WO2016003169A2 - 세포 리프로그래밍 유도용 조성물 - Google Patents
세포 리프로그래밍 유도용 조성물 Download PDFInfo
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- WO2016003169A2 WO2016003169A2 PCT/KR2015/006718 KR2015006718W WO2016003169A2 WO 2016003169 A2 WO2016003169 A2 WO 2016003169A2 KR 2015006718 W KR2015006718 W KR 2015006718W WO 2016003169 A2 WO2016003169 A2 WO 2016003169A2
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- 0 CCC(C)C(C1C2)*2CC1[S+2][S+2]/C=C(\C)/C* Chemical compound CCC(C)C(C1C2)*2CC1[S+2][S+2]/C=C(\C)/C* 0.000 description 12
- NPMRFSGSVFNPFQ-UHFFFAOYSA-N CCC1C=CC=CC1C Chemical compound CCC1C=CC=CC1C NPMRFSGSVFNPFQ-UHFFFAOYSA-N 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D231/00—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings
- C07D231/54—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings condensed with carbocyclic rings or ring systems
- C07D231/56—Benzopyrazoles; Hydrogenated benzopyrazoles
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
- A61K31/415—1,2-Diazoles
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
- A61K31/415—1,2-Diazoles
- A61K31/416—1,2-Diazoles condensed with carbocyclic ring systems, e.g. indazole
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P21/00—Drugs for disorders of the muscular or neuromuscular system
- A61P21/02—Muscle relaxants, e.g. for tetanus or cramps
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- 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
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- 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
- A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
- A61P25/16—Anti-Parkinson drugs
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- 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
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- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
- C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
- C07D401/06—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
- C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
- C07D401/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a chain containing hetero atoms as chain links
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
- C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
- C07D403/06—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
- C07D403/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing three or more hetero rings
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- 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
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0654—Osteocytes, Osteoblasts, Odontocytes; Bones, Teeth
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
- C12N2506/13—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells
- C12N2506/1323—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from skeletal muscle cells
Definitions
- the present invention relates to a composition for inducing cell reprogramming.
- De-differentiation technology enables the production of induced pluripotent stem cells of the same characteristics as human embryonic stem cells from a patient-somatic cell by securing self-somatic cells in a relatively easy way with little physical damage and discomfort to the patient.
- the strategy of establishing a custom self-differentiating stem cell line has been revolutionized.
- a virus may be used to combine a specific gene to induce undifferentiated stem cells, and various researchers have suggested a method of delivering the virus into a cell and expressing the protein in a form in which the gene is inserted into a cell genome. It became.
- the reprogrammed cells prepared by this method were found to have almost the same cellular characteristics and differentiation capacity as embryonic stem cells, but because the gene was transferred through the viral vector, the virus was transferred onto the cell genome. There is a problem that contains a large amount of genetic material derived. In addition, in vivo experiments showed a side effect of forming cancer from induced pluripotent stem cells, which means that the actual application of induced pluripotent stem cells may still be very dangerous.
- de-differentiated stem cells have a problem of using an oncogene in the reprogramming process.
- a direct reprogramming (di rect) that directly differentiates somatic cells into a cell line necessary for a patient is necessary, beyond the conventional reprogramming method. Reprogramming is being actively researched.
- an object of the present invention is to provide a composition for inducing cell reprogramming (re-progra ng i ng).
- Another object of the present invention is to provide a novel indazole derivative (i ndazo l e der i vat i ves).
