WO2010059401A2 - Compounds that expand hematopoietic stem cells - Google Patents

Compounds that expand hematopoietic stem cells Download PDF

Info

Publication number
WO2010059401A2
WO2010059401A2 PCT/US2009/062646 US2009062646W WO2010059401A2 WO 2010059401 A2 WO2010059401 A2 WO 2010059401A2 US 2009062646 W US2009062646 W US 2009062646W WO 2010059401 A2 WO2010059401 A2 WO 2010059401A2
Authority
WO
WIPO (PCT)
Prior art keywords
purin
ethyl
isopropyl
ylamino
pyridin
Prior art date
Application number
PCT/US2009/062646
Other languages
French (fr)
Other versions
WO2010059401A3 (en
Inventor
Anthony Boitano
Michael Cooke
Shifeng Pan
Peter G. Schultz
John Tellew
Yongqin Wan
Xing Wang
Original Assignee
Irm Llc
The Scripps Research Institute
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Family has litigation
First worldwide family litigation filed litigation Critical https://patents.darts-ip.com/?family=42111339&utm_source=google_patent&utm_medium=platform_link&utm_campaign=public_patent_search&patent=WO2010059401(A2) "Global patent litigation dataset” by Darts-ip is licensed under a Creative Commons Attribution 4.0 International License.
Priority to JP2011534784A priority Critical patent/JP5390626B2/en
Priority to EP09748626.0A priority patent/EP2350078B1/en
Priority to KR1020117012091A priority patent/KR101434719B1/en
Priority to LTEP09748626.0T priority patent/LT2350078T/en
Priority to MX2011004593A priority patent/MX347834B/en
Priority to AU2009317898A priority patent/AU2009317898B2/en
Priority to CA2740589A priority patent/CA2740589C/en
Priority to BRPI0921799A priority patent/BRPI0921799B8/en
Priority to EA201100663A priority patent/EA019872B1/en
Application filed by Irm Llc, The Scripps Research Institute filed Critical Irm Llc
Priority to NZ592435A priority patent/NZ592435A/en
Priority to CN200980143651.3A priority patent/CN102203096B/en
Priority to PL09748626T priority patent/PL2350078T3/en
Priority to SI200931987T priority patent/SI2350078T1/en
Priority to DK09748626.0T priority patent/DK2350078T3/en
Priority to EP19164770.0A priority patent/EP3524604B1/en
Priority to CU2011000096A priority patent/CU24495B1/en
Priority to RS20190863A priority patent/RS59027B1/en
Priority to ES09748626T priority patent/ES2736728T3/en
Priority to UAA201105290A priority patent/UA103206C2/en
Publication of WO2010059401A2 publication Critical patent/WO2010059401A2/en
Publication of WO2010059401A3 publication Critical patent/WO2010059401A3/en
Priority to TN2011000174A priority patent/TN2011000174A1/en
Priority to IL212368A priority patent/IL212368A/en
Priority to SM201100024T priority patent/SMP201100024B/en
Priority to MA33889A priority patent/MA32828B1/en
Priority to HRP20191238 priority patent/HRP20191238T1/en
Priority to CY20191100765T priority patent/CY1121857T1/en

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • 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
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A61K31/52—Purines, e.g. adenine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D473/00—Heterocyclic compounds containing purine ring systems
    • C07D473/02—Heterocyclic compounds containing purine ring systems with oxygen, sulphur, or nitrogen atoms directly attached in positions 2 and 6
    • C07D473/16—Heterocyclic compounds containing purine ring systems with oxygen, sulphur, or nitrogen atoms directly attached in positions 2 and 6 two nitrogen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D473/00—Heterocyclic compounds containing purine ring systems
    • C07D473/26—Heterocyclic compounds containing purine ring systems with an oxygen, sulphur, or nitrogen atom directly attached in position 2 or 6, but not in both
    • C07D473/32—Nitrogen atom
    • C07D473/34—Nitrogen atom attached in position 6, e.g. adenine
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/04—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D495/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms
    • C07D495/02—Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms in which the condensed system contains two hetero rings
    • C07D495/04—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0647—Haematopoietic stem cells; Uncommitted or multipotent progenitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K2035/124—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells the cells being hematopoietic, bone marrow derived or blood cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/30—Hormones
    • C12N2501/38—Hormones with nuclear receptors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/60—Transcription factors

