1
DESCRIPTION
TITLE OF INVENTION:
METHOD FOR PRODUCING MEGAKARYOCYTES AND/OR PLATELETS FROM
PLURIPOTENT STEM CELLS
5
TECHNICAL FIELD
The present invention relates to a method for producing megakaryocytes and/or
platelets from pluripotent stem cells. In particular, it relates to a method for efficiently
producing megakaryocytes and/or platelets by culturing hematopoietic progenitor cells
derived from iPS cells (induced pluripotent stem cells) or ES cells (Embryonic ste10 m
cells) in the presence of a compound having a platelet expanding activity.
BACKGROUND ART
For treatment of blood-related diseases including leukemia, it is extremely
15 important to supply therapeutically necessary amounts of blood cells stably by cell
expansion. Among blood cells, platelets are essential for blood coagulation and
hematostasis and, hence, are in high demand for leukemia, bone marrow
transplantation, thrombocytopenia, anticancer therapy and the like. To date, platelets
have been supplied from blood collected from blood donors. However, it is sometimes
20 difficult to supply platelets to patients stably by blood donation from donors because of
the risk of virus transmission, the chronic shortage of donors and the inviability of
collected platelets during long term storage. Apart from blood donation from donors ,
other approaches such as administration of thrombopoietin (TPO) to patients and
differentiation of megakaryocytes in umbilical cord blood or myelocytes were attempted
25 However, TPO administration to patients has not come into practical use because of
generation of antibodies neutralizing TPO after TPO administration. In recent years, ex
vivo platelet production techniques have been studied to replace blood transfusion by
returning platelets produced ex vivo by culturing hematopoietic stem cells and
hematopoietic progenitor cells into living bodies. Development of these techniques into
30 ex vivo production of large amounts of platelets is expected to dispense with the current
blood donation system and almost solve the problems of the shortage of platelet
products and the virus risk. Though as the source of hematopoietic stem cells and
hematopoietic progenitor cells, bone marrow, umbilical cord blood and peripheral blood
are known, it is difficult to stably produce and supply large amounts of platelets from
35 these sources, because hematopoietic stem cells and hematopoietic progenitor cells
which can produce megakaryocytes and platelets can be obtained only in small
numbers from these sources.
In recent years, for ex vivo platelet production, several reports have been made
on efficient differentiation of hematopoietic stem cells and hematopoietic progenitor cells
40 derived from ES cells (Embryonic stem cells) into magakaryocytes and platelets. Eto
et al. demonstrated that coculture with OP9 stromal cells induces mouse ES cells to
differentiate into megakaryocytes (Non-Patent Document 1). Fujimoto et al. reported
that they confirmed induction of platelets by using the same system as Eto et al. (Non-
Patent Document 2). Successful induction of differentiation of primate ES cells into
45 megakaryocytes (Non-Patent Document 3) and successful induction of platelets from
human ES cells (Non-Patent Document 4) were reported. However, even if production
of platelets from ES cells is established to a clinically applicable level, transfusion of ES
cell-derived platelets to patents still has the problem of human leukocyte antigen (HLA)
2
compatibility (in the cases of frequent transfusions into the same patient, though not in
the case of the initial transfusion).
iPS cells (Induced pluripotent stem cells) are also called artificial pluripotent stem
cells or induced pluripotent stem cells and are cells derived from somatic cells such as
fibroblasts which have acquired pluripotency equivalent to that of ES cells b5 y
transduction of several transcription factor genes. Mouse iPS cells were established
for the first time by Yamanaka et al. by transduction of four genes, Oct3/4, Sox2, Klf4
and c-Myc, into mouse fibroblasts, using the expression of Nanog gene important for
maintenance of pluripotency as a marker (Non-Patent Document 5). Later,
10 establishment of mouse iPS cells by similar methods was reported (Non-Patent
Document 6 and Non-Patent Document 7). Further, it was reported that iPS cells were
established by transduction of only the three genes other than c-Myc (Oct3/4, Sox2 and
Klf4) to solve the problem of tumorigenesis of iPS cells (Non-Patent Document 8).
