negative control. Expectedly, 80 μM leupeptin, 50 mM E64, 100 mM antipain, and both 10 and 100 μM ALLN inhibited degradation of α-DNP antibody in cells, while pepstatin did not (
First, the carboxylic acid-terminated MIF inhibitor 34 was synthesized (
An inhibitor of MIF's enzymatic activity was synthesized for use as a negative control compounds in protein depletion experiments. The morpholine-terminated MIF inhibitor 3w (105) was synthesized through adaptation of the procedure described above to synthesize carboxylic acid 34. The morpholine group was installed early in the synthesis through nucleophilic substitution of the terminal chloride of compound 101 (
Versions of the MIF-GN3 (76) bifunctional molecule with additional PEG spacers between the tri-GalNAc targeting motif and the MIF-binding moiety were synthesized. MIF-PEG2-GN3 (
A bifunctional molecule that binds to MIF was synthesized by incorporation of a MIF inhibitor that is structurally dissimilar from that utilized in the bifunctional molecule MIF-GN3. The azido-terminated MIF inhibitor was conjgated with the tri-GalNAc motif through copper-mediated triazole formation to afford final compound MIF-NVS-PEG3-GN3 (
Molecules which incorporated the high-affinity ASGPR ligand 15 to degrade MIF were synthesized. In addition, the impact of sugar valency on the ability of these molecules to degrade the MIF protein was explored. Through adaptation of previously discussed procedures, the bifunctional molecules MIF-AF1 (39, monovalent display of sugar 15,
Although numerous inhibitors of MIF's enzymatic activity have been reported, these small molecules have only been assayed against the human MIF protein. Therefore, whether reported MIF inhibitors were also effective at inhibiting the tautomerase reaction carried out by mouse MIF was studied. It was hypothesized that inhibitors found to inhibit both human and mouse MIF could be elaborated into bifunctional molecules with the ability to degrade MIF protein from both species. The primary sequence of MIF is highly conserved across rodents and mammals, with greater than 90% sequence conservation between species.
In order to determine whether reported inhibitors bind to mouse MIF, enzymatic activity assays were undertaken to measure the impact of these small molecules on mouse MIF's enzymatic activity. Both human MIF and mouse MIF were found to mediate the tautomerization of D-dopachrome. For all commercial preparations assayed, mouse MIF carried out the tautomerization reaction more slowly than human MIF when present at the same concentration. Under the assay conditions used, human MIF protein carried out complete tautomerization of its substrate in less than ten minutes. In contrast, the mouse MIF assays required up to 20 minutes to reach completion. The relative catalytic rates of human versus mouse MIF have not been investigated. One possible reason for the decreased enzymatic rate observed for mouse MIF could be the inefficient posttranslational processing of the protein. Post-translational cleavage of the N-terminal methionine residue from both human MIF and mouse MIF is necessary for the protein's enzymatic activity; in recombinant preparations, this modification is oftentimes not carried out. If the mouse MIF protein was not post-translationally modified to the same extent as the human MIF protein, a much smaller proportion of the protein would be expected to be enzymatically active. This would be consistent with the observed decrease in tautomerase rate. Several MIF inhibitors were assayed for their ability to inhibit mouse MIF's tautomerase activity (
The estimations of circulating MIF levels in humans range widely, from less than 1 ng/mL (80 pM) in healthy patients to up to 300 ng/mL (24 nM) in certain disease states. The levels of circulating MIF in mice range from 60-140 ng/mL.