Definitions

  • the present invention relates to compounds and compositions for expanding the number of CD34+ cells for transplantation.
  • the invention further relates to a cell population comprising expanded hematopoietic stem cells (HSCs) and its use in autologous or allogeneic transplantation for the treatment of patients with inherited immunodeficient and autoimmune diseases and diverse hematopoietic disorders to reconstitute the hematopoietic cell lineages and immune system defense.
  • HSCs expanded hematopoietic stem cells
  • Hematopoietic stem cells are capable of regenerating all blood products throughout the life of an individual, balancing their self -renewal with progeny differentiation. Hematopoietic stem cells have therapeutic potential as a result of their capacity to restore blood and immune cells in transplant recipients. Furthermore, HSCs have the potential to generate cells for other tissues such as brain, muscle and liver. Human autologous and allogeneic bone marrow transplantation methods are currently used as therapies for leukemia, lymphoma, and other life-threatening diseases. For these procedures, a large number of stem cells must be isolated to ensure that there are enough HSCs for engraftment. The number of HSCs available for treatment is a clinical limitation. [0004] The present invention relates to compounds and compositions for expanding hematopoietic stem cell populations and uses thereof.
  • the present invention provides a compound of Formula I:
  • G 1 is selected from N and CR 3 ;
  • G 2 , G 3 and G 4 are independently selected from CH and N; with the proviso that at least 1 of G3 and G4 is N; with the proviso that Gi and G 2 are not both N;
  • L is selected from -NR 5 a(CH 2 )o- 3 - (0-3 herein means 0, 1 , 2 or 3), -
  • R 53 and R 5b are independently selected from hydrogen and C]_ 4 alkyl
  • Ri is selected from hydrogen, phenyl, thiophenyl, furanyl, IH- benzoimidazolyl, isoquinolinyl, lH-imidazopyridinyl, benzothiophenyl, pyrimidinyl, IH- pyrazolyl, pyridinyl, lH-imidazolyl, pyrrolidinyl, pyrazinyl, pyridazinyl, lH-pyrrolyl and thiazolyl; wherein said phenyl, thiophenyl, furanyl, lH-benzoimidazolyl, isoquinolinyl, IH- imidazopyridinyl, benzothiophenyl, pyrimidinyl, lH-pyrazolyl, pyridinyl, lH-imidazolyl, pyrrolidinyl, pyrazinyl, pyridazinyl, pyrid
  • R 2 is selected from -S(O) 2 NR 63 R 5I ,, -NR 9a C(O)R 9b , -NR 6a C(O)NR 6b R 6c , phenyl, lH-pyrrolopyridin-3-yl, lH-indolyl, thiophenyl, pyridinyl, lH-l,2,4-triazolyl, 2- oxoimidazolidinyl, lH-pyrazolyl, 2-oxo-2,3-dihydro-lH-benzoimidazolyl and lH-indazolyl; wherein R 63 , R ⁇ b and R 6 c are independently selected from hydrogen and Ci_ 4 alkyl; wherein said phenyl, lH-pyrrolopyridin-3-yl, lH-indolyl, thiophenyl, pyridinyl, lH-l,2,4-
  • R 4 is selected from Ci-ioalkyl, prop-l-en-2-yl, cyclohexyl, cyclopropyl, 2-(2- oxopyrrolidin-l-yl)ethyl, oxetan-3-yl, benzhydryl, tetrahydro-2H-pyran-3-yl, tetrahydro-2H- pyran-4-yl, phenyl, tetrahydrofuran-3-yl, benzyl, (4-pentylphenyl)(phenyl)methyl and l-(l-(2- oxo-6,9,12-trioxa-3-azatetradecan-14-yl)-lH-l,2,3-triazol-4-yl)ethyl; wherein said alkyl, cyclopropyl, cyclohexyl, 2-(2-oxopyrrolidin-l-yl)ethyl, o
  • Fig 1 discloses the PXRD pattern of solid form modification A of 4-(2-(2-)
  • Figs 2 to 12 disclose the PXRD patterns of solid forms of 4-(2-(2-)
  • Fig 13 discloses the DSC pattern of the amorphous form of A-(I-(I-
  • alkyl as a group and as a structural element of other groups, for example halo-substituted-alkyl and alkoxy, can be either straight-chained or branched.
  • alkyl includes methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, t-butyl, etc.
  • Ci_ 4 - alkoxy includes methoxy, ethoxy, and the like.
  • Halo-substituted alkyl includes trifluoromethyl, pentafluoroethyl, and the like.
  • Aryl means a monocyclic or fused bicyclic aromatic ring assembly containing six to ten ring carbon atoms.
  • aryl may be phenyl or naphthyl, preferably phenyl.
  • Arylene means a divalent radical derived from an aryl group.
  • heteroaryl includes pyridyl, indolyl, indazolyl, quinoxalinyl, quinolinyl, benzofuranyl, benzopyranyl, benzothiopyranyl, benzo[l,3]dioxole, imidazolyl, benzo-imidazolyl, pyrimidinyl, furanyl, oxazolyl, isoxazolyl, triazolyl, tetrazolyl, pyrazolyl, thienyl, etc.
  • Cycloalkyl means a saturated or partially unsaturated, monocyclic, fused bicyclic or bridged polycyclic ring assembly containing the number of ring atoms indicated.
  • C 3 _iocycloalkyl includes cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, etc.
  • C 3 _ 8 heterocycloalkyl as used in this application to describe compounds of the invention includes morpholino, pyrrolidinyl, piperazinyl, piperidinyl, piperidinylone, 2-Oxo-pyrrolidin-l-yl, l,4-dioxa-8-aza-spiro[4.5]dec-8-yl, etc.
  • Halogen (or halo) preferably represents chloro or fluoro, but may also be bromo or iodo.
  • Hematopoietic stem cells as used herein refer to immature blood cells having the capacity to self-renew and to differentiate into more mature blood cells comprising granulocytes (e.g., promyelocytes, neutrophils, eosinophils, basophils), erythrocytes
  • granulocytes e.g., promyelocytes, neutrophils, eosinophils, basophils
  • erythrocytes e.g., promyelocytes, neutrophils, eosinophils, basophils
  • HSCs are interchangeably described as stem cells throughout the specification. It is known in the art that such cells may or may not include CD34 + cells.
  • CD34 + cells are immature cells that express the CD34 cell surface marker. CD34+ cells are believed to include a subpopulation of cells with the stem cell properties defined above.
  • HSCs include pluripotent stem cells, multipotent stem cells (e.g., a lymphoid stem cell), and/or stem cells committed to specific hematopoietic lineages.
  • the stem cells committed to specific hematopoietic lineages may be of T cell lineage, B cell lineage, dendritic cell lineage, Langerhans cell lineage and/or lymphoid tissue-specific macrophage cell lineage.
  • HSCs also refer to long term HSC (LT-HSC) and short term HSC (ST-HSC). ST-HSCs are more active and more proliferative than LT-HSCs.
  • Hematopoietic stem cells are optionally obtained from blood products.
  • a blood product includes a product obtained from the body or an organ of the body containing cells of hematopoietic origin. Such sources include un-fractionated bone marrow, umbilical cord, peripheral blood, liver, thymus, lymph and spleen.
  • “Expansion” in the context of cells refers to increase in the number of a characteristic cell type, or cell types, from an initial cell population of cells, which may or may not be identical.
  • the initial cells used for expansion may not be the same as the cells generated from expansion.
  • Cell population refers to eukaryotic mammalian, preferably human, cells isolated from biological sources, for example, blood product or tissues and derived from more than one cell.
  • CD34+ cells refers to cells that express at their surface CD34 marker. CD34+ cells can be detected and counted using for example flow cytometry and fluorescently labeled anti-CD34 antibodies.
  • CD34+ cells “Enriched in CD34+ cells” means that a cell population has been selected based on the presence of CD34 marker. Accordingly, the percentage of CD34+ cells in the cell population after selection method is higher than the percentage of CD34+ cells in the initial cell population before selecting step based on CD34 markers.
  • CD34+ cells may represent at least 50%, 60%, 70%, 80% or at least 90% of the cells in a cell population enriched in CD34+ cells.
  • Cord blood unit refers to the blood collected from umbilical cord of a single birth.
  • the present invention relates to methods and compositions for expanding
  • HSC populations using an agent capable of down-regulating the activity and/or expression of aryl hydrocarbon receptor and/or a downstream effector of aryl hydrocarbon receptor pathway is a compound of Formula I.
  • said agent capable of down-regulating the activity and/or expression of aryl hydrocarbon receptor is a compound of Formula I.
  • compounds of Formula I are compounds selected from Formulae Ia, Ib, Ic, Id and Ie:
  • L is selected from -NR 53 (CH 2 ) ⁇ -, -NR 53 CH(C(O)OCH 3 )CH 2 -, -
  • R] is selected from hydrogen, phenyl, thiophen-2-yl, thiophen-3-yl, furan-3- yl, lH-benzo[d]imidazol-l-yl, isoquinolin-4-yl, lH-imidazo[4,5-b]pyridin-l-yl, benzo[b]thiophen-3-yl, pyrimidin-5-yl, lH-pyrazol-4-yl, pyridin-2-yl, pyridin-4-yl, lH-imidazol- 1-yl, pyrrolidin-1-yl, pyrazin-2-yl, pyridin-3-yl, pyridazin-4-yl, lH-pyrrol-2-yl and thiazol-5-yl; [0036] wherein said phenyl, thiophen-2-yl, thiophen-3-yl, furan-3-yl, IH
  • R 3 is selected from hydrogen, and biphenyl
  • R 4 is selected from isopropyl, methyl, ethyl, prop-l-en-2-yl, isobutyl, cyclohexyl, sec-butyl, (S)-sec -butyl, (R)-sec-butyl, l-hydroxypropan-2-yl, (S)-l-hydroxypropan- 2-yl, (R)- l-hydroxypropan-2-yl, nonan-2-yl, 2-(2-oxopyrrolidin-l-yl)ethyl, oxetan-3-yl, oxetan- 2-yl, benzhydryl, tetrahydro-2H-pyran-2-yl, phenyl, tetrahydrofuran-3-yl and benzyl; wherein said cyclohexyl, 2-(2-oxopyrrolidin-l-yl)ethyl, oxetan-3-yl, o
  • R 53 and R 5b are independently selected from hydrogen and methyl; and R] is selected from hydrogen, phenyl, thiophen-2-yl, thiophen-3- yl, furan-3-yl, lH-benzo[d]imidazol-l-yl, isoquinolin-4-yl, lH-imidazo[4,5-b]pyridin-l-yl, benzo[b]thiophen-3-yl, pyrimidin-5-yl, lH-pyrazol-4-yl, pyridin-2-yl, pyridin-4-yl, l
  • NR 5a (CH 2 )i_ 3 (where 1-3 herein 1, 2 or 3).
  • R 2 is selected from urea, phenyl, lH-indol-2-yl, IH- indol-3-yl, thiophen-3-yl, piperidin-1-yl, pyridin-2-yl, pyridin-3-yl, pyridin-4-yl, 1H-1,2,4- triazol-3-yl, lH-l,2,4-triazol-5-yl, 2-oxoimidazolidin-l-yl, lH-pyrazol-3-yl, lH-pyrazol-4-yl, 2- oxo-2,3-dihydro-lH-benzo[d]imidazol-5-yl, lH-benzo[d]imidazol-5-yl and lH-imidazol-4-yl; wherein said phenyl, lH-indol-2-yl, lH-indol-3-
  • R 3 is selected from hydrogen, methyl and biphenyl; and R 4 is selected from isopropyl, methyl, ethyl, prop-l-en-2-yl, isobutyl, cyclohexyl, sec -butyl, (S)-sec -butyl, (R)-sec-butyl, l-hydroxypropan-2-yl, (S)-l-hydroxypropan-2-yl, (R)-I- hydroxypropan-2-yl, nonan-2-yl, 2-(2-oxopyrrolidin-l-yl)ethyl, oxetan-3-yl, oxetan-2-yl, benzhydryl, tetrahydro-2H-pyran-2-yl, phenyl, tetrahydrofuran-3-yl and benzyl; wherein said cyclohexyl, 2-(2-oxopyrrolidin-l-
  • L is selected from -NR 53 (CH 2 ) ⁇ -, -NR 53 CH(C(O)OCH 3 )CH 2 -, -
  • Ri is selected from hydrogen, phenyl, thiophen-2-yl, thiophen-3-yl, furan-2- yl, furan-3-yl, benzo[b]thiophen-2-yl, benzo[b]thiophen-3-yl, benzofuran-2-yl, benzofuran-3-yl, pyrimidin-4-yl, pyrimidin-5-yl, lH-pyrazol-4-yl, lH-pyrazol-3-yl, pyridin-2-yl, pyridazin-3-yl, pyridin-4-yl, lH-imidazol-1-yl, pyrrolidin-1-yl, pyrazin-2-yl, pyridin-3-yl, lH-pyrazol-1-yl, pyridazin-4-yl, lH-indol-2-yl, thiazol-4-yl, lH-ind
  • R 3 is selected from hydrogen, Ci_ 4 alkyl and biphenyl
  • R 4 is selected from isopropyl, isobutyl, sec -butyl, l-hydroxypropan-2-yl, cyclopropyl, oxetan-3-yl, oxetan-2-yl, benzhydryl, piperidin-4-yl, piperidin-3-yl, piperidin-2-yl, tetrahydro-2H-pyran-2-yl, tetrahydro-2H-pyran-3-yl, tetrahydro-2H-pyran-4-yl, phenyl, tetrahydrofuran-3-yl, tetrahydrofuran-2-yl, benzyl, (4-pentylphenyl)(phenyl)methyl and l-(l-(2- oxo-6,9,12-trioxa-3-azatetradecan-14-yl)-lH-l,2,3-triazol-4-yl)ethyl;
  • L is selected from -NR 5 a(CH 2 )o_ 3 -, -NR 53 CH(C(O)OCH 3 )CH 2 -, -NR 5 a(CH 2 ) 2 NR 5 b-, -NR 5a (CH 2 ) 2 S- , -NR 53 CH 2 CH(CH 3 )CH 2 -, -NR 53 CH(CH 3 )CH 2 -, -(CH 2 ) 3 - -CH 2 OCH 2 -, -CH 2 NR 53 CH 2 -, - NR 53 C(O)CH 2 - and -NR 53 Y-; wherein R 53 and R 5b are independently selected from hydrogen and methyl; Y is selected from isoxazole and 1,3,4-oxadiazole.
  • L is -NR 5a (CH 2 ) 0 - 3 , it
  • NR 5a (CH 2 )i_ 3 (where 1-3 herein means 1, 2 or 3).
  • R] is selected from hydrogen, phenyl, thiophen-3-yl, thiophen-2-yl, furan-3-yl, furan-2-yl, benzo[b]thiophen-3-yl, pyrimidin-5-yl, pyridin-4-yl, pyridin-2-yl, pyrrolidin-1-yl, lH-pyrazol-4-yl, pyrazin-2-yl, pyridazin-3-yl, pyridazin-4-yl, IH- pyrazol-1-yl, lH-pyrazol-3-yl, lH-imidazol-1-yl, thiazol-4-yl, lH-pyrrol-2-yl, thiazol-5-yl, and pyridin-3-yl; wherein said phenyl, thiophen-3-yl, thiophen-2-yl, furan-3-yl, furan-2-yl, be
  • R 2 is selected from amino-sulfonyl, methyl-carbonyl- amino, methyl-sulfonyl-amino, amino-sulfonyl-oxy, urea, phenyl, lH-indol-2-yl, lH-indol-3-yl, benzo[b]thiophen-2-yl, benzo[b]thiophen-3-yl, benzofuran-2-yl, benzofuran-3-yl, thiophen-2-yl, thiophen-3-yl, furan-2-yl, furan-3-yl, piperidin-4-yl, piperidin-3-yl, piperidin-2-yl, piperidin-1- yl, pyridin-2-yl, pyridin-3-yl, pyridin-4-yl, lH-l,2,4-triazol-3-yl, lH-l,2,4-triazol-3-yl,
  • R 3 is selected from hydrogen, methyl, and biphenyl; and R 4 is selected from isopropyl, isobutyl, sec-butyl, l-hydroxypropan-2-yl, cyclopropyl, oxetan-3-yl, oxetan-2-yl, benzhydryl, piperidin-4-yl, piperidin-3-yl, piperidin-2-yl, tetrahydro- 2H-pyran-2-yl, tetrahydro-2H-pyran-3-yl, tetrahydro-2H-pyran-4-yl, phenyl, tetrahydrofuran-3- yl, tetrahydrofuran-2-yl, benzyl, (4-pentylphenyl)(phenyl)methyl and l-(l-(2-oxo-6,9,12-trioxa- 3-azatetradecan-14-yl)-
  • R 2 is selected from lH-indol-3-yl and phenyl optionally substituted with hydroxy
  • R 4 is selected from isopropyl, sec -butyl, benzhydryl, nonan-2-yl, oxetan-3-yl and tetrahydrofuran-3-yl.
  • R 2 is selected from: lH-pyrrolo[2,3-b]pyridin-3-yl; lH-indol-3-yl optionally substituted with 1 to 2 radicals independently selected from halo, methyl and methoxy; and phenyl optionally substituted with 1 to 2 radicals independently selected from methyl, halo and hydroxy;
  • R 4 is selected from isopropyl, sec-butyl, l-hydroxypropan-2-yl, prop- l-en-2-yl, benzhydryl, nonan-2-yl, oxetan-3-yl and tetrahydrofuran-3-yl; and Ra, Rb and Rc are independently selected from hydrogen, cyano, methyl, halo, -SO 2 CH 3 and trifluoromethyl.
  • [0064] in another embodiment is a method of using a compound of Formula I to stimulate the expansion of stem cells by increasing the number of divisions; said method comprising contacting the stem cells with a compound of Formula I.
  • in another embodiment is a method in which the expansion of stem cells is in vivo, in vitro, or ex vivo.
  • stem cells are human hematopoietic stem cells.
  • [0067] in another embodiment is a cell population with expanded hematopoietic stem cells, as obtained or obtainable by the method of the invention.
  • compositions comprising a cell population with expanded HSCs derived from one or two cord blood units, preferably one cord blood unit, wherein said composition contains a total amount of cells of at least 10 5 cells, 10 7 cells, 10 8 cells or 10 9 cells, and wherein between 20-100% of total cells are CD34+ cells, for example between
  • CD34+ 40-80% of total cells are CD34+.
  • the administration is an autologous transplantation and the hematopoietic disorder is selected from Multiple myeloma, Non-Hodgkin lymphoma,
  • Hodgkin disease Acute myeloid leukemia, Neuroblastoma, Germ cell tumors, Autoimmune disorders and Amyloidosis.
  • the autoimmune disorders are selected from
  • SLE Systemic lupus erythematosus
  • the administration is an allogeneic transplantation and the hematopoietic disorder is selected from Acute myeloid leukemia, Acute lymphoblastic leukemia, Chronic myeloid leukemia, Chronic lymphocytic leukemia, Myeloproliferative disorders, Myelodysplastic syndromes, Multiple myeloma, Non-Hodgkin lymphoma,
  • Hodgkin disease Aplastic anemia, Pure red cell aplasia, Paroxysmal nocturnal hemoglobinuria, Fanconi anemi, Thalassemia major, Sickle cell anemia, Severe combined immunodeficiency (SCID), Wiskott-Aldrich syndrome, Hemophagocytic lymphohistiocytosis (HLH) and inborn errors of metabolism.
  • SCID Severe combined immunodeficiency
  • HHLH Hemophagocytic lymphohistiocytosis
  • the inborn errors of metabolism are selected from mucopolysaccharidosis, Gaucher disease, metachromatic leukodystrophies and adrenoleukodys trophies .
  • in another embodiment is a method for treating an inhereited immunodeficient disease, an autoimmune disease and/or a hematopoietic disorder comprising administration to a patient in need of such treatment hematopoietic stem cells expanded by a compound as described in the Summary of the Invention.
  • the administration is an autologous transplantation and the hematopoietic disorder is selected from Multiple myeloma, Non-Hodgkin lymphoma, Hodgkin disease, Acute myeloid leukemia, Neuroblastoma, Germ cell tumors, Autoimmune disorders and Amyloidosis.
  • the autoimmune disorders are selected from
  • SLE Systemic lupus erythematosus
  • the administration is an allogeneic transplantation and the hematopoietic disorder is selected from Acute myeloid leukemia, Acute lymphoblastic leukemia, Chronic myeloid leukemia, Chronic lymphocytic leukemia, Myeloproliferative disorders, Myelodysplastic syndromes, Multiple myeloma, Non-Hodgkin lymphoma,
  • Hodgkin disease Aplastic anemia, Pure red cell aplasia, Paroxysmal nocturnal hemoglobinuria, Fanconi anemi, Thalassemia major, Sickle cell anemia, Severe combined immunodeficiency (SCID), Wiskott-Aldrich syndrome, Hemophagocytic lymphohistiocytosis (HLH) and inborn errors of metabolism.
  • SCID Severe combined immunodeficiency
  • HHLH Hemophagocytic lymphohistiocytosis
  • the inborn errors of metabolism are selected from mucopolysaccharidosis, Gaucher disease, metachromatic leukodystrophies and adrenoleukodys trophies .
  • HSCs are primitive cells capable of regenerating all blood cells. During development, hematopoiesis translocates from the fetal liver to the bone marrow, which then remains the site of hematopoiesis throughout adulthood. Once hematopoiesis has been established in the bone marrow, the HSCs are not distributed randomly throughout the bone cavity. Instead, they are found in close proximity to the endosteal surfaces. The more mature stem cells increase in number as the distance from the bone surface increases. Finally, as the central longitudinal axis of the bone is approached terminal differentiation of mature cells occurs.
  • HSC numbers are increased ex vivo.
  • a method of increasing stem cell numbers is important as currently, approximately 25% of autologous donor transplants are prohibited for lack of sufficient stem cells. In addition, less than 25% of patients in need of allogeneic transplant can find a histocompatible donor.
  • Umbilical cord blood banks currently exist and cover the broad racial make-up of the general population, but these banks are currently restricted to use in children due to inadequate stem cell numbers in the specimens for adult recipients.
  • a method to increase stem cell numbers permits cord blood to be useful for adult patients, thereby expanding the use of allogeneic transplantation.
  • Compounds of the invention can also be used to expand the progenitor cell numbers which are clinically useful, for example, to speed engraftment and decrease the duration of neutopenia.
  • an increase in HSCs means that the subject has at least one more HSC, a 10% increase, a 20% increase, a 30% increase or greater.
  • HSCs may consist of a subset of CD34+ cells, increase of HSCs can be measured indirectly by counting the number of CD34+ cells in a cell population and, optionally, by assessing the differentiation properties of the CD34+ cells by analyzing the colony forming units (CFU) as described in the experimental part below:
  • CFU colony forming units
  • the expanded population of HSCs is harvested, for example, from a bone marrow sample of a subject or from a culture.
  • Harvesting HSCs is defined as the dislodging or separation of cells. This is accomplished using a number of methods, such as enzymatic, non-enzymatic, centrifugal, electrical, or size-based methods, or preferably, by flushing the cells using culture media (e.g., media in which cells are incubated) or buffered solution.
  • culture media e.g., media in which cells are incubated
  • buffered solution e.g., buffered solution.
  • the cells are optionally collected, separated, and further expanded generating even larger populations of HSCs and differentiated progeny.
  • a method for making an expanded population of HSCs comprises contacting an agent capable of down-regulating the activity and/or expression of AHR and/or a downstream effector of AHR, e.g., a compound of the invention, with a starting cell population (i.e., an unexpanded population of cells) comprising a mixture of HSCs and optionally HSC supporting cells.
  • the administration step occurs ex vivo, in vivo and/or in vitro.
  • the expanded population of HSCs is optionally administered to a subject.
  • agent for HSC expansion e.g.
  • a compound of the invention may be formulated in DMSO or some other suitable carrier, "washed" from the cells and the cells may be transferred, for example, into an infusion buffer.
  • a DMSO formulation for example, can contain 0.3mg/ml of a compound of the invention in 60% DMSO/40% water solution.
  • HSC supporting cell refers to cells naturally found in the vicinity of one or more HSCs such that factors released by HSC supporting cells reach the HSC by diffusion, for example.
  • HSC supporting cells include, but are not limited to, lymphoreticular stromal cells.
  • Lymphoreticular stromal cells as used herein include, but are not limited to, all cell types present in a lymphoid tissue which are not lymphocytes or lymphocyte precursors or progenitors.
  • lymphoreticular stromal cells include osteoblasts, epithelial cells, endothelial cells, mesothelial cells, dendritic cells, splenocytes and macrophages.
  • Lymphoreticular stromal cells also include cells that would not ordinarily function as lymphoreticular stromal cells, such as fibroblasts, which have been genetically altered to secrete or express on their cell surface the factors necessary for the maintenance, growth or differentiation of HSCs, including their progeny.
  • Lymphoreticular stromal cells are optionally derived from the disaggregation of a piece of lymphoid tissue. Such cells are capable of supporting in vitro or in vivo the maintenance, growth or differentiation of HSCs, including their progeny.
  • lymphoid tissue it is meant to include bone marrow, peripheral blood (including mobilized peripheral blood), umbilical cord blood, placental blood, fetal liver, embryonic cells (including embryonic stem cells), aortal-gonadal-mesonephros derived cells, and lymphoid soft tissue.
  • Lymphoid soft tissue as used herein includes, but is not limited to, tissues such as thymus, spleen, liver, lymph node, skin, tonsil, adenoids and Peyer's patch, and combinations thereof.
  • Lymphoreticular stromal cells provide the supporting microenvironment in the intact lymphoid tissue for the maintenance, growth or differentiation of HSCs, including their progeny.
  • the microenvironment includes soluble and cell surface factors expressed by the various cell types which comprise the lymphoreticular stroma.
  • the support which the lymphoreticular stromal cells provide is characterized as both contact-dependent and non- contact-dependent.
  • Lymphoreticular stromal cells for example, are autologous (self) or non- autologous (non-self, e.g., heterologous, allogeneic, syngeneic or xenogeneic) with respect to HSCs.
  • Autologous refers to cells from the same subject.
  • Allogeneic refers to cells of the same species that differ genetically.
  • Syngeneic refers to cells of a different subject that are genetically identical to the cell in comparison.
  • Xenogeneic refers to cells of a different species.
  • Lymphoreticular stroma cells are obtained, for example, from the lymphoid tissue of a human or a non-human subject at any time after the organ/tissue has developed to a stage (i.e., the maturation stage) at which it can support the maintenance, growth or differentiation of HSCs.
  • the lymphoid tissue from which lymphoreticular stromal cells are derived usually determines the lineage-commitment HSCs undertake, resulting in the lineage-specificity of the differentiated progeny.
  • HSCs and progeny thereof
  • lymphoreticular stromal cells usually occurs under conditions known in the art (e.g., temperature, CO 2 and O 2 content, nutritive media, duration, etc.).
  • the time sufficient to increase the number of cells is a time that can be easily determined by a person skilled in the art, and varies depending upon the original number of cells seeded.
  • the amounts of HSCs and lymphoreticular stromal cells initially introduced (and subsequently seeded) varies according to the needs of the experiment. The ideal amounts are easily determined by a person skilled in the art in accordance with needs.
  • a subject is meant an individual.
  • subjects include, for example, domesticated animals, such as cats and dogs, livestock (e.g., cattle, horses, pigs, sheep, and goats), laboratory animals (e.g., mice, rabbits, rats, and guinea pigs), mammals, non-human mammals, primates, non-human primates, rodents, birds, reptiles, amphibians, fish, and any other animal.
  • livestock e.g., cattle, horses, pigs, sheep, and goats
  • laboratory animals e.g., mice, rabbits, rats, and guinea pigs
  • mammals non-human mammals, primates, non-human primates, rodents, birds, reptiles, amphibians, fish, and any other animal.
  • the subject is optionally a mammal such as a primate or a human.
  • the invention therefore relates to a method for expanding hematopoietic stem cells, comprising (a) providing a starting cell population comprising hematopoietic stem cells and (b) culturing said starting cell population ex vivo in presence of an agent capable of down- regulating the activity and/or expression of aryl hydrocarbon receptor and/or a down-stream effector of aryl hydrocarbon receptor pathway, under suitable conditions for expanding hematopoietic stem cells.
  • aryl hydrocarbon (dioxin) receptor is a cytosolic ligand-activated transcription factor known to mediate a large number of toxic and carcinogenic effects in animals and possible in human (Safe S 2001 Toxicol Lett 120: 1-7).
  • phase I xenobiotic -metabolizing enzymes such as the cytochromes P450 CYPlAl, CYP 1A2, CYPlBl and CYP2S1, and the phase II enzymes UDP-glucuronosyltransf erase UGT1A6, NAD(P)H-dependent quinone oxidoreductase- 1 (NQOl), the aldehyde dehydrogenase ALDH3A1, and several glutathione-S-transferase.
  • phase I xenobiotic -metabolizing enzymes such as the cytochromes P450 CYPlAl, CYP 1A2, CYPlBl and CYP2S1, and the phase II enzymes UDP-glucuronosyltransf erase UGT1A6, NAD(P)H-dependent quinone oxidoreductase- 1 (NQOl), the aldehyde dehydrogenase ALDH3A1, and several glutathione
  • an agent capable of down-regulating the activity and/or expression of aryl hydrocarbon receptor and/or a down-stream effector of aryl hydrocarbon receptor pathway is selected among the group consisting of: (i) an organic compound; (ii) a small interference RNA (siRNA) molecule capable of down-regulating the expression of AHR; and (iii) antisense oligonucleotide capable of down-regulating the expression of AHR.
  • siRNA small interference RNA
  • said method for expanding hematopoietic stem cells comprises (a) providing a starting cell population comprising hematopoietic stem cells and (b) culturing said starting cell population ex vivo in the presence of an agent capable of down- regulating the activity and/or expression of aryl hydrocarbon receptor and/or a down-stream effector of aryl hydrocarbon receptor pathway, under suitable conditions for expanding hematopoietic stem cells, wherein said agent capable of down-regulating the activity and/or expression of aryl hydrocarbon receptor and/or a down-stream effector of aryl hydrocarbon receptor pathway is not alpha-napthoflavone or 3'-methoxy-4'-nitroflavone.
  • AHR antagonist Organic compound that inhibits AHR activity (also referred herein as AHR antagonist) have been described in the art, for example 2-methyl-2H-pyrazole-3-carboxylic acid (2-methyl-4-o-tolylazophenyl)amide (CH223191), alpha napthoflavone, resveratrol (Nutr. Metab. Cardiovasc. Dis., 2003 Apr; 13(2): 104-13), 3 ' -methoxy-4'-nitroflavone (Biochem. Pharmacol., 2007 May 15; 73(10): 1622-34, Epub 2007 Jan 30), and 6-methyl-l,3,8- trichlorodibenzofuran (Cancer Res., 2004, Apr 15;64(8):2889-97).
  • 2-methyl-2H-pyrazole-3-carboxylic acid (2-methyl-4-o-tolylazophenyl)amide CH223191
  • alpha napthoflavone alpha napthoflavone
  • resveratrol Nu
  • An inhibitor of AHR activity refers to a compound which decreases AHR activity to at least 10%, 20%, 30%, 50%, 60%, 70%, 80% or at least 90% the transcriptional activity of AHR as observed under activated conditions.
  • An assay to measure AHR inhibitory activity is for example the dioxin-induced AHR dependent luciferase reporter gene assay as described in the Examples.
  • an inhibitor of AHR activity is a compound that has an EC50 of less than lO ⁇ M, preferably less than 5 ⁇ M as measured in the dioxin-induced AHR dependent luciferase reporter gene assay.
  • AHR is a transcriptional factor regulating the transcription of various genes in human.
  • a downstream effector of AHR pathway is a gene which is directly regulated at the transcriptional level by AHR.
  • genes are selected from CyplBl, CyplAl, and AHRR.
  • AHR also functions in pathways outside of its well- characterized role in xenobiotic enzyme induction.
  • Xenobiotic ligands of AHR have been shown to regulate beta catenin, STAT5, STATl, HES-I, c-Myc, C/EBP, PU.l, ⁇ -catenin, p21, P27, pRb, deoxynucleotidyl transferase, CXCR4, and its chemokine ligand CXCL12 (SDF-I).
  • an agent capable of down-regulating the activity and/or expression of aryl hydrocarbon receptor is a compound as defined in the Summary of the Invention.
  • an agent capable of down-regulating the activity and/or expression of aryl hydrocarbon receptor is an antisense oligonucleotide or a small interfering RNA molecule (siRNA), capable of down-regulating AHR protein expression or the protein expression of one more down-stream effectors of AHR.
  • siRNA small interfering RNA molecule
  • RNAi molecules suitable for use with the present invention can be affected as follows: First, the AHR mRNA sequence (or one or more of its down-stream effectors) is scanned downstream of the AUG start codon for AA-dinucleotide sequences. Occurrence of each AA and the 19 3'-adjacent is recorded as a potential siRNA target site. Then, potential target sites are compared to an appropriate genomic database (e.g, human, mouse, rat, etc.) using any sequence alignment software. Putative target site that exhibit significant homology to other coding sequences are filtered out. Preferred sequences are then those including low G/C content, in particular sequences with G/C content lower than 55%.
  • an appropriate genomic database e.g, human, mouse, rat, etc.
  • target sites are then selected along the length of the target gene. Methods or algorithms to identify putative target site of siRNA are described for example in (Tilesi, et al., Curr. Opin. MoI. Ther. 11: 156, 2009).
  • siRNA molecules which are capable of down-regulating the expression of AHR are: AHR 11 IS, 5' GCG GCA TAG AGA CCG ACT TAA TTT CAA GAG AAT TAA GTC GGT CTC TAT GCC GCT TTT TTG G 3' ; AHR 11 IAS, 5' CGC GCC AAA AAA GCG GCA TAG AGA CCG ACT TAA TTC TCT TGA AAT TAA GTC GGT CTC TAT GCC GC 3' ; AHR 242S, 5' GGC TTC TTT GAT GTT GCA TTA ATT CAA GAG ATT AAT GCA ACA TCA AAG AAG CCT TTT TTG G 3' ; AHR 242AS, 5' CGC GCC AAA AAA GGC TTC TTT GAT GTT GCA TTA ATC TCT TGA ATT AAT GCA ACA TCA AAG AAG CCT TTT TTG G 3' ; AHR 242AS, 5' CGC GCC AAA AAA GGC TTC T
  • the starting cell population comprising hematopoietic stem cells will be selected by the person skilled in the art depending on the envisaged use.
  • Various sources of cells comprising hematopoietic stem cells have been described in the art, including bone marrow, peripheral blood, neonatal umbilical cord blood, placenta or other sources such as liver, particularly fetal liver.
  • the cell population may first be subjected to enrichment or purification steps, including negative and/or positive selection of cells based on specific cellular markers in order to provide the starting cell population.
  • Methods for isolating said starting cell population based on specific cellular markers may use fluorescent activated cell sorting (FACS) technology also called flow cytometry or solid or insoluble substrate to which is bound antibodies or ligands that interact with specific cell surface markers.
  • FACS fluorescent activated cell sorting
  • cells may be contacted with a solid substrate (e.g., column of beads, flasks, magnetic particles) containing the antibodies and any unbound cells are removed.
  • a solid substrate comprising magnetic or paramagnetic beads
  • cells bound to the beads can be readily isolated by a magnetic separator.
  • said starting cell population is enriched in a desirable cell marker phenotype (e.g., CD34+, CD133+, CD90+) or based on efflux of dyes such as rhodamine, Hoechst or aldehyde dehydrogenase activity.
  • a desirable cell marker phenotype e.g., CD34+, CD133+, CD90+
  • said starting cell population is enriched in CD34+ cells.
  • Methods for enriching blood cell population in CD34+ cells include kits commercialized by Miltenyi Biotec (CD34+ direct isolation kit,
  • the amount of cord blood from a single birth is often inadequate to treat an adult or an older child.
  • One advantage of the expansion methods using the compounds of the invention, or an agent capable of down-regulating the activity and/or expression of aryl hydrocarbon receptor and/or a down-stream effector of aryl hydrocarbon receptor pathway is that it enables the production of a sufficient amount of hematopoietic stem cells from only one cord blood unit.
  • the starting cell population is derived from neonatal umbilical cord blood cells which have been enriched in CD34+ cells.
  • said starting cell population is derived from one or two umbilical cord blood units.
  • the starting cell population is derived from human mobilized peripheral blood cells which have been enriched in CD34+ cells.
  • said starting cell population is derived from human mobilized peripheral blood cells isolated from only one patient.
  • Said starting cell population may preferably contain at least 50% CD34+ cells, in some embodiments, more than 90% of CD34+ cells, and may comprise between 10 5 and 10 9 nucleated cells.
  • the starting cell population may be used directly for expansion or frozen and stored for use at a later date.
  • Conditions for culturing the starting cell population for hematopoietic stem cell expansion will vary depending, inter alia, on the starting cell population, the desired final number of cells, and desired final proportion of HSCs.
  • the culturing conditions comprises the use of other cytokines and growth factors, generally known in the art for hematopoietic stem cell expansion.
  • cytokines and growth factors include without limitation IL-I, IL-3, IL-6, IL-I l,
  • analogs include any structural variants of the cytokines and growth factors having the biological activity as the naturally occurring forms, including without limitation, variants with enhanced or decreased biological activity when compared to the naturally occurring forms or cytokine receptor agonists such as an agonist antibody against the TPO receptor (for example, VB22B sc(Fv)2 as detailed in patent publication WO 2007/145227, and the like). Cytokine and growth factor combinations are chosen to expand HSC and progenitor cells while limiting the production of terminally differentiated cells.
  • one or more cytokines and growth factors are selected from the group consisting of SCF, Flt3-L and TPO.
  • at least TPO is used in a serum-free medium under suitable conditions for HSC expansion.
  • a mixture of IL6, SCF, Flt3-L and TPO is used in the method for expanding HSCs in combination with the compound of the invention or an agent capable of down-regulating the activity and/or expression of aryl hydrocarbon receptor and/or a down-stream effector of aryl hydrocarbon receptor pathway.
  • Human IL6 or interleukin-6 also known as B-cell stimulatory factor 2 has been described by (Kishimoto, Ann. review of Imm. 23: 1 2005) and is commercially available.
  • Human SCF or stem cell factor also known as c-kit ligand, mast cell growth factor or Steel factor has been described (Smith, MA et al., ACTA Haematologica, 105, 3: 143, 2001) and is commercially available.
  • Flt3-L or FLT-3 Ligand also referred as FL is a factor that binds to flt3- receptor. It has been described (Hannum C, Nature 368 (6472): 643-8) and is commercially available.
  • TPO or thrombopoietin also known as megakarayocyte growth factor (MGDF) or c- MpI ligand has been described (Kaushansky K (2006). N. Engl. J. Med. 354 (19): 2034-45) and is commercially available.
  • MGDF megakarayocyte growth factor
  • c- MpI ligand has been described (Kaushansky K (2006). N. Engl. J. Med. 354 (19): 2034-45) and is commercially available.
  • the expansion of HSC may be carried out in a basal medium, which is supplemented with the mixtures of cytokines and growth factors described above.
  • a basal medium typically comprises amino acids, carbon sources, vitamins, serum proteins (e.g. albumin), inorganic salts, divalent cations, buffers and any other element suitable for use in expansion of HSC.
  • the compound of the invention or the agent capable of down-regulating the activity and/or expression of aryl hydrocarbon receptor and/or a downstream effector of aryl hydrocarbon receptor pathway is administered during the expansion method of said starting cell population under a concentration appropriate for HSC expansion.
  • said compound or AhR modulating agent is administered at a concentration comprised between IpM and lOO ⁇ M, for example between lOpM and lO ⁇ M, or between lOOpM and l ⁇ M.
  • starting cell population essentially consists of CD34+ enriched cells from one or two cord blood units
  • the cells are grown under conditions for HSC expansion from about 3 days to about 90 days, for example between 7 and 2 days and/or until the indicated fold expansion and the characteristic cell populations are obtained.
  • the cells are grown under conditions for HSC expansion not more than 21 days, 14 days or 7 days.
  • the starting cell population is cultured during a time sufficient to reach an absolute number of CD34+ cells of at least 10 5 , 10 6 , 10 7 , 10 8 or 10 9 cells. In another embodiment, said starting cell population is cultured during a time sufficient for a 10 to 50000 fold expansion of CD34+ cells, for example between 100 and 10000 fold expansion.
  • the cell population obtained after the expansion method may be used without further purification or may be subject to further purification or selection steps.
  • the cell population may then be washed to remove the compound of invention or any other agent capable of down-regulating the activity and/or expression of aryl hydrocarbon receptor and/or a down-stream effector of aryl hydrocarbon receptor pathway and/or any other components of the cell culture and resuspended in an appropriate cell suspension medium for short term use or in a long-term storage medium, for example a medium suitable for cryopreservation.
  • the invention further provides a cell population with expanded HSCs, obtainable or obtained by the expansion method described above.
  • such cell population is resuspended in a pharmaceutically acceptable medium suitable for administration to a mammalian host, thereby providing a therapeutic composition.
  • the compound as defined in the Summary of the Invention or an agent capable of down-regulating the activity and/or expression of aryl hydrocarbon receptor and/or a down-stream effector of aryl hydrocarbon receptor pathway enables the expansion of HSCs, for example from only one or two cord blood units, to provide a cell population quantitatively and qualitatively appropriate for efficient short and long term engraftment in human patient in need thereof.
  • the invention relates to a composition
  • a composition comprising a cell population with expanded HSCs derived from not more than one or two cord blood units, wherein said therapeutic composition contains a total amount of cells of at least 10 5 , 10 6 , 10 7 , 10 8 or 10 9 cells, with between 20-100%, for example between 40-80% of total cells being CD34+ cells.
  • said composition contains between 0.1-40%, for example between 0.1-10% of total cells being CD34+ Thyl+ and 20-80% of cells being CD34+ CD45RA+.
  • said composition contains between 10-95% of cells being CD38+ and between 5-70% of cells being CD133+.
  • the invention further provides the cell population with expanded HSCs or its composition for use in allogeneic or autologous stem cell transplantation in a mammalian subject.
  • the subject referred to herein is, for example, a bone marrow donor or an individual with or at risk for depleted or limited blood cell levels.
  • the subject is a bone marrow donor prior to bone marrow harvesting or a bone marrow donor after bone marrow harvesting.
  • the subject is optionally a recipient of a bone marrow transplant.
  • the methods described herein are particularly useful in subjects that have limited bone marrow reserve such as elderly subjects or subjects previously exposed to an immune depleting treatment or myeloablative treatment such as chemotherapy, e.g., for treating leukemia or lymphomas.
  • the subject optionally, has a decreased blood cell level or is at risk for developing a decreased blood cell level as compared to a control blood cell level.
  • control blood cell level refers to an average level of blood cells in a subject prior to or in the substantial absence of an event that changes blood cell levels in the subject.
  • An event that changes blood cell levels in a subject includes, for example, anemia, trauma, chemotherapy, bone marrow transplant and radiation therapy.
  • the subject has anemia or blood loss due to, for example, trauma.
  • the expanded HSC population or the composition comprising the cell population with expanded HSCs is administered to the subject, for example, before, at the same time, or after chemotherapy, radiation therapy or a bone marrow transplant.
  • the subject optionally has depleted bone marrow related to, for example, congenital, genetic or acquired syndrome characterized by bone marrow loss or depleted bone marrow.
  • the subject is optionally a subject in need of hematopoiesis.
  • the subject is a bone marrow donor or is a subject with or at risk for depleted bone marrow.
  • Hematopoietic stem cell manipulation is useful as a supplemental treatment to chemotherapy or radiation therapy.
  • HSCs are localized into the peripheral blood and then isolated from a subject that will undergo chemotherapy, and after the therapy the cells are returned.
  • the subject is a subject undergoing or expected to undergo an immune cell depleting treatment such as chemotherapy, radiation therapy or serving as a donor for a bone marrow transplant.
  • Bone marrow is one of the most prolific tissues in the body and is therefore often the organ that is initially damaged by chemotherapy drugs and radiation. The result is that blood cell production is rapidly destroyed during chemotherapy or radiation treatment, and chemotherapy or radiation must be terminated to allow the hematopoietic system to replenish the blood cell supplies before a patient is re -treated with chemotherapy. Therefore, as described herein, HSCs or blood cells made by the methods described herein are optionally administered to such subjects in need of additional blood cells.
  • a therapeutic capable of enhancing the proliferation of HSCs in vivo, in vitro, or ex vivo for example, a small molecule, an antibody, or the like
  • a therapeutic capable of enhancing HSC proliferation is meant: an agonist antibody against the TPO receptor (for example, VB22B sc(Fv)2 as detailed in patent publication WO 2007/145227, and the like); a cytokine such as SCF, IL-6, Flt-3 ligand, TPO or a TPO mimetic (for example, such as described in WO/2007/022269; WO/2007/009120; WO/2004/054515; WO/2003/103686; WO/2002/085343; WO/2002/049413; WO/2001/089457; WO/2001/039773; WO/2001/034585; WO/2001/021180; WO/2001/021180; WO/2001/017349; WO/2000/066112; WO/2000/035446; WO/2000/028987; WO/2008/028645; and the like); granulocyte colony stimulating factor (G-CSF
  • pharmaceutically acceptable is meant a material that is not biologically or otherwise undesirable, i.e., the material may be administered to a subject or cell, without causing undesirable biological effects or interacting in a deleterious manner with the other components of the pharmaceutical composition in which it is contained.
  • the carrier or excipient is selected to minimize degradation of the active ingredient and to minimize adverse side effects in the subject or cell.
  • compositions are formulated in any conventional manner for use in the methods described herein. Administration is via any route known to be effective by one of ordinary skill.
  • the compositions is administered orally, parenterally (e.g., intravenously), by intramuscular injection, by intraperitoneal injection, transdermally, extracorporeally, intranasally or topically.
  • the preferred method of administration is intravenous infusion.
  • the number of cells transfused will take into consideration factors such as sex, age, weight, the types of disease or disorder, stage of the disorder, the percentage of the desired cells in the cell population and the amount of cells needed to produce a therapeutic benefit.
  • the composition is administered by intravenous infusion and comprises at least 10 4 cells/kg, from 10 5 to 5.10 7 cells/kg or more if necessary.
  • the infused cells are all deriving from expanded cord blood cells from a single birth.
  • a pharmaceutically acceptable carrier for infusion of a composition comprising cells into a patient typically comprise buffered saline with 5%HSA or unsupplemented basal medium or medium as known in the art.
  • compositions take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulphate).
  • binding agents e.g., pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose
  • fillers e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate
  • lubricants e.g., magnesium stearate, talc or silica
  • disintegrants e.g., potato starch or sodium star
  • Liquid preparations for oral administration take the form of, for example, solutions, syrups or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use.
  • Such liquid preparations are prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g., methyl or propyl -p-hydroxybenzoates or sorbic acid).
  • the preparations optionally contain buffer salts, flavoring, coloring and sweetening agents as appropriate.
  • compositions are formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion.
  • Formulations for injection are presented in unit dosage form, e.g., in ampules or in multi-dose containers, with or without an added preservative.
  • the compositions take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • the active ingredient is in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
  • water suitable oil, saline, aqueous dextrose (glucose), and related sugar solutions and glycols such as propylene glycol or polyethylene glycols are suitable carriers for parenteral solutions.
  • Solutions for parenteral administration contain, for example, a water soluble salt of the active ingredient, suitable stabilizing agents and, if necessary, buffer substances.
  • Antioxidizing agents such as sodium bisulfate, sodium sulfite or ascorbic acid, either alone or combined, are suitable stabilizing agents.
  • citric acid and its salts and sodium ethylenediaminetetraacetic acid (EDTA) are optionally used.
  • parenteral solutions optionally contain preservatives such as benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol.
  • preservatives such as benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol.
  • Suitable pharmaceutical carriers are described in Remington: The Science and Practice of Pharmacy, 21st Edition, David B. Troy, ed., Lippicott Williams & Wilkins (2005), which is incorporated by reference in its entirety at least for the material related to pharmaceutical carriers and compositions.
  • the compositions are optionally formulated as a depot preparation. Such long acting formulations are optionally administered by implantation.
  • compositions are formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
  • suitable polymeric or hydrophobic materials for example as an emulsion in an acceptable oil
  • ion exchange resins for example, as an ion exchange resin
  • sparingly soluble derivatives for example, as a sparingly soluble salt.
  • the compositions are applied to or embedded with implants concurrent with or after surgical implant.
  • control release preparations for example, polymers, polyesters, polyamino acids, polyvinyl, pyrolidone, ethylenevinylacetate, methyl cellulose, carboxymethyl cellulose or protamine sulfate.
  • concentration of macromolecules as well as the methods of incorporation are adjusted in order to control release.
  • the agent is incorporated into particles of polymeric materials such as polyesters, polyamino acids, hydrogels, poly (lactic acid) or ethylenevinylacetate copolymers. In addition to being incorporated, these agents are optionally used to trap the compound in microcapsules.
  • a composition for use in the methods described herein is optionally formulated as a sustained and/or timed release formulation.
  • sustained and/or timed release formulations are made by sustained release means or delivery devices that are well known to those of ordinary skill in the art.
  • the compositions are used to provide slow or sustained release of one or more of the active ingredients using, for example, hydropropylmethyl cellulose, other polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, liposomes, microspheres or a combination thereof to provide the desired release profile in varying proportions.
  • Suitable sustained release formulations are selected for use with the compositions described herein.
  • single unit dosage forms suitable for oral administration such as, but not limited to, tablets, capsules, gelcaps, caplets, powders that are adapted for sustained release are used.
  • compositions are optionally delivered by a controlled-release system.
  • the composition is administered using intravenous infusion, an implantable osmotic pump, liposomes, or other modes of administration.
  • a controlled release system is placed in proximity to the target.
  • the composition is administered by injection into the bone marrow of a long bone, for example.
  • Local administration is achieved, for example, by local infusion during surgery, topical application (e.g., in conjunction with a wound dressing after surgery), injection, catheter, suppository, or implant.
  • An implant is of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers.
  • compositions described herein are provided in a pharmaceutically acceptable form including pharmaceutically acceptable salts and derivatives thereof.
  • pharmaceutically acceptable form refers to compositions including the compounds described herein that are generally safe, relatively non-toxic and neither biologically nor otherwise undesirable. These compositions optionally include pharmaceutically acceptable carriers or stabilizers that are nontoxic to the cell or subject being exposed thereto at the dosages and concentrations employed.
  • physiologically acceptable carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as TWEENTM (Uniqema, United Kingdom), polyethylene glycol (PEG), and PLURONICSTM (BASF, Germany).
  • buffers such as phosphate, citrate, and other organic acids
  • antioxidants including ascorbic acid
  • pharmaceutically acceptable acid salts and derivatives refers to salts and derivatives of the compounds of Formula I described herein that retain the biological effectiveness and properties as described, and that are not biologically or otherwise undesirable.
  • Pharmaceutically acceptable salts are formed, for example, with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, and organic acids such as acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and the like.
  • compositions comprising a compound of Formula I or a pharmaceutically acceptable salt or ester thereof are enhanced by methods known to those of skill in the art. For example, an alkanoic acid ester of a polyethoxylated sorbitol (a polysorbate) is added to a composition containing a compound of Formula I in an amount effective to enhance the chemical stability of the compound.
  • a polyethoxylated sorbitol a polysorbate
  • the data obtained from the cell culture assays and animal studies are optionally used in formulating a range of dosage for use in humans.
  • the dosage of such compounds lies preferably within a range of circulating concentrations that include little or no toxicity.
  • the dosage varies within this range depending upon the dosage form employed and the route of administration utilized.
  • the therapeutically effective dose is estimated initially from cell culture assays.
  • a pack or kit comprising one or more containers filled with one or more of the ingredients described herein.
  • Such kits optionally comprise solutions and buffers as needed or desired.
  • the kit optionally includes an expanded population of stem cells made by the methods described above or can contain containers or compositions for making an expanded population of HSCs.
  • the invention provides a kit for expanding ex vivo hematopoietic stem cells, comprising a compound as defined in the Summary of Invention and instructions for use of such compound in a method for HSC expansion and, optionally, one ore more cytokines or growth factors, or media for cell growth, in particular media for hematopoietic stem cell growth as described above.
  • the kit may further comprise antibodies for monitoring production of the cells, such as anti-CD34, anti-CD133, anti-CD38, anti-CD45RA and/or anti- Thyl antibodies.
  • such kit further include one or more cytokines or growth factors selected from the group consisting of IL6, FLT3-L, SCF and TPO.
  • Optionally associated with such pack(s) or kit(s) are instructions for use.
  • kits for providing an effective amount of a compound of the invention to increase HSCs in a subject comprising one or more doses of the compound for use over a period of time, wherein the total number of doses of the compound of the invention in the kit equals the effective amount sufficient to increase HSCs in a subject.
  • the period of time is from about one to several days or weeks or months. Thus, the period of time is from at least about 5, 6, 7, 8, 10, 12, 14, 20, 21, 30 or 60 days or more or any number of days between one and 90.
  • the present invention also includes processes for the preparation of compounds of the invention.
  • reactive functional groups for example hydroxy, amino, imino, thio or carboxy groups, where these are desired in the final product, to avoid their unwanted participation in the reactions.
  • Conventional protecting groups can be used in accordance with standard practice, for example, see T.W. Greene and P. G. M. Wuts in "Protective Groups in Organic Chemistry", John Wiley and Sons, 1991.
  • a suitable catalyst e.g., Pd 2 (dba) 3 , or the like
  • an appropriate ligand e.g., l,3-bis(2,4,6-trimethylphenyl) imidazolium chloride
  • a suitable base e.g., CS 2 CO 3 , or the like
  • an appropriate solvent e.g., 1,4-dioxane
  • Compounds of Formula 2 in turn can be prepared by reacting a compound of Formula 4 with a slight excess of an amine compound of Formula 5 in an appropriate solvent (e.g.
  • Compounds of Formula 4 can be prepared by alkylation of a compound of Formula 6 with a suitable alkylating agent 7, in which X] is chlorine, bromine, iodine, or a sulfonate ester, in the presence of a suitable base (e.g. sodium hydride or potassium carbonate), in a suitable solvent (e.g. DMF), at a temperature of about 0 0 C to about 80 0 C.
  • a suitable base e.g. sodium hydride or potassium carbonate
  • a suitable solvent e.g. DMF
  • the reaction can be performed under Mitsunobu conditions using a suitable alcohol R 4 -OH in the presence of a suitable phosphine (e.g.
  • triphenylphosphine e.g. diethylazodicarboxylate
  • azodicarboxylate e.g. diethylazodicarboxylate
  • inert solvent such as THF or toluene
  • R 1 , R 2 , R 3 and R 4 are as defined for Formula I in the Summary of the Invention and L of Formula I is defined in the reaction scheme as -NH-Lj- which is equivalent to, for example, -NRs 3 (CH 2 ) O ⁇ - where R 5a is hydrogen and -(CH 2 ) 0-3 - is L 1 .
  • Compounds of Formula I can be prepared by reacting a compound of Formula
  • Compounds of Formula 8 can in turn be prepared by reacting a compound of Formula 9 with a compound of Formula 3 in the presence of a suitable catalyst (e.g., Pd(Ph 3 P) 4 , Pd 2 (dba) 3 , or the like), optionally in the presence of an appropriate ligand (e.g., l,3-bis(2,4,6-trimethylphenyl) imidazolium chloride), a suitable base (e.g., Cs 2 CO 3 , or the like) and an appropriate solvent (e.g., 1,4-dioxane) at a temperature of about 80 to 100 0 C for 2 to about 48 hours.
  • a suitable catalyst e.g., Pd(Ph 3 P) 4 , Pd 2 (dba) 3 , or the like
  • an appropriate ligand e.g., l,3-bis(2,4,6-trimethylphenyl) imidazolium chloride
  • a suitable base e.g.
  • Compounds of Formula 9 in turn can be prepared by reacting a compound of Formula 10 with a mixture of di-iodomethane, copper(I) iodide, and an alkyl nitrite (e.g. isoamylnitrite), optionally in the presence of an inert solvent, at a temperature of about 50 to 100 0 C.
  • Compounds of Formula 10 can be prepared by alkylation of a compound of Formula 11 with a suitable alkylating agent 7, in which X] is chlorine, bromine, iodine, or a sulfonate ester, in the presence of a suitable base (e.g. sodium hydride or potassium carbonate), in a suitable solvent (e.g.
  • reaction can be performed under Mitsunobu conditions using a suitable alcohol R 4 -OH in the presence of a suitable phosphine (e.g. triphenylphosphine) and azodicarboxylate (e.g. diethylazodicarboxylate), in an inert solvent such as THF or toluene, at a temperature from about 0 0 C to about room temperature.
  • a suitable phosphine e.g. triphenylphosphine
  • azodicarboxylate e.g. diethylazodicarboxylate
  • G 1 , G 2 , G 3 , G 4 , R 1 , R 2 and R 4 are as defined for Formula I in the Summary of the Invention and L of Formula I is defined in the reaction scheme as -NH-Lj - which is equivalent to, for example, where Rs 3 is hydrogen and -(CH 2 V 3 - is L 1 .
  • Compounds of Formula II can be prepared by reacting a compound of Formula 2 with a compound of Formula 20 in the presence of an excess of cyclic amine or NH-bearing heterocycle (for example, substituted pyrazole, substituted imidazole, and the like), at a temperature of about 50 0 C to about 250 0 C, for about 1 to about 24 hours, optionally in the presence of a base such as sodium hydride or DBU.
  • an excess of cyclic amine or NH-bearing heterocycle for example, substituted pyrazole, substituted imidazole, and the like
  • Compounds of Formula 10 can be prepared according to procedures described in J. Med. Chem, 1972, 456, and J. Med. Chem., 1992, 4180.
  • An orthoester compound of Formula 21 is reacted with a compound of Formula 22, optionally in the presence of an acid such as acetic acid, at a temperature of about room temperature to about 150 0 C, for about 1 to about 24 hr..
  • a compound of Formula 22 can in turn be prepared by reacting a compound of Formula 23 with a primary amine compound of Formula 24, optionally in the presence of an acid such as pTSA, or a base such as triethylamine or DBU, at a temperature of about 50 to about 200 0 C.
  • Compounds of Formula IV can be prepared as detailed in the following
  • a compound of Formula IV in which R 2 i is hydrogen, can be prepared from a compound of Formula III by treatment with a suitable reducing agent such as lithium aluminum hydride or di-isobutyl aluminum hydride, in a suitable solvent such as THF or toluene, at a temperature of about -78°C to about 50 0 C.
  • a suitable reducing agent such as lithium aluminum hydride or di-isobutyl aluminum hydride
  • a compound of Formula IV in which R 2I is lower alkyl, can be prepared by treatment of a compound of Formula III with an alkyl lithium or Grignard reagent, in a suitable solvent such as ether or tetrahydrofuran, at a temperature of about -78°C to about 50 0 C.
  • the reaction takes about 0.5 to about 16 hr to complete.
  • a compound of the invention can be prepared as a pharmaceutically acceptable acid addition salt by reacting the free base form of the compound with a pharmaceutically acceptable inorganic or organic acid.
  • a pharmaceutically acceptable base addition salt of a compound of the invention can be prepared by reacting the free acid form of the compound with a pharmaceutically acceptable inorganic or organic base.
  • the salt forms of the compounds of the invention can be prepared using salts of the starting materials or intermediates.
  • salt forms of 4-(2-(2-(benzo [blthiophen-3 -yl)-9-isopropyl-9H- purin-6-ylamino)ethyl)phenol were synthesized as follows: [00160]
  • the white suspension is allowed to cool over about 30 minutes with cooling to room temperature.
  • the slurry is stirred for 18 hours at room temperature and filtered.
  • the solid is washed with acetone (6 ml) in three portions and dried first for about 3 hours at 50 0 C / ca. 10 mbar and then for about 16 hours at 80 0 C /ca.lO mbar.
  • the material has a melting point at about 233°C with a melting enthalpy of 98g/J.
  • the material produced exhibited a loss on drying of 0.2%.
  • the water uptake was estimated by thermogravimetry after exposure to relative humidity (80%rh) during 24 hours. A water uptake of 0.4% was observed.
  • the invention provides a mesylate salt of the compound of Example 1.
  • the invention provides the mesylate salt of the compound of Example 1 comprising the following powder X-ray diffraction peaks (Angle 2- Theta °): 6.4, 6.7, 18.3, 18.6, 26.9; and which in an additional embodiment comprises the following powder X-ray diffraction peaks (Angle 2-Theta °): 6.4, 6.7, 10.3, 12.9, 16.4, 18.3, 25.8, 26.5, 26.9.
  • the invention provides the mesylate salt of the compound of Example 1 having the powder X-ray diffraction pattern shown in Figure 3 herein.
  • Tosylate salt 4-(2-(2-(benzorblthiophen-3-yl)-9-isopropyl-9H-purin-6- ylamino)ethyl)phenol free base (0.60g; 1.40 mmoles) are dissolved in 12 ml at 50 0 C. A solution of para-toluenesulfonic acid mono-hydrate (0.27 Ig; 1.40 mmoles) in acetone (1.2 ml) is added drop wise. The solution is seeded at 50 0 C and crystallization takes place quickly.
  • the invention provides a tosylate salt of the compound of Example 1.
  • the invention provides the tosylate salt of the compound of Example 1 comprising the following powder X-ray diffraction peaks (Angle 2-Theta °):6.2, 13.3, 16.7, 19.5, 25.4; and which in an additional embodiment comprises the following powder X-ray diffraction peaks: 6.2, 7.6, 12.4, 13.3, 15.1, 16.7, 17.7, 19.5, 20.2, 24.6, 24.9, 25.4, 25.6.
  • the invention provides the tosylate salt of the compound of Example 1 having the powder X-ray diffraction pattern shown in Figure 4 herein.
  • Sulfate salt 4-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6- ylamino)ethyl)phenol free base (0.60g; 1.40 mmoles) are dissolved in 10 ml acetone and 1 ml water at about 55°C. A solution of sulfuric acid (0.28Og; 2.79 mmoles) in 1 ml water is added drop wise. The crystallization takes place rapidly. The suspension is allowed to cool over about 30 minutes with cooling to room temperature, stirred for about 18 hours and filtered. The filter cake is washed with 6 ml acetone in three portions and dried first for about 3 hours at 50 0 C / ca.
  • the invention provides a sulfate salt of the compound of Example 1.
  • the invention provides the sulfate salt of the compound of Example 1 comprising the following powder X-ray diffraction peaks (Angle 2-Theta °): 6.5, 6.8, 10.7, 13.5, 26.4, 27.6; and which in an additional embodiment comprises the following powder X-ray diffraction peaks (Angle 2-Theta °): 6.5, 6.8, 10.7, 13.1, 13.5, 18.6, 18.8, 20.8, 26.4, 27.1, 27.6.
  • the invention provides the sulfate salt of the compound of Example 1 having the powder X-ray diffraction pattern shown in Figure 6 herein.
  • Esylate salt 4-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6- ylamino)ethyl)phenol free base (0.60g; 1.40 mmoles) are dissolved in 12 ml acetone at 50 0 C. Ethanesulfonic acid (0.155g; 1.40 mmoles) is added drop wise. The crystallization takes place quickly. The resulting white suspension is allowed to cool over about 30 minutes to room temperature.
  • the invention provides a esylate salt of the compound of Example 1.
  • the invention provides the esylate salt of the compound of Example 1 comprising the following powder X-ray diffraction peaks (Angle 2-Theta °): 6.3, 9.9, 18.4, 25.3, 26.1; and which in an additional embodiment comprises the following powder X- ray diffraction peaks (Angle 2-Theta °): 6.3, 9.9, 17.1, 17.9, 18.4, 19.0, 22.0, 25.3, 26.1, 27.1. [00171] In a yet further embodiment, the invention provides the esylate salt of the compound of Example 1 having the powder X-ray diffraction pattern shown in Figure 8 herein.
  • Hydrobromide salt 4-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6- ylamino)ethyl)phenol free base (0.60g; 1.40 mmoles) are dissolved in 6 ml DMF at 65°C. Hydrobromic acid 48% (0.235g; 1.40 mmoles) is added drop wise. The solution is allowed to cool over about 30 minutes to room temperature. Seeds are added at 55°C and crystallization takes place slowly. The suspension is stirred for about 18 hours at room temperature and filtered. The solid is washed with 4 ml DMF/water 1 : 1 and 6 ml water.
  • the salt is dried first for about 3 hours at 50 0 C / ca. 10 mbar and then for about 16 hours at 80 0 C /ca.lO mbar.
  • the material has a melting point at about 285 0 C with a melting enthalpy of 119g/J.
  • the material produced exhibited a loss on drying of 1.0%.
  • the water uptake was estimated by Thermogravimetry after exposure to relative humidity (80%rh) during 24 hours. No water uptake was observed.
  • the invention provides a hydrobromide salt of the compound of Example 1.
  • the invention provides the hydrobromide salt of the compound of Example 1 comprising the following powder X-ray diffraction peaks (Angle 2-Theta °): 7.0, 25.9, 26.8, 27.9; and which in an additional embodiment comprises the following powder X-ray diffraction peaks (Angle 2-Theta °): 7.0, 11.4, 13.3, 21.4, 23.4, 25.9, 26.4, 26.8, 27.9.
  • the invention provides the hydrobromide salt of the compound of Example 1 having the powder X-ray diffraction pattern shown in Figure 9 herein.
  • Orotate salt 4-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6- ylamino)ethyl)phenol free base (0.60g; 1.40 mmoles) and orotic acid (0.222g; 1.40 mmoles) are dissolved in 7.8 ml NMP (l-Methyl-2-pyrrolidone) at 85°C. The solution is cooled to 60 0 C and 6 ml water is added drop wise over about 5 minutes. The resulting white suspension is allowed to cool over about 30 minutes to room temperature and stirred for 18 hours.
  • NMP l-Methyl-2-pyrrolidone
  • the filter cake is washed with 4 ml NMP/water 1 : 1 in two portions and 6 ml water in three portions.
  • the solid is dried first for about 3 hours at 50 0 C / ca. 10 mbar and then for about 16 hours at 80 0 C /ca.lO mbar.
  • the material has a melting point at about 240 0 C with a melting enthalpy of 130g/J.
  • the material produced exhibited a loss on drying below 0.05%.
  • the water uptake was estimated by Thermogravimetry after exposure to relative humidity (80%rh) during 24 hours. A water uptake of 1.7% was observed.
  • the invention provides an orotate salt of the compound of Example 1.
  • the invention provides the orotate salt of the compound of Example 1 comprising the following powder X-ray diffraction peaks (Angle 2- Theta °): 7.1, 16.3, 19.2, 23.5, 25.6, 26.9; and which in an additional embodiment comprises the following powder X-ray diffraction peaks (Angle 2-Theta °): 7.1, 14.4, 16.3, 18.6, 19.2, 21.7, 23.0, 23.5, 25.6, 26.9, 28.7.
  • the invention provides the orotate salt of the compound of Example 1 having the powder X-ray diffraction pattern shown in Figure 10 herein.
  • Hemi-fumarate salt 4-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6- ylamino)ethyl)phenol free base (0.60g; 1.40 mmoles) are dissolved in 18 ml methanol at 65°C. Fumaric acid (0.164g; 1.40 mmoles) and 6 ml methanol are added. The solution is allowed to cool over about 30 minutes to room temperature.
  • Some seed crystals are added at 60 0 C and crystallization takes place slowly.
  • the suspension is stirred for 18 hours at room temperature and filtered.
  • the solid is washed with 6 ml methanol in three portions and dried first for about 3 hours at 50 0 C / ca. 10 mbar and then for about 16 hours at 80 0 C /ca.lO mbar.
  • the material has a melting point at about 232°C with a melting enthalpy of 83g/J.
  • the material produced exhibited a loss on drying below 0.05%.
  • the water uptake was estimated by Thermogravimetry after exposure to relative humidity (80%rh) during 24 hours. A water uptake of 0.3% was observed.
  • the invention provides a hemi-fumarate salt of the compound of Example 1.
  • the invention provides the hemi-fumarate salt of the compound of Example 1 comprising the following powder X-ray diffraction peaks (Angle 2-Theta °): 7.2, 8.7, 14.4, 15.8, 17.4, 19.0, 23.7; and which in an additional embodiment comprises the following powder X-ray diffraction peaks (Angle 2-Theta °): 7.2, 8.7, 10.8, 14.4,
  • the invention provides the hemi-fumarate salt of the compound of Example 1 having the powder X-ray diffraction pattern shown in Figure 11 herein.
  • Besylate salt 4-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6- ylamino)ethyl)phenol free base (0.60g; 1.40 mmoles) are dissolved in 12 ml acetone at 50 0 C. A solution of benzenesulfonic acid (0.225g; 2.79 mmoles) in 1.2 ml acetone is added drop wise. Seed crystals are added at 48°C and the crystallization takes place slowly. The suspension is allowed to cool over about 30 minutes to room temperature. The slurry is stirred for about 18 hours at room temperature and filtered.
  • the salt is washed with 6 ml acetone in three portions and dried first for about 3 hours at 50 0 C / ca. 10 mbar and then for about 16 hours at 80 0 C /ca.lO mbar.
  • the material has a melting point at about 219°C with a melting enthalpy of 92g/J.
  • the material produced exhibited a loss on drying of 0.3%.
  • the water uptake was estimated by Thermogravimetry after exposure to relative humidity (80%rh) during 24 hours. A water uptake of about 0.05% was observed.
  • the invention provides a besylate salt of the compound of Example 1.
  • the invention provides the besylate salt of the compound of Example 1 comprising the following powder X-ray diffraction peaks (Angle 2- Theta °): 6.2, 7.7, 17.7, 25.5; and which in an additional embodiment comprises the following powder X-ray diffraction peaks (Angle 2-Theta °): 6.2, 7.7, 15.2, 16.7, 17.1, 17.7, 19.8, 20.2,
  • the invention provides the besylate salt of the compound of Example 1 having the powder X-ray diffraction pattern shown in Figure 7 herein.
  • Napadisylate salt 4-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6- ylamino)ethyl)phenol free base (0.60g; 1.40 mmoles) and 0.259 g 1,5-naphthalenedisulfonic acid (0.70 mmoles) are dissolved in 9 ml DMF at 87°C. The clear solution is allowed to cool over about 30 minutes to room temperature. Seeds are added at 65°C and crystallization takes place slowly.
  • the suspension is stirred for about 18 hours at room temperature and filtered.
  • the solid is washed with 4 ml DMF/water 1 : 1 in two portions and 6 ml water in three portions.
  • the salt is dried first for about 3 hours at 50 0 C / ca. 10 mbar and then for about 16 hours at 80 0 C /ca.lO mbar.
  • the material has a melting point at about 304 0 C with a melting enthalpy of 83g/J.
  • a broad endothermic phenomenon is observed at 107 0 C that might be attributed to the loss of water.
  • the material produced exhibited a loss on drying of 6.1%.
  • the water uptake was estimated by Thermogravimetry after exposure to relative humidity (80%rh) during 24 hours. A water uptake less than 0.05% was observed.
  • the invention provides a napadysilate salt of the compound of Example 1.
  • the invention provides the napadysilate salt of the compound of Example 1 comprising the following powder X-ray diffraction peaks (Angle 2-Theta °): 6.4, 9.6, 13.1, 15.7, 16.1, 26.0; and which in an additional embodiment comprises the following powder X-ray diffraction peaks (Angle 2-Theta °): 9.6, 13.1, 15.7, 16.1, 16.4, 20.4, 20.9, 23.7, 26.0, 26.9.
  • the invention provides the napadysilate salt of the compound of Example 1 having the powder X-ray diffraction pattern shown in Figure 12 herein.
  • Hydrochloride salt 4-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6- ylamino)ethyl)phenol free base (0.60g; 1.40 mmoles) are dissolved in 12 ml acetone at 55°C. Hydrochloric acid 37% (0.138g; 1.40 mmoles) is added drop wise. The crystallization takes place quickly. The white suspension is allowed to cool over about 30 minutes to room temperature and stirred for 18 hours. After filtration the solid is washed with 6 ml acetone in three portions and dried first for about 3 hours at 50 0 C / ca.
  • the material is exhibiting an exothermic event at about 162°C with an enthalpy of -13.8J/g. This phenomenon might be attributed to a solid transformation into a more stable modification. An endothermic event is then seen at about 259°C with an enthalpy of 99.7J/g. The material produced exhibited a loss on drying of 0.6%.
  • the water uptake was estimated by Thermogravimetry after exposure to relative humidity (80%rh) during 24 hours. A water uptake of 0.3% was observed.
  • the invention provides a hydrochloride salt of the compound of Example 1.
  • the invention provides the hydrochloride salt of the compound of Example 1 comprising the following powder X-ray diffraction peaks (Angle 2-Theta °): 6.1, 7.0, 19.8, 26.1; and which in an additional embodiment comprises the following powder X-ray diffraction peaks (Angle 2-Theta °): 6.1, 7.0, 18.1, 19.8, 24.7, 26.1, 27.0, 27.7.
  • the invention provides the hydrochloride salt of the compound of Example 1 having the powder X-ray diffraction pattern shown in Figure 5 herein.
  • the free acid or free base forms of the compounds of the invention can be prepared from the corresponding base addition salt or acid addition salt form, respectively.
  • a compound of the invention in an acid addition salt form can be converted to the corresponding free base by treating with a suitable base (e.g., ammonium hydroxide solution, sodium hydroxide, and the like).
  • a compound of the invention in a base addition salt form can be converted to the corresponding free acid by treating with a suitable acid (e.g., hydrochloric acid, etc.).
  • a suitable acid e.g., hydrochloric acid, etc.
  • the nitrate salt of the compound of example 1 can be made using methods known to the skilled person.
  • the powder X-ray diffraction pattern is disclosed in Fig 2 herein.
  • Compounds of the invention in unoxidized form can be prepared from N- oxides of compounds of the invention by treating with a reducing agent (e.g., sulfur, sulfur dioxide, triphenyl phosphine, lithium borohydride, sodium borohydride, phosphorus trichloride, tribromide, or the like) in a suitable inert organic solvent (e.g. acetonitrile, ethanol, aqueous dioxane, or the like) at 0 to 80 0 C.
  • a reducing agent e.g., sulfur, sulfur dioxide, triphenyl phosphine, lithium borohydride, sodium borohydride, phosphorus trichloride, tribromide, or the like
  • a suitable inert organic solvent e.g. acetonitrile, ethanol, aqueous dioxane, or the like
  • Prodrug derivatives of the compounds of the invention can be prepared by methods known to those of ordinary skill in the art (e.g., for further details see Saulnier et al., (1994), Bioorganic and Medicinal Chemistry Letters, Vol. 4, p. 1985).
  • appropriate prodrugs can be prepared by reacting a non-derivatized compound of the invention with a suitable carbamylating agent (e.g., 1,1-acyloxyalkylcarbanochloridate, para-nitrophenyl carbonate, or the like).
  • Protected derivatives of the compounds of the invention can be made by means known to those of ordinary skill in the art. A detailed description of techniques applicable to the creation of protecting groups and their removal can be found in T. W. Greene, "Protecting Groups in Organic Chemistry", 3 rd edition, John Wiley and Sons, Inc., 1999.
  • Compounds of the present invention can be conveniently prepared, or formed during the process of the invention, as solvates (e.g., hydrates). Hydrates of compounds of the present invention can be conveniently prepared by recrystallization from an aqueous/organic solvent mixture, using organic solvents such as dioxin, tetrahydrofuran or methanol.
  • Compounds of the invention can be prepared as their individual stereoisomers by reacting a racemic mixture of the compound with an optically active resolving agent to form a pair of diastereoisomeric compounds, separating the diastereomers and recovering the optically pure enantiomers. While resolution of enantiomers can be carried out using covalent diastereomeric derivatives of the compounds of the invention, dissociable complexes are preferred (e.g., crystalline diastereomeric salts). Diastereomers have distinct physical properties (e.g., melting points, boiling points, solubilities, reactivity, etc.) and can be readily separated by taking advantage of these dissimilarities.
  • the diastereomers can be separated by chromatography, or preferably, by separation/resolution techniques based upon differences in solubility.
  • the optically pure enantiomer is then recovered, along with the resolving agent, by any practical means that would not result in racemization.
  • a more detailed description of the techniques applicable to the resolution of stereoisomers of compounds from their racemic mixture can be found in Jean Jacques, Andre Collet, Samuel H. Wilen, "Enantiomers, Racemates and Resolutions", John Wiley And Sons, Inc., 1981.
  • Compounds of the invention can also be prepared as their individual stereoisomers by using chiral chromatography techniques, in particular, by use of HPLC or SFC chromatography using a chiral stationary phase.
  • Powder X-ray diffraction spectra as enclosed herein were obtained using the instrument Bruker D8 Vario in transmission geometry, irradiation CuKa (30 kV, 40 mA), scan range 2°-40° (2 theta value), step time 90.3s.
  • Differential scanning calorimetry (DSC) of Example 1 amorphous material was carried out using the instrument Perkin Elmer DSC7 at a heating rate of 40°C/min.
  • the compounds of Formula I can be made by a process, which involves:
  • the flask was evacuated and backfilled with N 2 and anhydrous 1,4-dioxane (2 mL) was added. The flask was sealed and the reaction mixture was stirred at 80 0 C for 24 hours. The reaction mixture was concentrated and purified directly by column chromatography on silica gel, eluting with hexane/EtOAc (20: 1 to 1:4) to afford the title compound as a yellowish solid.
  • Example 1 [00204] Alternatively, the synthesis of Example 1 can be carried out as follows:
  • Example 1 [00207] Alternatively, the synthesis of Example 1 can be carried out as follows:
  • Example 1 The compound of Example 1 can be recrystallised using a toluene/ethanol mixture and washed at room temperature with NaHC ⁇ 3 aqueous solution.
  • the invention provides a compound of Example 1 in crystal form modification A, wherein modification A comprises the following powder X-ray diffraction peaks (angle 2-Theta °): 12.1, 16.9, 18.9, 21.3; and which in an additional embodiment comprises the following powder X-ray diffraction peaks (angle 2-Theta °): 12.1, 15.9, 16.9, 17.3, 18.9, 21.3, 22.1, 23.6, 24.4, 27.3.
  • the invention provides the compound of Example 1 in crystal form modification A, wherein modification A comprises the following powder X-ray diffraction peaks (angle 2-Theta °): 12.1, 16.9, 18.9, 21.3; and which in an additional embodiment comprises the following powder X-ray diffraction peaks (angle 2-Theta °): 12.1, 15.9, 16.9, 17.3, 18.9, 21.3, 22.1, 23.6, 24.4, 27.3.
  • the invention provides the compound of Example 1 in crystal form modification A, wherein modification A comprises the following powder X-ray
  • the powder X-ray diffraction pattern of the compound of Example 1, modification A, is shown in Fig 1 herein.
  • Amorphous material of the compound of Example l was produced in situ in a DSC (differential scanning calorimetry) crucible by heating the compound until melting and annealing/cooling. Upon the cooling cycle the glass transition could be observed but upon the reheating cycle is much more characterized at about 70-75 0 C.
  • the DSC pattern is shown in Fig 13 herein.
  • 6-Chloro-2-iodo-9-isopropyl-9H-purine (c): 6-chloro-9- isopropyl-9 ⁇ -purin-2-amine (2.68 g, 12.7 mmol) was dissolved in THF (64 mL) at rt. Iodine (1.61 g, 6.25 mmol), CH 2 I 2 (10.6 mL) and CuI (1.27 g, 6.66 mmol) were added. The mixture was stirred for 5min at room temperature. Isopentyl nitrite (5.33 mL) was added. The reaction mixture was refluxed for 45 min, and was then cooled to room temperature.
  • a microwave reaction tube was charged with 4-(2-(2-chloro-9-isopropyl-9H- purin-6-ylamino)ethyl)phenol (30 mg, 0.091 mmol), 2-methyl-lH-imidazole (59 mg, 0.73 mmol) and 0.5 ml of NMP.
  • the sealed tube was heated under microwave irradiation at 240 0 C for 2 hr.
  • the reaction mixture was purified by reverse-phase HPLC (C is column, eluting with ACN-H 2 O 0.05% TFA) to afford the title compound as an off-white solid.
  • Peaks 1 and 2 were eluted at 20 min and 22.5 min, respectively.
  • Analytical chromatography was performed on a 4.6 x 100 mm Lux_Cellulose-2 (Phenomenex) chiral column, eluting with 90/5/5 hexane/ethanol/methanol at 1 mL/min for 20 min. Peaks 1 and 2 were eluted at 17.45 and 18.14 min, respectively.
  • Example 153b (benzyloxy)propan-2-yl)-2-(5-fluoropyridin-3-yl)-9H-purin-6-amine
  • Example 153c (using tryptamine as reactant)
  • Example 153d using 5-fluoropyridin-3-ylboronic acid as reactant
  • Affinity probe compounds related to the compounds of the invention can also be prepared, as described in the following examples.
  • Example 210 showed an EC 50 value in the %CD34+ assay of 2.1 ⁇ M.
  • HSC hematopoietic stem cell
  • HSCs Primary adult CD34 + human hematopoietic stem cells
  • CD34 expression the desired phenotype
  • cytokines thrombopoietin, interleukin-6, Flt-3 ligand, and stem cell factor (all from R&D Systems, Minneapolis, MN), each at a final concentration of 50 ng/mL, with vehicle (DMSO) or a compound of the invention.
  • CD34 + cells Fresh human leukophoresed G-CSF mobilized peripheral blood from normal donors, CD34 + cells from adult bone marrow and cryopreserved human cord blood CD34 + cells are purchased from AllCells (Berkeley, CA). Human CD34 + cells are enriched from leukophoresed G-CSF mobilized peripheral blood using magnetic cell sorting (MACS, Direct CD34 Progenitor Cell Isolation Kit, Miltenyi Biotec, Bergisch Gladbach, Germany) and cryopreserved. CD34 + cell purity, checked by flow Cytometry, is higher than 90%. After thawing, the cell viability tested by trypan blue exclusion is higher than 70%.
  • MCS Magnetic cell sorting
  • the thawed cells are centrifuged and resuspended with StemSpan medium before being aliquoted for immediate culture.
  • Cells are plated at 10 4 cells/mL in a 384 well plate (Greiner Bio-One, Monroe, North Carolina) with 50 ⁇ L of medium per well for 7 days. Every 7 days the cells are transferred to larger well plates and fresh medium is added to keep the cell density between 10 4 and 5 x 10 5 cells/mL.
  • Cells were cultured at 37°C in 5% CO 2 . For transplantation, cells were cultured in 75 cm flasks before the cells were transplanted into mice.
  • Compounds of the invention were assayed in a dose response format (InM to lO ⁇ M) to determine the effective concentration that produced the desired effect in 50% of the cells (EC 50 ).
  • Compounds of the invention increased the total number and/or percent of CD34+ cells with an EC50 of less than 10 ⁇ M. The results are shown in Table 1 and examples, supra.
  • CFU-C Colony-Forming Units in Culture
  • the MethoCult is supplemented with the following human recombinant cytokines: thrombopoietin, and Flt-3 ligand (R&D Systems), each at a final concentration of 50 ng/mL. After stirring, the mixture is divided into three 35-mm dishes. The dishes are incubated for 14 days at 37°C in a humidified atmosphere of 5% CO 2 in air. At the end of the incubation period, myeloid and erythroid colonies are counted under an inverted microscope at 4OX magnification. CFU-C content of the expansion culture is calculated as follows: number of scored colonies per three dishes x total mononuclear cell number/input cell number.
  • Cells treated with a 4-(2-(2- (benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phenol generated more mixed colonies associated with a >10-fold increase in erythrocyte colonies, a >10-fold increase in granulocyte/macrophage colonies, and a >10-fold increase in macrophage colonies.
  • CAFC Cobblestone area-forming cell
  • the cultures are maintained in Iscove's medium supplemented with 10% fetal calf serum (FCS), 2.5% horse serum (HS), 1% L-glutamine, 1% penicillin- streptomycin, and 1 x 10 " M hydrocortisone at 37°C in a humidified atmosphere of 5% CO 2 in air.
  • FCS fetal calf serum
  • HS horse serum
  • 1% L-glutamine 1% penicillin- streptomycin
  • 1 x 10 " M hydrocortisone 1 x 10 " M hydrocortisone at 37°C in a humidified atmosphere of 5% CO 2 in air.
  • MNCs are added at 8 serial 1:3 dilutions (starting at 25,000 cells/well), with 10 wells for each cell dose.
  • the dilutions with wells with at least one phase-dark hematopoietic clone (cobblestone area) of at least five cells beneath the stromal layer are determined at week 4.
  • 4- (2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phenol (compound 1, table 1; example 1), stimulates a greater than 2-fold increase in the number of cobblestone area forming cells derived from mPB CD34 + HSCs after 5 days of culture, compared with control cultures that are treated with DMSO alone.
  • the cells are washed with staining media (Hanks balanced salt solution containing FBS (2%) and EDTA (2mM)) and stained (at 4°C for 30 minutes) with indicated primary conjugated antibodies.
  • the cells are washed in the previously described buffer and analyzed using a BD LSR II flow cytometer (Becton Dickinson, San Jose, CA).
  • the cells are passed at a rate of up to 1000 cells/second using 488-nm argon and 633-nm HeNe laser beams as the light source for excitation. Emission of 10 4 cells is measured using logarithmic amplification and analyzed using Flow Jo software (TreeStar Inc. Ashland, OR). Cells stained with primary conjugated isotype control antibodies are used to determine background fluorescence.
  • CD34 + cell subsets The percentages of CD34 + cell subsets are determined from aliquots of the cell culture. Cells were stained with APC anti-Thyl.l, PerCP anti-CD34, PECy7 anti CD45RA, FITC anti CD38, and PE anti-CD133 for determination of CD34 + Thyl.l + , CD34 + CD45RA " , CD34 + CD38 " , CD133 + CD38 " and CD34 + CD133 + cells. Antibodies to CD34, CD38, Thy 1.1 and CD45RA were purchased from Becton Dickinson and antibodies to CD133 were purchased Miltenyi Biotec. FACS analysis results of these subsets are given as percentage of the total population.
  • the absolute number of each population of cells in the culture is calculated from the total number of cells multiplied by the percentage of each population. Starting with CB CD34+ cell, after five weeks the total cell number in the cultures increased on average greater than 2-fold in the 4-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H- purin-6-ylamino)ethyl)phenol (compound 1, table 1; example 1) 1 micromolar treated cells compared to control cultures.
  • NOD/SCID To assess the in vivo repopulating capacity of CD34 + cells and their cultured progeny, uncultured CD34 + or the progenies of cultured CD34 + cells after 4 days (mPB) or 21 days (CB) with vehicle or a test compound were injected intravenously via the retro-orbital route into sub-lethally irradiated (3.0 Gy) 8- to 10-week-old NOD/SCID (for mPB HSC experiments) or NOD/SCIDgc-/- (for CB HSC experiments) mice.
  • erythrocyte lysis solution Qiagen, Valencia, CA
  • Engraftment was measured by detection of anti-human CD45 + cells in the blood.
  • the mice are sacrificed at 10 weeks post-transplantation; BM is collected from both femurs and tibiae. BM cells are washed in staining media and stained with anti-human antibodies.
  • the suspension is treated with erythrocyte lysis solution (Qiagen, Valencia, CA) to remove red blood cells, washed with staining media, and analyzed by flow Cytometry, as described earlier.
  • HSCs in an undifferentiated state a genome-wide transcriptional profiling of mPB-derived CD34 + cells treated for 24 hours with example 1 and a less active analog ( ⁇ 20-fold) of example 1 (2-(benzo[b]thiophen-3-yl)-N-(3-(3,5-dimethyl-lH-pyrazol-4-yl)propyl)-9-isopropyl-9H-purin- 6-amine).
  • Example 1 cytochrome P450 IBl [CYPlBl] and the aryl hydrocarbon receptor repressor [AHRR]) are transcriptionally regulated by the aryl hydrocarbon receptor (AHR). Therefore, compounds of the invention could be acting as an antagonist of AHR signaling.
  • TCDD 2,3,7, 8-tetrachlorodibenzo-p- dioxin
  • Example 1 Treatment with TCDD (3nM) caused a 4.5-fold increase in the level of CYPlBl mRNA compared with the vehicle control (0.01% toluene). This increase was inhibited by Example 1 in a dose-dependent manner indicating that compounds of the invention can antagonize AHR signaling.
  • Example 1 To determine the effects of Example 1 in AHR transcription the ability of Example 1 to inhibit a dioxin-induced AHR dependent luciferase reporter gene assay was tested. Inclusion of Example 1 (l ⁇ M) completely abolished dioxin- induced AHR dependent transcription when used on cells expressing human AHR. Titration of Example 1 revealed an EC 50 of 127nM, demonstrating that Example 1 is a potent AHR antagonist.
  • Example 1 only weakly inhibited dioxin induced transcription in murine cells and had no activity on rat cells, suggesting that Example 1 preferentially inhibits human AHR. This correlates with a lack of activity of Example 1 on murine HSC, and can explain the species selectivity of Example 1. Finally, Example 1 had only weak agonist activity on murine or rat cells, and failed to induce AHR dependent transcription in human cells.
  • Example 1 induced HSC expansion human CB-derived CD34 + HSCs were treated with lentiviral particles containing a shRNA-targeting AHR that co-expressed GFP or control virus. Forty-eight hours following transduction, CD34 + GFP + cells were purified by cell sorting and the levels of AHR were determined by qPCR. Both AHR targeting shRNAs led to decreases in AHR expression following transduction (81% with shl l l and 51% with sh242). These decreases were not seen in cells lacking GFP or in cells transduced with control virus. CB-derived CD34 + cells with decreased AHR expression displayed a phenotype similar to Example 1 treated cells with sustained expression of CD34 + . These data show that inhibition of AHR activity by a compound of the invention is sufficient to promote ex- vivo expansion of HSC.
  • the culture medium used is StemSpan SFEM (StemCell Technologies, Cat.
  • Compound dilution into media a 10,000x concentrate of a compound of the invention is used for the dilutions.
  • the addition of compound into the culture media occurs in two steps.
  • the first step is a 1:100 dilution (lO ⁇ L of 10,000x concentrate into 990 ⁇ L of complete culture media (containing cytokines) in a 1.5 mL effendorf tube [USA Scientific, Cat #1615-5500]) to generate a 10Ox solution of compound in the culture media.
  • the second step is a 1: 100 dilution into the culture media that will be used to initiate the cell culture.
  • the volume of the culture is variable depending on the input number of cord blood (CB) CD34 + cells.
  • IxIO 6 CB CD34 + cells are seeded into 20 mL of media (5xlO 4 cells/mL).
  • 200 ⁇ L of the 10Ox Example 1 solution is added to the 20 mL of media in a 50 mL conical tube (Becton Dickinson, Cat #352098) to reach the final concentration (see Table 3).
  • Cell culture initiation purified human CB CD34 + cells are used for the ex vivo expansion experiments. After thawing, the cell viability, tested by trypan blue exclusion, is higher than 50%.
  • the thawed cells are diluted 5-fold with culture media (no cytokines or compounds of the invention such as Example 1) and centrifuged at 300g at 25°C for 8 minutes. After aspirating the supernatant, the pellet is resuspended with the appropriate volume of culture medium (5 x 10 4 cells/mL, Table 3) before being injected (22 gauge needle, Air-Tite products; 20 mL syringe, BD cat # 309661) into AFC bags (Table 5) for immediate culture. Cells are cultured at 37 0 C in 5% CO 2 .
  • a composition comprising a population of cells with expanded HSCs appropriate for intravenous administration as an infusion can also be prepared.
  • cultured cells are pelletted by centrifugation for 10 minutes at 300g and resuspended in infusion buffer consisting of 5% HSA (Baxter) at a concentration of between 10 6 to 10 8 cells /ml.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Immunology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Hematology (AREA)
  • Biomedical Technology (AREA)
  • Epidemiology (AREA)
  • Zoology (AREA)
  • Biotechnology (AREA)
  • Developmental Biology & Embryology (AREA)
  • Cell Biology (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • Diabetes (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Dermatology (AREA)
  • Virology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Neurosurgery (AREA)
  • Neurology (AREA)
  • Oncology (AREA)
  • Obesity (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Plural Heterocyclic Compounds (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

The present invention relates to compounds and compositions for expanding the number of CD34+ cells for transplantation. The invention further relates to a cell population comprising expanded hematopoietic stem cells (HSCs) and its use in autologous or allogeneic transplantation for the treatment of patients with inherited immunodeficient and autoimmune diseases and diverse hematopoietic disorders to reconstitute the hematopoietic cell lineages and immune system defense.