With respect to human iPS cells, Thomson et al. established human iPS cells by
15 transduction of OCT3/4, SOX2, NANOG and LIN28 into human fibroblasts (Non-Patent
Document 9). Yamanaka et al. also established human iPS cells by transduction of
OCT3/4, SOX2, KLF4 and c-MYC into human fibroblasts (Non-Patent Document 9).
iPS cells are expected to solve the problems with ex vivo platelet production such as
insufficient quantities of hematopoietic stem cells and hematopoietic progenitor cells in
20 bone marrow and umbilical cord blood, ethical issues and the problem of rejection in
terms of using ES cells. In a study made from such a perspective, success in induction
of differentiation of human iPS cells into platelets was reported (Patent Document 1),
and addition of proteins such as TPO is effective for induction of differentiation into
megakaryocytes and platelets is suggested.
25 Recent years have seen reports that low-molecular-weight compounds
synthesized through organic chemistry are effective as therapeutic drugs for
thrombocytopenia (Patent Documents 2 and 3) and effective for ex vivo expansion of
hematopoietic stem cells (Patent Documents 4, 5, 6, 7 and 8).
30 PRIOR ART DOCUMENT
PATENT DOCUMENTS
Patent Document 1: WO 2009/122747
Patent Document 2: WO 2004/108683
Patent Document 3: WO 2007/010954
35 Patent Document 4: WO 2009/072624
Patent Document 5: WO 2009/072625
Patent Document 6: WO 2009/072626
Patent Document 7: WO 2009/072635
Patent Document 8: WO 2010/140685
40 NON-PATENT DOCUMENTS
Non-Patent Document 1: Eto et al., Proc. Acad. Sci. USA 2002, 99: 12819-
12824.
Non-Patent Document 2: Fujimoto et al., Blood 2003, 102: 4044-4051.
Non-Patent Document 3: Hiroyama et al. Exp. Hematol. 2006, 34: 760-769.
45 Non-Patent Document 4: Takayama et al., Blood 2008, 111: 5298-5306.
Non-Patent Document 5: Okita et al., Nature 2007, 448: 313-317.
Non-Patent Document 6: Wernig et al., Nature 2007, 448: 318-324.
Non-Patent Document 7: Maherali et al., Cell Stem Cell 2007, 1: 55-70.
3
Non-Patent Document 8: Nakagawa et al., Nat Biotechnol 2008, 26: 101-106.
Non-Patent Document 9: Yu et al., Science 2007, 318: 1917-1920.
Non-Patent Document 10: Takahashi et al., Cell 2007, 131: 861-872.
DISCLOSURE OF THE INVENTIO5 N
TECHNICAL PROBLEM
An object of the present invention is to establish a method for obtaining
megakaryocytes and platelets from pluripotent stem cells, in particular, to establish a
method for obtaining megakaryocytes and platelets with stable efficiency.
10
SOLUTION TO PROBLEMS
The present inventors have conducted intensive studies to solve the abovementioned
object in search for compounds capable of inducing megakaryopoiesis and
thrombopoiesis from pluripotent stem cells and found out that the compounds
15 represented by the following formula (I) have excellent megakaryopoietic and
thrombopoietic activity even in the absence of TPO and that megakaryocytes and/or
platelets can be produced ex vivo stably and efficiently. The present invention was
accomplished on the basis of this discovery.
Namely, the present invention provides the following methods [1] to [30],
20 megakaryocytes and/or platelets [31], blood preparation [32] and kit [33].