The ability of bifunctional MIF-binding molecules to mediate the depletion of human MIF from cell culture was investigated. In these assays, human MIF was present at a concentration of 100 nM, and a sandwich ELISA assay was utilized to measure the concentration of human MIF remaining in the cell culture supernatant. The closely related bifunctional molecules MIF-GN3, MIF-PEG2-GN3, and MIF-PEG4-GN3 were used, as well as the structurally dissimilar molecule MIF-NVS-PEG3-GN3. After an incubation of 24 hours, all bifunctional molecules tested were effective at mediating the depletion of human MIF from cell culture supernatant (
Bifunctional molecules containing optimized ASGPR-binding motifs are also capable of mediating MIF depletion from cell culture supernatant (
After demonstrating that MIF-binding bifunctional molecules can mediate the depletion of MIF from cell culture supernatant, whether endocytosed MIF protein accumulates in cells was determined. Human MIF protein was fluorescently labeled with Alexa 488 NHS ester, then incubated with HepG2 cells in the presence of varying levels of MIF-GN3. Increased intracellular fluorescence was observed with increasing concentrations of MIF-GN3, with maximal fluorescence observed at the highest concentration we investigated (1.0 μM) (
The ability of MIF-GN3 to mediate MIF endocytosis over a wide range of target protein concentrations was assessed. At a concentration of 100 nM MIF protein, several different concentrations of MIF-GN3 were found to mediate robust uptake of MIF-associated fluorophore (
Next, inhibitors of various endocytic pathways were utilized to determine if MIF-GN3 mediates MIF endocytosis in a manner consistent with ASGPR (
In order to investigate the subcellular localization of endocytosed MIF protein, colocalization studies were performed in HepG2 cells. After 12 hours of MIF-GN mediated endocytosis, several punctae displaying MIF-derived fluorescence were present in each cell. The location of these punctae did not overlap with the location of an antibody that detects the protein EEA1, which is present only in early endosomes (
The ability of MIF-GN3 to mediate the depletion of injected human MIF from serum in mice was studied. First, the pharmacokinetics of the bifunctional molecule were investigated. Following a one mpk dose of MIF-GN3 in male nude mice, a half-life of 0.43 hours in serum was observed, with a maximum plasma concentration of 586.87 ng/mL after 15 minutes.
In vivo human MIF depletion experiments were undertaken to determine whether MIF-GN3 can mediate the depletion of injected recombinant human MIF from serum in mice. Mice were injected with five μg of the human MIF protein. In this experiment, one group of mice was also injected with a single 10 mpk dose of MIF-GN3 along with human MIF protein. It was observed that after four hours, the average level of human MIF in serum in the PBS-treated mice was 1.76 ng/mL (
Next, an experiment was performed to determine whether MIF-GN3 could enhance MIF clearance at time points earlier than four hours. Mice were coinjected with recombinant human MIF and 10 mpk MIF-GN3 via either i.p. or i.v. routes. For the PBS control arms, human MIF was injected with PBS. It was observed that in the absence of MIF-GN3, there was a spike in huMIF levels in circulation after 30 minutes (
In order to confirm that mice injected with one or ten mpk MIF-GN3 had received the injection, and that the observed stability of MIF concentrations in MIF-GN3 treated mice was not the result of injection error, 200 μg of α-DNP antibody was coinjected with the MIF protein in this experiment. The levels of α-DNP antibody were not significantly changed by treatment with any bifunctional molecules (in all cases and time points, p>0.0702). Thus, the bifunctional molecule MIF-GN3 mediates MIF target protein depletion, while the control molecules 3w and DNP-GN3 do not. An investigation of the ability of MIF-GN3 to mediate the depletion of endogenous mouse MIF in vivo was next undertaken. Mice were treated with either PBS or 10 mpk of MIF-GN3. No significant decrease in mouse MIF concentrations was observed in the mice treated with MIF-GN3 (