- the present invention provides a composition for inducing cell reprogramming (re-progra ⁇ i ng) comprising a compound represented by the following Formula A:
- 3 ⁇ 4 is hydrogen, nitro, nitrooxy or Y-carbonyl;
- Y is hydrogen, hydroxy, halo, d-30 alkoxy, as a hetero atom nitrogen, oxygen or sulfur, C 2, including - 30 heterocycloalkyl, C 2 containing a nitrogen, oxygen or sulfur as the hetero atom-20 heteroaryl cycloalkenyl, C 6 - 30 aryl, CHO-alkyl, or C 2 - 30 alkenyl
- 3 ⁇ 4 is hydrogen, hydroxy, halo, d-
- R 3 is hydrogen, hydroxy-halo, 30-aryl-Cl 30 alkoxy, C M0 alkyl or C 2 - 30 alkenyl
- X is an amine, amido, sulfonamido, carbonyl, oxime or imideamido.
- the present invention comprises the steps of contacting the cell reprogramming composition of the invention to the differentiated cells; And (b) provides a method for producing a reprogrammed cells from the differentiated cells comprising the step of culturing the cells of step (a).
- the present inventors have diligently researched to develop compounds capable of performing cell reprogramming while exhibiting an improved biological profile than conventional indazole derivative compounds.
- One indazole derivative compound was molecularly designed and synthesized, and it was confirmed that these compounds exhibit excellent cell reprogramming ability without exhibiting cytotoxicity, unlike conventional indazole derivatives (eg, kinase inhibitors).
- the compound of the present invention represented by the formula (A) is a compound which reprograms differentiated cells into cells of a new trait, and is chemically synthesized using indazole as a mother nucleus.
- Conventional indazole derivatives compounds are not known at all as cell reprogramming applications, and the compounds of the present invention have no cytotoxicity or significantly lower cell reprogramming ability compared to conventional indazole derivative compounds. Is very excellent.
- the conventional direct reprogramming method has a drawback that it is difficult to apply the gene directly to the patient by introducing a gene using a virus, the efficiency of the induction process is low, and it is an immunological problem.
- the composition of the present invention is very effective for inducing reprogrammed cells from differentiated cells.
- the composition of the present invention can induce cell lines of various lineages, such as osteogenic lineage cells or adipogenic lineage cells, which are reprogrammed from differentiated cells of mammalian origin.
- the composition of the present invention induces differentiation from myoblast (osteoblast) or adipocyte (adipocyte).
- the differentiated cells are not particularly limited and include, for example, somatic cells or somatic stem cells.
- Somatic cells are cells constituting the adult means cells that are limited in differentiation and self-producing capacity, the somatic cells are human skin, hair, Somatic cells that make up fat or bone.
- the differentiated cells may be cells derived from various mammals such as humans, monkeys, pigs, horses, cows, sheep, dogs, cats, mice, and rabbits, and may preferably be cells derived from humans.
- reprogramming refers to a state having a new type of cell or a new type of differentiation potential from differentiation cells that exist in different aspects, such as non-differentiating cells or cells with partial differentiation ability. Means a process that can be restored or converted.
- differentiated cells can be reprogrammed into cells that exhibit 0% to 100% of other traits upon contact with the composition of the present invention.
- nitro as used to define an indazole derivative of formula A refers to a halogen group element and includes, for example, fluoro, chloro, bromo and iodo.
- carbonyl means a-(00)-functional group.
- hydroxy means a -0H functional group.
- halo refers to a halogen group element and includes, for example, fluoro, chloro, bromo and iodo.
- alkyl refers to a straight or branched unsubstituted or substituted saturated hydrocarbon group, for example methyl, ethyl, propyl, isobutyl, pentyl, nucleus, heptyl, octyl, nonyl, decyl, undecyl, tridecyl , Pentadecyl and heptadecyl and the like.
- d-so alkyl means an alkyl group having an alkyl unit having 1 to 30 carbon atoms, and when Ci- 30 alkyl is substituted, carbon atoms of the substituent are not included.
- the alkyl at position Y in formula A is Ci-i5 alkyl
- the Ci- 30 alkyl at position 3 ⁇ 4 in formula A is preferably d- 15 alkyl.