[1] A method for producing megakaryocytes and/or platelets, comprising culturing
hematopoietic progenitor cells derived from pluripotent stem cells ex vivo in the
presence of a compound represented by the formula (I), a tautomer, prodrug or
pharmaceutically acceptable salt of the compound or a solvate thereof and
25 differentiating the hematopoietic progenitor cells into megakaryocytes and/or platelets;
wherein W is a substituent represented by the formula (Ia) or a carboxy group:
each of R1, R2, R3 and R4 is independently a C1-10 alkyl group which may be substituted
with one or more halogen atoms or a hydrogen atom,
30 n is an integer of 0, 1, 2 or 3,
R5 is a C2-14 aryl group which may be substituted with one or more substituents
independently represented by V1, provided that when n is 2, R5 is not an unsubstituted
4
pyridyl group,
R6 is a C1-10 alkyl group which may be substituted with one or more halogen atoms or a
hydrogen atom,
R7 is a C2-14 aryl group which may be substituted with one or more substituents
independently represented by V25 ,
Ar1 is a C2-14 arylene group which may be substituted with one or more substituents
independently represented by V3,
X is -OR20,
each of Y and Z is independently an oxygen atom or a sulfur atom,
10 V1 is -(CH2)m1M1NR8R9, -(CH2)m6NR16R17, -M2NR18(CH2)m7R19 or -C(=O)-(piperazine-
1,4-diyl)-U,
each of V2, V3 and V4 is 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
15 sulfamoyl group, a sulfo group, a formyl group, a C1-3 alkoxy group which may be
substituted with one or more halogen atoms, a C1-10 alkyl group which may be
substituted with one or more halogen atoms, a C2-6 alkenyl group, a C2-6 alkynyl group,
a C1-10 alkylcarbonyloxy group, a C1-10 alkoxycarbonyl group, a C1-10 alkoxy group, a C1-
10 alkylcarbonyl group, a C1-10 alkylcarbonylamino group, a mono- or di-C1-10 alkylamino
20 group, a C1-10 alkylsulfonyl group, a C1-10 alkylaminosulfonyl group, a C1-10
alkylaminocarbonyl group, a C1-10 alkylsulfonylamino group or a C1-10 thioalkyl group,
each of M1 and M2 is independently -(C=O)- or -(SO2)-,
m1 is an integer of 0, 1 or 2,
each of m2, m3, m4, m5, m6 and m7 is independently an integer of 1 or 2,
25 R8 is a hydrogen atom or a C1-3 alkyl group,
each of R9 and U is independently -(CH2)m2OR10 or -(CH2)m4NR11R12, provided that
when m1 is 1 or 2, R9 may be any of those mentioned above or a hydrogen atom,
R10 is a hydrogen atom, a C1-3 alkyl group or -(CH2)m3T,
each of R11 and R12 is independently a hydrogen atom or -(CH2)m5Q, or N, R11 and R12
30 mean, as a whole, a substituent represented by the formula (II):
or a substituent represented by the formula (III):
T is a hydroxy group, a C1-6 alkoxy group or a C1-6 alkyl group,
Q is a hydroxy group, a C1-3 alkoxy group or -NR13R14,
each of R13 and R14 is independently a hydrogen atom or a C1-3 alkyl group,
35 R15 is a hydrogen atom, a C1-3 alkyl group or an amino-protecting group,
each of R16 and R17 is independently a hydrogen atom, a C1-3 alkylcarbonyl group or a
C1-3 alkylsulfonyl group,
R18 is a hydrogen atom or a C1-3 alkyl group,
R19 is a C2-9 heterocyclyl group or a C2-14 aryl group, and
40 R20 is a hydrogen atom, a C1-10 alkyl group which may be substituted with one or more
substituents independently represented by V4 or a C1-10 alkylcarbonyl group which may
be substituted with one or more substituents independently represented by V4.
N O (II)
,
5
[2] The method according to [1], wherein W is a substituent represented by the
formula (Ia):
.
[3] The method according to [2], wherein R1 is a hydrogen atom or a C1-6 alkyl group
which may be substituted with one or more halogen atom5 s,
each of R2,R3,R4 and R6 is independently a hydrogen atom or a C1-3 alkyl group,
n is an integer of 1 or 2,
Ar1 is represented by the formula (IV):
（IV)
S ,
10 R7 is a phenyl group which may be substituted with one or more substituents selected
from the group consisting of C1-10 alkyl groups which may be substituted with one or
more halogen atoms, C1-10 alkoxy groups, C1-3 alkoxy groups substituted with one or
more halogen atoms and halogen atoms,
X is -OH, and
15 Y and Z are oxygen atoms.
[4] The method according to [3], wherein R2, R3, R4 and R6 are hydrogen atoms.
[5] The method according to any one of [2] to [4] , wherein R5 is a phenyl group which
may be substituted with one or more substituents independently represented by V1.
[6] The method according to any one of [2] to [4], wherein R5 is a C2-9 heteroaryl
20 group which may be substituted with one or more substituents independently
represented by V1.
[7] The method according to [6], wherein the C2-9 heteroaryl group is a C2-9 nitrogencontaining
heteroaryl group.