Description

COMPOUNDS THAT EXPAND HEMATOPOIETIC STEM CELLS
CROSS-REFERENCED TO RELATED APPLICATIONS
[0001] This application claims the benefit of priority to U.S. Provisional Patent
Application Number 61/109,821, filed 30 October 2008 and U.S. Provisional Patent Application Number 61/242,765, filed 15 September 2009. The full disclosures of these applications are incorporated herein by reference in their entirety and for all purposes.
BACKGROUND OF THE INVENTION
Field of the Invention
[0002] The present invention relates to compounds and compositions for expanding the number of CD34+ cells for transplantation. The invention further relates to a cell population comprising expanded hematopoietic stem cells (HSCs) and its use in autologous or allogeneic transplantation for the treatment of patients with inherited immunodeficient and autoimmune diseases and diverse hematopoietic disorders to reconstitute the hematopoietic cell lineages and immune system defense.
Background
[0003] Hematopoietic stem cells (HSCs) are capable of regenerating all blood products throughout the life of an individual, balancing their self -renewal with progeny differentiation. Hematopoietic stem cells have therapeutic potential as a result of their capacity to restore blood and immune cells in transplant recipients. Furthermore, HSCs have the potential to generate cells for other tissues such as brain, muscle and liver. Human autologous and allogeneic bone marrow transplantation methods are currently used as therapies for leukemia, lymphoma, and other life-threatening diseases. For these procedures, a large number of stem cells must be isolated to ensure that there are enough HSCs for engraftment. The number of HSCs available for treatment is a clinical limitation. [0004] The present invention relates to compounds and compositions for expanding hematopoietic stem cell populations and uses thereof.
SUMMARY OF THE INVENTION
[0005] In one aspect, the present invention provides a compound of Formula I:
Figure imgf000004_0001
I
[0006] in which:
[0007] G1 is selected from N and CR3 ;
[0008] G2, G3 and G4 are independently selected from CH and N; with the proviso that at least 1 of G3 and G4 is N; with the proviso that Gi and G2 are not both N;
[0009] L is selected from -NR5a(CH2)o-3- (0-3 herein means 0, 1 , 2 or 3), -
NRSaCH(C(O)OCH3)CH2-, -NR5a(CH2)2NR5b-, -NR5a(CH2)2S-, -NR5aCH2CH(CH3)CH2-, -
NR53CH2CH(OH)- and -NR53CH(CH3)CH2-; wherein R53 and R5b are independently selected from hydrogen and C]_4alkyl;
[0010] Ri is selected from hydrogen, phenyl, thiophenyl, furanyl, IH- benzoimidazolyl, isoquinolinyl, lH-imidazopyridinyl, benzothiophenyl, pyrimidinyl, IH- pyrazolyl, pyridinyl, lH-imidazolyl, pyrrolidinyl, pyrazinyl, pyridazinyl, lH-pyrrolyl and thiazolyl; wherein said phenyl, thiophenyl, furanyl, lH-benzoimidazolyl, isoquinolinyl, IH- imidazopyridinyl, benzothiophenyl, pyrimidinyl, lH-pyrazolyl, pyridinyl, lH-imidazolyl, pyrrolidinyl, pyrazinyl, pyridazinyl, lH-pyrrolyl or thiazolyl of R] can be optionally substituted by 1 to 3 radicals independently selected from cyano, hydroxy, C]_4alkyl, C]_4alkoxy, halo, halo- substituted-Ci_4alkyl, halo-substituted-Ci_4alkoxy, hydroxy, amino, -C(O)Rs3, -S(0)o-2Rsa, -
C(O)ORs3 and -C(0)NRs3Rsb; wherein R83 and R8b are independently selected from hydrogen and Ci_4alkyl; with the proviso that R] and R3 are not both hydrogen;
[0011] R2 is selected from -S(O)2NR63R5I,, -NR9aC(O)R9b, -NR6aC(O)NR6bR6c, phenyl, lH-pyrrolopyridin-3-yl, lH-indolyl, thiophenyl, pyridinyl, lH-l,2,4-triazolyl, 2- oxoimidazolidinyl, lH-pyrazolyl, 2-oxo-2,3-dihydro-lH-benzoimidazolyl and lH-indazolyl; wherein R63, Rόb and R6c are independently selected from hydrogen and Ci_4alkyl; wherein said phenyl, lH-pyrrolopyridin-3-yl, lH-indolyl, thiophenyl, pyridinyl, lH-l,2,4-triazolyl, 2- oxoimidazolidinyl, lH-pyrazolyl, 2-oxo-2,3-dihydro-lH-benzoimidazolyl or lH-indazolyl of R2 is optionally substituted with 1 to 3 radicals independently selected from hydroxy, halo, methyl, methoxy, amino, -O(CH2)nNR7aR7b, -S(O)2NR7aR7b, -OS(O)2NR7aR7b and -NR7aS(O)2R7b; wherein R7a and R7b are independently selected from hydrogen and C]_4alkyl; [0012] R3 is selected from hydrogen, Ci_4alkyl and biphenyl; and
[0013] R4 is selected from Ci-ioalkyl, prop-l-en-2-yl, cyclohexyl, cyclopropyl, 2-(2- oxopyrrolidin-l-yl)ethyl, oxetan-3-yl, benzhydryl, tetrahydro-2H-pyran-3-yl, tetrahydro-2H- pyran-4-yl, phenyl, tetrahydrofuran-3-yl, benzyl, (4-pentylphenyl)(phenyl)methyl and l-(l-(2- oxo-6,9,12-trioxa-3-azatetradecan-14-yl)-lH-l,2,3-triazol-4-yl)ethyl; wherein said alkyl, cyclopropyl, cyclohexyl, 2-(2-oxopyrrolidin-l-yl)ethyl, oxetan-3-yl, oxetan-2-yl, benzhydryl, tetrahydro-2H-pyran-2-yl, tetrahydro-2H-pyran-3-yl, tetrahydro-2H-pyran-4-yl, phenyl, tetrahydrofuran-3-yl, tetrahydrofuran-2-yl, benzyl, (4-pentylphenyl)(phenyl)methyl or l-(l-(2- oxo-6,9,12-trioxa-3-azatetradecan-14-yl)-lH-l,2,3-triazol-4-yl)ethyl can be optionally substituted with 1 to 3 radicals independently selected from hydroxy, Ci_4alkyl and halo- substituted-Ci_4alkyl; or the N-oxide derivatives, prodrug derivatives, protected derivatives, individual isomers and mixture of isomers thereof; or the salts (preferably the pharmaceutically acceptable salts) and solvates (e.g. hydrates) of such compounds.
BRIEF DESCRIPTION OF DRAWINGS
[0014] Fig 1 discloses the PXRD pattern of solid form modification A of 4-(2-(2-
(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phenol.
[0015] Figs 2 to 12 disclose the PXRD patterns of solid forms of 4-(2-(2-
(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phenol salts, respectively the nitrate, mesylate, tosylate, hydrochloride, sulphate, besylate, esylate, hydrobromide, orotate, fumarate and napadysilate salts.
[0016] Fig 13 discloses the DSC pattern of the amorphous form of A-(I-(I-
(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phenol. DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0017] "Alkyl" as a group and as a structural element of other groups, for example halo-substituted-alkyl and alkoxy, can be either straight-chained or branched. For example, alkyl includes methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, t-butyl, etc. Ci_4- alkoxy includes methoxy, ethoxy, and the like. Halo-substituted alkyl includes trifluoromethyl, pentafluoroethyl, and the like.
[0018] "Aryl" means a monocyclic or fused bicyclic aromatic ring assembly containing six to ten ring carbon atoms. For example, aryl may be phenyl or naphthyl, preferably phenyl. "Arylene" means a divalent radical derived from an aryl group.
[0019] "Heteroaryl" is as defined for aryl where one or more of the ring members are a heteroatom or moiety selected from -O-, -N=, -NR-, -C(O) -, -S-, -S(O) - or -S(O)2-, wherein R is hydrogen, Ci_4alkyl or a nitrogen protecting group. For example heteroaryl includes pyridyl, indolyl, indazolyl, quinoxalinyl, quinolinyl, benzofuranyl, benzopyranyl, benzothiopyranyl, benzo[l,3]dioxole, imidazolyl, benzo-imidazolyl, pyrimidinyl, furanyl, oxazolyl, isoxazolyl, triazolyl, tetrazolyl, pyrazolyl, thienyl, etc.
[0020] "Cycloalkyl" means a saturated or partially unsaturated, monocyclic, fused bicyclic or bridged polycyclic ring assembly containing the number of ring atoms indicated. For example, C3_iocycloalkyl includes cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, etc.
"Heterocycloalkyl" means cycloalkyl, as defined in this application, provided that one or more of the ring carbons indicated, are replaced by a moiety selected from -O-, -N=, -NR-, -C(O) -, -S-, -S(O) - or -S(O)2-, wherein R is hydrogen, d_4alkyl or a nitrogen protecting group. For example, C3_8heterocycloalkyl as used in this application to describe compounds of the invention includes morpholino, pyrrolidinyl, piperazinyl, piperidinyl, piperidinylone, 2-Oxo-pyrrolidin-l-yl, l,4-dioxa-8-aza-spiro[4.5]dec-8-yl, etc.
[0021] "Halogen" (or halo) preferably represents chloro or fluoro, but may also be bromo or iodo.
[0022] "Hematopoietic stem cells" (HSCs) as used herein refer to immature blood cells having the capacity to self-renew and to differentiate into more mature blood cells comprising granulocytes (e.g., promyelocytes, neutrophils, eosinophils, basophils), erythrocytes
(e.g., reticulocytes, erythrocytes), thrombocytes (e.g., megakaryoblasts, platelet producing megakaryocytes, platelets), and monocytes (e.g., monocytes, macrophages). HSCs are interchangeably described as stem cells throughout the specification. It is known in the art that such cells may or may not include CD34+ cells. CD34+ cells are immature cells that express the CD34 cell surface marker. CD34+ cells are believed to include a subpopulation of cells with the stem cell properties defined above. It is well known in the art that HSCs include pluripotent stem cells, multipotent stem cells (e.g., a lymphoid stem cell), and/or stem cells committed to specific hematopoietic lineages. The stem cells committed to specific hematopoietic lineages may be of T cell lineage, B cell lineage, dendritic cell lineage, Langerhans cell lineage and/or lymphoid tissue-specific macrophage cell lineage. In addition, HSCs also refer to long term HSC (LT-HSC) and short term HSC (ST-HSC). ST-HSCs are more active and more proliferative than LT-HSCs. However, LT-HSC have unlimited self renewal (i.e., they survive throughout adulthood), whereas ST-HSC have limited self renewal (i.e., they survive for only a limited period of time). Any of these HSCs can be used in any of the methods described herein. Optionally, ST-HSCs are useful because they are highly proliferative and thus, quickly increase the number of HSCs and their progeny. Hematopoietic stem cells are optionally obtained from blood products. A blood product includes a product obtained from the body or an organ of the body containing cells of hematopoietic origin. Such sources include un-fractionated bone marrow, umbilical cord, peripheral blood, liver, thymus, lymph and spleen. All of the aforementioned crude or un-fractionated blood products can be enriched for cells having hematopoietic stem cell characteristics in ways known to those of skill in the art. [0023] "Treat", "treating" and "treatment" refer to a method of alleviating or abating a disease and/or its attendant symptoms.
[0024] "Expansion" in the context of cells refers to increase in the number of a characteristic cell type, or cell types, from an initial cell population of cells, which may or may not be identical. The initial cells used for expansion may not be the same as the cells generated from expansion.
[0025] "Cell population" refers to eukaryotic mammalian, preferably human, cells isolated from biological sources, for example, blood product or tissues and derived from more than one cell.
[0026] "Enriched" when used in the context of cell population refers to a cell population selected based on the presence of one or more markers, for example, CD34+. [0027] The term "CD34+ cells" refers to cells that express at their surface CD34 marker. CD34+ cells can be detected and counted using for example flow cytometry and fluorescently labeled anti-CD34 antibodies.
[0028] "Enriched in CD34+ cells" means that a cell population has been selected based on the presence of CD34 marker. Accordingly, the percentage of CD34+ cells in the cell population after selection method is higher than the percentage of CD34+ cells in the initial cell population before selecting step based on CD34 markers. For example, CD34+ cells may represent at least 50%, 60%, 70%, 80% or at least 90% of the cells in a cell population enriched in CD34+ cells.
[0029] "Cord blood unit" refers to the blood collected from umbilical cord of a single birth.
Description of the Preferred Embodiments
[0030] The present invention relates to methods and compositions for expanding
HSC populations using an agent capable of down-regulating the activity and/or expression of aryl hydrocarbon receptor and/or a downstream effector of aryl hydrocarbon receptor pathway. [0031] In one embodiment, said agent capable of down-regulating the activity and/or expression of aryl hydrocarbon receptor is a compound of Formula I. [0032] In one embodiment, with reference to compounds of Formula I, are compounds selected from Formulae Ia, Ib, Ic, Id and Ie:
Figure imgf000009_0001
Figure imgf000009_0002
[0033] in which:
[0034] L is selected from -NR53(CH2)^-, -NR53CH(C(O)OCH3)CH2-, -
NR5a(CH2)2NR5b-, -NR5a(CH2)2S-, -NR53CH2CH(CH3)CH2-, -NR53CH2CH(OH)- and - NR53CH(CH3)CH2-; wherein R53 and R5b are independently selected from hydrogen and C1- 4alkyl; wherein the right side of the L moiety as shown is attached to R2, for example: - NR5a(CH2)o-3-R2, -NR53CH(C(O)OCH3)CH2-R2, -NR5a(CH2)2NR5b-R2, -NR5a(CH2)2S-R2, - NR53CH2CH(CH3)CH2-R2, -NR53CH2CH(OH)-R2 and -NR53CH(CH3)CH2-R2. [0035] R] is selected from hydrogen, phenyl, thiophen-2-yl, thiophen-3-yl, furan-3- yl, lH-benzo[d]imidazol-l-yl, isoquinolin-4-yl, lH-imidazo[4,5-b]pyridin-l-yl, benzo[b]thiophen-3-yl, pyrimidin-5-yl, lH-pyrazol-4-yl, pyridin-2-yl, pyridin-4-yl, lH-imidazol- 1-yl, pyrrolidin-1-yl, pyrazin-2-yl, pyridin-3-yl, pyridazin-4-yl, lH-pyrrol-2-yl and thiazol-5-yl; [0036] wherein said phenyl, thiophen-2-yl, thiophen-3-yl, furan-3-yl, IH- benzo[d]imidazol-l-yl, isoquinolin-4-yl, lH-imidazo[4,5-b]pyridin-l-yl, benzo[b]thiophen-3-yl, pyrimidin-5-yl, pyridiny-2-yl, pyridin-4-yl, lH-imidazol-1-yl, pyrrolidin-1-yl, pyrazin-2-yl, pyridiny-3-yl, pyridazin-4-yl, lH-pyrrol-2-yl or thiazol-5-yl of Rj can be optionally substituted by 1 to 3 radicals independently selected from cyano, hydroxy,
Figure imgf000010_0001
halo, halo- substituted-Ci_4alkyl, -S(0)o-2Rsa and -C(O)ORs3; wherein Rg3 and Rgb are independently selected from hydrogen and C^alkyl; with the proviso that Ri and R3 are not both hydrogen; [0037] R2 is selected from -NR6aC(O)NR6bR6c, phenyl, lH-pyrrolo[2,3-b]pyridin-3- yl, lH-pyrrolo[2,3-b]pyridin-5-yl, lH-indol-3-yl, thiophen-3-yl, pyridin-2-yl, pyridin-3-yl, pyridin-4-yl, lH-l,2,4-triazol-5-yl, 2-oxoimidazolidin-l-yl, lH-pyrazol-3-yl, lH-pyrazol-4-yl, 2- oxo-2,3-dihydro-lH-benzo[d]imidazol-5-yl and lH-indazol-3-yl; wherein R6a, R6b and R& are independently selected from hydrogen and
Figure imgf000010_0002
wherein said phenyl, lH-pyrrolo[2,3- b]pyridin-3-yl, lH-pyrrolo[2,3-b]pyridin-5-yl, lH-indol-3-yl, thiophen-3-yl pyridin-2-yl, pyridin-3-yl, pyridin-4-yl, lH-l,2,4-triazol-5-yl, 2-oxoimidazolidin-l-yl, lH-pyrazol-3-yl, IH- pyrazol-4-yl, 2-oxo-2,3-dihydro-lH-benzo[d]imidazol-5-yl or lH-indazol-3-yl of R2 is optionally substituted with 1 to 3 radicals independently selected from hydroxy, halo, methoxy, amino, -OS(O)2NR7aR7b and -NRv3S(O)2RTb; wherein R7a and R7b are independently selected from hydrogen and Ci_4alkyl;
[0038] R3 is selected from hydrogen,
Figure imgf000010_0003
and biphenyl; and
[0039] R4 is selected from isopropyl, methyl, ethyl, prop-l-en-2-yl, isobutyl, cyclohexyl, sec-butyl, (S)-sec -butyl, (R)-sec-butyl, l-hydroxypropan-2-yl, (S)-l-hydroxypropan- 2-yl, (R)- l-hydroxypropan-2-yl, nonan-2-yl, 2-(2-oxopyrrolidin-l-yl)ethyl, oxetan-3-yl, oxetan- 2-yl, benzhydryl, tetrahydro-2H-pyran-2-yl, phenyl, tetrahydrofuran-3-yl and benzyl; wherein said cyclohexyl, 2-(2-oxopyrrolidin-l-yl)ethyl, oxetan-3-yl, oxetan-2-yl, benzhydryl, tetrahydro- 2H-pyran-4-yl, phenyl, tetrahydrofuran-3-yl or benzyl can be optionally substituted with 1 to 3 radicals independently selected from
Figure imgf000010_0004
and halo-substituted-Ci-4alkyl. [0040] In another embodiment, L is selected from -NR5a(CH2)o-3-, -
NR53CH(C(O)OCH3)CH2-, -NR5a(CH2)2NR5b-, -NR5a(CH2)2S- -NR53CH2CH(CH3)CH2-, - NR53CH(CH3)CH2- and -NR53CH2CH(OH)-; wherein R53 and R5b are independently selected from hydrogen and methyl; and R] is selected from hydrogen, phenyl, thiophen-2-yl, thiophen-3- yl, furan-3-yl, lH-benzo[d]imidazol-l-yl, isoquinolin-4-yl, lH-imidazo[4,5-b]pyridin-l-yl, benzo[b]thiophen-3-yl, pyrimidin-5-yl, lH-pyrazol-4-yl, pyridin-2-yl, pyridin-4-yl, lH-imidazol- 1-yl, pyrrolidin-1-yl, pyrazin-2-yl, pyridin-3-yl, pyridazin-4-yl, lH-pyrrol-2-yl and thiazol-5-yl; wherein said phenyl, thiophen-2-yl, thiophen-3-yl, furan-3-yl, lH-benzo[d]imidazol-l-yl, isoquinolin-4-yl, lH-imidazo[4,5-b]pyridin-l-yl, benzo[b]thiophen-3-yl, pyrimidin-5-yl, pyridiny-2-yl, pyridin-4-yl, lH-imidazol-1-yl, pyrrolidin-1-yl, pyrazin-2-yl, pyridiny-3-yl, pyridazin-4-yl, lH-pyrrol-2-yl or thiazol-5-yl of R] can be optionally substituted by 1 to 3 radicals independently selected from cyano, hydroxy, Ci_4alkyl,
Figure imgf000011_0001
halo, halo- substituted-Ci_4alkyl, -S(0)o-2R8a and -C(O)ORsa; wherein Rga and Rgb are independently selected from hydrogen and Ci_4alkyl; with the proviso that R] and R3 are not both hydrogen. [0041] In another embodiment, when L is -NR5a(CH2)o-3, it is preferably -
NR5a(CH2)i_3 (where 1-3 herein 1, 2 or 3).
[0042] In another embodiment, R2 is selected from urea, phenyl, lH-indol-2-yl, IH- indol-3-yl, thiophen-3-yl, piperidin-1-yl, pyridin-2-yl, pyridin-3-yl, pyridin-4-yl, 1H-1,2,4- triazol-3-yl, lH-l,2,4-triazol-5-yl, 2-oxoimidazolidin-l-yl, lH-pyrazol-3-yl, lH-pyrazol-4-yl, 2- oxo-2,3-dihydro-lH-benzo[d]imidazol-5-yl, lH-benzo[d]imidazol-5-yl and lH-imidazol-4-yl; wherein said phenyl, lH-indol-2-yl, lH-indol-3-yl, thiophen-3-yl, piperidin-1-yl, pyridin-2-yl, pyridin-3-yl, pyridin-4-yl, lH-l,2,4-triazol-3-yl, lH-l,2,4-triazol-5-yl, 2-oxoimidazolidin-l-yl, lH-pyrazol-3-yl, lH-pyrazol-4-yl, 2-oxo-2,3-dihydro-lH-benzo[d]imidazol-5-yl or IH- benzo[d]imidazol-5-yl of R2 is optionally substituted with hydroxy, methoxy, methyl, halo, amino and amino-sulfonyl.
[0043] In another embodiment, R3 is selected from hydrogen, methyl and biphenyl; and R4 is selected from isopropyl, methyl, ethyl, prop-l-en-2-yl, isobutyl, cyclohexyl, sec -butyl, (S)-sec -butyl, (R)-sec-butyl, l-hydroxypropan-2-yl, (S)-l-hydroxypropan-2-yl, (R)-I- hydroxypropan-2-yl, nonan-2-yl, 2-(2-oxopyrrolidin-l-yl)ethyl, oxetan-3-yl, oxetan-2-yl, benzhydryl, tetrahydro-2H-pyran-2-yl, phenyl, tetrahydrofuran-3-yl and benzyl; wherein said cyclohexyl, 2-(2-oxopyrrolidin-l-yl)ethyl, oxetan-3-yl, oxetan-2-yl, benzhydryl, tetrahydro-2H- pyran-4-yl, phenyl, tetrahydrofuran-3-yl or benzyl can be optionally substituted with 1 to 3 radicals independently selected from methyl and trifluoromethyl. [0044] In another embodiment are compounds selected from: 4- (2- (2-
(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phenol; 4-(2-(2- (benzo[b]thiophen-3-yl)-9-sec-butyl-9H-purin-6-ylamino)ethyl)phenol; 4-(2-(9-benzhydryl- 2-(benzo[b]thiophen-3-yl)-9H-purin-6-ylamino)ethyl)phenol; 4-(2-(2-(benzo[b]thiophen-3- yl)-9-(tetrahydro-2H-pyran-3-yl)-9H-purin-6-ylamino)ethyl)phenol; 4-(2-(9-isopropyl-2- (thiophen-2-yl)-9H-purin-6-ylamino)ethyl)phenol; 4-(2-(2-(benzo[b]thiophen-3-yl)-9-(4- (trifluoromethyl)benzyl)-9H-purin-6-ylamino)ethyl)phenol; 4-(2-(2-(benzo[b]thiophen-3-yl)- 9-isobutyl-9H-purin-6-ylamino)ethyl)phenol; 4-(2-(2-(benzo[b]thiophen-3-yl)-9-methyl-9H- purin-6-ylamino)ethyl)phenol; 4-(2-(2-(benzo[b]thiophen-3-yl)-9-(4-methylbenzyl)-9H- purin-6-ylamino)ethyl)phenol; N-(2-(lH-indol-3-yl)ethyl)-2-(benzo[b]thiophen-3-yl)-9- isopropyl-9H-purin-6-amine; 2-(benzo[b]thiophen-3-yl)-9-isopropyl-N-(2-(thiophen-3- yl)ethyl)-9H-purin-6-amine; 3-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6- ylamino)ethyl)phenol; 2-(benzo[b]thiophen-3-yl)-N-(4-fluorophenethyl)-9-isopropyl-9H- purin-6-amine; N-(4-aminophenethyl)-2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6- amine; 4-(2-(9-isopropyl-2-(pyrimidin-5-yl)-9H-purin-6-ylamino)ethyl)phenol; 4-(2-(9- isopropyl-2-(pyridin-3-yl)-9H-purin-6-ylamino)ethyl)phenol; 4-(2-(9-isopropyl-2-phenyl- 9H-purin-6-ylamino)ethyl)phenol; 4-(2-(9-isopropyl-2-(thiophen-3-yl)-9H-purin-6- ylamino)ethyl)phenol; 4-(2-(2-(furan-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phenol; 2- (benzo[b]thiophen-3-yl)-N-(4-fluorophenethyl)-9-phenyl-9H-purin-6-amine; N-benzyl-8- (biphenyl-4-yl)-9-isopropyl-9H-purin-6-amine; 4-(2-(2-(benzo[b]thiophen-3-yl)-9-(nonan-2- yl)-9H-purin-6-ylamino)ethyl)phenol; N-(2-(lH-indol-3-yl)ethyl)-2-(benzo[b]thiophen-3- yl)-9-sec-butyl-9H-purin-6-amine; 3-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6- ylamino)ethyl)-lH-indol-5-yl 5-((3aS,4S,6aR)-2-oxohexahydro-lH-thieno[3,4-d]imidazol-4- yl)pentanoate; N-(2-(2-(2-(2-(4-(l-(2-(benzo[b]thiophen-3-yl)-6-(4- hydroxyphenethylamino)-9H-purin-9-yl)ethyl)-lH-l,2,3-triazol-l- yl)ethoxy)ethoxy)ethoxy)ethyl)acetamide; 4-(2-(9-isopropyl-2-(pyridin-4-yl)-9H-purin-6- ylamino)ethyl)phenol; ethyl 5-(6-(4-hydroxyphenethylamino)-9-isopropyl-9H-purin-2- yl)nicotinate; ethyl 5-(6-(4-hydroxyphenethylamino)-9-isopropyl-9H-purin-2-yl)nicotinate; 4-(2-(2-(6-fluoropyridin-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phenol; 4-(2-(9- isopropyl-2-(4-methylpyridin-3-yl)-9H-purin-6-ylamino)ethyl)phenol; 5-(6-(4- hydroxyphenethylamino)-9-isopropyl-9H-purin-2-yl)nicotinonitrile; 4-(2-(9-isopropyl-2- (pyrrolidin-l-yl)-9H-purin-6-ylamino)ethyl)phenol; 4-(2-(2-(lH-imidazol-l-yl)-9-isopropyl- 9H-purin-6-ylamino)ethyl)phenol; 4-(2-(9-isopropyl-2-(pyridazin-4-yl)-9H-purin-6- ylamino)ethyl)phenol; 4-(2-(9-isopropyl-2-(pyrazin-2-yl)-9H-purin-6-ylamino)ethyl)phenol; 4-(2-(9-isopropyl-2-(pyridin-2-yl)-9H-purin-6-ylamino)ethyl)phenol; 4-(2-(9-isopropyl-2-(5- (methylsulfonyl)pyridin-3-yl)-9H-purin-6-ylamino)ethyl)phenol; 4-(2-(9-isopropyl-2-(5- methylpyridin-3-yl)-9H-purin-6-ylamino)ethyl)phenol; 4-(2-(2-(4-chloropyridin-3-yl)-9- isopropyl-9H-purin-6-ylamino)ethyl)phenol; 4-(2-(2-(5-fluoropyridin-3-yl)-9-isopropyl-9H- purin-6-ylamino)ethyl)phenol; 4-(2-(9-isopropyl-2-(l-methyl-lH-pyrazol-4-yl)-9H-purin-6- ylamino)ethyl)phenol; 4-(2-(9-isopropyl-2-(pyridin-3-yl)-9H-purin-6-ylamino)ethyl)-2- methoxyphenol; 4-(2-(9-isopropyl-2-(pyridin-3-yl)-9H-purin-6-ylamino)ethyl)-2- methoxyphenol; N-[2-(6-methoxy-lH-indol-3-yl)ethyl]-9-(propan-2-yl)-2-(pyridin-3-yl)-9H- purin-6-amine; N-[2-(5-methyl-lH-indol-3-yl)ethyl]-9-(propan-2-yl)-2-(pyridin-3-yl)-9H- purin-6-amine; l-(2-{ [9-(propan-2-yl)-2-(pyridin-3-yl)-9H-purin-6- yl]amino}ethyl)imidazolidin-2-one; N-(2-{ [9-(propan-2-yl)-2-(pyridin-3-yl)-9H-purin-6- yl]amino}ethyl)pyridin-2-amine; 9-(propan-2-yl)-N-[3-(lH-pyrazol-4-yl)propyl]-2-(pyridin- 3-yl)-9H-purin-6-amine; N-{2-[(3-methyl-lH-l,2,4-triazol-5-yl)sulfanyl]ethyl}-9-(propan-2- yl)-2-(pyridin-3-yl)-9H-purin-6-amine; l-(2-{ [2-(l-benzothiophen-3-yl)-9-(propan-2-yl)- 9H-purin-6-yl]amino}ethyl)imidazolidin-2-one; N-[2-(5-amino-lH-l,2,4-triazol-3-yl)ethyl]- 2-(l-benzothiophen-3-yl)-9-(propan-2-yl)-9H-purin-6-amine; N-(2-{ [2-(l-benzothiophen-3- yl)-9-(propan-2-yl)-9H-purin-6-yl]amino}ethyl)pyridin-2-amine; 2-(l-benzothiophen-3-yl)- 9-(propan-2-yl)-N-[3-(lH-pyrazol-4-yl)propyl]-9H-purin-6-amine; 2-(l-benzothiophen-3- yl)-N-[3-(3,5-dimethyl-lH-pyrazol-4-yl)propyl]-9-(propan-2-yl)-9H-purin-6-amine; (2-{ [2- (l-benzothiophen-3-yl)-9-(propan-2-yl)-9H-purin-6-yl]amino}ethyl)urea; 5-({ [2-(l- benzothiophen-3-yl)-9-(propan-2-yl)-9H-purin-6-yl]amino}methyl)-2,3-dihydro-lH-l,3- benzodiazol-2-one; N-[2-(lH-indol-3-yl)ethyl]-9-(propan-2-yl)-2-(pyridin-3-yl)-9H-purin-6- amine; N-(4-(2-(9-isopropyl-2-(pyridin-3-yl)-9H-purin-6-ylamino)ethyl)phenyl)methane- sulfonamide; 4-(2-(2-(pyridin-3-yl)-9-(tetrahydrofuran-3-yl)-9H-purin-6- ylamino)ethyl)phenol; 4-(2-(9-isopropyl-2-(pyridin-3-yl)-9H-purin-6- ylamino)propyl)phenol; 4-(2-(9-(oxetan-3-yl)-2-(pyridin-3-yl)-9H-purin-6- ylamino)ethyl)phenol; 5-(6-(4-hydroxyphenethylamino)-9-isopropyl-9H-purin-2-yl)-N- methylnicotinamide; 4-(2-(9-(l-hydroxypropan-2-yl)-2-(pyridin-3-yl)-9H-purin-6- ylamino)ethyl)phenol; 4-(2-(9-isopropyl-2-(pyridin-3-yl)-9H-purin-6-ylamino)ethyl)phenyl sulfamate; 4-(2-(2-(2-fluoropyridin-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phenol; 4- (2-(9-isopropyl-2-(l-methyl-lH-pyrrol-2-yl)-9H-purin-6-ylamino)ethyl)phenol; 4-(2-(9- isopropyl-2-(thiazol-5-yl)-9H-purin-6-ylamino)ethyl)phenol; 4-(2-(2-(lH-benzo[d]imidazol- l-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phenol; 4-(2-(2-(2,4-dimethyl-lH-imidazol-l- yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phenol; 4-(2-(9-isopropyl-2-(2-methyl-lH- imidazol-l-yl)-9H-purin-6-ylamino)ethyl)phenol; 5-(9-sec-butyl-6-(4-hydroxy-3- methylphenethylamino)-9H-purin-2-yl)nicotinonitrile; N-(2-(lH-pyrrolo[2,3-b]pyridin-5- yl)ethyl)-9-isopropyl-2-(pyridin-3-yl)-9H-purin-6-amine; 9-isopropyl-N-(2-(5-methyl-lH- pyrazol-3-yl)ethyl)-2-(pyridin-3-yl)-9H-purin-6-amine; 4-(2-(2-(5-fluoropyridin-3-yl)-9- (oxetan-3-yl)-9H-purin-6-ylamino)ethyl)phenol; 4-(2-(2-(5-chloropyridin-3-yl)-9-isopropyl- 9H-purin-6-ylamino)ethyl)phenol; 4-(2-(9-isopropyl-2-(5-(trifluoromethyl)pyridin-3-yl)-9H- purin-6-ylamino)ethyl)phenol; 5-(6-(2-(lH-indol-3-yl)ethylamino)-9-sec-butyl-9H-purin-2- yl)nicotinonitrile; N-(2-(lH-indol-3-yl)ethyl)-9-sec-butyl-2-(5-methylpyridin-3-yl)-9H- purin-6-amine; (R)-N-(2-(lH-indol-3-yl)ethyl)-9-sec-butyl-2-(5-fluoropyridin-3-yl)-9H- purin-6-amine; (S)-N-(2-(lH-indol-3-yl)ethyl)-9-sec-butyl-2-(5-fluoropyridin-3-yl)-9H- purin-6-amine; N-(2-(lH-indol-3-yl)ethyl)-9-sec-butyl-2-(5-fluoropyridin-3-yl)-9H-purin-6- amine; (R)-N-(2-(lH-indol-3-yl)ethyl)-9-sec-butyl-2-(5-methylpyridin-3-yl)-9H-purin-6- amine; (S)-N-(2-(lH-indol-3-yl)ethyl)-9-sec-butyl-2-(5-methylpyridin-3-yl)-9H-purin-6- amine; 5-(6-(4-hydroxyphenethylamino)-9-(oxetan-3-yl)-9H-purin-2-yl)nicotinonitrile; 4-(2- (6-(5-fluoropyridin-3-yl)-l-isopropyl-lH-pyrazolo[3,4-d]pyrimidin-4-ylamino)ethyl)phenol; 4-(2-(6-(benzo[b]thiophen-3-yl)-l-isopropyl-lH-pyrazolo[3,4-d]pyrimidin-4- ylamino)ethyl)phenol; (R)-4-(2-(2-(5-fluoropyridin-3-yl)-9-(tetrahydrofuran-3-yl)-9H-purin- 6-ylamino)ethyl)phenol; 4-(2-(9-isopropyl-2-(pyridin-3-yl)-9H-purin-6-ylamino)ethyl)-3- methylphenol; 5-(6-(4-hydroxyphenethylamino)-9-isopropyl-9H-purin-2-yl)picolinonitrile; 3-(6-(4-hydroxyphenethylamino)-9-isopropyl-9H-purin-2-yl)isonicotinonitrile; 4-(2-(2-(5- fluoropyridin-3-yl)-7-isopropyl-7H-pyrrolo[2,3-d]pyrimidin-4-ylamino)ethyl)phenol; 3-(6- (4-hydroxyphenethylamino)-9-isopropyl-9H-purin-2-yl)picolinonitrile; 4-(2-(9-isopropyl-2- (6-methylpyridin-3-yl)-9H-purin-6-ylamino)ethyl)phenol; 4-(2-(9-isopropyl-2-(isoquinolin- 4-yl)-9H-purin-6-ylamino)ethyl)phenol; 2-chloro-4-(2-(9-isopropyl-2-(pyridin-3-yl)-9H- purin-6-ylamino)ethyl)phenol; 3-fluoro-4-(2-(9-isopropyl-2-(pyridin-3-yl)-9H-purin-6- ylamino)ethyl)phenol; N-(2-(5-chloro-lH-indol-3-yl)ethyl)-9-isopropyl-2-(pyridin-3-yl)-9H- purin-6-amine; N-(2-(5-fluoro-lH-indol-3-yl)ethyl)-9-isopropyl-2-(pyridin-3-yl)-9H-purin- 6-amine; 4-(2-(9-isopropyl-2-(pyridin-3-yl)-9H-purin-6-ylamino)ethyl)-2-methylphenol; 4- (2-(2-(benzo[b]thiophen-3-yl)-9-(oxetan-3-yl)-9H-purin-6-ylamino)ethyl)phenol; (S)-4-(2- (2-(benzo[b]thiophen-3-yl)-9-(tetrahydrofuran-3-yl)-9H-purin-6-ylamino)ethyl)phenol; (R)- 4-(2-(2-(benzo[b]thiophen-3-yl)-9-(tetrahydrofuran-3-yl)-9H-purin-6-ylamino)ethyl)phenol; 2-(6-(2-(lH-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin-9-yl)propan-l-ol; (R)-2-(6-(2-(lH-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin-9-yl)propan-l-ol; (S)-2-(6-(2-(lH-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin-9-yl)propan-l-ol; (R)-N-(2-(lH-indol-3-yl)ethyl)-2-(5-fluoropyridin-3-yl)-9-(tetrahydrofuran-3-yl)-9H-purin- 6-amine; 4-(2-(2-(3H-imidazo[4,5-b]pyridin-3-yl)-9-isopropyl-9H-purin-6- ylamino)ethyl)phenol; 4-(2-(2-(lH-imidazo[4,5-b]pyridin-l-yl)-9-isopropyl-9H-purin-6- ylamino)ethyl)phenol; 4-(2-(6-(5-fluoropyridin-3-yl)-l-isopropyl-lH-imidazo[4,5-c]pyridin- 4-ylamino)ethyl)phenol; 4-(2-(2-(4,5-dimethyl-lH-imidazol-l-yl)-9-isopropyl-9H-purin-6- ylamino)ethyl)phenol; 2-(5-fluoropyridin-3-yl)-9-isopropyl-N-(2-(pyridin-3-yl)ethyl)-9H- purin-6-amine; 4-(2-(2-(5-fluoropyridin-3-yl)-9-isopropyl-9H-purin-6-ylamino)-l- hydroxyethyl)phenol; 2-(5-fluoropyridin-3-yl)-9-isopropyl-N-(2-(6-methoxy-lH-indol-3- yl)ethyl)-9H-purin-6-amine; N-(2-(lH-indol-3-yl)ethyl)-2-(5-fluoropyridin-3-yl)-9- isopropyl-9H-purin-6-amine; 2-(5-fluoropyridin-3-yl)-9-isopropyl-N-(2-(5-methoxy-lH- indol-3-yl)ethyl)-9H-purin-6-amine; N-(2-(lH-indol-3-yl)ethyl)-2-(5-fluoropyridin-3-yl)-9- (prop-l-en-2-yl)-9H-purin-6-amine; 5-(2-(2-(5-fluoropyridin-3-yl)-9-isopropyl-9H-purin-6- ylamino)ethyl)pyridin-2-ol; N-(2-(lH-pyrrolo[2,3-b]pyridin-3-yl)ethyl)-2-(5-fluoropyridin- 3-yl)-9-isopropyl-9H-purin-6-amine; N-(2-(6-(2-(diethylamino)ethoxy)-lH-indol-3- yl)ethyl)-2-(5-fluoropyridin-3-yl)-9-isopropyl-9H-purin-6-amine; 4-(2-(5-(5-fluoropyridin-3- yl)-3-isopropyl-3H-imidazo[4,5-b]pyridin-7-ylamino)ethyl)phenol; N-(2-(lH-indol-3- yl)ethyl)-9-sec-butyl-2-(2-methyl- lH-imidazol- l-yl)-9H-purin-6-amine; 4-(2-(2-(2-ethyl- lH-imidazol-l-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phenol; 4-(2-(9-isopropyl-2-(2- propyl-lH-imidazol-l-yl)-9H-purin-6-ylamino)ethyl)phenol; 3-(2-(2-(5-fluoropyridin-3-yl)- 9-isopropyl-9H-purin-6-ylamino)ethyl)-lH-indol-6-ol; N-(2-(lH-indol-3-yl)ethyl)-9- isopropyl-2-(5-methylpyridin-3-yl)-9H-purin-6-amine; N-(2-(lH-indol-3-yl)ethyl)-9- isopropyl-2-(2-methyl-lH-imidazol-l-yl)-9H-purin-6-amine; 2-(5-fluoropyridin-3-yl)-9- isopropyl-N-(2-(7-methyl-lH-indol-3-yl)ethyl)-9H-purin-6-amine; N-(2-(lH-indol-3- yl)ethyl)-2-(5-fluoropyridin-3-yl)-9-(oxetan-3-yl)-9H-purin-6-amine; N-(2-(lH-indol-3- yl)ethyl)-2-(5-methylpyridin-3-yl)-9-(oxetan-3-yl)-9H-purin-6-amine; N-(2-(6-fluoro-lH- indol-3-yl)ethyl)-2-(5-fluoropyridin-3-yl)-9-isopropyl-9H-purin-6-amine; 2-(5- fluoropyridin-3-yl)-9-isopropyl-N-(2-(6-methyl-lH-indol-3-yl)ethyl)-9H-purin-6-amine; 2- (5-fluoropyridin-3-yl)-9-isopropyl-N-(2-(2-methyl-lH-indol-3-yl)ethyl)-9H-purin-6-amine; N-(2-(4-fluoro-lH-indol-3-yl)ethyl)-2-(5-fluoropyridin-3-yl)-9-isopropyl-9H-purin-6-amine; N-(2-(7-fluoro-lH-indol-3-yl)ethyl)-2-(5-fluoropyridin-3-yl)-9-isopropyl-9H-purin-6-amine; 2-(5-fluoropyridin-3-yl)-9-isopropyl-N-(2-(4-methyl-lH-indol-3-yl)ethyl)-9H-purin-6- amine; 4-(2-(2-(benzo[b]thiophen-3-yl)-7-isopropyl-7H-pyrrolo[2,3-d]pyrimidin-4- ylamino)ethyl)phenol; 9-isopropyl-2-(pyridin-3-yl)-N-(2-(pyridin-4-yl)ethyl)-9H-purin-6- amine; N-(2-(lH-pyrrolo[2,3-b]pyridin-5-yl)ethyl)-9-isopropyl-2-(pyridin-3-yl)-9H-purin-6- amine; 4-(2-(2-(5-fluoropyridin-3-yl)-9-(l-hydroxypropan-2-yl)-9H-purin-6-ylamino)ethyl)- 2-methylphenol; 4-(2-(2-(benzo[b]thiophen-3-yl)-9-cyclohexyl-9H-purin-6- ylamino)ethyl)phenol; 4-(2-(9-isopropyl-2-(thiophen-3-yl)-9H-purin-6- ylamino)ethyl)phenol; and l-(2-(2-(benzo[b]thiophen-3-yl)-6-(4-hydroxyphenethylamino)- 9H-purin-9-yl)ethyl)pyrrolidin-2-one. Compounds of Formula I are detailed in the Examples and Table I, infra. [0045] In another embodiment are compounds of Formula Ia:
Figure imgf000016_0001
[0046] in which:
[0047] L is selected from -NR53(CH2)^-, -NR53CH(C(O)OCH3)CH2-, -
NR5a(CH2)2NR5b-, -NR5a(CH2)2S- -NR53CH2CH(CH3)CH2-, -NR53CH(CH3)CH2-, -(CH2),-, - CH2OCH2-, -CH2NR53CH2-, -NR53C(O)CH2- and -NR53Y-; wherein R53 and R5b are independently selected from hydrogen and
Figure imgf000016_0002
and Y is a 5 member heteroaryl ring containing up to 3 heteroatoms selected from O, N and S;
[0048] Ri is selected from hydrogen, phenyl, thiophen-2-yl, thiophen-3-yl, furan-2- yl, furan-3-yl, benzo[b]thiophen-2-yl, benzo[b]thiophen-3-yl, benzofuran-2-yl, benzofuran-3-yl, pyrimidin-4-yl, pyrimidin-5-yl, lH-pyrazol-4-yl, lH-pyrazol-3-yl, pyridin-2-yl, pyridazin-3-yl, pyridin-4-yl, lH-imidazol-1-yl, pyrrolidin-1-yl, pyrazin-2-yl, pyridin-3-yl, lH-pyrazol-1-yl, pyridazin-4-yl, lH-indol-2-yl, thiazol-4-yl, lH-indol-3-yl, lH-pyrrol-2-yl and thiazol-5-yl; wherein said phenyl, thiophen-2-yl, thiophen-3-yl, furan-2-yl, furan-3-yl, benzo[b]thiophen-2-yl, benzo[b]thiophen-3-yl, benzofuran-2-yl, benzofuran-3-yl, pyrimidin-4-yl, pyrimidin-5-yl, IH- pyrazol-4-yl, lH-pyrazol-3-yl, pyridiny-2-yl, pyridazin-3-yl, pyridin-4-yl, lH-imidazol-1-yl, pyrrolidin-1-yl, pyrazin-2-yl, pyridiny-3-yl, lH-pyrazol-1-yl, pyridazin-4-yl, lH-indol-2-yl, thiazol-4-yl, lH-indol-3-yl, lH-pyrrol-2-yl or thiazol-5-yl of R] can be optionally substituted by 1 to 3 radicals independently selected from cyano, hydroxy,
Figure imgf000017_0001
halo, halo- substituted-Ci_4alkyl, halo-substituted-Ci_4alkoxy, hydroxy, amino, -C(O)R8a, -S(0)o-2Rsa, - C(O)OR83 and -C(O)NR8aR8b; wherein R8a and R8b are independently selected from hydrogen and C]_4alkyl; with the proviso that Rj and R3 are not both hydrogen; [0049] R2 is selected from -S(O)2NR6aR5b, -NR9aC(O)R9b, -NR6aC(O)NR6bR6c, phenyl, lH-indol-2-yl, lH-indol-3-yl, benzo[b]thiophen-2-yl, benzo[b]thiophen-3-yl, benzofuran-2-yl, benzofuran-3-yl, thiophen-2-yl, thiophen-3-yl, furan-2-yl, furan-3-yl, piperidin- 4-yl, piperidin-3-yl, piperidin-2-yl, piperidin-1-yl, pyridin-2-yl, pyridin-3-yl, pyridin-4-yl, IH- l,2,4-triazol-3-yl, lH-l,2,4-triazol-5-yl, 2-oxoimidazolidin-l-yl, lH-pyrazol-3-yl, lH-pyrazol-4- yl, 3-oxopiperazin-l-yl, 2-oxo-2,3-dihydro-lH-benzo[d]imidazol-5-yl, 1,2,3,4- tetrahydronaphthalen-2-yl, indolin-5-yl, 2-oxoindolin-5-yl, lH-benzo[d]imidazol-5-yl, IH- indazol-5-yl and lH-imidazol-4-yl; wherein R6a, R6b and R& are independently selected from hydrogen and C^alkyl; wherein said phenyl, lH-indol-2-yl, lH-indol-3-yl, benzo[b]thiophen-2- yl, benzo[b]thiophen-3-yl, benzofuran-2-yl, benzofuran-3-yl, thiophen-2-yl, thiophen-3-yl or furan-2-yl, furan-3-yl, piperidin-4-yl, piperidin-3-yl, piperidin-2-yl, piperidin-1-yl, pyridin-2-yl, pyridin-3-yl, pyridin-4-yl, lH-l,2,4-triazol-3-yl, lH-l,2,4-triazol-5-yl, 2-oxoimidazolidin-l-yl, lH-pyrazol-3-yl, 3-oxopiperazin-l-yl, 2-oxo-2,3-dihydro-lH-benzo[d]imidazol-5-yl, 1,2,3,4- tetrahydronaphthalen-2-yl, indolin-5-yl, 2-oxoindolin-5-yl, lH-benzo[d]imidazol-5-yl, IH- indazol-5-yl or lH-imidazol-4-yl of R2 is optionally substituted with 1 to 3 radicals independently selected from hydroxy, halo, methyl, methoxy, amino, -S(O)2NR7aR7b, - OS(O)2NR7aR7b and -NR7aS(O)2R7b; wherein R7a and R7b are independently selected from hydrogen and Ci_4alkyl; or a single radical selected from 5-((3aS,4S,6aR)-2-oxohexahydro-lH- thieno[3,4-d]imidazol-4-yl)pentanoyloxy, 2-(2-(5-((3aS,4S,6aR)-2-oxohexahydro-lH- thieno[3,4-d]imidazol-4-yl)pentanamido)ethoxy)ethoxy and 2-(4-(4-hex-5- ynamidobenzoyl)phenylamino)-2-oxoethoxy;
[0050] R3 is selected from hydrogen, Ci_4alkyl and biphenyl; and
[0051] R4 is selected from isopropyl, isobutyl, sec -butyl, l-hydroxypropan-2-yl, cyclopropyl, oxetan-3-yl, oxetan-2-yl, benzhydryl, piperidin-4-yl, piperidin-3-yl, piperidin-2-yl, tetrahydro-2H-pyran-2-yl, tetrahydro-2H-pyran-3-yl, tetrahydro-2H-pyran-4-yl, phenyl, tetrahydrofuran-3-yl, tetrahydrofuran-2-yl, benzyl, (4-pentylphenyl)(phenyl)methyl and l-(l-(2- oxo-6,9,12-trioxa-3-azatetradecan-14-yl)-lH-l,2,3-triazol-4-yl)ethyl; wherein said cyclopropyl, oxetan-3-yl, oxetan-2-yl, benzhydryl, piperidin-4-yl, piperidin-3-yl, piperidin-2-yl, tetrahydro- 2H-pyran-2-yl, tetrahydro-2H-pyran-3-yl, tetrahydro-2H-pyran-4-yl, phenyl, tetrahydrofuran-3- yl, tetrahydrofuran-2-yl, benzyl, (4-pentylphenyl)(phenyl)methyl or l-(l-(2-oxo-6,9,12-trioxa-3- azatetradecan-14-yl)-lH-l,2,3-triazol-4-yl)ethyl can be optionally substituted with 1 to 3 radicals independently selected from Ci_4alkyl and halo-substituted-Ci_4alkyl; or the N-oxide derivatives, prodrug derivatives, protected derivatives, individual isomers and mixture of isomers thereof; or the pharmaceutically acceptable salts and solvates (e.g. hydrates) of such compounds. [0052] In a further embodiment, with reference to compounds of Formula Ia, L is selected from -NR5a(CH2)o_3-, -NR53CH(C(O)OCH3)CH2-, -NR5a(CH2)2NR5b-, -NR5a(CH2)2S- , -NR53CH2CH(CH3)CH2-, -NR53CH(CH3)CH2-, -(CH2)3- -CH2OCH2-, -CH2NR53CH2-, - NR53C(O)CH2- and -NR53Y-; wherein R53 and R5b are independently selected from hydrogen and methyl; Y is selected from isoxazole and 1,3,4-oxadiazole. [0053] In another embodiment, when L is -NR5a(CH2)0-3, it is preferably -
NR5a(CH2)i_3 (where 1-3 herein means 1, 2 or 3).
[0054] In another embodiment, R] is selected from hydrogen, phenyl, thiophen-3-yl, thiophen-2-yl, furan-3-yl, furan-2-yl, benzo[b]thiophen-3-yl, pyrimidin-5-yl, pyridin-4-yl, pyridin-2-yl, pyrrolidin-1-yl, lH-pyrazol-4-yl, pyrazin-2-yl, pyridazin-3-yl, pyridazin-4-yl, IH- pyrazol-1-yl, lH-pyrazol-3-yl, lH-imidazol-1-yl, thiazol-4-yl, lH-pyrrol-2-yl, thiazol-5-yl, and pyridin-3-yl; wherein said phenyl, thiophen-3-yl, thiophen-2-yl, furan-3-yl, furan-2-yl, benzo[b]thiophen-3-yl, pyrimidin-5-yl, pyridin-4-yl, pyridin-2-yl, pyrrolidin-1-yl, lH-pyrazol-4- yl, pyrazin-2-yl, pyridazin-3-yl, pyridazin-4-yl, lH-pyrazol-1-yl, lH-pyrazol-3-yl, lH-imidazol- 1-yl, thiazol-4-yl, lH-pyrrol-2-yl, thiazol-5-yl or pyridin-3-yl of R] is optionally substituted with 1 to 3 radicals independently selected from cyano, methyl, methyl-sulfonyl, methoxy, halo, hydroxy, carboxyl, ethoxy-carbonyl, methyl-amino-carbonyl and amino; with the proviso that R] and R3 are not both hydrogen.
[0055] In another embodiment, R2 is selected from amino-sulfonyl, methyl-carbonyl- amino, methyl-sulfonyl-amino, amino-sulfonyl-oxy, urea, phenyl, lH-indol-2-yl, lH-indol-3-yl, benzo[b]thiophen-2-yl, benzo[b]thiophen-3-yl, benzofuran-2-yl, benzofuran-3-yl, thiophen-2-yl, thiophen-3-yl, furan-2-yl, furan-3-yl, piperidin-4-yl, piperidin-3-yl, piperidin-2-yl, piperidin-1- yl, pyridin-2-yl, pyridin-3-yl, pyridin-4-yl, lH-l,2,4-triazol-3-yl, lH-l,2,4-triazol-5-yl, 2- oxoimidazolidin-1-yl, lH-pyrazol-3-yl, lH-pyrazol-4-yl, 3-oxopiperazin-l-yl, 2-oxo-2,3- dihydro-lH-benzo[d]imidazol-5-yl, l,2,3,4-tetrahydronaphthalen-2-yl, indolin-5-yl, 2- oxoindolin-5-yl, lH-benzo[d]imidazol-5-yl, lH-indazol-5-yl and lH-imidazol-4-yl; wherein said phenyl, lH-indol-2-yl, lH-indol-3-yl, benzo[b]thiophen-2-yl, benzo[b]thiophen-3-yl, benzofuran-2-yl, benzofuran-3-yl, thiophen-2-yl, thiophen-3-yl, furan-2-yl, furan-3-yl, piperidin- 4-yl, piperidin-3-yl, piperidin-2-yl, piperidin-1-yl, pyridin-2-yl, pyridin-3-yl, pyridin-4-yl, IH- l,2,4-triazol-3-yl, lH-l,2,4-triazol-5-yl, 2-oxoimidazolidin-l-yl, lH-pyrazol-3-yl, lH-pyrazol-4- yl, 3-oxopiperazin-l-yl, 2-oxo-2,3-dihydro-lH-benzo[d]imidazol-5-yl, 1,2,3,4- tetrahydronaphthalen-2-yl, indolin-5-yl, 2-oxoindolin-5-yl, lH-benzo[d]imidazol-5-yl, IH- indazol-5-yl and lH-imidazol-4-yl of R2 is optionally substituted with hydroxy, methoxy, methyl, halo, amino, amino-sulfonyl, 5-((3aS,4S,6aR)-2-oxohexahydro-lH-thieno[3,4- d]imidazol-4-yl)pentanoyloxy, 2-(2-(5-((3aS,4S,6aR)-2-oxohexahydro-lH-thieno[3,4- d]imidazol-4-yl)pentanamido)ethoxy)ethoxy and 2-(4-(4-hex-5-ynamidobenzoyl)phenylamino)- 2-oxoethoxy.
[0056] In another embodiment, R3 is selected from hydrogen, methyl, and biphenyl; and R4 is selected from isopropyl, isobutyl, sec-butyl, l-hydroxypropan-2-yl, cyclopropyl, oxetan-3-yl, oxetan-2-yl, benzhydryl, piperidin-4-yl, piperidin-3-yl, piperidin-2-yl, tetrahydro- 2H-pyran-2-yl, tetrahydro-2H-pyran-3-yl, tetrahydro-2H-pyran-4-yl, phenyl, tetrahydrofuran-3- yl, tetrahydrofuran-2-yl, benzyl, (4-pentylphenyl)(phenyl)methyl and l-(l-(2-oxo-6,9,12-trioxa- 3-azatetradecan-14-yl)-lH-l,2,3-triazol-4-yl)ethyl; wherein said cyclopropyl, oxetan-3-yl, oxetan-2-yl, benzhydryl, piperidin-4-yl, piperidin-3-yl, piperidin-2-yl, tetrahydro-2H-pyran-2-yl, tetrahydro-2H-pyran-3-yl, tetrahydro-2H-pyran-4-yl, phenyl, tetrahydrofuran-3-yl, tetrahydrofuran-2-yl, benzyl, (4-pentylphenyl)(phenyl)methyl or l-(l-(2-oxo-6,9,12-trioxa-3- azatetradecan-14-yl)-lH-l,2,3-triazol-4-yl)ethyl can be optionally substituted with 1 to 3 radicals independently selected from methyl and trifluoromethyl.
[0057] In another embodiment are compounds selected from: 4-(2-(2-
(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phenol; 4-(2-(2- (benzo[b]thiophen-3-yl)-9-sec-butyl-9H-purin-6-ylamino)ethyl)phenol; 4-(2-(9-benzhydryl-2- (benzo[b]thiophen-3-yl)-9H-purin-6-ylamino)ethyl)phenol; 4-(2-(2-(benzo[b]thiophen-3-yl)-9- (tetrahydro-2H-pyran-3-yl)-9H-purin-6-ylamino)ethyl)phenol; 4-(2-(2-(benzo[b]thiophen-3-yl)- 9-(4-(trifluoromethyl)benzyl)-9H-purin-6-ylamino)ethyl)phenol; 4-(2-(2-(benzo[b]thiophen-3- yl)-9-isobutyl-9H-purin-6-ylamino)ethyl)phenol; 4-(2-(2-(benzo[b]thiophen-3-yl)-9-methyl-9H- purin-6-ylamino)ethyl)phenol; 4-(2-(2-(benzo[b]thiophen-3-yl)-9-(4-methylbenzyl)-9H-purin-6- ylamino)ethyl)phenol; N-(2-(lH-indol-3-yl)ethyl)-2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H- purin-6-amine; 2-(benzo[b]thiophen-3-yl)-9-isopropyl-N-(2-(thiophen-3-yl)ethyl)-9H-purin-6- amine; 3-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phenol; 2- (benzo[b]thiophen-3-yl)-N-(4-fluorophenethyl)-9-isopropyl-9H-purin-6-amine; N-(4- aminophenethyl)-2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-amine; 4-(2-(9-isopropyl-2- (pyrimidin-5-yl)-9H-purin-6-ylamino)ethyl)phenol; 4-(2-(9-isopropyl-2-(pyridin-3-yl)-9H-purin- 6-ylamino)ethyl)phenol; 4-(2-(9-isopropyl-2-phenyl-9H-purin-6-ylamino)ethyl)phenol; 4-(2-(9- isopropyl-2-(thiophen-3 -yl)-9H-purin-6-ylamino)ethyl)phenol ; 4-(2-(2-(furan-3 -yl)-9-isopropyl- 9H-purin-6-ylamino)ethyl)phenol; 2-(benzo[b]thiophen-3-yl)-N-(4-fluorophenethyl)-9-phenyl- 9H-purin-6-amine; N-benzyl-8-(biphenyl-4-yl)-9-isopropyl-9H-purin-6-amine; 4-(2-(2- (benzo[b]thiophen-3-yl)-9-(nonan-2-yl)-9H-purin-6-ylamino)ethyl)phenol; 4-(2-(2- (benzo[b]thiophen-3-yl)-9-((4-pentylphenyl)(phenyl)methyl)-9H-purin-6-ylamino)ethyl)phenol; N-(2-( 1 H-indol-3 -yl)ethyl)-2-(benzo [b] thiophen-3 -yl)-9-sec-butyl-9H-purin-6-amine ; 4-(2-(2- (benzo[b]thiophen-3-yl)-9-sec-butyl-9H-purin-6-ylamino)ethyl)phenol; 3-(2-(2- (benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)-lH-indol-5-ol; 3-(2-(2- (benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)-lH-indol-5-yl 5-((3aS,4S,6aR)- 2-oxohexahydro-lH-thieno[3,4-d]imidazol-4-yl)pentanoate; N-(2-(2-(3-(2-(2-(benzo[b]thiophen- 3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)-lH-indol-5-yloxy)ethoxy)ethyl)-5-((3aS,4S,6aR)- 2-oxohexahydro- 1 H-thieno [3 ,4-d]imidazol-4-yl)pentanamide ; N-(4-(4-(2-(3 -(2-(2- (benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)-lH-indol-5- yloxy)acetamido)benzoyl)phenyl)hex-5-ynamide; N-(2-(2-(2-(2-(4-(l-(2-(benzo[b]thiophen-3- yl)-6-(4-hydroxyphenethylamino)-9H-purin-9-yl)ethyl)- IH- 1 ,2,3-triazol- 1- yl)ethoxy)ethoxy)ethoxy)ethyl)acetamide; 4-(2-(9-isopropyl-2-(pyridin-4-yl)-9H-purin-6- ylamino)ethyl)phenol; ethyl 5-(6-(4-hydroxyphenethylamino)-9-isopropyl-9H-purin-2- yl)nicotinate; ethyl 5-(6-(4-hydroxyphenethylamino)-9-isopropyl-9H-purin-2-yl)nicotinate; 4-(2- (2-(6-fluoropyridin-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phenol; 4-(2-(9-isopropyl-2-(4- methylpyridin-3-yl)-9H-purin-6-ylamino)ethyl)phenol; 4-(2-(9-isopropyl-2-(2-methoxypyridin- 3-yl)-9H-purin-6-ylamino)ethyl)phenol; 5-(6-(4-hydroxyphenethylamino)-9-isopropyl-9H-purin- 2-yl)nicotinonitrile; 4-(2-(9-isopropyl-2-(pyrrolidin-l-yl)-9H-purin-6-ylamino)ethyl)phenol; 4- (2-(9-isopropyl-2-( 1 H-pyrazol- 1 -yl)-9H-purin-6-ylamino)ethyl)phenol ; 4-(2-(2-( 1 H-imidazol- 1 - yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phenol; 4-(2-(9-isopropyl-2-(pyridazin-3-yl)-9H- purin-6-ylamino)ethyl)phenol; 4-(2-(9-isopropyl-2-(pyridazin-4-yl)-9H-purin-6- ylamino)ethyl)phenol; 4-(2-(9-isopropyl-2-(pyrazin-2-yl)-9H-purin-6-ylamino)ethyl)phenol; 4- (2-(9-isopropyl-2-(pyridin-2-yl)-9H-purin-6-ylamino)ethyl)phenol; 4-(2-(9-isopropyl-2-(5- (methylsulfonyl)pyridin-3-yl)-9H-purin-6-ylamino)ethyl)phenol; 4-(2-(9-isopropyl-2-(5- methylpyridin-3-yl)-9H-purin-6-ylamino)ethyl)phenol; 4-(2-(2-(2-chloropyridin-3-yl)-6- isopropyl-2,6-dihydroimidazo[4,5-c]pyrazol-3-ylamino)ethyl)phenol; 4-(2-(2-(4-chloropyridin- 3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phenol; 4-(2-(9-isopropyl-2-(4-methoxypyridin-3- yl)-9H-purin-6-ylamino)ethyl)phenol; 4-(2-(2-(5-fluoropyridin-3-yl)-9-isopropyl-9H-purin-6- ylamino)ethyl)phenol; 4-(2-(9-isopropyl-2-(thiazol-4-yl)-9H-purin-6-ylamino)ethyl)phenol; 4- (2-(9-isopropyl-2-(l -methyl- lH-pyrazol-4-yl)-9H-purin-6-ylamino)ethyl)phenol; 4-(2-(9- isopropyl-2-( 1 H-pyrazol-3 -yl)-9H-purin-6-ylamino)ethyl)phenol ; 4-(2-(9-isopropyl-2-( 1 H- pyrazol-4-yl)-9H-purin-6-ylamino)ethyl)phenol; 4-(2-(9-isopropyl-2-(thiophen-2-yl)-9H-purin- 6-ylamino)ethyl)phenol; 4-(6-(4-hydroxyphenethylamino)-9-isopropyl-9H-purin-2-yl)thiophene- 2-carboxylic acid; 4-(2-(2-(furan-2-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phenol; 4-(2-(9- isopropyl-2-(4-methylthiophen-3 -yl)-9H-purin-6-ylamino)ethyl)phenol ; 4-(2-(9-isopropyl-2- (pyridin-3-yl)-9H-purin-6-ylamino)ethyl)-2-methoxyphenol; 4-(2-(9-isopropyl-2-(pyridin-3-yl)- 9H-purin-6-ylamino)ethyl)-2-methoxyphenol; N-[2-(6-methoxy-lH-indol-3-yl)ethyl]-9-(propan- 2-yl)-2-(pyridin-3-yl)-9H-purin-6-amine; N-[2-(5-methyl-lH-indol-3-yl)ethyl]-9-(propan-2-yl)- 2-(pyridin-3-yl)-9H-purin-6-amine; N-[2-(piperidin-4-yl)ethyl]-9-(propan-2-yl)-2-(pyridin-3-yl)- 9H-purin-6-amine; l-(2-{ [9-(propan-2-yl)-2-(pyridin-3-yl)-9H-purin-6- yl]amino}ethyl)piperidin-4-ol; methyl (2S)-3-(4-hydroxyphenyl)-2-{ [9-(propan-2-yl)-2-(pyridin- 3-yl)-9H-purin-6-yl]amino}propanoate; 4-(2-{ [9-(propan-2-yl)-2-(pyridin-3-yl)-9H-purin-6- yl] amino } ethyl)benzene- 1 -sulfonamide; 2- { [2-( 1 -benzothiophen-3 -yl)-9-(propan-2-yl)-9H- purin-6-yl]amino}ethane-l-sulfonamide; 4-(2-{ [9-(propan-2-yl)-2-(pyridin-3-yl)-9H-purin-6- yl] amino } ethyl)benzene- 1 ,2-diol; N- [2-( 1 H-imidazol-4-yl)ethyl] -9-(propan-2-yl)-2-(pyridin-3 - yl)-9H-purin-6-amine; l-(2-{ [9-(propan-2-yl)-2-(pyridin-3-yl)-9H-purin-6- yl] amino } ethyl)imidazolidin-2-one; N- [2-(5-amino- IH-1 ,2,4-triazol-3 -yl)ethyl] -9-(propan-2-yl)- 2-(pyridin-3-yl)-9H-purin-6-amine; N-(2-{ [9-(propan-2-yl)-2-(pyridin-3-yl)-9H-purin-6- yl] amino } ethyl)pyridin-2-amine; 9-(propan-2-yl)-N- [3 -( 1 H-pyrazol-4-yl)propyl] -2-(pyridin-3 - yl)-9H-purin-6-amine; N-[2-({ [9-(propan-2-yl)-2-(pyridin-3-yl)-9H-purin-6- yl] amino} methyl )propyl]acetamide; 4-(2-{ [9-(propan-2-yl)-2-(pyridin-3-yl)-9H-purin-6- yl] amino } ethyl)piperazin-2-one; N- { 2- [(3 -methyl- IH-1 ,2,4-triazol-5-yl)sulfanyl]ethyl } -9- (propan-2-yl)-2-(pyridin-3-yl)-9H-purin-6-amine; N-[3-(3,5-dimethyl-lH-pyrazol-4-yl)propyl]- 9-(propan-2-yl)-2-(pyridin-3-yl)-9H-purin-6-amine; (2-{ [9-(propan-2-yl)-2-(pyridin-3-yl)-9H- purin-6-yl] amino }ethyl)urea; 5-({ [9-(propan-2-yl)-2-(pyridin-3-yl)-9H-purin-6- yl] amino } methyl)-2,3 -dihydro- 1 H- 1 ,3 -benzodiazol-2-one ; 2-( 1 -benzothiophen-3 -yl)-N- [2-( 1 H- imidazol-4-yl)ethyl] -9-(propan-2-yl)-9H-purin-6-amine ; 1 -(2- { [2-( 1 -benzothiophen-3 -yl)-9- (propan-2-yl)-9H-purin-6-yl] amino } ethyl)imidazolidin-2-one; N- [2-(5-amino- IH-1 ,2,4-triazol- 3 -yl)ethyl] -2-( 1 -benzothiophen-3 -yl)-9-(propan-2-yl)-9H-purin-6-amine ; N-(2- { [2-( 1 - benzothiophen-3-yl)-9-(propan-2-yl)-9H-purin-6-yl]amino}ethyl)pyridin-2-amine; 2-(l- benzothiophen-3 -yl)-9-(propan-2-yl)-N- [3 -( 1 H-pyrazol-4-yl)propyl] -9H-purin-6-amine; N- [2- ( { [2-( 1 -benzothiophen-3 -yl)-9-(propan-2-yl)-9H-purin-6-yl] amino } methyl )propyl] acetamide; 4- (2- { [2-( 1 -benzothiophen-3 -yl)-9-(propan-2-yl)-9H-purin-6-yl] amino } ethyl)piperazin-2-one; 2- ( 1 -benzothiophen-3 -yl)-N- { 2- [(3 -methyl- 1 H- 1 ,2,4-triazol-5-yl)sulfanyl]ethyl } -9-(propan-2-yl)- 9H-purin-6-amine; 2-(l-benzothiophen-3-yl)-N-[3-(3,5-dimethyl-lH-pyrazol-4-yl)propyl]-9- (propan-2-yl)-9H-purin-6-amine; (2- { [2-(l -benzothiophen-3 -yl)-9-(propan-2-yl)-9H-purin-6- yl] amino }ethyl)urea; 5-({ [2-(l-benzothiophen-3-yl)-9-(propan-2-yl)-9H-purin-6- yl] amino } methyl)-2,3 -dihydro- 1 H- 1 ,3 -benzodiazol-2-one ; N- [2-( 1 H-indol-3-yl)ethyl] -9- (propan-2-yl)-2-(pyridin-3-yl)-9H-purin-6-amine; N-(4-(2-(9-isopropyl-2-(pyridin-3-yl)-9H- purin-6-ylamino)ethyl)phenyl)methanesulfonamide; 4-(2-(2-(pyridin-3-yl)-9-(tetrahydrofuran-3- yl)-9H-purin-6-ylamino)ethyl)phenol; 4-(2-(9-isopropyl-2-(pyridin-3-yl)-9H-purin-6- ylamino)propyl)phenol; 4-(2-(9-(oxetan-3-yl)-2-(pyridin-3-yl)-9H-purin-6- ylamino)ethyl)phenol; 5-(6-(4-hydroxyphenethylamino)-9-isopropyl-9H-purin-2-yl)-N- methylnicotinamide; 6-(9-isopropyl-2-(pyridin-3-yl)-9H-purin-6-ylamino)-5, 6,7,8- tetrahydronaphthalen-2-ol; N-(2-(lH-indazol-3-yl)ethyl)-9-isopropyl-2-(pyridin-3-yl)-9H-purin- 6-amine; 4-(2-((9-isopropyl-2-(pyridin-3-yl)-9H-purin-6-yl)(methyl)amino)ethyl)phenol; 4-(2- (9-isopropyl-8-methyl-2-(pyridin-3-yl)-9H-purin-6-ylamino)ethyl)phenol; l-(2-(9-isopropyl-2- (pyridin-3 -yl)-9H-purin-6-ylamino)ethyl)- 1 H-benzo [d]imidazol-2(3H)-one ; 4-(3 -(9-isopropyl-2- (pyridin-3-yl)-9H-purin-6-yl)propyl)phenol; 4-((((9-isopropyl-2-(pyridin-3-yl)-9H-purin-6- yl)methyl)(methyl)amino)methyl)phenol; 4-(((9-isopropyl-2-(pyridin-3-yl)-9H-purin-6- yl)methylamino)methyl)phenol; 4-(((9-isopropyl-2-(pyridin-3-yl)-9H-purin-6- yl)methoxy)methyl)phenol; N-(2-(indolin-5-yl)ethyl)-9-isopropyl-2-(pyridin-3-yl)-9H-purin-6- amine; 4-(2-(9-(l-methylpiperidin-4-yl)-2-(pyridin-3-yl)-9H-purin-6-ylamino)ethyl)phenol; 4- (2-(9-(piperidin-4-yl)-2-(pyridin-3-yl)-9H-purin-6-ylamino)ethyl)phenol; N-(2-(lH-indazol-5- yl)ethyl)-9-isopropyl-2-(pyridin-3-yl)-9H-purin-6-amine; N-(2-(lH-benzo[d]imidazol-5- yl)ethyl)-9-isopropyl-2-(pyridin-3-yl)-9H-purin-6-amine; 5-(2-(9-isopropyl-2-(pyridin-3-yl)-9H- purin-6-ylamino)ethyl)indolin-2-one; 4-(2-(9-cyclopropyl-2-(pyridin-3-yl)-9H-purin-6- ylamino)ethyl)phenol; 4-(2-(9-(l-hydroxypropan-2-yl)-2-(pyridin-3-yl)-9H-purin-6- ylamino)ethyl)phenol; 4-(2-(9-isopropyl-2-(pyridin-3-yl)-9H-purin-6-ylamino)ethyl)phenyl sulfamate; 2-(4-hydroxyphenyl)-N-(9-isopropyl-2-(pyridin-3-yl)-9H-purin-6-yl)acetamide; 4-(5- (9-isopropyl-2-(pyridin-3-yl)-9H-purin-6-ylamino)isoxazol-3-yl)phenol; 4-(5-(9-isopropyl-2- (pyridin-3-yl)-9H-purin-6-ylamino)-l,3,4-oxadiazol-2-yl)phenol; 4-(2-(2-(2-fluoropyridin-3-yl)- 9-isopropyl-9H-purin-6-ylamino)ethyl)phenol; 4-(2-(9-isopropyl-2-(l -methyl- lH-pyrrol-2-yl)- 9H-purin-6-ylamino)ethyl)phenol; and 4-(2-(9-isopropyl-2-(thiazol-5-yl)-9H-purin-6- ylamino)ethyl)phenol. [0058] In another embodiment is a compound of formula If:
Figure imgf000023_0001
[0059] in which: R2 is selected from lH-indol-3-yl and phenyl optionally substituted with hydroxy; and R4 is selected from isopropyl, sec -butyl, benzhydryl, nonan-2-yl, oxetan-3-yl and tetrahydrofuran-3-yl.
[0060] In a further embodiment are compounds selected from: 4-(2-(2-
(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phenol; 4-(2-(2- (benzo[b]thiophen-3-yl)-9-sec-butyl-9H-purin-6-ylamino)ethyl)phenol; 4-(2-(9-benzhydryl-2- (benzo[b]thiophen-3-yl)-9H-purin-6-ylamino)ethyl)phenol; N-(2-(lH-indol-3-yl)ethyl)-2- (benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-amine; 4-(2-(2-(benzo[b]thiophen-3-yl)-9- (nonan-2-yl)-9H-purin-6-ylamino)ethyl)phenol; N-(2-(lH-indol-3-yl)ethyl)-2- (benzo[b]thiophen-3-yl)-9-sec-butyl-9H-purin-6-amine; 4-(2-(2-(benzo[b]thiophen-3-yl)-9- (oxetan-3 -yl)-9H-purin-6-ylamino)ethyl)phenol ; (S)-4-(2-(2-(benzo [b]thiophen-3 -yl)-9- (tetrahydrofuran-3-yl)-9H-purin-6-ylamino)ethyl)phenol; and (R)-4-(2-(2-(benzo[b]thiophen-3- yl)-9-(tetrahydromran-3-yl)-9H-purin-6-ylamino)ethyl)phenol. [0061] In another embodiment is a compound of formula Ig:
Figure imgf000024_0001
Ig
[0062] in which: R2 is selected from: lH-pyrrolo[2,3-b]pyridin-3-yl; lH-indol-3-yl optionally substituted with 1 to 2 radicals independently selected from halo, methyl and methoxy; and phenyl optionally substituted with 1 to 2 radicals independently selected from methyl, halo and hydroxy; R4 is selected from isopropyl, sec-butyl, l-hydroxypropan-2-yl, prop- l-en-2-yl, benzhydryl, nonan-2-yl, oxetan-3-yl and tetrahydrofuran-3-yl; and Ra, Rb and Rc are independently selected from hydrogen, cyano, methyl, halo, -SO2CH3 and trifluoromethyl. [0063] In a further embodiment are compounds selected from: 4-(2-(9-isopropyl-2-
(pyridin-3-yl)-9H-purin-6-ylamino)ethyl)phenol; 4-(2-(2-(6-fluoropyridin-3-yl)-9-isopropyl-9H- purin-6-ylamino)ethyl)phenol; 4-(2-(9-isopropyl-2-(4-methylpyridin-3-yl)-9H-purin-6- ylamino)ethyl)phenol; 5-(6-(4-hydroxyphenethylamino)-9-isopropyl-9H-purin-2- yl)nicotinonitrile; 4-(2-(9-isopropyl-2-(5-methylpyridin-3-yl)-9H-purin-6-ylamino)ethyl)phenol; 4-(2-(9-isopropyl-2-(5-(methylsulfonyl)pyridin-3-yl)-9H-purin-6-ylamino)ethyl)phenol; 4-(2-(2- (4-chloropyridin-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phenol; 4-(2-(2-(5-fluoropyridin-
99 3 -yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phenol ; 9-isopropyl-N-(2-(6-methoxy- 1 H-indol-3 - yl)ethyl)-2-(pyridin-3-yl)-9H-purin-6-amine; 9-isopropyl-N-(2-(5-methyl-lH-indol-3-yl)ethyl)- 2-(pyridin-3-yl)-9H-purin-6-amine; N-(2-(lH-indol-3-yl)ethyl)-9-isopropyl-2-(pyridin-3-yl)-9H- purin-6-amine; 4-(2-(9-(oxetan-3-yl)-2-(pyridin-3-yl)-9H-purin-6-ylamino)ethyl)phenol; 4-(2-(9- (l-hydroxypropan-2-yl)-2-(pyridin-3-yl)-9H-purin-6-ylamino)ethyl)phenol; 4-(2-(2-(2- fluoropyridin-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phenol; 5-(9-sec-butyl-6-(4-hydroxy- 3-methylphenethylamino)-9H-purin-2-yl)nicotinonitrile; 4-(2-(2-(5-fluoropyridin-3-yl)-9- (oxetan-3-yl)-9H-purin-6-ylamino)ethyl)phenol; 4-(2-(2-(5-chloropyridin-3-yl)-9-isopropyl-9H- purin-6-ylamino)ethyl)phenol; 4-(2-(9-isopropyl-2-(5-(trifluoromethyl)pyridin-3-yl)-9H-purin-6- ylamino)ethyl)phenol; 5-(6-(2-(lH-indol-3-yl)ethylamino)-9-sec-butyl-9H-purin-2- yl)nicotinonitrile; N-(2-(lH-indol-3-yl)ethyl)-9-sec-butyl-2-(5-methylpyridin-3-yl)-9H-purin-6- amine; (R)-N-(2-(lH-indol-3-yl)ethyl)-9-sec-butyl-2-(5-fluoropyridin-3-yl)-9H-purin-6-amine; (S)-N-(2-(lH-indol-3-yl)ethyl)-9-sec-butyl-2-(5-fluoropyridin-3-yl)-9H-purin-6-amine; N-(2- (lH-indol-3-yl)ethyl)-9-sec-butyl-2-(5-fluoropyridin-3-yl)-9H-purin-6-amine; (R)-N-(2-(lH- indol-3-yl)ethyl)-9-sec-butyl-2-(5-methylpyridin-3-yl)-9H-purin-6-amine; (S)-N-(2-(lH-indol-3- yl)ethyl)-9-sec-butyl-2-(5-methylpyridin-3-yl)-9H-purin-6-amine; 5-(6-(4- hydroxyphenethylamino)-9-(oxetan-3-yl)-9H-purin-2-yl)nicotinonitrile; 4-(2-(6-(5- fluoropyridin-3 -yl)- 1 -isopropyl- 1 H-pyrazolo [3 ,4-d]pyrimidin-4-ylamino)ethyl)phenol ; 3 -(6-(4- hydroxyphenethylamino)-9-isopropyl-9H-purin-2-yl)isonicotinonitrile; 4-(2-(2-(5-fluoropyridin- 3 -yl)-7-isopropyl-7H-pyrrolo [2,3 -d]pyrimidin-4-ylamino)ethyl)phenol ; 4-(2-(9-isopropyl-2-(6- methylpyridin-3-yl)-9H-purin-6-ylamino)ethyl)phenol; 2-chloro-4-(2-(9-isopropyl-2-(pyridin-3- yl)-9H-purin-6-ylamino)ethyl)phenol; 3-fluoro-4-(2-(9-isopropyl-2-(pyridin-3-yl)-9H-purin-6- ylamino)ethyl)phenol; N-(2-(5-fluoro-lH-indol-3-yl)ethyl)-9-isopropyl-2-(pyridin-3-yl)-9H- purin-6-amine; N-(2-(5-chloro-lH-indol-3-yl)ethyl)-9-isopropyl-2-(pyridin-3-yl)-9H-purin-6- amine; 4-(2-(9-isopropyl-2-(pyridin-3-yl)-9H-purin-6-ylamino)ethyl)-2-methylphenol; 2-(6-(2- ( 1 H-indol-3 -yl)ethylamino)-2-(5-fluoropyridin-3 -yl)-9H-purin-9-yl)propan- 1 -ol ; (R)-2-(6-(2- (lH-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin-9-yl)propan-l-ol; (S)-2-(6-(2- ( 1 H-indol-3 -yl)ethylamino)-2-(5-fluoropyridin-3 -yl)-9H-purin-9-yl)propan- 1 -ol ; (R)-N-(2-( 1 H- indol-3-yl)ethyl)-2-(5-fluoropyridin-3-yl)-9-(tetrahydrofuran-3-yl)-9H-purin-6-amine; 4-(2-(6- (5-fluoropyridin-3-yl)-l-isopropyl-lH-imidazo[4,5-c]pyridin-4-ylamino)ethyl)phenol; N-(2-(lH- indol-3-yl)ethyl)-2-(5-fluoropyridin-3-yl)-9-isopropyl-9H-purin-6-amine; 2-(5-fluoropyridin-3- yl)-9-isopropyl-N-(2-(6-methoxy-lH-indol-3-yl)ethyl)-9H-purin-6-amine; 2-(5-fluoropyridin-3- yl)-9-isopropyl-N-(2-(5-methoxy-lH-indol-3-yl)ethyl)-9H-purin-6-amine; N-(2-(lH-indol-3- yl)ethyl)-2-(5-fluoropyridin-3-yl)-9-(prop-l-en-2-yl)-9H-purin-6-amine; N-(2-(lH-pyrrolo[2,3- b]pyridin-3-yl)ethyl)-2-(5-fluoropyridin-3-yl)-9-isopropyl-9H-purin-6-amine; 4-(2-(5-(5- fluoropyridin-3-yl)-3-isopropyl-3H-imidazo[4,5-b]pyridin-7-ylamino)ethyl)phenol; N-(2-(lH- indol-3-yl)ethyl)-9-isopropyl-2-(5-methylpyridin-3-yl)-9H-purin-6-amine; 2-(5-fluoropyridin-3- yl)-9-isopropyl-N-(2-(7-methyl-lH-indol-3-yl)ethyl)-9H-purin-6-amine; N-(2-(lH-indol-3- yl)ethyl)-2-(5-fluoropyridin-3-yl)-9-(oxetan-3-yl)-9H-purin-6-amine; N-(2-(lH-indol-3- yl)ethyl)-2-(5-methylpyridin-3-yl)-9-(oxetan-3-yl)-9H-purin-6-amine; N-(2-(6-fluoro-lH-indol-
3-yl)ethyl)-2-(5-fluoropyridin-3-yl)-9-isopropyl-9H-purin-6-amine; 2-(5-fluoropyridin-3-yl)-9- isopropyl-N-(2-(2-methyl-lH-indol-3-yl)ethyl)-9H-purin-6-amine; 2-(5-fluoropyridin-3-yl)-9- isopropyl-N-(2-(6-methyl- 1 H-indol-3 -yl)ethyl)-9H-purin-6-amine; N-(2-(4-fluoro- 1 H-indol-3 - yl)ethyl)-2-(5-fluoropyridin-3-yl)-9-isopropyl-9H-purin-6-amine; 2-(5-fluoropyridin-3-yl)-9- isopropyl-N-(2-(4-methyl- 1 H-indol-3 -yl)ethyl)-9H-purin-6-amine; N-(2-(7-fluoro- 1 H-indol-3 - yl)ethyl)-2-(5-fluoropyridin-3-yl)-9-isopropyl-9H-purin-6-amine; and 4-(2-(2-(5-fluoropyridin-
3-yl)-9-(l-hydroxypropan-2-yl)-9H-purin-6-ylamino)ethyl)-2-methylphenol.
[0064] In another embodiment is a method of using a compound of Formula I to stimulate the expansion of stem cells by increasing the number of divisions; said method comprising contacting the stem cells with a compound of Formula I.
[0065] In another embodiment is a method in which the expansion of stem cells is in vivo, in vitro, or ex vivo.
[0066] In another embodiment is a method in which the stem cells are human hematopoietic stem cells.
[0067] In another embodiment is a cell population with expanded hematopoietic stem cells, as obtained or obtainable by the method of the invention.
[0068] In a further embodiment is a composition comprising a cell population with expanded HSCs derived from one or two cord blood units, preferably one cord blood unit, wherein said composition contains a total amount of cells of at least 105 cells, 107 cells, 108 cells or 109 cells, and wherein between 20-100% of total cells are CD34+ cells, for example between
40-80% of total cells are CD34+. [0069] In another embodiment is a method for treating a disease or disorder for which stem cell therapy would result in the prevention, treatment or eradication of said disorder.
[0070] It is anticipated that as stem cell use progresses the diseases that can be treated by stem cell transplantation will expand. A non-limiting list of examples follows, infra.
[0071] In another embodiment is the use of a compound of Formula I as defined in the Summary of the Invention, or a salt thereof, in the preparation of a composition for the treatment of an inhereited immunodeficient disease, an autoimmune disease and/or a hematopoietic disorder.
[0072] In a further embodiment, the administration is an autologous transplantation and the hematopoietic disorder is selected from Multiple myeloma, Non-Hodgkin lymphoma,
Hodgkin disease, Acute myeloid leukemia, Neuroblastoma, Germ cell tumors, Autoimmune disorders and Amyloidosis.
[0073] In a further embodiment, the autoimmune disorders are selected from
Systemic lupus erythematosus (SLE) and systemic sclerosis.
[0074] In a further embodiment, the administration is an allogeneic transplantation and the hematopoietic disorder is selected from Acute myeloid leukemia, Acute lymphoblastic leukemia, Chronic myeloid leukemia, Chronic lymphocytic leukemia, Myeloproliferative disorders, Myelodysplastic syndromes, Multiple myeloma, Non-Hodgkin lymphoma,
Hodgkin disease, Aplastic anemia, Pure red cell aplasia, Paroxysmal nocturnal hemoglobinuria, Fanconi anemi, Thalassemia major, Sickle cell anemia, Severe combined immunodeficiency (SCID), Wiskott-Aldrich syndrome, Hemophagocytic lymphohistiocytosis (HLH) and inborn errors of metabolism.
[0075] In a further embodiment, the inborn errors of metabolism are selected from mucopolysaccharidosis, Gaucher disease, metachromatic leukodystrophies and adrenoleukodys trophies .
[0076] In another embodiment is a method for treating an inhereited immunodeficient disease, an autoimmune disease and/or a hematopoietic disorder comprising administration to a patient in need of such treatment hematopoietic stem cells expanded by a compound as described in the Summary of the Invention.
[0077] In a further embodiment, the administration is an autologous transplantation and the hematopoietic disorder is selected from Multiple myeloma, Non-Hodgkin lymphoma, Hodgkin disease, Acute myeloid leukemia, Neuroblastoma, Germ cell tumors, Autoimmune disorders and Amyloidosis.
[0078] In a further embodiment, the autoimmune disorders are selected from
Systemic lupus erythematosus (SLE) and systemic sclerosis.
[0079] In a further embodiment, the administration is an allogeneic transplantation and the hematopoietic disorder is selected from Acute myeloid leukemia, Acute lymphoblastic leukemia, Chronic myeloid leukemia, Chronic lymphocytic leukemia, Myeloproliferative disorders, Myelodysplastic syndromes, Multiple myeloma, Non-Hodgkin lymphoma,
Hodgkin disease, Aplastic anemia, Pure red cell aplasia, Paroxysmal nocturnal hemoglobinuria, Fanconi anemi, Thalassemia major, Sickle cell anemia, Severe combined immunodeficiency (SCID), Wiskott-Aldrich syndrome, Hemophagocytic lymphohistiocytosis (HLH) and inborn errors of metabolism.
[0080] In a further embodiment, the inborn errors of metabolism are selected from mucopolysaccharidosis, Gaucher disease, metachromatic leukodystrophies and adrenoleukodys trophies .
Utility
[0081] HSCs are primitive cells capable of regenerating all blood cells. During development, hematopoiesis translocates from the fetal liver to the bone marrow, which then remains the site of hematopoiesis throughout adulthood. Once hematopoiesis has been established in the bone marrow, the HSCs are not distributed randomly throughout the bone cavity. Instead, they are found in close proximity to the endosteal surfaces. The more mature stem cells increase in number as the distance from the bone surface increases. Finally, as the central longitudinal axis of the bone is approached terminal differentiation of mature cells occurs.
[0082] Expanding the number of stem cells, whether from adult, umbilical cord blood, fetal, or embryonic sources, would have a huge impact on transplantation and other therapies for hematology and oncology diseases and disorders, the least of which would be increased safety and reduced costs. As described in the methods herein, HSC numbers are increased ex vivo. A method of increasing stem cell numbers is important as currently, approximately 25% of autologous donor transplants are prohibited for lack of sufficient stem cells. In addition, less than 25% of patients in need of allogeneic transplant can find a histocompatible donor. Umbilical cord blood banks currently exist and cover the broad racial make-up of the general population, but these banks are currently restricted to use in children due to inadequate stem cell numbers in the specimens for adult recipients. A method to increase stem cell numbers permits cord blood to be useful for adult patients, thereby expanding the use of allogeneic transplantation. Compounds of the invention can also be used to expand the progenitor cell numbers which are clinically useful, for example, to speed engraftment and decrease the duration of neutopenia.
[0083] Accordingly, a method for increasing the number of HSCs is provided. As used herein, an increase in HSCs means that the subject has at least one more HSC, a 10% increase, a 20% increase, a 30% increase or greater. HSCs may consist of a subset of CD34+ cells, increase of HSCs can be measured indirectly by counting the number of CD34+ cells in a cell population and, optionally, by assessing the differentiation properties of the CD34+ cells by analyzing the colony forming units (CFU) as described in the experimental part below: An increase of the number of CD34+ cells culture of a least 10%, preferably 20% increase or 30% increase or greater as compared with a control without expansion is indicative of HSC expansion. The expanded population of HSCs is harvested, for example, from a bone marrow sample of a subject or from a culture. Harvesting HSCs is defined as the dislodging or separation of cells. This is accomplished using a number of methods, such as enzymatic, non-enzymatic, centrifugal, electrical, or size-based methods, or preferably, by flushing the cells using culture media (e.g., media in which cells are incubated) or buffered solution. The cells are optionally collected, separated, and further expanded generating even larger populations of HSCs and differentiated progeny.
[0084] A method for making an expanded population of HSCs comprises contacting an agent capable of down-regulating the activity and/or expression of AHR and/or a downstream effector of AHR, e.g., a compound of the invention, with a starting cell population (i.e., an unexpanded population of cells) comprising a mixture of HSCs and optionally HSC supporting cells. The administration step occurs ex vivo, in vivo and/or in vitro. As described herein, the expanded population of HSCs is optionally administered to a subject. For ex vivo expansion, such agent for HSC expansion, e.g. a compound of the invention, may be formulated in DMSO or some other suitable carrier, "washed" from the cells and the cells may be transferred, for example, into an infusion buffer. A DMSO formulation, for example, can contain 0.3mg/ml of a compound of the invention in 60% DMSO/40% water solution. Thus, provided are methods of providing an expanded population of HSCs to a subject comprising administering to the subject the expanded population of HSCs described herein or made by the methods described herein. The expanded population of HSCs is optionally used to make blood cells. The blood cells are optionally administered to a subject in need. Optionally, the subject is the same subject from which the unexpanded population of HSCs or mixture of HSCs and HSC supporting cells was derived.
[0085] As used herein, the term HSC supporting cell refers to cells naturally found in the vicinity of one or more HSCs such that factors released by HSC supporting cells reach the HSC by diffusion, for example. HSC supporting cells include, but are not limited to, lymphoreticular stromal cells. Lymphoreticular stromal cells as used herein include, but are not limited to, all cell types present in a lymphoid tissue which are not lymphocytes or lymphocyte precursors or progenitors. Thus, lymphoreticular stromal cells include osteoblasts, epithelial cells, endothelial cells, mesothelial cells, dendritic cells, splenocytes and macrophages. Lymphoreticular stromal cells also include cells that would not ordinarily function as lymphoreticular stromal cells, such as fibroblasts, which have been genetically altered to secrete or express on their cell surface the factors necessary for the maintenance, growth or differentiation of HSCs, including their progeny. Lymphoreticular stromal cells are optionally derived from the disaggregation of a piece of lymphoid tissue. Such cells are capable of supporting in vitro or in vivo the maintenance, growth or differentiation of HSCs, including their progeny. By lymphoid tissue it is meant to include bone marrow, peripheral blood (including mobilized peripheral blood), umbilical cord blood, placental blood, fetal liver, embryonic cells (including embryonic stem cells), aortal-gonadal-mesonephros derived cells, and lymphoid soft tissue. Lymphoid soft tissue as used herein includes, but is not limited to, tissues such as thymus, spleen, liver, lymph node, skin, tonsil, adenoids and Peyer's patch, and combinations thereof. [0086] Lymphoreticular stromal cells provide the supporting microenvironment in the intact lymphoid tissue for the maintenance, growth or differentiation of HSCs, including their progeny. The microenvironment includes soluble and cell surface factors expressed by the various cell types which comprise the lymphoreticular stroma. Generally, the support which the lymphoreticular stromal cells provide is characterized as both contact-dependent and non- contact-dependent. [0087] Lymphoreticular stromal cells, for example, are autologous (self) or non- autologous (non-self, e.g., heterologous, allogeneic, syngeneic or xenogeneic) with respect to HSCs. Autologous, as used herein, refers to cells from the same subject. Allogeneic, as used herein, refers to cells of the same species that differ genetically. Syngeneic, as used herein, refers to cells of a different subject that are genetically identical to the cell in comparison. Xenogeneic, as used herein, refers to cells of a different species. Lymphoreticular stroma cells are obtained, for example, from the lymphoid tissue of a human or a non-human subject at any time after the organ/tissue has developed to a stage (i.e., the maturation stage) at which it can support the maintenance, growth or differentiation of HSCs. The lymphoid tissue from which lymphoreticular stromal cells are derived usually determines the lineage-commitment HSCs undertake, resulting in the lineage-specificity of the differentiated progeny. [0088] The co-culture of HSCs (and progeny thereof) with lymphoreticular stromal cells, usually occurs under conditions known in the art (e.g., temperature, CO2 and O2 content, nutritive media, duration, etc.). The time sufficient to increase the number of cells is a time that can be easily determined by a person skilled in the art, and varies depending upon the original number of cells seeded. The amounts of HSCs and lymphoreticular stromal cells initially introduced (and subsequently seeded) varies according to the needs of the experiment. The ideal amounts are easily determined by a person skilled in the art in accordance with needs. [0089] As used throughout, by a subject is meant an individual. Thus, subjects include, for example, domesticated animals, such as cats and dogs, livestock (e.g., cattle, horses, pigs, sheep, and goats), laboratory animals (e.g., mice, rabbits, rats, and guinea pigs), mammals, non-human mammals, primates, non-human primates, rodents, birds, reptiles, amphibians, fish, and any other animal. The subject is optionally a mammal such as a primate or a human.
Methods For Expanding Hematopoietic Stem Cells
[0090] The invention therefore relates to a method for expanding hematopoietic stem cells, comprising (a) providing a starting cell population comprising hematopoietic stem cells and (b) culturing said starting cell population ex vivo in presence of an agent capable of down- regulating the activity and/or expression of aryl hydrocarbon receptor and/or a down-stream effector of aryl hydrocarbon receptor pathway, under suitable conditions for expanding hematopoietic stem cells. [0091] The aryl hydrocarbon (dioxin) receptor (AHR) is a cytosolic ligand-activated transcription factor known to mediate a large number of toxic and carcinogenic effects in animals and possible in human (Safe S 2001 Toxicol Lett 120: 1-7). As a consequence of AHR activation by its ligands, many detoxification genes are transcriptionally induced, including those coding for phase I xenobiotic -metabolizing enzymes, such as the cytochromes P450 CYPlAl, CYP 1A2, CYPlBl and CYP2S1, and the phase II enzymes UDP-glucuronosyltransf erase UGT1A6, NAD(P)H-dependent quinone oxidoreductase- 1 (NQOl), the aldehyde dehydrogenase ALDH3A1, and several glutathione-S-transferase.
[0092] In one embodiment, an agent capable of down-regulating the activity and/or expression of aryl hydrocarbon receptor and/or a down-stream effector of aryl hydrocarbon receptor pathway is selected among the group consisting of: (i) an organic compound; (ii) a small interference RNA (siRNA) molecule capable of down-regulating the expression of AHR; and (iii) antisense oligonucleotide capable of down-regulating the expression of AHR. [0093] In one specific embodiment, said method for expanding hematopoietic stem cells, comprises (a) providing a starting cell population comprising hematopoietic stem cells and (b) culturing said starting cell population ex vivo in the presence of an agent capable of down- regulating the activity and/or expression of aryl hydrocarbon receptor and/or a down-stream effector of aryl hydrocarbon receptor pathway, under suitable conditions for expanding hematopoietic stem cells, wherein said agent capable of down-regulating the activity and/or expression of aryl hydrocarbon receptor and/or a down-stream effector of aryl hydrocarbon receptor pathway is not alpha-napthoflavone or 3'-methoxy-4'-nitroflavone. [0094] Organic compound that inhibits AHR activity (also referred herein as AHR antagonist) have been described in the art, for example 2-methyl-2H-pyrazole-3-carboxylic acid (2-methyl-4-o-tolylazophenyl)amide (CH223191), alpha napthoflavone, resveratrol (Nutr. Metab. Cardiovasc. Dis., 2003 Apr; 13(2): 104-13), 3 ' -methoxy-4'-nitroflavone (Biochem. Pharmacol., 2007 May 15; 73(10): 1622-34, Epub 2007 Jan 30), and 6-methyl-l,3,8- trichlorodibenzofuran (Cancer Res., 2004, Apr 15;64(8):2889-97). An inhibitor of AHR activity refers to a compound which decreases AHR activity to at least 10%, 20%, 30%, 50%, 60%, 70%, 80% or at least 90% the transcriptional activity of AHR as observed under activated conditions. An assay to measure AHR inhibitory activity is for example the dioxin-induced AHR dependent luciferase reporter gene assay as described in the Examples. In one embodiment, an inhibitor of AHR activity is a compound that has an EC50 of less than lOμM, preferably less than 5μM as measured in the dioxin-induced AHR dependent luciferase reporter gene assay. [0095] AHR is a transcriptional factor regulating the transcription of various genes in human. In one embodiment, a downstream effector of AHR pathway is a gene which is directly regulated at the transcriptional level by AHR. Examples of such genes are selected from CyplBl, CyplAl, and AHRR. AHR also functions in pathways outside of its well- characterized role in xenobiotic enzyme induction. Xenobiotic ligands of AHR have been shown to regulate beta catenin, STAT5, STATl, HES-I, c-Myc, C/EBP, PU.l, β-catenin, p21, P27, pRb, deoxynucleotidyl transferase, CXCR4, and its chemokine ligand CXCL12 (SDF-I). [0096] In one specific embodiment, an agent capable of down-regulating the activity and/or expression of aryl hydrocarbon receptor is a compound as defined in the Summary of the Invention.
[0097] In another embodiment, an agent capable of down-regulating the activity and/or expression of aryl hydrocarbon receptor is an antisense oligonucleotide or a small interfering RNA molecule (siRNA), capable of down-regulating AHR protein expression or the protein expression of one more down-stream effectors of AHR.
[0098] Design of antisense oligonucleotides which can be used to efficiently inhibit the AHR protein expression must be effected in a way that such oligonucleotides specifically binds the designated mRNA within cells in a way which inhibits translation thereof. Sequence suitable for use in design and synthesis of antisense oligonucleotides which specifically bind to AHR mRNA, genomic DNA and/or its promoter or other control sequences are available in published sequence of AHR, in particular human AHR. In addition, algorithms for identifying sequences with the highest predicted binding affinity for their target mRNA based on thermodynamic cycle that accounts for the energetics of structural alterations in both the target mRNA and the oligonucleotides are also available.
[0099] Synthesis of RNAi molecules suitable for use with the present invention can be affected as follows: First, the AHR mRNA sequence (or one or more of its down-stream effectors) is scanned downstream of the AUG start codon for AA-dinucleotide sequences. Occurrence of each AA and the 19 3'-adjacent is recorded as a potential siRNA target site. Then, potential target sites are compared to an appropriate genomic database (e.g, human, mouse, rat, etc.) using any sequence alignment software. Putative target site that exhibit significant homology to other coding sequences are filtered out. Preferred sequences are then those including low G/C content, in particular sequences with G/C content lower than 55%. Several target sites are then selected along the length of the target gene. Methods or algorithms to identify putative target site of siRNA are described for example in (Tilesi, et al., Curr. Opin. MoI. Ther. 11: 156, 2009). Examples of siRNA molecules which are capable of down-regulating the expression of AHR are: AHR 11 IS, 5' GCG GCA TAG AGA CCG ACT TAA TTT CAA GAG AAT TAA GTC GGT CTC TAT GCC GCT TTT TTG G 3' ; AHR 11 IAS, 5' CGC GCC AAA AAA GCG GCA TAG AGA CCG ACT TAA TTC TCT TGA AAT TAA GTC GGT CTC TAT GCC GC 3' ; AHR 242S, 5' GGC TTC TTT GAT GTT GCA TTA ATT CAA GAG ATT AAT GCA ACA TCA AAG AAG CCT TTT TTG G 3' ; AHR 242AS, 5' CGC GCC AAA AAA GGC TTC TTT GAT GTT GCA TTA ATC TCT TGA ATT AAT GCA ACA TCA AAG AAG CC 3'.
[00100] The starting cell population comprising hematopoietic stem cells will be selected by the person skilled in the art depending on the envisaged use. Various sources of cells comprising hematopoietic stem cells have been described in the art, including bone marrow, peripheral blood, neonatal umbilical cord blood, placenta or other sources such as liver, particularly fetal liver.
[00101] The cell population may first be subjected to enrichment or purification steps, including negative and/or positive selection of cells based on specific cellular markers in order to provide the starting cell population. Methods for isolating said starting cell population based on specific cellular markers may use fluorescent activated cell sorting (FACS) technology also called flow cytometry or solid or insoluble substrate to which is bound antibodies or ligands that interact with specific cell surface markers. For example, cells may be contacted with a solid substrate (e.g., column of beads, flasks, magnetic particles) containing the antibodies and any unbound cells are removed. When a solid substrate comprising magnetic or paramagnetic beads is used, cells bound to the beads can be readily isolated by a magnetic separator. [00102] In one embodiment, said starting cell population is enriched in a desirable cell marker phenotype (e.g., CD34+, CD133+, CD90+) or based on efflux of dyes such as rhodamine, Hoechst or aldehyde dehydrogenase activity. In one specific embodiment, said starting cell population is enriched in CD34+ cells. Methods for enriching blood cell population in CD34+ cells include kits commercialized by Miltenyi Biotec (CD34+ direct isolation kit,
Miltenyi Biotec, Bergisch, Gladbach, Germany) or by Baxter (Isolex 3000).
[00103] The amount of cord blood from a single birth is often inadequate to treat an adult or an older child. One advantage of the expansion methods using the compounds of the invention, or an agent capable of down-regulating the activity and/or expression of aryl hydrocarbon receptor and/or a down-stream effector of aryl hydrocarbon receptor pathway, is that it enables the production of a sufficient amount of hematopoietic stem cells from only one cord blood unit.
[00104] Accordingly, in one embodiment, the starting cell population is derived from neonatal umbilical cord blood cells which have been enriched in CD34+ cells. In one related embodiment, said starting cell population is derived from one or two umbilical cord blood units.
[00105] In another embodiment, the starting cell population is derived from human mobilized peripheral blood cells which have been enriched in CD34+ cells. In one related embodiment, said starting cell population is derived from human mobilized peripheral blood cells isolated from only one patient.
[00106] Said starting cell population may preferably contain at least 50% CD34+ cells, in some embodiments, more than 90% of CD34+ cells, and may comprise between 105 and 109 nucleated cells.
[00107] The starting cell population may be used directly for expansion or frozen and stored for use at a later date.
[00108] Conditions for culturing the starting cell population for hematopoietic stem cell expansion will vary depending, inter alia, on the starting cell population, the desired final number of cells, and desired final proportion of HSCs.
[00109] In one specific embodiment, in particular, using a starting cell population from umbilical cord blood cells enriched in CD34+ cells, the culturing conditions comprises the use of other cytokines and growth factors, generally known in the art for hematopoietic stem cell expansion. Such cytokines and growth factors include without limitation IL-I, IL-3, IL-6, IL-I l,
G-CSF, GM-CSF, SCF, F1T3-L, thrombopoietin (TPO), erythropoeitin, and analogs thereof. As used herein, "analogs" include any structural variants of the cytokines and growth factors having the biological activity as the naturally occurring forms, including without limitation, variants with enhanced or decreased biological activity when compared to the naturally occurring forms or cytokine receptor agonists such as an agonist antibody against the TPO receptor (for example, VB22B sc(Fv)2 as detailed in patent publication WO 2007/145227, and the like). Cytokine and growth factor combinations are chosen to expand HSC and progenitor cells while limiting the production of terminally differentiated cells. In one specific embodiment, one or more cytokines and growth factors are selected from the group consisting of SCF, Flt3-L and TPO. In one specific embodiment, at least TPO is used in a serum-free medium under suitable conditions for HSC expansion. In one related embodiment, a mixture of IL6, SCF, Flt3-L and TPO is used in the method for expanding HSCs in combination with the compound of the invention or an agent capable of down-regulating the activity and/or expression of aryl hydrocarbon receptor and/or a down-stream effector of aryl hydrocarbon receptor pathway.
[00110] Human IL6 or interleukin-6, also known as B-cell stimulatory factor 2 has been described by (Kishimoto, Ann. review of Imm. 23: 1 2005) and is commercially available. Human SCF or stem cell factor, also known as c-kit ligand, mast cell growth factor or Steel factor has been described (Smith, MA et al., ACTA Haematologica, 105, 3: 143, 2001) and is commercially available. Flt3-L or FLT-3 Ligand, also referred as FL is a factor that binds to flt3- receptor. It has been described (Hannum C, Nature 368 (6472): 643-8) and is commercially available. TPO or thrombopoietin, also known as megakarayocyte growth factor (MGDF) or c- MpI ligand has been described (Kaushansky K (2006). N. Engl. J. Med. 354 (19): 2034-45) and is commercially available.
[00111] The expansion of HSC may be carried out in a basal medium, which is supplemented with the mixtures of cytokines and growth factors described above. A basal medium typically comprises amino acids, carbon sources, vitamins, serum proteins (e.g. albumin), inorganic salts, divalent cations, buffers and any other element suitable for use in expansion of HSC. Examples of such basal medium appropriate for a method of expanding HSC include, without limitation, StemSpan® SFEM - Serum-Free Expansion Medium (StemCell Technologies, Vancouver, Canada), StemSpan® H3000 - Defined Medium (StemCell Technologies, Vancouver, Canada), CellGro® SCGM (CellGenix, Freiburg Germany), StemPro®-34 SFM (Invitrogen).
[00112] In one embodiment, the compound of the invention or the agent capable of down-regulating the activity and/or expression of aryl hydrocarbon receptor and/or a downstream effector of aryl hydrocarbon receptor pathway, is administered during the expansion method of said starting cell population under a concentration appropriate for HSC expansion. In one specific embodiment, said compound or AhR modulating agent is administered at a concentration comprised between IpM and lOOμM, for example between lOpM and lOμM, or between lOOpM and lμM.
[00113] In one specific embodiment where starting cell population essentially consists of CD34+ enriched cells from one or two cord blood units, the cells are grown under conditions for HSC expansion from about 3 days to about 90 days, for example between 7 and 2 days and/or until the indicated fold expansion and the characteristic cell populations are obtained. In one specific embodiment, the cells are grown under conditions for HSC expansion not more than 21 days, 14 days or 7 days.
[00114] In one embodiment, the starting cell population is cultured during a time sufficient to reach an absolute number of CD34+ cells of at least 105, 106, 107, 108 or 109 cells. In another embodiment, said starting cell population is cultured during a time sufficient for a 10 to 50000 fold expansion of CD34+ cells, for example between 100 and 10000 fold expansion. [00115] The cell population obtained after the expansion method may be used without further purification or may be subject to further purification or selection steps. [00116] The cell population may then be washed to remove the compound of invention or any other agent capable of down-regulating the activity and/or expression of aryl hydrocarbon receptor and/or a down-stream effector of aryl hydrocarbon receptor pathway and/or any other components of the cell culture and resuspended in an appropriate cell suspension medium for short term use or in a long-term storage medium, for example a medium suitable for cryopreservation.
Cell Population With Expanded HSCs As Obtained by the Expansion Method and Therapeutic Compositions
[00117] The invention further provides a cell population with expanded HSCs, obtainable or obtained by the expansion method described above. In one specific embodiment, such cell population is resuspended in a pharmaceutically acceptable medium suitable for administration to a mammalian host, thereby providing a therapeutic composition. [00118] The compound as defined in the Summary of the Invention or an agent capable of down-regulating the activity and/or expression of aryl hydrocarbon receptor and/or a down-stream effector of aryl hydrocarbon receptor pathway enables the expansion of HSCs, for example from only one or two cord blood units, to provide a cell population quantitatively and qualitatively appropriate for efficient short and long term engraftment in human patient in need thereof. In particular, the invention relates to a composition comprising a cell population with expanded HSCs derived from not more than one or two cord blood units, wherein said therapeutic composition contains a total amount of cells of at least 105, 106, 107, 108 or 109 cells, with between 20-100%, for example between 40-80% of total cells being CD34+ cells. In one related embodiment, said composition contains between 0.1-40%, for example between 0.1-10% of total cells being CD34+ Thyl+ and 20-80% of cells being CD34+ CD45RA+. In some specific embodiments, said composition contains between 10-95% of cells being CD38+ and between 5-70% of cells being CD133+.
Use of Therapeutic Compositions
[00119] The invention further provides the cell population with expanded HSCs or its composition for use in allogeneic or autologous stem cell transplantation in a mammalian subject.
[00120] The subject referred to herein is, for example, a bone marrow donor or an individual with or at risk for depleted or limited blood cell levels. Optionally, the subject is a bone marrow donor prior to bone marrow harvesting or a bone marrow donor after bone marrow harvesting. The subject is optionally a recipient of a bone marrow transplant. The methods described herein are particularly useful in subjects that have limited bone marrow reserve such as elderly subjects or subjects previously exposed to an immune depleting treatment or myeloablative treatment such as chemotherapy, e.g., for treating leukemia or lymphomas. The subject, optionally, has a decreased blood cell level or is at risk for developing a decreased blood cell level as compared to a control blood cell level. As used herein the term control blood cell level refers to an average level of blood cells in a subject prior to or in the substantial absence of an event that changes blood cell levels in the subject. An event that changes blood cell levels in a subject includes, for example, anemia, trauma, chemotherapy, bone marrow transplant and radiation therapy. For example, the subject has anemia or blood loss due to, for example, trauma. [00121] The expanded HSC population or the composition comprising the cell population with expanded HSCs is administered to the subject, for example, before, at the same time, or after chemotherapy, radiation therapy or a bone marrow transplant. The subject optionally has depleted bone marrow related to, for example, congenital, genetic or acquired syndrome characterized by bone marrow loss or depleted bone marrow. Thus, the subject is optionally a subject in need of hematopoiesis. Optionally, the subject is a bone marrow donor or is a subject with or at risk for depleted bone marrow.
[00122] Hematopoietic stem cell manipulation is useful as a supplemental treatment to chemotherapy or radiation therapy. For example, HSCs are localized into the peripheral blood and then isolated from a subject that will undergo chemotherapy, and after the therapy the cells are returned. Thus, the subject is a subject undergoing or expected to undergo an immune cell depleting treatment such as chemotherapy, radiation therapy or serving as a donor for a bone marrow transplant. Bone marrow is one of the most prolific tissues in the body and is therefore often the organ that is initially damaged by chemotherapy drugs and radiation. The result is that blood cell production is rapidly destroyed during chemotherapy or radiation treatment, and chemotherapy or radiation must be terminated to allow the hematopoietic system to replenish the blood cell supplies before a patient is re -treated with chemotherapy. Therefore, as described herein, HSCs or blood cells made by the methods described herein are optionally administered to such subjects in need of additional blood cells.
[00123] Provided are HSCs expanded by a compound of the invention or an agent capable of down-regulating the activity and/or expression of aryl hydrocarbon receptor and/or a down-stream effector of aryl hydrocarbon receptor pathway or the compositions with expanded HSCs as described above in combination with a therapeutic capable of enhancing the proliferation of HSCs in vivo, in vitro, or ex vivo (for example, a small molecule, an antibody, or the like) and optionally at least one pharmaceutically acceptable excipient or carrier. By a therapeutic capable of enhancing HSC proliferation is meant: an agonist antibody against the TPO receptor (for example, VB22B sc(Fv)2 as detailed in patent publication WO 2007/145227, and the like); a cytokine such as SCF, IL-6, Flt-3 ligand, TPO or a TPO mimetic (for example, such as described in WO/2007/022269; WO/2007/009120; WO/2004/054515; WO/2003/103686; WO/2002/085343; WO/2002/049413; WO/2001/089457; WO/2001/039773; WO/2001/034585; WO/2001/021180; WO/2001/021180; WO/2001/017349; WO/2000/066112; WO/2000/035446; WO/2000/028987; WO/2008/028645; and the like); granulocyte colony stimulating factor (G-CSF); granulyte macrophage colony stimulating factor (GM-CSF); a prostaglandin or a prostaglandin receptor agonist (for example, prostaglandin E2 receptor- 1 (EP-I) agonist, prostaglandin E2 receptor-2 (EP-2) agonist, prostaglandin E2 receptor-3 (EP-3) agonist and prostaglandin E2 receptor-4 (EP-4) agonists, as detailed in patent publication WO/2008/073748); tetraethylenepentamine (TEPA); Notch-ligands (Delta- 1); and/or a WNT agonist. In addition, culturing stem cells with mesenchymal stem cells (MSCs) prevents graft- versus-host disease (GVHD) and may help stem cell expansion. MSCs and stem cells can be transplanted as a whole culture.