- alkenyl refers to a straight-chain or branched unsubstituted or substituted unsaturated hydrocarbon group having the specified carbon number, for example ethenyl, vinyl, propenyl, allyl, isopropenyl, butenyl, isobutenyl, ⁇ butenyl, / ⁇ pentenyl and / nuclesenyl.
- C 2 alkenyl means an alkenyl group having an alkenyl group having 2 to 30 units, and, C 2 - in the case where the 30-alkenyl substituents substituted Carbon number is not included.
- C 2 in the Y position in the general formula A - 30 alkenyl is a C 2 - 15 alkenyl
- Al, C 2 of the R 2 position in formula A - 30 alkenyl is a C 2 - 15 alkenyl.
- alkoxy means an alkyl group or aryl bonded to one oxygen atom.
- d-3o alkoxy means an alkyl group having an alkoxy unit having 1 to 30 carbon atoms, and when d- 3Q alkoxy is substituted, the carbon number of the substituent is not included.
- alkoxyalkyl refers to an alkyl group substituted with an alkoxy group.
- C 2 -5 alkoxyalkyl means an alkoxyalkyl group is an alkoxyalkyl having units having 2 to 5, C 2 - 5 carbon atoms of an alkoxy substituent when the alkyl is substituted is not included.
- alkoxycarbonyl means carbonyl substituted with alkoxy.
- C 2 -5 alkoxycarbonyl means alkoxycarbonyl having an alkoxycarbonyl having 2 to unit 5, C 2 - 5 alkoxycarbonyl is that does not include carbon atoms of the substituents when substituted.
- heterocycloalkyl comprising nitrogen, oxygen or sulfur as a heteroatom means a non-aromatic cyclic hydrocarbon group comprising carbon and hydrogen and at least one heteroatom (nitrogen, oxygen or sulfur).
- the heteroatom is preferably nitrogen or oxygen, most preferably nitrogen. According to the present invention, the number of heteroatoms is 1-4, 1-3, or
- Heterocycloalkyl which is 2-30.
- the heterocycloalkyl is a wholly or partially substituted or unsubstituted carbon ringose
- the heterocycloalkyl is a hydroxy, halo, CrCs substituted or unsubstituted linear or branched alkyl, Ci-C 5 straight or branched chain alkoxy, dC 5 straight chain or branched chain alkenyl, C 2 - 8 alkoxyalkyl, C 2 - s alkoxycarbonyl, C 2 including nitrogen as a hetero atom-8 by interrogating cycloalkenyl alkyl, ⁇ a 0 3 ⁇ 4 - ' ⁇ . Or a combination thereof.
- heterocycloalkenyl comprising nitrogen, oxygen or sulfur as a heteroatom refers to a non-aromatic cyclic comprising carbon and hydrogen and at least one heteroatom (nitrogen, oxygen or sulfur) and at least one double bond. It means a hydrocarbon group.
- the heteroatom is preferably oxygen or nitrogen, most preferably nitrogen. According to the present invention, the number of heteroatoms is 1-4, 1-3, or 1-2.
- C 2-30 heterocycloalkenyl means heterocyclokenyl having 2 to 30 carbon atoms forming a ring structure.
- heterocycloalkenyl alkyl refers to an alkyl group substituted with a heterocycloalkenyl group.
- C 2 -8 hetero cycloalkenyl alkyl means a heterocycloalkyl alkenyl alkyl having a hetero cycloalkenyl alkyl unit having a carbon number of 2 to 5, C 2 - 5 carbon atoms of the heterocycle, if substituted alkenyl is the alkyl substituent comprises It is not.
- aryl refers to a substituted or unsubstituted monocyclic or polycyclic carbon ring which is wholly or partially unsaturated.
- C 6 -30 aryl group is not included in the carbon number of the substituent when the aryl group means a group having a carbon ring atom of a carbon number of 6 to 30, C 6 -30 aryl-substituted.
- aryl is monoaryl or biaryl.
- Monoaryl is 5-carbon
- biaryl has 9-10 carbon atoms.
- said aryl is substituted or unsubstituted phenyl.