[8] The method according to [7], wherein the C2-9 nitrogen-containing heteroaryl group
25 is selected from 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 [7], wherein the C2-9 nitrogen-containing heteroaryl group
is a 4-pyridyl group.
30 [10] The method according to any one of [2] to [9], wherein V1 is represented by any
one of the formulae (V) to (XXII):
7
O
HN
OH
（VIII）
.
[12] The method according to [3] or [4], wherein R5 is a 4-pyridyl group.
[13] The method according to any one of [2] to [12], wherein n is an integer of 1.
[14] The method according to any one of [2] to [13], wherein R7 is a phenyl group
substituted with one or more substituents selected from methyl groups, t-butyl group5 s,
halogen atoms, methoxy groups, trifluoromethyl groups and trifluoromethoxy groups.
[15] The method according to any one of [2] to [13], wherein R7 is a phenyl group
which may be substituted with one or two halogen atoms.
[16] The method according to any one of [2] to [15], wherein R1 is a methyl group.
10 [17] The method according to Claim 2, wherein the compound represented by the
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-
15 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 [1], wherein W is a carboxy group.
[19] The method according to [18], wherein R1 is a hydrogen atom or a C1-6 alkyl group
which may be substituted with one or more halogen atoms,
20 R6 is a hydrogen atom or a C1-3 alkyl group which may be substituted with one or more
halogen atoms,
R7 is a C2-14 aryl group
X is -OH,
Y is an oxygen atom or a sulfur atom, and
25 Ar1 is represented by the formula (IV):
（IV)
S .
[20] The method according to [19], wherein R1 is a hydrogen atom or a C1-6 alkyl group,
R6 is a hydrogen atom,
R7 is a substituent represented by any one of the formulae (A01) to (A15):
8
, and
Y is an oxygen atom.
[21] The method according to [20], wherein R1 is a C1 - 6 alkyl group, and
R7 is a substituent represented by the formula (A11):
[22] The method according to [1], wherein the compound represented by the formul5 a
(I) is (E)-5-(2-{1-[5-(2,3-dihydro-1H-indene-5-yl)-4-hydroxythiophen-3-
yl]ethylidene}hydrazinecarbonyl)thiophene-2-carboxylic acid.
[23] The method according to [1], wherein R1 is a hydrogen atom or a C1-6 alkyl group
which may be substituted with one or more halogen atoms,
10 each of R2, R3, R4 and R6 is independently a hydrogen atom or a C1-3 alkyl group,
n is an integer of 1 or 2,
R5 is a phenyl group or a C2-9 heteroaryl group which may be substituted with one or
more substituents independently represented by V1,
R7 is a phenyl group which may be substituted with one or more substituents selected
15 from C1-10 alkyl groups which may be substituted with one or more halogen atoms, C1-10
alkoxy groups, C1-3 alkoxy groups substituted with one or more halogen atoms and
halogen atoms or a substituent represented by any one of the formulae (A01) to (A15):
.
9
,
Ar1 is represented by the formula (IV):
（IV)
S ,
X is -OH, and
each of Y and Z is independently an oxygen atom or a sulfur atom5 .
[24] The method according to [22], wherein R1 is a hydrogen atom or a C1-6 alkyl group,
R2, R3, R4 and R6 are hydrogen atoms,
n is an integer of 1,
R5 is a pyridyl group, a pyrazinyl group or a phenyl group substituted with a substituent
10 represented by the formula (VII), (VIII), (XI) or (XII):
10
O
HN
N
OH
OH
O
NH2
（X Ｉ） （X Ｉ Ｉ）
O
HN
OH
（VIII）
O
HN
N NH
（VII）
,
R7 is a phenyl group which may be substituted with one or two halogen atoms or C1-10
alkyl groups or a substituent represented by the formula (A11), (A13) or (A15):
, an5 d
Y and Z are oxygen atoms.
[25] The method according to [24], wherein R1 is a C1 - 6 alkyl group, and
R7 is a group represented by the formula (A11):
[26] The method according to [1], wherein the compound represented by the formula
10 (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-
15 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-
20 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-
.
11
hydroxythiophen-3-yl]ethylidene}hydrazinecarbonyl)thiophene-2-carboxamide.
[27] The method according to any one of [1] to [26], wherein the pluripotent stem cells
are ES cells or iPS cells.