[00124] By pharmaceutically acceptable is meant a material that is not biologically or otherwise undesirable, i.e., the material may be administered to a subject or cell, without causing undesirable biological effects or interacting in a deleterious manner with the other components of the pharmaceutical composition in which it is contained. The carrier or excipient is selected to minimize degradation of the active ingredient and to minimize adverse side effects in the subject or cell.
[00125] The compositions are formulated in any conventional manner for use in the methods described herein. Administration is via any route known to be effective by one of ordinary skill. For example, the compositions is administered orally, parenterally (e.g., intravenously), by intramuscular injection, by intraperitoneal injection, transdermally, extracorporeally, intranasally or topically.
[00126] The preferred method of administration is intravenous infusion. The number of cells transfused will take into consideration factors such as sex, age, weight, the types of disease or disorder, stage of the disorder, the percentage of the desired cells in the cell population and the amount of cells needed to produce a therapeutic benefit. In one particular embodiment, the composition is administered by intravenous infusion and comprises at least 104 cells/kg, from 105 to 5.107 cells/kg or more if necessary. In one specific embodiment, the infused cells are all deriving from expanded cord blood cells from a single birth.
[00127] A pharmaceutically acceptable carrier for infusion of a composition comprising cells into a patient typically comprise buffered saline with 5%HSA or unsupplemented basal medium or medium as known in the art.
[00128] For oral administration, the compositions take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulphate). The tablets are coated by methods well known in the art. Liquid preparations for oral administration take the form of, for example, solutions, syrups or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations are prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g., methyl or propyl -p-hydroxybenzoates or sorbic acid). The preparations optionally contain buffer salts, flavoring, coloring and sweetening agents as appropriate.
[00129] The compositions are formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection are presented in unit dosage form, e.g., in ampules or in multi-dose containers, with or without an added preservative. The compositions take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient is in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use. In general, water, suitable oil, saline, aqueous dextrose (glucose), and related sugar solutions and glycols such as propylene glycol or polyethylene glycols are suitable carriers for parenteral solutions. Solutions for parenteral administration contain, for example, a water soluble salt of the active ingredient, suitable stabilizing agents and, if necessary, buffer substances. Antioxidizing agents such as sodium bisulfate, sodium sulfite or ascorbic acid, either alone or combined, are suitable stabilizing agents. Also citric acid and its salts and sodium ethylenediaminetetraacetic acid (EDTA) are optionally used. In addition, parenteral solutions optionally contain preservatives such as benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol. Suitable pharmaceutical carriers are described in Remington: The Science and Practice of Pharmacy, 21st Edition, David B. Troy, ed., Lippicott Williams & Wilkins (2005), which is incorporated by reference in its entirety at least for the material related to pharmaceutical carriers and compositions. [00130] The compositions are optionally formulated as a depot preparation. Such long acting formulations are optionally administered by implantation. Thus, for example, the compositions are formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt. The compositions are applied to or embedded with implants concurrent with or after surgical implant.
[00131] Additionally, standard pharmaceutical methods are employed to control the duration of action. These include control release preparations and appropriate macromolecules, for example, polymers, polyesters, polyamino acids, polyvinyl, pyrolidone, ethylenevinylacetate, methyl cellulose, carboxymethyl cellulose or protamine sulfate. The concentration of macromolecules as well as the methods of incorporation are adjusted in order to control release. Optionally, the agent is incorporated into particles of polymeric materials such as polyesters, polyamino acids, hydrogels, poly (lactic acid) or ethylenevinylacetate copolymers. In addition to being incorporated, these agents are optionally used to trap the compound in microcapsules. [00132] A composition for use in the methods described herein is optionally formulated as a sustained and/or timed release formulation. Such sustained and/or timed release formulations are made by sustained release means or delivery devices that are well known to those of ordinary skill in the art. The compositions are used to provide slow or sustained release of one or more of the active ingredients using, for example, hydropropylmethyl cellulose, other polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, liposomes, microspheres or a combination thereof to provide the desired release profile in varying proportions. Suitable sustained release formulations are selected for use with the compositions described herein. Thus, single unit dosage forms suitable for oral administration, such as, but not limited to, tablets, capsules, gelcaps, caplets, powders that are adapted for sustained release are used.
[00133] The compositions are optionally delivered by a controlled-release system. For example, the composition is administered using intravenous infusion, an implantable osmotic pump, liposomes, or other modes of administration. A controlled release system is placed in proximity to the target.
[00134] Optionally, it is desirable to administer the composition locally, i.e., to the area in need of treatment. For example, the composition is administered by injection into the bone marrow of a long bone, for example. Local administration is achieved, for example, by local infusion during surgery, topical application (e.g., in conjunction with a wound dressing after surgery), injection, catheter, suppository, or implant. An implant is of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers. [00135] The pharmaceutical compositions described herein are administered by any conventional means available for use in conjunction with pharmaceuticals, either as individual therapeutic active ingredients or in a combination of therapeutic active ingredients. They are optionally administered alone, but are generally administered with a pharmaceutical carrier selected on the basis of the chosen route of administration and standard pharmaceutical practice. [00136] The compounds described herein are provided in a pharmaceutically acceptable form including pharmaceutically acceptable salts and derivatives thereof. The term pharmaceutically acceptable form refers to compositions including the compounds described herein that are generally safe, relatively non-toxic and neither biologically nor otherwise undesirable. These compositions optionally include pharmaceutically acceptable carriers or stabilizers that are nontoxic to the cell or subject being exposed thereto at the dosages and concentrations employed. Examples of physiologically acceptable carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as TWEEN™ (Uniqema, United Kingdom), polyethylene glycol (PEG), and PLURONICS™ (BASF, Germany).
[00137] The term pharmaceutically acceptable acid salts and derivatives refers to salts and derivatives of the compounds of Formula I described herein that retain the biological effectiveness and properties as described, and that are not biologically or otherwise undesirable. Pharmaceutically acceptable salts are formed, for example, with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, and organic acids such as acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and the like. [00138] The chemical stability of a composition comprising a compound of Formula I or a pharmaceutically acceptable salt or ester thereof is enhanced by methods known to those of skill in the art. For example, an alkanoic acid ester of a polyethoxylated sorbitol (a polysorbate) is added to a composition containing a compound of Formula I in an amount effective to enhance the chemical stability of the compound.
[00139] The data obtained from the cell culture assays and animal studies are optionally used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include little or no toxicity. The dosage varies within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the provided methods, the therapeutically effective dose is estimated initially from cell culture assays. [00140] Also provided herein is a pack or kit comprising one or more containers filled with one or more of the ingredients described herein. Such kits optionally comprise solutions and buffers as needed or desired. The kit optionally includes an expanded population of stem cells made by the methods described above or can contain containers or compositions for making an expanded population of HSCs. In particular, the invention provides a kit for expanding ex vivo hematopoietic stem cells, comprising a compound as defined in the Summary of Invention and instructions for use of such compound in a method for HSC expansion and, optionally, one ore more cytokines or growth factors, or media for cell growth, in particular media for hematopoietic stem cell growth as described above. The kit may further comprise antibodies for monitoring production of the cells, such as anti-CD34, anti-CD133, anti-CD38, anti-CD45RA and/or anti- Thyl antibodies. In one specific embodiment, such kit further include one or more cytokines or growth factors selected from the group consisting of IL6, FLT3-L, SCF and TPO. Optionally associated with such pack(s) or kit(s) are instructions for use.
[00141] Also provided is a kit for providing an effective amount of a compound of the invention to increase HSCs in a subject comprising one or more doses of the compound for use over a period of time, wherein the total number of doses of the compound of the invention in the kit equals the effective amount sufficient to increase HSCs in a subject. The period of time is from about one to several days or weeks or months. Thus, the period of time is from at least about 5, 6, 7, 8, 10, 12, 14, 20, 21, 30 or 60 days or more or any number of days between one and 90. Processes for Making Compounds of the Invention
[00142] The present invention also includes processes for the preparation of compounds of the invention. In the reactions described, it can be necessary to protect reactive functional groups, for example hydroxy, amino, imino, thio or carboxy groups, where these are desired in the final product, to avoid their unwanted participation in the reactions. Conventional protecting groups can be used in accordance with standard practice, for example, see T.W. Greene and P. G. M. Wuts in "Protective Groups in Organic Chemistry", John Wiley and Sons, 1991.
[00143] The following reaction schemes 1-5 detail the preparation of compounds of the invention. It will be appreciated by one skilled in the art that, following introduction by the methods detailed below, any of the groups R1, R2, R3, R4, and Li may optionally be further elaborated by known transformations to arrive at the desired final compounds of Formula I. [00144] Compounds of Formula I can be prepared according the following Reaction
Scheme 1:
Reaction Scheme 1
Figure imgf000045_0001
(4)
Figure imgf000045_0002
(2) (I)
[00145] in which G1, G2, G3, G4, R1, R2 and R4 are as defined for Formula I in the
Summary of the Invention and L of Formula I is defined in the reaction scheme as -NH-Li- which is equivalent to, for example, -NR5a(CH2)o-3- where Rsa is hydrogen and -(CH2)o-3- is L1. [00146] Compounds of Formula I can be prepared by reacting a compound of Formula
2 with a compound of Formula 3 in the presence of a suitable catalyst (e.g., Pd2(dba)3, or the like) in the presence of an appropriate ligand (e.g., l,3-bis(2,4,6-trimethylphenyl) imidazolium chloride), a suitable base (e.g., CS2CO3, or the like) and an appropriate solvent (e.g., 1,4-dioxane) at a temperature of about 80 to 100 0C for 2 to about 48 hours. Compounds of Formula 2 in turn can be prepared by reacting a compound of Formula 4 with a slight excess of an amine compound of Formula 5 in an appropriate solvent (e.g. isopropanol) at a temperature of about room temperature to about 80 0C. Compounds of Formula 4 can be prepared by alkylation of a compound of Formula 6 with a suitable alkylating agent 7, in which X] is chlorine, bromine, iodine, or a sulfonate ester, in the presence of a suitable base (e.g. sodium hydride or potassium carbonate), in a suitable solvent (e.g. DMF), at a temperature of about 0 0C to about 80 0C. Alternatively, the reaction can be performed under Mitsunobu conditions using a suitable alcohol R4-OH in the presence of a suitable phosphine (e.g. triphenylphosphine) and azodicarboxylate (e.g. diethylazodicarboxylate), in an inert solvent such as THF or toluene, at a temperature from about 0 0C to about room temperature. [00147]
[00148] Compounds of Formula Ia, in which Gi is CR3 and in which all other G groups are N, can also be prepared by proceeding as in the following Reaction Scheme 2:
Reaction Scheme 2
Figure imgf000046_0001
(9) (8) (1a)
[00149] in which R1, R2, R3 and R4 are as defined for Formula I in the Summary of the Invention and L of Formula I is defined in the reaction scheme as -NH-Lj- which is equivalent to, for example, -NRs3(CH2)O^- where R5a is hydrogen and -(CH2)0-3- is L1. [00150] Compounds of Formula I can be prepared by reacting a compound of Formula
8 with an amine compound of Formula 5 in an appropriate solvent (e.g. isopropanol) at a temperature of about room temperature to about 100 0C. Compounds of Formula 8 can in turn be prepared by reacting a compound of Formula 9 with a compound of Formula 3 in the presence of a suitable catalyst (e.g., Pd(Ph3P)4 , Pd2(dba)3, or the like), optionally in the presence of an appropriate ligand (e.g., l,3-bis(2,4,6-trimethylphenyl) imidazolium chloride), a suitable base (e.g., Cs2CO3, or the like) and an appropriate solvent (e.g., 1,4-dioxane) at a temperature of about 80 to 100 0C for 2 to about 48 hours. Compounds of Formula 9 in turn can be prepared by reacting a compound of Formula 10 with a mixture of di-iodomethane, copper(I) iodide, and an alkyl nitrite (e.g. isoamylnitrite), optionally in the presence of an inert solvent, at a temperature of about 50 to 100 0C. Compounds of Formula 10 can be prepared by alkylation of a compound of Formula 11 with a suitable alkylating agent 7, in which X] is chlorine, bromine, iodine, or a sulfonate ester, in the presence of a suitable base (e.g. sodium hydride or potassium carbonate), in a suitable solvent (e.g. DMF), at a temperature of about 0 0C to about 80 0C. Alternatively, the reaction can be performed under Mitsunobu conditions using a suitable alcohol R4-OH in the presence of a suitable phosphine (e.g. triphenylphosphine) and azodicarboxylate (e.g. diethylazodicarboxylate), in an inert solvent such as THF or toluene, at a temperature from about 0 0C to about room temperature.
[00151] Compounds of Formula II, which are a subset of compounds of Formula I in which Ri is N-linked heterocyclyl or N-linked heteroaryl, can be prepared as detailed in the following Reaction Scheme 3 :
Reaction Scheme 3
Figure imgf000048_0001
(2) (II)
[00152] G1, G2, G3, G4, R1, R2 and R4 are as defined for Formula I in the Summary of the Invention and L of Formula I is defined in the reaction scheme as -NH-Lj - which is equivalent to, for example,
Figure imgf000048_0002
where Rs3 is hydrogen and -(CH2V3- is L1. Compounds of Formula II can be prepared by reacting a compound of Formula 2 with a compound of Formula 20 in the presence of an excess of cyclic amine or NH-bearing heterocycle (for example, substituted pyrazole, substituted imidazole, and the like), at a temperature of about 50 0C to about 250 0C, for about 1 to about 24 hours, optionally in the presence of a base such as sodium hydride or DBU.
[00153] Compounds of Formula 10 in which G1 is CR3, and in which all other G groups are N, can also be prepared by proceeding as in the following Reaction Scheme 4:
Reaction Scheme 4
Figure imgf000048_0003
[00154] in which R3 and R4 are as defined for Formula I in the Summary of the
Invention. Compounds of Formula 10 can be prepared according to procedures described in J. Med. Chem, 1972, 456, and J. Med. Chem., 1992, 4180. An orthoester compound of Formula 21 is reacted with a compound of Formula 22, optionally in the presence of an acid such as acetic acid, at a temperature of about room temperature to about 150 0C, for about 1 to about 24 hr.. A compound of Formula 22 can in turn be prepared by reacting a compound of Formula 23 with a primary amine compound of Formula 24, optionally in the presence of an acid such as pTSA, or a base such as triethylamine or DBU, at a temperature of about 50 to about 200 0C. [00155] Compounds of Formula IV can be prepared as detailed in the following
Reaction Scheme 5:
Reaction Scheme 5
R2
Figure imgf000049_0001
[00156] in which G1, G2, G3, G4, Ri and R2 are as defined for Formula I in the
Summary of the Invention and L of Formula I is defined in the reaction scheme as -NH-L1- which is equivalent to, for example, -NRsa(CH2)o-3- where R5a is hydrogen and -(CH2)0-3- is L1. R20 and R2I are independently selected from hydrogen and C^alkyl. A compound of Formula IV, in which R2i is hydrogen, can be prepared from a compound of Formula III by treatment with a suitable reducing agent such as lithium aluminum hydride or di-isobutyl aluminum hydride, in a suitable solvent such as THF or toluene, at a temperature of about -78°C to about 500C. The reaction takes about 0.5 to about 16 hr to complete. A compound of Formula IV, in which R2I is lower alkyl, can be prepared by treatment of a compound of Formula III with an alkyl lithium or Grignard reagent, in a suitable solvent such as ether or tetrahydrofuran, at a temperature of about -78°C to about 500C. The reaction takes about 0.5 to about 16 hr to complete.
[00157] Detailed examples of the synthesis of a compound of Formula I can be found in the Examples, infra.
Additional Processes for Making Compounds of the Invention [00158] A compound of the invention can be prepared as a pharmaceutically acceptable acid addition salt by reacting the free base form of the compound with a pharmaceutically acceptable inorganic or organic acid. Alternatively, a pharmaceutically acceptable base addition salt of a compound of the invention can be prepared by reacting the free acid form of the compound with a pharmaceutically acceptable inorganic or organic base. Alternatively, the salt forms of the compounds of the invention can be prepared using salts of the starting materials or intermediates.
[00159] For example, salt forms of 4-(2-(2-(benzo [blthiophen-3 -yl)-9-isopropyl-9H- purin-6-ylamino)ethyl)phenol (Example 1, infra) were synthesized as follows: [00160] Mesylate salt: 4-(2-(2-(benzorblthiophen-3-yl)-9-isopropyl-9H-purin-6- ylamino)ethyl)phenol free base (0.60g; 1.40 mmoles) are dissolved in 12 ml acetone at 500C. Methanesulfonic acid (0.137 g; 1.40 mmoles) is added drop wise. The crystallization takes place rapidly. The white suspension is allowed to cool over about 30 minutes with cooling to room temperature. The slurry is stirred for 18 hours at room temperature and filtered. The solid is washed with acetone (6 ml) in three portions and dried first for about 3 hours at 500C / ca. 10 mbar and then for about 16 hours at 800C /ca.lO mbar. The material has a melting point at about 233°C with a melting enthalpy of 98g/J. The material produced exhibited a loss on drying of 0.2%. The water uptake was estimated by thermogravimetry after exposure to relative humidity (80%rh) during 24 hours. A water uptake of 0.4% was observed. [00161] In another embodiment, the invention provides a mesylate salt of the compound of Example 1. In a further embodiment, the invention provides the mesylate salt of the compound of Example 1 comprising the following powder X-ray diffraction peaks (Angle 2- Theta °): 6.4, 6.7, 18.3, 18.6, 26.9; and which in an additional embodiment comprises the following powder X-ray diffraction peaks (Angle 2-Theta °): 6.4, 6.7, 10.3, 12.9, 16.4, 18.3, 25.8, 26.5, 26.9.
[00162] In a yet further embodiment, the invention provides the mesylate salt of the compound of Example 1 having the powder X-ray diffraction pattern shown in Figure 3 herein. [00163] Tosylate salt: 4-(2-(2-(benzorblthiophen-3-yl)-9-isopropyl-9H-purin-6- ylamino)ethyl)phenol free base (0.60g; 1.40 mmoles) are dissolved in 12 ml at 500C. A solution of para-toluenesulfonic acid mono-hydrate (0.27 Ig; 1.40 mmoles) in acetone (1.2 ml) is added drop wise. The solution is seeded at 500C and crystallization takes place quickly. The suspension is allowed to cool over about 30 minutes to room temperature and stirred for about 18 hours. After filtration the solid is washed with acetone (6 ml) in three portions and dried first for about 3 hours at 500C / ca. 10 mbar and then for about 16 hours at 800C /ca.lO mbar. The material has a melting point at about 233°C with a melting enthalpy of 88g/J. The material produced exhibited a loss on drying of 0.2%. The water uptake was estimated by Thermogravimetry after exposure to relative humidity (80%rh) during 24 hours. A water uptake of 0.4% was observed. [00164] In another embodiment, the invention provides a tosylate salt of the compound of Example 1. In a further embodiment, the invention provides the tosylate salt of the compound of Example 1 comprising the following powder X-ray diffraction peaks (Angle 2-Theta °):6.2, 13.3, 16.7, 19.5, 25.4; and which in an additional embodiment comprises the following powder X-ray diffraction peaks: 6.2, 7.6, 12.4, 13.3, 15.1, 16.7, 17.7, 19.5, 20.2, 24.6, 24.9, 25.4, 25.6. [00165] In a yet further embodiment, the invention provides the tosylate salt of the compound of Example 1 having the powder X-ray diffraction pattern shown in Figure 4 herein. [00166] Sulfate salt: 4-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6- ylamino)ethyl)phenol free base (0.60g; 1.40 mmoles) are dissolved in 10 ml acetone and 1 ml water at about 55°C. A solution of sulfuric acid (0.28Og; 2.79 mmoles) in 1 ml water is added drop wise. The crystallization takes place rapidly. The suspension is allowed to cool over about 30 minutes with cooling to room temperature, stirred for about 18 hours and filtered. The filter cake is washed with 6 ml acetone in three portions and dried first for about 3 hours at 500C / ca. 10 mbar and then for about 16 hours at 800C /ca.lO mbar. The material has a melting point at about 2240C with a melting enthalpy of 91g/J. The material produced exhibited a loss on drying below 0.05%. The water uptake was estimated by Thermogravimetry after exposure to relative humidity (80%rh) during 24 hours. A water uptake of 0.2% was observed. [00167] In another embodiment, the invention provides a sulfate salt of the compound of Example 1. In a further embodiment, the invention provides the sulfate salt of the compound of Example 1 comprising the following powder X-ray diffraction peaks (Angle 2-Theta °): 6.5, 6.8, 10.7, 13.5, 26.4, 27.6; and which in an additional embodiment comprises the following powder X-ray diffraction peaks (Angle 2-Theta °): 6.5, 6.8, 10.7, 13.1, 13.5, 18.6, 18.8, 20.8, 26.4, 27.1, 27.6.
[00168] In a yet further embodiment, the invention provides the sulfate salt of the compound of Example 1 having the powder X-ray diffraction pattern shown in Figure 6 herein. [00169] Esylate salt: 4-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6- ylamino)ethyl)phenol free base (0.60g; 1.40 mmoles) are dissolved in 12 ml acetone at 500C. Ethanesulfonic acid (0.155g; 1.40 mmoles) is added drop wise. The crystallization takes place quickly. The resulting white suspension is allowed to cool over about 30 minutes to room temperature. The suspension is stirred for about 18 hours at room temperature and filtered. The solid is washed with 6 ml acetone in three portions and dried first for about 3 hours at 500C / ca. 10 mbar and then for about 16 hours at 800C /ca.lO mbar. The material has a melting point at about 2310C with a melting enthalpy of 76g/J. The material produced exhibited a loss on drying of 0.6%. The water uptake was estimated by Thermogravimetry after exposure to relative humidity (80%rh) during 24 hours. A water uptake of 0.05% was observed. [00170] In another embodiment, the invention provides a esylate salt of the compound of Example 1. In a further embodiment, the invention provides the esylate salt of the compound of Example 1 comprising the following powder X-ray diffraction peaks (Angle 2-Theta °): 6.3, 9.9, 18.4, 25.3, 26.1; and which in an additional embodiment comprises the following powder X- ray diffraction peaks (Angle 2-Theta °): 6.3, 9.9, 17.1, 17.9, 18.4, 19.0, 22.0, 25.3, 26.1, 27.1. [00171] In a yet further embodiment, the invention provides the esylate salt of the compound of Example 1 having the powder X-ray diffraction pattern shown in Figure 8 herein. [00172] Hydrobromide salt: 4-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6- ylamino)ethyl)phenol free base (0.60g; 1.40 mmoles) are dissolved in 6 ml DMF at 65°C. Hydrobromic acid 48% (0.235g; 1.40 mmoles) is added drop wise. The solution is allowed to cool over about 30 minutes to room temperature. Seeds are added at 55°C and crystallization takes place slowly. The suspension is stirred for about 18 hours at room temperature and filtered. The solid is washed with 4 ml DMF/water 1 : 1 and 6 ml water. The salt is dried first for about 3 hours at 500C / ca. 10 mbar and then for about 16 hours at 800C /ca.lO mbar. The material has a melting point at about 2850C with a melting enthalpy of 119g/J. The material produced exhibited a loss on drying of 1.0%. The water uptake was estimated by Thermogravimetry after exposure to relative humidity (80%rh) during 24 hours. No water uptake was observed.
[00173] In another embodiment, the invention provides a hydrobromide salt of the compound of Example 1. In a further embodiment, the invention provides the hydrobromide salt of the compound of Example 1 comprising the following powder X-ray diffraction peaks (Angle 2-Theta °): 7.0, 25.9, 26.8, 27.9; and which in an additional embodiment comprises the following powder X-ray diffraction peaks (Angle 2-Theta °): 7.0, 11.4, 13.3, 21.4, 23.4, 25.9, 26.4, 26.8, 27.9. [00174] In a yet further embodiment, the invention provides the hydrobromide salt of the compound of Example 1 having the powder X-ray diffraction pattern shown in Figure 9 herein.
[00175] Orotate salt: 4-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6- ylamino)ethyl)phenol free base (0.60g; 1.40 mmoles) and orotic acid (0.222g; 1.40 mmoles) are dissolved in 7.8 ml NMP (l-Methyl-2-pyrrolidone) at 85°C. The solution is cooled to 600C and 6 ml water is added drop wise over about 5 minutes. The resulting white suspension is allowed to cool over about 30 minutes to room temperature and stirred for 18 hours. After filtration the filter cake is washed with 4 ml NMP/water 1 : 1 in two portions and 6 ml water in three portions. The solid is dried first for about 3 hours at 500C / ca. 10 mbar and then for about 16 hours at 800C /ca.lO mbar. The material has a melting point at about 2400C with a melting enthalpy of 130g/J. The material produced exhibited a loss on drying below 0.05%. The water uptake was estimated by Thermogravimetry after exposure to relative humidity (80%rh) during 24 hours. A water uptake of 1.7% was observed.
[00176] In another embodiment, the invention provides an orotate salt of the compound of Example 1. In a further embodiment, the invention provides the orotate salt of the compound of Example 1 comprising the following powder X-ray diffraction peaks (Angle 2- Theta °): 7.1, 16.3, 19.2, 23.5, 25.6, 26.9; and which in an additional embodiment comprises the following powder X-ray diffraction peaks (Angle 2-Theta °): 7.1, 14.4, 16.3, 18.6, 19.2, 21.7, 23.0, 23.5, 25.6, 26.9, 28.7.
[00177] In a yet further embodiment, the invention provides the orotate salt of the compound of Example 1 having the powder X-ray diffraction pattern shown in Figure 10 herein. [00178] Hemi-fumarate salt: 4-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6- ylamino)ethyl)phenol free base (0.60g; 1.40 mmoles) are dissolved in 18 ml methanol at 65°C. Fumaric acid (0.164g; 1.40 mmoles) and 6 ml methanol are added. The solution is allowed to cool over about 30 minutes to room temperature. Some seed crystals are added at 600C and crystallization takes place slowly. The suspension is stirred for 18 hours at room temperature and filtered. The solid is washed with 6 ml methanol in three portions and dried first for about 3 hours at 500C / ca. 10 mbar and then for about 16 hours at 800C /ca.lO mbar. The material has a melting point at about 232°C with a melting enthalpy of 83g/J. The material produced exhibited a loss on drying below 0.05%. The water uptake was estimated by Thermogravimetry after exposure to relative humidity (80%rh) during 24 hours. A water uptake of 0.3% was observed. [00179] In another embodiment, the invention provides a hemi-fumarate salt of the compound of Example 1. In a further embodiment, the invention provides the hemi-fumarate salt of the compound of Example 1 comprising the following powder X-ray diffraction peaks (Angle 2-Theta °): 7.2, 8.7, 14.4, 15.8, 17.4, 19.0, 23.7; and which in an additional embodiment comprises the following powder X-ray diffraction peaks (Angle 2-Theta °): 7.2, 8.7, 10.8, 14.4,
15.8, 17.4, 17.8, 19.0, 20.1, 23.7, 27.5.
[00180] In a yet further embodiment, the invention provides the hemi-fumarate salt of the compound of Example 1 having the powder X-ray diffraction pattern shown in Figure 11 herein.
[00181] Besylate salt: 4-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6- ylamino)ethyl)phenol free base (0.60g; 1.40 mmoles) are dissolved in 12 ml acetone at 500C. A solution of benzenesulfonic acid (0.225g; 2.79 mmoles) in 1.2 ml acetone is added drop wise. Seed crystals are added at 48°C and the crystallization takes place slowly. The suspension is allowed to cool over about 30 minutes to room temperature. The slurry is stirred for about 18 hours at room temperature and filtered. The salt is washed with 6 ml acetone in three portions and dried first for about 3 hours at 500C / ca. 10 mbar and then for about 16 hours at 800C /ca.lO mbar. The material has a melting point at about 219°C with a melting enthalpy of 92g/J. The material produced exhibited a loss on drying of 0.3%. The water uptake was estimated by Thermogravimetry after exposure to relative humidity (80%rh) during 24 hours. A water uptake of about 0.05% was observed.
[00182] In another embodiment, the invention provides a besylate salt of the compound of Example 1. In a further embodiment, the invention provides the besylate salt of the compound of Example 1 comprising the following powder X-ray diffraction peaks (Angle 2- Theta °): 6.2, 7.7, 17.7, 25.5; and which in an additional embodiment comprises the following powder X-ray diffraction peaks (Angle 2-Theta °): 6.2, 7.7, 15.2, 16.7, 17.1, 17.7, 19.8, 20.2,
24.9, 25.2, 25.5.
[00183] In a yet further embodiment, the invention provides the besylate salt of the compound of Example 1 having the powder X-ray diffraction pattern shown in Figure 7 herein. [00184] Napadisylate salt: 4-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6- ylamino)ethyl)phenol free base (0.60g; 1.40 mmoles) and 0.259 g 1,5-naphthalenedisulfonic acid (0.70 mmoles) are dissolved in 9 ml DMF at 87°C. The clear solution is allowed to cool over about 30 minutes to room temperature. Seeds are added at 65°C and crystallization takes place slowly. The suspension is stirred for about 18 hours at room temperature and filtered. The solid is washed with 4 ml DMF/water 1 : 1 in two portions and 6 ml water in three portions. The salt is dried first for about 3 hours at 500C / ca. 10 mbar and then for about 16 hours at 800C /ca.lO mbar. The material has a melting point at about 3040C with a melting enthalpy of 83g/J. A broad endothermic phenomenon is observed at 1070C that might be attributed to the loss of water. The material produced exhibited a loss on drying of 6.1%. The water uptake was estimated by Thermogravimetry after exposure to relative humidity (80%rh) during 24 hours. A water uptake less than 0.05% was observed.
[00185] In another embodiment, the invention provides a napadysilate salt of the compound of Example 1. In a further embodiment, the invention provides the napadysilate salt of the compound of Example 1 comprising the following powder X-ray diffraction peaks (Angle 2-Theta °): 6.4, 9.6, 13.1, 15.7, 16.1, 26.0; and which in an additional embodiment comprises the following powder X-ray diffraction peaks (Angle 2-Theta °): 9.6, 13.1, 15.7, 16.1, 16.4, 20.4, 20.9, 23.7, 26.0, 26.9.
[00186] In a yet further embodiment, the invention provides the napadysilate salt of the compound of Example 1 having the powder X-ray diffraction pattern shown in Figure 12 herein.
[00187] Hydrochloride salt: 4-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6- ylamino)ethyl)phenol free base (0.60g; 1.40 mmoles) are dissolved in 12 ml acetone at 55°C. Hydrochloric acid 37% (0.138g; 1.40 mmoles) is added drop wise. The crystallization takes place quickly. The white suspension is allowed to cool over about 30 minutes to room temperature and stirred for 18 hours. After filtration the solid is washed with 6 ml acetone in three portions and dried first for about 3 hours at 500C / ca. 10 mbar and then for about 16 hours at 800C /ca.lO mbar. The material is exhibiting an exothermic event at about 162°C with an enthalpy of -13.8J/g. This phenomenon might be attributed to a solid transformation into a more stable modification. An endothermic event is then seen at about 259°C with an enthalpy of 99.7J/g. The material produced exhibited a loss on drying of 0.6%. The water uptake was estimated by Thermogravimetry after exposure to relative humidity (80%rh) during 24 hours. A water uptake of 0.3% was observed.
[00188] In another embodiment, the invention provides a hydrochloride salt of the compound of Example 1. In a further embodiment, the invention provides the hydrochloride salt of the compound of Example 1 comprising the following powder X-ray diffraction peaks (Angle 2-Theta °): 6.1, 7.0, 19.8, 26.1; and which in an additional embodiment comprises the following powder X-ray diffraction peaks (Angle 2-Theta °): 6.1, 7.0, 18.1, 19.8, 24.7, 26.1, 27.0, 27.7. [00189] In a yet further embodiment, the invention provides the hydrochloride salt of the compound of Example 1 having the powder X-ray diffraction pattern shown in Figure 5 herein.
[00190] The free acid or free base forms of the compounds of the invention can be prepared from the corresponding base addition salt or acid addition salt form, respectively. For example a compound of the invention in an acid addition salt form can be converted to the corresponding free base by treating with a suitable base (e.g., ammonium hydroxide solution, sodium hydroxide, and the like). A compound of the invention in a base addition salt form can be converted to the corresponding free acid by treating with a suitable acid (e.g., hydrochloric acid, etc.). The nitrate salt of the compound of example 1 can be made using methods known to the skilled person. The powder X-ray diffraction pattern is disclosed in Fig 2 herein. [00191] Compounds of the invention in unoxidized form can be prepared from N- oxides of compounds of the invention by treating with a reducing agent (e.g., sulfur, sulfur dioxide, triphenyl phosphine, lithium borohydride, sodium borohydride, phosphorus trichloride, tribromide, or the like) in a suitable inert organic solvent (e.g. acetonitrile, ethanol, aqueous dioxane, or the like) at 0 to 800C.
[00192] Prodrug derivatives of the compounds of the invention can be prepared by methods known to those of ordinary skill in the art (e.g., for further details see Saulnier et al., (1994), Bioorganic and Medicinal Chemistry Letters, Vol. 4, p. 1985). For example, appropriate prodrugs can be prepared by reacting a non-derivatized compound of the invention with a suitable carbamylating agent (e.g., 1,1-acyloxyalkylcarbanochloridate, para-nitrophenyl carbonate, or the like).
[00193] Protected derivatives of the compounds of the invention can be made by means known to those of ordinary skill in the art. A detailed description of techniques applicable to the creation of protecting groups and their removal can be found in T. W. Greene, "Protecting Groups in Organic Chemistry", 3rd edition, John Wiley and Sons, Inc., 1999. [00194] Compounds of the present invention can be conveniently prepared, or formed during the process of the invention, as solvates (e.g., hydrates). Hydrates of compounds of the present invention can be conveniently prepared by recrystallization from an aqueous/organic solvent mixture, using organic solvents such as dioxin, tetrahydrofuran or methanol. [00195] Compounds of the invention can be prepared as their individual stereoisomers by reacting a racemic mixture of the compound with an optically active resolving agent to form a pair of diastereoisomeric compounds, separating the diastereomers and recovering the optically pure enantiomers. While resolution of enantiomers can be carried out using covalent diastereomeric derivatives of the compounds of the invention, dissociable complexes are preferred (e.g., crystalline diastereomeric salts). Diastereomers have distinct physical properties (e.g., melting points, boiling points, solubilities, reactivity, etc.) and can be readily separated by taking advantage of these dissimilarities. The diastereomers can be separated by chromatography, or preferably, by separation/resolution techniques based upon differences in solubility. The optically pure enantiomer is then recovered, along with the resolving agent, by any practical means that would not result in racemization. A more detailed description of the techniques applicable to the resolution of stereoisomers of compounds from their racemic mixture can be found in Jean Jacques, Andre Collet, Samuel H. Wilen, "Enantiomers, Racemates and Resolutions", John Wiley And Sons, Inc., 1981. Compounds of the invention can also be prepared as their individual stereoisomers by using chiral chromatography techniques, in particular, by use of HPLC or SFC chromatography using a chiral stationary phase. [00196] Powder X-ray diffraction spectra as enclosed herein were obtained using the instrument Bruker D8 Vario in transmission geometry, irradiation CuKa (30 kV, 40 mA), scan range 2°-40° (2 theta value), step time 90.3s. Differential scanning calorimetry (DSC) of Example 1 amorphous material was carried out using the instrument Perkin Elmer DSC7 at a heating rate of 40°C/min.
[00197] In summary, the compounds of Formula I can be made by a process, which involves:
(a) those of reaction schemes 1-5; and (b) optionally converting a compound of the invention into a pharmaceutically acceptable salt;
(c) optionally converting a salt form of a compound of the invention to a non-salt form;
(d) optionally converting an unoxidized form of a compound of the invention into a pharmaceutically acceptable N-oxide;
(e) optionally converting an N-oxide form of a compound of the invention to its unoxidized form;
(f) optionally resolving an individual isomer of a compound of the invention from a mixture of isomers;
(g) optionally converting a non-derivatized compound of the invention into a pharmaceutically acceptable prodrug derivative; and
(h) optionally converting a prodrug derivative of a compound of the invention to its non-derivatized form.
[00198] Insofar as the production of the starting materials is not particularly described, the compounds are known or can be prepared analogously to methods known in the art or as disclosed in the Examples hereinafter.
[00199] One of skill in the art will appreciate that the above transformations are only representative of methods for preparation of the compounds of the present invention, and that other well known methods can similarly be used.
Examples
[00200] The present invention is further exemplified, but not limited, by the following examples that illustrate the preparation of compounds of Formula I (Examples) according to the invention.
Example 1
4-(2-(2-(benzorblthiophen-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phenol
Figure imgf000059_0001
[00201] Synthesis of 2,6-dichloro-9-isopropyl-9H-purine (b): To 2,6-dichloro-9H- purine (a) (6.0 mmol) dissolved in anhydrous DMF (5.0 mL) was slowly added sodium hydride (7.8 mmol) with stirring at rt over 2 hr. 2-iodopropane was added and the mixture was stirred for 16 hr. The mixture was concentrated. The residue was purified by column chromatography on silica gel, eluting with hexane/EtOAc (20: 1 to 3: 1) to afford the title compound as a white solid. 1H NMR (500 MHz, CDCl3): δ 8.15 (s, IH), 4.91 (m, IH), 1.63 (d, 6H).
[00202] Synthesis of 4-(2-(2-chloro-9-isopropyl-9H-purin-6-ylamino)ethyl)phenol
(c): 2,6-dichloro-9-isopropyl-9H-purine (1.1 mmol) was mixed with tyramine (1.16 mmol) dissolved in i-PrOΗ (6 ml) and the mixture was stirred overnight. The reaction mixture was concentrated, and the residue purified by column chromatography on silica gel, eluting with hexane/EtOAc (5:1 to 1:2) to afford the title compound as a white solid. 1H NMR (500 MHz, CDCl3): δ 9.21 (br, IH), 8.49 (s, IH), 7.80 (s, IH), 7.10 (d, 2H), 6.73 (d, 2H), 4.87 (m, IH), 4.03 (t, 2H), 3.01 (t, 2H), 1.68 (d, 6H); HRMS (EI) calcd for Ci5H18ClN5O (M + H+) 332.1273, found 332.1278.
[00203] Synthesis of 4-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6- ylamino)ethyl)phenol (d): A flame-dried schlenk flask was charged with 4-(2-(2-chloro-9- isopropyl-9H-purin-6-ylamino)ethyl)phenol (0.62 mmol), thianaphthene-3-boronic acid (0.94 mmol), pd2(dba)3 (0.062 mmol), Cs2CO3 (1.25 mmol) and l,3-bis(2,4,6-trimethylphenyl) imidazolium chloride (0.125 mmol). The flask was evacuated and backfilled with N2 and anhydrous 1,4-dioxane (2 mL) was added. The flask was sealed and the reaction mixture was stirred at 80 0C for 24 hours. The reaction mixture was concentrated and purified directly by column chromatography on silica gel, eluting with hexane/EtOAc (20: 1 to 1:4) to afford the title compound as a yellowish solid.
[00204] Alternatively, the synthesis of Example 1 can be carried out as follows:
[00205] Synthesis of 2-(benzo[b]thiophen-3-yl)-6-chloro-9-isopropyl-9H-purine
(b): A round-bottom flask was charged with 6-chloro-2-iodo-9-isopropyl-9H-purine (prepared in Example 15c, 3.31 g, 0.0103 mol), benzo[b]thiophen-3-ylboronic acid (2.74 g, 0.0154 mol), and tetrakis(triphenylphosphine)palladium(0) (1.19 g, 0.0103 mol). To this mixture was added toluene (80 ml), ethanol (25 ml) and aqueous sodium carbonate solution (2M, 21 ml). The flask was sealed and the reaction mixture was stirred at 90 0C for 1 h. Water was added to the cooled mixture, which was extracted with ethyl acetate. The organic fractions were combined, dried over sodium sulfate, and concentrated. The residue was purified by column chromatography on silica gel, eluting with 20 to 50% EtOAc in hexane to afford the title compound as a solid, which was recrystallized from 1: 1 methanol/water. 1H NMR (400 MHz, DMSO-d6): δ = 9.15 (d, IH), 8.85 (s, 2H), 8.17 (d, IH), 7.62 (t, IH), 7.53 (t, IH), 5.06 (m, IH), 1.71 (d, 6H); MS m/z 329.0 (M + 1).
[00206] Synthesis of 4-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6- ylamino)ethyl)phenol (c): 2-(benzo[b]thiophen-3-yl)-6-chloro-9-isopropyl-9H-purine (2.2 g, 0.0067 mol) was suspended in anhydrous 2-propanol (70 mL) in a pressure tube. Tyramine (1.01 g, 0.0074 mol) was added. The tube was sealed and heated at 85 0C for 16 hr. Additional tyramine (0.50 g, 0.0037 mol) was added and the mixture was heted at 85 0C for 48 hr. The reaction was concentrated. Aqueous sodium bicarbonate solution was added to the residue, which was extracted with EtOAc. The combined organic extracts were dried over sodium sulfate, filtered, and concentrated. The residue was purified by column chromatography on silica gel, eluting with 50 to 85% EtOAc in hexane to afford a solid. The solid was triturated with methanol to provide the title compound as an off-white solid.