- substitutions may be made by various substituents at various positions, such as halo, hydroxy, nitro, cyano, d-C 5 substituted or unsubstituted straight or branched chains.
- aryl moieties are phenyl, benzyl, naphthyl, pyridyl, pyridinyl, pyridinyl, pyrimidinyl, furinyl, quinolinyl, isoquinolinyl, furyl, thiophenyl, imidazoryl, oxazolyl, Thiazolyl, pyrazoryl and thienyl, including but not limited to.
- amine refers to a functional group comprising a basic nitrogen atom having an unbonded pair (l one pa ir).
- Ci-io amine is -NH 2 or 1 carbon It means an amine group having an amine unit of 10 to 10, if du) the amine is substituted, the carbon number of the substituent is not included.
- the amine at position X in formula A is a d- 5 amine or NH 2 .
- amide means a functional group consisting of an acyl group bonded to a nitrogen atom.
- Cw 0 amide means an amide group having an amide unit having 1 to 10 carbon atoms, and when the Cwo amide is substituted, the carbon number of the substituent is not included.
- the amide is R-CO-NH-R ', sulfonamide or phosphoramide.
- Y is hydrogen, hydroxy, halo,
- Ci-i5 alkoxyalkyl C 2 containing a nitrogen, oxygen or sulfur as a hetero atom - 15 heterocycloalkyl, a heteroatom C 2 containing a nitrogen, oxygen or sulfur-alkenyl 15 heterocycloalkyl, C 6 - 15 aryl, 15 alkenyl - d- 15 alkyl or C 2.
- the heterocycloalkyl at Y is hydroxy, halo,
- the heterocycloalkenyl alkyl of Y is (1H—imidazole-1-ly) alkyl.
- the 3 ⁇ 4 is hydrogen, halo, d- 15 aryl, alkoxy, or d- d- 15 s alkyl or C 2 - 15 alkenyl, and;
- R 2 radicals of hydroxy, halo, d- 5-alkoxy, 5 alkyl, C 2 - may be replaced by a 5 alkenyl, or a combination thereof.
- X in the formula A is imideamido ()
- 3 ⁇ 4 may be bonded to C or N 'of the imideamido.
- the compounds of the present invention may have one or more chiral centers and / or geometric isomeric centers, so that the present invention includes all stereoisomers represented by Formula A, ie, optical isomers, diastereomers, and geometric isomers. do.
- composition for inducing cell reprogramming of the present invention is a compound represented by the formula selected from the group consisting of the following Chemical Formulas 1 to 45:
- composition for inducing cell reprogramming of the present invention is the following formula (1), (25), (30), (31), (33), (34), (35) and (45).
- the present invention provides a compound represented by the following formula A as indazole der ivat ives:
- 3 ⁇ 4 is hydrogen, nitro, nitrooxy or Y-carbonyl;
- Y is hydrogen, hydroxyl, methoxy, halo, d-30 alkoxy, as a hetero atom nitrogen, oxygen or sulfur, C 2, including - 30 heterocycloalkyl, C 2 containing a nitrogen, oxygen or sulfur as the hetero atom-20 heteroaryl cycloalkenyl, C 6 - 30 aryl, d- 30 alkyl or C 2 - 30 alkenyl, and
- R 2 is hydrogen, hydroxy, halo, d-
- R 3 is hydrogen, hydroxy halo, d- 30 aryl, d-30-alkoxy, Cwo alkyl or C 2 - 30 alkenyl, and; X is amine, amido, sulfonamido, carbonyl, oxime or imideamido; With the proviso that when said nitro and said X are amido, R 2 is not phenyl; And wherein R 3 is hydrogen, X is N-hydroxy-imideamido and said phenyl, 3 ⁇ 4 does not bond to the carbon atom of the imideamido (or R 2 is an N 'bonds to an atom).
