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WO2024254161A2 - Inhibiteurs de protéines de liaison aux acides gras (fabp), procédés d'utilisation et procédés de fabrication - Google Patents

Inhibiteurs de protéines de liaison aux acides gras (fabp), procédés d'utilisation et procédés de fabrication Download PDF

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WO2024254161A2
WO2024254161A2 PCT/US2024/032570 US2024032570W WO2024254161A2 WO 2024254161 A2 WO2024254161 A2 WO 2024254161A2 US 2024032570 W US2024032570 W US 2024032570W WO 2024254161 A2 WO2024254161 A2 WO 2024254161A2
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compound
structural formula
cancer
cells
cyclopropyl
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WO2024254161A3 (fr
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Liraz LEVI
Tej PAREEK
Elizabeth MEYERS
Seunghwan LIM
William J. Greenlee
Seong-Jin Kim
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Celloram Inc
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Celloram Inc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
    • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
    • C07D471/04—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
    • C07D405/14—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D413/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D413/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing three or more hetero rings

Definitions

  • the present disclosure relates to compounds that inhibit fatty acid binding proteins, such as FABP3, FABP4, FABP5, and/or FABP7, pharmaceutical compositions conta1ning these inhibitor compounds, and uses of these compounds and compositions for treating or preventing cancers that highly express any of these FABPs, in particular triple-negative breast cancer (TNBC), autoimmune diseases and disorders, viral infections, and other diseases linked to chronic inflammation including cardiovascular disease, obesity or an obesity-related disorders, diabetes, dyslipidemia, impaired glucose tolerance or impaired fasting glucose, vitiligo, psoriasis, pain and dementia.
  • TNBC triple-negative breast cancer
  • Fatty acid binding proteins are members of a family of small (12-15kDa), soluble proteins which contribute to the trafficking of fatty acids within the cytosolic compartments of cells.
  • the proteins are a multigene family, well-conserved, have no catalytic function but transport hydrophobic fatty acids within the aqueous environment of the cytosol to the various destinations enabling fatty acid oxidation, membrane homeostasis or nuclear signaling. In addition, they are involved in signaling processes which are so far poorly understood [1-4].
  • FABPs have unique tissue-expression pattern except FABP5 that is ubiquitously expressed in most tissues. Yet, in general, tissues with active lipid metabolism tend to express more than one isoform.
  • FABP3 is mainly expressed in muscle tissues, particularly in the heart and in neurons.
  • FABP4 is highly expressed in adipose tissue, macrophages and endothelial cells.
  • FABP5 is also expressed in macrophages and endothelial cells, as well as in skin, adipocytes, neurons, glia cells, and several other tissues [2, 5, 6].
  • FABP7 is expressed in the brain, specifically in glia cells and astrocytes.
  • Patient data that became available in recent years indicates FABP5 is highly upregulated in breast tumors, particularly in TNBC tumors.
  • the protein was reported to induce growth and metastasis of TNBC cells, and high levels of the protein are associated with poor survival of TNBC patients.
  • Genetic ablation of FABP5 in the breast cancer mouse model MMTV-NeuT markedly delayed formation of tumors and inhibited their growth rate [7].
  • chemical inhibition of FABP5 suppressed growth of tumors in xenograft models [8, 9].
  • the data indicates that inhibition of FABP5 is a promising novel approach for treatment of TNBC and perhaps other cancers that highly express this protein.
  • TNBC is the most aggressive and deadly breast cancer subtype and accounts for 10-20% of all breast cancer cases. Women diagnosed with TNBC are four times more likely to have cancer cells spread or metastasize to other organs within five years than patients with other types of breast cancer. TNBC are a heterogeneous group of breast tumors that are still poorly characterized at the molecular level and lack definitive prognostic markers and selective targets for therapy. This makes the treatment and management of TNBC a significant clinical problem and there is an urgent need for novel targeted therapies for this disease.
  • Current standard of care for TNBC includes neoadjuvant systemic treatment such as anthracyclines, taxanes, and cyclophosphamide. Platinum-based chemotherapy has been proposed but is not yet recommended by available guidelines.
  • TME tumor microenvironment
  • the tumor- infiltrating immune cells are involved in the regulation of tumor development, progression, and response to treatment.
  • the cellular and molecular profile of the immune TME impacts the disease response to therapy and its outcome by regulating the balance between suppressive versus cytotoxic responses in the vicinity of the tumor [13].
  • Specific immune cells such as T cells and natural killer cells, can help suppress tumors' growth and contribute to the elimination of cancer cells.
  • immunosuppressive immune cells such as regulatory T cells and myeloid- derived suppressor cells, can create an environment permissive to tumor growth and progression.
  • the balance between immune-stimulating and immune-suppressive cells in the tumor microenvironment is crucial to the success of immunotherapy and other cancer treatments.
  • Targeting immune cells in the tumor microenvironment is a promising strategy for improving the effectiveness of cancer treatments [13-15].
  • This approach aims to shift the balance of immune cells in the microenvironment from pro-tumor to anti-tumor.
  • One way to do this is by enhancing the activity of immune cells that can recognize and eliminate cancer cells, such as T cells, natural killer cells and the pro-inflammatory M1 macrophages (classically activated macrophages). This can be achieved through the use of immune checkpoint inhibitors, which release the brakes that generally prevent immune cells from attacking the tumor.
  • Another strategy is to reduce the number or activity of immune cells that suppress the immune response, such as regulatory T cells, myeloid-derived suppressor cells or the anti-inflammatory M2 macrophages (alternatively activated macrophages). This can be done through the use of drugs that specifically target these cell types. Combining these and other approaches, such as vaccines or CAR-T cell therapy, may provide a more comprehensive and effective way to target the immune cells in the tumor microenvironment and improve cancer treatment outcomes. [0010]
  • One of the mechanisms by which FABPs regulate immune cells was suggested to be through their role in mediating immune cell metabolism, which is critical for the proper functioning of the immune system. By affecting the utilization of fatty acids, FABPs regulate energy production and the signaling pathways involved in the activation and function of immune cells.
  • FABP5 was found to regulate lipid metabolism and function in T-cells in the TME by mediating the uptake and oxidation of long-chain FAs in the cells.
  • Activated T-cells mainly rely on aerobic glycolysis to facilitate their proliferation and anti-tumor function.
  • tumor-infiltrating T lymphocytes (TIL) that express high FABP5 levels usually exhibit an exhausted phenotype and impaired anti-tumor activity due to the limited availability of glucose and high levels of long-chain FAs. Inhibition of FABP5 in TILs is therefore expected to activate the anti-tumor activity of the cell by shifting their energy balance [16, 17].
  • Trm cells are a subset of memory T cells which are self-sustaining in non-lymphoid tissues such as the gut, lung, reproductive tract and skin for longer period without circulation and provide the first line of defense against antigens and pathogens through rapid recall responses [18-20]. Trm cells have the ability to regulate local immune homeostasis in tissues and participate in immune responses mediated by pathogens, cancer, and possibly autoantigens during autoimmunity [21].
  • Trm cell Due to their specialized function and location within tissues, gene expression signature of Trm cell, as well as their metabolic needs are distinctive from other types of T-cells [20, 22-24].
  • FFA exogenous free fatty acids
  • Trm cells selectively express the fatty acid binding proteins FABP4 and FABP5 that are essential for uptake of FFA into the cells [20].
  • T-cell-specific deficiency of FABP4/5 impaired uptake of FFA by Trm cells and significantly reduced their longevity and survival in vivo, while having no effect on survival of central memory T (TCM) cells in lymph nodes [20].
  • TCM central memory T
  • FABP4/5 inhibitors can be used to specifically target Trm in autoimmune diseases.
  • FABP4 and FABP5 are expressed in macrophages and were shown to regulate their function by facilitating uptake and metabolism of fatty acids and lipid in the cells [25].
  • FABP4 is highly expressed in Ly6C-MHCII-CD36 + circulating monocyte/macrophages to facilitate oxidative lipid uptake, foam cell formation, angiogenesis, tissue remodeling and pro-tumor functions [25].
  • FABP5 is highly expressed in Ly6C+MHCII+CD36 ⁇ macrophages where it was shown to be involved in formation of lipid droplets (LD) in macrophages [26], and in CD11c+ macrophages where it was shown to promote secretion of pro-inflammatory cytokine IL-1b [27, 28], and cause ER stress, exhaustion and ferroptosis [28, 29] and therefor affect immune cell fate and disease progression.
  • LD lipid droplets
  • FABP4/5 in macrophages will be beneficial in treating multiple diseases. It is expected to enhance anti-tumor responses in cancer, block formation of foam cells and chronic inflammation in obesity and atherosclerosis, and will promote anti-inflammatory response in cases of inflammatory and autoimmune disease as well as infections.
  • FABP4 was shown to be involved in virus replication and propagation in the case of SARS-Cov2 (COVID-19) and OCT43 (common cold coronavirus) viruses [30].
  • SAARS-Cov2 COVID-19
  • OCT43 common cold coronavirus
  • No.8,748,470 B2 (Lengyel et al.) describes methods for reducing or inhibiting cancer that include administering to a subject an inhibitor of FABP4 and/or FABP5, where the inhibitor is selected from a list of known compounds, including carbazole butanoic acid, aryl sulfonamide, sulfonylthiophene, 4-hydroxypyrimidine, 2,3-dimethylindole, benzoylbenzene, biphenyl-alkanoic acid, 2-oxazole-alkanoic acid, tetrahydropyrimidone, pyridone, pyrazinone, aryl carboxylic acid, tetrazole, triazolopyrimidinone, indole, or BMS480404.
  • the inhibitor is selected from a list of known compounds, including carbazole butanoic acid, aryl sulfonamide, sulfonylthiophene, 4-hydroxypyrimidine, 2,3
  • US Pat. No.8,815,875 B2 (Shipps, Jr. et al.) describes certain heterocyclic compounds that inhibit FABP, and the use of these compounds for the treatment of diseases or disorders including cardiovascular disease, a metabolic disorder, obesity, diabetes, dyslipidemia, and impaired glucose tolerance.
  • US Pat. No.9,278,918 B2 (Buettelmann, et al.) describes certain urea derivative compounds that inhibit FABP4 and/or FABP5, and the use of these compounds for the treatment of diseases or disorders including Type-2 diabetes, atherosclerosis, chronic kidney diseases, and cancer.
  • the present disclosure generally relates to compounds based on a substituted 2-amino- thiophene structure that are inhibitors of one or more of FABP3, FABP4, FABP5, and FABP7 (i.e., “FABP3/4/5/7 inhibitors”), and the use of these inhibitors, methods for preparing these inhibitors, and uses of these inhibitors in pharmaceutical compositions for treating diseases relating to fatty acid metabolism.
  • FABP3/4/5/7 inhibitors i.e., “FABP3/4/5/7 inhibitors”
  • the present disclosure provides a FABP3/4/5/7 inhibitor compound of structural formula I or pharmaceutically acceptable salt thereof: (I) wherein, R 1 is selected from hydrogen, cyano, and 5-membered heteroaryl ring; R 2 and R 3 are each independently selected from hydrogen, C 1 -C 4 linear or branched alkyl, cyclopropyl, cyclobutyl, phenyl, , trifluoromethyl, and benzyl, or R 2 and R 3 together form a 5- to 8-membered monocyclic, bicyclic, or spirocyclic carbocycle or heterocycle ring, or a 5- to 8- membered aryl or heteroaryl ring, wherein the carbocycle, heterocycle, aryl, or heteroaryl ring is optionally substituted with one or two substituents selected from C1-C4-alkyl, methoxy, or fluoro; X is a moiety of formula: wherein, Y is a
  • the compound of structural formula I of the present disclosure excludes the specific compounds as shown in Table 1 (shown elsewhere herein).
  • the chemical group at position R 1 is a cyano group, and the compound has a structural formula Ia: (Ia).
  • the compound of structural formula Ia is a compound having a structural formula selected from Ij, Ik, Il, Im, In, Io, Ip, Iq, Ir, Is, and It as shown in Table 2 (elsewhere herein).
  • the chemical substituents at positions R 2 and R 3 together form a 5- to 8-membered aryl or heteroaryl ring, and the compound has structural formula Ib wherein R 10 and R 11 are each independently selected from hydrogen, halogen, C 1 -C 4 linear or branched alkyl, cyclopropyl, and cyclobutyl.
  • the compound of structural formula Ib is a compound having a structural formula selected from Iu, Iv, and Iw as shown in Table 3 (elsewhere herein).
  • the chemical group at R 1 is a 5-membered heteroaryl ring (e.g., 3-substituted 1,2,4-oxadiazol), and the compound has a structural formula selected from Ic, Id, Ie, If, Ig, Ih, and Ii: wherein R 12 is selected from hydrogen, halogen, C 1 -C 4 linear or branched alkyl, cyclopropyl, cyclobutyl, phenyl.
  • the compound of structural formulas Ic, Id, Ie, If, Ig, Ih, and Ii include but are not limited to the compounds having structural formulas Ix, Iy, Iz, Iaa, Ibb, Icc, Idd, Iee, Iff, Igg, Ihh, Iii, Ijj, Ikk, Ill, Imm, Inn, Ioo, Ipp, Iqq, and Irr shown in Table 5 (shown elsewhere herein).
  • the X moiety is selected from the exemplary moieties shown in Tables 6, 7, or 8 (elsewhere herein).
  • the present disclosure provides a FABP3/4/5/7 inhibitor compound of structural formula II and any pharmaceutically acceptable salt thereof: wherein, R 1 is selected from hydrogen, cyano, and 5-membered heteroaryl ring; R 2 and R 3 are each independently selected from hydrogen, C 1 -C 4 linear or branched alkyl, cyclopropyl, cyclobutyl, phenyl, trifluoromethyl, and benzyl, or R 2 and R 3 together form a 5- to 8-membered monocyclic, bicyclic, or spirocyclic carbocycle or heterocycle ring, or a 5- to 8-membered aryl or heteroaryl ring, wherein the carbocycle, heterocycle, aryl, or heteroaryl ring is optionally substituted with one or two substituents selected from C1-C4-alkyl, methoxy, or fluoro; Y is a heteroatom selected from –S–, and –O–, or is
  • the compound of structural formula II of the present disclosure excludes the compounds as shown in Table 1.
  • the chemical group at position R 1 is a cyano group
  • the compound has structural formula IIa wherein the chemical groups R 2 , R 3 , R 4 , R 5 , R 6 and R 7 are as defined for the compound of structural formula II.
  • the compounds of having the substructure of structural formula IIa can have a structural formula IIj, IIk, IIl, IIm, IIn, IIo, IIp, IIq, IIr, IIs, or IIt as shown in Table 9 (elsewhere herein).
  • the chemical group at position R 1 is a cyano group
  • R 2 and R 3 together form a 6-membered aryl ring
  • the compound has structural formula IIb wherein the chemical groups R 2 , R 3 , R 4 , R 5 , R 6 and R 7 are as defined for the compound of structural formula II, and R 10 and R 11 are each independently selected from hydrogen, halogen, C 1 -C 4 linear or branched alkyl, cyclopropyl, and cyclobutyl.
  • the compounds of structural formula IIb can have a structural formula IIu, IIv, or IIw, as shown in Table 10 (elsewhere herein).