The ability of MIF-GN3 to mediate the depletion of MIF in a therapeutically relevant disease model was investigated. The human prostate cancer PC3 cell line has been demonstrated to grow more rapidly in the presence of human MIF homologs, as well as to be less proliferated in conditions in which MIF is depleted. An experiment was conducted to determine whether the bifunctional molecule MIF-GN3 can deplete sufficient human MIF from serum to slow PC3 tumor growth. Mice were injected with an aliquot of PC3 cells, and the size of the resultant tumors as well as the levels of human MIF in serum were monitored over five weeks. Tumor size arising from PC3 injection was observed to increase gradually over the course of the experiment. Five weeks after the initial PC3 cell injection, the first of the mice reached maximal tumor size (1000 mm2) and was sacrificed (
Mice were sacrificed when their tumor volumes reached 1000 mm3. Mice treated with 1 mpk MIF-GN3 showed 80% survival eight weeks after the initial PC3 cell injection, compared to 60% survival in the α-MIF antibody treated arm (
The bifunctional molecule FcIII-BCN-GN3 was synthesized in much the same manner as FcIII-GN3 (
The ability of the bifunctional molecule FcIII-GN3 to mediate IgG uptake by HepG2 cells was investigated. Cells were incubated with Alexa 488-labeled human IgG and treated with varying levels of FcIII-GN3. Two forms of FcIII-GN3 were investigated: both the reduced linear form and the oxidized cyclized form. The reduced form of FcIII has been previously reported to not bind strongly to human IgG (R. L. Dias et al., Journal of the American Chemical Society, 2006, 128:2726-2732). It was observed that antibody endocytosis was dependent on the concentration of FcIII-GN3, with maximal IgG uptake observed at a concentration of 200 nM (
In order to investigate the subcellular localization of endocytosed human IgG, fluorescence colocalization studies were performed. Cells were incubated with both fluorescently labeled human IgG and FcIII-GN3. In the absence of human IgG, no background fluorescence arising in the Alexa 568 channel was observed (
Synthesis of the TNF binder (
The following enumerated embodiments are provided, the numbering of which is not to be construed as designating levels of importance.
Embodiment 1 provides a compound comprising formula (I), or a salt, geometric isomer, stereoisomer, or solvate thereof:
[Protein binder]k′—[CON]h—[Linker]i—[CON]h′—[CRBM]j′ (I),
wherein: the Protein binder is a molecule that binds to an extracellular protein;
Embodiment 2 provides a compound comprising formula (II), or a salt, geometric isomer, stereoisomer, or solvate thereof:
[TNF binder]k′—[CON]h—[Linker]i—[CON]h′—[CRBM]j′ (II),
wherein: the TNF binder is a molecule that binds to TNF; the CRBM is a cellular receptor binding moiety that binds to at least one receptor on the surface of a degrading cell in a subject, whereby binding of (II) leads to endocytosis and degradation of TNF; each CON is independently a bond or a group that covalently links a TNF binder to an CRBM, a TNF binder to a Linker, and/or a Linker to a CRBM; the Linker is a group having a valence ranging from 1 to 15; k′ is an integer ranging from 1 to 15; h is an integer ranging from 0 to 15; i is an integer ranging from 0 to 15; h′ is an integer ranging from 0 to 15; j is an integer ranging from 1 to 15.
Embodiment 3 provides a compound comprising formula (III), or a salt, geometric isomer, stereoisomer, or solvate thereof:
[AATM]k′—[CON]h—[Linker]i—[CON]h′—[CRBM]j′ (III),
wherein: the AATM is a molecule that binds to an autoantibody; the CRBM is a cellular receptor binding moiety that binds to at least one receptor on the surface of a degrading cell in a subject, whereby binding of (III) leads to endocytosis and degradation of the autoantibody; each CON is independently a bond or a group that covalently links an AATM to an CRBM, an AATM to a Linker, and/or a Linker to a CRBM; the Linker is a group having a valence ranging from 1 to 15; k′ is an integer ranging from 1 to 15; h is an integer ranging from 0 to 15; i is an integer ranging from 0 to 15; h′ is an integer ranging from 0 to 15; j is an integer ranging from 1 to 15.
Embodiment 4 provides the compound of any one of Embodiments 1-3, wherein the valence of the Linker is 1, 2, or 3.
Embodiment 5 provides the compound of any one f Embodiments 1-4, wherein k′ is 1, 2, or 3.
