WO2023196640A1 - Oxindole derivatives as ampk activators for use in the treatment of rare blood disorders - Google Patents

Oxindole derivatives as ampk activators for use in the treatment of rare blood disorders Download PDF

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WO2023196640A1
WO2023196640A1 PCT/US2023/017952 US2023017952W WO2023196640A1 WO 2023196640 A1 WO2023196640 A1 WO 2023196640A1 US 2023017952 W US2023017952 W US 2023017952W WO 2023196640 A1 WO2023196640 A1 WO 2023196640A1
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group
substituted
unsubstituted
alkyl group
hydroxy
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Yannis HARA
Eykmar Fett
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Bioverativ Therapeutics Inc.
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/42—Oxazoles
    • A61K31/422—Oxazoles not condensed and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/17—Amides, e.g. hydroxamic acids having the group >N—C(O)—N< or >N—C(S)—N<, e.g. urea, thiourea, carmustine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/4439—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. omeprazole
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/06—Antianaemics

Definitions

  • AMPK 5 '-adenosine monophosphate-activated protein kinase
  • AMPK 5'AMP-activated protein kinases
  • AMPK When activated, AMPK blocks the metabolic pathways which consume ATP (such as fatty acid synthesis in adipocytes, cholesterol synthesis in the liver, and insulin secretion in ⁇ -cells) and activates the metabolic pathways which produce ATP (such as fatty acid absorption and beta-oxidation in various tissues, glycolysis in the heart, and the biogenesis of mitochondria in skeletal muscle). AMPK also modulates the transcription of genes which participate in energy metabolism, exerting a metabolic control to facilitate energy (Viollet et al.).
  • AMPK participates in the regulation of non-metabolic processes such as cell growth, progression of the cell cycle, and organization of the cytoskeleton (Wil liams T., et al., Proc Natl Acad Sci U S A, 2011. 108(14): p. 5849-54.). Although the activation of AMPK is an adaptive response to an energy stress in many biological systems, AMPK plays an important role in maintaining physiological functions and for adaptation to pathophysiological conditions. Furthermore, it has been reported that AMPK promotes anti-inflammatory effects in vitro in murine and human macrophages by promoting macrophage polarization to an anti-inflammatory functional phenotype (Sag, D., et al., J Immunol, 2008.
  • AMPK is an apy trimer of three subunits comprised of twelve known isoforms that are based on all possible combinations of 2 ⁇ , 2 ⁇ and 3 ⁇ s subunits.
  • Activators of AMPK including 5'AMP and pharmacological small molecule AMPK activators, bind the CBS sites in the y subunits and the ADAM site, or “allosteric drug and metabolite” binding site, that lies between the a and p subunits (Aledavood et al., J Chem Inf Model, 2019. 59(6): p. 2859-2870.).
  • AMPK can be activated both by activator binding to the ADAM: site and by phosphorylation of Thrl72 of the a-subunit.
  • Pharmacological activators of AMPK binding to the ADAM site are known to activate only AMPK isoforms containing the pl-subunit (pl-selective AMPK activators) or containing either a ⁇ 1-subunit or a p2-subunit (pan-selective AMPK activators).
  • Sickle cell disease also called sickle cell anemia
  • SCD is one of the most common monogenic diseases, with 330,000 affected individuals born annually worldwide (Piel, F.B., et al., Lancet, 2013. 381(9861): p. 142-51.). It is the result of substitution of glutamic acid by valine at position 6 of the ⁇ -globin subunit of hemoglobin. The presence of this mutant ⁇ -globin subunit leads to the production of abnormal hemoglobin S (HbS) that polymerizes in red blood cells under conditions of low oxygen, which affects blood flow rheology and ultimately leads to vaso-occlusive crises and end organ damage (Barabino et al. Annu Rev Biomed Eng, 2010.
  • HbS abnormal hemoglobin S
  • Oxidative stress in erythrocytes is also enhanced and causes haemolysis (Vona, R., et al., Antioxidants (Basel), 2021. 10(2), and inflammation involving monocytes and proinflammatory macrophages is exacerbated in tissues and blood and participates to the organ damage process (Van Beers, E.J., et al., Circ Res, 2015. 116(2): p. 298-306; and Allali, S., et al., Haematologica, 2020. 105(2): p. 273-283).
  • HbF fetal hemoglobin
  • F0X03 is the transcription factor forkhead box 0-3 hypothesized to be responsible for increasing HbF when overexpressed or activated in CD34+ erythroid cells (Zhang, Y., et al., Blood, 2018. 132(3): p. 321-333.).
  • F0X03 is in the forkhead/winged helix box gene, group 0 (FoxO) family of proteins that are evolutionarily conserved transcription factors.
  • FOXO transcription factors integrate many important cellular functions and were originally identified as regulators of insulin-related genes.
  • FOXO transcription factors regulate genes involved in biological processes, including substrate and energy metabolism, protein turnover, cell survival, oxidative stress, proteostasis, apoptosis and cell death, cell cycle regulation, metabolic processes, immunity, inflammation and stem cell maintenance (Morris, B.J., et al., Gerontology, 2015. 61(6): p. 515-25; and Stefanetti, R.J., et al., F1000Res, 2018. 7.).
  • FOXO3 is required for the maintenance of murine hematopoietic stem cells and functions (Yalcin, S., et al., J Biol Chem, 2008. 283(37): p. 25692-25705; and Rimmele, P., et al., EMBO Rep, 2015. 16(9): p. 1164-76.).
  • FOXO3 is a key mediator of erythroid terminal maturation, regulating cell cycle in early stages of erythropoiesis and critical for ROS regulation, enucleation and mitochondrial clearance in late stages (Marinkovic, D., et al., J Clin Invest, 2007. 117(8): p. 2133-44; and Liang, R., et al., PLoS Genet, 2015. 11(10): p. el005526.).
  • modulation of FOXO3 might influence erythroid disorders as has been reported specifically for hemoglobinopathies (Zhang, Y., et al., Blood, 2018. 132(3): p.
  • Sirtuin 1 is a homolog of the NAD-dependent protein silent information regulator 2 in yeast and is a deacetylase for histones and other proteins. Deacetylation of FOXO3 by Sirtl is an important regulatory control mechanism of FOXO3 in many cells and tissues (Giannakou, M.E., et al., Science, 2004. 305(5682): p. 361.).
  • HSC hematopoietic stem cells
  • oxindole ⁇ 1 -selective AMPK activators increase expression of HbF in differentiating human lineage erythroid cells without affecting terminal differentiation.
  • isoform ⁇ 1 is predominant in erythroid lineage in human bone marrow compared to 02, making oxindole ⁇ 1 -AMPK activators more specific to the erythroid lineage compared to pan-AMPK activators that potentially activate other cells lineage or tissues expressive 02.
  • oxindole ⁇ 1 -selective AMPK activators do not affect terminal differentiation as measured by enucleation and differentiation markers expression.
  • oxindole “Compound 2” is shown to cause anti-inflammatory responses by promoting macrophages polarization and differentiation to an anti-inflammatory M2 phenotype, potentially resulting in a decrease of the inflammatory response which is a key component of the sickle cell disease pathophysiology.
  • the present application relates to a method of treating a rare blood disorder, the method comprising administering to a patient in need thereof an effective amount of a compound of formula (I): wherein:
  • R 1 represents: ⁇ an (C 6 -C 10 )aryl group, unsubstituted or substituted with one or more substituents chosen from:
  • Ra represents a hydrogen atom, a (C 1 -C 3 )alkyl group or a -CF 3 group,
  • R e represents an hydrogen atom or a (C 1 - C 4 )alkyl group, said (C 1 -C 4 )alkyl group being unsubstituted or substituted with one or more (C 1 -C 4 )alkoxy or heterocycloalkyl group, and
  • R f and R f' independently, identical or different, represent a (C 1 -C 3 )alkyl group
  • R 2 represents:
  • a halogen atom a cyano group, a group, ⁇ a group,
  • R d and R d' independently, identical or different, represent a hydrogen atom, a (C 1 -C 3 )alkyl group, a hydroxy(C 1 -C 4 )alkyl or a (C 3 -C 6 )cycloalkyl group,
  • R g and Rg’ independently, identical or different, represent a (C 1 -C 3 )alkyl group
  • R 3 represents a halogen atom or a (C 1 -C 3 )alkyl group
  • R 4 represents a halogen atom or a hydrogen atom, or a pharmaceutically acceptable salt thereof
  • the rare blood disorder is a hemoglobinopathy.
  • the hemoglobinopathy is selected from ⁇ -thalassemia and sickle cell disease (SCD).
  • the compound of formula (I) is a selective ⁇ 1-AMPK activator that is a more selective activator of ⁇ 1-AMPK versus activation of ⁇ 2-AMPK or of activation of both ⁇ 1-AMPK and ⁇ 2-AMPK (pan-activation).
  • the selective ⁇ 1-AMPK activator may possess at least about a 10-fold selective activation, at least about a 50-fold selective activation, at least about a 100-fold activation, at least about a 300-fold selective activation, or at least about a 500 fold selective activation for ⁇ 1-AMPK relative to ⁇ 2-AMPK.
  • the selective ⁇ 1-AMPK activator has an EC 50 (half-maximal concentration required for full activation) for the activation of ⁇ 1-AMPK of about 100 nM or less. In other embodiments, the selective ⁇ 1-AMPK activator has an EC 50 of about 50 nM or less for the activation of ⁇ 1-AMPK. On other embodiments, the selective ⁇ 1-AMPK activator has an EC 50 of about 10 nM or less for the activation of ⁇ 1-AMPK. EC 50 values may be determined according to the enzymatic assays performed in Schmoll et al.
  • the selective ⁇ 1-AMPK activator increases the activity of AMPK above the baseline activity (as measured by autophosphorylation of the ⁇ -subunit of AMPK or phosphorylation of a peptide or protein substrate) by 50% or more. In other embodiments, the selective ⁇ 1-AMPK activator increases the activity of AMPK above the baseline activity by 100% or more. In other embodiments, the selective ⁇ 1-AMPK activator increases the activity of AMPK above the baseline activity by 150% or more.
  • FIG. 1A shows the result of fetal hemoglobin induction in human CD34+ cells when mobilized CD34+ HSPC from healthy individuals are cultured for 21 days under conditions to promote erythroid differentiation and are exposed to oxindoles at the indicated doses (pM). Exposure to oxindoles increases F-cell (HbF+) frequency in enucleated GlyA+ cells compared to control (DMSO indicated as Vehicle), measured by flowcytometry. Representative plots from a single donor are shown.
  • FIG. 1B oxindole “Compound 2,” induces fetal hemoglobin in a dose-dependent manner, compared to control. Representative plots from a single donor are shown.
  • FIG. 1C confirms the induction of fetal hemoglobin by oxindoles measured by western blot.
  • FIG. 1D are representative plots from a single donor showing fetal hemoglobin induction by Compound 2 in a dose-dependent manner, compared to control.
  • FIG. 1E shows RNA-seq analysis conducted on CD34+ cells treated with Compound 2 confirms the activation of gamma-globin genes HBG1 and HBG2.
  • FIG. 1F shows a proteomic analysis conducted on CD34+ cells from healthy donors and treated with Compound 2 that confirms the increase in HBG2 induced fetal hemoglobin protein expression.
  • FIG. 1C confirms the induction of fetal hemoglobin by oxindoles measured by western blot.
  • FIG. 1D are representative plots from a single donor showing fetal hemoglobin induction by Compound 2 in a dose-dependent manner, compared to control.
  • FIG. 1E shows RNA-seq analysis conducted on CD34
  • FIG. 1G RNA-seq and Proteomic integrative analysis shows a correlation in HBE1 and HBG2 gene activation and protein expression.
  • FIG 1H shows the solely expression of beta-1 subunit and the absence of beta-2 subunit expression in CD34+ HSPC before and during the erythroid differentiation.
  • FIG.2A shows the result of fetal hemoglobin induction in human CD34+ cells when CD34+ HSPC from sickle patients are cultured for 21 days under conditions to promote erythroid differentiation and are exposed to oxindoles at the indicated doses ( ⁇ M). Exposure to oxindoles increases F-cell (HbF+) frequency in enucleated GlyA+ cells compared to control (DMSO indicated as Vehicle), measured by flowcytometry. Representative plots from a single donor are shown.
  • FIG. 3 reveals that exposure of CD34+ HSPC from sickle patients to oxindoles prevents sickling of terminally differentiated CD34+ cells, measured by imaging flowcytometry Imagestream.
  • FIG. 4A shows the result of fetal hemoglobin induction in human CD34+ cells when mobilized CD34+ HSPC from healthy individuals are cultured for 14 days under conditions promoting erythroid differentiation and are exposed to hydroxyurea in combination or not with oxindoles. Exposure to hydroxyurea increases F-cell (HbF+) frequency in enucleated GlyA+ cells as expected, compared to control cells (DMSO indicated as Vehicle). The combination of hydroxyurea and oxindoles leads to a higher frequency of F-cell compared to hydroxyurea alone. Representative plots from one healthy donor are shown.
  • FIG. 4A shows the result of fetal hemoglobin induction in human CD34+ cells when mobilized CD34+ HSPC from healthy individuals are cultured for 14 days under conditions promoting erythroid differentiation and are exposed to hydroxyurea in combination or not with oxindoles. Exposure to hydroxyurea increases F-cell (HbF+) frequency in enucleated GlyA+ cells as
  • FIG. 5A shows the result of oxindoles exposure on human CD34+ cells maturation when mobilized CD34+ HSPC from healthy individuals are differentiated for 21 days into erythroid cells. Data confirms that oxindoles have no impact on enucleation of erythroid cells during differentiation compared to control cells (DMSO indicated as Vehicle), based on the frequency of enucleated cells as measured by flow cytometry. Representative plots from one healthy donor are shown.
  • FIG. 5B displays that oxindoles have no effect on the expression level of differentiation markers - Band-3, LRF, ALAS2 and GATA-1 - during maturation compared to control cells (DMSO indicated as Vehicle), measured by western blot.
  • FIG. 5A shows the result of oxindoles exposure on human CD34+ cells maturation when mobilized CD34+ HSPC from healthy individuals are differentiated for 21 days into erythroid cells. Data confirms that oxindoles have no impact on enucleation of erythroid cells during differentiation compared
  • FIG. 5C manifests that erythroid markers CD235a expression is not affected at terminal differentiated stage in CD34+ HSPC cells exposed to oxindoles. Representative plots from one healthy donor are shown.
  • FIG. 5D confirms that exposure CD34+ HSPC cells to oxindoles do not affect cells erythroid differentiation at day 14.
  • FIG. 6 shows the target engagement of oxindoles on AMPK in mobilized CD34+ HSPC cells from healthy individuals or in erythroid cell line HUDEP-2.
  • Target engagement is assessed by phosphorylation of residue Threonine 172 on the a-domain of AMPK at day 11 of erythroid differentiation.
  • FIG. 6A represents target engagement in mobilized CD34+ HSPC measured by Alpha SureFire Ultra Multiplex® bead-based assay technology. All data are expressed as a ratio of signals pAMPK and total AMPK and normalized to total protein concentration.
  • FIG. 6B represents target engagement in HUDEP-2 cells measured by Alpha SureFire Ultra Multiplex® bead-based assay technology.
  • FIG. 7 demonstrates that oxindole “Compound 2” promotes anti-inflammatory M2 macrophages polarization.
  • FIG. 7A shows the decrease of pro-inflammation markers CD86 in M-CSF polarized and activated Ml macrophages from healthy donor. Activation was triggered by the combination of LPS (lOmg/mL) and INF- ⁇ (50mg/mL). Thus, exposure to oxindole “Compound 2” prevents the increase of this pro-inflammatory marker induced by LPS and INF- ⁇ compared to control (DMSO indicated as Vehicle). Representative plots are shown.
  • FIG. 1 shows that oxindole “Compound 2” promotes anti-inflammatory M2 macrophages polarization.
  • FIG. 7A shows the decrease of pro-inflammation markers CD86 in M-CSF polarized and activated Ml macrophages from healthy donor. Activation was triggered by the combination of LPS (lOmg/mL) and INF- ⁇ (50
  • halogen atom as used herein, means a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom.
  • alkyl group means, unless otherwise mentioned in the text, a linear or branched saturated aliphatic group containing from 1 to 4 carbon atoms. Examples that may be mentioned include methyl, ethyl, propyl, isopropyl, butyl, isobutyl or tert-butyl groups, in particular methyl, ethyl or tert-butyl groups.
  • alkenyl group as used herein, means a linear or branched mono- or polyunsaturated aliphatic group containing, for example, one or two ethylenic unsaturations.
  • alkoxy group means a radical -O-alkyl in which the alkyl group is as defined previously.
  • the -O-alkyl group is a methoxy or an ethoxy group.
  • cycloalkyl group means a saturated mono or bi-cyclic aliphatic group comprising between 3 and 6 carbon atoms. Examples that may be mentioned include cyclopropyl, cyclobutyl or cyclohexyl group.
  • heterocycloalkyl group means a mono or bi-cyclic alkyl group comprising between 3 and 6 carbon atoms and comprising 1 or 2 heteroatoms, such as nitrogen or oxygen. Examples that may be mentioned include piperazinyl, morpholinyl, tetrahydropyranyl, piperidinyl or pyrrolidinyl groups.
  • aryl group as used herein, means a mono or bi-cyclic aromatic group comprising between 6 and 10 carbon atoms.
  • An example of an aryl group that may be mentioned is the phenyl or naphtalene group.
  • heteroaryl group means a mono or bi cyclic aromatic group comprising between 2 and 10 carbon atoms and comprising between 1 and 3 heteroatoms, such as nitrogen, oxygen, or sulfur. Examples that may be mentioned include pyridinyl, pyrazolyl, furanyl, isoxazolyl, thiazolyl, isothiazolyl, thiophenyl, indolyl, furopyridinyl, benzofuranyl, thienopyridinyl, pyrimidinyl and 1,3,4-oxadiazolyl groups.
  • the present disclosure relates to the treatment or prevention of the diseases and disorders discussed herein utilizing a compound of formula (I): wherein:
  • R 1 represents: an (C 6 -C 10 )aryl group, unsubstituted or substituted with one or more substituents chosen from: ⁇ a halogen atom,
  • Ra represents a hydrogen atom, a (C 1 -C 3 )alkyl group or a -CF 3 group,
  • R e represents an hydrogen atom or a (C 1 - C 4 )alkyl group, said (C 1 -C 4 )alkyl group being unsubstituted or substituted with one or more (C 1 -C 4 )alkoxy or heterocycloalkyl group, and
  • R f and R f' independently, identical or different, represent a (C 1 -C 3 )alkyl group
  • R 2 represents:
  • ⁇ a cyano group ⁇ a (C 1 -C 3 )alkyl group unsubstituted or substituted with one or more substituents chosen from a halogen atom, a hydroxyl group, and a (C 1 - C 4 )alkenyl group,
  • a (C 3 -C 6 )heterocycloalkyl group unsubstituted or substituted with one or more halogen atom, (C 1 -C 3 )alkyl group, hydroxyl group, hydroxy(C 1 -C 3 )alkyl group, (C 1 -C 4 )alkoxy group or (C 1 -C 4 )fluoroalkyl group, ⁇ an (C 6 -C 10 )aryl group, unsubstituted or substituted with one or more - OR c group, in which R c represents a hydrogen atom or a (C 1 -C 3 )alkyl group,
  • R d and R d' independently, identical or different, represent a hydrogen atom, a (C 1 -C 3 )alkyl group, a hydroxy(C 1 -C 4 )alkyl or a (C 3 -C 6 )cycloalkyl group,
  • R g and R g’ independently, identical or different, represent a (C 1 -C 3 )alkyl group
  • R 3 represents a halogen atom or a (C 1 -C 3 )alkyl group
  • R 4 represents a halogen atom or a hydrogen atom, or a pharmaceutically acceptable salt thereof.
  • R 1 represents:
  • halogen atom in particular a fluorine or a chlorine atom
  • Ra represents a hydrogen atom or a (C 1 -C 3 )alkyl group in particular a methyl group
  • a (C 1 -C 3 )alkyl group in particular a methyl group, optionally substituted by one or more halogen atoms, in particular a di or trifluoromethyl group,
  • a heteroaryl group in particular a pyridinyl group, a pyrazolyl group, an isoxazolyl group, athiazolyl group, an isothiazolyl group or a 1,3,4-oxadiazolyl group, optionally substituted by one or more substituents chosen from a halogen atom in particular a bromine atom, a (C 1 -C 4 )alkyl group, in particular a methyl group or a tert-butyl group, a cycloalkyl group, in particular a cyclopropyl group, an (C 1 -C 3 )alkoxy group in particular a methoxy group;
  • R 2 represents:
  • halogen atom in particular a fluorine or a chlorine atom
  • a cycloalkyl group in particular a cyclopropyl or a cyclobutyl group, optionally substituted by a hydroxy(C 1 -C 3 )alkyl group, in particular a hydroxymethyl group,
  • Rb represents a hydrogen atom, a -CF 3 group or an alkyl group in particular a (C 1 -C 3 )alkyl group, optionally substituted by one heterocycloalkyl group, in particular a piperazinyl group, the said heterocycloalkyl group being optionally substituted by a (C 1 - C 3 )alkyl group, in particular a methyl group;
  • a heterocycloalkyl group in particular a morpholinyl, a dihydropyranyl, a tetrahydropyranyl, a pyrrolidinyl, a tetrahydrofuranyl, a pip eridiny 1 or a piperazinyl group, optionally substituted by one or more a (C 1 - C 3 )alkyl group, in particular a methyl group,
  • R c represents a hydrogen atom or a (C 1 - C 3 )alkyl group, in particular a methyl group
  • a heteroaryl group in particular a pyridinyl, a thiazolyl or a furanyl group, optionally substituted by one or more -NH 2 group, ⁇ a -NR d R d' group, in which R d and R d’ , independently, identical or different, represent a hydrogen atom, a (C 1 -C 3 )alkyl group, in particular a methyl group,
  • a heteroaryl group in particular a thiophene group, optionally substituted by one or more cycloalkyl group in particular a cyclopropyl group, optionally substituted by a hydroxy(C 1 -C 3 )alkyl group in particular a hydroxymethyl group,
  • R 3 represents:
  • a halogen atom in particular a chlorine or a fluorine atom, or
  • R 4 represents:
  • halogen atom in particular a fluorine atom
  • R 1 represents:
  • R a represents a methyl group, a CF 3 group,
  • R e represents a methyl group, an ethyl group, an isopropyl group, a methoxy ethyl group, a morpholinoethyl group, and
  • R 2 represents:
  • a cyclopropyl or a cyclobutyl group unsubstituted or substituted with a hydroxyl group, a hydroxymethyl group or a methoxy group
  • R b represents a hydrogen atom, a - CF 3 group or an (C 1 -C 3 )alkyl group, unsubstituted or substituted with one piperazinyl group, the said piperazinyl group being unsubstituted or substituted with a methyl group, ⁇ a morpholinyl, a dihydropyranyl, a tetrahydropyranyl, a pyrrolidinyle, a tetrahydrofuranyl, a piperazinyl, a piperidinyl group, a dioxane group, an oxaazaspiro[3.3]heptanyl, unsubstituted or substituted with one or more methyl group, hydroxyl group, methoxy group, hydroxymethyl group or fluorine atom,
  • R c represents:
  • R d and R d' independently, identical or different, represent a methyl group, an hydroxypropyl group or a cyclopropyl group;
  • R 3 represents:
  • R 4 represents a fluorine or a hydrogen atom; or a pharmaceutically acceptable salt thereof.
  • Ri represents a (C 2 -C 10 )heteroaryl group, optionally substituted by one or more substituents chosen from a halogen atom, a (C 1 -C 3 )alkyl group, a (C 3 -C5)cycloalkyl group, and a (C 1 -C 4 )alkoxy group; or a pharmaceutically acceptable salt thereof.
  • R 1 represents an isoxazolyl group, optionally substituted by one substituent chosen from a (C 1 -C 4 )alkyl group in particular a methyl group, a (C 3 -C5)cycloalkyl group, in particular a cyclopropyl group or a cyclohexyl group or an alkoxy group, in particular a methoxy group; or a pharmaceutically acceptable salt thereof.
  • Ri represents a phenyl group, substituted with one substituent chosen from halogen atom and an a -ORa group, in which Ra represents a (C 1 -C 3 )alkyl group; or a pharmaceutically acceptable salt thereof.
  • R 2 represents:
  • a (C 3 -C 6 )heterocycloalkyl group in particular a morpholinyl, a dihydropyranyl, a tetrahydropyranyl, a pyrrolidinyl, a tetrahydrofuranyl, a piperidinyl or a piperazinyl group, optionally substituted by one or more (C 1 -C 3 )alkyl group, in particular a methyl group or an hydroxyl group,
  • an (C 6 -C 10 )aryl group in particular a phenyl group, optionally substituted by one or more -OR c group, in which R c represents a hydrogen atom or a (C 1 -C 3 )alkyl group,
  • a (C 2 -C 10 )heteroaryl group in particular a pyridinyl, a thiazolyl or a furanyl group, optionally substituted by one or more -NH 2 group, and
  • Rd and R d' independently, identical or different, represent a hydrogen atom, a (C 1 -C 3 )alkyl group, in particular a methyl group; or a pharmaceutically acceptable salt thereof
  • R 2 represents:
  • a (C 3 -C 6 )cycloalkyl group in particular a cyclopropyl group, optionally substituted by a hydroxy(C 1 -C 3 )alkyl group in particular a hydroxymethyl group,
  • R c represents a hydrogen atom or a (C 1 -C 3 )alkyl group in particular a methyl group
  • R d and R d' represent a (C 1 -C 3 )alkyl group, in particular a methyl group; or a pharmaceutically acceptable salt thereof.