  • the chemical group at position R 1 is a 5-membered heteroaryl ring
  • the chemical groups at R 2 and R 3 are each independently selected from hydrogen, C 1 -C 4 linear or branched alkyl, cyclopropyl, cyclobutyl, phenyl, and benzyl
  • the compound has structural formula IIc, IId, IIe, IIf, IIg, IIh, or IIi
  • the compound of structural formulas IIc, IId, IIe, IIf, IIg, IIh, and Iii is a compound having a structural formula IIx, IIy, IIz, IIaa, IIbb, IIcc, IIdd, IIee, IIff, IIgg, IIhh, IIii, IIjj, IIkk, IIll, IImm, IInn, IIoo, IIpp, IIqq, IIrr, and IIss as shown in Table 12 (elsewhere herein).
  • the compound is selected from any of exemplary compounds 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, and 65, as shown in Table 13 (elsewhere herein).
  • the present disclosure provides a pharmaceutical composition comprising a compound of structural formula I or formula II and one or more adjunct ingredients.
  • the present disclosure provides uses of the FABP3/4/5/7 inhibitor compounds of structural formula I or formula II in method of making medicament or pharmaceutical composition for the treatment of a disease or condition affected by any one or more of the FABPs, FABP3, FABP4, FABP5, and FABP7 (i.e., “conditions affected by FABP3/4/5/7”).
  • the present disclosure provides a method for treating a subject having a disease or condition affected by FABP3/4/5/7, comprising administering to a subject in need thereof a therapeutically effective amount of compound of structural formula I or formula II, or a pharmaceutical composition comprising a compound of structural formula I or formula II and one or more adjunct ingredients.
  • the diseases or conditions affected by FABP3/4/5/7 present in a subject which the compounds of structural formula I or formula II can be used to treat can be selected from: atherosclerosis, coronary atherosclerosis, arterial fibrosis, pulmonary hypertension, heart failure, obesity, Type-2 diabetes, Type-1-diabetes, gestational diabetes, polycystic ovary syndrome, endometriosis, conditions affected by lipid metabolism and free fatty acid serum levels, metabolic disorders, fatty liver disease, kidney fibrosis, systemic inflammation, acute inflammation, allergic inflammation, airway inflammation, viral infection (e.g., COVID-19, common cold), skin diseases (e.g., vitiligo, psoriasis, atopic dermatitis, allergic contact dermatitis, mycosis fungoides, alopecia areata, cicatricial alopecia, graft vs.
  • GvHD host disease
  • MS multiple sclerosis
  • Parkinson’s disease autoimmune diseases (e.g., experimental autoimmune encephalomyelitis (EAE), asthma, type-1-diabetes, autoimmune lung disease, autoimmune hepatitis, rheumatoid arthritis (RA), spondyloarthropathy, vesicular stomatitis virus infection, multiple sclerosis (MS), lupus nephritis, Crohn's disease, ulcerative colitis, and food allergy), ischemic stroke, graft versus host disease (GvHD) and cancer (e.g., breast cancer, prostate cancer
  • the present disclosure also provides methods for preparing the compounds of structural formula I or formula II, the method comprising: (a) combining in a solvent a substituted anhydride compound of formula III: wherein Y, R 4 , R 5 , R 6 and R 7 are as defined above for compounds of formula I and II; with a substituted 2-amino-thiophene compound of formula IV: wherein, R 1 , R 2 and R 3 are as defined above for compounds of formula I and II; and (b) removing the solvent to obtain a compound having the structural formula I or formula II.
  • the compound is a compound of structural formula IVa: wherein the chemical groups R 2 , R 3 , R 4 , R 5 , R 6 and R 7 are as defined for the compound of structural formula II.
  • the compounds of structural formula IVa is selected from the compounds 4a, 4b, 4c, 4d, 4e, 4f, 4g, 4h, 4i, 4j, and 4k, shown in Tables 14 and 15 (elsewhere herein).
  • the compound of formula IV has structural formula IVb: wherein the chemical groups R 2 , R 3 , R 4 , R 5 , R 6 and R 7 are as defined for the compound of structural formula II, and wherein R 10 and R 11 are each independently selected from hydrogen, halogen, C 1 -C 4 linear or branched alkyl, cyclopropyl, and cyclobutyl.
  • the compound of structural formula IVb is selected from compounds 4l, 4m, and 4n shown in Table 16 (elsewhere herein).
  • the chemical group at R 1 is a 5- membered heteroaryl ring (e.g., 3-substituted 1,2,4-oxadiazol), and the compound is selected from the compounds of structural formula IVc, IVd, IVe, IVf, IVg, IVh, and IVi shown in Table 17 (elsewhere herein).
  • the compounds of structural formulas IVc, IVd, IVe, IVf, IVg, IVh, and IVi are selected from compounds 4o, 4p, 4q, 4r, 4s, 4t, 4u, 4v, 4w, 4x, 4y, 4z, 4aa, 4bb, 4cc, 4dd, 4ee, 4ff, 4gg, 4hh, and 4ii shown in Table 18 (elsewhere herein).
  • FIG.1A, FIG.1B, and FIG.1C depict plots of results showing that inhibitor compounds FTS005, FTS030, FTS031, FTS037, and FTS039 do not activate transcription by PPAR ⁇ , PPAR ⁇ , or PPAR ⁇ .
  • Cells were treated with PPARs’ specific agonists Wy-134643 (5 ⁇ M), rosiglitazone (5 ⁇ M), and GW0742 (5 ⁇ M) or one of the compounds FTS005, FTS030, FTS031, FTS037, and FTS039 (10 ⁇ M). Data is mean ⁇ SD from 3 independent experiments.
  • FIG.2A, FIG.2B, and FIG.2C depict plots of results showing that the FABP3/4/5/7 inhibitor compound FTS005, which exhibits specificity for FABP4/5, inhibits growth of TNBC cells in an FABP5-dependent manner and more efficiently than the FABP5/7 inhibitor, SBF-I26.
  • Cells in all experiments were treated with one of the denoted compounds at indicated concentrations for 4 days. Cells confluency was measured using Incucyte software.
  • FIG.2A Results indicating that compound FTS005 at different concentrations inhibits proliferation of MB-231 and BT-549 cell lines. IC 50 values were calculated using GraphPad fitting algorithms.
  • FIG.2B Plots of results indicating that compound FTS005 at different concentrations inhibits proliferation of wild type MB-231 cell line but not those cells stably expressing the FABP5 shRNA (“shF5”).
  • FIG.2C Plots of results indicating that compound FTS005 at different concentrations inhibits proliferation of MB-231 cell lines better than the commercially available FABP5/7 inhibitor SBFI-26. Data is mean ⁇ SD from 3 independent experiments. [0042]
  • FIG.3A, FIG.3B, and FIG.3C depict plots of results indicating that the FABP3/4/5/7 inhibitor compound FTS005 inhibits growth of mouse mammary carcinoma cell lines expressing high levels of FABP5 relative to those expressing low levels of the gene.
  • FIG.3A Levels of FABP5 mRNA in the donated cell lines was measured by QPCR.
  • FIG.3B Results indicating that compound FTS005 inhibits proliferation of MB- 231 and 4T1 cell lines in correlation to expression levels of FABP5 in the cells. IC 50 values were calculated using GraphPad fitting algorithms.
  • FIG.3C Results indicating that compound FTS005 inhibit cell proliferation only in cells that express FABP5. Data is mean ⁇ SD from 3 independent experiments.
  • FIG.4A and FIG.4B depict plots of results indicating that the FABP3/4/5/7 inhibitors compounds FTS005, FTS040, FTS041, FTS042, FTS043, FTS044, FTS045, and FTS049 inhibit proliferation of ovarian cancer cells OVCAR8.
  • Cells in all experiments were treated with the denoted compounds at indicated concentrations for 4 days. Cells confluency was measured using Incucyte software.: FIG.4A Results indicating that compounds FTS005, FTS040, FTS041, FTS042, FTS043, FTS044, FTS045, and FTS049 inhibit proliferation of OVCAR8 cell line.
  • FIG.4B IC 50 values for all compounds were calculated using GraphPad fitting algorithms.
  • FIG.5A, FIG.5B, and FIG.5C depict results showing that the FABP3/4/5/7 inhibitor FTS005 inhibits growth of neuroblastoma and sensitize cells to all-trans retinoic acid (atRA) treatments.
  • NPG human neuroblastoma cells were treated with FTS005 at indicated concentrations in the presence or absence of retinoic acid (1 mM) for 4 days. Cells confluency was measured using Incucyte software.
  • FIG.5A Results indicating that compound FTS005 inhibits proliferation of NPG cells but shows synergistic effect when combined with atRA.
  • FIG.5B, FIG.5C Results indicating that compound FTS005 in combination with atRA inhibit cell proliferation more efficiently than atRA mono- treatment. IC 50 values were calculated using GraphPad fitting algorithms. Data is mean ⁇ SD from 3 independent experiments.
  • FIG.6A, FIG.6B, FIG.6C, FIG.6D, FIG.6E, FIG.6F, and FIG.6G depict results showing FABP3/4/5/7 inhibitor FTS005 suppresses tumor growth in vivo in xenograft model.
  • FIG. 6A Tumor growth in MB-231 xenograft model. MB-231 cells (5x10 6 ) were transplanted into the right flank of 7-week-old female NOD scid gamma (NSG) mice.
  • NSG NOD scid gamma
  • FIG.6B Data plotted represents the weight of tumors in each mouse at the end point (day 24). Statistical significance between the control and treated mice in all experiments was evaluated using a Student's t-test.
  • FIG.6C Representative histological sections of paraffin embedded section from tumors stained with antibodies for Ki67, VEGFA, and F4/80.
  • FIG. 6D, FIG.6E, and FIG.6F Plots indicating the intensity of the immunohistology staining for each sample.
  • FIG.6G Expression levels of the denoted PPAR ⁇ target genes in samples from collected tumors. Data in FIG 5C, FIG 5D, and FIG 5G is mean ⁇ SD of 3 mice/group. Statistical analyses were carried out using two-tailed Student’s t-test. *p ⁇ 0.05, **p ⁇ 00.1. [0046] FIG.7A, FIG.7B, FIG.7C, FIG.7D, FIG.7E, and FIG.7F depict results showing FABP3/4/5/7 inhibitor FTS005 suppresses tumor growth in vivo in syngeneic mouse model.
  • FIG.7B Data plotted represents the weight of individual tumors in each mouse at the end point (day 32). Statistical significance between the control and treated mice in all experiments was evaluated using a Student's t-test.
  • FIG.7C Expression levels of the known PPAR ⁇ direct targets VEGFA, ACSL1, and PLIN2 that are involved in tumor growth and FA storage and oxidation, in tumor samples collected from treated and untreated mice.
  • FIG.7D Representative histological sections of paraffin embedded section from treated and untreated tumors stained with antibodies for the proliferation marker Ki67 and VEGFA.
  • FIG.7E and FIG.7F Plots indicating the percentage of positive immunohistology staining of Ki67 (FIG.7E) and VEGFA (FIG.6F) out of the total section area for each sample. *p ⁇ 0.05.
  • FIG.8A, FIG.8B, FIG.8C, and FIG.8D depict results of metabolomic analysis for metabolites of glycolysis, TCA cycle, FA oxidation, long-chain fatty acids, and ADP and ATP, that were measured by LC/MS/MS in tumor samples collected from the treated and untreated mice.
  • FIG. 8A Amounts of long chain fatty acids in the tumor cells.
  • FIG.8B Amount of TCA cycle metabolites in treated and untreated tumors.
  • FIG.8C Amount of ADP, ATP and their calculated ratio in treated and untreated tumors.
  • FIG.8D Amount of glycolysis metabolites in treated and untreated tumors. Data is mean ⁇ SD of 3 mice/group.
  • FIG.9A, FIG.9B, FIG.9C, FIG.9D, FIG.9E, and FIG.9F depict results showing FABP3/4/5/7 inhibitor FTS005 regulates tumor associated macrophages within the tumor microenvironment in 4T1 syngeneic mouse model.
  • FIG.9A, FIG.9B, and FIG.9C Representative histological sections of paraffin embedded section from treated and untreated tumors stained with antibodies for F4/80 (FIG.9A), CD68 (FIG.9B), and CD163 (FIG.9C).
  • FIG.9D, FIG.9E, and FIG.9F Plots indicating the percentage of positive immunohistology staining with each antibody for each sample.
  • Data in FIG 9D, FIG 9E, and FIG 9F is mean ⁇ SD of 3 mice/group.
  • Statistical analyses were carried out using two-tailed Student’s t-test. *p ⁇ 0.05.
  • FIG.10A, FIG.10B, FIG.10C, FIG.10D, FIG.10E, and FIG.10F depict results showing FABP3/4/5/7 inhibitor FTS005 regulates T cells within the tumor microenvironment in 4T1 syngeneic mouse model.
  • FIG.10A Representative histological sections of paraffin embedded section from treated and untreated tumors stained with antibodies for CD3 (A), CD4 (B), and CD8 (C).
  • FIG. 10B Plots indicating the percentage of positive immunohistology staining with each antibody for each sample. Data is mean ⁇ SD of 3 mice/group. Statistical analyses were carried out using two-tailed Student’s t-test. *p ⁇ 0.05.
  • FIG.10C, FIG.10D Plots indicating the frequency of CD4 and CD8 splenic T-cells (FIG.10C) and activated CD4 and CD8 T-cells (TNF ⁇ + CD4 CD8T cells) (FIG. 10D) harvested from untreated and treated syngeneic mice.
  • FIG.11 depict results of immune cell profiling done by utilizing nCounter PanCancer Immune Profiling Panel showing that treatment with FABP3/4/5/7 inhibitor FTS005 modulates multiple immune cells within the tumor microenvironment in 4T1 syngeneic mouse model.
  • FIG.11 immune cells profile indicating all immune cell types that were found to be significantly different in the analysis (P-value ⁇ 0.05, fold change>1.5).
  • FIG.12A and FIG.12B depict results showing that FTS005 inhibits lipid uptake in cell culture models of steatosis and mature adipocytes.
  • FIG.12A Plots indicating the intensity of Nile Red staining in hepatic HepG2 cells that were treated with designated concentrations of FTS005 or the known FABP4 inhibitor BMS309403 for 4 h followed by oleic acid treatment (OA) (1mM, 24 h). Lipid accumulation was only measure in live cells that were positive for Dapi staining. Quantification of total lipid uptake was done using Biotech Cytation 5 plate reader.
  • FIG.12B Histogram depicting the color intensity of Nile Red staining in viable mature adipocytes following treatment with FTS005.
  • 3T3-L1 pre-adipocytes were differentiated in culture.
  • Cells were treated with FTS005 or the known FABP4 inhibitor BMS309403 (BMS) on day six and stained with Nile Red on day 12.
  • FIG.13A, FIG.13B, FIG.13C, FIG.13D, FIG.13E, FIG.13F, FIG.13G, FIG.13H, FIG.13I, FIG.13J, and FIG.13K depict results showing that FABP inhibitor FTS005 modulate differentiation of macrophages in culture.
  • FIG.13A, FIG.13C, FIG.13D Frequency of the M1 marker MHC-II (FIG.13A), and the M2 markers CD36 (FIG.13C) and CD206 (FIG.13D) in na ⁇ ve macrophages following their differentiated into M1- or M2-macrophages.
  • FIG.13B, FIG.13E Levels of the cytokines IL-12 (FIG.13B) and IL-10 (FIG.13E) secreted from M1 and M2 macrophages, respectively, during differentiation.
  • FIG.13F, FIG.13G, FIG.13H Expression levels of CD206 (FIG.13F), and levels of IL-10 (FIG.13G) and IL-12 (FIG.13H) in macrophages differentiated from na ⁇ ve status to M2-macrophages in the absence and presence of FABP4/5 inhibitor FTS005.