- Y is hydrogen, hydroxy, halo, C W5 alkoxy, C 2 containing a nitrogen, oxygen or sulfur as a hetero atom - C, including 15 heterocycloalkyl, nitrogen as a hetero atom, oxygen or sulfur 2 - 15 by interrogating cycloalkenyl, C 6 - 15-alkenyl-15-aryl, Ci-i5 alkyl or C 2.
- the heterocycloalkyl is a hydroxy halo, d- 5 alkyl, C 2 in the Y of the formula A - 5 alkenyl, d- 5 alkoxy, C 2 - 8 alkoxyalkyl, C 2 - 8 alkoxy Tero cycloalkenyl Kenyl alkyl, or
- heterocycloalkenyl alkyl is (1H-imidazole-1-ly) alkyl.
- R 2 is hydrogen, halo, d- 15 aryl, d- 15 alkoxy or d- 15 alkyl or C 2 — 15 alkenyl, wherein aryl in R 2 is hydroxy, halo , C- 5 alkoxy, d- 5 alkyl, C 2 - may be replaced by a 5 alkenyl, or a combination thereof.
- the hetero atom in Formula A is nitrogen.
- the indazole derivative compound of the present invention is a chemical formula selected from the group consisting of the following Chemical Formula 1, Chemical Formula 2 and Chemical Formulas 4 to 45 Is displayed:
- the indazole derivative compound of the present invention is represented by a formula selected from the group consisting of the following Formula 1, Formula 25, Formula 30, Formula 31, Formula 33, Formula 34, Formula 35 and Formula 45:
- the invention provides a method of inducing cell reprogramming by contacting a cell comprising a composition comprising a compound represented by Formula A:
- R is hydrogen, nitro, nitrooxy or Y- Carbonyl;
- Y is C 2 containing a nitrogen, oxygen or sulfur as hydrogen, hydroxy, halo, alkoxy, heteroatom-20 hetero cycloalkenyl-C 2 containing 30 heterocycloalkyl, nitrogen as a hetero atom, oxygen or sulfur , C 6 - 30 aryl, d- 30 alkyl or C 2 - 30 alkenyl, and; 3 ⁇ 4 is hydrogen, hydroxy, halo, C r-
- aryl (30 alkoxy, Cl-30 alkyl or C 2 - 30 alkenyl
- R 3 is hydrogen, hydroxy halo, 30 aryl, alkoxy, alkyl, or C 2 eu 30 alkenyl
- X is an amine, an amido , Sulfonamido, carbonyl, oxime or imideamido.
- the method of inducing cell reprogramming of the present invention is to use the cell reprogramming inducing composition of the present invention described above, and the common content between the two is to avoid excessive complexity of the specification according to the repetitive description. The description thereof is omitted.
- the present invention relates to a cell reprogramming inducing composition.
- the indazole derivative compounds of the present invention exhibit an improved biological profile and can perform efficient cell reprogramming.
- indazole derivative compounds reprogramming inducing compound conventional low molecular cell of the invention e.g., a river new or BI0
- cytotoxicity cytotoxicity
- FIGS. 1 and 2 show C2C12 mouse myoblasts of the present invention. The result of reprogramming into osteoblasts after contact with the compound is shown. After contact with the compound of the present invention LDD-1821, LDD-1664, LDD-1945, LDD-2199 or LDD-2200, it was observed by allenzarin red staining (ATDC5: cartilage progenitor cell line).
- Figure 3 shows C2C12 mouse myoblasts (myob l ast) in contact with a compound of the present invention, showing the results of allenzarin red assay.
- the graph shows the absorbance when each compound was treated.
- step (b-2) Synthetic Scheme 1, compound of Chemical Formula 2 (50 mg, 0.28 ⁇ l ol) was dissolved in pyridine (0.3 mL). Thereafter, R 2 -benzoylchloride (16.5 pL, 0.28 mmol) was added to the reaction mixture and stirred for 1 hour, followed by extraction with ethyl acetate and IN HC1.