  • R 3 represents a halogen atom, in particular a chlorine or a fluorine atom.
  • R 4 represents a fluorine atom.
  • R 4 when R 4 represents a fluorine then R 3 also represents a fluorine atom.
  • the compound of formula (I) is selected from the group consisting of:
  • the compound of formula (I) is 6-chloro-3-[l-hydroxy-l-(3-methoxy- isoxazol-5-yl)-methylidene]-5-[4-(l-hydroxymethyl-cyclopropyl)-phenyl]-l,3-dihydro-indol-
  • the compound of formula (I) is 6-chloro-3-[(3-cyclopropylisoxazol-5- yl)-hydroxy-methylene]-5-[4-[l-(hydroxymethyl)cyclopropyl]phenyl]indolin-2-one, which has the following structure:
  • the compound of formula (I) is 6-chloro-3-[(3-fluoro-4-pyridyl)- hydroxy-methylene]-5-[4-[l-(hydroxymethyl)cyclopropyl]phenyl]indolin-2-one, which has the following structure: (“Compound 3”), or a pharmaceutically acceptable salt thereof.
  • the compound of formula (I) is 6-chloro-5-(2'-hydroxy-3'-methoxy- biphenyl-4-yl)-3-[l-hydroxy-l-(3-methyl-isoxazol-5-yl)-methylidene]-l,3-dihydro-indol-2- one, which has the following structure: (“Compound 4”), or a pharmaceutically acceptable salt thereof.
  • any of the compounds disclosed herein that are basic in nature are generally capable of forming a wide variety of different salts with various inorganic and/or organic acids.
  • such salts are generally pharmaceutically acceptable for administration to animals and humans, it is often desirable in practice to initially isolate a compound from the reaction mixture as a pharmaceutically unacceptable salt and then simply convert the latter back to the free base compound by treatment with an alkaline reagent, and subsequently convert the free base to a pharmaceutically acceptable acid addition salt.
  • the acid addition salts of the base compounds can be readily prepared using conventional techniques, e.g.
  • the base compound by treating the base compound with a substantially equivalent amount of the chosen mineral or organic acid in an aqueous solvent medium or in a suitable organic solvent such as, for example, methanol or ethanol. Upon careful evaporation of the solvent, the desired solid salt is obtained.
  • a suitable organic solvent such as, for example, methanol or ethanol.
  • the desired solid salt is obtained.
  • Presently disclosed compounds that are positively charged, e.g. containing a quaternary ammonium may also form salts with the anionic component of various inorganic and/or organic acids.
  • Acids which can be used to prepare pharmaceutically acceptable salts of a compound of formula (I) are those which can form non-toxic acid addition salts, e.g. salts containing pharmacologically acceptable anions, such as chloride, bromide, iodide, nitrate, sulfate or bisulfate, phosphate or acid phosphate, acetate, lactate, citrate or acid citrate, tartrate or bitartrate, succinate, malate, maleate, fumarate, gluconate, saccharate, benzoate, methanesulfonate and pamoate [i.e. 1, 1'-methylene-bis-(2-hydroxy-3-naphthoate)] salts.
  • non-toxic acid addition salts e.g. salts containing pharmacologically acceptable anions, such as chloride, bromide, iodide, nitrate, sulfate or bisulfate, phosphate or acid phosphate, acetate
  • Presently disclosed compounds that are acidic in nature are generally capable of forming a wide variety of different salts with various inorganic and/or organic bases.
  • such salts are generally pharmaceutically acceptable for administration to animals and humans, it is often desirable in practice to initially isolate a compound from the reaction mixture as a pharmaceutically unacceptable salt and then simply convert the latter back to the free acid compound by treatment with an acidic reagent, and subsequently convert the free acid to a pharmaceutically acceptable base addition salt.
  • These base addition salts can be readily prepared using conventional techniques, e.g.
  • Bases which can be used to prepare the pharmaceutically acceptable base addition salts of a compound of formula (I) are those which can form non-toxic base addition salts, e.g. salts containing pharmacologically acceptable cations, such as, alkali metal cations (e.g. potassium and sodium), alkaline earth metal cations (e.g. calcium and magnesium), ammonium or other water-soluble amine addition salts such as A-methylglucamine (meglumine), lower alkanolammonium, and other such bases of organic amines.
  • pharmacologically acceptable cations such as, alkali metal cations (e.g. potassium and sodium), alkaline earth metal cations (e.g. calcium and magnesium), ammonium or other water-soluble amine addition salts such as A-methylglucamine (meglumine), lower alkanolammonium, and other such bases of organic amines.
  • the present disclosure further embraces stereoisomers and mixture of stereoisomers of the compounds disclosed herein.
  • Stereoisomers e.g. cis and trans isomers
  • all optical isomers of a presently disclosed compound e.g. R- and S- enantiomers
  • racemic, diastereomeric and other mixtures of such isomers are within the scope of the present disclosure.
  • the compound of formula (I), and pharmaceutical compositions containing them, such as those described herein, are useful in therapy, in particular in the therapeutic treatment of blood disorders, including hemoglobinopathies.
  • Subjects to be treated according to the methods described herein include vertebrates, such as mammals.
  • a hemoglobinopathy is a condition that involves a mutation in human beta-globin or an expression control sequence thereof, such as sickle cell disease (SCD) or beta-thalassemia.
  • SCD sickle cell disease
  • beta-thalassemia beta-thalassemia
  • SCD typically arises from a mutation substituting thymine for adenine in the sixth codon of the beta-chain gene of hemoglobin (i.e., GAG to GTG of the HBB gene). This mutation causes glutamate to valine substitution in position 6 of the Hb beta chain.
  • the resulting Hb referred to as HbS, has the physical properties of forming polymers under conditions of low oxygen tension.
  • SCD is typically an autosomal recessive disorder.
  • Subjects with SCD may experience a range of medical complications including acute pain episodes, also known as vaso-occlusive crises or vaso-occlusive episodes, that require hospitalization and may progress to more severe complications such as acute chest syndrome.
  • SCD is associated with vascular disease and stroke and SCD subjects may experience cerebrovascular accidents including transient ischemic attack, overt strokes and silent cerebral infarctions.
  • Retinopathy and seizures are also associated with SCD.
  • Proliferative sickle cell retinopathy is a frequent vision-threatening complication in sickle cell anemia, leading to visual impairment. In PSR, the blood vessels become blocked and divert away from the retina causing the retina to starve and die, leading to vision loss.
  • Subjects with SCD may experience both chronic and acute complications including bone pain crisis as a complication of vaso-occlusive pain, bone and bone marrow infarction, osteonecrosis and vascular necrosis.
  • Subjects with SCD may experience chronic and acute cardiopulmonary complications including acute chest syndrome (ACS), pulmonary hypertension and left-sided heart disease.
  • SCD subjects may experience chronic and acute reticuloendothelial complications including splenic sequestration, which is more prevalent in subjects who have had a first acute pain episode. Splenic sequestration can result in worsened anemia in SCD subjects.
  • Subjects with SCD may experience chronic and acute gastrointestinal and urogenital complications including cholelithiasis, acute cholecystitis, biliary sludge, acute choledocholi thiasis and gallstones.
  • Urogenital complications, including renal dysfunction may occur at an early age and lead to chronic renal failure.
  • a priapism may occurs as a severe urogenital complication.
  • ⁇ -Thalassemia are a group of inherited blood disorders caused by a variety of mutational mechanisms that result in a reduction or absence of synthesis of ⁇ -globin and leading to accumulation of aggregates of unpaired, insoluble a-chains that cause ineffective erythropoiesis, accelerated red cell destruction, and severe anemia.
  • Subjects with beta- thalassemia exhibit variable phenotypes ranging from severe anemia to clinically asymptomatic individuals.
  • the genetic mutations present in ⁇ -Thalassemia are diverse, and can be caused by a number of different mutations.
  • the mutations can involve a single base substitution or deletions or inserts within, near or upstream of the ⁇ globin gene. For example, mutations occur in the promoter regions preceding the beta-globin genes or cause production of abnormal splice variants.
  • ⁇ 0 is used to indicate a mutation or deletion which results in no functional ⁇ globin being produced.
  • ⁇ + is used to indicate a mutation in which the quantity or ⁇ globin is reduced or in which the ⁇ -globin produced has a reduced functionality.
  • ⁇ -Thalassemia examples include thalassemia minor, thalassemia intermedia, and thalassemia major.
  • 0- Thalassemia minor refers to thalassemia where only one of ⁇ -globin alleles bears a mutation. Individuals typically suffer from microcytic anemia. Detection usually involves lower than normal MCV value ( ⁇ 80 fL) plus an increase in fraction of hemoglobin A2 (>3.5%) and a decrease in fraction of hemoglobin A ( ⁇ 97.5%). Genotypes can be ⁇ + / ⁇ or ⁇ 0 / ⁇ .
  • ⁇ -Thalassemia intermedia refers to a ⁇ -Thalassemia intermediate between the major and minor forms.
  • Genotypes can be ⁇ + / ⁇ + or ⁇ 0 / ⁇ .
  • ⁇ -Thalassemia major refers to a ⁇ -Thalassemia where both ⁇ -globin alleles have thalassemia mutations. This is a severe microcytic, hypochromic anemia. If left untreated, it causes anemia, splenomegaly, and severe bone deformities and typically leads to death before age 20. Treatment consists of periodic blood transfusion; splenectomy if splenomegaly is present, and treatment of transfusion-caused iron overload.
  • Genotypes include ⁇ + / ⁇ 0 or ⁇ 0 / ⁇ 0 or ⁇ + / ⁇ + .
  • Mediterranean anemia or Cooley's anemia has a genotype of ⁇ 0 / ⁇ 0 so that no hemoglobin A is produced. It is the most severe form of ⁇ -Thalassemia.
  • HbC hemoglobin C
  • HbC Hemoglobin C
  • Hb C is an abnormal hemoglobin in which substitution of a glutamic acid residue with a lysine residue at the 6th position of the ⁇ -globin chain has occurred.
  • HbSC disease A subject that is a double heterozygote for HbS and HbC (HbSC disease) is typically characterized by symptoms of moderate clinical severity.
  • HbE hemoglobin E
  • HbE is an abnormal hemoglobin in which substitution of a glutamic acid residue with a lysine residue at the 26th position of the ⁇ -globin chain has occurred.
  • a subject that is a double heterozygote for HbS and HbE has HbS/HbE syndrome, which usually causes a phenotype similar to HbS/b+ thalassemia, discussed below.
  • a subject that is a double heterozygote for HbS and 30 thalassemia i.e., HbS/ ⁇ 0 thalassemia
  • HbS/ ⁇ 0 thalassemia can suffer symptoms clinically indistinguishable from sickle cell anemia.
  • a subject that is a double heterozygote for HbS and ⁇ + thalassemia i.e., HbS/ ⁇ + thalassemia
  • HbS Rare combinations of HbS with other abnormal hemoglobins include HbD Los Angeles, G-Philadelphia, HbO Arab, and others.
  • a compound of formula (I), or a pharmaceutically acceptable salt thereof is used to treat a hemoglobinopathy, such as SCD or thalassemia (e.g. ⁇ -Thalassemia), including those that involve a mutation in human ⁇ -globin or an expression control sequence thereof, as described above.
  • a hemoglobinopathy such as SCD or thalassemia (e.g. ⁇ -Thalassemia), including those that involve a mutation in human ⁇ -globin or an expression control sequence thereof, as described above.
  • methods of treating a hemoglobinopathy comprising administering an effective amount of a compound of formula (I), or pharmaceutically acceptable salt thereof, to a patient in need thereof.
  • the compound of formula (I) or, pharmaceutically acceptable salt thereof is a ⁇ 1 -selective AMPK activator.
  • the compound of Formula (I) is selected from the group comprising Compound 1, Compound 2, Compound 3, and Compound 4.
  • the compound of formula (I) is used to treat a subject with an HbS/ ⁇ 0 genotype, an HbS/ ⁇ + genotype, an HBSC genotype, an HbS/HbE genotype, an HbD Los Angeles genotype, a G-Philadelphia genotype, or an abHbO Arab genotype.
  • the ⁇ 1 -selective AMPK activators are administered to a subject in need thereof in an effective amount to treat one or more symptoms of sickle cell disease, a thalassemia (e.g. ⁇ -Thalassemia), or a related disorder.
  • a thalassemia e.g. ⁇ -Thalassemia
  • physiological changes in RBCs can result in a disease with the following signs: (1) hemolytic anemia; (2) vaso-occlusive crisis; and (3) multiple organ damage from microinfarcts, including heart, skeleton, spleen, and central nervous system.
  • Thalassemia can include symptoms such as anemia, fatigue and weakness, pale skin or jaundice (yellowing of the skin), protruding abdomen with enlarged spleen and liver, dark urine, abnormal facial bones and poor growth, and poor appetite.
  • Retinopathy due to SCD can also be treated by administering an effective amount of ⁇ 1 -AMPK activator.
  • Sickle retinopathy occurs when the retinal blood vessels get occluded by sickle red blood cells and the retina becomes ischemic, angiogenic factors are made in retina. In sickle cell disease, this occurs mostly in the peripheral retina, which does not obscure vision at first. Eventually, the entire peripheral retina of the sickle cell patient becomes occluded and many neovascular formations occur.
  • Administration of a ⁇ 1 -selective AMPK activator can reduce or inhibit the formation of occlusions in the peripheral retina of a sickle cell patient.
  • the ⁇ 1 -selective AMPK activators are used to increase HbF expression in a patient in need thereof.
  • ⁇ 1-selective AMPK activator is a compound of Formula (I) or a pharmaceutically acceptable salt thereof.
  • the ⁇ 1-selective AMPK selected from the group comprising Compound 1, Compound 2, Compound 3, and Compound 4, or a pharmaceutically acceptable salt thereof.
  • the ⁇ 1 -AMPK activators are used to decrease inflammation in a patient with a ⁇ -hemoglobinopathy.
  • ⁇ 1-selective AMPK activator is a compound of Formula (I) or a pharmaceutically acceptable salt thereof.
  • the ⁇ 1-selective AMPK activator is selected from the group comprising Compound 1, Compound 2, Compound 3, and Compound 4, or a pharmaceutically acceptable salt thereof.
  • the ⁇ 1-AMPK activators are used to decrease oxidative stress in a patient with a p-hemoglobinopathy.
  • ⁇ 1-selective AMPK activator is a compound of Formula (I) or a pharmaceutically acceptable salt thereof.
  • the ⁇ 1-selective AMPK activator is selected from the group comprising Compound 1, Compound 2, Compound 3, and Compound 4, or a pharmaceutically acceptable salt thereof.
  • the ⁇ 1 -selective AMPK activator is administered in combination with hydroxyurea.
  • a method of treating a hemoglobinopathy comprising administering an effective amount of a ⁇ 1-selective AMPK activator to a patient in need thereof in combination with an effective amount of hydroxyurea.
  • the ⁇ 1- selective AMPK activator is a compound of Formula (I) or a pharmaceutically acceptable salt thereof.
  • the ⁇ 1 -selective AMPK activator is selected from the group comprising Compound 1, Compound 2, Compound 3, and Compound 4, or a pharmaceutically acceptable salt thereof.
  • ⁇ 1 -selective AMPK activator is a compound of Formula (I) or a pharmaceutically acceptable salt thereof.
  • the ⁇ 1 -selective AMPK activator is selected from the group comprising Compound 1, Compound 2, Compound 3, and Compound 4, or a pharmaceutically acceptable salt thereof.
  • compositions comprising at least one ⁇ 1- selective AMPK activator as described herein and at least one pharmaceutically acceptable excipient, e.g. for use according to the methods disclosed herein.
  • the pharmaceutically acceptable excipient can be any such excipient known in the art including those described in, for example, Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985).
  • Pharmaceutical compositions of the pl-selective AMPK activator may be prepared by conventional means known in the art including, for example, mixing at least one ⁇ 1 -selective AMPK activator with a pharmaceutically acceptable excipient.
  • the present disclosure provides a pharmaceutical dosage form comprising a ⁇ 1 -selective AMPK activator as described herein and a pharmaceutically acceptable excipient, wherein the dosage form is formulated to provide, when administered (e.g. when administered orally), an amount of said compound sufficient to treat a disease or disorder as described herein.
  • a pharmaceutical composition or dosage form of the invention can include an agent and another carrier, e.g. compound or composition, inert or active, such as a detectable agent, label, adjuvant, diluent, binder, stabilizer, buffers, salts, lipophilic solvents, preservative, adjuvant or the like.
  • Carriers also include pharmaceutical excipients and additives, for example, proteins, peptides, amino acids, lipids, and carbohydrates (e.g.
  • sugars including monosaccharides, di-, tri-, tetra-, and oligosaccharides; derivatized sugars such as alditols, aldonic acids, esterified sugars and the like; and polysaccharides or sugar polymers), which can be present singly or in combination, comprising alone or in combination 1 to 99.99% by weight or volume.
  • exemplary protein excipients include serum albumin such as human serum albumin (HSA), recombinant human albumin (rHA), gelatin, casein, and the like.
  • amino acid/antibody components which can also function in a buffering capacity, include alanine, glycine, arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine, phenylalanine, aspartame, and the like.
  • Carbohydrate excipients are also intended within the scope of this invention, examples of which include but are not limited to monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbose, and the like; disaccharides, such as lactose, sucrose, trehalose, cellobiose, and the like; polysaccharides, such as raffinose, melezitose, maltodextrins, dextrans, starches, and the like; and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitol sorbitol (glucitol) and myoinositol.
  • monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbose, and the like
  • disaccharides such as lactose, sucrose
  • Carriers which may be used include a buffer or a pH adjusting agent; typically, the buffer is a salt prepared from an organic acid or base.
  • Representative buffers include organic acid salts such as salts of citric acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid, or phthalic acid; Tris, tromethamine hydrochloride, or phosphate buffers.
  • Additional carriers include polymeric excipients/additives such as polyvinylpyrrolidones, ficolls (a polymeric sugar), dextrates (e.g.
  • cyclodextrins such as 2-hydroxypropyl- ⁇ - cyclodextrin
  • polyethylene glycols such as 2-hydroxypropyl- ⁇ - cyclodextrin
  • flavoring agents such as 2-hydroxypropyl- ⁇ - cyclodextrin
  • antimicrobial agents such as “STYPES”
  • sweeteners such as “STYPES”
  • antioxidants such as “TWEEN 20” and “TWEEN 80”
  • surfactants e.g. polysorbates such as “TWEEN 20” and “TWEEN 80”
  • lipids e.g. phospholipids, fatty acids
  • steroids e.g. cholesterol
  • chelating agents e.g. EDTA
  • ⁇ 1 -selective AMPK activators and pharmaceutical compositions can be used in an animal or human.
  • a ⁇ 1-selective AMPK activator can be formulated as an active ingredient in a pharmaceutical composition for oral, buccal, parenteral (e.g. intravenous, intramuscular or subcutaneous), topical, rectal or intranasal administration or in a form suitable for administration by inhalation or insufflation.
  • the ⁇ 1-AMPK activator or pharmaceutical composition is formulated for systemic administration, e.g. via a non-parenteral route.
  • the ⁇ 1-AMPK activator or pharmaceutical composition is formulated for oral administration, e.g. in solid form.
  • Such modes of administration and the methods for preparing appropriate pharmaceutical compositions are described, for example, in Gibaldi’s Drug Delivery Systems in Pharmaceutical Care (1st ed., American Society of Health-System Pharmacists 2007).
  • the pharmaceutical compositions can be formulated so as to provide slow, extended, or controlled release of the active ingredient therein using, for example, hydroxypropyl methyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and/or microspheres.
  • the pharmaceutical compositions can also optionally contain opacifying agents and may be of a composition that releases the active ingredient(s) only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner, e.g. by using an enteric coating.
  • embedding compositions include polymeric substances and waxes.
  • the active ingredient can also be in micro-encapsulated form, if appropriate, with one or more pharmaceutically acceptable carriers, excipients, or diluents well known in the art (see, e.g., Remington’s).
  • the ⁇ 1 -selective AMPK activator may be formulated for sustained delivery according to methods well known to those of ordinary skill in the art. Examples of such formulations can be found in United States Patents 3,119,742; 3,492,397; 3,538,214; 4,060,598; and 4,173,626.
  • the active ingredient is mixed with one or more pharmaceutically acceptable carriers, excipients, or diluents, such as sodium citrate or dicalcium phosphate, and/or any of the following: (1) fdlers or extenders, such as starches, lactose, sucrose, glucose, mannitol, microcrystalline cellulose, calcium phosphate and/or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, pregelatinized maize starch, polyvinyl pyrrolidone, hydroxypropyl methylcellulose, sucrose and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, sodium starch glycolate, potato or tapioca starch, alginic acid, certain silicates, and
  • the pharmaceutical compositions can also comprise buffering agents.
  • Solid compositions of a similar type can also be prepared using fillers in soft and hard- filled gelatin capsules, and excipients such as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.
  • a tablet can be made by compression or molding, optionally with one or more accessory ingredients.
  • Compressed tablets can be prepared using binders (for example, gelatin or hydroxypropyl methyl cellulose), lubricants, inert diluents, preservatives, disintegrants (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface- actives, and/ or dispersing agents.
  • Molded tablets can be made by molding in a suitable machine a mixture of the powdered active ingredient moistened with an inert liquid diluent.
  • the tablets and other solid dosage forms, such as dragees, capsules, pills, and granules can optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the art.
  • the pharmaceutical compositions are administered orally in a liquid form.
  • Liquid dosage forms for oral administration of an active ingredient include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs.
  • Liquid preparations for oral administration may be presented as a dry product for constitution with water or other suitable vehicle before use.
  • the liquid dosage forms can contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (e.g. cottonseed, groundnut, com, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
  • inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol
  • the liquid pharmaceutical compositions can include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents, and the like.
  • Suspensions in addition to the active ingredient(s) can contain suspending agents such as, but not limited to, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.
  • Suitable liquid preparations may be prepared by conventional means with a pharmaceutically acceptable additive(s) such as a suspending agent (e.g.
  • sorbitol syrup methyl cellulose or hydrogenated edible fats
  • emulsifying agent e.g. lecithin or acacia
  • non-aqueous vehicle e.g. almond oil, oily esters or ethyl alcohol
  • preservative e.g. methyl or propyl p-hydroxybenzoates or sorbic acid
  • the active ingredient(s) can also be administered as a bolus, electuary, or paste.
  • the composition may take the form of tablets or lozenges formulated in a conventional manner.
  • the pharmaceutical compositions are administered by non-oral means such as by topical application, transdermal application, injection, and the like.
  • the pharmaceutical compositions are administered parenterally by injection, infusion, or implantation (e.g. intravenous, intramuscular, intra-arterial, subcutaneous, and the like).
  • the ⁇ 1 -selective AMPK activator may be formulated for parenteral administration by injection, including using conventional catheterization techniques or infusion.
  • Formulations for injection may be presented in unit dosage form, e.g. in ampules or in multi-dose containers, with an added preservative.
  • the compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain a formulating agent such as a suspending, stabilizing and/or dispersing agent recognized by those of skill in the art.
  • the active ingredient may be in powder form for reconstitution with a suitable vehicle, e.g. sterile pyrogen-free water, before use.
  • compositions may be administered directly to the central nervous system. Accordingly, in certain embodiments the compositions are administered directly to the central nervous system so as to avoid the blood brain barrier. In some embodiments, the composition can be administered via direct spinal cord injection. In some embodiments, the composition is administered by intrathecal injection. In some embodiments, the composition is administered via intracerebroventricular injection. In some embodiments, the composition is administered into a cerebral lateral ventricle. In some embodiments, the composition is administered into both cerebral lateral ventricles. In additional embodiments, the composition is administered via intrahippocampal injection. The compositions may be administered in one injection or in multiple injections. In other embodiments, the composition is administered to more than one location (e.g. to two sites in the central nervous system).
  • the pharmaceutical compositions can be in the form of sterile injections.
  • the pharmaceutical compositions can be sterilized by, for example, filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved in sterile water, or some other sterile injectable medium immediately before use.
  • the active ingredient is dissolved or suspended in a parenterally acceptable liquid vehicle.
  • exemplary vehicles and solvents include, but are not limited to, water, water adjusted to a suitable pH by addition of an appropriate amount of hydrochloric acid, sodium hydroxide or a suitable buffer, 1,3 -butanediol, Ringer’s solution and isotonic sodium chloride solution.
  • the pharmaceutical composition can also contain one or more preservatives, for example, methyl, ethyl or n-propyl p-hydroxybenzoate.
  • a dissolution enhancing or solubilizing agent can be added or the solvent can contain 10-60% w/w of propylene glycol or the like.
  • the pharmaceutical compositions can contain one or more pharmaceutically acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders, which can be reconstituted into sterile injectable solutions or dispersions just prior to use.
  • Such pharmaceutical compositions can contain antioxidants; buffers; bacteriostats; solutes, which render the formulation isotonic with the blood of the intended recipient; suspending agents; thickening agents; preservatives; and the like.
  • aqueous and nonaqueous carriers examples include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate.
  • polyols such as glycerol, propylene glycol, polyethylene glycol, and the like
  • vegetable oils such as olive oil
  • injectable organic esters such as ethyl oleate.
  • Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
  • Controlled release parenteral compositions can be in form of aqueous suspensions, microspheres, microcapsules, magnetic microspheres, oil solutions, oil suspensions, emulsions, or the active ingredient can be incorporated in biocompatible carrier(s), liposomes, nanoparticles, implants or infusion devices.
