METHOD FOR PRODUCING MEGAKARYOCYTES AND/OR PLATELETS FROM PLURIPOTENT STEM CELLS
An agent for inducing production of megakaryocytes and/or platelets from pluripotent stem cells which is useful for treatment of disease accompanied by a decrease in platelets is provided. A method for producing megakaryocytes and/or platelets, including separating hematopoietic progenitor cells from the septal cells in sac-like structures produced by pluripotent stem cells, and culturing the hematopoietic progenitor cells ex vivo in the presence of a compound represented by the formula (I) where R 1 to R 7 , W, X, Y, Z, Ar 1 and n are as defined in the description to differentiate them into megakaryocytes and/or platelets.
1 . A method for producing a megakaryocyte, a platelet, or both, comprising:
culturing a hematopoietic progenitor cell derived from a pluripotent stem cell ex vivo in the presence of a compound of formula (I), a tautomer, prodrug or pharmaceutically acceptable salt of the compound or a solvate thereof; and
differentiating the hematopoietic progenitor cell into a megakaryocyte, a platelet, or both,
wherein W is a substituent of formula (Ia) or a carboxy group:
R 1 , R 2 , R 3 and R 4 are each independently a C 1-10 alkyl group which may be substituted with a halogen atom or a hydrogen atom,
n is an integer of 0, 1, 2 or 3,
R 5 is a C 2-14 aryl group which may be substituted with a substituent independently represented by V 1 , where when n is 2, R 5 is not an unsubstituted pyridyl group,
R 6 is a C 1-10 alkyl group which may be substituted with a halogen atom or a hydrogen atom,
R 7 is a C 2-14 aryl group which may be substituted with a substituent independently represented by V 2 ,
Ar 1 is a C 2-14 arylene group which may be substituted with a substituent independently represented by V 3 ,
X is —OR 20 ,
Y and Z are ach independently an oxygen atom or a sulfur atom,
V 1 is —(CH 2 )m 1 M 1 NR 8 R 9 , —(CH 2 )m 6 NR 16 R 17 , -M 2 NR 18 (CH 2 )m 7 R 19 or —C(═O)-(piperazine-1,4-diyl)-U,
V 2 , V 3 and V 4 are each independently a hydroxy group, a protected hydroxy group, an amino group, a protected amino group, a thiol group, a protected thiol group, a nitro group, a cyano group, a halogen atom, a carboxy group, a carbamoyl group, a sulfamoyl group, a sulfo group, a formyl group, a C 1-3 alkoxy group which may be substituted with a halogen atom, a C 1-10 alkyl group which may be substituted with a halogen atom, a C 2-6 alkenyl group, a C 2-6 alkynyl group, a C 1-10 alkylcarbonyloxy group, a C 1-10 alkoxycarbonyl group, a C 1-10 alkoxy group, a C 1-10 alkylcarbonyl group, a C 1-10 alkylcarbonylamino group, a mono- or di-C 1-10 alkylamino group, a C 1-10 alkylsulfonyl group, a C 1-10 alkylaminosulfonyl group, a C 1-10 alkylaminocarbonyl group, a C 1-10 alkylsulfonylamino group or a C 1-10 thioalkyl group,
M 1 and M 2 are each independently —(C═O)— or —(SO 2 )—,
m 1 is an integer of 0, 1 or 2,
m 2 , m 3 , m 4 , m 5 , m 6 and m 7 are each independently an integer of 1 or 2,
R 8 is a hydrogen atom or a C 1-3 alkyl group,
R 9 and U are each independently —(CH 2 )m 2 OR 16 or —(CH 2 )m 4 NR 11 R 12 , provided that when m 1 is 1 or 2, R 9 may be any of those mentioned above or a hydrogen atom,
R 10 is a hydrogen atom, a C 1-3 alkyl group or —(CH 2 )m 3 T,
R 11 and R 12 are each independently a hydrogen atom or —(CH 2 )m 5 Q, or N, R 11 and R 12 mean, as a whole, a substituent of formula (II):
or a substituent of formula (III):
T is a hydroxy group, a C 1-6 alkoxy group or a C 1-6 alkyl group,
Q is a hydroxy group, a C 1-3 alkoxy group or —NR 13 R 14 ,
R 13 and R 14 are each independently a hydrogen atom or a C 1-3 alkyl group,
R 15 is a hydrogen atom, a C 1-3 alkyl group or an amino-protecting group,
R 16 and R 17 are each independently a hydrogen atom, a C 1-3 alkylcarbonyl group or a C 1-3 alkylsulfonyl group,
R 18 is a hydrogen atom or a C 1-3 alkyl group,
R 19 is a C 2-9 heterocyclyl group or a C 2-14 aryl group, and
R 20 is a hydrogen atom, a C 1-10 alkyl group which may be substituted with a substituent independently represented by V 4 or a C 1-10 alkylcarbonyl group which may be substituted with a substituent independently represented by V 4 .