[28] The method according to any one of [1] to [27], wherein the hematopoietic
progenitor cells derived from pluripotent stem cells are hematopoietic progenitor cell5 s
obtained from a sac-like structure formed by differentiating pluripotent stem cells into
hematopoietic progenitor cells.
[29] The method according to any one of [1] to [28], wherein the hematopoietic
progenitor cells derived from pluripotent stem cells have one or more introduced genes
10 selected from oncogenes, polycomb genes, apoptosis suppressor genes and genes
which suppress a tumor suppressor gene and have proliferative and/or differentiative
capability enhanced by regulation of expression of the introduced genes.
[30] The method according to any one of [1] to [29], wherein the hematopoietic
progenitor cells derived from pluripotent stem cells are hematopoietic progenitor cells
15 which have one or more introduced genes selected from MYC family genes, Bmi1
genes, BCL2 family genes and genes which suppress the p53 gene expression and
have proliferative and/or differentiative capability enhanced by regulation of expression
of the introduced genes.
[31] Megakaryocytes and/or platelets obtained by the method as defined in any one of
20 [1] to [30].
[32] A blood preparation containing platelets obtained by the method as defined in any
one of [1] to [30], as an active ingredient.
[32] A kit for producing platelets by the method as defined in any one of [1] to [30].
25 ADVANTAGEOUS EFFECT(S) OF INVENTION
The present invention makes it possible to induce megakaryocytes and platelets
from hematopoietic progenitor cells derived from pluripotent stem cells (especially,
human iPS cells or human ES cells) by using the compounds represented by the
formula (I), tautomers, prodrugs or pharmaceutically acceptable salts of the compounds
30 or solvates thereof (which will be collectively referred to as specific compounds).
When used in culture of hematopoietic progenitor cells derived from pluripotent stem
cells, the specific compounds induce megakaryocytes and platelets more stably and
more efficiently than proteins such as TPO. Namely, the method of the present
invention realizes stable blood preparations containing platelets as an active ingredient.
35 The specific compounds are low-molecular-weight compounds obtainable by
ordinary processes for organic synthesis and hence, are easy to produce under
conditions which preclude microbial cell survival. Therefore, the method for producing
platelet using the specific compounds can prevent contamination with an unknown
pathogen or a biomaterial from an nonhuman animal more easily than conventional
40 methods using proteins such as cytokines and growth factors obtained by gene
recombination technology. Namely, platelets produced by the method of the present
invention can avoid infections, contamination with foreign genes or immune response to
foreign proteins. While being proteins, cytokines and growth factors can be stored or
used within very narrow optimal ranges in terms of pH, temperature and ion strength,
45 the specific compounds can be used and stored under relatively broad ranges of
conditions. In addition, because the specific compounds can be produced
continuously at low costs, unlike proteins, it is possible to eventually reduce treatment
cost.
12
DESCRIPTION OF DRAWING(S)
[Fig. 1] A graph showing that megakaryocytes (CD41a+ CD42b+ cells) were expanded
more remarkably in a culture of iPS cell-induced hematopoietic progenitor cells in the
presence of specific compounds than in the presence of TPO. The ordinance of th5 e
graph is the number of megakaryocytes (CD41a+ CD42b+ cells) in the presence of the
specific compounds relative to that in the absence of the compounds.
[Fig. 2] A graph showing megakaryocytes (CD41a+ CD42b+ cells) were expanded more
remarkably in a culture of iPS cell-induced hematopoietic progenitor cells in the
10 presence of specific compounds than in the presence of TPO. The ordinance of the
graph is the number of megakaryocytes (CD41a+ CD42b+ cells) in the presence of the
specific compounds relative to that in the presence of TPO.
[Fig. 3] A graph showing that platelets (CD41a+ CD42b+ cells) were expanded more
remarkably in a culture of iPS cell-derived hematopoietic progenitor cells in the
15 presence of specific compounds than in the presence of TPO. The ordinance of the
graph is the number of platelets (CD41a+ CD42b+ cells) in the presence of the specific
compounds relative to that in the absence of the compounds.
[Fig. 4] A graph showing platelets (CD41a+ CD42b+ cells) were expanded more
remarkably in a culture of iPS cell-derived hematopoietic progenitor cells in the
20 presence of specific compounds than in the presence of TPO. The ordinance of the
graph is the number of platelets (CD41a+ CD42b+ cells) in the presence of the specific
compounds relative to that in the presence of TPO.