  • Materials for use in the preparation of microspheres and/or microcapsules include, but are not limited to, biodegradable/bioerodible polymers such as polyglactin, poly-(isobutyl cyanoacrylate), poly(2-hydroxyethyl-L- glutamine) and poly(lactic acid).
  • Biocompatible carriers which can be used when formulating a controlled release parenteral formulation include carbohydrates such as dextrans, proteins such as albumin, lipoproteins or antibodies.
  • Materials for use in implants can be non- biodegradable, e.g. polydimethylsiloxane, or biodegradable such as, e.g., poly(caprolactone), poly(lactic acid), poly(glycolic acid) or poly(ortho esters).
  • the compound of formula (I) may be formulated as an ointment, cream, or liquid eye drops.
  • a compound of formula (I) may also be formulated in rectal compositions such as suppositories or retention enemas, e.g. containing conventional suppository bases such as cocoa butter or other glycerides.
  • a compound of formula (I) may be conveniently delivered in the form of a solution or suspension from a pump spray container that is squeezed or pumped by the patient or as an aerosol spray presentation from a pressurized container or a nebulizer, with the use of a suitable propellant, e.g. dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • a suitable propellant e.g. dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • the dosage unit may be determined by providing a valve to deliver a metered amount.
  • the pressurized container or nebulizer may contain a solution or suspension of the compound of formula (I).
  • Capsules and cartridges for use in an inhaler or insufflator may be formulated containing a powder mix of a ⁇ 1 -selective AMPK activator and a suitable powder base such as lactose or starch.
  • EXAMPLES Example 1 - Induction of fetal hemoglobin HbF by oxindoles in human CD34+ cells from healthy donors during differentiation in vitro measured by flow cytometry and western blot.
  • HSPC Human Stem/Progenitor Cells
  • Step 1 media consisting of Iscove’s modified Dulbecco’s medium (IMDM) (ThermoFisher) supplemented with IX GlutaMAX, 100 U/mL penicillin-streptomycin (ThermoFisher), 5% human AB+plasma, 330 ug/mL human holo- transferrin, 10 ug/mL human insulin, 2 U/mL heparin, 1 uM/mL hydrocortisone (Sigma- Aldrich), 3 U/mL recombinant human erythropoietin (EPO) (ThermoFisher), 100 ng/mL SCF (ThermoFisher) and 5 ng/mL interleukin 3 (IL-3)
  • IMDM Iscove’s modified Dulbecco’s medium
  • IX GlutaMAX 100 U/mL penicillin-streptomycin
  • human AB+plasma 330 ug/mL
  • step 7 cells were transferred to step 2 media, a step 1 media without hydrocortisone and IL-3, and cultured for 3-4 days. Then cells were cultured for 8-9 days in step 3 media, a step 2 media without SCF.
  • mobilized CD34+ HSPC were exposed to ⁇ 1-selective AMPK activator oxindoles.
  • F- cells differentiated cells were fixed and permeabilized using a fixation kit (ThermoFisher). Cells were stained with PE-Cy7-conjugated anti-CD235 or PE-conjugated anti-CD71 antibodies.
  • HbF levels were detected using Allophycocyanin (APC)-conjugated anti-HbF antibody (ThermoFisher). The acquisition of stained cells was performed on BD FACSCantoTM and the analysis was run using FlowJoTM Software.
  • FIG.1A data shows an increase in the frequency of F-cells when CD34+ cells were exposed to oxindoles compared to vehicle (DMSO), after 21 days of differentiation.
  • FIG. IB high performance liquid chromatography (HPLC) analysis of globin proteins isolated from CD34+ cells treated with Compound 2 confirms an increase in fetal hemoglobin protein level.
  • FIG 1C confirms the induction of fetal hemoglobin in CD34+ cells treated with oxindoles assessed by Western blot.
  • RNA-Seq analysis was conducted at day 15 after treatment with Compound 2. Volcano plot reveals an increase in mRNA level of HBG1 and HBG2, confirming the activation of the gamma globin genes by Compound 2 (FIG. IE).
  • FIG. IF shows the RNA-seq and Proteomics integrative analysis. HBE1 and HBG2 values are increased, confirming the activation of HBG2 gene and the subsequent increase in fetal hemoglobin protein expression.
  • a western blot was realized.
  • Beta-1 and Beta-2 subunits were measured and signals were normalized to ⁇ -actin (Cell Signaling) (FIG.1H). Beta-1 is the only subunit expressed in CD34+ cells..
  • a Vi- CELL BLU Cell Viability Analyzer (Beckman Coulter) was used, confirming the absence of cytotoxicity effect by oxindoles (FIG.1I).
  • Example 2 Induction of fetal hemoglobin HbF by oxindoles in human CD34+ cells from sickle donors during differentiation in vitro measured by flow cytometry and western blot. Circulating CD34+ progenitor cells were isolated from total blood obtained from sickle cell patients, by performing a positive selection for CD34+ cells with magnetic beads (Miltenyi Biotec). Then cells were differentiated into erythroid cells using a three-step differentiation protocol developed by the Luc Douay group (Giarratana et al. 2005).
  • CD34+ cells were cultured for 7 days in Step 1 media, consisting of Iscove’s modified Dulbecco’s medium (IMDM) (ThermoFisher) supplemented with IX GlutaMAX, 100 U/mL penicillin- streptomycin (ThermoFisher), 5% human AB+plasma, 330 ug/mL human holo-transferrin, 10 ug/mL human insulin, 2 U/mL heparin, 1 uM/mL hydrocortisone (Sigma-Aldrich), 3 U/mL recombinant human erythropoietin (EPO) (ThermoFisher), 100 ng/mL SCF (ThermoFisher) and 5 ng/mL interleukin 3 (IL-3) (Sigma-Aldrich).
  • IMDM Iscove’s modified Dulbecco’s medium
  • IX GlutaMAX 100 U/mL penicillin
  • step 7 cells were transferred to step 2 media, a step 1 media without hydrocortisone and IL-3, and cultured for 3-4 days. Then cells were cultured for 8-9 days in step 3 media, a step 2 media without SCF.
  • mobilized CD34+ HSPC were exposed to ⁇ 1 -selective AMPK activator oxindoles.
  • F-cells differentiated cells were fixed and permeabilized using a fixation kit (ThermoFisher). Cells were stained with PE-Cy7-conjugated anti-CD235 or PE-conjugated anti-CD71 antibodies.
  • HbF levels were detected using Allophycocyanin (APC)-conjugated anti-HbF antibody (ThermoFisher).
  • APC Allophycocyanin
  • ThermoFisher ThermoFisher
  • the acquisition of stained cells was performed on BD FACSCantoTM and the analysis was run using FlowJoTM Software.
  • FIG.2A data shows an increase in the frequency of F-cells when CD34+ cells were exposed to oxindoles compared to vehicle (DMSO), after 21 days of differentiation.
  • DMSO vehicle
  • FIG.2B Western blot were conducted (FIG.2B).
  • Total cell lysates from CD34+ cells were generated and the total protein concentration was determined using a Bradford protein assay (ThermoFisher Scientific).
  • a cell sickling assay was conducted upon completion of erythroid differentiation in culture.
  • Fully differentiated CD34+ cells from sickle donors were incubated under hypoxia (2% O2) for 4 hours and then abnormal shaped cells were analyzed using the ImageStream®X Mk II Imaging Flow Cytometer (FIG.3A).
  • FOG.3A Imaging Flow Cytometer
  • cells were determined using RMS of brightfield image.
  • Single cells were determined using Area vs Aspect Ratio of brightfield image.
  • Live cells were determined using Live Dead NIR stain.
  • Single, Live, in focus cells were analyzed using the following masking of the brightfield image: Skeleton (Ml, Chi, Thin) and Object (Ml, Chi, Tight).
  • Sickling factor was calculated using the following feature: Length Skeleton (Ml, CHI, Thin) / Width (Ml, Chi, Tight). Cells with the sickle factor feature of 1.4 or higher were sickled. Cells with the sickle factor feature less than 1.4 were not sickled.
  • FIG3 A data shows that oxindoles-activated AMPK prevents sickling in terminally differentiated CD34+ cells from sickle donors. This result was confirmed by microscopy, using a Nikon Eclipse Ti microscope (FIG.3B).
  • Example 4 - Oxindoles and Hydroxyurea combination shows additive effect on fetal hemoglobin induction in human CD34+ cells during differentiation in vitro.
  • Example 5 - AMPK activation by oxindoles in human CD34+ does not affect CD34+ cells maturation to erythrocytes in vitro.
  • Flowcytometry data confirms a normal level of CD235a expression in cells for most of oxindoles compared to control (DMSO expressed as vehicle).
  • CD34+ cells were stained at day 14 of differentiation using the Wright- Giemsa method. Cells were spun onto glass slides with a Cytospin4 (ThermoFisher Scientific) at 300rpm for 3 min. Slides were allowed to dry for 5 minutes before staining with May Grunwald (Scy-Tek) for 6 minutes followed by 1 :20 diluted Giemsa stain for 13 minutes.
  • Example 6 - AMPK target engagement in human CD34+ and human HUDEP-2 cells after exposure to oxindoles in vitro.
  • CD34+ cells from a healthy donor or HUDEP-2 cells were exposed to oxindoles at the indicated doses (pM), harvested and lysed at the indicated time points.
  • Phosphorylation of AMPK at threonine 172 (Thrl72) on the a-subunit of AMPK was assessed by HTRF using the Alpha SureFire Ultra Multiplex p-AMPK ⁇ 1/2 (Thrl72) + Total AMPKal/2 Assay Kit from Perkin Elmer as a target engagement assay.
  • the assay kit contains antibodies, coupled with fluorophore Europium, which recognize the phospho-Thrl72 epitope and a distal epitope on ⁇ -AMPK of human or mouse AMPK.
  • the kit also contains antibodies coupled with the fluorophore Terbium to measure the total levels of AMPK.
  • cells were collected on Day 11 of differentiation then exposed to oxindoles.
  • human HUDEP-2 cells line cells were undifferentiated during exposure to oxindoles. According to the time course, cells were collected and lysed using RIPA buffer mixed with a phosphatase and protease inhibitors cocktail (Thermofisher).
  • Phospho-AMPK signal was divided by total AMPK signal and the ratio was normalized to the total protein concentration.
  • Samples protein concentrations were measured with the Pierce BSA Protein Assay (ThermoFisher). Data show a peak of signal at 30 minutes after exposure to AMPK activators in CD34+ cells (FIG. 6 A), and at Ih after exposure in HUDEP-2 cells (FIG. 6B), confirming target engagement in AMPK when cells are treated with AMPK activators oxindoles.
  • AMPK downstream pathway activation was verified by measuring phosphorylation of FOXO3 and ULK-1, which are direct targets of activated AMPK (FIG.6C).
  • Total cell lysates from human erythroid CD34+ cells were generated and the total protein concentration was determined using a Bradford protein assay (ThermoFisher Scientific).
  • Reduced and denaturated protein (40pg) was loaded and separated by SDS-PAGE (12% gel), blotted on nitrocellulose membranes (BioRad) and finally incubated with FOXO3, Phospho- FOXO3 (Ser413), ULK-1, Phospho-ULK-1 (Ser317) and a-tubulin antibodies (Cell Signaling).
  • the a-tubulin antibody served as an internal control. Immunoreactive proteins were visualized by using an ECL® (enhanced chemiluminescence) detection system (BioRad). Blot confirm the phosphorylation of FOXO3 at Serine 413 and ULK-1 at Serine 317 in CD34+ cells exposed to oxindoles, confirming the upstream activation of AMPK by oxindoles.
  • ECL® enhanced chemiluminescence
  • Example 7 - AMPK activation by oxindole promotes human macrophage polarization to an anti-inflammatory functional phenotype in vitro.
  • monocytes and macrophages were isolated from total blood collected from a healthy donor by performing a magnetic positive selection for CD 14+ cells with magnetic beads (Miltenyi Biotec).
  • Ml macrophages were induced by stimulation with M- CSF (50ng/mL) (Sigma- Aldrich) for 6 days, then activated by IFN- ⁇ (Sigma-Aldrich) and LPS (Sigma-Aldrich) for 24h, fixed, stained for Ml polarization pro-inflammatory marker CD86 (BD Biosciences) and for M2 polarization anti-inflammatory marker CD 163 (BD Biosciences).

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Abstract

This disclosure to methods for treating or preventing particular symptoms and disorders which are associated with blood disorders using oxindole derivatives as AMPK activators. Also disclosed are pharmaceutical composition comprising an oxindole derivative as a AMPK activator for use in said methods.

Description

OXINDOLE DERIVATIVES AS AMPK ACTIVATORS FOR USE IN THE TREATMENT OF RARE BLOOD DISORDERS
Disclosed herein are compounds that activate 5 '-adenosine monophosphate-activated protein kinase (AMPK), pharmaceutical compositions containing these compounds, and methods for treating or preventing blood disorders such as hemoglobinopathies.
BACKGROUND
The 5'AMP-activated protein kinases (AMPK) are sensors of the overall energy level in mammalian cells and organs. AMPK is activated by an increase in the intracellular AMP/ATP ratio, induced for example by a metabolic stress, hormones, or nutrient signaling pathways (Viollet et al., Crit Rev Biochem Mol Biol, 2010. 45(4): p. 276-95). When activated, AMPK blocks the metabolic pathways which consume ATP (such as fatty acid synthesis in adipocytes, cholesterol synthesis in the liver, and insulin secretion in β-cells) and activates the metabolic pathways which produce ATP (such as fatty acid absorption and beta-oxidation in various tissues, glycolysis in the heart, and the biogenesis of mitochondria in skeletal muscle). AMPK also modulates the transcription of genes which participate in energy metabolism, exerting a metabolic control to facilitate energy (Viollet et al.). Moreover, AMPK participates in the regulation of non-metabolic processes such as cell growth, progression of the cell cycle, and organization of the cytoskeleton (Wil liams T., et al., Proc Natl Acad Sci U S A, 2011. 108(14): p. 5849-54.). Although the activation of AMPK is an adaptive response to an energy stress in many biological systems, AMPK plays an important role in maintaining physiological functions and for adaptation to pathophysiological conditions. Furthermore, it has been reported that AMPK promotes anti-inflammatory effects in vitro in murine and human macrophages by promoting macrophage polarization to an anti-inflammatory functional phenotype (Sag, D., et al., J Immunol, 2008. 181(12): p. 8633-41.) AMPK is an apy trimer of three subunits comprised of twelve known isoforms that are based on all possible combinations of 2 α, 2 β and 3 γs subunits. Activators of AMPK, including 5'AMP and pharmacological small molecule AMPK activators, bind the CBS sites in the y subunits and the ADAM site, or “allosteric drug and metabolite” binding site, that lies between the a and p subunits (Aledavood et al., J Chem Inf Model, 2019. 59(6): p. 2859-2870.). AMPK can be activated both by activator binding to the ADAM: site and by phosphorylation of Thrl72 of the a-subunit. Pharmacological activators of AMPK binding to the ADAM site are known to activate only AMPK isoforms containing the pl-subunit (pl-selective AMPK activators) or containing either a β1-subunit or a p2-subunit (pan-selective AMPK activators).
Sickle cell disease (SCD), also called sickle cell anemia, is one of the most common monogenic diseases, with 330,000 affected individuals born annually worldwide (Piel, F.B., et al., Lancet, 2013. 381(9861): p. 142-51.). It is the result of substitution of glutamic acid by valine at position 6 of the β-globin subunit of hemoglobin. The presence of this mutant β-globin subunit leads to the production of abnormal hemoglobin S (HbS) that polymerizes in red blood cells under conditions of low oxygen, which affects blood flow rheology and ultimately leads to vaso-occlusive crises and end organ damage (Barabino et al. Annu Rev Biomed Eng, 2010. 12: p. 345-67.). Oxidative stress in erythrocytes is also enhanced and causes haemolysis (Vona, R., et al., Antioxidants (Basel), 2021. 10(2), and inflammation involving monocytes and proinflammatory macrophages is exacerbated in tissues and blood and participates to the organ damage process (Van Beers, E.J., et al., Circ Res, 2015. 116(2): p. 298-306; and Allali, S., et al., Haematologica, 2020. 105(2): p. 273-283).
It has been reported that increased fetal hemoglobin (HbF), whether endogenous or drug induced, ameliorates the symptoms and complications of SCD by preventing red blood cell sickling through dilution of the concentration of HbS in erythrocytes and inhibiting the polymerization of HbS (Piel et al. Lancet, 2013. 381(9861): p. 142-51.). Although hydroxyurea (HU) is the only US Food and Drug Administration approved HbF-inducing agent for use in adults with SCD (Steinberg, M.H., et al., JAMA, 2003. 289(13): p. 1645-51.), up to 50% of patients do not experience clinical improvement on HU. This variability of HbF- inducing response to HU is still under investigation (Platt, et al., N Engl J Med, 2008. 358(13): p. 1362-9.). Increasing HbF in erythrocytes is also beneficial to treat clinical manifestations of other P-hemoglobinopathies, including β-Thalassemia (Ye, L., et al., Proc Natl Acad Sci U S A, 2016. 113(38): p. 10661-5.). Finally, decreasing inflammation and oxidative stress in SCD leads to beneficial clinical effects in patients (Kato, G.J., et al., Nat Rev Dis Primers, 2018. 4: p. 18010.). F0X03 is the transcription factor forkhead box 0-3 hypothesized to be responsible for increasing HbF when overexpressed or activated in CD34+ erythroid cells (Zhang, Y., et al., Blood, 2018. 132(3): p. 321-333.). F0X03 is in the forkhead/winged helix box gene, group 0 (FoxO) family of proteins that are evolutionarily conserved transcription factors. FOXO transcription factors integrate many important cellular functions and were originally identified as regulators of insulin-related genes. FOXO transcription factors regulate genes involved in biological processes, including substrate and energy metabolism, protein turnover, cell survival, oxidative stress, proteostasis, apoptosis and cell death, cell cycle regulation, metabolic processes, immunity, inflammation and stem cell maintenance (Morris, B.J., et al., Gerontology, 2015. 61(6): p. 515-25; and Stefanetti, R.J., et al., F1000Res, 2018. 7.). In particular, FOXO3 is required for the maintenance of murine hematopoietic stem cells and functions (Yalcin, S., et al., J Biol Chem, 2008. 283(37): p. 25692-25705; and Rimmele, P., et al., EMBO Rep, 2015. 16(9): p. 1164-76.).
In addition, FOXO3 is a key mediator of erythroid terminal maturation, regulating cell cycle in early stages of erythropoiesis and critical for ROS regulation, enucleation and mitochondrial clearance in late stages (Marinkovic, D., et al., J Clin Invest, 2007. 117(8): p. 2133-44; and Liang, R., et al., PLoS Genet, 2015. 11(10): p. el005526.). In this context, modulation of FOXO3 might influence erythroid disorders as has been reported specifically for hemoglobinopathies (Zhang, Y., et al., Blood, 2018. 132(3): p. 321-333; Pourfarzad, F., et al., Cell Rep, 2013. 4(3): p. 589-600; and Franco, S.S., et al., 2014. 99(2): p. 267-75.). Sirtuin 1 (Sirtl) is a homolog of the NAD-dependent protein silent information regulator 2 in yeast and is a deacetylase for histones and other proteins. Deacetylation of FOXO3 by Sirtl is an important regulatory control mechanism of FOXO3 in many cells and tissues (Giannakou, M.E., et al., Science, 2004. 305(5682): p. 361.). Increased acetylation of FOXO3 by elimination of the deacetylase Sirtl in hematopoietic stem cells (HSC) results in a defective lineage specification biased toward the myeloid lineage and a phenotype that resembles aged HSC (Rimmele, P., et al., Stem Cell Reports. 2014 Jul 8; 3(1): 44-59.).
An in vitro study in differentiating human CD34+ erythroid cells showed that decreasing FOXO3 by shRNA delivery decreased expression of HbF, and conversely, over-expression of FOXO3 increased HbF expression in these cells. It was theorized that AMPK phosphorylates and activates F0X03, and metformin was used to increase cellular AMP and indirectly activate AMPK to increase HbF expression (Zhang, Y., et al., Blood, 2018. 132(3): p. 321- 333.).
New HbF inducing drugs are therefore urgently needed. Surprisingly we found and demonstrated herein below that oxindole β1 -selective AMPK activators increase expression of HbF in differentiating human lineage erythroid cells without affecting terminal differentiation. Moreover, isoform β1 is predominant in erythroid lineage in human bone marrow compared to 02, making oxindole β1 -AMPK activators more specific to the erythroid lineage compared to pan-AMPK activators that potentially activate other cells lineage or tissues expressive 02. In support of the benefit of the oxindole β1 -selective AMPK activation to induce HbF in erythroid lineage, we show that oxindole β1 -selective AMPK activators do not affect terminal differentiation as measured by enucleation and differentiation markers expression. Finally, oxindole “Compound 2” is shown to cause anti-inflammatory responses by promoting macrophages polarization and differentiation to an anti-inflammatory M2 phenotype, potentially resulting in a decrease of the inflammatory response which is a key component of the sickle cell disease pathophysiology.
SUMMARY OF THE INVENTION
Thus, in one aspect, the present application relates to a method of treating a rare blood disorder, the method comprising administering to a patient in need thereof an effective amount of a compound of formula (I):
Figure imgf000005_0001
wherein:
R1 represents: ・ an (C6-C10)aryl group, unsubstituted or substituted with one or more substituents chosen from:
■ a halogen atom,
■ a — ORa group, in which Ra represents a hydrogen atom, a (C1-C3)alkyl group or a -CF3 group,
■ a (C1-C3)alkyl group, unsubstituted or substituted with one or more halogen atoms,
■ a carboxyl group,
■ a cyano group, and
■ a (C3-C6)heterocycloalkyl group, unsubstituted or substituted with one or more (C1-C3)alkyl group; or
• a (C2-C10)heteroaryl group, unsubstituted or substituted with one or more substituents chosen from:
■ a halogen atom,
■ a (C1-C4)alkyl group,
■ a (C3-C6)cycloalkyl group,
■ a — ORe group, in which Re represents an hydrogen atom or a (C1- C4)alkyl group, said (C1-C4)alkyl group being unsubstituted or substituted with one or more (C1-C4)alkoxy or heterocycloalkyl group, and
■ a -NRfRr group, in which Rf and Rf', independently, identical or different, represent a (C1-C3)alkyl group;
R2 represents:
• an (C6-C10)aryl group, unsubstituted or substituted with one or more substituents chosen from:
■ a halogen atom, a cyano group, a
Figure imgf000006_0001
group, ■ a
Figure imgf000007_0001
group,
■ a (C1-C3)alkyl group unsubstituted or substituted with one or more substituents chosen from a halogen atom, a hydroxyl group, and a (C1- C4)alkenyl group,
■ a (C3-C6)cycloalkyl group, unsubstituted or substituted with a hydroxy(C1-C3)alkyl group, a hydroxyl group, or an (C1-C4)alkoxy group,
■ a -ORb group, in which Rb represents:
• a hydrogen atom,
• a -CF3 group,
• a (C3-C6)cycloalkyl group, unsubstituted or substituted with a hydroxyl group,
• a (C3-C6)heterocycloalkyl group, said (C3-C6)heterocycloalkyl being unsubstituted or substituted with a (C1-C3)alkyl group, or
• or an (C1-C3)alkyl group, said (C1-C3)alkyl group being unsubstituted or substituted with one or more groups selected from: o hydroxyl group, o (C1-C4)alkoxy group, o (C2-C10)heteroaryl group, o acetamido group, o di(C1-C3)alkyl-amino group, o (C3-C6)cycloalkyl group, said (C3-C6)cycloalkyl group being unsubstituted or substituted with one or more hydroxyl group, and o (C3-C6)heterocycloalkyl group, said (C3- C6)heterocycloalkyl group being unsubstituted or substituted with a (C1-C3)alkyl group; ■ a (C3-C6)heterocycloalkyl group unsubstituted or substituted with one or more halogen atom, (C1-C3)alkyl group, hydroxyl group, hydroxy(C1-C3)alkyl group, (C1-C4)alkoxy group or (C1-C4)fluoroalkyl group,
■ an (C6-C10)aryl group, unsubstituted or substituted with one or more - ORc group, in which Rc represents a hydrogen atom or a (C1-C3)alkyl group,
■ a (C2-C10)heteroaryl group, unsubstituted or substituted with one or more -NH2 group,
■ a -NRdRd’ group, in which Rd and Rd', independently, identical or different, represent a hydrogen atom, a (C1-C3)alkyl group, a hydroxy(C1-C4)alkyl or a (C3-C6)cycloalkyl group,
• a (C2-C10)heteroaryl group, unsubstituted or substituted with one or more substituents chosen from:
• a (C1-C3)alkyl group
• a (C3-C6)cycloalkyl group, unsubstituted or substituted with a hydroxy(C1-C3)alkyl group,
• a -NRgRg’ group, in which Rg and Rg’, independently, identical or different, represent a (C1-C3)alkyl group;
R3 represents a halogen atom or a (C1-C3)alkyl group;
R4 represents a halogen atom or a hydrogen atom, or a pharmaceutically acceptable salt thereof
General methods for the preparation of the compounds of formula (I), are described in WO2015/091937 and US Patent No. 10,077,237, which is incorporated herein in its entirety.
In some embodiments, the rare blood disorder is a hemoglobinopathy. In some embodiments, the hemoglobinopathy is selected from β-thalassemia and sickle cell disease (SCD).