  • FIG.13I, FIG.13J, FIG.13K Frequency (FIG.13I) and expression levels of CD206 (FIG.13J), and levels of IL-12 (FIG.13K) in M1-macrophages differentiated into M2-macrophages in the absence and presence of FTS005.
  • any embodiment of any of the disclosed methods or compositions can consist of or consist essentially of – rather than comprise/include/contain/have – any of the described steps, elements, and/or features.
  • the term “consisting of” or “consisting essentially of” can be substituted for any of the open-ended linking verbs recited above, in order to change the scope of a given claim from what it would otherwise be using the open-ended linking verb.
  • any embodiment of any of the disclosed compounds or methods can consist of or consist essentially of – rather than comprise/include/contain/have – any of the described steps, elements, and/or features.
  • the term “consisting of” or “consisting essentially of” can be substituted for any of the open-ended linking verbs recited above, in order to change the scope of a given claim from what it would otherwise be using the open-ended linking verb.
  • the term “subject” refers to a human or an animal that would benefit from being administered with the FABP3/4/5/7 inhibitor compounds discussed in the present application, such as those suffering from, without limitation a disease affected by expression of one or more of the FABPs, FABP3, FABP4, FABP5, and FABP7, lack of control of free fatty acid serum levels, cancer, metabolic syndrome, or atherosclerosis.
  • the terms “treat,” “treating,” “treatment,” and the like refer to reducing or ameliorating a disorder and/or symptoms associated therewith.
  • treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated.
  • the terms “prevent,” “preventing,” “prevention,” “prophylactic treatment” and the like are encompassed within the term “treating,” and refer to reducing the probability of developing a disorder or condition in a subject, who does not have, but is at risk of or susceptible to developing a disorder or condition.
  • “pharmaceutically acceptable” means physiologically tolerable, for either human or veterinary applications.
  • pharmaceutically acceptable is meant a material that is not biologically or otherwise undesirable, i.e., the material may be administered to a subject without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained.
  • the pharmaceutically acceptable material is nontoxic to the recipient.
  • the carrier would naturally be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject, as would be well known to one of skill in the art.
  • pharmaceutically acceptable carriers and other components of pharmaceutical compositions see, e.g., Remington’s Pharmaceutical Sciences, 18th ed., Mack Publishing Company, 1990.
  • Test agents refers to an agent or compound that is to be screened in one or more of the assays described herein.
  • Test agents include compounds of a variety of general types including, but not limited to, small organic molecules, known pharmaceuticals, polypeptides; carbohydrates such as oligosaccharides and polysaccharides; polynucleotides; lipids or phospholipids; fatty acids; steroids; or amino acid analogs.
  • Test agents can be obtained from libraries, such as natural product libraries and combinatorial libraries.
  • methods of automating assays are known that permit screening of several thousands of compounds in a short period.
  • Non-limiting examples of substitutions for hydrogen include the following: [0068]
  • Substituted and unsubstituted linear, branched, or cyclic alkyl units include the following non-limiting examples: methyl (C 1 ), ethyl (C 2 ), n-propyl (C 3 ), iso-propyl (C 3 ), cyclopropyl (C 3 ), n- butyl (C 4 ), sec-butyl (C 4 ), iso-butyl (C 4 ), tert-butyl (C 4 ), cyclobutyl (C 4 ), cyclopentyl (C 5 ), cyclohexyl (C 6 ), and the like; whereas substituted linear, branched, or cyclic alkyl, non-limiting examples of which includes, hydroxymethyl (C 1 ), chloromethyl (C 1 ), trifluoromethyl (C 1 ), aminomethyl (C 1 ), 1- chloroethyl (C 2
  • Substituted and unsubstituted linear, branched, or cyclic alkenyl include, ethenyl (C 2 ), 3- propenyl (C 3 ), 1-propenyl (also 2-methylethenyl) (C 3 ), isopropenyl (also 2-methylethen-2-yl) (C 3 ), buten-4-yl (C 4 ), and the like; substituted linear or branched alkenyl, non-limiting examples of which include, 2-chloroethenyl (also 2-chlorovinyl) (C 2 ), 4-hydroxybuten-1-yl (C 4 ), 7-hydroxy-7-methyloct- 4-en-2-yl (C 9 ), 7-hydroxy-7-methyloct-3,5-dien-2-yl (C 9 ), and the like.
  • Substituted and unsubstituted linear or branched alkynyl include, ethynyl (C 2 ), prop-2-ynyl (also propargyl) (C 3 ), propyn-1-yl (C 3 ), and 2-methyl-hex-4-yn-1-yl (C 7 ); substituted linear or branched alkynyl, non-limiting examples of which include, 5-hydroxy-5-methylhex-3-ynyl (C 7 ), 6- hydroxy-6-methylhept-3-yn-2-yl (C8), 5-hydroxy-5-ethylhept-3-ynyl (C9), and the like.
  • Substituted and unsubstituted “alkoxy” are used herein denotes a unit having the general formula –OR 100 wherein R 100 is an alkyl, alkylenyl, or alkynyl unit as defined herein above, for example, methoxy, methoxymethyl, methoxymethyl.
  • Substituted and unsubstituted “haloalkyl” are used herein denotes an alkyl unit having a hydrogen atom substituted by one or more halogen atoms, for example, trifluoromethyl, 1,2- dicloroethyl, and 3,3,3-trifluoropropyl.
  • aryl denotes cyclic organic units that comprise at least one benzene ring having a conjugated and aromatic six-membered ring, non-limiting examples of which include phenyl (C 6 ), naphthylen-1-yl (C 10 ), naphthylen-2-yl (C 10 ).
  • Aryl rings can have one or more hydrogen atoms substituted by another organic or inorganic radical.
  • Non-limiting examples of substituted aryl rings include: 4-fluorophenyl (C 6 ), 2-hydroxyphenyl (C 6 ), 3-methylphenyl (C 6 ), 2-amino-4- fluorophenyl (C 6 ), 2-(N,N-diethylamino)phenyl (C 6 ), 2-cyanophenyl (C 6 ), 2,6-di-tert-butylphenyl (C 6 ), 3-methoxyphenyl (C 6 ), 8-hydroxynaphthylen-2-yl (C 10 ), 4,5-dimethoxynaphthylen-1-yl (C 10 ), and 6-cyanonaphthylen-1-yl (C 10 ).
  • heteroaryl denotes an organic unit comprising a five or six membered conjugated and aromatic ring wherein at least one of the ring atoms is a heteroatom selected from nitrogen, oxygen, or sulfur.
  • the heteroaryl rings can comprise a single ring, for example, a ring having 5 or 6 atoms wherein at least one ring atom is a heteroatom not limited to nitrogen, oxygen, or sulfur, such as a pyridine ring, a furan ring, or thiofuran ring.
  • heteroaryl can also be a fused multicyclic and heteroaromatic ring system having wherein at least one of the rings is an aromatic ring and at least one atom of the aromatic ring is a heteroatom including nitrogen, oxygen, or sulfur.
  • the following are non-limiting examples of heteroaryl rings according to the present disclosure:
  • heterocyclic denotes a ring system having from 3 to 10 atoms wherein at least one of the ring atoms is a heteroatom not limited to nitrogen, oxygen, or sulfur.
  • the rings can be single rings, fused rings, or bicyclic rings.
  • Non-limiting examples of heterocyclic rings include: [0076] All of the aforementioned heteroaryl or heterocyclic rings can be optionally substituted with one or more substitutes for hydrogen as described herein further.
  • thiophene-2-yl and thiophene-3-yl are used to describe the heteroaryl units having the respective formulae: whereas in naming the compounds of the present disclosure, the chemical nomenclature for these moieties are typically spelled “thiophen-2-yl and thiophen-3-yl” respectively.
  • thiophene-2-yl and thiophene-3-yl are used when describing these rings as units or moieties which make up the compounds of the present disclosure solely to make it unambiguous to the artisan of ordinary skill which rings are referred to herein.
  • the terms “compound,” “analog,” and “composition of matter” stand equally well for the HIF-1 ⁇ prolyl hydroxylase enzyme inhibitors described herein, including all enantiomeric forms, diastereomeric forms, salts, and the like, and the terms “compound,” “analog,” and “composition of matter” are used interchangeably throughout the present specification.
  • the compounds disclosed herein include all salt forms, for example, salts of both basic groups, inter alia, amines, as well as salts of acidic groups, inter alia, carboxylic acids.
  • anions that can form pharmaceutically acceptable salts with basic groups: chloride, bromide, iodide, sulfate, bisulfate, carbonate, bicarbonate, phosphate, formate, acetate, propionate, butyrate, pyruvate, lactate, oxalate, malonate, maleate, succinate, tartrate, fumarate, citrate, and the like.
  • cations that can form pharmaceutically acceptable salts of the anionic form of acidic substituent groups on the compounds described herein: sodium, lithium, potassium, calcium, magnesium, zinc, bismuth, and the like.
  • FABP3/4/5/7 Inhibitor Compounds Disclosed herein are compounds that inhibit one or more of the FABP3, FABP4, FABP5, and FABP7 (i.e., an “FABP3/4/5/7 inhibitor”).
  • FABP3/4/5/7 inhibitor compounds of the present disclosure are based on a substituted thiophene ring having the general structural formula I: wherein, R 1 is selected from hydrogen, cyano, and 5-membered heteroaryl ring; R 2 and R 3 are each independently selected from hydrogen, C 1 -C 4 linear or branched alkyl, cyclopropyl, cyclobutyl, phenyl, trifluoromethyl, and benzyl, or R 2 and R 3 together form a 5- to 8- membered monocyclic, bicyclic, or spirocyclic carbocycle or heterocycle ring, or a 5- to 8- membered aryl or heteroaryl ring, wherein the carbocycle, heterocycle, aryl, or heteroaryl ring is optionally substituted with one or two substituents selected from C 1 -C 4 -alkyl, methoxy, or fluoro; X is a moiety of formula: wherein, Y is a heteroatom selected
  • the compound of structural formula I includes any pharmaceutically acceptable salt thereof.
  • the compound of structural formula I of the present disclosure excludes the compounds shown in Table 1 below.
  • the chemical group at position R 1 is a cyano group.
  • the compound of formula I can have formula Ia: [0086] Exemplary compounds of formula Ia, include but are not limited to the compounds having structural formulas Ij, Ik, Il, Im, In, Io, Ip, Iq, Ir, Is, and It as shown in Table 2 below. [0087] TABLE 2
  • the chemical substituents at positions R 2 and R 3 together form a 5- to 8-membered aryl or heteroaryl ring.
  • the compound of formula I can have formula Ib
  • R 10 and R 11 are each independently selected from hydrogen, halogen, C 1 -C 4 linear or branched alkyl, cyclopropyl, and cyclobutyl.
  • Exemplary compounds of formula Ib include but are not limited to the compounds having structural formulas Iu, Iv, and Iw shown in Table 3 below.
  • the chemical group at R 1 is a 5-membered heteroaryl ring (e.g., 3-substituted 1,2,4- oxadiazol).
  • the compound of formula I can be a compound structural formula Ic, Id, Ie, If, Ig, Ih, or Ii as shown in Table 4 below. [0092] TABLE 4
  • R 12 is selected from hydrogen, halogen, C 1 -C 4 linear or branched alkyl, cyclopropyl, cyclobutyl, phenyl.
  • Exemplary compounds of formulas Ic, Id, Ie, If, Ig, Ih, or Ii include but are not limited to the compounds having structural formulas Ix, Iy, Iz, Iaa, Ibb, Icc, Idd, Iee, Iff, Igg, Ihh, Iii, Ijj, Ikk, Ill, Imm, Inn, Ioo, Ipp, Iqq, and Irr shown in Table 5 below. [0094] TABLE 5
  • the FABP3/4/5/7 inhibitor compounds having structural formula I of the present disclosure comprise a moiety X attached to the amine group of the thiophene.
  • the X moiety is a chemical group of formula: wherein, Y is a heteroatom selected from –S–, and –O–, or is –CR 8 R 9 –, wherein R 8 and R 9 are each independently selected from hydrogen, C 1 -C 4 linear or branched alkyl, phenyl, and benzyl, or R 8 and R 9 together form a cyclopropyl, cyclobutyl, cyclopentyl, or oxetanyl ring; R 4 , R 5 , R 6 and R 7 is each independently selected from hydrogen, C 1 -C 4 linear or branched alkyl, phenyl, and benzyl, and/or R 4 and R 5 together or R 6 and R 7 together form a cyclopropyl ring or a cyclobutyl ring
  • the chemical group Y of the X moiety is selected from –S– or –O– and R 4 , R 5 , R 6 and R 7 are each independently hydrogen or C 1 -C 4 linear or branched alkyl.
  • R 4 , R 5 , R 6 and R 7 are each independently hydrogen or C 1 -C 4 linear or branched alkyl.
  • Exemplary X moieties where Y is a sulfur atom (-S-) can include any of the moieties shown in Table 6 below. [0098] TABLE 6
  • Exemplary X moieties where Y is an oxygen atom (-O-) can include any of the moieties shown in Table 7 below. [0100] TABLE 7
  • the chemical group Y of the X moiety is –CR 8 R 9 –, wherein R 8 and R 9 are each independently selected from hydrogen, C 1 -C 4 linear or branched alkyl, phenyl, and benzyl, or R 8 and R 9 together form a cyclopropyl, cyclobutyl, cyclopentyl, or oxetanyl ring, and the chemical groups at positions R 4 , R 5 , R 6 and R 7 are each independently hydrogen or C 1 -C 4 linear or branched alkyl, and.
  • Exemplary X moieties where Y is –CR 8 R 9 – can include any of the moieties shown in Table 8 below.
  • the various inhibitor compounds of structural formula I provided in the present disclosure include a range of compounds with various substituted thiophene ring moieties combined with various X moieties.
  • the various FABP3/4/5/7 inhibitor compounds can be represented as compounds of structural formula II and any pharmaceutically acceptable salt thereof: wherein, R 1 is selected from hydrogen, cyano, and 5-membered heteroaryl ring; R 2 and R 3 are each independently selected from hydrogen, C 1 -C 4 linear or branched alkyl, cyclopropyl, cyclobutyl, phenyl, trifluoromethyl, and benzyl, or R 2 and R 3 together form a 5- to 8-membered monocyclic, bicyclic, or spirocyclic carbocycle or heterocycle ring, or a 5- to 8-membered aryl or heteroaryl ring, wherein the carbocycle, heterocycle, aryl, or heteroaryl ring is optionally substituted with one or two substituents selected from C 1 -
  • the compound of structural formula II of the present disclosure excludes the compounds of Table 1 (see above).
  • the compounds of structural formula II include a range of compounds represented by sub-structures.
  • the chemical group at position R 1 is a cyano group
  • the compound has structural formula (IIa) wherein the chemical groups R 2 , R 3 , R 4 , R 5 , R 6 and R 7 are as defined for the compound of structural formula II.
  • the compounds of having the substructure of structural formula IIa can have a structural formula IIj, IIk, IIl, IIm, IIn, IIo, IIp, IIq, IIr, IIs, and IIt as shown in Table 9 below. [0108] TABLE 9
  • the chemical groups R 4 , R 5 , R 6 and R 7 are as defined for the compound of structural formula II.