Embodiment 6 provides the compound of any one of Embodiments 1-5, wherein j is 1, 2, or 3.
Embodiment 7 provides the compound of any one of Embodiments 1-6, wherein h is 1, 2, or 3.
Embodiment 8 provides the compound of any one of Embodiments 1-7, wherein h′ is 1, 2, or 3.
Embodiment 9 provides the compound of any one of Embodiments 1-8, wherein i is 1, 2, or 3.
Embodiment 10 provides the compound of any one of Embodiments 1-9, wherein at least one of h, h′, and i is at least 1.
Embodiment 11 provides the compound of any one of Embodiments 1-10, wherein k′, j′, h, h′, and i are each independently 1, 2, or 3.
Embodiment 12 provides the compound of any one of Embodiments 1-11, wherein k′ is 1, and j′ is 1, 2, or 3.
Embodiment 13 provides the compound of any one of Embodiments 1 or 4-12, which is:
[Protein binder]—[CON]0-1—[Linker]—[CON]0-1—[CRBM] (Ia).
Embodiment 14 provides the compound of any one of Embodiments 2 or 4-12, which is:
[TNF binder]—[CON]-1—[Linker]—[CON]0-1—[CRBM] (IIa).
Embodiment 15 provides the compound of any one of Embodiments 3-12, which is:
[AATM]—[CON]0-1—[Linker]—[CON]0.1—[CRBM] (IIIa).
Embodiment 16 provides the compound of any one of Embodiments 1-15, wherein the degrading cell comprises a hepatocyte.
Embodiment 17 provides the compound of any one of Embodiments 1-16, wherein the CRBM is a folic acid (folate) receptor binder, mannose receptor binder, mannose-6-phosphate (M6P) receptor binder, low density lipoprotein receptor-related protein 1 (LRP1) receptor binder, low density lipoprotein receptor (LDLR) binder, FcγRI receptor binder, transferrin receptor binder, macrophage scavenger receptor binder, G-Protein coupled receptor binder, or asialoglycoprotein receptor (ASGPR) binder.
Embodiment 18 provides the compound of any one of Embodiments 1-17, wherein the CRBM is:
and each occurrence of ‘n’ is independently 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20; and
a polymeric molecule selected from the group consisting of:
wherein
m is an integer from 1 to 100,
r, s, t, and o are each independently an integer from 0 to 100, and the COOH in the polymeric molecule is derivatized with the Protein binder, the TNF binder, or the AATM;
wherein n is an integer from 1 to 100;
a compound selected from:
a compound selected from:
wherein each occurrence of R is independently H or C1-C6 alkyl;
wherein:
L1-≡-, L1-(CH2)K—, and CYC—(CH2)K—,
wherein:
optionally substituted with 1-3 C1-C3 alkyl groups optionally substituted with 1-3 independently selected halogens, or RTA is
wherein each —(CH2)K group is optionally substituted with 1-4 C1-C3 alkyl groups optionally substituted with 1-3 fluoro groups or 1-2 hydroxyl groups.
Embodiment 19 provides the compound of Embodiment 18, wherein:
Embodiment 20 provides the compound of any one of Embodiments 18-19, wherein X is OCH2 and RN1 is H, or wherein X is CH2O and RN1 is H.
Embodiment 21 provides the compound of any one of Embodiments 18-20, wherein the ASGPRBM comprises the structure:
Embodiment 22 provides the compound of any one of Embodiments 18-21, wherein the ASGPRBM group comprises:
wherein:
or
Embodiment 23 provides the compound of any one of Embodiments 1-22, wherein the Linker is a polyethylene glycol containing linker having 1-12 ethylene glycol residues.
Embodiment 24 provides the compound of any one of Embodiments 1-23,
—(CH2)i—C(R2)═C(R2)— (cis or trans), —(CH2)i—≡—, or —Y—C(═O)—Y—;
wherein R′ and R″ are each independently H, methyl, or a bond; or
Embodiment 26 provides the compound of any one of Embodiments 1 or 4-25, wherein the Protein binder that binds to CD40OL comprises:
NH-TVFTSWEEYLDWV-X (SEQ ID NO:66), wherein X═OH or NH2;
each of which can be acyclic or cyclic.