In some embodiments, the compound of formula (I) is a selective β1-AMPK activator that is a more selective activator of β1-AMPK versus activation of β2-AMPK or of activation of both β1-AMPK and β2-AMPK (pan-activation). In some embodiments, the selective β1-AMPK activator may possess at least about a 10-fold selective activation, at least about a 50-fold selective activation, at least about a 100-fold activation, at least about a 300-fold selective activation, or at least about a 500 fold selective activation for β1-AMPK relative to β2-AMPK.
In some embodiments, the selective β1-AMPK activator has an EC50 (half-maximal concentration required for full activation) for the activation of β1-AMPK of about 100 nM or less. In other embodiments, the selective β1-AMPK activator has an EC50 of about 50 nM or less for the activation of β1-AMPK. On other embodiments, the selective β1-AMPK activator has an EC50 of about 10 nM or less for the activation of β1-AMPK. EC50 values may be determined according to the enzymatic assays performed in Schmoll et al.
In some embodiments, the selective β1-AMPK activator increases the activity of AMPK above the baseline activity (as measured by autophosphorylation of the α-subunit of AMPK or phosphorylation of a peptide or protein substrate) by 50% or more. In other embodiments, the selective β1-AMPK activator increases the activity of AMPK above the baseline activity by 100% or more. In other embodiments, the selective β1-AMPK activator increases the activity of AMPK above the baseline activity by 150% or more.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1: FIG. 1A shows the result of fetal hemoglobin induction in human CD34+ cells when mobilized CD34+ HSPC from healthy individuals are cultured for 21 days under conditions to promote erythroid differentiation and are exposed to oxindoles at the indicated doses (pM). Exposure to oxindoles increases F-cell (HbF+) frequency in enucleated GlyA+ cells compared to control (DMSO indicated as Vehicle), measured by flowcytometry. Representative plots from a single donor are shown. FIG. 1B: oxindole “Compound 2,” induces fetal hemoglobin in a dose-dependent manner, compared to control. Representative plots from a single donor are shown. FIG. 1C: confirms the induction of fetal hemoglobin by oxindoles measured by western blot. FIG. 1D: are representative plots from a single donor showing fetal hemoglobin induction by Compound 2 in a dose-dependent manner, compared to control. FIG. 1E: shows RNA-seq analysis conducted on CD34+ cells treated with Compound 2 confirms the activation of gamma-globin genes HBG1 and HBG2. FIG. 1F shows a proteomic analysis conducted on CD34+ cells from healthy donors and treated with Compound 2 that confirms the increase in HBG2 induced fetal hemoglobin protein expression. FIG. 1G: RNA-seq and Proteomic integrative analysis shows a correlation in HBE1 and HBG2 gene activation and protein expression. FIG 1H shows the solely expression of beta-1 subunit and the absence of beta-2 subunit expression in CD34+ HSPC before and during the erythroid differentiation. FIG. 1I: confirms that oxindoles do not affect CD34+ HSPC viability during the erythroid differentiation. All data are expressed as mean ± SEM (NS = not significant, * p<0.05, ** p<0.01, n=3).
FIG. 2: FIG.2A shows the result of fetal hemoglobin induction in human CD34+ cells when CD34+ HSPC from sickle patients are cultured for 21 days under conditions to promote erythroid differentiation and are exposed to oxindoles at the indicated doses (μM). Exposure to oxindoles increases F-cell (HbF+) frequency in enucleated GlyA+ cells compared to control (DMSO indicated as Vehicle), measured by flowcytometry. Representative plots from a single donor are shown. FIG. 2B confirms the induction of fetal hemoglobin by oxindoles measured by western blot. All data are expressed as mean ± SEM (NS = not significant, * p<0.05, ** p<0.01, n=3).
FIG. 3: FIG. 3A reveals that exposure of CD34+ HSPC from sickle patients to oxindoles prevents sickling of terminally differentiated CD34+ cells, measured by imaging flowcytometry Imagestream. FIG. 3B confirms the reduction of sickle cells number after CD34+ HSCP cells exposure to oxindoles during the erythroid differentiation, assessed by microscopy. Representative pictures from a single donor are shown. All data are expressed as mean ± SEM (* p<0.05, n=3).
FIG. 4: FIG. 4A shows the result of fetal hemoglobin induction in human CD34+ cells when mobilized CD34+ HSPC from healthy individuals are cultured for 14 days under conditions promoting erythroid differentiation and are exposed to hydroxyurea in combination or not with oxindoles. Exposure to hydroxyurea increases F-cell (HbF+) frequency in enucleated GlyA+ cells as expected, compared to control cells (DMSO indicated as Vehicle). The combination of hydroxyurea and oxindoles leads to a higher frequency of F-cell compared to hydroxyurea alone. Representative plots from one healthy donor are shown. FIG. 4B shows the result of fetal hemoglobin induction in human CD34+ cells when CD34+ HSPC from sickle patients are cultured for 14 days under conditions promoting erythroid differentiation and are exposed to hydroxyurea in combination or not with oxindoles. Exposure to hydroxyurea increases F- cell (HbF+) frequency in enucleated GlyA+ cells as expected, compared to control cells (DMSO indicated as Vehicle). The combination of hydroxyurea and oxindoles leads to a higher frequency of F-cell compared to hydroxyurea alone. Representative plots from one healthy donor are shown. All data are expressed as mean ± SEM (* p < 0.05, n = 3).
FIG. 5: FIG. 5A shows the result of oxindoles exposure on human CD34+ cells maturation when mobilized CD34+ HSPC from healthy individuals are differentiated for 21 days into erythroid cells. Data confirms that oxindoles have no impact on enucleation of erythroid cells during differentiation compared to control cells (DMSO indicated as Vehicle), based on the frequency of enucleated cells as measured by flow cytometry. Representative plots from one healthy donor are shown. FIG. 5B displays that oxindoles have no effect on the expression level of differentiation markers - Band-3, LRF, ALAS2 and GATA-1 - during maturation compared to control cells (DMSO indicated as Vehicle), measured by western blot. FIG. 5C manifests that erythroid markers CD235a expression is not affected at terminal differentiated stage in CD34+ HSPC cells exposed to oxindoles. Representative plots from one healthy donor are shown. FIG. 5D confirms that exposure CD34+ HSPC cells to oxindoles do not affect cells erythroid differentiation at day 14. Cellular phenotypic characteristics, phenotypic assessed by May-Grunwald staining, are similar between control and oxindoles-treated cells. All data are expressed as mean ± SEM (NS = not significant, n = 3).
FIG. 6 shows the target engagement of oxindoles on AMPK in mobilized CD34+ HSPC cells from healthy individuals or in erythroid cell line HUDEP-2. Target engagement is assessed by phosphorylation of residue Threonine 172 on the a-domain of AMPK at day 11 of erythroid differentiation. FIG. 6A represents target engagement in mobilized CD34+ HSPC measured by Alpha SureFire Ultra Multiplex® bead-based assay technology. All data are expressed as a ratio of signals pAMPK and total AMPK and normalized to total protein concentration. FIG. 6B represents target engagement in HUDEP-2 cells measured by Alpha SureFire Ultra Multiplex® bead-based assay technology. FIG. 6C: Western blot confirms target engagement of oxindoles on AMPK downstream pathway by measuring the phosphorylation of FOXO3 on Ser413 and of ULK-1 on Ser317, which are substrates of AMPK. All data are expressed as mean ± SEM (NS = not significant, * p < 0.05, ** p<0.01, *** p<0.001, n = 3).
FIG. 7 demonstrates that oxindole “Compound 2” promotes anti-inflammatory M2 macrophages polarization. FIG. 7A shows the decrease of pro-inflammation markers CD86 in M-CSF polarized and activated Ml macrophages from healthy donor. Activation was triggered by the combination of LPS (lOmg/mL) and INF-γ (50mg/mL). Thus, exposure to oxindole “Compound 2” prevents the increase of this pro-inflammatory marker induced by LPS and INF-γ compared to control (DMSO indicated as Vehicle). Representative plots are shown. FIG. 7B confirms the anti-inflammatory effect of oxindole “Compound 2” by showing the increase of anti-inflammatory marker CD 163 in M-CSF polarized and activated Ml macrophages from healthy donor. Activation was triggered by the combination of LPS (10mg/mL) and INF-γ (50mg/mL). Exposure to oxindole “Compound 2” triggers the increase of this anti-inflammatory marker compared to control (DMSO indicated as Vehicle). Representative plots are shown. All data are expressed as percentage of total cells (n = 1).
As used above, and throughout the description of the invention, the following terms, unless otherwise indicated, shall be understood to have the following meanings:
The term “halogen atom” as used herein, means a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom.
The term “alkyl group” as used herein, means, unless otherwise mentioned in the text, a linear or branched saturated aliphatic group containing from 1 to 4 carbon atoms. Examples that may be mentioned include methyl, ethyl, propyl, isopropyl, butyl, isobutyl or tert-butyl groups, in particular methyl, ethyl or tert-butyl groups.
The term "alkenyl group” as used herein, means a linear or branched mono- or polyunsaturated aliphatic group containing, for example, one or two ethylenic unsaturations.
The term “alkoxy group” as used herein, means a radical -O-alkyl in which the alkyl group is as defined previously. In particular, the -O-alkyl group is a methoxy or an ethoxy group. The term “cycloalkyl group” as used herein, means a saturated mono or bi-cyclic aliphatic group comprising between 3 and 6 carbon atoms. Examples that may be mentioned include cyclopropyl, cyclobutyl or cyclohexyl group.
The term “heterocycloalkyl group” as used herein, means a mono or bi-cyclic alkyl group comprising between 3 and 6 carbon atoms and comprising 1 or 2 heteroatoms, such as nitrogen or oxygen. Examples that may be mentioned include piperazinyl, morpholinyl, tetrahydropyranyl, piperidinyl or pyrrolidinyl groups.
The term “aryl group” as used herein, means a mono or bi-cyclic aromatic group comprising between 6 and 10 carbon atoms. An example of an aryl group that may be mentioned is the phenyl or naphtalene group.
The term “heteroaryl group” as used herein, means a mono or bi cyclic aromatic group comprising between 2 and 10 carbon atoms and comprising between 1 and 3 heteroatoms, such as nitrogen, oxygen, or sulfur. Examples that may be mentioned include pyridinyl, pyrazolyl, furanyl, isoxazolyl, thiazolyl, isothiazolyl, thiophenyl, indolyl, furopyridinyl, benzofuranyl, thienopyridinyl, pyrimidinyl and 1,3,4-oxadiazolyl groups.
Compounds
The present disclosure relates to the treatment or prevention of the diseases and disorders discussed herein utilizing a compound of formula (I):
Figure imgf000013_0001
wherein:
R1 represents: an (C6-C10)aryl group, unsubstituted or substituted with one or more substituents chosen from: ■ a halogen atom,
■ a — ORa group, in which Ra represents a hydrogen atom, a (C1-C3)alkyl group or a -CF3 group,
■ a (C1-C3)alkyl group, unsubstituted or substituted with one or more halogen atoms,
■ a carboxyl group,
■ a cyano group, and
■ a (C3-C6)heterocycloalkyl group, unsubstituted or substituted with one or more (C1-C3)alkyl group; or
• a (C2-C10)heteroaryl group, unsubstituted or substituted with one or more substituents chosen from:
■ a halogen atom,
■ a (C1-C4)alkyl group,
■ a (C3-C6)cycloalkyl group,
■ a — ORe group, in which Re represents an hydrogen atom or a (C1- C4)alkyl group, said (C1-C4)alkyl group being unsubstituted or substituted with one or more (C1-C4)alkoxy or heterocycloalkyl group, and
■ a -NRfRr group, in which Rf and Rf', independently, identical or different, represent a (C1-C3)alkyl group;
R2 represents:
• an (C6-C10)aryl group, unsubstituted or substituted with one or more substituents chosen from:
■ a halogen atom,
■ a cyano group,
Figure imgf000014_0001
■ a (C1-C3)alkyl group unsubstituted or substituted with one or more substituents chosen from a halogen atom, a hydroxyl group, and a (C1- C4)alkenyl group,
■ a (C3-C6)cycloalkyl group, unsubstituted or substituted with a hydroxy(C1-C3)alkyl group, a hydroxyl group, or an (C1-C4)alkoxy group,
■ a -ORb group, in which Rb represents:
• a hydrogen atom,
• a -CF3 group,
• a (C3-C6)cycloalkyl group, unsubstituted or substituted with a hydroxyl group,
• a (C3-C6)heterocycloalkyl group, said (C3-C6)heterocycloalkyl being unsubstituted or substituted with a (C1-C3)alkyl group, or
• or an (C1-C3)alkyl group, said (C1-C3)alkyl group being unsubstituted or substituted with one or more groups selected from: o hydroxyl group, o (C1-C4)alkoxy group, o (C2-C10)heteroaryl group, o acetamido group, o di(C1-C3)alkyl-amino group, o (C3-C6)cycloalkyl group, said (C3-C6)cycloalkyl group being unsubstituted or substituted with one or more hydroxyl group, and o (C3-C6)heterocycloalkyl group, said (C3- C6)heterocycloalkyl group being unsubstituted or substituted with a (C1-C3)alkyl group;
■ a (C3-C6)heterocycloalkyl group unsubstituted or substituted with one or more halogen atom, (C1-C3)alkyl group, hydroxyl group, hydroxy(C1-C3)alkyl group, (C1-C4)alkoxy group or (C1-C4)fluoroalkyl group, ■ an (C6-C10)aryl group, unsubstituted or substituted with one or more - ORc group, in which Rc represents a hydrogen atom or a (C1-C3)alkyl group,
■ a (C2-C10)heteroaryl group, unsubstituted or substituted with one or more -NH2 group,
■ a -NRdRd' group, in which Rd and Rd', independently, identical or different, represent a hydrogen atom, a (C1-C3)alkyl group, a hydroxy(C1-C4)alkyl or a (C3-C6)cycloalkyl group,
• a (C2-C10)heteroaryl group, unsubstituted or substituted with one or more substituents chosen from:
• a (C1-C3)alkyl group
• a (C3-C6)cycloalkyl group, unsubstituted or substituted with a hydroxy(C1-C3)alkyl group,
• a -NRgRg’ group, in which Rg and Rg’, independently, identical or different, represent a (C1-C3)alkyl group;
R3 represents a halogen atom or a (C1-C3)alkyl group;
R4 represents a halogen atom or a hydrogen atom, or a pharmaceutically acceptable salt thereof.
In some aspects, for the compound of formula (I):
R1 represents:
• an aryl group, in particular a phenyl group, optionally substituted with one or more substituents chosen from
■ a halogen atom, in particular a fluorine or a chlorine atom,
■ a — ORa group, in which Ra represents a hydrogen atom or a (C1-C3)alkyl group in particular a methyl group,
■ a (C1-C3)alkyl group, in particular a methyl group, optionally substituted by one or more halogen atoms, in particular a di or trifluoromethyl group,
■ a carboxyl group, and ■ a cyano group, or
• a heteroaryl group, in particular a pyridinyl group, a pyrazolyl group, an isoxazolyl group, athiazolyl group, an isothiazolyl group or a 1,3,4-oxadiazolyl group, optionally substituted by one or more substituents chosen from a halogen atom in particular a bromine atom, a (C1-C4)alkyl group, in particular a methyl group or a tert-butyl group, a cycloalkyl group, in particular a cyclopropyl group, an (C1-C3)alkoxy group in particular a methoxy group;
R2 represents:
• an aryl group, in particular a phenyl or naphtalene group, optionally substituted by one or more substituents chosen from:
■ a halogen atom, in particular a fluorine or a chlorine atom,
■ a cyano group,
■ a (C1-C3)alkyl group, in particular an ethyl group,
■ a cycloalkyl group, in particular a cyclopropyl or a cyclobutyl group, optionally substituted by a hydroxy(C1-C3)alkyl group, in particular a hydroxymethyl group,
■ a -ORb group, in which Rb represents a hydrogen atom, a -CF3 group or an alkyl group in particular a (C1-C3)alkyl group, optionally substituted by one heterocycloalkyl group, in particular a piperazinyl group, the said heterocycloalkyl group being optionally substituted by a (C1- C3)alkyl group, in particular a methyl group;
■ a heterocycloalkyl group, in particular a morpholinyl, a dihydropyranyl, a tetrahydropyranyl, a pyrrolidinyl, a tetrahydrofuranyl, a pip eridiny 1 or a piperazinyl group, optionally substituted by one or more a (C1- C3)alkyl group, in particular a methyl group,
■ an aryl group, in particular a phenyl group, optionally substituted by one or more -ORc group, in which Rc represents a hydrogen atom or a (C1- C3)alkyl group, in particular a methyl group,
■ a heteroaryl group, in particular a pyridinyl, a thiazolyl or a furanyl group, optionally substituted by one or more -NH2 group, ■ a -NRdRd' group, in which Rd and Rd’, independently, identical or different, represent a hydrogen atom, a (C1-C3)alkyl group, in particular a methyl group,
• a heteroaryl group, in particular a thiophene group, optionally substituted by one or more cycloalkyl group in particular a cyclopropyl group, optionally substituted by a hydroxy(C1-C3)alkyl group in particular a hydroxymethyl group,
R3 represents:
• a halogen atom, in particular a chlorine or a fluorine atom, or
• a (C1-C3)alkyl group, in particular a methyl group,
R4 represents:
• a halogen atom, in particular a fluorine atom or
• a hydrogen atom; or a pharmaceutically acceptable salt thereof.
In some aspects, for the compound of formula (I):
R1 represents:
• a phenyl group, unsubstituted or substituted with one or more substituents chosen from:
■ a fluorine atom or a chlorine atom,
■ a — ORa group, in which Ra represents a methyl group, a CF3 group,
■ a di or trifluoro-methyl group,
■ a carboxyl group,
■ a cyano group, and
■ a morpholine group, a methylpiperazine group; or
• a pyridinyl group, a pyrazolyl group, a pyrimidinyl group, an isoxazolyl group, a thiazolyl group, an isothiazolyl group, a 1,3,4-oxadiazolyle group, a thiazol- 2onyl group, a thienyl group, a furyl group, a furopyridinyl group, a benzofuran- 2-yl group, a thienopyridinyl gtoup, an indolynonyl group, unsubstituted or substituted with one or more substituents chosen from:
■ a bromine atom, a chlorine atom, a fluorine atom,
■ a methyl or tert-butyl group,
■ a hydroxyl group,
■ a cyclopropyl group or a cyclohexyl group,
■ a — ORe group , in which Re represents a methyl group, an ethyl group, an isopropyl group, a methoxy ethyl group, a morpholinoethyl group, and
■ a dimethylamino group;
R2 represents:
• a phenyl or a naphtalene group, unsubstituted or substituted with one or more substituents chosen from:
■ a fluorine atom or a chlorine atom,
Figure imgf000019_0001
■ a cyano group,
■ an ethyl group, unsubsituted or substituted with one or two hydroxyl groups,
■ an propyl group, unsubsituted or substituted with two or more groups chosen from a chlorine atom, an hydroxyl group and a propenyl group,
■ a cyclopropyl or a cyclobutyl group, unsubstituted or substituted with a hydroxyl group, a hydroxymethyl group or a methoxy group,
■ a -ORb group, in which Rb represents a hydrogen atom, a - CF3 group or an (C1-C3)alkyl group, unsubstituted or substituted with one piperazinyl group, the said piperazinyl group being unsubstituted or substituted with a methyl group, ■ a morpholinyl, a dihydropyranyl, a tetrahydropyranyl, a pyrrolidinyle, a tetrahydrofuranyl, a piperazinyl, a piperidinyl group, a dioxane group, an oxaazaspiro[3.3]heptanyl, unsubstituted or substituted with one or more methyl group, hydroxyl group, methoxy group, hydroxymethyl group or fluorine atom,
■ an azetidinyl group unsubstituted or substituted with one or two fluorine atoms, methyl groups, or hydroxymethyl group,
■ a pyrrolidinyl group substituted with a trifluoroethyl group,
■ a phenyl group, unsubstituted or substituted with one or more -ORc group, in which Rc represents:
• a hydrogen atom,
• a methyl group,
• an hydroxypropyl group,
• an hydroxyxyxlohexyl group,
• a methoxypropyl group,
• a dimethylaminopropyl group,
• a morpholinoethyl group, a morpholinopropyl group,
• a hydroxypropyl group, a methoxyethyl group,
• a tetrahydropyranyl,
• a pyridylmethyl group,
• a pyrimidinyl group,
• a methylpiperidyl group,
• a thiazolylmethyl gtoup,
• an acetamidoethyl group,
• an oxetanylmethyl group, or
• an hydroxycyxlobutylmethyl group,
■ a pyridinyl, a thiazolyl, a furanyl group, unsubstituted or substituted with one -NH2 group, and ■ a -NRdRd' group, in which Rd and Rd', independently, identical or different, represent a methyl group, an hydroxypropyl group or a cyclopropyl group;
• a thiophene group, unsubstituted or substituted with a cyclopropyl group, unsubstituted or substituted with a hydroxymethyl group;
• a pyrimidinyl or a pyridinyl group substituted with a dimethylamino group; or
• an indolyl group, substituted with a methyl group;
R3 represents:
• a chlorine or a fluorine atom, or
• a methyl group,
R4 represents a fluorine or a hydrogen atom; or a pharmaceutically acceptable salt thereof.
In some aspects, for the compound of formula (I), Ri represents a (C2-C10)heteroaryl group, optionally substituted by one or more substituents chosen from a halogen atom, a (C1-C3)alkyl group, a (C3-C5)cycloalkyl group, and a (C1-C4)alkoxy group; or a pharmaceutically acceptable salt thereof.
In some aspects, for the compound of formula (I), R1 represents an isoxazolyl group, optionally substituted by one substituent chosen from a (C1-C4)alkyl group in particular a methyl group, a (C3-C5)cycloalkyl group, in particular a cyclopropyl group or a cyclohexyl group or an alkoxy group, in particular a methoxy group; or a pharmaceutically acceptable salt thereof.
In some aspects, for the compound of formula (I), Ri represents a phenyl group, substituted with one substituent chosen from halogen atom and an a -ORa group, in which Ra represents a (C1-C3)alkyl group; or a pharmaceutically acceptable salt thereof.
In some aspects, for the compound of formula (I):
R2 represents:
• an aryl group, in particular a phenyl group, optionally substituted by one or more substituents chosen from: ■ a (C1-C3)alkyl group,
■ a (C3-C6)cycloalkyl group, optionally substituted by a hydroxy(C1- C3)alkyl group,
■ a (C3-C6)heterocycloalkyl group, in particular a morpholinyl, a dihydropyranyl, a tetrahydropyranyl, a pyrrolidinyl, a tetrahydrofuranyl, a piperidinyl or a piperazinyl group, optionally substituted by one or more (C1-C3)alkyl group, in particular a methyl group or an hydroxyl group,
■ an (C6-C10)aryl group, in particular a phenyl group, optionally substituted by one or more -ORc group, in which Rc represents a hydrogen atom or a (C1-C3)alkyl group,
■ a (C2-C10)heteroaryl group, in particular a pyridinyl, a thiazolyl or a furanyl group, optionally substituted by one or more -NH2 group, and
■ a -NRdRd' group, in which Rd and Rd', independently, identical or different, represent a hydrogen atom, a (C1-C3)alkyl group, in particular a methyl group; or a pharmaceutically acceptable salt thereof
In some aspects, for the compound of formula (I):
R2 represents:
• a phenyl group, optionally substituted by one substituent chosen from:
■ a halogen atom,
■ a (C3-C6)cycloalkyl group, in particular a cyclopropyl group, optionally substituted by a hydroxy(C1-C3)alkyl group in particular a hydroxymethyl group,
■ a (C3-C6)heterocycloalkyl group, in particular a dihydropyranyl group,
■ a phenyl group, optionally substituted by several -ORc group, in which Rc represents a hydrogen atom or a (C1-C3)alkyl group in particular a methyl group,
■ a pyridinyl group, and ■ a -NRdRd' group, in which Rd and Rd', identical, represent a (C1-C3)alkyl group, in particular a methyl group; or a pharmaceutically acceptable salt thereof.
In some aspects, for the compound of formula (I), R3 represents a halogen atom, in particular a chlorine or a fluorine atom.
In some aspects, for the compound of formula (I), R4 represents a fluorine atom.
In some aspects, for the compound of formula (I), when R4 represents a fluorine then R3 also represents a fluorine atom.