  • the chemical group at position R 1 is a cyano group
  • R 2 and R 3 together form a 6-membered aryl ring
  • the compound has structural formula (IIb) wherein the chemical groups R 2 , R 3 , R 4 , R 5 , R 6 and R 7 are as defined for the compound of structural formula II, and wherein R 10 and R 11 are each independently selected from hydrogen, halogen, C 1 -C 4 linear or branched alkyl, cyclopropyl, and cyclobutyl.
  • the compounds of having the substructure of structural formula IIb can have a structural formula IIu, IIv, and IIw as shown in Table 10 below. [0111] TABLE 10
  • the chemical group at position R 1 is a 5-membered heteroaryl ring (e.g., 3-substituted 1,2,4-oxadiazol), and the chemical groups at R 2 and R 3 are each independently selected from hydrogen, C 1 -C 4 linear or branched alkyl, cyclopropyl, cyclobutyl, phenyl, and benzyl.
  • the compound can have structural formula IIc, IId, IIe, IIf, IIg, IIh, and IIi shown in Table 11 below. [0113] TABLE 11
  • chemical groups R 4 , R 5 , R 6 and R 7 are as defined for the compound of structural formula II and the chemical group R 12 in IIc, IId, IIe, IIf, IIg, and IIh can be a hydrogen, halogen, C 1 -C 4 linear or branched alkyl, cyclopropyl, cyclobutyl, or phenyl.
  • the compounds having the substructures of structural formulas IIc, IId, IIe, IIf, IIg, and IIh can have a structural formula IIx, IIy, IIz, IIaa, IIbb, IIcc, IIdd, IIee, IIff, IIgg, IIhh, IIii, IIjj, IIkk, IIll, IImm, IInn, IIoo, IIpp, IIqq, IIrr, and IIss as shown in Table 12 below. [0115] TABLE 12
  • each of the various substructure embodiments of the compound of structural formula II including the compounds of structural formulas IIa, IIb, IIc, IId, IIe, IIf, IIg, IIh, IIi, IIj, IIk, IIl, IIm, IIn, IIo, IIp, IIq, IIr, IIs, IIt, IIu, IIv, IIw, IIx, IIy, IIz, IIaa, IIbb, IIcc, IIdd, IIee, IIff, IIgg, IIhh, IIii, IIjj, IIkk, IIll, IImm, IInn, IIoo, IIpp, IIqq, IIrr, and IIss, it is contemplated that the atom or chemical group represented by Y can be–S– or –O–, and the chemical groups R 4 , R 5 , R 6 and R 7 can each independently be hydrogen or C 1 -C 4 linear or branched alkyl.
  • exemplary FABP3/4/5/7 inhibitor compounds of structural formulas I and II include, but are not limited to, compounds 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, and 65, which are shown in Table 13 below. [0117] TABLE 13
  • the FABP3/4/5/7 compounds provided herein can exist in various well-known closely-related and/or equivalent forms not explicitly described by the chemical structures and formulae. It is intended that the FABP3/4/5/7 inhibitor compounds of structural formulas I and II of the present disclosure (including the compounds of Tables 2-5, and 9-13, and as described the Examples) includes these closely-related forms of the compounds defined by the chemical structures and formulae including, but not limited to, pharmaceutically acceptable salts of the compounds, mixture of stereoisomers of the compounds, single stereoisomers of the compounds, tautomeric forms of the compounds, and/or prodrug forms of the compounds.
  • the progress of the reaction can be followed by one or more analytical methods, for example, thin layer chromatography (TLC), gas chromatography, and the like.
  • TLC thin layer chromatography
  • the solvent is removed in vacuo to afford the desired FABP3/4/5/7 inhibitor of structural formula II.
  • the process for preparing the disclosed FABP3/4/5/7 inhibitor compounds of structural formula II comprising: (a) combining in a solvent a substituted anhydride compound of formula III: wherein Y is a heteroatom selected from –S–, and –O–, or is –CR 8 R 9 –, wherein R 8 and R 9 are each independently selected from hydrogen, C 1 -C 4 linear or branched alkyl, phenyl, and benzyl, or R 8 and R 9 together form a cyclopropyl, cyclobutyl, cyclopentyl, or oxetanyl ring; R 4 , R 5 , R 6 and R 7 is each independently selected from hydrogen, C 1 -C 4 linear or branched alkyl, phenyl, and benzyl, and/or R 4 and R 5 together or R 6 and R 7 together form a cyclopropyl ring or a cyclobutyl ring, or
  • the chemical group Y is a sulfur atom. In another embodiment of the anhydride of formula III, the chemical group Y is an oxygen atom. In a further embodiment of the anhydride of formula III, the chemical group Y is – CR 5 R 6 – wherein R 5 and R 6 are each independently chosen from hydrogen, C 1 -C 4 linear or branched alkyl. In a still further embodiment when Y is R 5 and R 6 are each independently chosen from C 1 -C 4 linear alkyl, R 5 and R 6 can be taken together to form a spirocyclic ring having from 4 to 7 atoms.
  • R 1 and R 4 can be taken together to form a heterocyclic ring having from 4 to 6 carbon atoms.
  • a range of specific anhydride compounds of formula III that can be used in the preparation of compound of formula II are further described in the Examples.
  • the compound is a compound of structural formula IVa: wherein, the chemical groups at R 2 and R 3 are each independently selected from hydrogen, C 1 -C 4 linear or branched alkyl, cyclopropyl, cyclobutyl, phenyl, and benzyl, optionally substituted with one or two substituents selected from C 1 -C 4 -alkyl, methoxy, chloro or fluoro.
  • Such exemplary compounds of structural formula IVa include but are not limited to the compounds 4a, 4b, 4c, 4d, 4e, and 4f shown in Table 14 below.
  • the chemical substituents at positions R 2 and R 3 together form a 5- to 8-membered monocyclic, bicyclic, or spirocyclic carbocycle or heterocycle ring,.
  • Such exemplary compounds of structural formula IVa include but are not limited to the compounds 4g, 4h, 4i, 4j, and 4k, shown in Table 15 below.
  • the chemical substituents at positions R 2 and R 3 together form a 5- to 8-membered aryl or heteroaryl ring, wherein the carbocycle, heterocycle, aryl, or heteroaryl ring is optionally substituted with one or two substituents selected from C 1 -C 4 -alkyl, methoxy, or fluoro, as depicted by the compound of structural formula IVb: wherein R 10 and R 11 are each independently selected from hydrogen, halogen, C 1 -C 4 linear or branched alkyl, cyclopropyl, and cyclobutyl.
  • Such compounds of structural formula IVb include but are not limited to the compounds 4l, 4m, and 4n shown in Table 16 below.
  • the chemical group at R 1 is a 5-membered heteroaryl ring (e.g., 3-substituted 1,2,4-oxadiazol) and the compound is selected from the compounds of structural formula IVc, IVd, IVe, IVf, IVg, IVh, and IVi shown in Table 17 below.
  • the chemical groups at R 2 and R 3 are each independently selected from hydrogen, C 1 -C 4 linear or branched alkyl, cyclopropyl, cyclobutyl, phenyl, and benzyl, or R 2 and R 3 together form a 5- to 8-membered monocyclic, bicyclic, or spirocyclic carbocycle or heterocycle ring, or a 5- to 8- membered aryl or heteroaryl ring, wherein the carbocycle, heterocycle, aryl, or heteroaryl ring is optionally substituted with one or two substituents selected from C 1 -C 4 -alkyl, methoxy, or fluoro; and the chemical group at R 12 is selected from hydrogen, halogen, C 1 -C 4 linear or branched alkyl, cyclopropyl, cyclobutyl, and phenyl.
  • Such compounds of structural formulas IVc, IVd, IVe, IVf, IVg, IVh, and IVi include but are not limited to the compounds 4o, 4p, 4q, 4r, 4s, 4t, 4u, 4v, 4w, 4x, 4y, 4z, 4aa, 4bb, 4cc, 4dd, 4ee, 4ff, 4gg, 4hh, and 4ii shown in Table 18 below. [0132] TABLE 18
  • the compound of structural formula IVd may be synthesized using the routes shown in Scheme B below.
  • Scheme B [0135] Another route by which the compound of structural formula IVd may be synthesized has been described by J. Sarvanan, et al., (Indian Journal of Heterocyclic Chemistry 1998, 7, 285-288) and is shown in Scheme C below.
  • Scheme C [0136] A route by which a compound of structural formula IVe may be synthesized starting with a 2- amino-3-cyano-thiopene has been described R. W. Sabnis et al. (J. Het. Chem.1992, 4, 285-288), and is shown in Scheme D below.
  • Another synthetic route by which the compound of structural formula IVe may be synthesized has been described J. K. Augustine et al., (Tetrahedron 2009, 65, 9989-9996) and is shown in Scheme E below.
  • Scheme E
  • the resulting crystals are isolated via vacuum filtration and rinsed with ice cold dichloromethane. The crystals are allowed to air dry via vacuum filtration for 30 minutes. A small sample of isolated crystal is dissolved in acetone and purity is verified with silica thin-layer chromatography using a solvent system of 40% ethyl acetate in hexanes with 0.1% acetic acid. Plates are stained with PMA as a general stain and the formation of the carboxylic acid is verified with bromocresol green stain. Structure of the purified crystals is verified via 1 H NMR. The crystals are transferred to a clean, pre-weighed glass dram vial and yield is calculated.
  • methods for treating a subject having a disease or condition affected by FABP3, FABP4, FABP5, and/or FABP7 using a FABP3/4/5/7 inhibitor compound of the present disclosure comprises administering to the subject in need thereof, a therapeutically effective amount of a compound of structural formula I and II, or a pharmaceutical composition comprising such a compound and one or more pharmaceutically acceptable adjunct ingredients.
  • the conditions and diseases known to be affected by one or more of FABP4, FABP5, FABP3 and FABP7 which are contemplated for treatment using the FABP3/4/5/7 inhibitor compounds of structural formula I and II include, but are not limited to, the following: atherosclerosis, coronary atherosclerosis, arterial fibrosis, pulmonary hypertension, heart failure, obesity, Type-2 diabetes, Type-1-diabetes, gestational diabetes, polycystic ovary syndrome, endometriosis, conditions affected by lipid metabolism and free fatty acid serum levels, metabolic disorders, fatty liver disease, kidney fibrosis, systemic inflammation, acute inflammation, allergic inflammation, airway inflammation, viral infection (e.g., COVID-19, common cold), skin diseases (e.g., vitiligo, psoriasis, atopic dermatitis, allergic contact dermatitis, mycosis fungoides, alopecia areata, cicatricial alopecia, graft vs.
  • GvHD host disease
  • MS multiple sclerosis
  • Parkinson’s disease autoimmune diseases (e.g., experimental autoimmune encephalomyelitis (EAE), asthma, type-1-diabetes, autoimmune lung disease, autoimmune hepatitis, rheumatoid arthritis (RA), spondyloarthropathy, vesicular stomatitis virus infection, multiple sclerosis (MS), lupus nephritis, Crohn's disease, ulcerative colitis, and food allergy), ischemic stroke, graft versus host disease (GvHD) and cancer (e.g., breast cancer, prostate cancer
  • TNBC Triple-Negative Breast Cancer
  • HEGR-2 Human Epidermal Growth Factor Receptor 2
  • TNBC tumors of TNBC lack definitive prognostic markers and selective targets for therapy
  • the treatment and management of this disease is a significant clinical problem and warrants an urgent need for a direct approach to inhibiting the biological processes which regulate development and metastasis of tumors.
  • data disclosed herein indicate that the disclosed FABP inhibitor compounds can provide inhibition of FABP5 and can thereby modulate the level of TNBC.
  • the FABP3/4/5/7 inhibitor compounds of structural formula I and II can be used in methods treating cancer in a subject, wherein the methods comprise administering to a subject in need thereof a composition, comprising: (a) an effective amount of one or more of the disclosed FABP3/4/5/7 inhibitors or a pharmaceutically acceptable salt thereof; and (b) a pharmaceutically acceptable carrier or other adjunct ingredients.
  • the subject can be diagnosed with, suffering from, and/or undergoing treatment for one or more cancers selected from breast cancer, prostate cancer, ovarian cancer, skin cancer, gastric cancer, glioma, cholangiocarcinoma, bladder cancer, multiple myeloma, colorectal cancer, hepatocellular cancer, cervical cancer, oral squamous cell carcinoma, and/or non-small cell lung cancer (NSCLC).
  • cancer treatment method relates to breast cancer.
  • the disclose methods relate to preventing the metastasis of TNBC cells in a subject diagnosed with cancer.
  • Another still further aspect of the disclosed methods relates to methods treating cancer in a subject, comprising administering to a subject in need a composition, comprising: (a) an effective amount of one or more of the disclosed FABP3/4/5/7 inhibitors or a pharmaceutically acceptable salt thereof; and (b) a pharmaceutically acceptable carrier or other adjunct ingredients.
  • the cancer is chosen from breast cancer, prostate cancer, ovarian cancer, hepatocellular cancer, multiple myeloma, neuroblastoma, lung adenocarcinoma or gastric carcinoma.
  • the cancer is breast cancer.
  • the cancer is prostate cancer.
  • the cancer is ovarian cancer.
  • the cancer is hepatocellular cancer.
  • the cancer is multiple myeloma.
  • the cancer is neuroblastoma.
  • the cancer is lung adenocarcinoma.
  • the cancer is gastric carcinoma.
  • the FABP3/4/5/7 inhibitors of the present disclosure can be used in a method of sensitizing cancer cells for treatment with other chemotherapeutic agents.
  • Such agents can include standard chemotherapeutic compounds, such as doxorubicin, gemcitabine, cisplatin, paclitaxel, all-trans retinoic acid (atRA), a PARP inhibitor compound, and an immune checkpoint inhibitor compound, including but not limited to, an antibody that targets PD-1, or PD-L1.
  • chemotherapeutic compounds such as doxorubicin, gemcitabine, cisplatin, paclitaxel, all-trans retinoic acid (atRA), a PARP inhibitor compound, and an immune checkpoint inhibitor compound, including but not limited to, an antibody that targets PD-1, or PD-L1.
  • the present disclosure provides a method of sensitizing cancer cells for treatment with other chemotherapeutic agents, wherein the method comprises contacting the cancer cells with one or more of the disclosed FABP3/4/5/7 inhibitors and contacting the cells with one or more chemotherapeutic agents.
  • the present disclosure also provides the use of a FABP3/4/5/7 inhibitor compounds of structural formula I or II, or a pharmaceutical composition comprising such a compound, for the manufacture of a medicament for treating a cancer in a subject.
  • the cancer treated by the use or medicament is chosen from breast cancer, prostate cancer, ovarian cancer, hepatocellular cancer, multiple myeloma, neuroblastoma, lung adenocarcinoma or gastric carcinoma.
  • One aspect of the disclosed uses and methods relates to methods for inhibiting one or more of FABP3, FABP4, FABP5, and FABP7 in a subject, comprising administering to a subject in need a composition, comprising: (a) an effective amount of one or more of the FABP3/4/5/7 inhibitor compounds of structural formula I or II, or a pharmaceutically acceptable salt thereof; and (b) a pharmaceutically acceptable carrier or other adjunct ingredients.
  • a further aspect of the disclosed uses and methods relates to methods for controlling the free fatty acid serum levels in a subject, comprising administering to a subject in need a composition, comprising: (a) an effective amount of one or more of the disclosed FABP3/4/5/7 inhibitors of structural formula I or II, or a pharmaceutically acceptable salt thereof; and (b) a pharmaceutically acceptable carrier or other adjunct ingredients.