[00207] Alternatively, the synthesis of Example 1 can be carried out as follows:
[00208] Synthesis of 2,6-dichloro-9-isopropyl-9H-purine (b): To 2,6-dichloro-9H- purine (a) (998 g, 5.28 mol) dissolved in anhydrous DMF (5.0 L) was added sodium hydride (60% dispersion, 254 g, 6.35 mol) with stirring at 10 0C over 1 hr. 2-iodopropane (1595 g) was added and the mixture was stirred at rt for 24 hr. Water (5.0 L) was added, and the resulting solid precipitate was collected and washed with water (500 ml) and heptane (2 x 2.5 L). The crude solid was crystallized from isopropyl acetate (2.1 L) to provide the title compound as a solid. [00209] Synthesis of 4-(2-(2-chloro-9-isopropyl-9H-purin-6-ylamino)ethyl)phenol
(c): 2,6-dichloro-9-isopropyl-9H-purine (500 g) was added in portions to a stirred mixture of tyramine (593g), triethylamine (262 g), and i-PrOΗ (5.0 L) at 50 0C. The mixture was stirred at that temperature for 4 hr, then the reaction mixture was concentrated. The residue was taken up in isopropyl acetate (6.0 L) and was washed with 20% citric acid solution (2.0 L) and water (2.0 L). The organic layer was concentrated to dryness, then was taken up in ethanol (2.0 L) and again concentrated to dryness. The crude solid was crystallized from ethanol (3.2 L) to provide the title compound as a solid.
[00210] Synthesis of 4-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9Η-purin-6- ylamino)ethyl)phenol (d): A mixture of 4-(2-(2-chloro-9-isopropyl-9H-purin-6- ylamino)ethyl)phenol (950 g), thianaphthene-3-boronic acid (561 g), dichlorobis(triphenylphosphine)palladium(II) (10.1 g), potassium carbonate (791 g), water (3.25 L), and DMA (3.25 L) was stirred under a nitrogen atmosphere for 10 min. The stirred mixture was then heated at 70 0C for 14 h. Ethyl acetate (6.5 L) and water (3.25 L) were added, and the mixture was filtered through Celite (125 g) at 50 0C, rinsing with ethyl acetate (1.0 L). The layers were separated, and the aqueous layer was extracted at 50 0C with additional ethyl acetate (7.5 L). The combined organic layers were washed with water (3 x 2.5 L), then distilled to remove about 2.5 L of solvent. Tetrahydrofuran (2.5 L) and silica bond thio silica gel (200 g) were added. The mixture was stirred at 70 0C for 16 hr, then was filtered, washing the pad with ethyl acetate (1.0 L). The combined filtrates were concentrated at atmospheric pressure to a volume of about 5 L, then the mixture was allowed to cool. The resulting solid was collected and washed with ethyl acetate (2 x 1.0 L) to provide the title compound. [00211] The compound of Example 1 can be recrystallised using a toluene/ethanol mixture and washed at room temperature with NaHCθ3 aqueous solution. [00212] In another embodiment, the invention provides a compound of Example 1 in crystal form modification A, wherein modification A comprises the following powder X-ray diffraction peaks (angle 2-Theta °): 12.1, 16.9, 18.9, 21.3; and which in an additional embodiment comprises the following powder X-ray diffraction peaks (angle 2-Theta °): 12.1, 15.9, 16.9, 17.3, 18.9, 21.3, 22.1, 23.6, 24.4, 27.3. [00213] In a yet further embodiment, the invention provides the compound of Example
1 as solid form modification A comprising the following powder X-ray diffraction peaks (angle 2-Theta °): 9.0, 12.1, 13.0, 13.1, 13.6, 14.4, 14.7, 15.1, 15.9, 16.9, 17.3, 17.7, 18.0, 18.9, 19.0, 20.1, 21.3, 22.1, 22.3, 22.6, 22.8, 23.4, 23.6, 24.4, 25.3, 26.3, 26.5, 27.3, 27.8, 28.2, 29.5, 29.7, 30.4, 30.7, 31.0, 31.4, 32.2, 32.8, 33.3, 34.3, 35.5, 36.4, 37.5, 38.4, 39.0, 39.4. [00214] The powder X-ray diffraction pattern of the compound of Example 1, modification A, is shown in Fig 1 herein. Amorphous material of the compound of Example lwas produced in situ in a DSC (differential scanning calorimetry) crucible by heating the compound until melting and annealing/cooling. Upon the cooling cycle the glass transition could be observed but upon the reheating cycle is much more characterized at about 70-750C. The DSC pattern is shown in Fig 13 herein.
Example 15
4-(2-(Pyridin-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phenol
Figure imgf000062_0001
[00215] Synthesis of 2-Amino-6-chloro-9-isopropyl-9H-purine (b): Sodium hydride (1.5 g of 60% dispersion in mineral oil, 38 mmol) was added in portions over 10 min to a stirred suspension of 2-amino-6-chloro-9H-purine (5.34 g, 31.5 mmol) in anhydrous DMF (50 mL) at rt. After 45 min, the mixture was cooled in an ice bath, then 2-iodopropane was added. The cooling bath was removed and the stirred mixture was allowed to warm to rt over 16 h. The mixture was cooled in ice, then water was added. The mixture was concentrated, and the residue was treated with hot ethyl acetate. The cooled mixture was filtered, and the filtrate was concentrated. The residue was purified by column chromatography on silica gel, eluting with 0 to 50% EtOAc in hexane to afford the title compound as a solid. 1H NMR (400 MHz, CDCl3) δ 7.83(s, IH), 5.17(s, 2H), 4.71-4.66(m, IH), 1.57(d, 6H). MS m/z 212.1 (M + 1). [00216] Synthesis of 6-Chloro-2-iodo-9-isopropyl-9H-purine (c): 6-chloro-9- isopropyl-9Η-purin-2-amine (2.68 g, 12.7 mmol) was dissolved in THF (64 mL) at rt. Iodine (1.61 g, 6.25 mmol), CH2I2 (10.6 mL) and CuI (1.27 g, 6.66 mmol) were added. The mixture was stirred for 5min at room temperature. Isopentyl nitrite (5.33 mL) was added. The reaction mixture was refluxed for 45 min, and was then cooled to room temperature. Saturated aqueous sodium bicarbonate solution was added, and the mixture was extracted with EtOAc three times. The combined organic phase was washed with brine, dried with MgSO4 and concentrated. The residue was purified by column chromatography on silica gel, eluting with 0 to 30% ethyl acetate in hexane to afford the title compound as a solid. 1H NMR (400 MHz, CDCl3) δ 8.09(s, IH), 4.95-4.88(m, IH), 1.65(d, 6H). MS m/z 323.0 (M + 1).
[00217] Synthesis of 6-Chloro-2-(pyridin-3-yl)-9-isopropyl-9H-purine (d): A round-bottom flask was charged with 6-chloro-2-iodo-9-isopropyl-9Η-purine (1.2 g, 3.7 mmol), pyridine-3-boronic acid 1,3 -propanediol cyclic ester (0.91 g, 5.6 mmol), and tetrakis(triphenylphosphine)palladium(0) (430 mg, 0.37 mmol). To this mixture was added toluene (60 ml), ethanol (6 ml) and aqueous sodium carbonate solution (2M, 15 ml). The flask was sealed and the reaction mixture was stirred at 80 0C for 4 h. Water was added to the cooled mixture, which was extracted with ethyl acetate (50 ml x 3). The organic fractions were combined, dried over sodium sulfate, and concentrated. The residue was purified by column chromatography on silica gel, eluting with 30 to 70% EtOAc in hexane to afford the title compound as a solid. 1H NMR (400 MHz, CD3OD) δ 9.60(d, IH), 8.90-8.87(m, IH), 8.68(s, IH), 8.67(d, IH), 7.63-7.60(m, IH), 5.12-5.05(m, IH), 1.74(d, 6H). MS m/z 274.1 (M + 1). [00218] Synthesis of 4-(2-(pyridin-3-yl)-9-isopropyl-9H-purin-6- ylamino)ethyl)phenol (e): 6-chloro-9-isopropyl-2-(pyridin-3-yl)-9H-purine (300 mg, 1.1 mmol) was suspended in anhydrous 2-propanol (40 mL) in a pressure tube. Tyramine (300 mg, 2.2 mmol) was added. The tube was sealed and heated to 85 0C for 16 hr. The reaction was concentrated and the residue was purified by column chromatography on silica gel, eluting with 0 to 70% EtOAc in hexane to afford the title compound as a solid. Example 123
4-(2-(9-Isopropyl-2-(2-methyl-lH-imidazol-l-yl)-9H-purin-6-ylamino)ethyl)phenol
Figure imgf000064_0001
[00219] A microwave reaction tube was charged with 4-(2-(2-chloro-9-isopropyl-9H- purin-6-ylamino)ethyl)phenol (30 mg, 0.091 mmol), 2-methyl-lH-imidazole (59 mg, 0.73 mmol) and 0.5 ml of NMP. The sealed tube was heated under microwave irradiation at 240 0C for 2 hr. The reaction mixture was purified by reverse-phase HPLC (C is column, eluting with ACN-H2O 0.05% TFA) to afford the title compound as an off-white solid.
Example 128
4-(2-(2-(5-Chloropyridin-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phenol
Figure imgf000064_0002
[00220] Synthesis of 4-(2-(2-iodo-9-isopropyl-9H-purin-6-ylamino)ethyl)phenol
(b): A mixture of 6-chloro-2-iodo-9-isopropyl-9H-purine (a) (1.0 g, 3.1 mmol), tyramine (0.64 g, 4.65 mmol), triethylamine (0.63 g, 6.2 mmol) and 2-propanol (30 mL) was heated at 85 0C for 2 hr. The reaction mixture was concentrated and saturated aqueous sodium bicarbonate solution was added. The mixture was extracted with ethyl acetate (50 ml x 3). The combined organic layers were dried over sodium sulfate, filtered, and concentrated. The residue was purified by column chromatography on silica gel (25 to 75% ethyl acetate in hexane eluant) to afford the title compound as a solid. MS m/z 424.1 (M + 1). [00221] Synthesis of 4-(2-(2-(5-chloropyridin-3-yl)-9-isopropyl-9H-purin-6- ylamino)ethyl)phenol (c): Following the procedure of Example 15d, 4-(2-(2-iodo-9-isopropyl- 9H-purin-6-ylamino)ethyl)phenol (b) was reacted with S-chloropyridin-3-ylboronic acid. The crude product was purified by re verse -phase HPLC (C is column, eluting with ACN-H2O 0.05% TFA) to afford the title compound as an off-white solid.
Example 134
4-(2-(6-(5-Fluoropyridin-3-yl)-l-isopropyl-lH-pyrazolor3,4-dlpyrimidin-4- vlamino)ethvl)phenol
Figure imgf000065_0001
[00222] Synthesis of 4-(2-(6-chloro-l-isopropyl-lH-pyrazolo[3,4-d]pyrimidin-4- ylamino)ethyl)phenol (b): Following the procedure of Example 128b, 4,6-dichloro-l- isopropyl-lH-pyrazolo[3,4-d]pyrimidine (US3399196) (a) (0.184 g, 0.795 mmol) was reacted with tyramine. The crude residue was purified by silica gel chromatography (25 to 75% ethyl acetate in hexane eluant) to afford the title compound as a solid. MS m/z 332.1 (M + 1). [00223] Synthesis of 4-(2-(6-(5-fluoropyridin-3-yl)-l-isopropyl-lH-pyrazolo[3,4- d]pyrimidin-4-ylamino)ethyl)phenol (c): Following the procedure of Example 15d, 4-(2-(6- chloro-l-isopropyl-lH-pyrazolo[3,4-d]pyrimidin-4-ylamino)ethyl)phenol (b) was reacted with 5- fluoropyridin-3-ylboronic acid. The crude residue was purified by re verse -phase HPLC (C is column, eluting with ACN-H2O 0.05% TFA) to afford the title compound as an off-white solid. Example 141
4-(2-(2-(5-Fluoropyridin-3-yl)-7-isopropyl-7H-pyrrolor23-dlpyrimidin-4- ylamino)ethyl)phenol
Figure imgf000066_0001
[00224] Synthesis of 2,4-dichloro-7-isopropyl-7H-pyrrolo[2,3-d]pyrimidine (b):
Following the procedure of Example 15b, 2,4-dichloro-7H-pyrrolo[2,3-d]pyrimidine (0.5 g, 2.67 mmol) was reacted with 2-iodopropane. The crude residue was purified by silica gel chromatography (15 to 25% ethyl acetate in hexane eluant) to afford the title compound as a solid. MS m/z 230.2 (M + 1).
[00225] Synthesis of 4-(2-(2-chloro-7-isopropyl-7H-pyrrolo[2,3-d]pyrimidin-4- ylamino)ethyl)phenol (c): Following the procedure of Example 128b, 2,4-dichloro-7- isopropyl-7H-pyrrolo[2,3-d]pyrimidine (b) (0.278 g, 1.21 mmol) was reacted with tyramine. The crude residue was purified by silica gel chromatography (25 to 75% ethyl acetate in hexane eluant) to afford the title compound as a solid. MS m/z 331.1 (M + 1). [00226] Synthesis of 4-(2-(2-(5-fluoropyridin-3-yl)-7-isopropyl-7H-pyrrolo[2,3- d]pyrimidin-4-ylamino)ethyl)phenol (d): Following the procedure of Example 15d, 4-(2-(2- chloro-7-isopropyl-7H-pyrrolo[2,3-d]pyrimidin-4-ylamino)ethyl)phenol (20 mg, 0.06 mmol) was reacted with 5-fluoropyridin-3-ylboronic acid. The crude residue was purified by reverse-phase HPLC (C18 column, eluting with ACN-H2O 0.05% TFA) to afford the title compound as an off- white solid. Example 153
(R)-4-(2-(2-(benzorblthiophen-3-yl)-9-(tetrahydrofuran-3-yl)-9H-purin-6- ylamino)ethyl)phenol
Figure imgf000067_0001
[00227] Synthesis of (R)-2,6-dichloro-9-(tetrahydrofuran-3-yl)-9H-purine (b): A solution of 5,7- 2,6-dichloro-9H-purine (400 mg, 2.12mmol), (S)-tetrahydrofuran-3-ol (88 mg, 2.5 mmol) and triphenylphosphine (1.0 g, 3.8 mmol) in anhydrous THF (30 mL) was treated at - 78 0C with diisopropyl azodicarboxylate (856 mg, 4.23 mmol). The reaction was allowed to warmed to rt and was stirred for 16 hr. Saturated aqueous sodium bicarbonate solution was added and the mixture was extracted with ethyl acetate. The organic layers were combined, dried over sodium sulfate, and concentrated. The residue was purified by silica gel chromatography (10 to 80% ethyl acetate in hexane eluant) to afford a white solid which consisted of the title compound contaminated with triphenylphosphoxide. MS m/z 258.0 (M + 1). [00228] Synthesis of (R)-4-(2-(2-chloro-9-(tetrahydrofuran-3-yl)-9H-purin-6- ylamino)ethyl)phenol (c): Following the procedure of Example 128b, (R)-2,6-dichloro-9- (tetrahydrofuran-3-yl)-9H-purine (b) was reacted with tyramine. The crude reaction mixture was purified by re verse -phase preparative HPLC. MS m/z 360.1 (M + 1).
[00229] Synthesis of (R)-4-(2-(2-(benzo[b]thiophen-3-yl)-9-(tetrahydrofuran-3-yl)-
9H-purin-6-ylamino)ethyl)phenol: Following the procedure of Example 15d, (R)-4-(2-(2- chloro-9-(tetrahydrofuran-3-yl)-9H-purin-6-ylamino)ethyl)phenol (c) was reacted with benzo[b]thiophen-3-ylboronic acid (22.3 mg, 0.125 mmol). The crude residue was purified by reverse-phase preparative HPLC to afford the title compound as an off-white solid. Example 157
2-(6-(2-(lH-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin-9-yl)propan-l-ol
Figure imgf000068_0001
[00230] Synthesis of methyl 2-(2,6-dichloro-9H-purin-9-yl)propanoate (b): A mixture of 2,6-dichloro-9H-purine (5.0 g, 26.5 mmol), methyl 2-bromopropanoate (5.3 g, 31.7 mmol) and potassium carbonate (11.0 g, 79.4 mmol) in anhydrous DMF (100 mL) was heated at 100 0C for 15h. Sat. aqueous sodium bicarbonate solution was added and reaction was extracted with ethyl acetate (150 ml X 3). The organic layers were combined, washed with brine, dried over sodium sulfate, and concentrated. The residue was purified by silica gel chromatography (10 to 80% ethyl acetate in hexane eluant) to afford the title compound as a white solid. MS m/z 275.0 (M + 1).
[00231] Synthesis of methyl 2-(6-(2-(lH-indol-3-yl)ethylamino)-2-chloro-9H- purin-9-yl)propanoate (c): A mixture of methyl 2-(2,6-dichloro-9H-purin-9-yl)propanoate (b) (600 mg, 2.2 mmol), tryptamine (420 mg, 2.6 mmol) and 2-propanol (30 mL) was heated at 85°C in a sealed tube for 16 h. The reaction mixture was cooled to room temperature and concentrated. The residue was purified by silica gel chromatography (10 to 80% ethyl acetate in hexane eluant) to afford the title compound as a white solid. MS m/z 360.1 (M + 1). [00232] Synthesis of methyl 2-(6-(2-(lH-indol-3-yl)ethylamino)-2-(5- fluoropyridin-3-yl)-9H-purin-9-yl)propanoate (d): A 150 ml pressure tube was charged with methyl 2-(6-(2-(lH-indol-3-yl)ethylamino)-2-chloro-9H-purin-9-yl)propanoate (c) (300 mg, 0.75 mmol), 5-fluoropyridin-3-ylboronic acid (159 mg, 1.1 mmol), tetrakis(triphenylphosphine)- palladium(O) (87 mg, 0.075 mmol), K3PO4 (638 mg, 3.0 mmol), and anhydrous dioxane (15mL). The pressure tube was sparged with nitrogen and was sealed, then the reaction mixture was heated at 1300C for 6 h with stirring. Water was added to the cooled mixture, and the mixture was extracted with ethyl acetate (50 ml x 3). The combined organic layers were washed with brine, dried over sodium sulfate, and concentrated. The residue was purified by silica gel chromatography (10 to 80% ethyl acetate in hexane eluant) to afford the title compound contaminated with a small amount of triphenylphosphine oxide. MS m/z 460.1 (M + 1). [00233] Synthesis of 2-(6-(2-(lH-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-
9H-purin-9-yl)propan-l-ol: Lithium aluminum hydride (230 mg, 6.1 mmol) was added in portions to a 0 0C solution of methyl 2-(6-(2-(lH-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3- yl)-9H-purin-9-yl)propanoate (282 mg, 0.61 mmol) in anhydrous THF (15 mL). The stirred reaction mixture was allowed to warm to rt over 2h, then water was added carefully. The mixture was extracted with EtOAc (50 ml x 3). The organic fractions were combined, washed with brine, dried over sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography (0 to 5% solvent B in dichloromethane; solvent B = 2M ammonia in methanol) to afford the partially purified title product. This was further purified by preparative TLC (5% solvent B in dichloromethane) to provide the title compound as a white solid.
Examples 157R & 157S
(R)-2-(6-(2-(lH-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin-9-yl)propan-l-ol & (S)-2-(6-(2-(lH-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin-9-yl)propan-l-ol
Figure imgf000069_0001
[00234] (R/S)-(6-(2-(lH-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H- purin-9-yl)propan-l-ol was separated into the individual enantiomers using preparative chiral HPLC on a 21x250 mm Lux-Cellulose-2 (Phenomenex) chiral column. A 3mg/ml solution of the racemate in methanol was prepared and loaded onto the column with 0.5 ml solution per injection. The column was eluted with 85/7.5/7.5 hexane/ethanol/methanol at a flow rate of 20 mL/min for 25 min. Peaks 1 and 2 were eluted at 20 min and 22.5 min, respectively. Analytical chromatography was performed on a 4.6 x 100 mm Lux_Cellulose-2 (Phenomenex) chiral column, eluting with 90/5/5 hexane/ethanol/methanol at 1 mL/min for 20 min. Peaks 1 and 2 were eluted at 17.45 and 18.14 min, respectively.
Example 157R
(R)-2-(6-(2-(lH-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin-9-yl)propan-l-ol
Figure imgf000070_0001
[00235] Synthesis of (R)-N-(2-(lH-indol-3-yl)ethyl)-9-(l-
(benzyloxy)propan-2-yl)-2-(5-fluoropyridin-3-yl)-9H-purin-6-amine (b): Following, in succession, the procedures of Example 153b (using 2,6-dichloro-9H-purine and (S)-I- (benzyloxy)propan-2-ol as reactants), Example 153c (using tryptamine as reactant), and Example 153d (using 5-fluoropyridin-3-ylboronic acid as reactant), the title compound was obtained. [00236] Synthesis of (R)-2-(6-(2-(lH-indol-3-yl)ethylamino)-2-(5- fluoropyridin-3-yl)-9H-purin-9-yl)propan-l-ol (c): A solution of (R)- N-(2-(lH-indol-3- yl)ethyl)-9-(l-(benzyloxy)propan-2-yl)-2-(5-fluoropyridin-3-yl)-9H-purin-6-amine (b) (0.15 g, 0.29 mmol) in DCM (10 ml) was treated with BCl3 (IM, 2.9 ml, 2.9 mmol) in DCM (10 ml) at - 78 0C for 2 hr. IN aqueous sodium hydroxide solution was added, and the mixture was extracted with DCM. The combined organic extracts were dried over sodium sulfate, filtered, and concentrated and the residue was purified by silica gel column chromatography (5% MeOH in DCM eluant) to provide the title compound. MS m/z 432.2 (M + 1).
Example 157S (S)-2-(6-(2-(lH-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin-9-yl)propan-l-ol
Figure imgf000071_0001
[00237] Following the procedure of Example 157R, but employing (R)-I-
(benzyloxy)propan-2-ol in place of (S)-l-(benzyloxy)propan-2-ol, the title compound was prepared. MS m/z 432.2 (M + 1).
Example 161
4-(2-(6-(5-Fluoropyridin-3-yl)-l-isopropyl-lH-imidazor4,5-clpyridin-4- ylamino)ethyl)phenol
Figure imgf000071_0002
[00238] Synthesis of 4,6-dichloro-l-isopropyl-lH-imidazo[4,5-c]pyridine
(b): Following the procedure of Example 15b, 4,6-dichloro-lH-imidazo[4,5-c]pyridine (J. Het. Chem. 1965, 196-201) (0.19 g, 1.0 mmol) was reacted with 2-iodopropane. The residue was purified by silica gel chromatography (25 to 35% ethyl acetate in hexane eluant) to afford the title compound as a solid. MS m/z 230.2 (M + 1).
[00239] Synthesis of 4-(2-(6-chloro-l-isopropyl-lH-imidazo[4,5-c]pyridin-
4-ylamino)ethyl)phenol (c): A mixture of 4,6-dichloro-l-isopropyl-lH-imidazo[4,5-c]pyridine (b) (40 mg, 0.17 mmol), tyramine (120 mg, 0.86 mmol), and 2-butanol (2 mL) was heated under microwave irradiation at 140 0C for 8 hr. The mixture was concentrated and the residue was purified by reverse-phase HPLC (C18 column, eluting with ACN-H2O 0.05% TFA) to afford the title compound as an off-white solid. MS m/z 331.1 (M + 1).
[00240] Synthesis of 4-(2-(6-(5-fluoropyridin-3-yl)-l-isopropyl-lH- imidazo[4,5-c]pyridin-4-ylamino)ethyl)phenol (d): A 5 ml microwave reaction vial was charged with 4-(2-(6-chloro-l-isopropyl-lH-imidazo[4,5-c]pyridin-4-ylamino)ethyl)phenol (c): (17 mg, 0.051 mmol), 5-fluoropyridin-3-ylboronic acid (72 mg, 0.51 mmol), and tetrakis(triphenylphosphine)palladium(0) (36 mg, 0.031 mmol). To this mixture was added toluene (1 ml), ethanol (0.5 ml) and aqueous sodium carbonate solution (2M, 0.5 ml). The vial was sealed and the reaction mixture was stirred at 140 0C under microwave irradiation for 2 hours. Water was added to the cooled mixture, which was extracted with ethyl acetate (5 ml x 3). The organic fractions were combined, dried over sodium sulfate, and concentrated. The residue was purified by reverse-phase HPLC (C18 column, eluting with ACN-H2O 0.05% TFA) to afford the title compound as an off-white solid.
Example 177
4-(2-(5-(5-Fluoropyridin-3-yl)-3-isopropyl-3H-imidazor4,5-blpyridin-7- vlamino)ethvl)phenol
Figure imgf000072_0001
[00241] Synthesis of 5,7-dichloro-3-isopropyl-3H-imidazo[4,5-b]pyridine
(b): Following the procedure of Example 15b, 5,7-dichloro-3H-imidazo[4,5-b]pyridine (J. Med. Chem., 2007, 50, 828-834) (0.118 g, 0.624 mmol) was reacted with 2-iodopropane. The crude product mixture was purified by silica gel chromatography (25 to 35% ethyl acetate in hexane eluant) to afford a mixture of the title compound (major) and an isomeric product as a solid. MS m/z 230.2 (M + 1).
[00242] Synthesis of 4-(2-(5-chloro-3-isopropyl-3H-imidazo[4,5-b]pyridin-
7-ylamino)ethyl)phenol (c): The product mixture containing 5,7-dichloro-3-isopropyl-3H- imidazo[4,5-b]pyridine (b) (40 mg, 0.17 mmol), tyramine (120 mg, 0.87 mmol), and 2-propanol (2 mL) was heated in a sealed vial at 140 0C for 72 hr. The mixture was concentrated, and the residue was purified by preparative TLC (1:2 hexanes/ethyl acetate eluant) to afford the title compound as an off-white solid. MS m/z 331.1 (M + 1).
[00243] Synthesis of 4-(2-(5-(5-fluoropyridin-3-yl)-3-isopropyl-3H- imidazo[4,5-b]pyridin-7-ylamino)ethyl)phenol (d): Following the procedure of Example 16 Id, 4-(2-(5-chloro-3-isopropyl-3H-imidazo[4,5-b]pyridin-7-ylamino)ethyl)phenol (c) (15 mg, 0.047 mmol) was reacted with 5-fluoropyridin-3-ylboronic acid. The crude residue was purified preparative TLC (1: 1 hexanes/ethyl acetate eluant) to afford the title compound as an off-white solid.
[00244] By repeating the procedures described in the above examples, using appropriate starting materials, the following compounds of Formula I, as identified in Table 1, are obtained.
Table 1
Figure imgf000073_0001
Figure imgf000074_0001
Figure imgf000075_0001
Figure imgf000076_0001
Figure imgf000077_0001
Figure imgf000078_0001
Figure imgf000079_0001
Figure imgf000080_0001
Figure imgf000081_0001
Figure imgf000082_0001
Figure imgf000083_0001
Figure imgf000084_0001
Figure imgf000085_0001
Figure imgf000086_0001
Figure imgf000087_0001
Figure imgf000088_0001
Figure imgf000089_0001
Figure imgf000090_0001
Figure imgf000091_0001
Figure imgf000092_0001
Figure imgf000093_0001
Figure imgf000094_0001
Figure imgf000095_0001
Figure imgf000096_0001
Figure imgf000097_0001
Figure imgf000098_0001
Figure imgf000099_0001
Figure imgf000100_0001
Figure imgf000101_0001
Figure imgf000102_0001
Figure imgf000103_0001
Figure imgf000104_0001
Figure imgf000105_0001
[00245] Affinity probe compounds related to the compounds of the invention can also be prepared, as described in the following examples.
Example 210 -(2-(2-(benzorblthiophen-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)-lH-indol-5-yl 6-(5- ((3aS,4S,6aR)-2-oxohexahydro-lH-thienor3,4-dlimidazol-4-yl)pentanamido)hexanoate
Figure imgf000106_0001
[00246] Synthesis of 3-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6- ylamino)ethyl)-lH-indol-5-yl 6-aminohexanoate (e): To a solution of 3-(2-(2- (benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)-lH-indol-5-yl 6-(tert- butoxycarbonylamino)hexanoate (a) (80 mg, 0.117 mmol) in DCM (20 ml) was added TFA (5 ml). The reaction was stirred at rt for 3 hr. It was concentrated. Aqueous sodium carbonate solution was added and the mixture was extracted with DCM. The organic fractions were combined, dried over sodium sulfate, and concentrated to afford the product as an oil. MS m/z 582.2 (M + 1).
[00247] Synthesis of 3-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6- ylamino)ethyl)-lH-indol-5-yl 6-(5-((3aS,4S,6aR)-2-oxohexahydro-lH-thieno[3,4- d]imidazol-4-yl)pentanamido)hexanoate: To a solution of (+)-biotin (35 mg, 0.14 mmol) and Et3N (36 mg, 0.35 mmol) in DMF (1 ml) was added HATU (90 mg, 0.24 mmol). The mixture was stirred for 10 min, and then was added to a solution of (3-(2-(2-(benzo[b]thiophen-3-yl)-9- isopropyl-9H-purin-6-ylamino)ethyl)-lH-indol-5-yl 6-aminohexanoate (b) (68 mg, 0.12 mmol) in DMF (1 ml). The reaction mixture was stirred for 16 hr at rt and then was concentrated. The residue was purified by reverse-phase HPLC (C is column, eluting with MeOH-H2O 0.05% TFA) to afford the title compound as an off-white solid. Example 210 showed an EC50 value in the %CD34+ assay of 2.1 μM.
Example 211
3-(2-(2-(benzorblthiophen-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)-lH-indol-5-yl 6-
(tert-butoxycarbonylamino)hexanoate
Figure imgf000107_0001
[00248] Synthesis of 2-(benzo[b]thiophen-3-yl)-6-chloro-9-isopropyl-9H-purine
(b): Following the procedure of Example 15d, 6-chloro-2-iodo-9-isopropyl-9H-purine (3.31 g, 0.0103 mol) was reacted with benzo[b]thiophen-3-ylboronic acid. The crude product was purified by silica gel chromatography (20 to 50% ethyl acetate in hexane) to afford the title compound as a solid. MS m/z 329.0 (M + 1). 1H NMR (400 MHz, DMSO-d6): δ = 9.15 (d, IH), 8.85 (s, 2H), 8.17 (d, IH), 7.62 (t, IH), 7.53 (t, IH), 5.06 (m, IH), 1.71 (d, 6H). [00249] Synthesis of 3-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6- ylamino)ethyl)-lH-indol-5-ol (c): Following the procedure of Example 15e, 2- (benzo[b]thiophen-3-yl)-6-chloro-9-isopropyl-9H-purine (b) (80 mg, 0.243 mmol) was reacted with serotonin. The reaction mixture was concentrated, then aqueous sodium bicarbonate solution was added. The mixture was extracted with ethyl acetate. The organic fractions were combined, dried over sodium sulfate, and concentrated. The residue was purified by silica gel chromatography (0 to 5% MeOH in DCM eluant) to afford the title compound as an off-white solid. MS m/z 469.2 (M + 1).
[00250] Synthesis of S^-CZ-CbenzotbJthiophen-S-yD^-isopropyl-rø-purin-β- ylamino)ethyl)-lH-indol-5-yl 6-(tert-butoxycarbonylamino)hexanoate (d): To a solution of 3-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)-lH-indol-5-ol (55.5 mg, 0.119 mmol) and 6-(tert-butoxycarbonylamino)hexanoic acid (30 mg, 0.113 mmol) in DMF (3 ml ) was added Et3N (24 mg, 0.237 mmol) and HATU (90 mg, 0.237 mmol). The mixture was stirred at rt for 16 hr, and then was concentrated. Water was added and the reaction mixture was extracted with ethyl acetate. The organic fractions were combined, dried over sodium sulfate, and concentrated. The residue was purified by silica gel chromatography (0 to 5% MeOH in DCM eluant) to afford the title compound as an off-white solid.
[00251] By repeating the procedures described in the above examples, using appropriate starting materials, the following affinity probes, as identified in Table 2, are obtained:
Table 2
Figure imgf000109_0001
Figure imgf000110_0001
Figure imgf000111_0001
Assays
[00252] The following assays are used to assess the activity of the compounds of the invention to facilitate hematopoietic stem cell (HSC) expansion.
[00253] Primary adult CD34+ human hematopoietic stem cells (HSCs) are cultured and screened to identify compounds of the invention that facilitate HSC expansion. The cells are analyzed for the presence of the desired phenotype (CD34 expression). Compounds of the invention promote HSC expansion in a dose dependent manner.
[00254] Culture Medium: StemSpan SFEM medium is serum-free medium (StemCell
Technologies, Vancouver, BC) supplemented with the following human recombinant cytokines: thrombopoietin, interleukin-6, Flt-3 ligand, and stem cell factor (all from R&D Systems, Minneapolis, MN), each at a final concentration of 50 ng/mL, with vehicle (DMSO) or a compound of the invention.
[00255] Human Cell Culture: Fresh human leukophoresed G-CSF mobilized peripheral blood from normal donors, CD34+ cells from adult bone marrow and cryopreserved human cord blood CD34+ cells are purchased from AllCells (Berkeley, CA). Human CD34+ cells are enriched from leukophoresed G-CSF mobilized peripheral blood using magnetic cell sorting (MACS, Direct CD34 Progenitor Cell Isolation Kit, Miltenyi Biotec, Bergisch Gladbach, Germany) and cryopreserved. CD34+ cell purity, checked by flow Cytometry, is higher than 90%. After thawing, the cell viability tested by trypan blue exclusion is higher than 70%. The thawed cells are centrifuged and resuspended with StemSpan medium before being aliquoted for immediate culture. Cells are plated at 104 cells/mL in a 384 well plate (Greiner Bio-One, Monroe, North Carolina) with 50 μL of medium per well for 7 days. Every 7 days the cells are transferred to larger well plates and fresh medium is added to keep the cell density between 104 and 5 x 105 cells/mL. Cells were cultured at 37°C in 5% CO2. For transplantation, cells were cultured in 75 cm flasks before the cells were transplanted into mice. At a concentration of 1 micromolar, 4-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phenol (compound 1, table 1; example 1), 4-(2-(2-(benzo[b]thiophen-3-yl)-9-sec-butyl-9H-purin-6- ylamino)ethyl)phenol (compound 2, table 1), and N-(2-(lH-indol-3-yl)ethyl)-2- (benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-amine (compound 9, table 1) each gives rise to a greater than 10-fold increase in the number of CD34+CD45RA~ cells derived from 1000 mPB CD34+ HSCs after 21 days compared to vehicle. Compounds of the invention were assayed in a dose response format (InM to lOμM) to determine the effective concentration that produced the desired effect in 50% of the cells (EC50). Compounds of the invention increased the total number and/or percent of CD34+ cells with an EC50 of less than 10 μM. The results are shown in Table 1 and examples, supra.
[00256] Colony-Forming Units in Culture (CFU-C) Assay: Mononuclear cells at 1000 per mL for cord blood 5 week and mPB 3 week culture and 100 cells per mL for CB 3 week and mPB 1 week culture were added to MethoCult SF H4436, serum-free methylcellulose medium containing methylcellulose in Iscove's MDM, bovine serum albumin, 2-mercaptoethanol, L- glutamine, human transferring (iron saturated), recombinant human insulin, and recombinant human cytokines: stem cell factor, GM-CSF, IL-3, IL-6, G-CSF, and erythropoietin (StemCell Technologies). The MethoCult is supplemented with the following human recombinant cytokines: thrombopoietin, and Flt-3 ligand (R&D Systems), each at a final concentration of 50 ng/mL. After stirring, the mixture is divided into three 35-mm dishes. The dishes are incubated for 14 days at 37°C in a humidified atmosphere of 5% CO2 in air. At the end of the incubation period, myeloid and erythroid colonies are counted under an inverted microscope at 4OX magnification. CFU-C content of the expansion culture is calculated as follows: number of scored colonies per three dishes x total mononuclear cell number/input cell number. Up to one week, total mononuclear cells are determined by multiplying the number of cells per milliliter by the culture volume. From week 1 and on, the number of passages is also taken into account. Cultures treated with 4-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6- ylamino)ethyl)phenol (compound 1, table 1; example 1) at a concentration of 1 micromolar, generated a greater than 10-fold increase in the number of colony forming cells after 21 days of culture of mPB CD34+ cells compared to vehicle. Using 1 x 103 CB CD34+ cells treated with 4- (2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phenol (compound 1, table 1; example 1) at a concentration of 1 micromolar taken from the 5 week culture showed a >10- fold increase in colony forming units compared to control. Cells treated with a 4-(2-(2- (benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phenol (compound 1, table 1; example 1) generated more mixed colonies associated with a >10-fold increase in erythrocyte colonies, a >10-fold increase in granulocyte/macrophage colonies, and a >10-fold increase in macrophage colonies. Cells treated with 4-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin- 6-ylamino)ethyl)phenol (compound 1, table 1; example 1) also give rise mixed granulocyte/erythrocyte/monocyte/macrophage colonies, which are not observed in colonies derived from untreated cultures.
[00257] Cobblestone area-forming cell (CAFC) assays: The FBMD-I stromal cells are maintained in 25-cm flasks and are trypsinized after 1/3 confluence. Since this non-transformed line ages, and therefore gradually loses its potential to support CAFC growth at late stages, all feeders are used below passage 20. For supporting CAFC growth in 96-well plates, 1 x 103 stromal cells are seeded per well. The cultures are maintained in Iscove's medium supplemented with 10% fetal calf serum (FCS), 2.5% horse serum (HS), 1% L-glutamine, 1% penicillin- streptomycin, and 1 x 10" M hydrocortisone at 37°C in a humidified atmosphere of 5% CO2 in air. After stromal layers reach confluency, they are inoculated with CD34+ HSCs that have been cultured for 5 days with vehicle or a compound of the invention. MNCs are added at 8 serial 1:3 dilutions (starting at 25,000 cells/well), with 10 wells for each cell dose. The dilutions with wells with at least one phase-dark hematopoietic clone (cobblestone area) of at least five cells beneath the stromal layer are determined at week 4. At a test concentration of 1 micromilar, 4- (2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phenol (compound 1, table 1; example 1), stimulates a greater than 2-fold increase in the number of cobblestone area forming cells derived from mPB CD34+ HSCs after 5 days of culture, compared with control cultures that are treated with DMSO alone.
[00258] Surface Antigen Analysis: The cells are washed with staining media (Hanks balanced salt solution containing FBS (2%) and EDTA (2mM)) and stained (at 4°C for 30 minutes) with indicated primary conjugated antibodies. The cells are washed in the previously described buffer and analyzed using a BD LSR II flow cytometer (Becton Dickinson, San Jose, CA). The cells are passed at a rate of up to 1000 cells/second using 488-nm argon and 633-nm HeNe laser beams as the light source for excitation. Emission of 104 cells is measured using logarithmic amplification and analyzed using Flow Jo software (TreeStar Inc. Ashland, OR). Cells stained with primary conjugated isotype control antibodies are used to determine background fluorescence.
[00259] Determination of CD34+ cell subsets: The percentages of CD34+ cell subsets are determined from aliquots of the cell culture. Cells were stained with APC anti-Thyl.l, PerCP anti-CD34, PECy7 anti CD45RA, FITC anti CD38, and PE anti-CD133 for determination of CD34+Thyl.l+, CD34+CD45RA", CD34+CD38", CD133+CD38" and CD34+CD133+ cells. Antibodies to CD34, CD38, Thy 1.1 and CD45RA were purchased from Becton Dickinson and antibodies to CD133 were purchased Miltenyi Biotec. FACS analysis results of these subsets are given as percentage of the total population. The absolute number of each population of cells in the culture is calculated from the total number of cells multiplied by the percentage of each population. Starting with CB CD34+ cell, after five weeks the total cell number in the cultures increased on average greater than 2-fold in the 4-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H- purin-6-ylamino)ethyl)phenol (compound 1, table 1; example 1) 1 micromolar treated cells compared to control cultures. More importantly, >50% of 4-(2-(2-(benzo[b]thiophen-3-yl)-9- isopropyl-9H-purin-6-ylamino)ethyl)phenol (compound 1, table 1; example 1) cultured cells were CD34+ compared to < 10% of vehicle cultured cells resulting in a greater than 10-fold expansion of CD34+ cells compared to control and a greater than 10,000-fold expansion compared to input cells. In addition, the presence of 1 micromolar 4-(2-(2-(benzo[b]thiophen-3- yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phenol (compound 1, table 1; example 1) increased both the percentage and total numbers of the CD34+ subpopulations, CD34+CD45RA~, CD34+CD38~, CD133+CD38~, and CD34+CD133+ resulting in a net expansion of greater than 30- fold for each subset.
[00260] Transplantation of human CD34+ cells into NOD.CB lV-Prkdc80"1 mice
(NOD/SCID): To assess the in vivo repopulating capacity of CD34+ cells and their cultured progeny, uncultured CD34+ or the progenies of cultured CD34+ cells after 4 days (mPB) or 21 days (CB) with vehicle or a test compound were injected intravenously via the retro-orbital route into sub-lethally irradiated (3.0 Gy) 8- to 10-week-old NOD/SCID (for mPB HSC experiments) or NOD/SCIDgc-/- (for CB HSC experiments) mice. To monitor engraftment blood was drawn weekly via the retro-orbital and treated with erythrocyte lysis solution (Qiagen, Valencia, CA) to remove red blood cells, washed with staining media, and analyzed by flow Cytometry. Engraftment was measured by detection of anti-human CD45+ cells in the blood. The mice are sacrificed at 10 weeks post-transplantation; BM is collected from both femurs and tibiae. BM cells are washed in staining media and stained with anti-human antibodies. Following incubation, the suspension is treated with erythrocyte lysis solution (Qiagen, Valencia, CA) to remove red blood cells, washed with staining media, and analyzed by flow Cytometry, as described earlier. Both mPB and CB derived HSCs cultured with 4-(2-(2-(benzo[b]thiophen-3- yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phenol (compound 1, table 1; example 1) at a concentration of 1 micromolar give rise to a statistically significant increase in the percentage of human cells 10 weeks after engraftment.
Target identification
[00261] To identify the mechanism whereby a compound of the invention expands
HSCs in an undifferentiated state, a genome-wide transcriptional profiling of mPB-derived CD34+ cells treated for 24 hours with example 1 and a less active analog (~20-fold) of example 1 (2-(benzo[b]thiophen-3-yl)-N-(3-(3,5-dimethyl-lH-pyrazol-4-yl)propyl)-9-isopropyl-9H-purin- 6-amine). Of the >50,000 probe sets analyzed, only 5 genes were up-regulated greater than 3- fold upon treatment with Example 1 and most were also induced to some degree by the inactive analog. In addition, 5 genes were down-regulated by >70% upon treatment with 1 μM of Example 1. All were down-regulated in a dose dependent fashion and none were significantly affected by the inactive analog. The two genes that were the most highly repressed by treatment with Example 1 (cytochrome P450 IBl [CYPlBl] and the aryl hydrocarbon receptor repressor [AHRR]) are transcriptionally regulated by the aryl hydrocarbon receptor (AHR). Therefore, compounds of the invention could be acting as an antagonist of AHR signaling. [00262] Further, the ability of Example 1 to block 2,3,7, 8-tetrachlorodibenzo-p- dioxin (TCDD, dioxin) -mediated CYPlBl mRNA expression by qPCR in mPB-derived CD34+ cells was determined. Treatment with TCDD (3nM) caused a 4.5-fold increase in the level of CYPlBl mRNA compared with the vehicle control (0.01% toluene). This increase was inhibited by Example 1 in a dose-dependent manner indicating that compounds of the invention can antagonize AHR signaling. To determine the effects of Example 1 in AHR transcription the ability of Example 1 to inhibit a dioxin-induced AHR dependent luciferase reporter gene assay was tested. Inclusion of Example 1 (lμM) completely abolished dioxin- induced AHR dependent transcription when used on cells expressing human AHR. Titration of Example 1 revealed an EC50 of 127nM, demonstrating that Example 1 is a potent AHR antagonist. Interestingly, Example 1 only weakly inhibited dioxin induced transcription in murine cells and had no activity on rat cells, suggesting that Example 1 preferentially inhibits human AHR. This correlates with a lack of activity of Example 1 on murine HSC, and can explain the species selectivity of Example 1. Finally, Example 1 had only weak agonist activity on murine or rat cells, and failed to induce AHR dependent transcription in human cells.
[00263] To further explore the role of AHR signaling in HSCs two other AHR antagonists (alpha-naphthoflavone and CH-223191) were tested. Both compounds lead to dose dependent increases in the number of CD34+ cells when cultured with mPB-derived CD34+ cell for 7 days: inclusion of 1 μM CH223191 afforded a 2.2-fold expansion of CD34+ cells; 0.75 μM α-naphthoflavone afforded a 1.9-fold expansion of CD34+ cells while 0.75 μM Example 1 afforded a 3.4-fold expansion of total CD34+ cells. To show a direct role for the AHR in Example 1 induced HSC expansion, human CB-derived CD34+ HSCs were treated with lentiviral particles containing a shRNA-targeting AHR that co-expressed GFP or control virus. Forty-eight hours following transduction, CD34+GFP+ cells were purified by cell sorting and the levels of AHR were determined by qPCR. Both AHR targeting shRNAs led to decreases in AHR expression following transduction (81% with shl l l and 51% with sh242). These decreases were not seen in cells lacking GFP or in cells transduced with control virus. CB-derived CD34+ cells with decreased AHR expression displayed a phenotype similar to Example 1 treated cells with sustained expression of CD34+. These data show that inhibition of AHR activity by a compound of the invention is sufficient to promote ex- vivo expansion of HSC.
Method of expanding HSCs from human neonatal umbilical cord blood [00264] The culture medium used is StemSpan SFEM (StemCell Technologies, Cat.
#09650) supplemented with the following recombinant human cytokines: TPO, IL6, Flt3 ligand, and SCF each at a final concentration of 50 ng/mL. The culture media is prepared fresh the day of use.
[00265] Compound dilution into media: a 10,000x concentrate of a compound of the invention is used for the dilutions. The addition of compound into the culture media occurs in two steps. The first step is a 1:100 dilution (lOμL of 10,000x concentrate into 990μL of complete culture media (containing cytokines) in a 1.5 mL effendorf tube [USA Scientific, Cat #1615-5500]) to generate a 10Ox solution of compound in the culture media. The second step is a 1: 100 dilution into the culture media that will be used to initiate the cell culture. The volume of the culture is variable depending on the input number of cord blood (CB) CD34+ cells. For example, IxIO6 CB CD34+ cells are seeded into 20 mL of media (5xlO4 cells/mL). In this case, 200μL of the 10Ox Example 1 solution is added to the 20 mL of media in a 50 mL conical tube (Becton Dickinson, Cat #352098) to reach the final concentration (see Table 3). [00266] Cell culture initiation: purified human CB CD34+ cells are used for the ex vivo expansion experiments. After thawing, the cell viability, tested by trypan blue exclusion, is higher than 50%. The thawed cells are diluted 5-fold with culture media (no cytokines or compounds of the invention such as Example 1) and centrifuged at 300g at 25°C for 8 minutes. After aspirating the supernatant, the pellet is resuspended with the appropriate volume of culture medium (5 x 104 cells/mL, Table 3) before being injected (22 gauge needle, Air-Tite products; 20 mL syringe, BD cat # 309661) into AFC bags (Table 5) for immediate culture. Cells are cultured at 37 0C in 5% CO2.
[00267] Addition of media to the cell culture: for media volumes up to 80 mL the procedure above (compound dilution into media) is used. For media volumes larger than 80 mL the first 1:100 dilution is carried out in 10 mL conical tubes (Corning, Cat #430052, Table 4). The second step is a 1:100 dilution into the culture media, in sterile containers, (BD Falcon, Cat #354015) that is added to the AFC bag (22 gauge needle, Air-Tite products; 60 mL syringe, BD cat # 309653).
Figure imgf000118_0001
Table 4. Example 1 dilutions for adding media to the cord blood derived CD34+ cell ex ansion
Figure imgf000118_0002
Table 5. Volume restrictions for American Fluoroseal Corporation bags.
Figure imgf000119_0001
[00268] The same protocol can be used starting from mobilized peripheral blood cells from a patient for autologous graft transplantation.
[00269] A composition comprising a population of cells with expanded HSCs appropriate for intravenous administration as an infusion can also be prepared. To prepare cells for infusion, cultured cells are pelletted by centrifugation for 10 minutes at 300g and resuspended in infusion buffer consisting of 5% HSA (Baxter) at a concentration of between 106 to 108 cells /ml.
[00270] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference for all purposes.