In some aspects, the compound of formula (I) is selected from the group consisting of:
6-Chloro-5-(4-dimethylamino-phenyl)-3-[l-hydroxy-l-(3-methyl-isoxazol-5-yl)- methylidene]-l,3-dihydro-indol-2-one;
6-Chloro-5-(4-dimethylamino-phenyl)-3-[l-hydroxy-l-(3-methyl-isoxazol-5-yl)- methylidene]-l,3-dihydro-indol-2-one, sodium salt;
6-Chloro-3-[l-hydroxy-l-(3-hydroxy-phenyl)-methylidene]-5-(4-morpholin-4-yl- phenyl)-l ,3 -dihydro-indol-2-one;
3-{[6-Chloro-5-(4-morpholin-4-yl-phenyl)-2-oxo-l,2-dihydro-indolylidene]-hydroxy- m ethyl} -benzoic acid;
6-Chloro-5-(2'-hydroxy-3'-methoxy-biphenyl-4-yl)-3-[l-hydroxy-l-(3-methoxy-isoxazol-
5-yl)-methylidene]-l,3-dihydro-indol-2-one;
6-Chloro-5-[4-(l-hydroxymethyl-cyclopropyl)-phenyl]-3-[l-hydroxy-l-(3-methyl- isoxazol-5-yl)-methylidene]-l,3-dihydro-indol-2-one;
5-Biphenyl-4-yl-6-chloro-3-[l-hydroxy-l-phenyl-methylidene]-l,3-dihydro-indol-2-one;
3-{[6-Chloro-5-(4-dimethylamino-phenyl)-2-oxo-l,2-dihydro-indolylidene]-hydroxy- methyl} -benzoic acid;
4-{[6-Chloro-5-(4-dimethylamino-phenyl)-2-oxo-l,2-dihydro-indolylidene]-hydroxy- methyl} -benzoic acid;
6-Chloro-5-(4-dimethylamino-phenyl)-3-[l-hydroxy-l-(2-methyl-thiazol-5-yl)- methylidene]-l,3-dihydro-indol-2-one; 6-Chloro-3-[l-(3-fluoro-phenyl)-l-hydroxy-methylidene]-5-naphthalen-2-yl-l,3-dihydro- indol-2-one;
6-Chloro-5-(2'-hydroxy-3'-methoxy-biphenyl-4-yl)-3-[l-hydroxy-l-(3-methyl-isoxazol- 5-yl)-methylidene]-l,3-dihydro-indol-2-one;
6-Chloro-3-[l-hydroxy-l-(3-methyl-isothiazol-5-yl)-methylidene]-5-(4-morpholin-4-yl- phenyl)-l ,3 -dihydro-indol-2-one;
6-Chloro-3-[l-hydroxy-l-(3-methyl-isoxazol-5-yl)-methylidene]-5-(4-morpholin-4-yl- phenyl)-l ,3 -dihydro-indol-2-one;
6-Chloro-3-[l-(2,4-dimethyl-thiazol-5-yl)-l-hydroxy-methylidene]-5-(4-morpholin-4-yl- phenyl)-l ,3 -dihydro-indol-2-one;
6-Chloro-3-[l-hydroxy-l-(5-methyl-[l,3,4]oxadiazol-2-yl)-methylidene]-5-(4-morpholin-
4-yl-phenyl)- 1 ,3 -dihydro-indol-2-one;
6-Chloro-3-[l-hydroxy-l-(3-methoxy-isoxazol-5-yl)-methylidene]-5-(4-morpholin-4-yl- phenyl)-l ,3 -dihydro-indol-2-one;
3-{[6-Chloro-5-(4-morpholin-4-yl-phenyl)-2-oxo-l,2-dihydro-indolylidene]-hydroxy- m ethyl} -benzonitrile;
6-Chloro-3-[l-(3-fluoro-phenyl)-l-hydroxy-methylidene]-5-(4-morpholin-4-yl-phenyl)- l,3-dihydro-indol-2-one;
6-Chloro-3-[l-hydroxy-l-(3-methyl-isoxazol-5-yl)-methylidene]-5-(4-methoxy-phenyl)- l,3-dihydro-indol-2-one;
6-Chloro-5-[4-(3,6-dihydro-2H-pyran-4-yl)-phenyl]-3-[l-hydroxy-l-(3-methyl-isoxazol-
5-yl)-methylidene]-l,3-dihydro-indol-2-one;
6-Chloro-5-(4-cyclopropyl-phenyl)-3-[l-hydroxy-l-(3-methyl-isoxazol-5-yl)- methylidene]-l,3-dihydro-indol-2-one;
6-Chloro-5-[4-(l-hydroxymethyl-cyclopropyl)-phenyl]-3-[l-hydroxy-l-(3-methyl- isothiazol-5-yl)-methylidene]-l,3-dihydro-indol-2-one;
6-Chloro-3-[l-hydroxy-l-(3-methyl-isoxazol-5-yl)-methylidene]-5-(4-pyrrolidin-l-yl- phenyl)-l ,3 -dihydro-indol-2-one;
6-Chloro-3-[l-hydroxy-l-pyridin-3-yl-methylidene]-5-(4-morpholin-4-yl-phenyl)-l,3- dihydro-indol-2-one; 6- Chi oro-5 -(4-cy clopropyl-phenyl)-3 - [ 1 -(3 -fluoro-phenyl)- 1 -hy droxy-methylidene]- 1 , 3 - dihydro-indol-2-one;
6-Chloro-3-[l-(3-fluoro-phenyl)-l-hydroxy-methylidene]-5-[4-(l-hydroxymethyl- cyclopropyl)-phenyl]-l,3-dihydro-indol-2-one;
6-Chloro-3-[l-hydroxy-l-(5-methyl-isoxazol-3-yl)-methylidene]-5-(4-morpholin-4-yl- phenyl)-l ,3 -dihydro-indol-2-one;
6-Fluoro-3-[l-hydroxy-l-(3-methyl-isoxazol-5-yl)-methylidene]-5-(4-morpholin-4-yl- phenyl)-l ,3 -dihydro-indol-2-one;
6-Chloro-5-(4-fluoro-phenyl)-3-[l-hydroxy-l-(3-methyl-isoxazol-5-yl)-methylidene]- l,3-dihydro-indol-2-one;
3-[l-Hydroxy-l-(3-methyl-isoxazol-5-yl)-methylidene]-6-methyl-5-(4-morpholin-4-yl- phenyl)-l ,3 -dihydro-indol-2-one;
6-Chloro-3-[l-(2-fluoro-phenyl)-l-hydroxy-methylidene]-5-(4-morpholin-4-yl-phenyl)- l,3-dihydro-indol-2-one;
6-Chloro-3-[l-hydroxy-l-(3-methyl-isoxazol-5-yl)-methylidene]-5-(4-hydroxy-phenyl)- l,3-dihydro-indol-2-one;
6-Chl oro-3 -[ 1 -(3 -chloro-phenyl)- 1 -hydroxy -methylidene]-5-(4-morpholin-4-yl-phenyl)- l,3-dihydro-indol-2-one;
6-Chloro-3-[l-hydroxy-l-(3-methyl-isoxazol-5-yl)-methylidene]-5-(4-pyridin-4-yl- phenyl)-l,3-dihydro-indol-2-one, hydrochloride;
6-Chloro-3-[l-(4-fluoro-phenyl)-l-hydroxy-methylidene]-5-(4-morpholin-4-yl-phenyl)- l,3-dihydro-indol-2-one;
5-[4-(2-Amino-thiazol-4-yl)-phenyl]-6-chloro-3-[l-hydroxy-l-(3-methyl-isoxazol-5-yl)- methylidene]-l,3-dihydro-indol-2-one;
4-{6-Chloro-3-[l-hydroxy-l-(3-methyl-isoxazol-5-yl)-methylidene]-2-oxo-2,3-dihydro- lH-indol-5-yl}-benzonitrile;
6-Chloro-3-[l-(3-difluoromethyl-phenyl)-l-hydroxy-methylidene]-5-(4-morpholin-4-yl- phenyl)-l ,3 -dihydro-indol-2-one;
6-Chloro-3-[l-hydroxy-l-(3-trifluoromethyl-phenyl)-methylidene]-5-(4-morpholin-4-yl- phenyl)-! ,3 -dihydro-indol-2-one; 3-[l-(3-Bromo-isoxazol-5-yl)-l-hydroxy-methylidene]-6-chloro-5-(4-morpholin-4-yl- phenyl)-l ,3 -dihydro-indol-2-one;
6-Chloro-5-(2'-hydroxy-3'-methoxy-biphenyl-4-yl)-3-[l-hydroxy-l-(3-methyl-isothiazol- 5-yl)-methylidene]-l,3-dihydro-indol-2-one;
6-Chloro-3-[l-hydroxy-l-phenyl-methylidene]-5-(4-morpholin-4-yl-phenyl)-l,3-dihydro- indol-2-one;
6-Chloro-5-(3-fluoro-4-hydroxy-phenyl)-3-[l-hydroxy-l-(3-methyl-isoxazol-5-yl)- methylidene]-l,3-dihydro-indol-2-one;
6-Chloro-5-[5-(l-hydroxymethyl-cyclopropyl)-thiophen-2-yl]-3-[l-hydroxy-l-(3-methyl- isoxazol-5-yl)-methylidene]-l,3-dihydro-indol-2-one;
6-Chloro-3-[l-(2-fluoro-phenyl)-l-hydroxy-methylidene]-5-[4-(l-hydroxymethyl- cyclopropyl)-phenyl]-l,3-dihydro-indol-2-one;
6-Chloro-3-[l-(2-fluoro-phenyl)-l-hydroxy-methylidene]-5-[4-(l-hydroxymethyl- cyclopropyl)-phenyl]-l,3-dihydro-indol-2-one, sodium salt;
6-Chloro-5-[4-(3,6-dihydro-2H-pyran-4-yl)-phenyl]-3-[l-hydroxy-l-(3-methyl- isothiazol-5-yl)-methylidene]-l,3-dihydro-indol-2-one;
6-Chloro-5-[4-(l-hydroxymethyl-cyclobutyl)-phenyl]-3-[l-hydroxy-l-(3-methyl- isoxazol-5-yl)-methylidene]-l,3-dihydro-indol-2-one;
6-Chloro-3-[l-hydroxy-l-(3-methoxy-phenyl)-methylidene]-5-(4-morpholin-4-yl- phenyl)-l ,3 -dihydro-indol-2-one;
6-Chloro-5-(2'-hydroxy-3'-methoxy-biphenyl-4-yl)-3-[l-hydroxy-l-(2-methyl-thiazol-5- yl)-methylidene]-l,3-dihydro-indol-2-one;
6-Chloro-5-[4-(3,6-dihydro-2H-pyran-4-yl)-phenyl]-3-[l-(3-fluoro-phenyl)-l-hydroxy- methylidene]-l,3-dihydro-indol-2-one;
6-Chloro-3-[l-hydroxy-l-(l-methyl-lH-pyrazol-3-yl)-methylidene]-5-(4-morpholin-4-yl- phenyl)-l ,3 -dihydro-indol-2-one;
6-Chloro-3-[l-hydroxy-l-(3-methoxy-isoxazol-5-yl)-methylidene]-5-[4-(l- hydroxymethyl-cyclopropyl)-phenyl]-l,3-dihydro-indol-2-one;
6-Chloro-5-(3-fluoro-4-methoxy-phenyl)-3-[l-hydroxy-l-(3-methyl-isoxazol-5-yl)- methylidene]-!, 3-dihydro-indol-2-one; 6-Chloro-5-(4-fluoro-2-hydroxy-phenyl)-3-[l-hydroxy-l-(3-methyl-isoxazol-5-yl)- methylidene]-l,3-dihydro-indol-2-one;
6-Chloro-3-[l-hydroxy-l-(2-methyl-thiazol-4-yl)-methylidene]-5-(4-morpholin-4-yl- phenyl)-l ,3 -dihydro-indol-2-one;
6-Chloro-3-[l-hydroxy-l-(3-methyl-isoxazol-5-yl)-methylidene]-5-(4-trifluoromethoxy- phenyl)-l ,3 -dihydro-indol-2-one;
6-Chloro-3-[l-hydroxy-l-isoxazol-5-yl-methylidene]-5-(4-morpholin-4-yl-phenyl)-l,3- dihydro-indol-2-one;
6-Chloro-3-[l-hydroxy-l-(3-methyl-isoxazol-4-yl)-methylidene]-5-(4-morpholin-4-yl- phenyl)-l ,3 -dihydro-indol-2-one;
6-Chloro-3-[l-hydroxy-l-(3-methyl-isoxazol-5-yl)-methylidene]-5-[4-(tetrahydro-pyran- 4-yl)-phenyl] - 1 , 3 -dihy dro-indol-2-one;
6-Chloro-3-[l-(5-cyclopropyl-isoxazol-3-yl)-l-hydroxy-methylidene]-5-(4-morpholin-4- yl-phenyl)- 1 ,3 -dihydro-indol-2-one;
6-Chloro-5-(4-chloro-phenyl)-3-[l-hydroxy-l-(3-methyl-isoxazol-5-yl)-methylidene]- l,3-dihydro-indol-2-one;
6-Chloro-5-(4-ethyl-phenyl)-3-[l-hydroxy-l-(3-methyl-isoxazol-5-yl)-methylidene]-l,3- dihydro-indol-2-one;
6-Chloro-5-(4-furan-2-yl-phenyl)-3-[l-hydroxy-l-(3-methyl-isoxazol-5-yl)- methylidene]-l,3-dihydro-indol-2-one;
6-Chloro-5-(4-ethoxy-phenyl)-3-[l-hydroxy-l-(3-methyl-isoxazol-5-yl)-methylidene]- l,3-dihydro-indol-2-one;
6-Chloro-5-[4-(3,6-dihydro-2H-pyran-4-yl)-phenyl]-3-[l-hydroxy-l-(3-methoxy- isoxazol-5-yl)-methylidene]-l,3-dihydro-indol-2-one;
6-Chloro-3-[l-hydroxy-l-(3-methyl-isoxazol-5-yl)-methylidene]-5-[4-(tetrahydro-furan-
2-yl)-phenyl] - 1 , 3 -dihy dro-indol-2-one;
6-Chloro-3-[l-hydroxy-l-(3-methyl-isoxazol-5-yl)-methylidene]-5-[4-(3-piperazin-l-yl- propoxy)-phenyl]- 1,3 -dihy dro-indol-2-one, hydrochloride;
6-Chloro-3-[l-hydroxy-l-(3-methyl-isoxazol-5-yl)-methylidene]-5-{4-[3-(4-methyl- piperazin- 1 -yl)-propoxy ]-phenyl } - 1 , 3 -dihydro-indol-2-one, hydrochloride; 6-Fluoro-5 -(2'-hydroxy-3 '-methoxy -biphenyl-4-yl)-3 - [ 1 -hydroxy- 1 -(3 -methoxy-i soxazol- 5-yl)-methylidene]-l,3-dihydro-indol-2-one;
6-Fluoro-5-[4-(l-hydroxymethyl-cyclopropyl)-phenyl]-3-[l-hydroxy-l-(3-methyl- isoxazol-5-yl)-methylidene]-l,3-dihydro-indol-2-one;
3-[l-(3-Bromo-isoxazol-5-yl)-l-hydroxy-methylidene]-6-chloro-5-[4-(l-hydroxymethyl- cyclopropyl)-phenyl]-l,3-dihydro-indol-2-one;
6-Chloro-3-[l-hydroxy-l-(3-methyl-isoxazol-5-yl)-methylidene]-5-[4-(4-methyl- piperazin-l-yl)-phenyl]-l,3-dihydro-indol-2-one, hydrochloride;
6-Chloro-5-[4-(l -hydroxymethyl-cyclopropyl)-phenyl]-3 -[ 1 -hydroxy- 1 -(2-methyl- thiazol-5-yl)-methylidene]-l,3-dihydro-indol-2-one;
3 -[ 1 -(3 -tert-Butyl-i soxazol-5 -yl)- 1 -hy droxy-methylidene]-6-chl oro-5 - [4-( 1 - hydroxymethyl-cyclopropyl)-phenyl]-l,3-dihydro-indol-2-one;
6-Chloro-3-[l-(3-fluoro-4-methoxy-phenyl)-l-hydroxy-methylidene]-5-(4-morpholin-4- yl-phenyl)- 1 ,3 -dihydro-indol-2-one;
6-Chloro-3-[l-hydroxy-l-(3-methyl-isoxazol-5-yl)-methylidene]-5-[4-(l-methyl- piperidin-4-yl)-phenyl]-l,3-dihydro-indol-2-one, hydrochloride;
6-chloro-3-[hydroxy-[3-(2-methoxy ethoxy )isoxazol-5-yl]methylene]-5-[4-(2-hydroxy-3- methoxy-phenyl)phenyl]indolin-2-one;
6-chloro-5-[4-[l-(chloromethyl)-l,2-dihydroxy-ethyl]phenyl]-3-[hydroxy-(3- methylisoxazol-5-yl)methylene]indolin-2-one;
6-chloro-5-(4-hydroxy-3-methoxy-phenyl)-3-[hydroxy-(3-methylisoxazol-5- yl)methylene]indolin-2-one;
6-chloro-3-[hydroxy-(3-methylisoxazol-5-yl)methylene]-5-[4-(2- morpholinoethoxy)phenyl]indolin-2-one hydrochloride;
4,6-difluoro-3-[hydroxy-(3-methoxyisoxazol-5-yl)methylene]-5-[4-[l-
(hydroxymethyl)cyclopropyl]phenyl]indolin-2-one;
6-chloro-3-[(3-fluoro-4-methoxy-phenyl)-hydroxy-methylene]-5-[4-(2-hydroxy-3- methoxy-phenyl)phenyl]indolin-2-one;
6-chloro-5-[2-fluoro-4-[l-(hydroxymethyl)cyclopropyl]phenyl]-3-[hydroxy-(3- methylisoxazol-5-yl)methylene]indolin-2-one; 6-chloro-3-[(3-cyclohexylisoxazol-5-yl)-hydroxy-methylene]-5-(4- morpholinophenyl)indolin-2-one;
6-chloro-3-[(3-cyclopropylisoxazol-5-yl)-hydroxy-methylene]-5-(4- morpholinophenyl)indolin-2-one;
6-chloro-3-[(3-fluoro-4-methoxy-phenyl)-hydroxy-methylene]-5-[4-[l-
(hydroxymethyl)cyclopropyl]phenyl]indolin-2-one;
6-chloro-3-[hydroxy-(3-methoxyisoxazol-5-yl)methylene]-5-[4-(4- hydroxytetrahydropyran-4-yl)phenyl]indolin-2-one;
6-chloro-5-[4-(2-hydroxy-3-methoxy-phenyl)phenyl]-3-[hydroxy-[3-(2- morpholinoethoxy)isoxazol-5-yl]methylene]indolin-2-one;
6-chloro-3-[hydroxy-(3-methylisoxazol-5-yl)methylene]-5-[4-(3- hydroxypropoxy)phenyl]indolin-2-one;
4,6-difluoro-3-[hydroxy-(3-methoxyisoxazol-5-yl)methylene]-5-[4-(2-hydroxy-3- methoxy-phenyl)phenyl]indolin-2-one;
6-chloro-3-[hydroxy-[3-(4-methylpiperazin-l-yl)phenyl]methylene]-5-(4- morpholinophenyl)indolin-2-one hydrochloride;
6-chloro-3-[hydroxy-(3-morpholinophenyl)methylene]-5-(4-morpholinophenyl)indolin-2- one;
6-chloro-3-[[3-fluoro-4-(trifluoromethoxy)phenyl]-hydroxy-methylene]-5-(4- morpholinophenyl)indolin-2-one;
6-chloro-5-[3-fluoro-4-[l-(hydroxymethyl)cyclopropyl]phenyl]-3-[hydroxy-(3- methoxyisoxazol-5-yl)methylene]indolin-2-one;
6-chloro-3-[(3-chloro-5-fluoro-4-methoxy-phenyl)-hydroxy-methylene]-5-(4- morpholinophenyl)indolin-2-one;
6-chloro-3-[(2,3-difluoro-4-methoxy-phenyl)-hydroxy-methylene]-5-(4- morpholinophenyl)indolin-2-one;
6-chloro-3-[hydroxy-(3-methoxyisoxazol-5-yl)methylene]-5-[4-(3- morpholinopropoxy)phenyl]indolin-2-one;
6-chloro-3-[hydroxy-(3-methoxyisoxazol-5-yl)methylene]-5-[4-(3- pyridylmethoxy)phenyl]indolin-2-one; 6-chloro-3-[hydroxy-(3-methylisoxazol-5-yl)methylene]-5-[4-(pyrimidin-2- ylmethoxy)phenyl]indolin-2-one;
6-chloro-3-[hydroxy-(3-hydroxyisoxazol-5-yl)methylene]-5-(4- morpholinophenyl)indolin-2-one;
6-chloro-3-[hydroxy-(3-methylisoxazol-5-yl)methylene]-5-[4-(2- methoxyethoxy)phenyl]indolin-2-one;
N-[2-[4-[6-chloro-3-[hydroxy-(3-methylisoxazol-5-yl)methylene]-2-oxo-indolin-5- yl]phenoxy]ethyl]acetamide;
6-chloro-3-[(3-ethoxyisoxazol-5-yl)-hydroxy-methylene]-5-[4-(2-hydroxy-3-methoxy- phenyl)phenyl]indolin-2-one;
6-chloro-3-[(3-cyclopropylisoxazol-5-yl)-hydroxy-methylene]-5-[4-[l- (hydroxymethyl)cyclopropyl]phenyl]indolin-2-one;
5-[-[6-chloro-5-(4-morpholinophenyl)-2-oxo-indolin-3-ylidene]-hydroxy-methyl]-4- methyl-3H-thiazol-2-one;
6-chloro-3-[(2,5-difluoro-4-methoxy-phenyl)-hydroxy-methylene]-5-(4- morpholinophenyl)indolin-2-one;
6-chloro-3-[hydroxy-(3-isopropoxyisoxazol-5-yl)methylene]-5-[4-(2-hydroxy-3- methoxy-phenyl)phenyl]indolin-2-one;
6-chloro-3-[hydroxy-(3-methoxyisoxazol-5-yl)methylene]-5-[4-(l- methoxycyclobutyl)phenyl]indolin-2-one;
6-chloro-5-[2-fluoro-4-[l-(hydroxymethyl)cyclopropyl]phenyl]-3-[hydroxy-(3- methoxyisoxazol-5-yl)methylene]indolin-2-one;
3-[(3-tert-butylisoxazol-5-yl)-hydroxy-methylene]6-chloro-5-(4- morpholinophenyl)indolin-2-one;
6-chloro-3-[(3-cyclopropylisoxazol-5-yl)-hydroxy-methylene]-5-[2-fluoro-4-[l- (hydroxymethyl)cyclopropyl]phenyl]indolin-2-one;
6-chloro-5-[4-[l-(hydroxymethyl)cyclopropyl]phenyl]-3-[hydroxy(2- thienyl)methylene]indolin-2-one;
6-chloro-3-[hydroxy-(3-methylisoxazol-5-yl)methylene]-5-[4-(oxetan-2- ylmethoxy)phenyl]indolin-2-one; 6-chloro-3-[hydroxy-(6-methoxy-3-pyridyl)methylene]-5-[4-[ l - (hydroxymethyl)cyclopropyl]phenyl]indolin-2-one hydrochloride;
6-chl oro-3 -[(3 -cyclopropyli soxazol-5 -yl)-hy droxy-methylene] -5 - [4 - (2-hy droxy-3 - methoxy-phenyl)phenyl]indolin-2-one;
6-fluoro-3-[hydroxy-(3-methoxyisoxazol-5-yl)methylene]-5-[4-[l-
(hydroxymethyl)cyclopropyl]phenyl]indolin-2-one;
3-[(3-cyclopropylisoxazol-5-yl)-hydroxy-methylene]6-fluoro-5-[4-[l-
(hydroxymethyl)cyclopropyl]phenyl]indolin-2-one;
3-[(3-cyclopropylisoxazol-5-yl)-hydroxy-methylene]-4,6-difluoro-5-[4-[l-
(hydroxymethyl)cyclopropyl]phenyl]indolin-2-one;
6-chloro-5-[4-[2-hydroxy-l-(hydroxymethyl)ethyl]phenyl]-3-[hydroxy-(3- methoxyisoxazol-5-yl)methylene]indolin-2-one;
6-chloro-5-[4-(l,2-dihydroxyethyl)phenyl]-3-[hydroxy-(3-methoxyisoxazol-5- yl)methylene]indolin-2-one;
6-chloro-5-[4-[l-(hydroxymethyl)cyclopropyl]phenyl]-3-[hydroxy-(5-methyl-2- thienyl)methylene]indolin-2-one;
6-chloro-3-[hydroxy-(3-methoxyisoxazol-5-yl)methylene]-5-[4-[(l-methyl-4- piperidyl)oxy]phenyl]indolin-2-one hydrochloride;
6-fluoro-3 - [(3 -fluoro-4-methoxy-phenyl)-hy droxy-m ethyl ene] -5 - [4- [ 1 - (hydroxymethyl)cyclopropyl]phenyl]indolin-2-one;
6-chloro-3-[2-furyl(hydroxy)methylene]-5-[4-[l-
(hydroxymethyl)cyclopropyl]phenyl]indolin-2-one;
6-chloro-5-(4-dimethylaminophenyl)-3-[hydroxy-(3-methoxyisoxazol-5- yl)methylene]indolin-2-one;
6-chloro-3-[hydroxy-(3-methoxyisoxazol-5-yl)methylene]-5-(4-tetrahydropyran-4- yloxyphenyl)indolin-2-one;
6-chloro-3-[hydroxy-(3-methoxyisoxazol-5-yl)methylene]-5-[4-(3- hydroxypropoxy)phenyl]indolin-2-one;
6-chloro-3-[(5-chloro-2-thienyl)-hydroxy-methylene]-5-[4-[l- (hydroxymethyl)cyclopropyl]phenyl]indolin-2-one; 6-chloro-3-[hydroxy-(3-methoxyisoxazol-5-yl)methylene]-5-[4-(3- methoxypropoxy)phenyl]indolin-2-one;
6-chloro-3-[hydroxy-(3-methylisoxazol-5-yl)methylene]-5-[4-(oxetan-3- ylmethoxy)phenyl]indolin-2-one;
6-chloro-3-[hydroxy-(3-methoxyisoxazol-5-yl)methylene]-5-[4-(l-methylpyrrolidin-3- yl)phenyl]indolin-2-one hydrochloride;
6-chloro-3-[hydroxy-(3-methoxyisoxazol-5-yl)methylene]-5-(4-pyrrolidin-3- ylphenyl)indolin-2-one hydrochloride;
6-chloro-5-[4-(3-hydroxycyclobutyl)phenyl]-3-[hydroxy-(3-methoxyisoxazol-5- yl)methylene]indolin-2-one;
6-chloro-3-[hydroxy-(3-methoxyisoxazol-5-yl)methylene]-5-[4-(thiazol-5- ylmethoxy)phenyl]indolin-2-one;
6-chloro-3-[hydroxy-(3-methoxyisoxazol-5-yl)methylene]-5-(4-pyrrolidin-l- ylphenyl)indolin-2-one;
6-chloro-3-[(2,3-difluorophenyl)-hydroxy-methylene]-5-[4-[l- (hydroxymethyl)cyclopropyl]phenyl]indolin-2-one;
6-chloro-5-[4-[(3-hydroxycyclobutyl)methoxy]phenyl]-3-[hydroxy-(3-methoxyisoxazol-
5-yl)methylene]indolin-2-one;
6-chloro-3-[hydroxy-(5-methoxy-2-pyridyl)methylene]-5-(4-morpholinophenyl)indolin- 2-one;
6-chloro-3-[(2,4-difluorophenyl)-hydroxy-methylene]-5-[4-[l-
(hydroxymethyl)cyclopropyl]phenyl]indolin-2-one;
6-chloro-3-[(5-fluoro-3-pyridyl)-hydroxy-methylene]-5-[4-[l-
(hydroxymethyl)cyclopropyl]phenyl]indolin-2-one;
6-chloro-3-[(3,4-difluorophenyl)-hydroxy-methylene]-5-[4-[l-
(hydroxymethyl)cyclopropyl]phenyl]indolin-2-one;
6-chloro-3-[(3,5-difluorophenyl)-hydroxy-methylene]-5-[4-[l-
(hydroxymethyl)cyclopropyl]phenyl]indolin-2-one;
6-chloro-5-[2-(dimethylamino)pyrimidin-5-yl]-3-[hydroxy-(3-methoxyisoxazol-5- yl)methylene]indolin-2-one; 6-chloro-5-[6-(dimethylamino)-3-pyridyl]-3-[hydroxy-(3-methoxyisoxazol-5- yl)methylene]indolin-2-one;
6-chloro-5-[4-(dimethylamino)-3-fluoro-phenyl]-3-[hydroxy-(3-methoxyisoxazol-5- yl)methylene]indolin-2-one;
6-chloro-5-[4-(dimethylamino)-2-fluoro-phenyl]-3-[hydroxy-(3-methoxyisoxazol-5- yl)methylene]indolin-2-one;
6-chloro-5-(3-fluoro-4-morpholino-phenyl)-3-[hydroxy-(3-methoxyisoxazol-5- yl)methylene]indolin-2-one;
6-chloro-3-[(3,5-difluorophenyl)-hydroxy-methylene]-5-[4-(l-methylpyrrolidin-3- yl)phenyl]indolin-2-one hydrochloride;
6-chloro-3-[hydroxy-(6-methoxy-3-pyridyl)methylene]-5-(4-morpholinophenyl)indolin- 2-one;
6-chloro-5-[4-(4-hydroxycyclohexoxy)phenyl]-3-[hydroxy-(3-methoxyisoxazol-5- yl)methylene]indolin-2-one;
6-chloro-5-[4-[cyclopropyl(methyl)amino]phenyl]-3-[hydroxy-(3-methoxyisoxazol-5- yl)methylene]indolin-2-one;
6-chloro-3-[hydroxy-(3-methoxyisoxazol-5-yl)methylene]-5-(l-methylindol-5-yl)indolin- 2-one;
6-chloro-3-[(2,4-difluorophenyl)-hydroxy-methylene]-5-[4-(3- hydroxycyclobutyl)phenyl]indolin-2-one;
6-chloro-3-[hydroxy-(3-methoxyisoxazol-5-yl)methylene]-5-[4-(3-hydroxypyrrolidin-l- yl)phenyl]indolin-2-one;
6-chloro-3-[furo[2,3-b]pyridin-2-yl(hydroxy)methylene]-5-[4-[l-
(hydroxymethyl)cyclopropyl]phenyl]indolin-2-one;
6-chloro-3-[hydroxy-(2-methoxy-4-pyridyl)methylene]-5-[4-[l-
(hydroxymethyl)cyclopropyl]phenyl]indolin-2-one;
6-chloro-3-[(2,5-difluoro-4-methoxy-phenyl)-hydroxy-methylene]-5-[4-(3- hydroxycyclobutyl)phenyl]indolin-2-one;
6-chloro-3-[(3-fluoro-4-pyridyl)-hydroxy-methylene]-5-[4-[l-
(hydroxymethyl)cyclopropyl]phenyl]indolin-2-one; 6-chloro-3-[(3,5-difluorophenyl)-hydroxy-methylene]-5-[4-(3- hydroxycyclobutyl)phenyl]indolin-2-one;
5-[4-(azetidin-l-yl)phenyl]6-chloro-3-[hydroxy-(3-methoxyisoxazol-5- yl)methylene]indolin-2-one;