Embodiment 27 provides the compound of any one of Embodiments 2 and 4-25, wherein the TNF binder comprises the amino acid sequence of at least one of:
or tetra branched (SEQ ID NO:128) peptide,
or
or
or
or
or
or
or
or
or
or
or
Embodiment 28 provides the compound of Embodiment 27, wherein the compound of formula (1a) comprises one of the following:
wherein:
or
wherein G is N or CH; Z is CH or CF; R1 is selected from the group consisting of
and R2 is selected from the group consisting of
Embodiment 29 provides the compound of any one of Embodiments 3-25, wherein the AATM comprises one of the following:
Embodiment 30 provides the compound of Embodiment 29, wherein the FcRn antagonist comprises rozanolixizumab or efgartigimod.
Embodiment 31 provides a pharmaceutical composition comprising at least one pharmaceutically acceptable excipient at least one compound of any one of Embodiments 1-30.
Embodiment 32 provides the pharmaceutical composition of Embodiment 31, further comprising another therapeutically agent that treats, ameliorates, and/or prevents a disease or disorder.
Embodiment 33 provides a method of treating, ameliorating, and/or preventing a disease or disorder in a subject, the method comprising administering a therapeutically effective amount of at least one compound of any one of Embodiments 1-30 and/or at least one pharmaceutical composition of any one of Embodiments 31-32.
Embodiment 34 provides the method of Embodiment 33, wherein the disease or disorder comprises an autoimmune disease, cancer, or inflammation.
Embodiment 35 provides the method of Embodiment 34, wherein the autoimmune disease comprises Addison's Disease, Autoimmune polyendodrine syndrome (APS) types 1, 2 and 3, autoimmune pancreatitis (AIP), diabetes mellitus type 1, autoimmune thyroiditis, Ord's thyroiditis, Grave's disease, autoimmune oophoritis, endometriosis, autoimmune orchitis, Sjogren's syndrome, autoimmune enteropathy, coeliac disease, Crohn's disease, microscopic colitis, ulcerative colitis, autophospholipid syndrome (APlS), aplastic anemia, autoimmune hemolytica anemia, autoimmune lymphoproliferative syndrome, autoimmune neutropenia, autoimmune thrombocytopenic purpura, cold agglutinin disease, essential mixed cryoglulinemia, Evans syndrome, pernicious anemia, pure red cell aplasia, thrombocytopenia, adiposis dolorosa, adult-onset Still's disease, ankylosing spondylitis, CREST syndrome, drug-induced lupus, enthesitis-related arthritis, eosinophilic fasciitis, Felty syndrome, AgG4-related disease, juvenile arthritis, Lyme disease (chronic), mixed connective tissue disease (MCTD), palindromic rheumatism, Parry Romberg syndrome, Parsonage-Turner syndrome, psoriatic arthritis, reactive arthritis, relapsing polychondritis, retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis, sarcoidosis, Schnitzler syndrome, systemic lupus erythematosus, undifferentiated connective tissue disease (UCTD), dermatomyositis, fibromyalgia, myositis, inclusion body myositis, myasthenia gravis, neuromyotonia, paraneoplastic cerebellar degeneration, polymyositis, acute disseminated encephalomyelitis (ADEM), acute motor axonic neuropathy, anti-NMDA receptor encephalitis, Balo concentric sclerosis, Bickerstaff's encephalitis, chronic inflammatory demyelinating polyneuropathy, Guillain-Barré syndrome, Hashimoto's encephalopathy, idiopathic inflammatory demyelinating diseases, Lambert-Eaton myasthenic syndrome, multiple sclerosis, pattern II, Oshtoran Syndrome, Pediatric Autoimmune Neuropsychiatric Disorder Associated with Streptococcus (PANDAS), progressive inflammatory neuropathy, restless leg syndrome, stiff person syndrome, Syndenham chorea, transverse myelitis, autoimmune retinopathy, autoimmune uveitis, Cogan syndrome, Graves ophthalmopathy, intermediate