- Synthetic Scheme 3 compound of Chemical Formula 11 (500 mg, 2.63 Korean ol) was dissolved in DMF (13 niL). Then, hydroxylamine hydrochloride (550 mg, 7.89 ⁇ ol) and triethylamine (110 L, 7.89 ⁇ ol) were added to the reaction mixture and stirred at 80 ° C. for 12 hours, followed by ethyl acetate and IN HC1 Extracted with. The combined organic layers are washed with brine and dried over anhydrous Na 2 S0 4 . After drying, it was concentrated in vacuo. The target compound was obtained for performing the next reaction without further purification. Synthesis Example 17: 5—Nitro-1H-indazole-3-carboxaldehyde oxime (Compound 17)
- 5-nitro-1H-indazole-3-carboxaldehyde oxime 500 mg, 2.44 ⁇ l ol was dissolved in DMF (12 mL). N-chlorosuccinimide (320 mg, 2.44 dl ol) was added to the reaction mixture and the mixture was stirred at 40 ° C. for 1 hour. The obtained residue was extracted with ethyl acetate and water. The combined organic layers were washed with brine, dried over anhydrous Na 2 SO 4 and concentrated in vacuo. The desired compound was obtained (yield 80%) without further purification.
- N-hydroxy-5-nitro-l ⁇ -indazole-3-carlimidoyl chloride (30 mg, 0.13 dl ol) was dissolved in ethanol (1.5 mL).
- Aniline 35 yL, 0.38 mmol was added and the mixture was stirred at 80 ° C for 2 h.
- the obtained residue was concentrated by removing ethanol and purified by silica gel chromatography to give the target compound (yield 33%).
- N-hydroxy-5-nitro-lH-indazole-3-carimidoyl chloride (30 mg, 0.13 mmol) was dissolved in ethanol (1.5 mL). Then, 4-polooaniline (37 yL, 0.38 mmol) was added and the mixture was stirred at 80 ° C. for 2 hours. The obtained residue was concentrated by removing ethanol and purified by silica gel chromatography to give the target compound (yield 33%).
- N-hydroxy-5nitro-lH-indazole-3-carlimidoyl chloride (30 mg, 0.13 dl ol) was dissolved in ethanol (1.5 mL). Subsequently, 4-hydroxy aniline (41 mg, 0.38 ⁇ l ol) was added and the mixture was stirred at 80 ° C. for 2 hours. The obtained residue was concentrated by removing ethanol and purified by silica gel chromatography to give the target compound (yield 33%).
- N-hydroxy-5-nitro-1H-indazole-3-carlimidoyl chloride (30 mg, 0.13 dl ol) was dissolved in ethanol (1.5 mL). Then, 4-methoxy aniline (41 mg, 47 ⁇ l ol) was added and the mixture was stirred at 80 ° C. for 2 hours. The obtained residue was concentrated by removing ethanol and purified by silica gel chromatography to give the target compound (yield 33%).
- Synthetic Scheme 4 compound of formula 2 (20 mg, 0.11 ⁇ l ol) was dissolved in 1,4-dioxane (1 mL). Then, R 2 -N-hydroxybenzimidyl chloride (0.17 mmol) and TEA (0.22 ⁇ L ol) were added to the solution. The mixture was stirred at room temperature for 18 hours and the residue obtained was extracted with ethyl acetate and saturated aqueous NH 4 C1 solution. Then purified by silica gel chromatography to give the target compound.
- Synthesis Example 42 (LDD-2194): 3- ( ⁇ '-hydroxybenzimidamido) -1 ⁇ -indazole-5-carboxylic acid (Compound 42)
- the method for finding compounds that induce cell reprogramming is based on previous studies of bone cell transformation of muscle cells through bone induction PMID: 23044010 and 18974773 and the reversine paper (Chen S 2004).