6-chloro-3-[(2,4-dimethoxyphenyl)-hydroxy-methylene]-5-[4-[l- (hydroxymethyl)cyclopropyl]phenyl]indolin-2-one;
3-[(3-tert-butylisoxazol-5-yl)-hydroxy-methylene]6-chloro-5-[4-(3- hydroxycyclobutyl)phenyl]indolin-2-one;
6-chloro-3-[hydroxy-(3-methoxyisoxazol-5-yl)methylene]-5-[4-(3- methoxycyclobutyl)phenyl]indolin-2-one;
6-chloro-5-[4-(3-hydroxycyclobutyl)phenyl]-3-[hydroxy-(3-isopropoxyisoxazol-5- yl)methylene]indolin-2-one;
6-chloro-3-[hydroxy-(3-methoxyisoxazol-5-yl)methylene]-5-[4-[l-(2,2,2- trifluoroethyl)pyrrolidin-3-yl]phenyl]indolin-2-one hydrochloride;
3-[benzofuran-2-yl(hydroxy)methylene]6-chloro-5-[4-[l-
(hydroxymethyl)cyclopropyl]phenyl]indolin-2-one;
6-chloro-3-[furo[3,2-b]pyridin-2-yl(hydroxy)methylene]-5-[4-[l-
(hydroxymethyl)cyclopropyl]phenyl]indolin-2-one;
6-chloro-3-[(5-chlorobenzofuran-2-yl)-hydroxy-methylene]-5-[4-[l-
(hydroxymethyl)cyclopropyl]phenyl]indolin-2-one;
6-chloro-3-[(2-fluorophenyl)-hydroxy-methylene]-5-[4-(3- hydroxycyclobutyl)phenyl]indolin-2-one;
6-chloro-5-[4-[3-(dimethylamino)propoxy]phenyl]-3-[hydroxy-(3-methoxyisoxazol-5- yl)methylene]indolin-2-one;
6-chloro-3-[(3-cyclopropylisoxazol-5-yl)-hydroxy-methylene]-5-[4-(3- hydroxycyclobutyl)phenyl]indolin-2-one;
6-chloro-3-[hydroxy-(3-methoxy-4-pyridyl)methylene]-5-[4-[l- (hydroxymethyl)cyclopropyl]phenyl]indolin-2-one;
6-chloro-5-[4-(2,2-dimethyl-l,3-dioxan-5-yl)phenyl]-3-[hydroxy-(3-methoxyisoxazol-5- yl)methylene]indolin-2-one; 6-chloro-3-[hydroxy-(3-methoxyisoxazol-5-yl)methylene]-5-[4-(6-oxa-2- azaspiro[3.3]heptan-2-yl)phenyl]indolin-2-one;
6-chloro-3-[hydroxy-(2-methoxy-3-pyridyl)methylene]-5-[4-[l-
(hydroxymethyl)cyclopropyl]phenyl]indolin-2-one;
6-chloro-3-[hydroxy-(3-methoxyisoxazol-5-yl)methylene]-5-[4-[3- hydroxypropyl(methyl)amino]phenyl]indolin-2-one;
6-chloro-5-[4-(3-fluoroazetidin-l-yl)phenyl]-3-[hydroxy-(3-methoxyisoxazol-5- yl)methylene]indolin-2-one;
6-chloro-5-[4-(3,3-difluoroazetidin-l-yl)phenyl]-3-[hydroxy-(3-methoxyisoxazol-5- yl)methylene]indolin-2-one;
6-chloro-3-[furo[3,2-b]pyridin-2-yl(hydroxy)methylene]-5-[4-(3-hydroxypyrrolidin-l- yl)phenyl]indolin-2-one;
6-chloro-3-[hydroxy-(6-methoxy-2-pyridyl)methylene]-5-[4-[l-
(hydroxymethyl)cyclopropyl]phenyl]indolin-2-one;
6-chloro-3-[hydroxy-(3-methoxy-2-pyridyl)methylene]-5-[4-[l-
(hydroxymethyl)cyclopropyl]phenyl]indolin-2-one;
6-chloro-3-[(3-ethoxyisoxazol-5-yl)-hydroxy-methylene]-5-[4-(3- hydroxycyclobutyl)phenyl]indolin-2-one;
6-chloro-3-[hydroxy-(3-methoxyisoxazol-5-yl)methylene]-5-[4-(l-methylazetidin-3- yl)phenyl]indolin-2-one;
6-chloro-3-[hydroxy-(3-methoxyisoxazol-5-yl)methylene]-5-[4-[(3R)-3- hydroxypyrrolidin-l-yl]phenyl]indolin-2-one;
6-chloro-3-[hydroxy-(3-methoxyisoxazol-5-yl)methylene]-5-[4-[(3S)-3- hydroxypyrrolidin-l-yl]phenyl]indolin-2-one;
6-chloro-3-[(2-fluorophenyl)-hydroxy-methylene]-5-[4-(3-hydroxypyrrolidin-l- yl)phenyl]indolin-2-one;
6-chloro-3-[[2-(dimethylamino)pyrimidin-5-yl]-hydroxy-methylene]-5-[4-(3- hydroxypyrrolidin-l-yl)phenyl]indolin-2-one;
6-chloro-5-[4-(3,3-dimethylazetidin-l-yl)phenyl]-3-[hydroxy-(3-methoxyisoxazol-5- yl)methylene]indolin-2-one; 6-chloro-3-[(3-cyclopropylisoxazol-5-yl)-hydroxy-methylene]-5-[4-(3- hydroxypyrrolidin-l-yl)phenyl]indolin-2-one;
6-chloro-3-[hydroxy-(3-methoxyisoxazol-5-yl)methylene]-5-[4-[3- (hydroxymethyl)azetidin-l-yl]phenyl]indolin-2-one;
6-chloro-5-[4-[l-(hydroxymethyl)cyclopropyl]phenyl]-3-[hydroxy(thieno[2,3-b]pyridin- 2-yl)methylene]indolin-2-one hydrochloride;
6-chloro-3-[hydroxy-(3-methoxyisoxazol-5-yl)methylene]-5-[4-[(3S,4S)-3-hydroxy-4- methoxy-pyrrolidin-l-yl]phenyl]indolin-2-one;
6-chloro-5-[4-[l-(2-chloroethyl)-2-methyl-prop-l-enyl]phenyl]-3-[hydroxy-(3- methoxyisoxazol-5-yl)methylene]indolin-2-one;
6-chloro-3-[hydroxy-(3-methoxyisoxazol-5-yl)methylene]-5-[4-(4-hydroxy-l- piperidyl)phenyl]indolin-2-one;
6-chloro-5-[4-[(3R)-3-fluoropyrrolidin-l-yl]phenyl]-3-[hydroxy-(3-methoxyisoxazol-5- yl)methylene]indolin-2-one;
6-chloro-3-[hydroxy-(l-methylindol-5-yl)methylene]-5-[4-(3-hydroxypyrrolidin-l- yl)phenyl]indolin-2-one;
6-chloro-5-(2,6-difluoro-4-morpholino-phenyl)-3-[hydroxy-(3-methoxyisoxazol-5- yl)methylene]indolin-2-one;
6-chloro-3-[hydroxy-(3-methoxyisoxazol-5-yl)methylene]-5-[4-[2- (hydroxymethyl)azetidin-l-yl]phenyl]indolin-2-one;
6-chloro-3-[hydroxy-(3-methoxyisoxazol-5-yl)methylene]-5-[4-[(2S)-2-
(hydroxymethyl)pyrrolidin- 1 -yl]phenyl]indolin-2-one;
6-chloro-3-[hydroxy-(3-methoxyisoxazol-5-yl)methylene]-5-[4-[(2R)-2-
(hydroxymethyl)pyrrolidin- 1 -yl]phenyl]indolin-2-one;
4-[4-[6-chloro-3-[hydroxy-(3-methoxyisoxazol-5-yl)methylene]-2-oxo-indolin-5- yl]phenyl]cyclohex-3-ene-l -carboxylic acid; trans-6-chloro-3-[hydroxy-(3-methoxyisoxazol-5-yl)methylene]-5-[4-[(3S,4R)-4- hydroxytetrahydrofuran-3-yl]phenyl]indolin-2-one; cis-6-chloro-3-[hydroxy-(3-methoxyisoxazol-5-yl)methylene]-5-[4-[(2S,4S)-4- hydroxytetrahydrofuran-2-yl]phenyl]indolin-2-one; trans-6-chloro-3-[hydroxy-(3-methoxyisoxazol-5-yl)methylene]-5-[4-[(2R,4S)-4- hydroxytetrahydrofuran-2-yl]phenyl]indolin-2-one;
6-chloro-3-[hydroxy-(3-methoxyisoxazol-5-yl)methylene]-5-[4-(4-hydroxy-4-methyl-l- piperidyl)phenyl]indolin-2-one; 6-chloro-3-[hydroxy-(3-methoxyisoxazol-5-yl)methylene]-5-[4-[2-
(hydroxymethyl)morpholin-4-yl]phenyl]indolin-2-one; and
6-chloro-3-[hydroxy-(3-methoxyisoxazol-5-yl)methylene]-5-[4-[3-
(hydroxymethyl)morpholin-4-yl]phenyl]indolin-2-one. In some embodiments, the compound of formula (I) is 6-chloro-3-[l-hydroxy-l-(3-methoxy- isoxazol-5-yl)-methylidene]-5-[4-(l-hydroxymethyl-cyclopropyl)-phenyl]-l,3-dihydro-indol-
2-one, which has the following structure:
Figure imgf000037_0002
(“Compound 1”), or a pharmaceutically acceptable salt thereof: In some embodiments, the compound of formula (I) is 6-chloro-3-[(3-cyclopropylisoxazol-5- yl)-hydroxy-methylene]-5-[4-[l-(hydroxymethyl)cyclopropyl]phenyl]indolin-2-one, which has the following structure:
Figure imgf000037_0001
(“Compound 2”), or a pharmaceutically acceptable salt thereof. In some embodiments, the compound of formula (I) is 6-chloro-3-[(3-fluoro-4-pyridyl)- hydroxy-methylene]-5-[4-[l-(hydroxymethyl)cyclopropyl]phenyl]indolin-2-one, which has the following structure:
Figure imgf000038_0001
(“Compound 3”), or a pharmaceutically acceptable salt thereof.
In some embodiments, the compound of formula (I) is 6-chloro-5-(2'-hydroxy-3'-methoxy- biphenyl-4-yl)-3-[l-hydroxy-l-(3-methyl-isoxazol-5-yl)-methylidene]-l,3-dihydro-indol-2- one, which has the following structure:
Figure imgf000038_0002
(“Compound 4”), or a pharmaceutically acceptable salt thereof.
The presently disclosed compounds, e.g., any of the compounds disclosed herein that are basic in nature are generally capable of forming a wide variety of different salts with various inorganic and/or organic acids. Although such salts are generally pharmaceutically acceptable for administration to animals and humans, it is often desirable in practice to initially isolate a compound from the reaction mixture as a pharmaceutically unacceptable salt and then simply convert the latter back to the free base compound by treatment with an alkaline reagent, and subsequently convert the free base to a pharmaceutically acceptable acid addition salt. The acid addition salts of the base compounds can be readily prepared using conventional techniques, e.g. by treating the base compound with a substantially equivalent amount of the chosen mineral or organic acid in an aqueous solvent medium or in a suitable organic solvent such as, for example, methanol or ethanol. Upon careful evaporation of the solvent, the desired solid salt is obtained. Presently disclosed compounds that are positively charged, e.g. containing a quaternary ammonium, may also form salts with the anionic component of various inorganic and/or organic acids.
Acids which can be used to prepare pharmaceutically acceptable salts of a compound of formula (I) are those which can form non-toxic acid addition salts, e.g. salts containing pharmacologically acceptable anions, such as chloride, bromide, iodide, nitrate, sulfate or bisulfate, phosphate or acid phosphate, acetate, lactate, citrate or acid citrate, tartrate or bitartrate, succinate, malate, maleate, fumarate, gluconate, saccharate, benzoate, methanesulfonate and pamoate [i.e. 1, 1'-methylene-bis-(2-hydroxy-3-naphthoate)] salts.
Presently disclosed compounds that are acidic in nature, e.g. compounds containing a thiol moiety, are generally capable of forming a wide variety of different salts with various inorganic and/or organic bases. Although such salts are generally pharmaceutically acceptable for administration to animals and humans, it is often desirable in practice to initially isolate a compound from the reaction mixture as a pharmaceutically unacceptable salt and then simply convert the latter back to the free acid compound by treatment with an acidic reagent, and subsequently convert the free acid to a pharmaceutically acceptable base addition salt. These base addition salts can be readily prepared using conventional techniques, e.g. by treating the corresponding acidic compounds with an aqueous solution containing the desired pharmacologically acceptable cations, and then evaporating the resulting solution to dryness, e.g. under reduced pressure. Alternatively, they also can be prepared by mixing lower alkanolic solutions of the acidic compounds and the desired alkali metal alkoxide together, and then evaporating the resulting solution to dryness in the same manner as before. In either case, stoichiometric quantities of reagents may be employed in order to ensure completeness of reaction and maximum product yields of the desired solid salt.
Bases which can be used to prepare the pharmaceutically acceptable base addition salts of a compound of formula (I) are those which can form non-toxic base addition salts, e.g. salts containing pharmacologically acceptable cations, such as, alkali metal cations (e.g. potassium and sodium), alkaline earth metal cations (e.g. calcium and magnesium), ammonium or other water-soluble amine addition salts such as A-methylglucamine (meglumine), lower alkanolammonium, and other such bases of organic amines.
The present disclosure further embraces stereoisomers and mixture of stereoisomers of the compounds disclosed herein. Stereoisomers (e.g. cis and trans isomers) and all optical isomers of a presently disclosed compound (e.g. R- and S- enantiomers), as well as racemic, diastereomeric and other mixtures of such isomers are within the scope of the present disclosure.
The compound of formula (I), and pharmaceutical compositions containing them, such as those described herein, are useful in therapy, in particular in the therapeutic treatment of blood disorders, including hemoglobinopathies. Subjects to be treated according to the methods described herein include vertebrates, such as mammals.
A hemoglobinopathy is a condition that involves a mutation in human beta-globin or an expression control sequence thereof, such as sickle cell disease (SCD) or beta-thalassemia.
SCD typically arises from a mutation substituting thymine for adenine in the sixth codon of the beta-chain gene of hemoglobin (i.e., GAG to GTG of the HBB gene). This mutation causes glutamate to valine substitution in position 6 of the Hb beta chain. The resulting Hb, referred to as HbS, has the physical properties of forming polymers under conditions of low oxygen tension. SCD is typically an autosomal recessive disorder.
Subjects with SCD may experience a range of medical complications including acute pain episodes, also known as vaso-occlusive crises or vaso-occlusive episodes, that require hospitalization and may progress to more severe complications such as acute chest syndrome. SCD is associated with vascular disease and stroke and SCD subjects may experience cerebrovascular accidents including transient ischemic attack, overt strokes and silent cerebral infarctions. Retinopathy and seizures are also associated with SCD. Proliferative sickle cell retinopathy (PSR) is a frequent vision-threatening complication in sickle cell anemia, leading to visual impairment. In PSR, the blood vessels become blocked and divert away from the retina causing the retina to starve and die, leading to vision loss.
Subjects with SCD may experience both chronic and acute complications including bone pain crisis as a complication of vaso-occlusive pain, bone and bone marrow infarction, osteonecrosis and vascular necrosis. Subjects with SCD may experience chronic and acute cardiopulmonary complications including acute chest syndrome (ACS), pulmonary hypertension and left-sided heart disease. SCD subjects may experience chronic and acute reticuloendothelial complications including splenic sequestration, which is more prevalent in subjects who have had a first acute pain episode. Splenic sequestration can result in worsened anemia in SCD subjects.
Subjects with SCD may experience chronic and acute gastrointestinal and urogenital complications including cholelithiasis, acute cholecystitis, biliary sludge, acute choledocholi thiasis and gallstones. Urogenital complications, including renal dysfunction, may occur at an early age and lead to chronic renal failure. In male subjects with SCD a priapism may occurs as a severe urogenital complication.
Although children with SCD may or may not experience a vaso-occlusive crisis before they reach adolescence, even infants with SCD may develop symptoms. Infants with SCD may develop a syndrome that develops suddenly and lasts several weeks called hand-foot syndrome. Hand-foot syndrome is a dactylitis that presents as exquisite pain and soft tissue swelling of the dorsum of the hands and feet. β-Thalassemia are a group of inherited blood disorders caused by a variety of mutational mechanisms that result in a reduction or absence of synthesis of β-globin and leading to accumulation of aggregates of unpaired, insoluble a-chains that cause ineffective erythropoiesis, accelerated red cell destruction, and severe anemia. Subjects with beta- thalassemia exhibit variable phenotypes ranging from severe anemia to clinically asymptomatic individuals. The genetic mutations present in β-Thalassemia are diverse, and can be caused by a number of different mutations. The mutations can involve a single base substitution or deletions or inserts within, near or upstream of the β globin gene. For example, mutations occur in the promoter regions preceding the beta-globin genes or cause production of abnormal splice variants. β0 is used to indicate a mutation or deletion which results in no functional β globin being produced. β+ is used to indicate a mutation in which the quantity or β globin is reduced or in which the β-globin produced has a reduced functionality. Examples of β-Thalassemia include thalassemia minor, thalassemia intermedia, and thalassemia major. 0- Thalassemia minor refers to thalassemia where only one of β-globin alleles bears a mutation. Individuals typically suffer from microcytic anemia. Detection usually involves lower than normal MCV value (<80 fL) plus an increase in fraction of hemoglobin A2 (>3.5%) and a decrease in fraction of hemoglobin A (<97.5%). Genotypes can be β+/β or β0/β. β-Thalassemia intermedia refers to a β-Thalassemia intermediate between the major and minor forms. Affected individuals can often manage a normal life but may need occasional transfusions, e.g., at times of illness or pregnancy, depending on the severity of their anemia. Genotypes can be β+/β+ or β0/β. β-Thalassemia major refers to a β-Thalassemia where both β-globin alleles have thalassemia mutations. This is a severe microcytic, hypochromic anemia. If left untreated, it causes anemia, splenomegaly, and severe bone deformities and typically leads to death before age 20. Treatment consists of periodic blood transfusion; splenectomy if splenomegaly is present, and treatment of transfusion-caused iron overload. Cure is possible by bone marrow transplantation. Genotypes include β+/ β0 or β0/ β0 or β+/β+. Mediterranean anemia or Cooley's anemia has a genotype of β0/ β0 so that no hemoglobin A is produced. It is the most severe form of β-Thalassemia.
Although carriers of sickle cell trait do not suffer from SCD, individuals with one copy of HbS and one copy of a gene that codes for another abnormal variant of hemoglobin, such as HbC or Hb beta-thalassemia, typically will have a less severe form of sickle cell disease. For example, another specific defect in β-globin causes another structural variant, hemoglobin C (HbC). Hemoglobin C (abbreviated as Hb C or HbC) is an abnormal hemoglobin in which substitution of a glutamic acid residue with a lysine residue at the 6th position of the β-globin chain has occurred. A subject that is a double heterozygote for HbS and HbC (HbSC disease) is typically characterized by symptoms of moderate clinical severity. Another common structural variant of β-globin is hemoglobin E (HbE). HbE is an abnormal hemoglobin in which substitution of a glutamic acid residue with a lysine residue at the 26th position of the β-globin chain has occurred. A subject that is a double heterozygote for HbS and HbE has HbS/HbE syndrome, which usually causes a phenotype similar to HbS/b+ thalassemia, discussed below.
A subject that is a double heterozygote for HbS and 30 thalassemia (i.e., HbS/ β0 thalassemia) can suffer symptoms clinically indistinguishable from sickle cell anemia.
A subject that is a double heterozygote for HbS and β+ thalassemia (i.e., HbS/ β+ thalassemia) can have mild-to-moderate severity of clinical symptoms with variability among different ethnicities.
Rare combinations of HbS with other abnormal hemoglobins include HbD Los Angeles, G-Philadelphia, HbO Arab, and others.
In some embodiments, a compound of formula (I), or a pharmaceutically acceptable salt thereof, is used to treat a hemoglobinopathy, such as SCD or thalassemia (e.g. β-Thalassemia), including those that involve a mutation in human β-globin or an expression control sequence thereof, as described above. Accordingly, provided herein are methods of treating a hemoglobinopathy comprising administering an effective amount of a compound of formula (I), or pharmaceutically acceptable salt thereof, to a patient in need thereof. In some aspects, the compound of formula (I) or, pharmaceutically acceptable salt thereof, is a β1 -selective AMPK activator. In some aspects, the compound of Formula (I) is selected from the group comprising Compound 1, Compound 2, Compound 3, and Compound 4.