uveitis, ligneous conjunctivitis, Mooren's ulcer, neuromyelitis optica, opsoclonus myoclonus syndrome, optic neuritis, scleritis, Susac's syndrome, sympathetic ophthalmia, Tolosa-Hunt syndrome, autoimmune inner ear disease (AIED), Méniére's disease, Behçet's disease, Eosinophilic granulomatosis with polyangiitis (EGPA), giant cell arteritis, granulomatosis with polyangiitis (GPA), IgA vasculitis (IgAV), IgA nephropathy, Kawasaki's disease, leukocytoclastic vasculitis, lupus vasculitis, rheumatoid vasculitis, microscopic polyangiitis (MPA), polyarteritis nodosa (PAN), polymyalgia rheumatica, urticarial vasculitis, vasculitis, primary immune deficiency, chronic fatigue syndrome, complex regional pain syndrome, eosinophilic esophagitis, gastritis, interstitial lung disease, POEMS syndrome, Raynaud's syndrome, primary immunodeficiency, or pyoderma gangrenosum.
Embodiment 36 provides the method of Embodiment 34, wherein the cancer comprises prostate cancer, metastatic prostate cancer, stomach cancer, colon cancer, rectal cancer, liver cancer, pancreatic cancer, lung cancer, breast cancer, cervix uteri cancer, corpus uteri cancer, ovary cancer, testis cancer, bladder cancer, renal cancer, brain/CNS cancer, head and neck cancer, throat cancer, Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma, leukemia, melanoma, non-melanoma skin cancer, acute lymphocytic leukemia, acute myelogenous leukemia, Ewing's sarcoma, small cell lung cancer, choriocarcinoma, rhabdomyosarcoma, Wilms' tumor, neuroblastoma, hairy cell leukemia, mouth/pharynx, oesophagus, larynx, kidney cancer, or lymphoma.
Embodiment 37 provides the method of Embodiment 34, wherein the inflammation comprises inflammatory diseases of neurodegeneration, diseases of compromised immune response causing inflammation, chronic inflammatory diseases, hyperglycemic disorders, diabetes (I and II), pancreatic β-cell death and related hyperglycemic disorders, liver disease, renal disease, cardiovascular disease, muscle degeneration and atrophy, low grade inflammation, gout, silicosis, atherosclerosis and associated conditions, stroke and spinal cord injury, or arteriosclerosis.
Embodiment 38 provides the method of any one of Embodiments 33-37, wherein the subject is further administered at least one additional therapeutic agent that treats, ameliorates, and/or prevents the disease or disorder.
Embodiment 39 provides the method of any one of Embodiments 33-38, wherein the subject is a mammal.
Embodiment 40 provides the method of any one of Embodiments 33-39, wherein the subject is a human.
The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety. While this disclosure has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this disclosure may be devised by others skilled in the art without departing from the true spirit and scope of the disclosure. The appended claims are intended to be construed to include all such embodiments and equivalent variations.
This application is a continuation-in-part of, and claims priority to, International Application No. PCT/US2020/055078, Oct. 9, 2020, which claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Applications No. 62/913,665, filed Oct. 10, 2019, No. 62/913,668, filed Oct. 10, 2019, and No. 62/913,683, filed Oct. 10, 2019, all of which applications are incorporated herein by reference in their entireties.
This invention was made with government support under GM067543 awarded by National Institutes of Health and under W81XWH-13-1-0062 awarded by United States Army Medical Research and Material Command. The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 62913665 | Oct 2019 | US | |
| 62913668 | Oct 2019 | US | |
| 62913683 | Oct 2019 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/US20/55078 | Oct 2020 | US |
| Child | 17654990 | US |