  • the present disclosure also provides the use of a FABP3/4/5/7 inhibitor compound of the present disclosure, or a pharmaceutical composition comprising a FABP3/4/5/7 inhibitor compound of structural formula I or II, for the manufacture of a medicament for treating a disease or condition affected by FABP3/4/5/7 in a subject.
  • the disease or condition relates to control of the free fatty acid serum levels in a subject.
  • C. Metabolic Disorders Treatment [0159] Additionally, FABP4 and FABP5 are members of a family of small, soluble proteins which contribute to the trafficking of fatty acids within the cytosolic compartments of cells. These proteins have no catalytic function but transport hydrophobic fatty acids within the aqueous environment of the cytosol to the various destinations enabling fatty acid oxidation, membrane homeostasis or nuclear signaling. In addition, they are involved in signaling processes which are so far poorly understood. FABP4 is highly expressed in adipose tissue, macrophages, and endothelial cells.
  • FABP5 is also expressed in macrophages, adipocytes and endothelial cells, as well as in skin and several other tissues.
  • plasma levels of FABP4 are increased in patients with metabolic syndrome and atherosclerosis.
  • FABP4 in angiogenesis. More than a quarter of the population suffers from an aggregation of co- morbidities, including obesity, atherosclerosis, insulin resistance, dyslipidemias, coagulopathies, hypertension, and a pro-inflammatory state known as the metabolic syndrome.
  • Patients with metabolic syndrome have high risk of atherosclerosis as well as Type-2 diabetes and other health problems. Like obesity, atherosclerosis has very limited therapeutic options.
  • a yet further aspect of the disclosed methods relates to methods for regulating insulin sensitivity in a subject, comprising administering to a subject in need a composition, comprising: (a) an effective amount of one or more of the FABP3/4/5/7 inhibitor compounds of structural formula I or II, or a pharmaceutically acceptable salt thereof; and (b) a pharmaceutically acceptable carrier or other adjunct ingredients.
  • a yet still further aspect of the disclosed methods relates to methods for treating Type-2 diabetes in a subject, comprising administering to a subject in need thereof a composition, comprising: (a) an effective amount of one or more of the FABP3/4/5/7 inhibitor compounds of structural formula I or II, or a pharmaceutically acceptable salt thereof; and (b) a pharmaceutically acceptable carrier or other adjunct ingredients.
  • a still yet further aspect of the disclosed methods relates to methods for the glucose plasma level in a subject, comprising administering to a subject in need thereof a composition, comprising: (a) an effective amount of one or more of the FABP3/4/5/7 inhibitor compounds of structural formula I or II, or a pharmaceutically acceptable salt thereof; and (b) a pharmaceutically acceptable carrier or other adjunct ingredients.
  • a yet still further aspect of the disclosed methods relates to methods for treating atherosclerosis in a subject, comprising administering to a subject in need thereof a composition, comprising: (a) an effective amount of one or more of the FABP3/4/5/7 inhibitor compounds of structural formula I or II, or a pharmaceutically acceptable salt thereof; and (b) a pharmaceutically acceptable carrier or other adjunct ingredients.
  • a yet further aspect of the disclosed methods relates to methods for treating liver steatosis in a subject, comprising administering to a subject in need thereof a composition, comprising: (a) an effective amount of one or more of the FABP3/4/5/7 inhibitor compounds of structural formula I or II, or a pharmaceutically acceptable salt thereof; and (b) a pharmaceutically acceptable carrier or other adjunct ingredients.
  • the present disclosure also provides the use of a FABP3/4/5/7 inhibitor compound of structural formula I or II, or a pharmaceutical composition comprising a FABP3/4/5/7 inhibitor compound of the present disclosure, for the manufacture of a medicament for treating a metabolic disorder in a subject.
  • FABPs are involved in the regulation of immune cell activity. Without intending to be limited by mechanism, FABPs are believed to mediate immune cell metabolism, which is critical for the proper functioning of the immune system. More specifically, the activity of FABPs affects the utilization of fatty acids, thereby regulating energy production and the signaling pathways involved in the activation and function of immune cells. For example, FABP5 was found to regulate lipid metabolism and function in T-cells in the tumor microenvironment (TME) by mediating the uptake and oxidation of long-chain FAs in the cells.
  • TAE tumor microenvironment
  • TIL tumor-infiltrating T lymphocytes
  • TIL tumor-infiltrating T lymphocytes
  • ADBP4 is highly expressed in Ly6C-MHCII-CD36 + circulating monocyte/macrophages to facilitate oxidative lipid uptake, foam cell formation, angiogenesis, tissue remodeling and pro-tumor functions. This high expression suggests that inhibition of FABP4 can enhance anti-tumor immune responses in cancer cells and can also block formation of foam cells and chronic inflammation in obesity.
  • FABP4/5 Another function of FABP4/5 in immune cells is their role in maintenance of CD8+ tissue resident memory T-cells (Trm). Specifically, it was reported elsewhere that skin Trm, that relay on fatty acids as energy source, express high levels of FABP4/5 that are necessary for uptake of fatty acids into the cells and transport them to the mitochondria for metabolism. The energy produced in this process is required for survival of Trm cells. Targeting Trm cells by inhibition of FABP4/5 is expected to have therapeutic effect in autoimmune diseases. Overall, the role of FABPs in immune cell regulation highlights the importance of lipid metabolism in the regulation of the immune system and imply that targeting FABPs may be a promising strategy for improving immune cell function and treating a wide range of diseases and conditions caused by chronic inflammation and cancer.
  • a method for modulating immune cell populations and/or immune cell activity in a subject in need thereof comprising administering to a subject in need thereof a therapeutically effective amount of a compound of structural formula I or II, or a pharmaceutical composition of such a compound.
  • the subject in need thereof has a disease or disorder caused by, affected by, and/or characterized by immune cell populations and/or immune cell activity, for example, wherein the immune cells are M2 macrophages or tumor associated macrophages (TAMs).
  • TAMs tumor associated macrophages
  • the subject in need of a treatment for modulating immune cell populations and/or immune cell activity has been diagnosed with, is suffering from, or is being treated for cancer.
  • compositions [0171] The present disclosure also provides uses and methods in which a FABP4/5 inhibitor compounds, such as a compound of structural formula I or II, is administered to a subject in the form of a pharmaceutical composition.
  • the pharmaceutical composition includes a therapeutically effective amount of the FABP3/4/5/7 inhibitor compound (e.g., compound of Table 11), or a pharmaceutically acceptable salt or ester of such a compound and one or more pharmaceutically acceptable carriers.
  • compositions can be prepared using methods well known in the pharmaceutical art (see, e.g., Remington’s Pharmaceutical Sciences, Mace Publishing Co., Philadelphia, PA 17th Ed. (1985) and Modern Pharmaceutics, Marcel Dekker, Inc. 3rd Ed. (G.S. Banker & C.T. Rhodes, Eds.). Methods of preparing pharmaceutical compositions of FABP3/4/5/7 inhibitor compounds are described in the present disclosure, including the Examples disclosed herein. [0172] In at least one embodiment, the present disclosure provides a pharmaceutical composition comprising: an effective amount of one or more of the disclosed FABP3/4/5/7 inhibitors, such as a compound of structural formula I or II; and one or more adjunct ingredients, such as a pharmaceutically acceptable carrier.
  • compositions can comprise from about 10% to about 95% by weight of one or more of the disclosed FABP3/4/5/7 inhibitors. In another embodiment, the compositions comprise from about 10% to about 80% by weight of one or more of the disclosed FABP3/4/5/7 inhibitors. In a further embodiment the compositions comprise from about 20% to about 50% by weight of one or more of the disclosed FABP3/4/5/7 inhibitors. In a still further embodiment, the compositions comprise from about 50% to about 90% by weight of one or more of the disclosed FABP3/4/5/7 inhibitors. In a yet another embodiment the compositions comprise from about 70% to about 90% by weight of one or more of the disclosed FABP3/4/5/7 inhibitors.
  • compositions comprise from about 80% to about 95% by weight of one or more of the disclosed FABP3/4/5/7 inhibitors. In a still yet further embodiment, the compositions comprise from about 90% to about 95% by weight of one or more of the disclosed FABP3/4/5/7 inhibitors.
  • the pharmaceutical compositions can be prepared by diluting the active ingredient(s) with an excipient and/or enclosing it within a carrier in the form of a capsule, sachet, paper or other container.
  • the excipient serves as a diluent, it can be a solid, semi-solid, or liquid material (as above), which acts as a vehicle, carrier or medium for the active ingredient.
  • the pharmaceutical composition(s) suitable for administering in the methods of the disclosure can be in the dosage form of tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium), ointments containing, for example, up to 10% by weight of the active compound, soft and hard gelatin capsules, sterile injectable solutions, and sterile packaged powders.
  • the carriers used in the preparation of the pharmaceutical compositions can include excipients such as inert solid diluents and fillers, diluents, including sterile aqueous solution and various organic solvents, permeation enhancers, solubilizers and adjuvants.
  • excipients such as inert solid diluents and fillers, diluents, including sterile aqueous solution and various organic solvents, permeation enhancers, solubilizers and adjuvants.
  • Suitable excipients for use in the pharmaceutical compositions comprising a celastrol derivative of the present disclosure are well known in the art and include lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, sterile water, syrup, and methyl cellulose.
  • the pharmaceutical compositions can additionally include: lubricating agents such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifying and suspending agents; preserving agents such as methyl- and propylhydroxy-benzoates; sweetening agents; and flavoring agents.
  • a pharmaceutical composition comprising an celastrol derivative can be administered using a variety of different modes including oral administration, intravenous administration, topical administration, parenteral administration, intraperitoneal administration, intramuscular administration, intrathecal administration, intralesional administration, intracranial administration, intranasal administration, intraocular administration, intracardiac administration, intravitreal administration, intraosseous administration, intracerebral administration, intraarterial administration, intraarticular administration, intradermal administration, transdermal administration, transmucosal administration, sublingual administration, enteral administration, sublabial administration, insufflation administration, suppository administration, inhaled administration, or subcutaneous administration.
  • oral administration intravenous administration, topical administration, parenteral administration, intraperitoneal administration, intramuscular administration, intrathecal administration, intralesional administration, intracranial administration, intranasal administration, intraocular administration, intracardiac administration, intravitreal administration, intraosseous administration, intracerebral administration, intraarterial administration, intraarticular administration
  • the pharmaceutical compositions including the FABP3/4/5/7 inhibitor compounds of the present disclosure can be used in a range of therapeutic methods of treatment and a range of dosages are contemplated for administration of a pharmaceutically effective amount.
  • the dosage and frequency (single or multiple doses) of administration of the pharmaceutical composition to a subject can vary depending upon a range of factors, such as, the route of administration; the subject’s size, age, sex, health, body mass, and/or diet; the state of the disease being treated; whether the subject is suffering from any other diseases, and any concurrent treatment being received.
  • adjustment of established dosages e.g., frequency and duration
  • to obtain the therapeutically effective amount may be required depending on the subject.
  • the amount of a pharmaceutical composition containing a FABP3/4/5/7 inhibitor compound to be administered to a subject in a therapeutic method of treatment will be determined by a physician, in view of relevant circumstances of the subject being so treated, the chosen route of administration, and of course, the age, the weight, the severity of symptoms, the response of the individual subject to the treatment, and the like.
  • a therapeutically effective amount is the amount sufficient for the administered composition to accomplish a desired therapeutic purpose relative to the absence of the compound.
  • the therapeutically effective amount can be the amount determined to be sufficient to contribute to the treatment, prevention, or reduction of a symptom or symptoms of a disease.
  • Methods for determining the dosage providing a therapeutically effective amount of a compound are well-known to those of ordinary skill in the art, and typically are based on analysis of amounts determined in cellular assays and/or animal models.
  • a dosage for administration to humans can be formulated to achieve a concentration that has been observed as therapeutically effective in an animal model.
  • the dosage in the pharmaceutical composition for humans can further be adjusted by monitoring the effectiveness and adjusting upwards or downwards.
  • One of ordinary skill can used methods well known in the art to adjust the dosage in a pharmaceutical composition of the present disclosure to achieve maximal therapeutic efficacy for humans.
  • methods for therapeutic treatment are developed by starting with a pharmaceutical composition containing less than the optimal dose of the FABP3/4/5/7 inhibitor compound.
  • the dosage of the compound is increased incrementally until optimal efficacy is attained.
  • a key factor considered in developing the optimal dose is the ratio between the toxicity and the therapeutic efficacy of the active ingredient.
  • This ratio referred to as the compound’s therapeutic index, is typically described as the ratio of the active ingredient’s LD 50 (the amount of compound lethal in 50% of the population) to its ED 50 (the amount of compound effective in 50% of the population).
  • LD 50 the amount of compound lethal in 50% of the population
  • ED 50 the amount of compound effective in 50% of the population.
  • Therapeutic index data can be obtained from cell culture assays and/or animal model studies and then used to determine a safe range of dosages of the active ingredient in a pharmaceutical composition for administration to humans. Ideally the dosage determined provides the active ingredient at its ED 50 level in the subject with little or no toxicity.
  • Solid form preparations of pharmaceutical compositions can include, for example, powders, tablets, dispersible granules, capsules, cachets, and suppositories.
  • a solid carrier can be one or more substances that can also act as diluents, flavoring agents, solubilizers, lubricants, suspending agents, binders, or tablet disintegrating agents; it can also be an encapsulating material.
  • the carrier is a finely divided solid that is in an admixture with the finely divided active component, e.g., a disclosed FABP3/4/5/7 inhibitor.
  • the active ingredient is mixed with the carrier having the necessary binding properties in suitable proportions and compacted in the shape and size desired.
  • the binding agent is ethylcellulose.
  • the solid compositions can comprise from about 0.5% to about 10% by weight of a carrier.
  • solid carriers include: starch such as tapioca starch, corn starch, potato starch, gelatin, dextrin, inulin, cyclodextrin, oxidized starch, starch ester, starch ether, crosslinked starch, alpha starch, octenyl-succinate ester, and processed starch obtained by treating a starch by an acid, heat, or enzyme, or an emulsifier such as gum arabic, modified starch, pectin, xanthan gum, gum ghatti, gum tragacanth, fenugreek gum, mesquite gum, mono-glycerides and di- glycerides of long chain fatty acids, sucrose monoesters, sorbitan esters, polyethoxylated glycerol
  • the disclosed compositions can comprise from about 25 mg to about 1200 mg of one or more of the disclosed FABP3/4/5/7 inhibitor.
  • the disclosed single dose compositions of a disclosed FABP3/4/5/7 inhibitor can comprise any amount from about 25 mg to about 500 mg.
  • the disclosed single dose compositions of a disclosed FABP3/4/5/7 inhibitor can comprise any amount from about 100 mg to about 500 mg.
  • the disclosed single dose compositions of a disclosed FABP3/4/5/7 inhibitor can comprise any amount from about 500 mg to about 1000 mg.
  • the single dose compositions can comprise any amount of FABP3/4/5/7 inhibitor from about 25 mg to about 250 mg.