2 . The method according to claim 1 , wherein W is a substituent of formula (Ia):
3 . The method according to claim 2 , wherein R 1 is a hydrogen atom or a C 1-6 alkyl group which may be substituted with a halogen atom,
R 2 , R 3 , R 4 and R 6 are each independently a hydrogen atom or a C 1-3 alkyl group,
n is an integer of 1 or 2,
Ar 1 is of formula (IV):
R 7 is a phenyl group which may be substituted with at least one substituent selected from the group consisting of C 1-10 alkyl groups which may be substituted with a halogen atom, C 1-10 alkoxy groups, C 1-3 alkoxy groups substituted with a halogen atom and halogen atoms,
X is —OH, and
Y and Z are oxygen atoms.
4 . The method according to claim 3 , wherein R 2 , R 3 , R 4 and R 6 are hydrogen atoms.
5 . The method according to claim 2 , wherein R 5 is a phenyl group which may be substituted with a substituent independently represented by V 1 .
6 . The method according to claim 2 , wherein R 5 is a C 2-9 heteroaryl group which may be substituted with a substituent independently represented by V 1 .
7 . The method according to claim 6 , wherein the C 2-9 heteroaryl group is a C 2-9 nitrogen-containing heteroaryl group.
8 . The method according to claim 7 , wherein the C 2-9 nitrogen-comprising heteroaryl group is selected from the group consisting of a 2-pyridyl group, a 3-pyridyl group, a 4-pyridyl group, a 3-pyridazinyl group, a 4-pyridazinyl group, a 2-pyrimidinyl group, a 4-pyrimidinyl group, a 5-pyrimidinyl group and a 2-pyrazinyl group.
9 . The method according to claim 7 , wherein the C 2-9 nitrogen-containing heteroaryl group is a 4-pyridyl group.
10 . The method according to claim 2 , wherein V 1 is any one of formulae (V) to (XXII):
11 . The method according to claim 3 , wherein R 5 is a phenyl group substituted with a substituent of formula (VIII):
12 . The method according to claim 3 , wherein R 5 is a 4-pyridyl group.
13 . The method according to claim 2 , wherein n is an integer of 1.
14 . The method according to claim 2 , wherein R 7 is a phenyl group substituted with at least one substituent selected from the group consisting of methyl groups, t-butyl groups, halogen atoms, methoxy groups, trifluoromethyl groups and trifluoromethoxy groups.
15 . The method according to claim 2 , wherein R 7 is a phenyl group which may be substituted with one or two halogen atoms.
16 . The method according to claim 2 , wherein R 1 is a methyl group.
17 . The method according to claim 2 , wherein the compound of formula (I) is (E)-5-(2-{1-[5-(3,4-dichlorophenyl)-4-hydroxythiophen-3-yl]ethylidene}hydrazinecarbonyl)-N-[4-(2-hydroxyethylcarbamoyl)benzyl]thiophene-2-carboxamide, (E)-5-(2-{1-[5-(4-bromophenyl)-4-hydroxythiophen-3-yl]ethylidene}hydrazinecarbonyl)-N-[4-(2-hydroxyethylcarbamoyl)benzyl]thiophene-2-carboxamide or (E)-5-(2-{1-[5-(3,4-dichlorophenyl)-4-hydroxylthiophen-3-yl]ethylidene}hydrazinecarbonyl)-N-(pyridin-4-ylmethyl)thiophene-2-carboxamide.
18 . The method according to claim 1 , wherein W is a carboxy group.
19 . The method according to claim 18 , wherein R 1 is a hydrogen atom or a C 1-6 alkyl group which may be substituted with a halogen atom,
R 6 is a hydrogen atom or a C 1-3 alkyl group which may be substituted with a halogen atom,
R 7 is a C 2-14 aryl group
X is —OH,
Y is an oxygen atom or a sulfur atom, and
Ar 1 is of formula (IV):
20 . The method according to claim 19 , wherein R 1 is a hydrogen atom or a C 1-6 alkyl group,
R 6 is a hydrogen atom,
R 7 is a substituent of any one of formulae (A01) to (A15):
and
Y is an oxygen atom.
21 . The method according to claim 1 , wherein the compound of formula (I) is (E)-5-(2-{1-[5-(2,3-dihydro-1H-indene-5-yl)-4-hydroxythiophen-3-yl]ethylidene}hydrazinecarbonyl)thiophene-2-carboxylic acid.