- C2C12 rat skeletal muscle cells were harvested at 1 ⁇ 10 4 per well in a 24-well cell culture dish. Dispense at density. 24 hours after dispensing, the compound of interest was added to three wells at a concentration of 1.
- As positive control cells were treated with 100 nM reversin or 2 uM BIO. Both compounds are known to induce bone cell turnover of myoblasts (Chen S 2004 and Kim WH recapitulate papers).
- Negative control treated the cells with DMS0 or none.
- Bone induction media were based on those previously described in direct cell reprogramming Oe ers / e papers, Chen S 2004 and Chen S 3 ⁇ 4? Direct cell reprogramming from muscle cells to bone cells was detected through alizarin red staining, which detects the presence or absence of calcium deposits (Gregory CA 2004: PMID: 15136169) in bone lineage cells.
- the cells were washed once with PBS and fixed for 20 minutes using phosphate-buffered formal in.
- the fixed cells were washed with distilled water and then immersed in 1% alizarin red-S (Sigma-Aldrich) dissolved in water for 5 minutes.
- the remaining dye solution was washed with distilled water.
- the cells were dried and photographed of the stained cells using an optical microscope (CKX41 Olympus). Stained cells appear as bright red areas, which were selected as 'hit' compounds for further analysis.
- the cell reprogramming ability of the synthesized indazole compound was evaluated using the assay method described above.
- LDD-1664, LDD-1821, LDD-1945, LDD-2199 and LDD-2200 showed reprogramming ability as strong as reversin, a positive control in differentiation into osteoblasts.
- LDD-1986, LDD-1987, LDD-1988 Also shown is the reprogramming ability. Differentiation into adipocytes In oil red 0 staining, LDD-1821, LDD-1986, LDD-1987 and LDD-1988 materials showed strong reprogramming ability.
- LDD-1821 showed strong reprogramming ability in differentiation into osteoblasts and adipocytes (see FIGS. 1, 2 and 3).
- HTRF assay is an assay method for confirming phosphorylation of peptide material in the presence of ATP. Phosphorylated material is TR-FRET (Time Resolved- Fluorescence Resonance) signal was detected.
- Recombinant GSK-3P and Aurora A kinase were purchased from Millipore (Bi 1 lerica, MA).
- Assays were carried out using the HTRF KinEASE kit (Cisbio), and the degree of phosphorylation inhibited after 1 ⁇ treatment of indazole was applied to GSK-3P and Aurora A, respectively.
- the assay consists of a substance-enzyme mixture and peptide material dissolved in kinase reaction buffer (250 mM HEPES (pH 7.0), 0.5 mM orthovanadate, 0.05% BSA, 0.1% NaN 3 ). After adding the detection buffer, the TR-FRET signal was detected by the EnVision multi-label reader.
- LDD-1664, LDD-1667, LDD-1820, and LDD-1821 were treated with GSK-3P and Aurora A by 1 ⁇ M, respectively, and the extent to which enzyme activity was inhibited is shown in the following table. As shown in Table 2, all four derivatives showed less than 50% inhibitory activity against both GSK-3P and Aurora A at 1 ⁇ concentration. In addition, LDD-1821, which showed excellent cell reprogramming ability, showed no inhibitory activity at 1 ⁇ M concentration for both enzymes. Therefore, it has been shown to induce cell reprogramming through different mechanisms from the inhibitors of GSK-3P or Aurora A, BI0 and reversin.
- C2C12 myoblasts were dispensed into 96 well plates at a density of 3xl0 3 cells per well. The cells were incubated for one day and then treated with the material. Then incubated for 2 days, then 3- (4, 5-dimethylthiazol-2-yl) -2,5- Serum-free DMEM containing diphenyltetrazolium bromide (MTT) was treated with cells for 3 hours. The medium was removed and incubated for 10 minutes at DMS0 with shaking. The absorbance is measured at 570 nm with a microreader (VersaMax,
- the cytotoxicity of the compounds summarized in the table below was evaluated through the ⁇ assay, and all of the indazole derivatives evaluated showed IC 50 of 10 ⁇ or more.