  • the disclosed compositions can comprise 25 mg, 26 mg, 27 mg, 28 mg, 29 mg, 30 mg 31 mg, 32 mg, 33 mg, 34 mg, 35 mg, 36 mg, 37 mg, 38 mg, 39 mg, 40 mg, 41 mg, 42 mg, 43 mg, 44 mg, 45 mg, 46 mg, 47 mg, 48 mg, 49 mg, 50 mg, 51 mg, 52 mg, 53 mg, 54 mg, 55 mg, 56 mg, 57 mg, 58 mg, 59 mg, 60 mg, 61 mg, 62 mg, 63 mg, 64 mg, 65 mg, 66 mg, 67 mg, 68 mg, 69 mg, 70 mg, 71 mg, 72 mg, 73 mg, 74 mg, 75 mg, 76 mg, 77 mg, 78 mg, 79 mg, 80 mg, 81 mg, 82 mg, 83 mg, 84 mg, 85 mg, 86 mg, 87 mg, 88 mg, 89 mg, 90 mg, 90 mg, 91 mg, 92 mg, 93 mg, 94 mg, 95 mg, 96
  • Liquid forms of the pharmaceutical compositions can include, for example, solutions suitable for oral or parenteral administration, suspensions, and emulsions suitable for oral administration.
  • Sterile water solutions of the active component or sterile solutions of the active component in solvents comprising water, buffered water, saline, PBS, ethanol, or propylene glycol are examples of liquid compositions suitable for parenteral administration.
  • the compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents, detergents, and the like.
  • the disclosed liquid compositions can comprise from about 5% to about 25% by weight of a liquid carrier.
  • the targeted cells for example, cancer cells or tumor cells can be contacted with an aqueous solution comprising from about 0.5 ⁇ g/mL to about 250 ⁇ g/mL.
  • the compositions can comprise from about 1 ⁇ g/mL to about 100 ⁇ g/mL.
  • the compositions can comprise from about 10 ⁇ g/mL to about 100 ⁇ g/mL.
  • the compositions can comprise from about 5 ⁇ g/mL to about 20 ⁇ g/mL.
  • the compositions can comprise from about 1 ⁇ g/mL to about 50 ⁇ g/mL.
  • compositions can comprise from about 1 ⁇ g/mL to about 10 ⁇ g/mL. In a still further embodiment, the compositions can comprise from about 15 ⁇ g/mL to about 50 ⁇ g/mL. In still another embodiment the compositions can comprise from about 20 ⁇ g/mL to about 200 ⁇ g/mL.
  • the disclosed compositions can provide a single dose of a disclosed FABP3/4/5/7 inhibitor based upon the body mass of the subject being treated. Therefore, a single dose of a disclosed FABP3/4/5/7 inhibitor can range from about 0.35 mg/kg to about 20 mg/kg of the subject’s body mass.
  • the amount of a disclosed FABP3/4/5/7 inhibitor in a single dose is from about 1 mg/kg to about 8 mg/kg of the subject’s body mass. In another embodiment, the amount of a disclosed FABP3/4/5/7 inhibitor in a single dose is from about 2 mg/kg to about 5 mg/kg of the subject’s body mass. In a further embodiment, the amount of a disclosed FABP3/4/5/7 inhibitor in a single dose is from about 1.5 mg/kg to about 4 mg/kg of the subject’s body mass. In a yet further embodiment, the amount of a disclosed FABP3/4/5/7 inhibitor in a single dose is from about 4 mg/kg to about 10 mg/kg of the subject’s body mass.
  • the amount of a disclosed FABP3/4/5/7 inhibitor in a single dose is from about 5 mg/kg to about 8 mg/kg of the subject’s body mass.
  • the dose can comprise any amount from about 0.5 mg/kg to about 10 mg/kg on the body mass of the subject being treated.
  • Example 2 Preparation of 2-((2-((3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)amino)- 2-oxoethyl)thio)-2-methylpropanoic acid (compound FTS003) [0196] This example illustrates the preparation of the FAB3/4/5/7 inhibitor compound FTS003 via the synthetic method of Scheme 2 as shown below.
  • Example 7 Preparation of 5-((3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2- yl)carbamoyl)tetrahydrofuran-2-carboxylic acid (compound FTS013) [0221] This example illustrates the preparation of the FAB3/4/5/7 inhibitor compound FTS013 via the synthetic method of Scheme 7 as shown below.
  • Step 1 synthesis of 2-amino-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carbonitrile: In a 250 mL single necked dried round bottom flask under nitrogen atmosphere cyclohexanone (5.0 g, 50.9 mmol) was dissolved in dioxane (100 mL) and malononitrile (3.37 g, 50.9 mmol), sulfur (1.633 g, 50.9 mmol) was added and heated to 50 °C and morpholine (4.44 g, 50.9 mmol) was added and stirred for 16h at same temperature. Progress of the reaction was monitored by LCMS & TLC.
  • Step 2 synthesis of 2-chloro-N-(3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2- yl)acetamide: To a stirred solution of 2-amino-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carbonitrile (2.0 g, 11.22 mmol) in Dioxane (25 mL) and 2-chloroacetyl chloride (1.521 g, 13.46 mmol) was added in a 100 mL single necked dried round bottom flask under nitrogen atmosphere and stirred at RT for 16h.Progress of the reaction was monitored by TLC.(20% EtOAc in hexane, 0.7 rf )
  • Step 3 synthesis of methyl 2-(2-((3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)amino)- 2-oxoethoxy)-2-methylpropanoate: To a stirred solution of methyl 2-hydroxy-2-methylpropanoate (0.464 g, 3.93 mmol) in THF (25 mL) and methyl 2-hydroxy-2-methylpropanoate (0.464 g, 3.93 mmol), 2-chloro-N-(3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)acetamide (0.500 g, 1.963 mmol) was added in a 100 mL single necked dried round bottom flask under nitrogen atmosphere and heated to 70 °C for 16h.
  • Step 4 Synthesis of 2-(2-((3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)amino)-2- oxoethoxy)-2-methylpropanoic acid: In a 100 mL single necked dried round bottom flask under nitrogen atmosphere methyl 2-(2-((3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)amino)-2- oxoethoxy)-2-methylpropanoate (0.180 g, 0.535 mmol) was dissolved in THF (5 mL).
  • Example 10 Preparation of 1-((2-((3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2- yl)amino)-2-oxoethyl)thio)cyclopropanecarboxylic acid (compound FTS028) [0242] This example illustrates the preparation of the FAB3/4/5/7 inhibitor compound FTS028 via the seven step synthetic method of Scheme 10 as shown below.
  • Step 1 Synthesis of ethyl 2-((bis(4-methoxyphenyl)(phenyl)methyl)thio)acetate: To a solution of ethyl 2-sulfanylacetate (10.0 g, 83.22 mmol, 1.0 eq) in DCM (120 mL) was added NaHCO 3 (8.3 g, 99.86 mmol, 1.2 eq). The mixture was stirred at 25°C for 20 min.
  • Step 2 Synthesis of ethyl 1- ((bis(4methoxyphenyl)(phenyl)methyl)thio)cyclopropanecarboxylate: To a solution of ethyl 2-[bis(4- methoxyphenyl)-phenyl-methyl]sulfanylacetate (10.0 g, 23.67 mmol, 1.0 eq) in THF (100 mL) was added dropwise LDA (2 M, 29.58 mL, 2.5 eq) at -60°C.
  • Step 3 Synthesis of ethyl 1-mercaptocyclopropanecarboxylate: To a solution of ethyl 1- [bis(4-methoxyphenyl)-phenyl-methyl]sulfanylcyclopropanecarboxylate (3.0 g, 6.69 mmol, 1.0 eq) in DCM (30 mL), was added triethylsilane (933.2 mg, 8.03 mmol, 1.28 mL, 1.2 eq) at 0°C. After addition, TFA (762.5 mg, 6.69 mmol, 495.16 uL, 1.0 eq) was added, the mixture was stirred at 25°C for 16h.
  • TFA 762.5 mg, 6.69 mmol, 495.16 uL, 1.0 eq
  • Step 4 Synthesis of ethyl 1-((2-(tert-butoxy)-2-oxoethyl)thio)cyclopropanecarboxylate: To the solution above was added THF (300 mL), K 2 CO 3 (4.4 g, 32.49 mmol, 5.0 eq) and tert-butyl 2- bromoacetate (1.3 g, 6.50 mmol, 960.12 uL, 1.0 eq) at 0°C.
  • Step 5 Synthesis of 2-((1-(ethoxycarbonyl)cyclopropyl)thio)acetic acid: To a solution of ethyl 1-(2-tert-butoxy-2-oxo-ethyl)sulfanylcyclopropanecarboxylate (1.6 g, 6.49 mmol, 1.0 eq) in DCM (10 mL) was added TFA (15.4 g, 135.06 mmol, 10 mL, 20.8 eq).
  • Step 6-Sythesis of ethyl 2-(1-((2-((3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)amino)- 2-oxoethyl)thio)cyclopropyl)-2-oxoacetate To a solution of 2-(1- ethoxycarbonylcyclopropyl)sulfanylacetic acid (1.15 g, 5.61 mmol, 5.0 eq) in DMF (2.0 mL) was added DIEA (1.16 g, 8.98 mmol, 1.56 mL, 8.0 eq).
  • Example 11 Preparation of 2-(1-(2-((3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2- yl)amino)-2-oxoethyl)cyclopropyl)acetic acid) (compound FTS029) [0256] This example illustrates the preparation of the FAB3/4/5/7 inhibitor compound FTS029 via the synthetic method of Scheme 11 as shown below.
  • Step 1 Synthesis of ethyl 1-((2-(tert-butoxy)-2-oxoethyl)thio)cyclobutanecarboxylate: To a solution of ethyl 1-bromocyclobutanecarboxylate (2.0 g, 9.66 mmol, 1.56 mL, 1.0 eq) in THF (10 mL) was added tert-butyl 2-sulfanylacetate (1.43 g, 9.66 mmol, 1.0 eq) and KOH (541.9 mg, 9.66 mmol, 1.0 eq). The mixture was stirred at 25°C for 2h.
  • Step 2 2-((1-(ethoxycarbonyl)cyclobutyl)thio)acetic acid: To a solution of ethyl 1-(2-tert- butoxy-2-oxo-ethyl)sulfanylcyclobutanecarboxylate (1.0 g, 3.64 mmol, 1.0 eq) in DCM (15 mL) was added TFA (5.9 g, 51.94 mmol, 3.85 mL, 14.2 eq). The mixture was stirred at 25°C for 16 h.
  • Step 3 Ethyl 1-((2-((3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)amino)-2- oxoethyl)thio)cyclobutanecarboxylate: To a solution of 2-(1-ethoxycarbonylcyclobutyl)sulfanylacetic acid (0.75 g, 3.44 mmol, 1.0 eq) in DMF (1 mL) was added DIEA (2.22 g, 17.18 mmol, 2.99 mL, 5.0 eq).
  • Step 4 Synthesis of 1-((2-((3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)amino)-2- oxoethyl)thio)cyclobutanecarboxylic acid: To a solution of ethyl 1-[2-[(3-cyano-4,5,6,7- tetrahydrobenzothiophen-2-yl)amino]-2-oxo-ethyl]sulfanylcyclobutanecarboxylate (800.0 mg, 2.11 mmol, 1.0 eq) in a mixed solvent of MeOH (10 mL) and H 2 O (2 mL) was added LiOH.H 2 O (443.4 mg, 10.57
  • Example 13 Preparation of 3-((2-((3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)amino)- 2-oxoethyl)thio)oxetane-3-carboxylic acid (FTS035).
  • FTS035 3-((2-((3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)amino)- 2-oxoethyl)thio)oxetane-3-carboxylic acid
  • Example 14 Preparation of 2-(1-(2-((3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2- yl)amino)-2-oxoethyl)cyclobutyl)acetic acid (compound FTS031) [0274] This example illustrates a preparation of the FAB3/4/5/7 inhibitor compound FTS031 via the synthetic method of Scheme 14 as shown below.
  • Example 15 Preparation of (1s,3s)-3-((3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2- yl)carbamoyl)cyclobutane-1-carboxylic acid (compound FTS032) [0278]
  • This example illustrates the preparation of the FAB3/4/5/7 inhibitor compound FTS032 via the two step synthetic method of Scheme 15 as shown below.
  • reaction mixture (1s,3s)-3-(methoxycarbonyl) cyclobutane-1-carboxylic acid (266 mg, 1.683 mmol)) and DIPEA (1.176 mL, 6.73 mmol) were added at 25 °C under nitrogen atmosphere. Then reaction mixture was cooled to 0 °C and POCl 3 (0.314 mL, 3.37 mmol) was added dropwise and stirred at 25 °C for 16 h under nitrogen atmosphere. Progress of reaction was monitored by TLC (20 % EtOAc in pet-ether, 0.3 rf). After completion of reaction, reaction mixture was quenched with NaHCO 3 solution (100mL). Reaction mixture was extracted with DCM (3 x 100 mL).
  • Step 2 Synthesis of (1s,3s)-3-((3- ano-4,5,6,7-tetrahydrobenzo[b]thiophen-2- yl)carbamoyl)cyclobutane-1-carboxylic acid: In a 100 mL single necked dried round bottom flask under nitrogen atmosphere, methyl(1s,3s)-3-((3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl) carbamoyl)cyclobutane-1-carboxylate (150 mg, 0.471 mmol) were dissolved in THF (10 mL), and water (5 mL).To this reaction mixture Lithium hydroxide monohydrate (59.4 mg, 1.413 mmol) was added at 25 °C under nitrogen atmosphere.
  • Example 16 Preparation of (1r,3r)-3-((3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2- yl)carbamoyl)cyclo-butane-1-carboxylic acid (compound FTS033) [0285]
  • This example illustrates the preparation of the FAB3/4/5/7 inhibitor compound FTS033 via the two-step synthetic method of Scheme 16 as shown below.
  • Scheme 16 Step 1 Step 1
  • Step 1 Synthesis of methyl (1r,3r)-3-((3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2- yl)carbamoyl)cyclobutane-1-carboxylate: In a 100 mL two necked dried round bottom flask under nitrogen 2-amino-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carbonitrile (400 mg, 2.244 mmol) was dissolved in DCM (15 mL).
  • Step 2 Synthesis of (1r,3r)-3-((3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2- yl)carbamoyl)cyclobutane-1-carboxylic acid: In a 50 mL single necked dried round bottom flask under nitrogen atmosphere, methyl(1r,3r)-3-((3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl) carbamoyl)cyclobutane-1-carboxylate (48 mg, 0.151 mmol) was dissolved in THF (2 mL) and water (1 mL).
  • Example 17 Preparation of 3-((3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2- yl)carbamoyl)bicyclo[1.1.1]pentane-1-carboxylic acid (compound FTS034) [0292] This example illustrates the preparation of the FAB3/4/5/7 inhibitor compound FTS034 via the two-step synthetic method of Scheme 17 as shown below.
  • Scheme 17 illustrates the preparation of the FAB3/4/5/7 inhibitor compound FTS034 via the two-step synthetic method of Scheme 17 as shown below.
  • Step 1 Synthesis of methyl 3-((3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2- yl)carbamoyl)bicyclo[1.1.1]pentane-1-carboxylate: In a 100 mL two necked dried round bottom flask under nitrogen 2-amino-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carbonitrile (200 mg, 1.122 mmol) was dissolved in CH2Cl2 (10 mL).
  • Step 2 Synthesis of 3-((3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2- yl)carbamoyl)bicyclo[1.1.1]pentane-1-carboxylic acid: In a 25 mL single necked dried round bottom flask under nitrogen atmosphere, methyl3-((3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2- yl)carbamoyl)bicyclo[1.1.1]pentane-1-carboxylate (100 mg, 0.303 mmol) were dissolved in THF (3.00 mL), water (3 mL), and MeOH (1 mL).To this reaction mixture, LiOH (21.74 mg, 0.908 mmol) was added at 25 °C under nitrogen atmosphere.