Claims

WE CLAIM:
1. A compound of Formula I:
Figure imgf000120_0001
I
in which:
Gi is selected from N and CR3;
G2, G3 and G4 are independently selected from CH and N; with the proviso that at least 1 of
G3 and G4 is N; with the proviso that Gi and G2 are not both N;
L is selected from -NR5a(CH2)M- -NR53CH(C(O)OCH3)CH2-, -
NR5a(CH2)2NR5b-, -NR5a(CH2)2S- -NR5aCH2CH(CH3)CH2-, -NR5aCH2CH(OH)- and -
NRs3CH(CH3)CH2-; wherein R5a and R5b are independently selected from hydrogen and C1- 4alkyl;
Ri is selected from hydrogen, phenyl, thiophenyl, furanyl, lH-benzoimidazolyl, isoquinolinyl, lH-imidazopyridinyl, benzothiophenyl, pyrimidinyl, lH-pyrazolyl, pyridinyl, lH-imidazolyl, pyrrolidinyl, pyrazinyl, pyridazinyl, lH-pyrrolyl and thiazolyl; wherein said phenyl, thiophenyl, furanyl, lH-benzoimidazolyl, isoquinolinyl, lH-imidazopyridinyl, benzothiophenyl, pyrimidinyl, lH-pyrazolyl, pyridinyl, lH-imidazolyl, pyrrolidinyl, pyrazinyl, pyridazinyl, lH-pyrrolyl or thiazolyl of Ri can be optionally substituted by 1 to 3 radicals independently selected from cyano, hydroxy, Ci_4alkyl, Ci_4alkoxy, halo, halo- substituted-Ci_4alkyl, halo-substituted-Ci_4alkoxy, hydroxy, amino, -C(O)R83, -S(0)o-2R8a, -
C(O)ORs3 and -C(O)NRs3RSb; wherein R83 and R8b are independently selected from hydrogen and Ci_4alkyl; with the proviso that Ri and R3 are not both hydrogen;
R2 is selected from -S(O)2NR6aR6b, -NR9aC(O)R9b, -NR63C(O)NR6bR5C, phenyl, lH-pyrrolopyridin-3-yl, lH-indolyl, thiophenyl, pyridinyl, lH-l,2,4-triazolyl, 2- oxoimidazolidinyl, lH-pyrazolyl, 2-oxo-2,3-dihydro-lH-benzoimidazolyl and lH-indazolyl; wherein R6a, R6b and R6c are independently selected from hydrogen and Ci_4alkyl; wherein said phenyl, lH-pyrrolopyridin-3-yl, lH-indolyl, thiophenyl, pyridinyl, lH-l,2,4-triazolyl, 2- oxoimidazolidinyl, lH-pyrazolyl, 2-oxo-2,3-dihydro- lH-benzoimidazolyl or lH-indazolyl of R2 is optionally substituted with 1 to 3 radicals independently selected from hydroxy, halo, methyl, methoxy, amino, -O(CH2)nNR7aR7b, -S(O)2NR7aR7b, -OS(O)2NR7aR7b and - NR7aS(O)2R7b; wherein R7a and R7b are independently selected from hydrogen and Ci_4alkyl; R3 is selected from hydrogen, C]_4alkyl and biphenyl; and
R4 is selected from Ci_ioalkyl, prop-l-en-2-yl, cyclohexyl, cyclopropyl, 2-(2- oxopyrrolidin-l-yl)ethyl, oxetan-3-yl, benzhydryl, tetrahydro-2H-pyran-3-yl, tetrahydro-2H- pyran-4-yl, phenyl, tetrahydrofuran-3-yl, benzyl, (4-pentylphenyl)(phenyl)methyl and 1-(1- (2-0X0-6, 9, 12-trioxa-3-azatetradecan-14-yl)- IH- l,2,3-triazol-4-yl)ethyl; wherein said alkyl, cyclopropyl, cyclohexyl, 2-(2-oxopyrrolidin-l-yl)ethyl, oxetan-3-yl, oxetan-2-yl, benzhydryl, tetrahydro-2H-pyran-2-yl, tetrahydro-2H-pyran-3-yl, tetrahydro-2H-pyran-4-yl, phenyl, tetrahydrofuran-3-yl, tetrahydrofuran-2-yl, benzyl, (4-pentylphenyl)(phenyl)methyl or l-(l-(2-oxo-6,9,12-trioxa-3-azatetradecan-14-yl)-lH-l,2,3-triazol-4-yl)ethyl can be optionally substituted with 1 to 3 radicals independently selected from hydroxy, Ci_4alkyl and halo-substituted-C]_4alkyl; or a salt thereof.
2. The compound of claim 1 selected from Formulae Ia, Ib, Ic, Id and Ie:
Figure imgf000122_0001
Figure imgf000122_0002
in which:
L is selected from -NR5a(CH2)M- -NR53CH(C(O)OCH3)CH2-, -
NR5a(CH2)2NR5b-, -NR5a(CH2)2S-, -NR53CH2CH(CH3)CH2-, -NR53CH2CH(OH)- and - NR53CH(CH3)CH2-; wherein R53 and R5b are independently selected from hydrogen and C1- 4alkyl;
Ri is selected from hydrogen, phenyl, thiophen-2-yl, thiophen-3-yl, furan-3-yl, lH-benzo[d]imidazol-l-yl, isoquinolin-4-yl, lH-imidazo[4,5-b]pyridin-l-yl, benzo[b]thiophen-3-yl, pyrimidin-5-yl, lH-pyrazol-4-yl, pyridin-2-yl, pyridin-4-yl, IH- imidazol-1-yl, pyrrolidin-1-yl, pyrazin-2-yl, pyridin-3-yl, pyridazin-4-yl, lH-pyrrol-2-yl and thiazol-5-yl; wherein said phenyl, thiophen-2-yl, thiophen-3-yl, furan-3-yl, IH- benzo[d]imidazol-l-yl, isoquinolin-4-yl, lH-imidazo[4,5-b]pyridin-l-yl, benzo[b]thiophen- 3-yl, pyrimidin-5-yl, pyridiny-2-yl, pyridin-4-yl, lH-imidazol-1-yl, pyrrolidin-1-yl, pyrazin- 2-yl, pyridiny-3-yl, pyridazin-4-yl, lH-pyrrol-2-yl or thiazol-5-yl of Ri can be optionally substituted by 1 to 3 radicals independently selected from cyano, hydroxy, Ci_4alkyl, C1- 4alkoxy, halo, halo-substituted-Ci_4alkyl, -S(0)o-2R8a and -C(O)OR83; wherein R8a and R8b are independently selected from hydrogen and Ci_4alkyl; with the proviso that Ri and R3 are not both hydrogen;
R2 is selected from -NR6aC(O)NR6bR6c, phenyl, lH-pyrrolo[2,3-b]pyridin-3-yl, lH-pyrrolo[2,3-b]pyridin-5-yl, lH-indol-3-yl, thiophen-3-yl, pyridin-2-yl, pyridin-3-yl, pyridin-4-yl, lH-l,2,4-triazol-5-yl, 2-oxoimidazolidin-l-yl, lH-pyrazol-3-yl, lH-pyrazol-4- yl, 2-oxo-2,3-dihydro-lH-benzo[d]imidazol-5-yl and lH-indazol-3-yl; wherein R63, K(,b and R6c are independently selected from hydrogen and Ci_4alkyl; wherein said phenyl, IH- pyrrolo[2,3-b]pyridin-3-yl, lH-pyrrolo[2,3-b]pyridin-5-yl, lH-indol-3-yl, thiophen-3-yl pyridin-2-yl, pyridin-3-yl, pyridin-4-yl, lH-l,2,4-triazol-5-yl, 2-oxoimidazolidin-l-yl, IH- pyrazol-3-yl, lH-pyrazol-4-yl, 2-oxo-2,3-dihydro-lH-benzo[d]imidazol-5-yl or lH-indazol- 3-yl of R2 is optionally substituted with 1 to 3 radicals independently selected from hydroxy, halo, methoxy, amino, -OS(O)2NR7aR7b and -NR73S(O)2R?!,; wherein R7a and R7b are independently selected from hydrogen and Ci_4alkyl; R3 is selected from hydrogen, Ci_4alkyl and biphenyl; and
R4 is selected from isopropyl, methyl, ethyl, prop-l-en-2-yl, isobutyl, cyclohexyl, sec -butyl, (S)-sec-butyl, (R)-sec -butyl, l-hydroxypropan-2-yl, (S)-l-hydroxypropan-2-yl, (R)-l-hydroxypropan-2-yl, nonan-2-yl, 2-(2-oxopyrrolidin-l-yl)ethyl, oxetan-3-yl, oxetan-2- yl, benzhydryl, tetrahydro-2H-pyran-2-yl, phenyl, tetrahydrofuran-3-yl and benzyl; wherein said cyclohexyl, 2-(2-oxopyrrolidin-l-yl)ethyl, oxetan-3-yl, oxetan-2-yl, benzhydryl, tetrahydro-2H-pyran-4-yl, phenyl, tetrahydrofuran-3-yl or benzyl can be optionally substituted with 1 to 3 radicals independently selected from Ci_4alkyl and halo-substituted- C^alkyl.
3. The compounds of claim 2 in which L is selected from -NR5a(CH2)o-3-, -
NR53CH(C(O)OCH3)CH2-, -NR5a(CH2)2NR5b-, -NR5a(CH2)2S- -NR53CH2CH(CH3)CH2-, -NR53CH(CH3)CH2- and -NR53CH2CH(OH)-; wherein R53 and R5b are independently selected from hydrogen and methyl; and Ri is selected from hydrogen, phenyl, thiophen-2- yl, thiophen-3-yl, furan-3-yl, lH-benzo[d]imidazol-l-yl, isoquinolin-4-yl, lH-imidazo[4,5- b]pyridin-l-yl, benzo[b]thiophen-3-yl, pyrimidin-5-yl, lH-pyrazol-4-yl, pyridin-2-yl, pyridin-4-yl, lH-imidazol-1-yl, pyrrolidin-1-yl, pyrazin-2-yl, pyridin-3-yl, pyridazin-4-yl, lH-pyrrol-2-yl and thiazol-5-yl; wherein said phenyl, thiophen-2-yl, thiophen-3-yl, furan-3- yl, lH-benzo[d]imidazol-l-yl, isoquinolin-4-yl, lH-imidazo[4,5-b]pyridin-l-yl, benzo[b]thiophen-3-yl, pyrimidin-5-yl, pyridiny-2-yl, pyridin-4-yl, lH-imidazol-1-yl, pyrrolidin-1-yl, pyrazin-2-yl, pyridiny-3-yl, pyridazin-4-yl, lH-pyrrol-2-yl or thiazol-5-yl of R] can be optionally substituted by 1 to 3 radicals independently selected from cyano, hydroxy, Ci_4alkyl, Ci_4alkoxy, halo, halo-substituted-Ci_4alkyl, -S(0)o-2R8a and -C(O)OR83; wherein R83 and R8b are independently selected from hydrogen and C]_4alkyl; with the proviso that R] and R3 are not both hydrogen.
4. The compounds of claim 3 in which L is selected from -NR5a(CH2)i-3-, - NR53CH(C(O)OCH3)CH2-, -NR53(CH2)2NR5b-, -NR53(CH2)2S-, -NR53CH2CH(CH3)CH2-, -NR53CH(CH3)CH2- and -NR53CH2CH(OH)-; wherein R53 and R5b are independently selected from hydrogen and methyl.
5. The compound of claim 4 in which R2 is selected from urea, phenyl, lH-indol-2-yl, IH- indol-3-yl, thiophen-3-yl, piperidin-1-yl, pyridin-2-yl, pyridin-3-yl, pyridin-4-yl, 1H-1,2,4- triazol-3-yl, lH-l,2,4-triazol-5-yl, 2-oxoimidazolidin-l-yl, lH-pyrazol-3-yl, lH-pyrazol-4- yl, 2-oxo-2,3-dihydro-lH-benzo[d]imidazol-5-yl, lH-benzo[d]imidazol-5-yl and IH- imidazol-4-yl; wherein said phenyl, lH-indol-2-yl, lH-indol-3-yl, thiophen-3-yl, piperidin-1- yl, pyridin-2-yl, pyridin-3-yl, pyridin-4-yl, lH-l,2,4-triazol-3-yl, lH-l,2,4-triazol-5-yl, 2- oxoimidazolidin-1-yl, lH-pyrazol-3-yl, lH-pyrazol-4-yl, 2-oxo-2,3-dihydro-lH- benzo[d]imidazol-5-yl or lH-benzo[d]imidazol-5-yl of R2 is optionally substituted with hydroxy, methoxy, methyl, halo, amino and amino-sulfonyl.
6. The compound of claim 5 in which R3 is selected from hydrogen, methyl and biphenyl; and R4 is selected from isopropyl, methyl, ethyl, prop-l-en-2-yl, isobutyl, cyclohexyl, sec -butyl, (S)-sec -butyl, (R)-sec-butyl, l-hydroxypropan-2-yl, (S)-l-hydroxypropan-2-yl, (R)-I- hydroxypropan-2-yl, nonan-2-yl, 2-(2-oxopyrrolidin-l-yl)ethyl, oxetan-3-yl, oxetan-2-yl, benzhydryl, tetrahydro-2H-pyran-2-yl, phenyl, tetrahydrofuran-3-yl and benzyl; wherein said cyclohexyl, 2-(2-oxopyrrolidin-l-yl)ethyl, oxetan-3-yl, oxetan-2-yl, benzhydryl, tetrahydro- 2H-pyran-4-yl, phenyl, tetrahydrofuran-3-yl or benzyl can be optionally substituted with 1 to 3 radicals independently selected from methyl and trifluoromethyl.
7. The compound of claim 6 selected from: 4-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl- 9H-purin-6-ylamino)ethyl)phenol; 4-(2-(2-(benzo[b]thiophen-3-yl)-9-sec-butyl-9H- purin-6-ylamino)ethyl)phenol; 4-(2-(9-benzhydryl-2-(benzo[b]thiophen-3-yl)-9H-purin- 6-ylamino)ethyl)phenol; 4-(2-(2-(benzo[b]thiophen-3-yl)-9-(tetrahydro-2H-pyran-3-yl)- 9H-purin-6-ylamino)ethyl)phenol; 4-(2-(9-isopropyl-2-(thiophen-2-yl)-9H-purin-6- ylamino)ethyl)phenol; 4-(2-(2-(benzo[b]thiophen-3-yl)-9-(4-(trifluoromethyl)benzyl)- 9H-purin-6-ylamino)ethyl)phenol; 4-(2-(2-(benzo[b]thiophen-3-yl)-9-isobutyl-9H-purin- 6-ylamino)ethyl)phenol; 4-(2-(2-(benzo[b]thiophen-3-yl)-9-methyl-9H-purin-6- ylamino)ethyl)phenol; 4-(2-(2-(benzo[b]thiophen-3-yl)-9-(4-methylbenzyl)-9H-purin-6- ylamino)ethyl)phenol; N-(2-(lH-indol-3-yl)ethyl)-2-(benzo[b]thiophen-3-yl)-9- isopropyl-9H-purin-6-amine; 2-(benzo[b]thiophen-3-yl)-9-isopropyl-N-(2-(thiophen-3- yl)ethyl)-9H-purin-6-amine; 3-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6- ylamino)ethyl)phenol; 2-(benzo[b]thiophen-3-yl)-N-(4-fluorophenethyl)-9-isopropyl-9H- purin-6-amine; N-(4-aminophenethyl)-2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin- 6-amine; 4-(2-(9-isopropyl-2-(pyrimidin-5-yl)-9H-purin-6-ylamino)ethyl)phenol; 4-(2- (9-isopropyl-2-(pyridin-3-yl)-9H-purin-6-ylamino)ethyl)phenol; 4-(2-(9-isopropyl-2- phenyl-9H-purin-6-ylamino)ethyl)phenol; 4-(2-(9-isopropyl-2-(thiophen-3-yl)-9H-purin- 6-ylamino)ethyl)phenol; 4-(2-(2-(furan-3-yl)-9-isopropyl-9H-purin-6- ylamino)ethyl)phenol; 2-(benzo[b]thiophen-3-yl)-N-(4-fluorophenethyl)-9-phenyl-9H- purin-6-amine; N-benzyl-8-(biphenyl-4-yl)-9-isopropyl-9H-purin-6-amine; 4-(2-(2- (benzo[b]thiophen-3-yl)-9-(nonan-2-yl)-9H-purin-6-ylamino)ethyl)phenol; N-(2-(lH- indol-3-yl)ethyl)-2-(benzo[b]thiophen-3-yl)-9-sec-butyl-9H-purin-6-amine; 3-(2-(2- (benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)-lH-indol-5-yl 5- ((3aS,4S,6aR)-2-oxohexahydro-lH-thieno[3,4-d]imidazol-4-yl)pentanoate; N-(2-(2-(2- (2-(4-(l-(2-(benzo[b]thiophen-3-yl)-6-(4-hydroxyphenethylamino)-9H-purin-9-yl)ethyl)- lH-l,2,3-triazol-l-yl)ethoxy)ethoxy)ethoxy)ethyl)acetamide; 4-(2-(9-isopropyl-2- (pyridin-4-yl)-9H-purin-6-ylamino)ethyl)phenol; ethyl 5-(6-(4-hydroxyphenethylamino)- 9-isopropyl-9H-purin-2-yl)nicotinate; ethyl 5-(6-(4-hydroxyphenethylamino)-9- isopropyl-9H-purin-2-yl)nicotinate; 4-(2-(2-(6-fluoropyridin-3-yl)-9-isopropyl-9H-purin- 6-ylamino)ethyl)phenol; 4-(2-(9-isopropyl-2-(4-methylpyridin-3-yl)-9H-purin-6- ylamino)ethyl)phenol; 5-(6-(4-hydroxyphenethylamino)-9-isopropyl-9H-purin-2- yl)nicotinonitrile; 4-(2-(9-isopropyl-2-(pyrrolidin-l-yl)-9H-purin-6- ylamino)ethyl)phenol; 4-(2-(2-(lH-imidazol-l-yl)-9-isopropyl-9H-purin-6- ylamino)ethyl)phenol; 4-(2-(9-isopropyl-2-(pyridazin-4-yl)-9H-purin-6- ylamino)ethyl)phenol; 4-(2-(9-isopropyl-2-(pyrazin-2-yl)-9H-purin-6- ylamino)ethyl)phenol; 4-(2-(9-isopropyl-2-(pyridin-2-yl)-9H-purin-6- ylamino)ethyl)phenol; 4-(2-(9-isopropyl-2-(5-(methylsulfonyl)pyridin-3-yl)-9H-purin-6- ylamino)ethyl)phenol; 4-(2-(9-isopropyl-2-(5-methylpyridin-3-yl)-9H-purin-6- ylamino)ethyl)phenol; 4-(2-(2-(4-chloropyridin-3-yl)-9-isopropyl-9H-purin-6- ylamino)ethyl)phenol; 4-(2-(2-(5-fluoropyridin-3-yl)-9-isopropyl-9H-purin-6- ylamino)ethyl)phenol; 4-(2-(9-isopropyl-2-(l-methyl-lH-pyrazol-4-yl)-9H-purin-6- ylamino)ethyl)phenol; 4-(2-(9-isopropyl-2-(pyridin-3-yl)-9H-purin-6-ylamino)ethyl)-2- methoxyphenol; 4-(2-(9-isopropyl-2-(pyridin-3-yl)-9H-purin-6-ylamino)ethyl)-2- methoxyphenol; N-[2-(6-methoxy-lH-indol-3-yl)ethyl]-9-(propan-2-yl)-2-(pyridin-3-yl)- 9H-purin-6-amine; N-[2-(5-methyl-lH-indol-3-yl)ethyl]-9-(propan-2-yl)-2-(pyridin-3- yl)-9H-purin-6-amine; l-(2-{ [9-(propan-2-yl)-2-(pyridin-3-yl)-9H-purin-6- yl]amino}ethyl)imidazolidin-2-one; N-(2-{ [9-(propan-2-yl)-2-(pyridin-3-yl)-9H-purin-6- yl]amino}ethyl)pyridin-2-amine; 9-(propan-2-yl)-N-[3-(lH-pyrazol-4-yl)propyl]-2- (pyridin-3-yl)-9H-purin-6-amine; N-{2-[(3-methyl-lH-l,2,4-triazol-5-yl)sulfanyl]ethyl}- 9-(propan-2-yl)-2-(pyridin-3-yl)-9H-purin-6-amine; l-(2-{ [2-(l-benzothiophen-3-yl)-9- (propan-2-yl)-9H-purin-6-yl]amino}ethyl)imidazolidin-2-one; N-[2-(5-amino- IH- 1,2,4- triazol-3-yl)ethyl]-2-(l-benzothiophen-3-yl)-9-(propan-2-yl)-9H-purin-6-amine; N-(2- { [2-(l-benzothiophen-3-yl)-9-(propan-2-yl)-9H-purin-6-yl]amino}ethyl)pyridin-2- amine; 2-(l-benzothiophen-3-yl)-9-(propan-2-yl)-N-[3-(lH-pyrazol-4-yl)propyl]-9H- purin-6-amine; 2-(l-benzothiophen-3-yl)-N-[3-(3,5-dimethyl-lH-pyrazol-4-yl)propyl]-9- (propan-2-yl)-9H-purin-6-amine; (2-{ [2-(l-benzothiophen-3-yl)-9-(propan-2-yl)-9H- purin-6-yl]amino}ethyl)urea; 5-({ [2-(l-benzothiophen-3-yl)-9-(propan-2-yl)-9H-purin-6- yl]amino}methyl)-2,3-dihydro-lH-l,3-benzodiazol-2-one; N-[2-(lH-indol-3-yl)ethyl]-9- (propan-2-yl)-2-(pyridin-3-yl)-9H-purin-6-amine; N-(4-(2-(9-isopropyl-2-(pyridin-3-yl)- 9H-purin-6-ylamino)ethyl)phenyl)methane-sulfonamide; 4-(2-(2-(pyridin-3-yl)-9- (tetrahydrofuran-3-yl)-9H-purin-6-ylamino)ethyl)phenol; 4-(2-(9-isopropyl-2-(pyridin-3- yl)-9H-purin-6-ylamino)propyl)phenol; 4-(2-(9-(oxetan-3-yl)-2-(pyridin-3-yl)-9H-purin- 6-ylamino)ethyl)phenol; 5-(6-(4-hydroxyphenethylamino)-9-isopropyl-9H-purin-2-yl)- N-methylnicotinamide; 4-(2-(9-(l-hydroxypropan-2-yl)-2-(pyridin-3-yl)-9H-purin-6- ylamino)ethyl)phenol; 4-(2-(9-isopropyl-2-(pyridin-3-yl)-9H-purin-6- ylamino)ethyl)phenyl sulfamate; 4-(2-(2-(2-fluoropyridin-3-yl)-9-isopropyl-9H-purin-6- ylamino)ethyl)phenol; 4-(2-(9-isopropyl-2-(l-methyl-lH-pyrrol-2-yl)-9H-purin-6- ylamino)ethyl)phenol; 4-(2-(9-isopropyl-2-(thiazol-5-yl)-9H-purin-6- ylamino)ethyl)phenol; 4-(2-(2-(lH-benzo[d]imidazol-l-yl)-9-isopropyl-9H-purin-6- ylamino)ethyl)phenol; 4-(2-(2-(2,4-dimethyl- lH-imidazol- l-yl)-9-isopropyl-9H-purin-6- ylamino)ethyl)phenol; 4-(2-(9-isopropyl-2-(2-methyl-lH-imidazol-l-yl)-9H-purin-6- ylamino)ethyl)phenol; 5-(9-sec-butyl-6-(4-hydroxy-3-methylphenethylamino)-9H-purin- 2-yl)nicotinonitrile; N-(2-(lH-pyrrolo[2,3-b]pyridin-5-yl)ethyl)-9-isopropyl-2-(pyridin- 3-yl)-9H-purin-6-amine; 9-isopropyl-N-(2-(5-methyl-lH-pyrazol-3-yl)ethyl)-2-(pyridin- 3-yl)-9H-purin-6-amine; 4-(2-(2-(5-fluoropyridin-3-yl)-9-(oxetan-3-yl)-9H-purin-6- ylamino)ethyl)phenol; 4-(2-(2-(5-chloropyridin-3-yl)-9-isopropyl-9H-purin-6- ylamino)ethyl)phenol; 4-(2-(9-isopropyl-2-(5-(trifluoromethyl)pyridin-3-yl)-9H-purin-6- ylamino)ethyl)phenol; 5-(6-(2-(lH-indol-3-yl)ethylamino)-9-sec-butyl-9H-purin-2- yl)nicotinonitrile; N-(2-(lH-indol-3-yl)ethyl)-9-sec-butyl-2-(5-methylpyridin-3-yl)-9H- purin-6-amine; (R)-N-(2-(lH-indol-3-yl)ethyl)-9-sec-butyl-2-(5-fluoropyridin-3-yl)-9H- purin-6-amine; (S)-N-(2-(lH-indol-3-yl)ethyl)-9-sec-butyl-2-(5-fluoropyridin-3-yl)-9H- purin-6-amine; N-(2-(lH-indol-3-yl)ethyl)-9-sec-butyl-2-(5-fluoropyridin-3-yl)-9H- purin-6-amine; (R)-N-(2-(lH-indol-3-yl)ethyl)-9-sec-butyl-2-(5-methylpyridin-3-yl)-9H- purin-6-amine; (S)-N-(2-(lH-indol-3-yl)ethyl)-9-sec-butyl-2-(5-methylpyridin-3-yl)-9H- purin-6-amine; 5-(6-(4-hydroxyphenethylamino)-9-(oxetan-3-yl)-9H-purin-2- yl)nicotinonitrile; 4-(2-(6-(5-fluoropyridin-3-yl)-l-isopropyl-lH-pyrazolo[3,4- d]pyrimidin-4-ylamino)ethyl)phenol; 4-(2-(6-(benzo[b]thiophen-3-yl)-l-isopropyl-lH- pyrazolo[3,4-d]pyrimidin-4-ylamino)ethyl)phenol; (R)-4-(2-(2-(5-fluoropyridin-3-yl)-9- (tetrahydrofuran-3-yl)-9H-purin-6-ylamino)ethyl)phenol; 4-(2-(9-isopropyl-2-(pyridin-3- yl)-9H-purin-6-ylamino)ethyl)-3-methylphenol; 5-(6-(4-hydroxyphenethylamino)-9- isopropyl-9H-purin-2-yl)picolinonitrile; 3-(6-(4-hydroxyphenethylamino)-9-isopropyl- 9H-purin-2-yl)isonicotinonitrile; 4-(2-(2-(5-fluoropyridin-3-yl)-7-isopropyl-7H- pyrrolo[2,3-d]pyrimidin-4-ylamino)ethyl)phenol; 3-(6-(4-hydroxyphenethylamino)-9- isopropyl-9H-purin-2-yl)picolinonitrile; 4-(2-(9-isopropyl-2-(6-methylpyridin-3-yl)-9H- purin-6-ylamino)ethyl)phenol; 4-(2-(9-isopropyl-2-(isoquinolin-4-yl)-9H-purin-6- ylamino)ethyl)phenol; 2-chloro-4-(2-(9-isopropyl-2-(pyridin-3-yl)-9H-purin-6- ylamino)ethyl)phenol; 3-fluoro-4-(2-(9-isopropyl-2-(pyridin-3-yl)-9H-purin-6- ylamino)ethyl)phenol; N-(2-(5-chloro-lH-indol-3-yl)ethyl)-9-isopropyl-2-(pyridin-3-yl)- 9H-purin-6-amine; N-(2-(5-fluoro-lH-indol-3-yl)ethyl)-9-isopropyl-2-(pyridin-3-yl)-9H- purin-6-amine; 4-(2-(9-isopropyl-2-(pyridin-3-yl)-9H-purin-6-ylamino)ethyl)-2- methylphenol; 4-(2-(2-(benzo[b]thiophen-3-yl)-9-(oxetan-3-yl)-9H-purin-6- ylamino)ethyl)phenol; (S)-4-(2-(2-(benzo[b]thiophen-3-yl)-9-(tetrahydrofuran-3-yl)-9H- purin-6-ylamino)ethyl)phenol; (R)-4-(2-(2-(benzo[b]thiophen-3-yl)-9-(tetrahydrofuran- 3-yl)-9H-purin-6-ylamino)ethyl)phenol; 2-(6-(2-(lH-indol-3-yl)ethylamino)-2-(5- fluoropyridin-3-yl)-9H-purin-9-yl)propan-l-ol; (R)-2-(6-(2-(lH-indol-3-yl)ethylamino)- 2-(5-fluoropyridin-3-yl)-9H-purin-9-yl)propan-l-ol; (S)-2-(6-(2-(lH-indol-3- yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin-9-yl)propan-l-ol; (R)-N-(2-(lH-indol- 3-yl)ethyl)-2-(5-fluoropyridin-3-yl)-9-(tetrahydrofuran-3-yl)-9H-purin-6-amine; 4-(2-(2- (3H-imidazo[4,5-b]pyridin-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phenol; 4-(2-(2- (lH-imidazo[4,5-b]pyridin-l-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phenol; 4-(2-(6- (5-fluoropyridin-3-yl)- 1-isopropyl- lH-imidazo[4,5-c]pyridin-4-ylamino)ethyl)phenol; 4- (2-(2-(4,5-dimethyl-lH-imidazol-l-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phenol; 2- (5-fluoropyridin-3-yl)-9-isopropyl-N-(2-(pyridin-3-yl)ethyl)-9H-purin-6-amine; 4-(2-(2- (5-fluoropyridin-3-yl)-9-isopropyl-9H-purin-6-ylamino)-l-hydroxyethyl)phenol; 2-(5- fluoropyridin-3-yl)-9-isopropyl-N-(2-(6-methoxy-lH-indol-3-yl)ethyl)-9H-purin-6- amine; N-(2-(lH-indol-3-yl)ethyl)-2-(5-fluoropyridin-3-yl)-9-isopropyl-9H-purin-6- amine; 2-(5-fluoropyridin-3-yl)-9-isopropyl-N-(2-(5-methoxy-lH-indol-3-yl)ethyl)-9H- purin-6-amine; N-(2-(lH-indol-3-yl)ethyl)-2-(5-fluoropyridin-3-yl)-9-(prop-l-en-2-yl)- 9H-purin-6-amine; 5-(2-(2-(5-fluoropyridin-3-yl)-9-isopropyl-9H-purin-6- ylamino)ethyl)pyridin-2-ol; N-(2-(lH-pyrrolo[2,3-b]pyridin-3-yl)ethyl)-2-(5- fluoropyridin-3-yl)-9-isopropyl-9H-purin-6-amine; N-(2-(6-(2-(diethylamino)ethoxy)- lH-indol-3-yl)ethyl)-2-(5-fluoropyridin-3-yl)-9-isopropyl-9H-purin-6-amine; 4-(2-(5-(5- fluoropyridin-3-yl)-3-isopropyl-3H-imidazo[4,5-b]pyridin-7-ylamino)ethyl)phenol; N- (2-(lH-indol-3-yl)ethyl)-9-sec-butyl-2-(2-methyl-lH-imidazol-l-yl)-9H-purin-6-amine; 4-(2-(2-(2-ethyl-lH-imidazol-l-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phenol; 4- (2- (9-isopropyl-2-(2-propyl-lH-imidazol-l-yl)-9H-purin-6-ylamino)ethyl)phenol; 3- (2- (2- (5-fluoropyridin-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)-lH-indol-6-ol; N-(2-(lH- indol-3-yl)ethyl)-9-isopropyl-2-(5-methylpyridin-3-yl)-9H-purin-6-amine; N-(2-(lH- indol-3-yl)ethyl)-9-isopropyl-2-(2-methyl- lH-imidazol- l-yl)-9H-purin-6-amine; 2-(5- fluoropyridin-3-yl)-9-isopropyl-N-(2-(7-methyl-lH-indol-3-yl)ethyl)-9H-purin-6-amine; N-(2-(lH-indol-3-yl)ethyl)-2-(5-fluoropyridin-3-yl)-9-(oxetan-3-yl)-9H-purin-6-amine; N-(2-(lH-indol-3-yl)ethyl)-2-(5-methylpyridin-3-yl)-9-(oxetan-3-yl)-9H-purin-6-amine; N-(2-(6-fluoro-lH-indol-3-yl)ethyl)-2-(5-fluoropyridin-3-yl)-9-isopropyl-9H-purin-6- amine; 2-(5-fluoropyridm-3-yl)-9-isopropyl-N-(2-(6-methyl-lH-mdol-3-yl)ethyl)-9H- purin-6-amine; 2-(5-fluoropyridin-3-yl)-9-isopropyl-N-(2-(2-methyl-lH-indol-3- yl)ethyl)-9H-purin-6-amine; N-(2-(4-fluoro-lH-indol-3-yl)ethyl)-2-(5-fluoropyridin-3- yl)-9-isopropyl-9H-purin-6-amine; N-(2-(7-fluoro-lH-indol-3-yl)ethyl)-2-(5- fluoropyridin-3-yl)-9-isopropyl-9H-purin-6-amine; 2-(5-fluoropyridin-3-yl)-9-isopropyl- N-(2-(4-methyl-lH-indol-3-yl)ethyl)-9H-purin-6-amine; 4-(2-(2-(benzo[b]thiophen-3- yl)-7-isopropyl-7H-pyrrolo[2,3-d]pyrimidin-4-ylamino)ethyl)phenol; 9-isopropyl-2- (pyridin-3-yl)-N-(2-(pyridin-4-yl)ethyl)-9H-purin-6-amine; N-(2-(lH-pyrrolo[2,3- b]pyridin-5-yl)ethyl)-9-isopropyl-2-(pyridin-3-yl)-9H-purin-6-amine; 4-(2-(2-(5- fluoropyridin-3-yl)-9-(l-hydroxypropan-2-yl)-9H-purin-6-ylamino)ethyl)-2- methylphenol; 4-(2-(2-(benzo[b]thiophen-3-yl)-9-cyclohexyl-9H-purin-6- ylamino)ethyl)phenol; 4-(2-(9-isopropyl-2-(thiophen-3-yl)-9H-purin-6- ylamino)ethyl)phenol; and l-(2-(2-(benzo[b]thiophen-3-yl)-6-(4- hydroxyphenethylamino)-9H-purin-9-yl)ethyl)pyrrolidin-2-one; or a salt thereof.
8. A method of increasing the number of stem and progenitor cells; said method comprising contacting the stem cells with an agent capable of antagonizing the activity and/or expression of aryl hydrocarbon receptor and/or a down-stream effector of aryl hydrocarbon receptor pathway.
9. The method of Claim 8, wherein said agent capable of antagonizing the activity and/or expression of aryl hydrocarbon receptor and/or a down-stream effector of aryl hydrocarbon receptor pathway is selected from:
(i) an organic compound,
(ii) a small interfering RNA molecule inhibiting the expression of aryl hydrocarbon receptor, and,
(iii) an antisense oligonucleotide inhibiting the expression of aryl hydrocarbon receptor.
10. The method of Claim 8, wherein said agent capable of antagonizing the activity and/or expression of aryl hydrocarbon receptor is a compound of claim 1.
11. The method of claim 8 in which the method is carried out in vivo, in vitro or ex vivo.
12. The method of claim 11 in which the stem cells are human.
13. The method of claim 12 in which the stem cells are derived from bone marrow.
14. The method of claim 12 in which the stem cells are derived from umbilical cord blood.
15. The methods of claim 13 or 14 in which the stem cells are hematopoietic stem cells.
16. A method of expanding hematopoietic stem cells, comprising (a) providing a starting cell population comprising hematopoietic stem cells and (b) culturing said starting cell population ex vivo in presence of an agent capable of antagonizing the activity and/or expression of aryl hydrocarbon receptor and/or a down-stream effector of aryl hydrocarbon receptor pathway, under suitable conditions for expanding hematopoietic stem cells.
17. The method of Claim 16, wherein said agent capable of down-regulating the activity of aryl hydrocarbon receptor is selected from: (i) an organic compound,
(ii) a small interfering RNA molecule inhibiting the expression of aryl hydrocarbon receptor, and,
(iii) an antisense oligonucleotide inhibiting the expression of aryl hydrocarbon receptor.
18. The method of Claim 17, wherein said organic compound is 4-(2-(2-(benzo[b]thiophen-3- yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phenol.
19. The method of Claim 16, wherein a down-stream effector of aryl hydrocarbon receptor pathway is selected from CyplBl, CyplAl, Beta catenin, AHRR, STAT5 and STATl.
20. The method of Claim 16, wherein said agent capable of down-regulating the activity of aryl hydrocarbon receptor is a compound of any one of claims 1-7.
21. The method of Claim 16 or 18, wherein said starting cell population is enriched in CD34+ cells.
22. The method of Claim 16 or 18, wherein said starting cell population is derived from umbilical cord blood cells.
23. The method of Claim 16 or 18, wherein said starting cell population is derived from one or two cord blood units, preferably one cord blood unit.
24. The method of Claim 16 or 18, wherein said starting cell population is derived from mobilized peripheral blood cells.
25. The method of claim 24 wherein the mobilized peripheral blood cells are obtained from a single mammalian subject.
26. The method of Claim 16 or 18, wherein said starting cell population essentially consists of CD34+ cells purified from one or two cord blood units, preferably one cord blood unit.
27. The method of Claim 16 or 18, wherein said starting cell population essentially consists of CD34+ cells purified from one or two cord blood units using affinity chromatography columns or magnetic beads comprising CD34 antigen binding antibody fragments.
28. The method of Claim 16 or 18, wherein said conditions for hematopoietic stem cell expansion include culturing said starting cell population in presence of a sufficient amount of IL6, Flt3-L, TPO and SCF and a compound of claim 1.
29. The method of Claim 16 or 18, wherein said compound of Claim 1 is administered in the cell culture medium at a concentration between IpM and lOμM, for example between lOpM and lOOμM.
30. The method of Claim 16 or 18, wherein said starting cell population essentially consists of CD34+ cells purified from one or two cord blood units, preferably one cord blood unit, and said starting cell population is cultured in presence of a compound of claim 1 from about 3 days to about 90 days, preferably between 7 to 35 days.
31. The method of Claim 16 or 18, wherein said starting cell population is cultured in presence of a compound of Claim 1 during a time sufficient for a 10 to 50000 fold expansion of CD34+ cells.
32. A kit for expanding hematopoietic stem cells, comprising a compound of Claim 1 and instructions for use in a method of Claim 16 and, optionally, one or more of the following: cytokines, growth factors and cell growth media.
33. The kit according to Claim 32, wherein said one or more cytokines or growth factors is selected from the group consisting of IL6, Flt3-L, SCF and TPO .
34. A cell population with expanded hematopoietic stem cells, obtainable or as obtained by the method of Claim 16 or 18.
35. A composition comprising the cell population of Claim 34 resuspended in a pharmaceutically acceptable medium suitable for administration to a mammalian host.
36. A composition comprising a cell population with expanded hematopoietic stem cells derived from one or two cord blood units, preferably one cord blood unit, wherein said composition contains a total amount of cells of at least 105 cells, 107 cells, 108 cells or 109 cells, and wherein between 20-100% of total cells are CD34+ cells.
37. The composition of Claim 36, wherein said composition contains between 0.1%-40% of total cells expressing CD34 and Thyl markers; 20-80% of total cells expressing CD34 and CD45RA markers; 10-95% of cells being CD38+; and 5-70% of cells being CD133+.
38. The composition according to any one of Claims 35-37 for use in allogeneic hematopoietic stem cell transplantation in mammalian subject.
39. The composition according to any one of Claims 35-37, for treating a patient suffering from impaired hematopoiesis.
40. The composition of Claim 39, wherein the patient is suffering from an inherited immunodeficient disease, an autoimmune disorder or hematopoietic disorder.
41. The composition of Claim 40, wherein said hematopoietic disorder is selected from Acute myeloid leukemia, Acute lymphoblastic leukemia, Chronic myeloid leukemia, Chronic lymphocytic leukemia, Myeloproliferative disorders, Myelodysplastic syndromes, Multiple myeloma, Non-Hodgkin lymphoma, Hodgkin disease, Aplastic anemia, Pure red cell aplasia, Paroxysmal nocturnal hemoglobinuria, Fanconi anemi, Thalassemia major, Sickle cell anemia, Severe combined immunodeficiency, Wiskott-Aldrich syndrome, Hemophagocytic lymphohistiocytosis and inborn errors of metabolism.
42. The composition of any on Claims 35-37, wherein said composition is appropriate for intravenous infusion and said composition comprises at least 104 cells / kg transfused in the patient, preferably between 105cells / kg and 109 cells / kg.
43. A method of treating an inherited immunodeficient disease, an autoimmune disease and/or a hematopoietic disorder comprising administration to a patient in need of such treatment hematopoietic stem cells expanded by an agent capable of down-regulating the activity and/or expression of aryl hydrocarbon receptor and/or a down-stream effector of AHR pathway.
44. The method of claim 43, wherein said agent capable of down-regulating the activity and/or expression of aryl hydrocarbon receptor is a compound of claim 1.
45. The method of claim 43, wherein the administration is an autologous transplantation and the hematopoietic disorder is selected from Multiple myeloma, Non-Hodgkin lymphoma, Hodgkin disease, Acute myeloid leukemia, Neuroblastoma, Germ cell tumors, Autoimmune disorders and Amyloidosis.
46. The method of claim 45 wherein the autoimmune disorders are selected from Systemic lupus erythematosus and systemic sclerosis.
47. The method of claim 46 wherein the administration is an allogeneic transplantation and the hematopoietic disorder is selected from Acute myeloid leukemia, Acute lymphoblastic leukemia, Chronic myeloid leukemia, Chronic lymphocytic leukemia, Myeloproliferative disorders, Myelodysplastic syndromes, Multiple myeloma, Non- Hodgkin lymphoma, Hodgkin disease, Aplastic anemia, Pure red cell aplasia, Paroxysmal nocturnal hemoglobinuria, Fanconi anemi, Thalassemia major, Sickle cell anemia, Severe combined immunodeficiency, Wiskott-Aldrich syndrome, Hemophagocytic lymphohistiocytosis and inborn errors of metabolism.
8. The method of claim 47 wherein the inborn errors of metabolism are selected from mucopolysaccharidosis, Gaucher disease, metachromatic leukodystrophies and adrenoleukodystrophies .
PCT/US2009/062646 2008-10-30 2009-10-29 Compounds that expand hematopoietic stem cells WO2010059401A2 (en)