In some embodiments, the compound of formula (I) is used to treat a subject with an HbS/ β0 genotype, an HbS/ μ+ genotype, an HBSC genotype, an HbS/HbE genotype, an HbD Los Angeles genotype, a G-Philadelphia genotype, or an abHbO Arab genotype.
In some embodiments, the β1 -selective AMPK activators are administered to a subject in need thereof in an effective amount to treat one or more symptoms of sickle cell disease, a thalassemia (e.g. β-Thalassemia), or a related disorder. In subjects with sickle cell disease, or a related disorder, physiological changes in RBCs can result in a disease with the following signs: (1) hemolytic anemia; (2) vaso-occlusive crisis; and (3) multiple organ damage from microinfarcts, including heart, skeleton, spleen, and central nervous system. Thalassemia can include symptoms such as anemia, fatigue and weakness, pale skin or jaundice (yellowing of the skin), protruding abdomen with enlarged spleen and liver, dark urine, abnormal facial bones and poor growth, and poor appetite.
Retinopathy due to SCD can also be treated by administering an effective amount of β1 -AMPK activator. Sickle retinopathy occurs when the retinal blood vessels get occluded by sickle red blood cells and the retina becomes ischemic, angiogenic factors are made in retina. In sickle cell disease, this occurs mostly in the peripheral retina, which does not obscure vision at first. Eventually, the entire peripheral retina of the sickle cell patient becomes occluded and many neovascular formations occur. Administration of a β1 -selective AMPK activator can reduce or inhibit the formation of occlusions in the peripheral retina of a sickle cell patient.
In some embodiments, the β1 -selective AMPK activators are used to increase HbF expression in a patient in need thereof.
Accordingly, provided herein are methods of increasing HbF expression comprising administering an effective amount of a β1-selective AMPK activator to a patient in need thereof. In some aspects, the β1-selective AMPK activator is a compound of Formula (I) or a pharmaceutically acceptable salt thereof. In some aspects, the β1-selective AMPK selected from the group comprising Compound 1, Compound 2, Compound 3, and Compound 4, or a pharmaceutically acceptable salt thereof.
In some embodiments, the β1 -AMPK activators are used to decrease inflammation in a patient with a β-hemoglobinopathy.
Accordingly, provided herein are decreasing inflammation in 0-hemoglobinopathy comprising administering an effective amount of a β1-selective AMPK activator to a patient in need thereof. In some aspects, the β1-selective AMPK activator is a compound of Formula (I) or a pharmaceutically acceptable salt thereof. In some aspects, the β1-selective AMPK activator is selected from the group comprising Compound 1, Compound 2, Compound 3, and Compound 4, or a pharmaceutically acceptable salt thereof. In some embodiments, the β1-AMPK activators are used to decrease oxidative stress in a patient with a p-hemoglobinopathy.
Accordingly, provided herein are decreasing oxidative stress in P-hemoglobinopathy comprising administering an effective amount of a β1 -selective AMPK activator to a patient in need thereof. In some aspects, the β1-selective AMPK activator is a compound of Formula (I) or a pharmaceutically acceptable salt thereof. In some aspects, the β1-selective AMPK activator is selected from the group comprising Compound 1, Compound 2, Compound 3, and Compound 4, or a pharmaceutically acceptable salt thereof.
In some embodiments, the β1 -selective AMPK activator is administered in combination with hydroxyurea.
Accordingly, provided herein is a method of treating a hemoglobinopathy comprising administering an effective amount of a β1-selective AMPK activator to a patient in need thereof in combination with an effective amount of hydroxyurea. In some aspects, the β1- selective AMPK activator is a compound of Formula (I) or a pharmaceutically acceptable salt thereof. In some aspects, the β1 -selective AMPK activator is selected from the group comprising Compound 1, Compound 2, Compound 3, and Compound 4, or a pharmaceutically acceptable salt thereof.
In another aspect, provided herein are methods of treating a hemoglobinopathy comprising administering an effective amount of a combination of a β1 -selective AMPK activator and hydroxyurea to a patient in need thereof . In some aspects, the β1 -selective AMPK activator is a compound of Formula (I) or a pharmaceutically acceptable salt thereof. In some aspects, the β1 -selective AMPK activator is selected from the group comprising Compound 1, Compound 2, Compound 3, and Compound 4, or a pharmaceutically acceptable salt thereof.
Pharmaceutical compositions
The present disclosure also provides pharmaceutical compositions comprising at least one β1- selective AMPK activator as described herein and at least one pharmaceutically acceptable excipient, e.g. for use according to the methods disclosed herein. The pharmaceutically acceptable excipient can be any such excipient known in the art including those described in, for example, Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985). Pharmaceutical compositions of the pl-selective AMPK activator may be prepared by conventional means known in the art including, for example, mixing at least one β1 -selective AMPK activator with a pharmaceutically acceptable excipient.
Thus, in one aspect the present disclosure provides a pharmaceutical dosage form comprising a β1 -selective AMPK activator as described herein and a pharmaceutically acceptable excipient, wherein the dosage form is formulated to provide, when administered (e.g. when administered orally), an amount of said compound sufficient to treat a disease or disorder as described herein.
A pharmaceutical composition or dosage form of the invention can include an agent and another carrier, e.g. compound or composition, inert or active, such as a detectable agent, label, adjuvant, diluent, binder, stabilizer, buffers, salts, lipophilic solvents, preservative, adjuvant or the like. Carriers also include pharmaceutical excipients and additives, for example, proteins, peptides, amino acids, lipids, and carbohydrates (e.g. sugars, including monosaccharides, di-, tri-, tetra-, and oligosaccharides; derivatized sugars such as alditols, aldonic acids, esterified sugars and the like; and polysaccharides or sugar polymers), which can be present singly or in combination, comprising alone or in combination 1 to 99.99% by weight or volume. Exemplary protein excipients include serum albumin such as human serum albumin (HSA), recombinant human albumin (rHA), gelatin, casein, and the like. Representative amino acid/antibody components, which can also function in a buffering capacity, include alanine, glycine, arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine, phenylalanine, aspartame, and the like. Carbohydrate excipients are also intended within the scope of this invention, examples of which include but are not limited to monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbose, and the like; disaccharides, such as lactose, sucrose, trehalose, cellobiose, and the like; polysaccharides, such as raffinose, melezitose, maltodextrins, dextrans, starches, and the like; and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitol sorbitol (glucitol) and myoinositol. Carriers which may be used include a buffer or a pH adjusting agent; typically, the buffer is a salt prepared from an organic acid or base. Representative buffers include organic acid salts such as salts of citric acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid, or phthalic acid; Tris, tromethamine hydrochloride, or phosphate buffers. Additional carriers include polymeric excipients/additives such as polyvinylpyrrolidones, ficolls (a polymeric sugar), dextrates (e.g. cyclodextrins, such as 2-hydroxypropyl-β- cyclodextrin), polyethylene glycols, flavoring agents, antimicrobial agents, sweeteners, antioxidants, antistatic agents, surfactants (e.g. polysorbates such as “TWEEN 20” and “TWEEN 80”), lipids (e.g. phospholipids, fatty acids), steroids (e.g. cholesterol), and chelating agents (e.g. EDTA).
The β1 -selective AMPK activators and pharmaceutical compositions can be used in an animal or human. Thus, a β1-selective AMPK activator can be formulated as an active ingredient in a pharmaceutical composition for oral, buccal, parenteral (e.g. intravenous, intramuscular or subcutaneous), topical, rectal or intranasal administration or in a form suitable for administration by inhalation or insufflation. In particular embodiments, the β1-AMPK activator or pharmaceutical composition is formulated for systemic administration, e.g. via a non-parenteral route. In one embodiment, the β1-AMPK activator or pharmaceutical composition is formulated for oral administration, e.g. in solid form. Such modes of administration and the methods for preparing appropriate pharmaceutical compositions are described, for example, in Gibaldi’s Drug Delivery Systems in Pharmaceutical Care (1st ed., American Society of Health-System Pharmacists 2007).
The pharmaceutical compositions can be formulated so as to provide slow, extended, or controlled release of the active ingredient therein using, for example, hydroxypropyl methyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and/or microspheres. The pharmaceutical compositions can also optionally contain opacifying agents and may be of a composition that releases the active ingredient(s) only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner, e.g. by using an enteric coating. Examples of embedding compositions include polymeric substances and waxes. The active ingredient can also be in micro-encapsulated form, if appropriate, with one or more pharmaceutically acceptable carriers, excipients, or diluents well known in the art (see, e.g., Remington’s). The β1 -selective AMPK activator may be formulated for sustained delivery according to methods well known to those of ordinary skill in the art. Examples of such formulations can be found in United States Patents 3,119,742; 3,492,397; 3,538,214; 4,060,598; and 4,173,626.
In solid dosage forms for oral administration (e.g. capsules, tablets, pills, dragees, powders, granules and the like), the active ingredient is mixed with one or more pharmaceutically acceptable carriers, excipients, or diluents, such as sodium citrate or dicalcium phosphate, and/or any of the following: (1) fdlers or extenders, such as starches, lactose, sucrose, glucose, mannitol, microcrystalline cellulose, calcium phosphate and/or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, pregelatinized maize starch, polyvinyl pyrrolidone, hydroxypropyl methylcellulose, sucrose and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, sodium starch glycolate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as, for example, sodium lauryl sulphate, acetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such as talc, silica, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and (10) coloring agents. In the case of capsules, tablets, and pills, the pharmaceutical compositions can also comprise buffering agents. Solid compositions of a similar type can also be prepared using fillers in soft and hard- filled gelatin capsules, and excipients such as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.
A tablet can be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets can be prepared using binders (for example, gelatin or hydroxypropyl methyl cellulose), lubricants, inert diluents, preservatives, disintegrants (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface- actives, and/ or dispersing agents. Molded tablets can be made by molding in a suitable machine a mixture of the powdered active ingredient moistened with an inert liquid diluent. The tablets and other solid dosage forms, such as dragees, capsules, pills, and granules, can optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the art.
In some embodiments, the pharmaceutical compositions are administered orally in a liquid form. Liquid dosage forms for oral administration of an active ingredient include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. Liquid preparations for oral administration may be presented as a dry product for constitution with water or other suitable vehicle before use. In addition to the active ingredient, the liquid dosage forms can contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (e.g. cottonseed, groundnut, com, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. In addition to inert diluents, the liquid pharmaceutical compositions can include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents, and the like. Suspensions, in addition to the active ingredient(s) can contain suspending agents such as, but not limited to, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof. Suitable liquid preparations may be prepared by conventional means with a pharmaceutically acceptable additive(s) such as a suspending agent (e.g. sorbitol syrup, methyl cellulose or hydrogenated edible fats); emulsifying agent (e.g. lecithin or acacia); non-aqueous vehicle (e.g. almond oil, oily esters or ethyl alcohol); and/or preservative (e.g. methyl or propyl p-hydroxybenzoates or sorbic acid). The active ingredient(s) can also be administered as a bolus, electuary, or paste.
For buccal administration, the composition may take the form of tablets or lozenges formulated in a conventional manner.
In some embodiments, the pharmaceutical compositions are administered by non-oral means such as by topical application, transdermal application, injection, and the like. In related embodiments, the pharmaceutical compositions are administered parenterally by injection, infusion, or implantation (e.g. intravenous, intramuscular, intra-arterial, subcutaneous, and the like).
The β1 -selective AMPK activator may be formulated for parenteral administration by injection, including using conventional catheterization techniques or infusion. Formulations for injection may be presented in unit dosage form, e.g. in ampules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain a formulating agent such as a suspending, stabilizing and/or dispersing agent recognized by those of skill in the art. Alternatively, the active ingredient may be in powder form for reconstitution with a suitable vehicle, e.g. sterile pyrogen-free water, before use.
The pharmaceutical compositions may be administered directly to the central nervous system. Accordingly, in certain embodiments the compositions are administered directly to the central nervous system so as to avoid the blood brain barrier. In some embodiments, the composition can be administered via direct spinal cord injection. In some embodiments, the composition is administered by intrathecal injection. In some embodiments, the composition is administered via intracerebroventricular injection. In some embodiments, the composition is administered into a cerebral lateral ventricle. In some embodiments, the composition is administered into both cerebral lateral ventricles. In additional embodiments, the composition is administered via intrahippocampal injection. The compositions may be administered in one injection or in multiple injections. In other embodiments, the composition is administered to more than one location (e.g. to two sites in the central nervous system).
The pharmaceutical compositions can be in the form of sterile injections. The pharmaceutical compositions can be sterilized by, for example, filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved in sterile water, or some other sterile injectable medium immediately before use. To prepare such a composition, the active ingredient is dissolved or suspended in a parenterally acceptable liquid vehicle. Exemplary vehicles and solvents include, but are not limited to, water, water adjusted to a suitable pH by addition of an appropriate amount of hydrochloric acid, sodium hydroxide or a suitable buffer, 1,3 -butanediol, Ringer’s solution and isotonic sodium chloride solution. The pharmaceutical composition can also contain one or more preservatives, for example, methyl, ethyl or n-propyl p-hydroxybenzoate. To improve solubility, a dissolution enhancing or solubilizing agent can be added or the solvent can contain 10-60% w/w of propylene glycol or the like.
The pharmaceutical compositions can contain one or more pharmaceutically acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders, which can be reconstituted into sterile injectable solutions or dispersions just prior to use. Such pharmaceutical compositions can contain antioxidants; buffers; bacteriostats; solutes, which render the formulation isotonic with the blood of the intended recipient; suspending agents; thickening agents; preservatives; and the like.
Examples of suitable aqueous and nonaqueous carriers, which can be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. In some embodiments, in order to prolong the effect of an active ingredient, it is desirable to slow the absorption of the compound from subcutaneous or intramuscular injection. This can be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility. The rate of absorption of the active ingredient then depends upon its rate of dissolution which, in turn, can depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally-administered active ingredient is accomplished by dissolving or suspending the compound in an oil vehicle. In addition, prolonged absorption of the injectable pharmaceutical form can be brought about by the inclusion of agents that delay absorption such as aluminum monostearate and gelatin.
Controlled release parenteral compositions can be in form of aqueous suspensions, microspheres, microcapsules, magnetic microspheres, oil solutions, oil suspensions, emulsions, or the active ingredient can be incorporated in biocompatible carrier(s), liposomes, nanoparticles, implants or infusion devices. Materials for use in the preparation of microspheres and/or microcapsules include, but are not limited to, biodegradable/bioerodible polymers such as polyglactin, poly-(isobutyl cyanoacrylate), poly(2-hydroxyethyl-L- glutamine) and poly(lactic acid). Biocompatible carriers which can be used when formulating a controlled release parenteral formulation include carbohydrates such as dextrans, proteins such as albumin, lipoproteins or antibodies. Materials for use in implants can be non- biodegradable, e.g. polydimethylsiloxane, or biodegradable such as, e.g., poly(caprolactone), poly(lactic acid), poly(glycolic acid) or poly(ortho esters).
For topical administration, the compound of formula (I) may be formulated as an ointment, cream, or liquid eye drops. A compound of formula (I) may also be formulated in rectal compositions such as suppositories or retention enemas, e.g. containing conventional suppository bases such as cocoa butter or other glycerides.
For intranasal administration or administration by inhalation, a compound of formula (I) may be conveniently delivered in the form of a solution or suspension from a pump spray container that is squeezed or pumped by the patient or as an aerosol spray presentation from a pressurized container or a nebulizer, with the use of a suitable propellant, e.g. dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. The pressurized container or nebulizer may contain a solution or suspension of the compound of formula (I). Capsules and cartridges (made, for example, from gelatin) for use in an inhaler or insufflator may be formulated containing a powder mix of a β1 -selective AMPK activator and a suitable powder base such as lactose or starch.
Having been generally described herein, the follow non-limiting examples are provided to further illustrate this invention.
EXAMPLES Example 1 - Induction of fetal hemoglobin HbF by oxindoles in human CD34+ cells from healthy donors during differentiation in vitro measured by flow cytometry and western blot.
Mobilized CD34+ Human Stem/Progenitor Cells (HSPC) from healthy individuals were cultured for 3 days in a maintenance media consisting of X-VIVO 10 (VWR), 100 U/mL penicillin-streptomycin (ThermoFisher), 2mM L-glutamine (Fisher Scientific), 100 ng/mL Recombinant Human Stem Cell Factor (SCF), 100 ng/mL Recombinant Human Thrombopoietin (TPO) and 100 ng/mL Recombinant Human Fit- 3 Ligand (Flt-3L) (all three from ThermoFisher). Cells were differentiated into erythroid cells using a three-step differentiation protocol developed by the Luc Douay group (Giarratana et al. 2005). In brief, CD34+ cells were cultured for 7 days in Step 1 media, consisting of Iscove’s modified Dulbecco’s medium (IMDM) (ThermoFisher) supplemented with IX GlutaMAX, 100 U/mL penicillin-streptomycin (ThermoFisher), 5% human AB+plasma, 330 ug/mL human holo- transferrin, 10 ug/mL human insulin, 2 U/mL heparin, 1 uM/mL hydrocortisone (Sigma- Aldrich), 3 U/mL recombinant human erythropoietin (EPO) (ThermoFisher), 100 ng/mL SCF (ThermoFisher) and 5 ng/mL interleukin 3 (IL-3) (Sigma-Aldrich). On day 7, cells were transferred to step 2 media, a step 1 media without hydrocortisone and IL-3, and cultured for 3-4 days. Then cells were cultured for 8-9 days in step 3 media, a step 2 media without SCF. During the entire process of differentiation, mobilized CD34+ HSPC were exposed to β1-selective AMPK activator oxindoles. To determine the percentage of HbF-positive cells (F- cells), differentiated cells were fixed and permeabilized using a fixation kit (ThermoFisher). Cells were stained with PE-Cy7-conjugated anti-CD235 or PE-conjugated anti-CD71 antibodies. HbF levels were detected using Allophycocyanin (APC)-conjugated anti-HbF antibody (ThermoFisher). The acquisition of stained cells was performed on BD FACSCanto™ and the analysis was run using FlowJo™ Software. In FIG.1A, data shows an increase in the frequency of F-cells when CD34+ cells were exposed to oxindoles compared to vehicle (DMSO), after 21 days of differentiation. In FIG. IB, high performance liquid chromatography (HPLC) analysis of globin proteins isolated from CD34+ cells treated with Compound 2 confirms an increase in fetal hemoglobin protein level. Human CD34+ cells were lysed in MilliQ H2O then hemolysates were centrifuged and hemoglobin variants HbF and HbAO analyzed by cation exchange HPLC on a Waters Acquity system with a Thermo ProPac WCX-10 Analytical Column (4 x 250 mm). Proteins were eluted in 20mM bis tris pH 6.95 using a 0 to 200mM NaCl gradient and hemoglobin peak absorbance areas at 410nm were integrated. FIG 1C confirms the induction of fetal hemoglobin in CD34+ cells treated with oxindoles assessed by Western blot. Total cell lysates from CD34+ cells were generated and the total protein concentration was determined using a Bradford protein assay (ThermoFisher Scientific). Reduced and denaturated protein (40pg) was loaded and separated by SDS-PAGE (12% gel), blotted on nitrocellulose membranes (BioRad) and finally incubated with hemoglobin beta and hemoglobin gamma antibodies (Cell Signaling). The a-tubulin antibody served as an internal control. Immunoreactive proteins were visualized by using an ECL® (enhanced chemiluminescence) detection system (BioRad). In FIG. ID, data shows an increase in the frequency of F-cells when CD34+ cells were exposed to oxindole “Compound 2” in a dose-response manner. To determine which genes are upregulated and downregulated when AMPK is activated by oxindoles in CD34+ cells from healthy donors, a RNA-Seq analysis was conducted at day 15 after treatment with Compound 2. Volcano plot reveals an increase in mRNA level of HBG1 and HBG2, confirming the activation of the gamma globin genes by Compound 2 (FIG. IE). To understand which proteins are upregulated and downregulated when AMPK is activated by oxindoles in CD34+ cells from healthy donors, a Proteomic analysis was conducted at day 15 after treatment with Compound 2 (FIG. IF). Volcano plot reveals an increase in HBG2 induced fetal hemoglobin protein. FIG.1G shows the RNA-seq and Proteomics integrative analysis. HBE1 and HBG2 values are increased, confirming the activation of HBG2 gene and the subsequent increase in fetal hemoglobin protein expression. To confirm the solely expression of beta-1 subunit in CD34+ HSPC cells, a western blot was realized. Beta-1 and Beta-2 subunits were measured and signals were normalized to β-actin (Cell Signaling) (FIG.1H). Beta-1 is the only subunit expressed in CD34+ cells.. Finally, to measure the effect of oxindoles on CD34+ cells viability during erythroid differentiation, a Vi- CELL BLU Cell Viability Analyzer (Beckman Coulter) was used, confirming the absence of cytotoxicity effect by oxindoles (FIG.1I).
Example 2 - Induction of fetal hemoglobin HbF by oxindoles in human CD34+ cells from sickle donors during differentiation in vitro measured by flow cytometry and western blot. Circulating CD34+ progenitor cells were isolated from total blood obtained from sickle cell patients, by performing a positive selection for CD34+ cells with magnetic beads (Miltenyi Biotec). Then cells were differentiated into erythroid cells using a three-step differentiation protocol developed by the Luc Douay group (Giarratana et al. 2005). In brief, CD34+ cells were cultured for 7 days in Step 1 media, consisting of Iscove’s modified Dulbecco’s medium (IMDM) (ThermoFisher) supplemented with IX GlutaMAX, 100 U/mL penicillin- streptomycin (ThermoFisher), 5% human AB+plasma, 330 ug/mL human holo-transferrin, 10 ug/mL human insulin, 2 U/mL heparin, 1 uM/mL hydrocortisone (Sigma-Aldrich), 3 U/mL recombinant human erythropoietin (EPO) (ThermoFisher), 100 ng/mL SCF (ThermoFisher) and 5 ng/mL interleukin 3 (IL-3) (Sigma-Aldrich). On day 7, cells were transferred to step 2 media, a step 1 media without hydrocortisone and IL-3, and cultured for 3-4 days. Then cells were cultured for 8-9 days in step 3 media, a step 2 media without SCF. During the entire process of differentiation, mobilized CD34+ HSPC were exposed to β1 -selective AMPK activator oxindoles. To determine the percentage of HbF-positive cells (F-cells), differentiated cells were fixed and permeabilized using a fixation kit (ThermoFisher). Cells were stained with PE-Cy7-conjugated anti-CD235 or PE-conjugated anti-CD71 antibodies. HbF levels were detected using Allophycocyanin (APC)-conjugated anti-HbF antibody (ThermoFisher). The acquisition of stained cells was performed on BD FACSCanto™ and the analysis was run using FlowJo™ Software. In FIG.2A, data shows an increase in the frequency of F-cells when CD34+ cells were exposed to oxindoles compared to vehicle (DMSO), after 21 days of differentiation. Moreover, to confirm the induction of fetal hemoglobin in CD34+ cells treated with oxindoles, a western blot were conducted (FIG.2B). Total cell lysates from CD34+ cells were generated and the total protein concentration was determined using a Bradford protein assay (ThermoFisher Scientific). Reduced and denaturated protein (40pg) was loaded and separated by SDS-PAGE (12% gel), blotted on nitrocellulose membranes (BioRad) and finally incubated with hemoglobin beta and hemoglobin gamma antibodies (Cell Signaling). The α- Tubulin antibody served as an internal control. Immunoreactive proteins were visualized by using an ECL® (enhanced chemiluminescence) detection system (BioRad). Optical density was measured with ImageJ software (National Institutes of Health, Bethesda, MD). Blot confirms the induction of fetal hemoglobin in CD34+ cells exposed to oxindoles. Example 3 - AMPK activation by oxindoles in human CD34+ from sickle donors prevents sickling at terminal differentiation stage in vitro.
To evaluate the effect of AMPK activation by oxindoles on erythrocytes sickling, a cell sickling assay was conducted upon completion of erythroid differentiation in culture. Fully differentiated CD34+ cells from sickle donors were incubated under hypoxia (2% O2) for 4 hours and then abnormal shaped cells were analyzed using the ImageStream®X Mk II Imaging Flow Cytometer (FIG.3A). In focus, cells were determined using RMS of brightfield image. Single cells were determined using Area vs Aspect Ratio of brightfield image. Live cells were determined using Live Dead NIR stain. Single, Live, in focus cells were analyzed using the following masking of the brightfield image: Skeleton (Ml, Chi, Thin) and Object (Ml, Chi, Tight). Sickling factor was calculated using the following feature: Length Skeleton (Ml, CHI, Thin) / Width (Ml, Chi, Tight). Cells with the sickle factor feature of 1.4 or higher were sickled. Cells with the sickle factor feature less than 1.4 were not sickled. In FIG3 A, data shows that oxindoles-activated AMPK prevents sickling in terminally differentiated CD34+ cells from sickle donors. This result was confirmed by microscopy, using a Nikon Eclipse Ti microscope (FIG.3B).
Example 4 - Oxindoles and Hydroxyurea combination shows additive effect on fetal hemoglobin induction in human CD34+ cells during differentiation in vitro.
To assess the effect of the combination of hydroxyurea (Sigma-Aldrich) and oxindoles, human CD34+ cells from healthy individuals and sickle donors were incubated with oxindoles and hydroxyurea during the differentiation process. After 14 days of differentiation, cells were fixed and stained with CD235a, CD71 and fetal hemoglobin antibodies for flowcytometry analysis. Data show that the combination of hydroxyurea and oxindoles leads to a synergetic effect in fetal hemoglobin induction compared to hydroxyurea alone in CD34+ cells from healthy individuals (FIG.4A) and from sickle donors (FIG.4B).