- the indazole derivatives have excellent advantages compared to BI0 and reversin, which have previously exhibited high cytotoxicity.
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ES15815352T ES2768803T3 (es) | 2014-07-01 | 2015-06-30 | Composición para inducir la reprogramación celular |
JP2016575756A JP6511075B2 (ja) | 2014-07-01 | 2015-06-30 | 細胞リプログラミング誘導用組成物 |
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US5244907A (en) * | 1992-03-26 | 1993-09-14 | A. H. Robins Company, Incorporated | Carbocyclic carboxylic acid amides and esters of azabicyclic compounds as gastric prokinetic, antiemetic, anxiolytic and antiarrhythmic agents |
JP3289177B2 (ja) * | 1995-07-07 | 2002-06-04 | コニカ株式会社 | 新規な写真用カプラー及びハロゲン化銀カラー写真感光材料 |
DE19810018A1 (de) * | 1998-03-09 | 1999-09-16 | Bayer Ag | Benzoheterocyclyloxime |
US7642278B2 (en) * | 2001-07-03 | 2010-01-05 | Novartis Vaccines And Diagnostics, Inc. | Indazole benzimidazole compounds |
US6897208B2 (en) | 2001-10-26 | 2005-05-24 | Aventis Pharmaceuticals Inc. | Benzimidazoles |
TW200306819A (en) * | 2002-01-25 | 2003-12-01 | Vertex Pharma | Indazole compounds useful as protein kinase inhibitors |
FR2836915B1 (fr) * | 2002-03-11 | 2008-01-11 | Aventis Pharma Sa | Derives d'aminoindazoles, procede de preparation et intermediaires de ce procede a titre de medicaments et compositions pharmaceutiques les renfermant |
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AU2003255767A1 (en) * | 2002-08-10 | 2004-02-25 | Astex Technology Limited | 3-(carbonyl) 1h-indazole compounds as cyclin dependent kinases (cdk) inhibitors |
FR2845382A1 (fr) * | 2002-10-02 | 2004-04-09 | Sanofi Synthelabo | Derives d'indazolecarboxamides, leur preparation et leur utilisation en therapeutique |
RU2417225C2 (ru) * | 2004-03-25 | 2011-04-27 | Мемори Фармасьютиклз Корпорейшн | Индазолы, бензотиазолы, бензоизотиазолы, бензизоксазолы и их получение и применение |
DE102004028862A1 (de) | 2004-06-15 | 2005-12-29 | Merck Patent Gmbh | 3-Aminoindazole |
US8008481B2 (en) * | 2006-03-31 | 2011-08-30 | Ericsson Anna M | Indazole compounds |
AU2007317435A1 (en) | 2006-11-02 | 2008-05-15 | Vertex Pharmaceuticals Incorporated | Aminopyridines and aminopyrimidines useful as inhibitors of protein kinases |
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CA2815169C (en) * | 2010-10-29 | 2015-10-06 | Pfizer Inc. | N1/n2-lactam acetyl-coa carboxylase inhibitors |
KR101744607B1 (ko) * | 2011-12-28 | 2017-06-08 | 후지필름 가부시키가이샤 | 신규인 니코틴아미드 유도체 또는 그 염 |
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US20130281399A1 (en) * | 2012-04-19 | 2013-10-24 | Rvx Therapeutics Inc. | Treatment of diseases by epigenetic regulation |
US20160096850A9 (en) * | 2013-08-22 | 2016-04-07 | Genentech, Inc. | Alkynyl alcohols and methods of use |
ES2703851T3 (es) * | 2014-08-22 | 2019-03-12 | Merck Patent Gmbh | Indazoles |
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US10858321B2 (en) | 2020-12-08 |
ES2768803T3 (es) | 2020-06-23 |
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US20170158642A1 (en) | 2017-06-08 |
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