Priority Applications (25)

Application Number Priority Date Filing Date Title
PL09748626T PL2350078T3 (en) 2008-10-30 2009-10-29 Compounds that expand hematopoietic stem cells
SI200931987T SI2350078T1 (en) 2008-10-30 2009-10-29 Compounds that expand hematopoietic stem cells
KR1020117012091A KR101434719B1 (en) 2008-10-30 2009-10-29 Compounds that expand hematopoietic stem cells
LTEP09748626.0T LT2350078T (en) 2008-10-30 2009-10-29 Compounds that expand hematopoietic stem cells
MX2011004593A MX347834B (en) 2008-10-30 2009-10-29 Compounds that expand hematopoietic stem cells.
AU2009317898A AU2009317898B2 (en) 2008-10-30 2009-10-29 Compounds that expand hematopoietic stem cells
CA2740589A CA2740589C (en) 2008-10-30 2009-10-29 Compounds that expand hematopoietic stem cells
BRPI0921799A BRPI0921799B8 (en) 2008-10-30 2009-10-29 compounds that expand hematopoietic stem cells
EA201100663A EA019872B1 (en) 2008-10-30 2009-10-29 Compounds that expand hematopoietic stem cells
EP09748626.0A EP2350078B1 (en) 2008-10-30 2009-10-29 Compounds that expand hematopoietic stem cells
NZ592435A NZ592435A (en) 2008-10-30 2009-10-29 Compounds that expand hematopoietic stem cells
CN200980143651.3A CN102203096B (en) 2008-10-30 2009-10-29 Compounds that expand hematopoietic stem cells
UAA201105290A UA103206C2 (en) 2008-10-30 2009-10-29 Compounds that expand hematopoietic stem cells
JP2011534784A JP5390626B2 (en) 2008-10-30 2009-10-29 Compounds that increase hematopoietic stem cells
ES09748626T ES2736728T3 (en) 2008-10-30 2009-10-29 Compounds that expand hematopoietic stem cells
EP19164770.0A EP3524604B1 (en) 2008-10-30 2009-10-29 Expanded hematopoietic stem cells from cord blood and their therapeutic use
CU2011000096A CU24495B1 (en) 2008-10-30 2009-10-29 COMPOUNDS THAT EXPAND HEMATOPOIETIC CELLS AND EX VIVO METHOD TO EXPAND HEMATOPOIETIC CELLS
RS20190863A RS59027B1 (en) 2008-10-30 2009-10-29 Compounds that expand hematopoietic stem cells
DK09748626.0T DK2350078T3 (en) 2008-10-30 2009-10-29 CONNECTIONS RELATING TO HUMATOPOITIC STAMPS
TN2011000174A TN2011000174A1 (en) 2008-10-30 2011-04-13 Compounds that expand hematopoietic stem cells
IL212368A IL212368A (en) 2008-10-30 2011-04-14 Compounds that expand hematopoietic stem cells, in vitro methods for increasing the number of stem and progenitor cells, cell population comprising expanded hematopoietic stem cells, compositions comprising same and use of said cells in the manufacture of medicaments
SM201100024T SMP201100024B (en) 2008-10-30 2011-05-13 Compounds that expand hematopoietic stem cells
MA33889A MA32828B1 (en) 2008-10-30 2011-05-25 COMPOUNDS THAT PROMOTE THE DEVELOPMENT OF HEMATOPOIETIC STEM CELLS
HRP20191238 HRP20191238T1 (en) 2008-10-30 2019-07-10 Compounds that expand hematopoietic stem cells
CY20191100765T CY1121857T1 (en) 2008-10-30 2019-07-17 COMPOUNDS THAT EXPAND HEMAPOPOIETIC STEM CELLS

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US10982108P 2008-10-30 2008-10-30
US61/109,821 2008-10-30
US24276509P 2009-09-15 2009-09-15
US61/242,765 2009-09-15

Publications (2)

Publication Number Publication Date
WO2010059401A2 true WO2010059401A2 (en) 2010-05-27
WO2010059401A3 WO2010059401A3 (en) 2010-09-23

Family

ID=42111339

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2009/062646 WO2010059401A2 (en) 2008-10-30 2009-10-29 Compounds that expand hematopoietic stem cells

Country Status (41)

Country Link
US (4) US8927281B2 (en)
EP (2) EP2350078B1 (en)
JP (1) JP5390626B2 (en)
KR (1) KR101434719B1 (en)
CN (1) CN102203096B (en)
AR (1) AR074063A1 (en)
AU (1) AU2009317898B2 (en)
BR (1) BRPI0921799B8 (en)
CA (3) CA2740589C (en)
CL (2) CL2009002010A1 (en)
CO (1) CO6410309A2 (en)
CR (1) CR20110192A (en)
CU (1) CU24495B1 (en)
CY (1) CY1121857T1 (en)
DK (1) DK2350078T3 (en)
DO (1) DOP2011000114A (en)
EA (1) EA019872B1 (en)
EC (1) ECSP11011090A (en)
ES (2) ES2948567T3 (en)
GE (1) GEP20146053B (en)
HN (1) HN2011001195A (en)
HR (1) HRP20191238T1 (en)
HU (1) HUE044802T2 (en)
IL (1) IL212368A (en)
JO (1) JO3593B1 (en)
LT (1) LT2350078T (en)
MA (1) MA32828B1 (en)
MX (1) MX347834B (en)
MY (1) MY155137A (en)
NI (1) NI201100083A (en)
NZ (1) NZ592435A (en)
PE (1) PE20100362A1 (en)
PL (1) PL2350078T3 (en)
PT (1) PT2350078T (en)
RS (1) RS59027B1 (en)
SI (1) SI2350078T1 (en)
SM (1) SMP201100024B (en)
SV (1) SV2011003888A (en)
TN (1) TN2011000174A1 (en)
TW (1) TWI532486B (en)
WO (1) WO2010059401A2 (en)

Cited By (34)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012102937A3 (en) * 2011-01-25 2012-11-29 Irm Llc Benz imidazole compounds that expand hematopoietic stem cells
WO2013019857A3 (en) * 2011-08-01 2013-06-06 Alnylam Pharmaceuticals, Inc. Method for improving the success rate of hematopoietic stem cell transplants
WO2013086436A1 (en) * 2011-12-08 2013-06-13 Fred Hutchinson Cancer Research Center Compositions and methods for enhanced generation of hematopoietic stem/progenitor cells
WO2013110198A1 (en) 2012-01-27 2013-08-01 Université de Montréal Pyrimido[4,5-b]indole derivatives and use thereof in the expansion of hematopoietic stem cells
EP2647697A1 (en) * 2010-12-01 2013-10-09 Nissan Chemical Industries, Ltd. Method for producing hematopoietic stem cells using pyrazole compound
WO2014138485A1 (en) 2013-03-08 2014-09-12 Irm Llc Ex vivo production of platelets from hematopoietic stem cells and the product thereof
US9353115B2 (en) 2011-06-01 2016-05-31 Janus Biotherapeutics, Inc. Immune system modulators
WO2017115268A1 (en) 2015-12-28 2017-07-06 Novartis Ag Compositions and methods for the treatment of hemoglobinopathies
WO2017202816A1 (en) 2016-05-25 2017-11-30 Bayer Pharma Aktiengesellschaft 3-oxo-2,6-diphenyl-2,3-dihydropyridazine-4-carboxamides
WO2018048346A1 (en) * 2016-08-18 2018-03-15 National University Of Singapore Substituted azole derivatives for generation, proliferation and differentiation of hematopoietic stem and progenitor cells
WO2018064387A1 (en) 2016-09-28 2018-04-05 Novartis Ag Porous membrane-based macromolecule delivery system
WO2018142364A1 (en) 2017-02-06 2018-08-09 Novartis Ag Compositions and methods for the treatment of hemoglobinopathies
WO2018146010A1 (en) 2017-02-09 2018-08-16 Bayer Aktiengesellschaft 2-heteroaryl-3-oxo-2,3-dihydropyridazine-4-carboxamides for the treatment of cancer
WO2018191476A1 (en) * 2017-04-12 2018-10-18 Magenta Therapeutics, Inc. Aryl hydrocarbon receptor antagonists and uses thereof
WO2019067999A1 (en) 2017-09-29 2019-04-04 Intellia Therapeutics, Inc. In vitro method of mrna delivery using lipid nanoparticles
WO2019101642A1 (en) 2017-11-21 2019-05-31 Bayer Aktiengesellschaft Sulphur substituted 3-oxo-2,3-dihydropyridazine-4-carboxamides
WO2019101647A1 (en) 2017-11-21 2019-05-31 Bayer Aktiengesellschaft 2-phenylpyrimidine-4-carboxamides as ahr inhibitors
WO2019101641A1 (en) 2017-11-21 2019-05-31 Bayer Aktiengesellschaft 2-hetarylpyrimidine-4-carboxamides as aryl hydrocarbon receptor anatgonists
WO2019101643A1 (en) 2017-11-21 2019-05-31 Bayer Aktiengesellschaft 3-oxo-6-heteroaryl-2-phenyl-2,3-dihydropyridazine-4-carboxamides
WO2019136159A1 (en) * 2018-01-03 2019-07-11 Magenta Therapeutics Inc. Compositions and methods for the expansion of hematopoietic stem and progenitor cells and treatment of inherited metabolic disorders
WO2019156989A1 (en) * 2018-02-06 2019-08-15 Ideaya Biosciences, Inc. COMPOUNDS AND METHODS FOR THE MODULATION OF AhR
WO2020039093A1 (en) 2018-08-24 2020-02-27 Jaguahr Therapeutics Pte Ltd Tetrahydropyridopyrimidine derivatives as ahr modulators
WO2020043880A1 (en) 2018-08-31 2020-03-05 Jaguahr Therapeutics Pte Ltd Heterocyclic compounds as ahr modulators
WO2020050409A1 (en) 2018-09-07 2020-03-12 Otsuka Pharmaceutical Co., Ltd. Heterocyclic compound
US10617721B2 (en) 2013-10-24 2020-04-14 Ospedale San Raffaele S.R.L. Methods for genetic modification of stem cells
WO2021028382A1 (en) 2019-08-12 2021-02-18 Bayer Aktiengesellschaft [1,2,4]triazolo[1,5-c]quinazolin-5-amines
WO2021117733A1 (en) 2019-12-09 2021-06-17 Otsuka Pharmaceutical Co., Ltd. Acrylamide compounds
WO2021123920A1 (en) 2019-12-18 2021-06-24 Novartis Ag Compositions and methods for the treatment of hemoglobinopathies
WO2021173082A1 (en) 2020-02-26 2021-09-02 Jaguahr Therapeutics Pte Ltd Pyridopyrimidine derivatives useful in modulation of ahr signalling
WO2022029063A1 (en) 2020-08-04 2022-02-10 Bayer Aktiengesellschaft Pyrido[1,2,4]triazolo[1,5-c]pyrimidin-5-amines
CN114369097A (en) * 2020-10-15 2022-04-19 山东轩竹医药科技有限公司 Heteroaromatic AhR inhibitors
WO2022269518A2 (en) 2021-06-23 2022-12-29 Novartis Ag Compositions and methods for the treatment of hemoglobinopathies
WO2024076300A1 (en) 2022-10-03 2024-04-11 Jaguahr Therapeutics Pte Ltd Compounds useful in modulation of ahr signalling
US12077542B2 (en) 2017-04-21 2024-09-03 Ikena Oncology, Inc. Indole AHR inhibitors and uses thereof

Families Citing this family (49)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8846393B2 (en) * 2005-11-29 2014-09-30 Gamida-Cell Ltd. Methods of improving stem cell homing and engraftment
EP2142546B1 (en) * 2007-03-28 2017-06-07 Saniona A/S Purinyl derivatives and their use as potassium channel modulators
ZA200905796B (en) * 2007-03-28 2010-10-27 Neurosearch As Purinyl derivatives and their use as potassium channel modulators
JP2011521968A (en) * 2008-05-30 2011-07-28 ジェネンテック， インコーポレイテッド Purine PI3K inhibitor compounds and methods of use
US20110237607A1 (en) * 2008-09-26 2011-09-29 Neurosearch A/S Substituted purinyl-pyrazol derivatives and their use as potassium channel modulators
WO2010034707A1 (en) * 2008-09-26 2010-04-01 Neurosearch A/S Substituted purinyl-pyrazol derivatives and their use as potassium channel modulators
PE20100362A1 (en) * 2008-10-30 2010-05-27 Irm Llc PURINE DERIVATIVES THAT EXPAND HEMATOPOYETIC STEM CELLS
EP2729560A4 (en) * 2011-07-06 2014-12-03 Cellerant Therapeutics Inc Megakaryocyte progenitor cells for production of platelets
US9593062B2 (en) * 2011-09-07 2017-03-14 Deutschland Krebsforschungszentrum Means and methods for treating and/or preventing natural AHR ligand-dependent cancer
CA2863795A1 (en) 2012-02-13 2013-08-22 Gamida-Cell Ltd. Culturing of mesenchymal stem cells
BR112014032501A2 (en) 2012-06-26 2017-06-27 Saniona Aps compound and pharmaceutical composition.
US9567569B2 (en) * 2012-07-23 2017-02-14 Gamida Cell Ltd. Methods of culturing and expanding mesenchymal stem cells
US9175266B2 (en) 2012-07-23 2015-11-03 Gamida Cell Ltd. Enhancement of natural killer (NK) cell proliferation and activity
US9074186B2 (en) 2012-08-15 2015-07-07 Boston Medical Center Corporation Production of red blood cells and platelets from stem cells
WO2014110020A1 (en) 2013-01-08 2014-07-17 Fred Hutchinson Cancer Research Center Compositions and methods for expansion of embryonic hematopoietic stem cells
CA2848575C (en) * 2013-05-17 2021-01-26 Universite De Montreal Methods to modulate acute myeloid leukemia stem/progenitor cell expansion and/or differentiation
MX2016005689A (en) 2013-10-31 2016-08-08 Hutchinson Fred Cancer Res Modified hematopoietic stem/progenitor and non-t effector cells, and uses thereof.
UA115388C2 (en) 2013-11-21 2017-10-25 Пфайзер Інк. 2,6-substituted purine derivatives and their use in the treatment of proliferative disorders
US10350245B2 (en) 2015-01-21 2019-07-16 Fred Hutchinson Cancer Research Center Point-of-care and/or portable platform for gene therapy
EP3288569A4 (en) 2015-04-29 2018-12-19 Fred Hutchinson Cancer Research Center Modified hematopoietic stem/progenitor and non-t effector cells, and uses thereof
EP3288570A4 (en) 2015-04-29 2018-11-21 Fred Hutchinson Cancer Research Center Modified stem cells and uses thereof
NZ741612A (en) * 2015-10-15 2022-12-23 Celularity Inc Natural killer cells and ilc3 cells and uses thereof
IL258405B (en) 2015-10-23 2022-09-01 Eureka Therapeutics Inc Antibody/t-cell receptor chimeric constructs and uses thereof
CN108463548B (en) 2015-10-30 2023-04-18 加利福尼亚大学董事会 Method for producing T cell from stem cell and immunotherapy method using the T cell
AU2017250295B2 (en) 2016-04-14 2022-08-25 Fred Hutchinson Cancer Center Compositions and methods to program therapeutic cells using targeted nucleic acid nanocarriers
BR112018072701A2 (en) * 2016-05-07 2019-02-19 Celularity, Inc. Methods of treating acute myeloid leukemia and multiple myeloma using natural killer cells
WO2018013430A2 (en) 2016-07-12 2018-01-18 Arisan Therapeutics Inc. Heterocyclic compounds for the treatment of arenavirus infection
US11613573B2 (en) 2017-04-26 2023-03-28 Eureka Therapeutics, Inc. Chimeric antibody/T-cell receptor constructs and uses thereof
US11548867B2 (en) 2017-07-19 2023-01-10 Idea Ya Biosciences, Inc. Amido compounds as AhR modulators
EP3661500A1 (en) 2017-07-31 2020-06-10 Novartis AG Use of mavoglurant in the reduction of cocaine use or in preventing relapse into cocaine use
US20200283444A1 (en) * 2017-08-02 2020-09-10 Northwestern University Substituted fused pyrimidine compounds and uses thereof
CA3077325A1 (en) 2017-09-28 2019-04-04 Celularity Inc. Tumor suppression using human placenta-derived intermediate natural killer (pink) cells in combination with an antibody
CN111683669A (en) 2017-10-31 2020-09-18 美真达治疗公司 Compositions and methods for hematopoietic stem and progenitor cell transplantation therapy
WO2019089826A1 (en) 2017-10-31 2019-05-09 Magenta Therapeutics Inc. Compositions and methods for the expansion of hematopoietic stem and progenitor cells
AU2018378804A1 (en) 2017-12-06 2020-06-11 Ensoma, Inc. Dosing regimens for the mobilization of hematopoietic stem and progenitor cells
US11260079B2 (en) 2017-12-06 2022-03-01 Magenta Therapeutics, Inc. Dosing regimens for the mobilization of hematopoietic stem and progenitor cells
US20220340875A1 (en) * 2018-07-19 2022-10-27 Ideaya Biosciences, Inc. Methods of culturing and/or expanding stem cells and/or lineage committed progenitor cells using amido compounds
EP4268831A3 (en) 2018-09-12 2024-05-22 Fred Hutchinson Cancer Center Reducing cd33 expression to selectively protect therapeutic cells
CN113272305A (en) * 2018-10-16 2021-08-17 医肯纳肿瘤学公司 Indole AHR inhibitors and uses thereof
WO2020092694A2 (en) * 2018-10-31 2020-05-07 Magenta Therapeutics Inc. Methods for hematopoietic stem and progenitor cell transplant therapy
US20220401481A1 (en) 2019-11-01 2022-12-22 Magenta Therapeutics, Inc. Dosing regimens for the mobilization of hematopoietic stem and progenitor cells
AU2020394424A1 (en) 2019-11-26 2022-06-16 Ikena Oncology, Inc. Polymorphic carbazole derivatives and uses thereof
US20230193212A1 (en) 2020-05-06 2023-06-22 Orchard Therapeutics (Europe) Limited Treatment for neurodegenerative diseases
CN115715203A (en) 2020-05-06 2023-02-24 塞勒克提斯公司 Methods of genetically modifying cells to deliver therapeutic proteins
CA3177093A1 (en) 2020-05-06 2021-11-11 Cellectis S.A. Methods for targeted insertion of exogenous sequences in cellular genomes
WO2022004864A1 (en) 2020-07-01 2022-01-06 ネクスジェン株式会社 Human long-term hematopoietic stem cell marker
EP4308694A1 (en) 2021-03-16 2024-01-24 Magenta Therapeutics, Inc. Dosing regimens for hematopoietic stem cell mobilization for stem cell transplants in multiple myeloma patients
WO2023150393A2 (en) 2022-02-07 2023-08-10 Ensoma, Inc. Inhibitor-resistant mgmt modifications and modification of mgmt-encoding nucleic acids
WO2024020429A1 (en) 2022-07-22 2024-01-25 Lyell Immunopharma, Inc. Immune cell therapy

Family Cites Families (54)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3399196A (en) 1959-01-22 1968-08-27 Ciba Geigy Corp Nu-substituted pyrazolo-pyrimidines
DE3150486A1 (en) * 1981-12-19 1983-08-25 Merck Patent Gmbh, 6100 Darmstadt IMIDAZO (4,5-C) PYRIDINE, PHARMACEUTICAL PREPARATIONS CONTAINING THE SAME AND METHOD FOR THE PRODUCTION THEREOF
US5437994A (en) 1989-06-15 1995-08-01 Regents Of The University Of Michigan Method for the ex vivo replication of stem cells, for the optimization of hematopoietic progenitor cell cultures, and for increasing the metabolism, GM-CSF secretion and/or IL-6 secretion of human stromal cells
US5117830A (en) 1990-11-08 1992-06-02 Whitby Research, Inc. Method of determining viability of tissue
EP0682027B1 (en) * 1994-05-03 1997-10-15 Novartis AG Pyrrolopyrimidine derivatives with antiproliferative action
FR2732604B1 (en) * 1995-04-07 1997-06-06 Vacsyn Sa DERIVATIVES AND CONJUGATES OF MDP HAVING STIMULATORY ACTIVITY OF HEMATOPOIETIC FUNCTION AND COMPOSITIONS CONTAINING THEM
FR2741881B1 (en) 1995-12-01 1999-07-30 Centre Nat Rech Scient NOVEL PURINE DERIVATIVES HAVING IN PARTICULAR ANTI-PROLIFERATIVE PRORIETES AND THEIR BIOLOGICAL APPLICATIONS
US6232320B1 (en) 1998-06-04 2001-05-15 Abbott Laboratories Cell adhesion-inhibiting antiinflammatory compounds
EP1133291B1 (en) 1998-11-17 2006-01-11 Smithkline Beecham Corporation Cyclic polyamines for treating thrombocytopenia
GC0000177A (en) 1998-12-17 2006-03-29 Smithkline Beecham Thrombopoietin mimetics
MXPA01007851A (en) * 1999-02-01 2003-06-04 Cv Therapeutics Inc PURINE INHIBITORS OF CYCLIN DEPENDENT KINASE 2 AND Ikappa-Aalpha.
WO2000066112A1 (en) 1999-05-03 2000-11-09 Smithkline Beecham Corporation Cxcr-4 receptor antagonists - thrombopoietin mimetics
US6960439B2 (en) * 1999-06-28 2005-11-01 Source Precision Medicine, Inc. Identification, monitoring and treatment of disease and characterization of biological condition using gene expression profiles
CN1110552C (en) * 1999-07-13 2003-06-04 中国人民解放军第二军医大学 Method of extracorporeal cloning hemopoietic stem cell
WO2001017349A1 (en) 1999-09-10 2001-03-15 Smithkline Beckman Corporation Thrombopoietin mimetics
US6552008B1 (en) 1999-09-24 2003-04-22 Smithkline Beecham Corporation Thrombopoietin mimetics
JP2003513965A (en) 1999-11-05 2003-04-15 スミスクライン・ビーチャム・コーポレイション Semicarbazone derivatives and their use as thrombopoietin mimetics
WO2001039773A1 (en) 1999-12-06 2001-06-07 Smithkline Beecham Corporation Thrombopoietin mimetics
ATE327242T1 (en) 1999-12-17 2006-06-15 Ariad Pharma Inc NEW PURINES
ATE322494T1 (en) * 2000-01-07 2006-04-15 Universitaire Instelling Antwe PURINE DERIVATIVES, THEIR PRODUCTION AND USE
CY2010012I2 (en) 2000-05-25 2020-05-29 Novartis Ag THROMBOPOIETIN MIMETICS
CA2377666A1 (en) * 2000-07-18 2002-01-24 Minoru Takebe Stem cell reinforcing material
WO2002049413A2 (en) 2000-12-19 2002-06-27 Smithkline Beecham Corporation Thrombopoietin mimetics
WO2002085343A1 (en) 2001-03-01 2002-10-31 Smithkline Beecham Corporation Thrombopoietin mimetics
AU2002342051B2 (en) * 2001-10-12 2009-06-11 Irm Llc Kinase inhibitor scaffolds and methods for their preparation
BR0207725A (en) 2001-12-28 2004-03-23 Daiichi Suntory Biomedical Res Hematopoietic stem cell growth and / or differentiation promoters and / or hematopoietic progenitors
WO2003103686A1 (en) 2002-06-06 2003-12-18 Smithkline Beecham Corporation Thrombopoietin mimetics
US20030139427A1 (en) 2002-08-23 2003-07-24 Osi Pharmaceuticals Inc. Bicyclic pyrimidinyl derivatives and methods of use thereof
JP2004089068A (en) * 2002-08-30 2004-03-25 Kobe University Ah RECEPTOR LIGAND-SPECIFIC GENE EXPRESSION INDUCER AND APPLICATION TECHNOLOGY OF HETEROGENE INDUCTION AND EXPRESSION SYSTEM BASED ON THE FUNCTION OF THE INDUCER
WO2004054515A2 (en) 2002-12-13 2004-07-01 Smithkline Beecham Corporation Thrombopoietin mimetics
WO2005020892A2 (en) 2003-08-08 2005-03-10 Mitochroma Research, Inc. Pharmaceutical compositions and methods for metabolic modulation
EP1670795A1 (en) * 2003-09-18 2006-06-21 ALTANA Pharma AG Pharmacologically active imidazo 4,5-c pyridines
MXPA06007095A (en) 2003-12-22 2006-09-04 Gilead Sciences Inc Kinase inhibitor phosphonate conjugates.
EP1724268A4 (en) 2004-02-20 2010-04-21 Kirin Pharma Kk Compound having tgf-beta inhibitory activity and pharmaceutical composition containing same
US7622108B2 (en) * 2004-04-23 2009-11-24 Bioe, Inc. Multi-lineage progenitor cells
JP2008511553A (en) * 2004-09-03 2008-04-17 プロメティック バイオサイエンシズ インコーポレーテッド Substituted purinyl derivatives with immunomodulatory and chemoprotective activities and their use alone or in combination with medium chain fatty acids or glycerides
GB0420719D0 (en) 2004-09-17 2004-10-20 Addex Pharmaceuticals Sa Novel allosteric modulators
AR052771A1 (en) * 2004-09-30 2007-04-04 Tibotec Pharm Ltd HCV INHIBITING BICYCLE PYRIMIDINS
FR2876583B1 (en) 2004-10-15 2007-04-13 Centre Nat Rech Scient Cnrse USE OF PURINE DERIVATIVES FOR THE MANUFACTURE OF MEDICAMENTS FOR THE TREATMENT OF MUCOVISCIDOSIS AND DISEASES ASSOCIATED WITH A DEFECT OF ADDRESSING PROTEINS IN CELLS
WO2006061380A2 (en) * 2004-12-09 2006-06-15 Altana Pharma Ag SUBSTITUTED IMIDAZO[4,5-b]PYRIDINES AS INHIBITORS OF GASTRIC ACID SECRETION
US20060281791A1 (en) 2005-04-29 2006-12-14 Children's Medical Center Corporation Methods of increasing proliferation of adult mammalian cardiomyocytes through p38 map kinase inhibition
WO2007009120A2 (en) 2005-07-14 2007-01-18 Irm Llc Heterotetracyclic compounds as tpo mimetics
CA2618634A1 (en) 2005-08-15 2007-02-22 Irm Llc Compounds and compositions as tpo mimetics
NZ571429A (en) * 2006-04-21 2011-09-30 Novartis Ag Purine derivatives for use as adenosine A2A receptor agonists
EP2044956A4 (en) 2006-06-14 2010-08-11 Chugai Pharmaceutical Co Ltd Hematopoietic stem cell proliferation promoter
US8372797B2 (en) * 2006-06-22 2013-02-12 Creative Medical Health, Inc. Treatment of erectile dysfunction by stem cell therapy
EP1889846A1 (en) * 2006-07-13 2008-02-20 Novartis AG Purine derivatives as A2a agonists
WO2008028645A1 (en) 2006-09-05 2008-03-13 Aplagen Gmbh Peptides binding the tpo receptor
WO2008051502A1 (en) 2006-10-19 2008-05-02 Genzyme Corporation Purine derivatives for the treatment of cystic diseases
EP2094839B1 (en) 2006-12-08 2020-02-05 University of Rochester Expansion of hematopoietic stem cells
EP2142546B1 (en) * 2007-03-28 2017-06-07 Saniona A/S Purinyl derivatives and their use as potassium channel modulators
CN101981037B (en) 2008-01-30 2013-09-04 吉宁特有限公司 Pyrazolopyrimidine PI3K inhibitor compounds and methods of use
PE20100362A1 (en) * 2008-10-30 2010-05-27 Irm Llc PURINE DERIVATIVES THAT EXPAND HEMATOPOYETIC STEM CELLS
US9175266B2 (en) * 2012-07-23 2015-11-03 Gamida Cell Ltd. Enhancement of natural killer (NK) cell proliferation and activity

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
None

Cited By (63)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2647697A1 (en) * 2010-12-01 2013-10-09 Nissan Chemical Industries, Ltd. Method for producing hematopoietic stem cells using pyrazole compound
US9212348B2 (en) 2010-12-01 2015-12-15 Nissan Chemical Industries, Ltd. Method for producing hematopoietic stem cells using pyrazole compounds
EP2647697A4 (en) * 2010-12-01 2014-04-30 Nissan Chemical Ind Ltd Method for producing hematopoietic stem cells using pyrazole compound
WO2012102937A3 (en) * 2011-01-25 2012-11-29 Irm Llc Benz imidazole compounds that expand hematopoietic stem cells
US9353115B2 (en) 2011-06-01 2016-05-31 Janus Biotherapeutics, Inc. Immune system modulators
AU2012262021B2 (en) * 2011-06-01 2016-07-28 Janus Biotherapeutics, Inc. Novel immune system modulators
WO2013019857A3 (en) * 2011-08-01 2013-06-06 Alnylam Pharmaceuticals, Inc. Method for improving the success rate of hematopoietic stem cell transplants
WO2013086436A1 (en) * 2011-12-08 2013-06-13 Fred Hutchinson Cancer Research Center Compositions and methods for enhanced generation of hematopoietic stem/progenitor cells
US9834755B2 (en) 2011-12-08 2017-12-05 Fred Hutchinson Cancer Research Center Compositions and methods for enhanced generation of hematopoietic stem/progenitor cells
AU2012347534B2 (en) * 2011-12-08 2018-01-25 Fred Hutchinson Cancer Research Center Compositions and methods for enhanced generation of hematopoietic stem/progenitor cells
WO2013110198A1 (en) 2012-01-27 2013-08-01 Université de Montréal Pyrimido[4,5-b]indole derivatives and use thereof in the expansion of hematopoietic stem cells
US10336747B2 (en) 2012-01-27 2019-07-02 Université de Montréal Pyrimido[4,5-B]indole derivatives and use thereof in the expansion of hematopoietic stem cells
JP2015504902A (en) * 2012-01-27 2015-02-16 ユニヴェルスィテ・ドゥ・モントリオール Pyrimido [4,5-b] indole derivatives and their use in the proliferation of hematopoietic stem cells
US9409906B2 (en) 2012-01-27 2016-08-09 Universite De Montreal Pyrimido[4,5-B]indole derivatives and use thereof in the expansion of hematopoietic stem cells
EP2807165A4 (en) * 2012-01-27 2015-08-26 Univ Montreal Pyrimido[4,5-b]indole derivatives and use thereof in the expansion of hematopoietic stem cells
WO2014138485A1 (en) 2013-03-08 2014-09-12 Irm Llc Ex vivo production of platelets from hematopoietic stem cells and the product thereof
US10617721B2 (en) 2013-10-24 2020-04-14 Ospedale San Raffaele S.R.L. Methods for genetic modification of stem cells
WO2017115268A1 (en) 2015-12-28 2017-07-06 Novartis Ag Compositions and methods for the treatment of hemoglobinopathies
JP2021166514A (en) * 2015-12-28 2021-10-21 ノバルティス アーゲー Compositions and methods for treatment of hemoglobinopathies
JP2019500043A (en) * 2015-12-28 2019-01-10 ノバルティス アーゲー Compositions and methods for the treatment of abnormal hemoglobinosis
WO2017202816A1 (en) 2016-05-25 2017-11-30 Bayer Pharma Aktiengesellschaft 3-oxo-2,6-diphenyl-2,3-dihydropyridazine-4-carboxamides
AU2017325511B2 (en) * 2016-08-18 2021-12-09 National University Of Singapore Substituted azole derivatives for generation, proliferation and differentiation of hematopoietic stem and progenitor cells
WO2018048346A1 (en) * 2016-08-18 2018-03-15 National University Of Singapore Substituted azole derivatives for generation, proliferation and differentiation of hematopoietic stem and progenitor cells
WO2018064387A1 (en) 2016-09-28 2018-04-05 Novartis Ag Porous membrane-based macromolecule delivery system
US11466271B2 (en) 2017-02-06 2022-10-11 Novartis Ag Compositions and methods for the treatment of hemoglobinopathies
WO2018142364A1 (en) 2017-02-06 2018-08-09 Novartis Ag Compositions and methods for the treatment of hemoglobinopathies
WO2018146010A1 (en) 2017-02-09 2018-08-16 Bayer Aktiengesellschaft 2-heteroaryl-3-oxo-2,3-dihydropyridazine-4-carboxamides for the treatment of cancer
US11795164B2 (en) 2017-02-09 2023-10-24 Bayer Aktiengesellschaft 2-heteroaryl-3-oxo-2,3-dihydropyridazine-4-carboxamides for the treatment of cancer
US10457683B2 (en) 2017-04-12 2019-10-29 Magenta Therapeutics Inc. Aryl hydrocarbon receptor antagonists and uses thereof
US10919900B2 (en) 2017-04-12 2021-02-16 Magenta Therapeutics Inc. Aryl hydrocarbon receptor antagonists and uses thereof
US10351572B2 (en) 2017-04-12 2019-07-16 Magenta Therapeutics Inc. Aryl hydrocarbon receptor antagonists and uses thereof
WO2018191476A1 (en) * 2017-04-12 2018-10-18 Magenta Therapeutics, Inc. Aryl hydrocarbon receptor antagonists and uses thereof
CN110785419A (en) * 2017-04-12 2020-02-11 美真达治疗公司 Aromatic receptor antagonists and uses thereof
CN110785419B (en) * 2017-04-12 2024-03-01 博雅缉因（北京）生物科技有限公司 Aromatic hydrocarbon receptor antagonist and application thereof
US12077542B2 (en) 2017-04-21 2024-09-03 Ikena Oncology, Inc. Indole AHR inhibitors and uses thereof
WO2019067999A1 (en) 2017-09-29 2019-04-04 Intellia Therapeutics, Inc. In vitro method of mrna delivery using lipid nanoparticles
WO2019101641A1 (en) 2017-11-21 2019-05-31 Bayer Aktiengesellschaft 2-hetarylpyrimidine-4-carboxamides as aryl hydrocarbon receptor anatgonists
WO2019101647A1 (en) 2017-11-21 2019-05-31 Bayer Aktiengesellschaft 2-phenylpyrimidine-4-carboxamides as ahr inhibitors
US11591311B2 (en) 2017-11-21 2023-02-28 Bayer Aktiengesellschaft 3-oxo-6-heteroaryl-2-phenyl-2,3-dihydropyridazine-4-carboxamides
US11304946B2 (en) 2017-11-21 2022-04-19 Bayer Aktiengesellschaft 2-hetarylpyrimidine-4-carboxamides as aryl hydrocarbon receptor antagonists
WO2019101642A1 (en) 2017-11-21 2019-05-31 Bayer Aktiengesellschaft Sulphur substituted 3-oxo-2,3-dihydropyridazine-4-carboxamides
US11459312B2 (en) 2017-11-21 2022-10-04 Bayer Aktiengesellschaft Sulphur substituted 3-oxo-2,3-dihydropyridazine-4-carboxamides
US11524944B2 (en) 2017-11-21 2022-12-13 Bayer Aktiengesellschaft 2-phenylpyrimidine-4-carboxamides as AHR inhibitors
WO2019101643A1 (en) 2017-11-21 2019-05-31 Bayer Aktiengesellschaft 3-oxo-6-heteroaryl-2-phenyl-2,3-dihydropyridazine-4-carboxamides
WO2019136159A1 (en) * 2018-01-03 2019-07-11 Magenta Therapeutics Inc. Compositions and methods for the expansion of hematopoietic stem and progenitor cells and treatment of inherited metabolic disorders
WO2019156989A1 (en) * 2018-02-06 2019-08-15 Ideaya Biosciences, Inc. COMPOUNDS AND METHODS FOR THE MODULATION OF AhR
US11530220B2 (en) 2018-02-06 2022-12-20 Ideaya Biosciences, Inc. Substituted imidazo[1,5-a]pyrazines and [1,2,4]triazolo[4,3-a]pyrazines for the modulation of AhR
CN112739698A (en) * 2018-08-24 2021-04-30 捷豹治疗有限公司 Tetrahydropyrimidine derivatives as AHR modulators
WO2020039093A1 (en) 2018-08-24 2020-02-27 Jaguahr Therapeutics Pte Ltd Tetrahydropyridopyrimidine derivatives as ahr modulators
WO2020043880A1 (en) 2018-08-31 2020-03-05 Jaguahr Therapeutics Pte Ltd Heterocyclic compounds as ahr modulators
US11932657B2 (en) 2018-09-07 2024-03-19 Otsuka Pharmaceutical Co., Ltd. Heterocyclic compound
JP7448527B2 (en) 2018-09-07 2024-03-12 大塚製薬株式会社 heterocyclic compound
KR20210056373A (en) 2018-09-07 2021-05-18 오츠카 세이야쿠 가부시키가이샤 Heterocyclic compound
WO2020050409A1 (en) 2018-09-07 2020-03-12 Otsuka Pharmaceutical Co., Ltd. Heterocyclic compound
WO2021028382A1 (en) 2019-08-12 2021-02-18 Bayer Aktiengesellschaft [1,2,4]triazolo[1,5-c]quinazolin-5-amines
KR20220113392A (en) 2019-12-09 2022-08-12 오츠카 세이야쿠 가부시키가이샤 acrylamide compound
WO2021117733A1 (en) 2019-12-09 2021-06-17 Otsuka Pharmaceutical Co., Ltd. Acrylamide compounds
WO2021123920A1 (en) 2019-12-18 2021-06-24 Novartis Ag Compositions and methods for the treatment of hemoglobinopathies
WO2021173082A1 (en) 2020-02-26 2021-09-02 Jaguahr Therapeutics Pte Ltd Pyridopyrimidine derivatives useful in modulation of ahr signalling
WO2022029063A1 (en) 2020-08-04 2022-02-10 Bayer Aktiengesellschaft Pyrido[1,2,4]triazolo[1,5-c]pyrimidin-5-amines
CN114369097A (en) * 2020-10-15 2022-04-19 山东轩竹医药科技有限公司 Heteroaromatic AhR inhibitors
WO2022269518A2 (en) 2021-06-23 2022-12-29 Novartis Ag Compositions and methods for the treatment of hemoglobinopathies
WO2024076300A1 (en) 2022-10-03 2024-04-11 Jaguahr Therapeutics Pte Ltd Compounds useful in modulation of ahr signalling

Also Published As

Publication number Publication date
PE20100362A1 (en) 2010-05-27
CL2009002010A1 (en) 2010-06-25
JO3593B1 (en) 2020-07-05
IL212368A0 (en) 2011-06-30
US8927281B2 (en) 2015-01-06
TWI532486B (en) 2016-05-11
KR20110075042A (en) 2011-07-05
RS59027B1 (en) 2019-08-30
SV2011003888A (en) 2011-07-05
TN2011000174A1 (en) 2012-12-17
EP3524604A1 (en) 2019-08-14
CU24495B1 (en) 2021-01-12
GEP20146053B (en) 2014-03-10
SMP201100024B (en) 2012-03-05
EP2350078A2 (en) 2011-08-03
ECSP11011090A (en) 2011-09-30
CU20110096A7 (en) 2012-01-31
MX2011004593A (en) 2011-06-16
CA3061937A1 (en) 2010-05-27
MX347834B (en) 2016-10-07
DK2350078T3 (en) 2019-07-15
NI201100083A (en) 2011-11-03
SI2350078T1 (en) 2019-08-30
AU2009317898A1 (en) 2010-05-27
EP3524604B1 (en) 2023-03-29
MA32828B1 (en) 2011-11-01
ES2948567T3 (en) 2023-09-14
CN102203096B (en) 2014-09-17
EP2350078B1 (en) 2019-04-17
CL2013001874A1 (en) 2014-04-04
BRPI0921799B1 (en) 2020-09-29
EA019872B1 (en) 2014-06-30
US20210187033A1 (en) 2021-06-24
CA2943540C (en) 2020-10-27
BRPI0921799A2 (en) 2016-01-12
SMAP201100024A (en) 2011-07-11
CO6410309A2 (en) 2012-03-30
MY155137A (en) 2015-09-15
PT2350078T (en) 2019-07-19
US20100183564A1 (en) 2010-07-22
ES2736728T3 (en) 2020-01-07
IL212368A (en) 2015-02-26
CA2740589C (en) 2017-11-21
CY1121857T1 (en) 2020-07-31
JP2012507554A (en) 2012-03-29
US20140114070A1 (en) 2014-04-24
CN102203096A (en) 2011-09-28
US20170239296A1 (en) 2017-08-24
HRP20191238T1 (en) 2019-11-29
JP5390626B2 (en) 2014-01-15
PL2350078T3 (en) 2019-09-30
NZ592435A (en) 2013-01-25
DOP2011000114A (en) 2017-09-29
KR101434719B1 (en) 2014-08-27
AR074063A1 (en) 2010-12-22
HN2011001195A (en) 2014-01-13
US9580426B2 (en) 2017-02-28
CR20110192A (en) 2011-06-10
LT2350078T (en) 2019-07-25
CA2740589A1 (en) 2010-05-27
TW201028153A (en) 2010-08-01
WO2010059401A3 (en) 2010-09-23
CA2943540A1 (en) 2010-05-27
AU2009317898B2 (en) 2013-07-11
BRPI0921799B8 (en) 2021-05-25
HUE044802T2 (en) 2019-11-28
EA201100663A1 (en) 2011-12-30

Similar Documents

Publication Publication Date Title
US20210187033A1 (en) Compounds that expand hematopoietic stem cells
US10336747B2 (en) Pyrimido[4,5-B]indole derivatives and use thereof in the expansion of hematopoietic stem cells
US9175266B2 (en) Enhancement of natural killer (NK) cell proliferation and activity
WO2014138485A1 (en) Ex vivo production of platelets from hematopoietic stem cells and the product thereof
EP2788476A1 (en) Compositions and methods for enhanced generation of hematopoietic stem/progenitor cells
US9567569B2 (en) Methods of culturing and expanding mesenchymal stem cells
WO2012102937A2 (en) Compounds that expand hematopoietic stem cells
AU2013203936A1 (en) Compounds that expand hematopoietic stem cells

Legal Events

Date Code Title Description
WWE Wipo information: entry into national phase

Ref document number: 200980143651.3

Country of ref document: CN

121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 09748626

Country of ref document: EP

Kind code of ref document: A2

WWE Wipo information: entry into national phase

Ref document number: 2009317898

Country of ref document: AU

WWE Wipo information: entry into national phase

Ref document number: 2740589

Country of ref document: CA

Ref document number: 2741/DELNP/2011

Country of ref document: IN

ENP Entry into the national phase

Ref document number: 2009317898

Country of ref document: AU

Date of ref document: 20091029

Kind code of ref document: A

WWE Wipo information: entry into national phase

Ref document number: 212368

Country of ref document: IL

Ref document number: CR2011-000192

Country of ref document: CR

WWE Wipo information: entry into national phase

Ref document number: 2009748626

Country of ref document: EP

WWE Wipo information: entry into national phase

Ref document number: 592435

Country of ref document: NZ

WWE Wipo information: entry into national phase

Ref document number: D2011096

Country of ref document: CU

WWE Wipo information: entry into national phase

Ref document number: 12011500831

Country of ref document: PH

WWE Wipo information: entry into national phase

Ref document number: MX/A/2011/004593

Country of ref document: MX

Ref document number: 12209/1

Country of ref document: GE

NENP Non-entry into the national phase

Ref country code: DE

WWE Wipo information: entry into national phase

Ref document number: 2011534784

Country of ref document: JP

WWE Wipo information: entry into national phase

Ref document number: DZP2011000314

Country of ref document: DZ

WWE Wipo information: entry into national phase

Ref document number: 11058070

Country of ref document: CO

WWE Wipo information: entry into national phase

Ref document number: 201100663

Country of ref document: EA

ENP Entry into the national phase

Ref document number: 20117012091

Country of ref document: KR

Kind code of ref document: A

WWE Wipo information: entry into national phase

Ref document number: A201105290

Country of ref document: UA

ENP Entry into the national phase

Ref document number: PI0921799

Country of ref document: BR

Kind code of ref document: A2

Effective date: 20110429