  • Step 1 Synthesis of ethyl 1-((2-((3-cyano-4,5-dimethylthiophen-2-yl)amino)-2- oxoethyl)thio)cyclopropanecarboxylate: To a solution of 2-(1- ethoxycarbonylcyclopropyl)sulfanylacetic acid (1.0 g, 4.93 mmol, 5.0 eq) in DMF (2 mL) was added DIEA (891.5 mg, 6.90 mmol, 1.20 mL, 7.0 eq).
  • Example 19 Preparation of 1-((2-((3-cyano-4,5-dimethylthiophen-2-yl)amino)-2- oxoethyl)thio)cyclobutanecarboxylic acid (compound FTS037) [0306] This example illustr ates the preparation of the FAB3/4/5/7 inhibitor compound FTS037 via the two-step synthetic method of Scheme 19 as shown below.
  • the crude mixture is purified by dissolving impurities in ethyl acetate and acetone.
  • the resultant white powder is filtered and washed with more ethyl acetate to yield 2-((2-((3-cyano-4,5-dimethylthiophen-2-yl)amino)-2-oxoethyl)thio)-2-methylpropanoic acid as a light yellow powder.
  • NMR analysis confirmed preparation of the desired product compound.
  • Step 1 2-butanone (0.680 mmol), 2-(3-cyclopropyl-1,2,4-oxadiazol-5-yl)acetonitrile (1.36 mmol, 2 eq.) and ammonium acetate (1.36 mmol, 2 eq.) were charged into a 100mL 2-neck round bottom flask. The flask was purged with argon and 10mL dry toluene is added. The reaction mixture is heated to reflux for 18h.
  • the reaction is cooled and poured onto 30mL 10% aq. NaHCO 3 and 30mL EtOAc.
  • the aqueous layer is separated and extracted once more with 30mL EtOAc.
  • the organic layers are combined, washed with brine and dried over anhydrous sodium sulfate. Excess solvent is removed via rotary evaporation.
  • the crude mixture is purified via silica column in 90% hexanes in ethyl acetate to yield a mixture of (Z)-2-(3-cyclopropyl-1,2,4-oxadiazol-5-yl)-3-methylpent-2- enenitrile and (E)-2-(3-cyclopropyl-1,2,4-oxadiazol-5-yl)-3-methylpent-2-enenitrile.
  • Step 2 A mixture of (Z)-2-(3-cyclopropyl-1,2,4-oxadiazol-5-yl)-3-methylpent-2-enenitrile and (E)-2-(3-cyclopropyl-1,2,4-oxadiazol-5-yl)-3-methylpent-2-enenitrile (1.12 mmol), DBU (2.8 mmol, 2.5eq.) and elemental sulfur (1.12 mmol, 1eq.) were charged into a 100mL 2-neck round bottom flask. The flask was purged with argon and 20mL dry EtOH. The reaction mixture was heated to 65 o C under reflux for 2 hours.
  • the reaction mixture is cooled, then poured onto 30mL 10% aq. NaHCO 3 and 30mL EtOAc. The aqueous layer is separated and extracted once more with 30mL EtOAc. The organic layers are combined, washed with brine and dried over anhydrous sodium sulfate. Excess solvent is removed via rotary evaporation. The crude mixture is purified via silica column in 90% hexanes in ethyl acetate to yield 3-(3-cyclopropyl-1,2,4-oxadiazol-5-yl)-4,5-dimethylthiophen-2- amine as a pale yellow powder (42mg, 15.9%).
  • Step 3 3,3-dimethyl-1,4-oxathiane-2,6-dione (0.227 mmol) and 3-(3-cyclopropyl-1,2,4- oxadiazol-5-yl)-4,5-dimethylthiophen-2-amine (0.170, 0.75 eq.) were charged into a 50mL round bottom flask. The flask was purged with argon and 15mL dry dichloromethane was added. The mixture was allowed to stir overnight under positive pressure of argon.
  • Step 1 Synthesis of 2-(3-cyclopropyl-1,2,4-oxadiazol-5-yl)acetonitrile: To a stirred solution of 3-(3,5-dimethyl-1H-pyrazol-1-yl)-3-oxopropanenitrile (0.5 g, 3.06 mmol) in Dioxane (25 mL) and N-hydroxy cyclopropane carboximidamide (0.368 g, 3.68 mmol) was added in a single necked dried round bottom flask under nitrogen atmosphere and heated to 105 °C for 3h. Progress of the reaction was monitored by TLC. (10%EtOAc in pet ether, 0.5 rf).
  • Step 2- Synthesis of 3-(3-cyclopropyl-1,2,4-oxadiazol-5-yl)-4,5-dimethylthiophen-2-amine: To a stirred solution of 2-(3-cyclopropyl-1,2,4-oxadiazol-5-yl)acetonitrile (1 g crude) in Ethanol (30 mL) and butan-2-one (0.218 g, 3.06 mmol), sulfur (0.097 g, 3.06 mmol) was added in a single necked dried round bottom flask under nitrogen atmosphere and heated to 50 °C and morpholine (0.263 mL, 3.06 mmol) was added at 50 °C and stirred at 50 °C for 16h.
  • Step 3- Synthesis of methyl (1s,3s)-3-((3-(3-cyclopropyl-1,2,4-oxadiazol-5-yl)-4,5- dimethylthiophen-2-yl)carbamoyl)cyclobutane-1-carboxylate: To a stirred solution of 3-(3- cyclopropyl-1,2,4-oxadiazol-5-yl)-4,5-dimethylthiophen-2-amine (200 mg, 0.850 mmol) was dissolved in DCM (40 mL, (1s,3s)-3-(methoxycarbonyl)cyclobutane-1-carboxylic acid (202 mg, 1.275 mmol) and DIPEA (0.891 mL, 5.10 mmol) were added in two necked dried round bottom flask under nitrogen at 25 °C .
  • reaction mixture was cooled to 0 °C and POCl3 (0.238 mL, 2.55 mmol) was added dropwise at 0 °C and stirred at 25 °C for 16 h under nitrogen atmosphere. Progress of reaction was monitored by TLC (20 % EtOAc in pet ether, 0.5 rf). After completion of reaction, reaction mixture was quenched with NaHCO3 solution (100 mL). Reaction mixture was extracted with DCM (3 x 100 mL). Combined organic layers were dried over sodium sulphate and concentrated over rotary evaporator under reduced pressure (Bath Temperature 45 °C) to get crude product.
  • Step 4- Synthesis of (1s,3s)-3-((3-(3-cyclopropyl-1,2,4-oxadiazol-5-yl)-4,5-dimethylthiophen- 2-yl)carbamoyl)cyclobutane-1-carboxylic acid: To a stirred solution of methyl (1s,3s)-3-((3-(3- cyclopropyl-1,2,4-oxadiazol-5-yl)-4,5-dimethylthiophen-2-yl)carbamoyl)cyclobutane-1-carboxylate (0.120 g, 0.320 mmol) was dissolved in THF (6 mL), water (5 mL) and lithium hydroxide monohydrate (0.040 g, 0.959 mmol) was added in single necked dried round bottom flask and stirred at RT for 1hr.
  • Example 23 Preparation of (1r,3r)-3-((3-(3-cyclopropyl-1,2,4-oxadiazol-5-yl)-4,5- dimethylthiophen-2-yl)carbamoyl)cyclobutane-1-carboxylic acid (compound FTS041) [0335]
  • This example illustrates the preparation of the FAB3/4/5/7 inhibitor compound FTS041 via the two-step synthetic method of Scheme 23 as shown below.
  • Step 1 Synthesis of methyl -3-((3-(3-cyclopropyl-1,2,4-oxadiazol-5-yl)-4,5- dimethylthiophen-2-yl)carbamoyl)cyclobutane-1-carboxylate: In a 50 mL two necked dried round bottom flask under nitrogen 3-(3-cyclopropyl-1,2,4-oxadiazol-5-yl)-4,5-dimethylthiophen-2-amine (0.170 g, 0.722 mmol) was dissolved in DCM (10 mL).
  • Step 2 Synthesis of (1r,3r)-3-((3-(3-cyclopropyl-1,2,4-oxadiazol-5-yl)-4,5- dimethylthiophen-2-yl)carbamoyl)cyclobutane-1-carboxylic acid: In a 25 mL single necked dried round bottom flask under nitrogen atmosphere methyl (1r,3r)-3-((3-(3-cyclopropyl-1,2,4-oxadiazol-5- yl)-4,5-dimethylthiophen-2-yl)carbamoyl)cyclobutane-1-carboxylate (0.050 g, 0.133 mmol) was dissolved in THF (3 mL), water (2 mL) and lithium hydroxide (9.57 mg, 0.400 mmol) was added and stirred at RT for 1hr.Progress of the reaction was monitored by TLC (5% MeOH in DCM).
  • Example 24 Preparation of 3-((3-(3-cyclopropyl-1,2,4-oxadiazol-5-yl)-4,5- dimethylthiophen-2-yl)carbamoyl)-bicyclo[1.1.1]pentane-1-carboxylic acid (compound FTS042) FTS042 [0342] This example illustrates the preparation of the FAB3/4/5/7 inhibitor compound FTS042 via the synthetic method of Scheme 24 as shown below.
  • Step 1 Synthesis of methyl 3-((3-(3-cyclopropyl-1,2,4-oxadiazol-5-yl)-4,5- dimethylthiophen-2-yl)carbamoyl)bicyclo[1.1.1]pentane-1-carboxylate: In a 100 mL two necked dried round bottom flask under nitrogen 3-(3-cyclopropyl-1,2,4-oxadiazol-5-yl)-4,5-dimethylthiophen-2- amine (500 mg, 2.125 mmol) was dissolved in DCM (15 mL).
  • Step 2 Synthesis of 3-((3-(3-cyclopropyl-1,2,4-oxadiazol-5-yl)-4,5-dimethylthiophen-2- yl)carbamoyl)bicyclo[1.1.1]pentane-1-carboxylic acid: In a 50 mL single necked dried round bottom flask under nitrogen atmosphere, methyl 3-((3-(3-cyclopropyl-1,2,4-oxadiazol-5-yl)-4,5- dimethylthiophen-2-yl)carbamoyl)bicyclo[1.1.1]-pentane-1-carboxylate (250 mg, 0.645 mmol) were dissolved in water (5 mL) ,THF (5 mL), and MeOH (1 mL).To this reaction mixture Lithium hydroxide monohydrate (81 mg, 1.936 mmol) was added at 25 °C under nitrogen atmosphere.
  • Example 25 Preparation of 2-(1-(2-((3-(3-cyclopropyl-1,2,4-oxadiazol-5-yl)-4,5- dimethylthiophen-2-yl)amino)-2-oxoethyl)cyclopentyl)acetic acid (compound FTS043) [0350] This example illustrates the preparation of the FAB3/4/5/7 inhibitor compound FTS043 via the synthetic method of Scheme 25 as shown below.
  • Example 26 Preparation of 2-((2-((3-cyanobenzo[b]thiophen-2-yl)amino)-2-oxoethyl)thio)- 2-methylpropanoic acid (compound [0355]
  • This example illustrates the preparation of the FAB3/4/5/7 inhibitor compound FTS044 via the two-step synthetic method of Scheme 26 as shown below.
  • Step 1 Scheme 26 Step 1 FTS044
  • 2-aminobenzo[b]thio-phene-3-carbonitrile (0.200 g, 1.148 mmol) was dissolved in dioxane (10 mL) and 2-chloroacetyl chloride (0.130 g, 1.148 mmol) was added at RT and stirred for 16h at same temperature. Completion of the reaction was monitored by TLC.
  • Step 2 Synthesis of 2-((2-((3-cyanobenzo[b]thiophen-2-yl)amino)-2-oxoethyl)thio)-2- methylpropanoic acid: To a stirred solution of 2-chloro-N-(3-cyanobenzo[b]thiophen-2-yl)acetamide (0.220 g, 0.878 mmol), in a 50 mL single necked dried round bottom flask under nitrogen atmosphere, methyl 2-mercapto-2-methylpropanoate (0.141 g, 1.053 mmol) and cesium carbonate (0.572 g, 1.755 mmol) were added and stirred at RT for 16h.
  • Example 27 Preparation of 2-((2-((6-chloro-3-cyanobenzo[b]thiophen-2-yl)amino)-2- oxoethyl)thio)-2-methylpropanoic acid (compound FTS045) FTS045 [0362] This example illustrates the preparation of the FAB3/4/5/7 inhibitor compound FTS045 via the three-step synthetic method of Scheme 27 as shown below.
  • FTS047 [0376] This example illustrates a preparation of the FAB3/4/5/7 inhibitor compound FTS047 via the synthetic method of Scheme 29 as shown below.
  • Scheme 29 FTS047 [0377] Materials and methods [0378] 3,3-dimethyl-1,4-oxathiane-2,6-dione (0.697 mmol) and 2-amino-4,5,6,7-tetrahydro-4,7- methanobenzo[b]thiophene-3-carbonitrile (0.523 mmol, 0.75eq.) was charged into a 50 mL round bottom flask. The flask was purged with argon and 20mL dry dichloromethane was added. The mixture was allowed to stir overnight under positive pressure of argon.
  • Example 30 Preparation of 2-((2-((3-cyano-4-cyclopropylthiophen-2-yl)amino)-2- oxoethyl)thio)-2-methylpropanoic acid (compound FTS048)
  • FTS048 [0380] This example illustrates the preparation of the FAB3/4/5/7 inhibitor compound FTS048 via the synthetic method of Scheme 30 as shown below.
  • Scheme 30 FTS048 [0381] Materials and methods [0382] 3,3-dimethyl-1,4-oxathiane-2,6-dione (0.787 mmol) and 2-amino-4-cyclopropylthiophene-3- carbonitrile (0.590 mmol, 0.75 eq.) were charged into a 50mL round bottom flask. The flask was purged with argon and 15mL dry dichloromethane was added. The mixture was allowed to stir overnight under positive pressure of argon.
  • White powder forms under rapid cooling recrystallization and is isolated via vacuum filtration and washed with cold CH2Cl2. Residual solvent is removed via rotary evaporation to yield 2-((2-((3-cyano-4-cyclopropylthiophen-2-yl)amino)-2-oxoethyl)thio)-2- methylpropanoic acid as a white powder (45mg, 23.4%).
  • FTS049 [0385] This example illustrates the preparation of the FAB3/4/5/7 inhibitor compound FTS049 via the synthetic method of Scheme 31 as shown below.
  • Scheme 31 Step 1 FTS049 [0386] Materials and methods [0387] Step 1-Synthesis of 2-chloro-N-(3-cyano-4-methyl-5-phenylthiophen-2-yl)acetamide: To a solution of 2-amino-4-methyl-5-phenylthiophene-3-carbonitrile (500 mg, 2.333 mmol) in THF (15 mL) was added Et3N (826 mg, 8.17 mmol) followed by 2-chloroacetyl chloride (659 mg, 5.83 mmol) at 0 oC.
  • reaction mixture was stirred at RT for 2h. Progress of reaction was monitored by TLC (10% EtOAc in pet ether) and LCMS. After completion of reaction, reaction mixture was quenched with water. Reaction mixture was extracted with ethyl acetate (3 x 20 mL). Combined organic layers were separated and washed with brine, dried over sodium sulphate and concentrated in rotary- evaporator under reduced pressure to get crude.