Example 5 - AMPK activation by oxindoles in human CD34+ does not affect CD34+ cells maturation to erythrocytes in vitro.
During human CD34+ cells differentiation, effect of oxindoles on maturation of CD34+ cells is measured by quantification of enucleation (FIG.5A), expression of erythroid differentiation markers Band-3, LRF, ALAS2, GATA-1 (FIG.5B), and expression of erythroid markers CD71 and CD235a (FIG.5C). Enucleation and expression of CD235a were measured by flowcytometry. To determine the enucleation rate of the erythroid differentiated cells at day 21, cells were stained using NucRed (living cells marker). The acquisition was performed on BD FACSCanto™ and the analysis was done using FlowJo™ Software. Data shows there was no effect of oxindoles on the enucleation of CD34+ cells after 21 days of differentiation (FIG.5 A). As to the erythroid differentiation markers Band-3, LRF, ALAS2, GATA-1, a western blot was conducted to measure their expression (FIG.5B). Antibody anti -band-3 (Cell Signaling), anti-LRF (Cell Signaling), anti-ALAS2 (Abeam), and GATA-1 (Cell Signaling) were used. p-Actin antibody served as an internal control. The blot confirmed there were no effect of oxindoles in the erythroid differentiation markers in CD34+ cells. Furthermore, markers CD235a was measured at terminal stage of differentiation (FIG.5C). Flowcytometry data confirms a normal level of CD235a expression in cells for most of oxindoles compared to control (DMSO expressed as vehicle). Finally, to confirm normal cell phenotype in CD34 treated with oxindoles, CD34+ cells were stained at day 14 of differentiation using the Wright- Giemsa method. Cells were spun onto glass slides with a Cytospin4 (ThermoFisher Scientific) at 300rpm for 3 min. Slides were allowed to dry for 5 minutes before staining with May Grunwald (Scy-Tek) for 6 minutes followed by 1 :20 diluted Giemsa stain for 13 minutes. The stained slides were rinsed in water and allowed to dry for 10 minutes before a coverslip was sealed on the preparation with Cytoseal 60 (ThermoFisher Scientific). The images were captured at 40X resolution on ikon Eclipse Ti (FIG.5D). Data shows a normal shape and cells population in CD34+ cells treated with oxindole “Compound 2”.
Example 6 - AMPK target engagement in human CD34+ and human HUDEP-2 cells after exposure to oxindoles in vitro.
To verify AMPK target engagement by AMPK activators, CD34+ cells from a healthy donor or HUDEP-2 cells (Riken Research Institute, Ibaraki, lapan) were exposed to oxindoles at the indicated doses (pM), harvested and lysed at the indicated time points. Phosphorylation of AMPK at threonine 172 (Thrl72) on the a-subunit of AMPK was assessed by HTRF using the Alpha SureFire Ultra Multiplex p-AMPKα1/2 (Thrl72) + Total AMPKal/2 Assay Kit from Perkin Elmer as a target engagement assay. The assay kit contains antibodies, coupled with fluorophore Europium, which recognize the phospho-Thrl72 epitope and a distal epitope on α-AMPK of human or mouse AMPK. The kit also contains antibodies coupled with the fluorophore Terbium to measure the total levels of AMPK. As to the in vitro study with human CD34+ cells, cells were collected on Day 11 of differentiation then exposed to oxindoles. As to the human HUDEP-2 cells line, cells were undifferentiated during exposure to oxindoles. According to the time course, cells were collected and lysed using RIPA buffer mixed with a phosphatase and protease inhibitors cocktail (Thermofisher). Phospho-AMPK signal was divided by total AMPK signal and the ratio was normalized to the total protein concentration. Samples protein concentrations were measured with the Pierce BSA Protein Assay (ThermoFisher). Data show a peak of signal at 30 minutes after exposure to AMPK activators in CD34+ cells (FIG. 6 A), and at Ih after exposure in HUDEP-2 cells (FIG. 6B), confirming target engagement in AMPK when cells are treated with AMPK activators oxindoles.
Furthermore, AMPK downstream pathway activation was verified by measuring phosphorylation of FOXO3 and ULK-1, which are direct targets of activated AMPK (FIG.6C). Total cell lysates from human erythroid CD34+ cells were generated and the total protein concentration was determined using a Bradford protein assay (ThermoFisher Scientific). Reduced and denaturated protein (40pg) was loaded and separated by SDS-PAGE (12% gel), blotted on nitrocellulose membranes (BioRad) and finally incubated with FOXO3, Phospho- FOXO3 (Ser413), ULK-1, Phospho-ULK-1 (Ser317) and a-tubulin antibodies (Cell Signaling). The a-tubulin antibody served as an internal control. Immunoreactive proteins were visualized by using an ECL® (enhanced chemiluminescence) detection system (BioRad). Blot confirm the phosphorylation of FOXO3 at Serine 413 and ULK-1 at Serine 317 in CD34+ cells exposed to oxindoles, confirming the upstream activation of AMPK by oxindoles.
Example 7 - AMPK activation by oxindole promotes human macrophage polarization to an anti-inflammatory functional phenotype in vitro.
To evaluate the effect of AMPK activation by oxindoles on macrophage polarization to pro- inflammatory Ml phenotype, monocytes and macrophages were isolated from total blood collected from a healthy donor by performing a magnetic positive selection for CD 14+ cells with magnetic beads (Miltenyi Biotec). Ml macrophages were induced by stimulation with M- CSF (50ng/mL) (Sigma- Aldrich) for 6 days, then activated by IFN-γ (Sigma-Aldrich) and LPS (Sigma-Aldrich) for 24h, fixed, stained for Ml polarization pro-inflammatory marker CD86 (BD Biosciences) and for M2 polarization anti-inflammatory marker CD 163 (BD Biosciences). Cells were acquired by flowcytometry. Data shows that activation of AMPK by oxindole “Compound 2” in Ml-polarized macrophages decreased the expression of proinflammatory Ml marker CD86 (FIG. 7A) and increased the expression of anti- inflammatory M2 marker CD 163 (FIG.7B). Overall, this data demonstrates that oxindoles have anti-inflammatory effect by promoting a M2 macrophages polarization.

Claims

CLAIMS What is claimed is:
1. A method of treating or preventing a β-hemoglobinopathy, the method comprising administering to a patient in need thereof a therapeutically effective amount of a compound of formula (I):
Figure imgf000060_0001
wherein:
Ri represents:
• an (C6-C10)aryl group, unsubstituted or substituted with one or more substituents chosen from:
■ a halogen atom,
■ a — ORa group, in which Ra represents a hydrogen atom, a (C1-C3)alkyl group or a -CF3 group,
■ a (C1-C3)alkyl group, unsubstituted or substituted with one or more halogen atoms,
■ a carboxyl group,
■ a cyano group, and
■ a (C3-C6)heterocycloalkyl group, unsubstituted or substituted with one or more (C1-C3)alkyl group; or
• a (C2-C10)heteroaryl group, unsubstituted or substituted with one or more substituents chosen from:
■ a halogen atom,
■ a (C1-C4)alkyl group,
■ a (C3-C6)cycloalkyl group, ■ a — ORe group, in which Re represents an hydrogen atom or a (C1- C4)alkyl group, said (C1-C4)alkyl group being unsubstituted or substituted with one or more (C1-C4)alkoxy or heterocycloalkyl group, and
■ a -NRfRf' group, in which Rf and Rf', independently, identical or different, represent a (C1-C3)alkyl group;
R2 represents:
• an (C6-C10)aryl group, unsubstituted or substituted with one or more substituents chosen from:
■ a halogen atom,
■ a cyano group,
Figure imgf000061_0001
Figure imgf000061_0002
■ a (C1-C3)alkyl group unsubstituted or substituted with one or more substituents chosen from a halogen atom, a hydroxyl group, and a (C1- C4)alkenyl group,
■ a (C3-C6)cycloalkyl group, unsubstituted or substituted with a hydroxy(C1-C3)alkyl group, a hydroxyl group, or an (C1-C4)alkoxy group,
■ a -ORb group, in which Rb represents:
• a hydrogen atom,
• a -CF3 group,
• a (C3-C6)cycloalkyl group, unsubstituted or substituted with a hydroxyl group,
• a (C3-C6)heterocycloalkyl group, said (C3-C6)heterocycloalkyl being unsubstituted or substituted with a (C1-C3)alkyl group, or • or an (C1-C3)alkyl group, said (C1-C3)alkyl group being unsubstituted or substituted with one or more groups selected from: o hydroxyl group, o (C1-C4)alkoxy group, o (C2-C10)heteroaryl group, o acetamido group, o di(C1-C3)alkyl-amino group, o (C3-C6)cycloalkyl group, said (C3-C6)cycloalkyl group being unsubstituted or substituted with one or more hydroxyl group, and o (C3-C6)heterocycloalkyl group, said (C3- C6)heterocycloalkyl group being unsubstituted or substituted with a (C1-C3)alkyl group;
■ a (C3-C6)heterocycloalkyl group unsubstituted or substituted with one or more halogen atom, (C1-C3)alkyl group, hydroxyl group, hydroxy(C1-C3)alkyl group, (C1-C4)alkoxy group or (C1-C4)fluoroalkyl group,
■ an (C6-C10)aryl group, unsubstituted or substituted with one or more - ORc group, in which Rc represents a hydrogen atom or a (C1-C3)alkyl group,
■ a (C2-C10)heteroaryl group, unsubstituted or substituted with one or more -NH2 group,
■ a -NRdRd' group, in which Rd and Rd', independently, identical or different, represent a hydrogen atom, a (C1-C3)alkyl group, a hydroxy(C1-C4)alkyl or a (C3-C6)cycloalkyl group,
• a (C2-C10)heteroaryl group, unsubstituted or substituted with one or more substituents chosen from: a (C1-C3)alkyl group a (C3-C6)cycloalkyl group, unsubstituted or substituted with a hydroxy(C1-C3)alkyl group, • a -NRgRg’ group, in which Rg and Rg’, independently, identical or different, represent a (C1-C3)alkyl group;
R3 represents a halogen atom or a (C1-C3)alkyl group; R4 represents a halogen atom or a hydrogen atom, or a pharmaceutically acceptable salt thereof.
2. A method of increasing HbF expression, the method comprising administering to a patient in need thereof a therapeutically effective amount of a compound of formula (I): formula (I):
Figure imgf000063_0001
wherein:
R1 represents:
• an (C6-C10)aryl group, unsubstituted or substituted with one or more substituents chosen from:
■ a halogen atom,
■ a — ORa group, in which Ra represents a hydrogen atom, a (C1-C3)alkyl group or a -CF3 group,
■ a (C1-C3)alkyl group, unsubstituted or substituted with one or more halogen atoms,
■ a carboxyl group,
■ a cyano group, and
■ a (C3-C6)heterocycloalkyl group, unsubstituted or substituted with one or more (C1-C3)alkyl group; or
• a (C2-C10)heteroaryl group, unsubstituted or substituted with one or more substituents chosen from: ■ a halogen atom,
■ a (C1-C4)alkyl group,
■ a (C3-C6)cycloalkyl group,
■ a - ORe group, in which Re represents an hydrogen atom or a (C1- C4)alkyl group, said (C1-C4)alkyl group being unsubstituted or substituted with one or more (C1-C4)alkoxy or heterocycloalkyl group, and
■ a -NRfRf' group, in which Rf and Rf', independently, identical or different, represent a (C1-C3)alkyl group;
R2 represents:
• an (C6-C10)aryl group, unsubstituted or substituted with one or more substituents chosen from:
■ a halogen atom,
■ a cyano group,
Figure imgf000064_0001
■ a (C1-C3)alkyl group unsubstituted or substituted with one or more substituents chosen from a halogen atom, a hydroxyl group, and a (C1- C4)alkenyl group,
■ a (C3-C6)cycloalkyl group, unsubstituted or substituted with a hydroxy(C1-C3)alkyl group, a hydroxyl group, or an (C1-C4)alkoxy group,
■ a -ORb group, in which Rb represents:
• a hydrogen atom,
• a -CF3 group,
• a (C3-C6)cycloalkyl group, unsubstituted or substituted with a hydroxyl group, • a (C3-C6)heterocycloalkyl group, said (C3-C6)heterocycloalkyl being unsubstituted or substituted with a (C1-C3)alkyl group, or
• or an (C1-C3)alkyl group, said (C1-C3)alkyl group being unsubstituted or substituted with one or more groups selected from: o hydroxyl group, o (C1-C4)alkoxy group, o (C2-C10)heteroaryl group, o acetamido group, o di(C1-C3)alkyl-amino group, o (C3-C6)cycloalkyl group, said (C3-C6)cycloalkyl group being unsubstituted or substituted with one or more hydroxyl group, and o (C3-C6)heterocycloalkyl group, said (C3- C6)heterocycloalkyl group being unsubstituted or substituted with a (C1-C3)alkyl group;
■ a (C3-C6)heterocycloalkyl group unsubstituted or substituted with one or more halogen atom, (C1-C3)alkyl group, hydroxyl group, hydroxy(C1-C3)alkyl group, (C1-C4)alkoxy group or (C1-C4)fluoroalkyl group,
■ an (C6-C10)aryl group, unsubstituted or substituted with one or more - ORc group, in which Rc represents a hydrogen atom or a (C1-C3)alkyl group,
■ a (C2-C10)heteroaryl group, unsubstituted or substituted with one or more -NH2 group,
■ a -NRdRd' group, in which Rd and Rd', independently, identical or different, represent a hydrogen atom, a (C1-C3)alkyl group, a hydroxy(C1-C4)alkyl or a (C3-C6)cycloalkyl group,
• a (C2-C10)heteroaryl group, unsubstituted or substituted with one or more substituents chosen from: a (C1-C3)alkyl group • a (C3-C6)cycloalkyl group, unsubstituted or substituted with a hydroxy(C1-C3)alkyl group,
• a -NRgRg’ group, in which Rg and Rg’, independently, identical or different, represent a (C1-C3)alkyl group; R3 represents a halogen atom or a (C1-C3)alkyl group; R4 represents a halogen atom or a hydrogen atom, or a pharmaceutically acceptable salt thereof
3. A method of decreasing inflammation in β-hemoglobinopathy, the method comprising administering to a patient in need thereof a therapeutically effective amount of a compound of formula (I): formula (I):
Figure imgf000066_0001
wherein:
R1 represents: an (C6-C10)aryl group, unsubstituted or substituted with one or more substituents chosen from:
■ a halogen atom,
■ a - ORa group, in which Ra represents a hydrogen atom, a (C1-C3)alkyl group or a -CF3 group,
■ a (C1-C3)alkyl group, unsubstituted or substituted with one or more halogen atoms,
■ a carboxyl group,
■ a cyano group, and ■ a (C3-C6)heterocycloalkyl group, unsubstituted or substituted with one or more (C1-C3)alkyl group; or
• a (C2-C10)heteroaryl group, unsubstituted or substituted with one or more substituents chosen from:
■ a halogen atom,
■ a (C1-C4)alkyl group,
■ a (C3-C6)cycloalkyl group,
■ a — ORe group, in which Re represents an hydrogen atom or a (C1- C4)alkyl group, said (C1-C4)alkyl group being unsubstituted or substituted with one or more (C1-C4)alkoxy or heterocycloalkyl group, and
■ a -NRfRf' group, in which Rf and Rf', independently, identical or different, represent a (C1-C3)alkyl group;
R2 represents:
• an (C6-C10)aryl group, unsubstituted or substituted with one or more substituents chosen from:
■ a halogen atom,
■ a cyano group,
Figure imgf000067_0001
■ a (C1-C3)alkyl group unsubstituted or substituted with one or more substituents chosen from a halogen atom, a hydroxyl group, and a (C1- C4)alkenyl group,
■ a (C3-C6)cycloalkyl group, unsubstituted or substituted with a hydroxy(C1-C3)alkyl group, a hydroxyl group, or an (C1-C4)alkoxy group,
■ a -ORb group, in which Rb represents: • a hydrogen atom,
• a -CF3 group,
• a (C3-C6)cycloalkyl group, unsubstituted or substituted with a hydroxyl group,
• a (C3-C6)heterocycloalkyl group, said (C3-C6)heterocycloalkyl being unsubstituted or substituted with a (C1-C3)alkyl group, or
• or an (C1-C3)alkyl group, said (C1-C3)alkyl group being unsubstituted or substituted with one or more groups selected from: o hydroxyl group, o (C1-C4)alkoxy group, o (C2-C10)heteroaryl group, o acetamido group, o di(C1-C3)alkyl-amino group, o (C3-C6)cycloalkyl group, said (C3-C6)cycloalkyl group being unsubstituted or substituted with one or more hydroxyl group, and o (C3-C6)heterocycloalkyl group, said (C3- C6)heterocycloalkyl group being unsubstituted or substituted with a (C1-C3)alkyl group;
■ a (C3-C6)heterocycloalkyl group unsubstituted or substituted with one or more halogen atom, (C1-C3)alkyl group, hydroxyl group, hydroxy(C1-C3)alkyl group, (C1-C4)alkoxy group or (C1-C4)fluoroalkyl group,
■ an (C6-C10)aryl group, unsubstituted or substituted with one or more - ORc group, in which Rc represents a hydrogen atom or a (C1-C3)alkyl group,
■ a (C2-C10)heteroaryl group, unsubstituted or substituted with one or more -NH2 group, ■ a -NRdRd' group, in which Rd and Rd', independently, identical or different, represent a hydrogen atom, a (C1-C3)alkyl group, a hydroxy(C1-C4)alkyl or a (C3-C6)cycloalkyl group,
• a (C2-C10)heteroaryl group, unsubstituted or substituted with one or more substituents chosen from:
• a (C1-C3)alkyl group
• a (C3-C6)cycloalkyl group, unsubstituted or substituted with a hydroxy(C1-C3)alkyl group,
• a -NRgRg’ group, in which Rg and Rg’, independently, identical or different, represent a (C1-C3)alkyl group; R3 represents a halogen atom or a (C1-C3)alkyl group; R4 represents a halogen atom or a hydrogen atom, or a pharmaceutically acceptable salt thereof
4. The method according to any one of claims 1 to 3, wherein the compound of formula (I) is selected from the group consisting of:
Figure imgf000069_0001
Figure imgf000070_0001
and
Figure imgf000070_0002
or a pharmaceutically acceptable salt thereof.
5. The method according to any one of claims 1 to 4, wherein the compound of formula (I) is a β1 -selective AMPK activator.
6. The method according to claim 5, wherein the β1-selective AMPK activator possesses at least about a 10-fold selective activation for β1-AMPK relative to β2-AMPK.
7. The method according to claim 5, wherein the β1-selective AMPK activator possesses at least about a 50-fold selective activation for β1-AMPK relative to β2-AMPK.
8. The method according to claim 5, wherein the β1-selective AMPK activator possesses at least about a 100-fold selective activation for β1-AMPK relative to β2-AMPK.
9. The method according to claim 5, wherein the β1-selective AMPK activator possesses at least about a 300-fold selective activation for β1-AMPK relative to β2-AMPK.
10. The method according to any one of claims 1 to 9, wherein the β-hemoglobinopathy is sickle cell disease (SCD).
11. The method according to any one of claims 1 to 9, wherein the β-hemoglobinopathy is β - thalassemia.
12. The method according to any one of claims 1 to 1, wherein the patient has an HbS/ β0 genotype, an HbS/μ+ genotype, an HBSC genotype, an HbS/HbE genotype, an HbD Los Angeles genotype, a G-Philadelphia genotype, or an abHbO Arab genotype.
13. The method according to any one of the preceding claims, wherein the compound of formula (I) is administered in combination with hydroxyurea.
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Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3119742A (en) 1962-12-19 1964-01-28 Smith Kline French Lab Method of preparing sustained release pharmaceutical pellets and product thereof
US3492397A (en) 1967-04-07 1970-01-27 Warner Lambert Pharmaceutical Sustained release dosage in the pellet form and process thereof
US3538214A (en) 1969-04-22 1970-11-03 Merck & Co Inc Controlled release medicinal tablets
US4060598A (en) 1967-06-28 1977-11-29 Boehringer Mannheim G.M.B.H. Tablets coated with aqueous resin dispersions
US4173626A (en) 1978-12-11 1979-11-06 Merck & Co., Inc. Sustained release indomethacin
WO2015091937A1 (en) 2013-12-19 2015-06-25 Sanofi Oxindole derivatives, preparation thereof and therapeutic use in the treatment of ampk-related diseases
WO2022072397A1 (en) * 2020-09-30 2022-04-07 Bioverativ Therapeutics Inc. Ampk activators and methods of use thereof

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3119742A (en) 1962-12-19 1964-01-28 Smith Kline French Lab Method of preparing sustained release pharmaceutical pellets and product thereof
US3492397A (en) 1967-04-07 1970-01-27 Warner Lambert Pharmaceutical Sustained release dosage in the pellet form and process thereof
US4060598A (en) 1967-06-28 1977-11-29 Boehringer Mannheim G.M.B.H. Tablets coated with aqueous resin dispersions
US3538214A (en) 1969-04-22 1970-11-03 Merck & Co Inc Controlled release medicinal tablets
US4173626A (en) 1978-12-11 1979-11-06 Merck & Co., Inc. Sustained release indomethacin
WO2015091937A1 (en) 2013-12-19 2015-06-25 Sanofi Oxindole derivatives, preparation thereof and therapeutic use in the treatment of ampk-related diseases
US10077237B2 (en) 2013-12-19 2018-09-18 Sanofi Oxindole derivatives, preparation thereof and therapeutic use in the treatment of AMPK-related diseases
WO2022072397A1 (en) * 2020-09-30 2022-04-07 Bioverativ Therapeutics Inc. Ampk activators and methods of use thereof

Non-Patent Citations (26)

* Cited by examiner, † Cited by third party
Title
"Gibaldi's Drug Delivery Systems in Pharmaceutical Care", 2007, AMERICAN SOCIETY OF HEALTH-SYSTEM PHARMACISTS
ALEDAVOOD ET AL., J CHEM INF MODEL, vol. 59, no. 6, 2019, pages 2859 - 2870
ALLALI, S. ET AL., HAEMATOLOGICA, vol. 105, no. 2, 2020, pages 273 - 283
BARABINO ET AL., ANNU REV BIOMED ENG, vol. 12, 2010, pages 345 - 67
CHANG ALICIA ET AL: "The Role of Adenosine Monophosphate Activated Protein Kinase Alpha 1 (AMPK) in Gamma-Globin Regulation", BLOOD, AMERICAN SOCIETY OF HEMATOLOGY, US, vol. 132, 29 November 2018 (2018-11-29), pages 413, XP086595885, ISSN: 0006-4971, DOI: 10.1182/BLOOD-2018-99-117092 *
GIANNAKOU, M.E. ET AL., SCIENCE, vol. 305, no. 5682, 2004, pages 361
KATO, G.J. ET AL., NAT REV DIS PRIMERS, vol. 4, 2018, pages 18010
LADLI MERIEM ET AL: "Finely-tuned regulation of AMP-activated protein kinase is crucial for human adult erythropoiesis", HAEMATOLOGICA, vol. 104, no. 5, 1 May 2019 (2019-05-01), IT, pages 907 - 918, XP055877914, ISSN: 0390-6078, Retrieved from the Internet <URL:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6518903/pdf/1040907.pdf> DOI: 10.3324/haematol.2018.191403 *
LIANG, R. ET AL., PLOS GENET, vol. 11, no. 10, 2015, pages el005526
MARINKOVIC, D. ET AL., J CLIN INVEST, vol. 117, no. 8, 2007, pages 2133 - 44
MORRIS, B.J. ET AL., GERONTOLOGY, vol. 61, no. 6, 2015, pages 515 - 25
PIEL, F.B. ET AL., LANCET, vol. 381, no. 9861, 2013, pages 142 - 51
PLATT ET AL., N ENGL J MED, vol. 358, no. 13, 2008, pages 1362 - 9
POURFARZAD, F. ET AL., CELL REP, vol. 4, no. 3, 2013, pages 589 - 600
RIMMELE, P. ET AL., EMBO REP, vol. 16, no. 9, 2015, pages 1164 - 76
RIMMELE, P. ET AL., STEM CELL REPORTS, vol. 3, no. 1, 8 July 2014 (2014-07-08), pages 44 - 59
SAG, D. ET AL., J IMMUNOL, vol. 181, no. 12, 2008, pages 8633 - 41
STEFANETTI, R.J ET AL., F1000RES, 2018, pages 7
STEINBERG, M.H. ET AL., JAMA, vol. 289, no. 13, 2003, pages 1645 - 51
VAN BEERS, E.J. ET AL., CIRC RES, vol. 116, no. 2, 2015, pages 298 - 306
VIOLLET ET AL., CRIT REV BIOCHEM MOL BIOL, vol. 45, no. 4, 2010, pages 276 - 95
VONA, R. ET AL., ANTIOXIDANTS (BASEL, vol. 10, no. 2, 2021
WIL LIAMS T. ET AL., PROC NATL ACAD SCI U S A, vol. 108, no. 14, 2011, pages 5849 - 54
YALCIN, S. ET AL., J BIOL CHEM, vol. 283, no. 37, 2008, pages 25692 - 25705
YE, L. ET AL., PROC NATL ACAD SCI U S A, vol. 113, no. 38, 2016, pages 10661 - 5
ZHANG, Y. ET AL., BLOOD, vol. 132, no. 3, 2018, pages 321 - 333

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