22 . The method according to claim 1 , wherein R 1 is a hydrogen atom or a C 1-6 alkyl group which may be substituted with a halogen atom,
R 2 , R 3 , R 4 and R 6 are each independently a hydrogen atom or a C 1-3 alkyl group,
n is an integer of 1 or 2,
R 5 is a phenyl group or a C 2-9 heteroaryl group which may be substituted with a substituent independently represented by V 1 ,
R 7 is a phenyl group which may be substituted with at least one substituent selected from the group consisting of C 1-10 alkyl groups which may be substituted with a halogen atom, C 1-10 alkoxy groups, C 1-3 alkoxy groups substituted with a halogen atom and halogen atoms or a substituent of any one of formulae (A01) to (A15):
Ar 1 is represented by the formula (IV):
X is —OH, and
Y and Z are each independently an oxygen atom or a sulfur atom.
23 . The method according to claim 22 , wherein R 1 is a hydrogen atom or a C 1-6 alkyl group,
R 2 , R 3 , R 4 and R 6 are hydrogen atoms,
n is an integer of 1,
R 5 is a pyridyl group, a pyrazinyl group or a phenyl group substituted with a substituent of formula (VII), (VIII), (XI) or (XII):
R 7 is a phenyl group which may be substituted with a halogen atom or C 1-10 alkyl groups or a substituent of formula (A11), (A13) or (A15):
and
Y and Z are oxygen atoms.
24 . The method according to claim 1 , wherein the compound of formula (I) is (E)-5-(2-{1-[5-(3,4-dichlorophenyl)-4-hydroxythiophen-3-yl]ethylidene}hydrazinecarbonyl)-N-[4-(2-hydroxyethylcarbamoyl)benzyl]thiophene-2-carboxamide, (E)-5-(2-{1-[5-(4-bromophenyl)-4-hydroxythiophen-3-yl]ethylidene}hydrazinecarbonyl)-N-[4-(2-hydroxyethylcarbamoyl)benzyl]thiophene-2-carboxamide, (E)-5-(2-{1-[5-(3,4-dichlorophenyl)-4-hydroxythiophen-3-yl]ethylidene}hydrazinecarbonyl)-N-(pyridin-4-ylmethyl)thiophene-2-carboxamide, (E)-5-(2-{1-[5-(2,3-dihydro-1H-inden-5-yl)-4-hydroxythiophen-3-yl]ethylidene}hydrazinecarbonyl)thiophene-2-carboxylic acid, potassium (E)-2-(3,4-dichlorophenyl)-4-[1-(2-{5-[(pyrazin-2-ylmethyl)carbamoyl]thiophene-2-carbonyl}hydrazono)ethyl]thiophen-3-olate, (E)-5-(2-{1-[5-(4-bromophenyl)-4-hydroxythiophen-3-yl]ethylidene}hydrazinecarbonyl)-N-{4-[2-(piperazin-1-yl)ethylcarbamoyl]benzyl}thiophene-2-carboxamide, (E)-N-[4-(2-amino-2-oxoethyl)benzyl]-5-(2-{1-[5-(3,4-dichlorophenyl)-4-hydroxythiophen-3-yl]ethylidene}hydrazinecarbonyl)thiophene-2-carboxamide or (E)-N-(4-{2-[bis(2-hydroxyethyl)amino]ethylcarbamoyl} benzyl)-5-(2-{1-[5-(4-t-butylphenyl)-4-hydroxythiophen-3-yl]ethylidene}hydrazinecarbonyl)thiophene-2-carboxamide.
25 . The method according to claim 1 , wherein the pluripotent stem cell is an ES cell or iPS cell.
26 . The method according to claim 1 , wherein the hematopoietic progenitor cell derived from the pluripotent stem cell is a hematopoietic progenitor cell obtained from a sac-like structure formed by differentiating a pluripotent stem cell into a hematopoietic progenitor cell.
27 . The method according to claim 1 , wherein the hematopoietic progenitor cell derived from the pluripotent stem cell has at least one introduced gene selected from the group consisting of oncogene, polycomb gene, apoptosis suppressor gene and a gene which suppresses a tumor suppressor gene and has proliferative capability, differentiative capability, or both, enhanced by regulation of expression of the introduced genes.
28 . The method according to claim 1 , wherein the hematopoietic progenitor cell derived from the pluripotent stem cell is a hematopoietic progenitor cell which has at least one introduced gene selected from the group consisting of MYC family gene, Bmi1 gene, BCL2 family gene and a gene which suppress a p53 gene expression and has proliferative capability, differentiative capability, or both, enhanced by regulation of expression of the introduced genes.
29 . A megakaryocyte, platelet, or both, obtained by the method according to claim 1 .
30 . A blood preparation comprising a platelet obtained by the method according to claim 1 , as an active ingredient.
31 . A kit suitable for producing a platelet by the method according to claim 1 .