  • Step 2-Synthesis of 2-((2-((3-cyano-4-methyl-5-phenylthiophen-2-yl)amino)-2- oxoethyl)thio)-2-methylpropanoic acid To a solution of 2-chloro-N-(3-cyano-4-methyl-5- phenylthiophen-2-yl)acetamide (350 mg, 1.204 mmol) and cesium carbonate (588 mg, 1.806 mmol) in ACN (15.0 mL) was added methyl 2-mercapto-2-methylpropanoate (242 mg, 1.806 mmol). The resulting mixture was stirred at RT for 16h.
  • Example 32 Screening of FABP3/4/5/7 Inhibitor Compounds [0392] This example illustrates a two-step fluorescence binding assay study used to determine the binding affinity of the various inhibitor compounds disclosed herein with the various FABPs: FABP3, FABP4, FABP5 and FABP7.
  • Binding assays for FABP3, FABP4, FABP5 and FABP7 were carried out by fluorescence titrations. His-tagged FABPs were bacterially expressed in E. coli, purified using Ni Sepharose beads, and the equilibrium dissociation constants (Kd) that characterize their interactions with different inhibitor compounds were measured by fluorescence competition assays. The method entails two steps as described in e.g., Lin, Q.
  • Kd for the association of the protein with the fluorescent fatty acid probe ANS was measured. Protein (2 ⁇ M) was titrated with ANS from a concentrated solution in DMSO. Ligand binding was monitored by following the increase in the fluorescence of the ligand upon binding to the protein, and Kd for the association of ANS with the each FABP was computed from titration curves as described in e.g., Norris, A. W.
  • Kds for binding of non-fluorescent ligands were measured by monitoring their ability to displace ANS in the binding pocket of the protein.
  • Each FABP was precomplexed with ANS at 1:1 molar ratio and titrated with the different compounds whose binding was reflected by a decrease in probe fluorescence.
  • Kds were extracted from the EC50 of the competition curve and the measured Kd for ANS.
  • COS-7 were cultured in 6-well plates and co-transfected with either a luciferase reporter driven by 3 copies of a PPRE and expression vector for either PPAR ⁇ , PPAR ⁇ or PPAR ⁇ together with a vector harboring cDNA for ⁇ -galactosidase, serving as a transfection control.
  • a luciferase reporter driven by 3 copies of a PPRE and expression vector for either PPAR ⁇ , PPAR ⁇ or PPAR ⁇ together with a vector harboring cDNA for ⁇ -galactosidase, serving as a transfection control.
  • a luciferase reporter driven by 3 copies of a PPRE and expression vector for either PPAR ⁇ , PPAR ⁇ or PPAR ⁇ together with a vector harboring cDNA for ⁇ -galactosidase, serving as a transfection control.
  • FABP4 or FABP5 mediate activation of their cognate receptors PPAR ⁇ and PPAR
  • Example 33 Biological Studies of FABP3/4/5/7 Inhibitor Compounds FTS005, FTS030, FTS031, FTS037, and FTS039 [0402] This example illustrates studies of the biological function of the FABP3/4/5/7 inhibitor compounds FTS005, FTS030, FTS031, FTS037, and FTS039 in cancer models and in models of metabolic diseases. [0403] Materials and methods [0404] A.
  • COS-7, MDA-MB-231, NPG, HepG2, 3T3-L1 were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (Invitrogen Life Sciences, Carlsbad, CA).4T1, 4T01, NR67, MDA-MB-157, MB-549 and OVCAR8 cells were cultured in L-Glutamine containing RPMI medium supplemented with 10% fetal calf serum (Invitrogen Life Sciences, Carlsbad, CA).
  • DMEM Dulbecco's modified Eagle's medium
  • 4T01, NR67, MDA-MB-157, MB-549 and OVCAR8 cells were cultured in L-Glutamine containing RPMI medium supplemented with 10% fetal calf serum (Invitrogen Life Sciences, Carlsbad, CA).
  • B. Transcriptional activation assays [0407] Transcriptional activation assays were carried out utilizing COS
  • mice were scarified 24 days after injection. Statistical significance between the control and treated mice in both experiments was evaluated using a Student's t-test. Mouse experiments were conducted after approval by the institutional animal care and use committee at Case Western Reserve University. [0417]
  • G. TNBC syngeneic mouse model [0418] Seven-week-old BALB/c females were injected subcutaneously into the mammary fat pad with 1 x 10 5 4T1 cells 1:1 with Matrigel. Treatments started the day after cells injections 5 days a week. Mice were orally treated by gavage with either vehicle (sesame oil), or 40 mg/kg FTS005 dissolved in vehicle. Tumor size was assessed twice per week using a digital caliper.
  • Compound FTS005 suppresses growth of TNBC cells that express FABP5 more effectively than the commercially available inhibitor SBF-I26 [0427]
  • the TNBC lines MB-231 and BT-549 were used to calculate the efficacy of compound FTS005 in inhibiting proliferation of the cancer cells. Cells were treated with serial dilution of the compound and proliferation was measured in Incucyte by calculating percentage of confluency every 4 hours over 4 days. As shown in FIG.2A, FTS005 inhibited proliferation of the 2 human TNBC lines MB- 231 and BT-549 very effectively with calculated IC 50 of 0.145 mM, and IC 50 of 0.25 mM, respectively.
  • FTS005 To verify the inhibitory effect of FTS005 on TNBC cells is mediated through FABP5 MB- 231 line stably expressing FABP5 shRNA were utilized. As shown in FIG.2B, FTS005 only inhibited proliferation of the WT MB-231 and BT-549 lines that express FABP5 but did not affect proliferation of the F5_KD MB-231 cell line in which levels of FABP5 are low. [0429] The efficiency of FTS005 in inhibiting proliferation of MB-231 cells was also compared to the known FABP5/7 inhibitor compound, SBF-I26.
  • the data clearly show that treatment of MB-231 cells with the thiophene compound suppress proliferation of the TNBC cells 10 to 20-fold more effectively than the SBF-I26.
  • FTS005 was also tested on proliferation of the mouse mammary carcinoma lines 67NR, 4T07, and 4T1 that serve as a model for human TNBC. Lines 4T07 and 4T1 originated from NR67 with 4T1 being the most metastatic and aggressive of them all. Expression levels of FABP5 were measured in all lines and compared to the human line MB-231. Levels of FABP5 were found to be positively correlated with the aggressiveness of the cells with MB-231 having the highest levels then 4T1 and 4T07.
  • Compounds FTS005, FTS040, FTS041, FTS042, FTS043, FTS044, FTS045, and FTS049 suppress growth of human ovarian cancer cells OVCAR8 [0432]
  • the ovarian cancer cell line OVCAR8 was used to calculate the efficacy of compounds FTS005, FTS040, FTS041, FTS042, FTS043, FTS044, FTS045, and FTS049 in inhibiting proliferation of the ovarian cancer cells.
  • Cells were treated with serial dilution of the compounds and proliferation was measured in Incucyte by calculating percentage of confluency every 4 hours over 4 days. As shown in FIG.4A, all compounds inhibited proliferation of the cancer line very effectively.
  • TNBC xenograft model was used to test the efficacy of FTS005 in suppressing growth on tumors in vivo.5x10 6 MB-231 cells were subcutaneously injected into the right flank of NSG mice. Mice were treated with FTS005 (20 mg/kg, or 40 mg/kg) or a vehicle by gavage 5 times a week and tumor growth was monitored. As shown in FIG.6A, FTS005 significantly inhibited growth of MB- 231 tumors as determined by tumor volume and tumor weight (FIGS.6A and 6B).
  • F. Compound FTS005 suppresses growth of TNBC tumors in vivo in syngeneic mouse model
  • 4T1 cells were utilized in TNBC syngeneic model to test the efficacy of FTS005 in suppressing growth on tumors in vivo in an immunocompetent model.1x10 5 4T1 cells were injected into the mammary fat pad of BALB/c mice.
  • FTS005 40 mg/kg or a vehicle by gavage 5 times a week and tumor growth was monitored.
  • FTS005 significantly inhibited growth of 4T1 tumors as determined by tumor volume and tumor weight (see FIG.7A and FIG.7B).
  • Expression levels of the angiogenesis marker that is also a known PPAR ⁇ target genes VEGFA were measured by QPCR and were found to be significantly lower in treated tumors (FIG.7C).
  • levels of the genes ACSL1 and PLIN2 that are involved in FA metabolism and lipid accumulation and are also known PPAR ⁇ targets were significantly reduced in treated tumors (FIG .7C).
  • the metabolites profiles measured in tumors treated with FTS005 significantly changed following the treatment.
  • Amount of long-chain fatty acids (FIG.8A) measured in treated tumors was significantly lower than in untreated tumors and similarly, lower levels of TCA cycle metabolites were detected (FIG.8B).
  • TAMs tumor infiltrating macrophages
  • FIG.10C untreated mice
  • FIG.10D The 4T1 cells utilized in this experiment stably express Luc2 and therefore, to stimulate an immune response by the harvested splenocytes, harvested cells were treated with Luc2 peptides in the growth media for 2 weeks and cell number was measured.
  • FIG.10E the number of T-cells harvested from FTS005-treated spleens was markedly higher than those harvested from untreated mice, indicating treated T-cells proliferated more in response to the Luc2 antigen exposure.
  • the live CFSE+ cells were counted by flow cytometry and the percentage of lysed cells was calculated.
  • FIG.10F the number of lysed cells was markedly higher after incubation with T-cells harvested from treated mice.
  • the data suggests that treatment with FABP3/4/5/7 inhibitor FTS005 stimulates formation of memory T-cells.
  • RNA samples extracted from untreated and treated tumors were used to for comprehensive profiling of the immune response by utilizing nCounter PanCancer Immune Profiling Panel.
  • treatment of tumor-bearing mice with FTS005 resulted in change in immune cells’ profile in the tumors.
  • higher number of CD45 cells, macrophages, B-cells, dendritic cells, cytotoxic cells, T-cells, CD8 T-cells, NK cells and NK CD56dim cells was found in treated tumors indicating a more immune-active TME.
  • mice treated with FTS005 suggests that inhibition of FABP3/4/5/7 modulates immune cells in the TME in two ways: 1) suppressing the immune-suppressive M2 tumor associated macrophages and, 2) stimulating tumor infiltrating lymphocytes (TILs) into the tumors including activated CD4 and CD8, NK cells, cytotoxic and dendritic cells, all are known to activate the immune-repressed tumors and turning them into “hot” tumors that can be recognized by the immune system.
  • TILs tumor infiltrating lymphocytes
  • FTS005 inhibits uptake of lipids into hepatocytes in in vitro model for liver steatosis
  • HepG2 cells were used to test effect of FTS005 on uptake of lipid into hepatic cells in an in vitro liver steatosis model.
  • Cells were treated with oleic acid (OA) (1 mM) in the presence or absence of the aniline compounds or the known FABP4 inhibitor BMS-309403 (BMS) and lipid accumulation in the cells was quantified using Nile Red.
  • OA oleic acid
  • BMS FABP4 inhibitor BMS-309403
  • Uptake of lipid into hepatic cells treated with FTS005 was markedly inhibited (FIG.12A). Inhibition of lipid uptake was more efficient by FTS005 compared with BMS.
  • lipids accumulated in the cells following treatment with 5 ⁇ M FTS005 was comparable to that established after treatment with 25 ⁇ M BMS (FIG.12A), indicating the thiophene compound is more efficient than BMS.
  • G. FTS005 inhibits uptake of lipids into mature adipocytes
  • Mouse 3T3-L1 cells were differentiated in culture to become mature adipocytes. Compounds FTS005 or the known FABP4 inhibitor BMS in varying concentrations were added to the cells starting at day 6 of the differentiation. On day 12, the lipid droplets were stained by Nile Red and quantified (FIG.12B).
  • BMDMs Mouse bone marrow-derived macrophages
  • M ⁇ cells were treated with either LPS (10 pM) and INFg (20 ng/mL) to promote differentiation into M1 macrophages, or IL-4 (20 ng/mL) and IL-13 (20 ng/mL) to promote differentiation into M2 macrophages.
  • LPS 10 pM
  • INFg 20 ng/mL
  • IL-4 20 ng/mL
  • IL-13 20 ng/mL
  • Cells were incubated with the cytokines for 3 days in the presence or absence of FABP3/4/5/7 inhibitors then lysed and immnunostained for further analysis.
  • FACS Fluorescence-activated cell sorting
  • T-cells frequency of macrophages
  • F4/80 total macrophages
  • CD11b total macrophages
  • MHC-II M1 macrophages
  • CD36 M2 macrophages
  • CD206 M2 macrophages
  • T-cells frequency the following markers were used: CD4, CD8, CD25 and FoxP3 (Treg), and TNFa (activated T-cells). Data was analyzed using FlowJo software.
  • IL-10 and IL-12 levels of the cytokines interleukin-10 (IL-10) and interleukin-12 p70 (IL-12) that were secreted from macrophages were measured in the media of treated cells using ELISA assay kits and according to manufacturer’s protocol.
  • Results [0461] A. FABP3/4/5/7 inhibitor treatment of macrophages during differentiation supports M1 phenotype while suppressing M2 phenotype
  • Bone marrow-derived macrophages (BMDM) were isolated from mice and differentiated in culture. Cells treated with M-CSF for 7 days were established as na ⁇ ve macrophages (M ⁇ ).
  • Na ⁇ ve macrophages were then treated with either LPS and IFNg to promote differentiation into M1 macrophages, or with IL-4 and IL-13 to promote differentiation into M2 macrophages. This was done in the absence or presence of FABP3/4/5/7 inhibitors.
  • High frequency of the marker MHC-II (FIG. 13A) and high levels of IL-12 (FIG.13B) were used to verify M1 polarization of macrophages.
  • High frequency of the markers CD36 (FIG.13C) and CD206 (FIG.13D), and high levels of IL-10 (FIG. 13E) were used to verify M2 polarization of macrophages.
  • the data indicate that FABP3/4/5/7 inhibitors can be utilized to block differentiation of macrophages from na ⁇ ve to M2 state as indicated in by both cell surface markers and secreted cytokines.
  • cells were initially differentiated into M1 macrophages then into M2 macrophages in the presence of FABP3/4/5/7 inhibitors.
  • Frequency of CD206+ macrophage population (FIG.13I) as well as expression levels of CD206 (FIG.13J) in differentiated cells were significantly decrease in the presence of FABP3/4/5/7 inhibitors and levels of IL-12 was markedly increased (FIG.13K).

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Abstract

L'invention concerne des composés inhibiteurs de FABP et leur utilisation dans des compositions pharmaceutiques pour le traitement de maladies comprenant des cancers qui expriment fortement l'une quelconque de ces FABP, en particulier le cancer du sein triple négatif (TNBC) et d'autres maladies induites par inflammation, y compris les maladies cardiovasculaires, l'obésité ou un trouble lié à l'obésité, le diabète, la dyslipidémie, une tolérance au glucose altérée ou le glucose à jeun altéré, le vitiligo, le psoriasis, les troubles auto-immuns, la douleur et la démence. La divulgation concerne également des procédés de préparation des composés divulgués.
PCT/US2024/032570 2023-06-05 2024-06-05 Inhibiteurs de protéines de liaison aux acides gras (fabp), procédés d'utilisation et procédés de fabrication Pending WO2024254161A2 (fr)

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MA52941A (fr) * 2018-06-21 2021-04-28 UCB Biopharma SRL Dérivés de thiophène pour le traitement de troubles provoqués par ige
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