[Fig. 5] A graph showing integrin activation (PAC-1 binding to platelets) by ADP on
platelets (CD41a+ CD42b+ cells) prepared from iPS cells in the presence of specific
25 compounds. The ordinate of the graph is the PAC-binding to the platelets relative to
the PAC-binding to platelets from peripheral blood.
[Fig. 6] A graph showing platelets (CD41a+ CD42b+ cells) were expanded more
remarkably in a culture of ES cell-derived hematopoietic progenitor cells in the presence
of a specific compound than in the presence of TPO. The ordinance of the graph is the
30 number of platelets (CD41a+ CD42b+ cells) in the presence of the specific compounds
relative to that in the absence of the specific compound.
[Fig. 7] A graph showing platelets (CD41a+ CD42b+ cells) were expanded more
remarkably in a culture of genetically manipulated hematopoietic progenitor cells with
enhanced proliferative and differentiative capability in the presence of a specific
35 compound than in the presence of TPO. The ordinance of the graph is the number of
platelets (CD41a+ CD42b+ cells) in the presence of the specific compound relative to
that in the presence of TPO.
DESCRIPTION OF EMBODIMENT(S)
40 Now, the present invention will be described in detail.
The terms herein are defined as follows.
Pluripotent stem cells are cells having both pluripotency which allows them to
differentiate into various kinds of cells in the body such as those in the endoderm
(interior stomach lining, gastrointestinal tract, the lungs), in the mesoderm (muscle,
45 bone, blood, urogenital) and in the ectoderm (epidermal tissues and nervous system)
and self-renewal ability to proliferate through cell division while maintaining the
pluripotency, and as examples , ES cells, iPS cells, embryonic germ cells (EG cells) and
Muse cells may be mentioned. ES cells are pluripotent stem cells derived from an
13
embryo at an early stage in the development of animals called the blastocysto stage.
iPS cells are also called artificial pluripotent stem cells or induced pluripotent stem cells
and are cells derived from somatic cells such as fibroblasts which have acquired
pluripotency equivalent to that of ES cells by transduction of several transcription factor
genes. EG cells are pluripotent stem cells derived from spermatogonial cells (see5 :
Nature. 2008, 456, 344-49). Muese cells are pluripotent stem cells separated from
mesenchymal cell populations (see: Proc Natl Acad Sci U S A. 2010, 107, 8639-43).
Hematopoietic stem cells are defined as cells having both pluripotency which
allows them to differentiate into blood cells of all lineages and the ability to renew
10 themselves while maintaining the pluripotency. Multipotential hematopoietic progenitor
cells are cells which can differentiate into a plurality of blood cell lineages, though not
into all blood cell lineages Unipotential hematopoietic progenitor cells are cells which
can differentiate into only one blood cell lineage.
Hematopoietic progenitor cells are a group of cells which covers both pluripotent
15 hematopoietic progenitor cells and unipotent hematopoietic progenitor cells. For
example, the hematopoietic progenitor cells in the present invention may be
granulocyte-macrophage colony forming cells (CFU-GM), eosinophil colony forming
cells (EO-CFC), erythroid burst forming cells (BFU-E) as erythroid progenitor cells,
megakaryocyte colony forming cells (CFU-MEG), megakaryocyte progenitor cells,
20 megakaryoblasts, promegakaryocytes, megakaryocyte/erythroid progenitor cells (MEP
cells) or myeloid stem cells (mixed colony forming cells, CFU-GEMM). Among them,
hematopoietic progenitor cells which differentiate into megakaryocytes and platelets are
megakaryocyte colony forming cells (CFU-MEG), megakaryocyte progenitor cells,
megakaryoblasts, promegakaryocytes, megakaryocyte/erythroid progenitor cells (MEP
25 cells) and myeloid progentor cells (mixed colony forming cells, CFU-GEMM).
Megakaryocytes are differentiated cells which develop through myeloid progenitor
cells, MEP cells, megakaryocyte progenitor cells, megakaryoblasts and
promegakaryocytes with an increase in cell size during the cytoplasmic maturation
events such as polyploidization, development of the demarcation membrane system
30 and granulation and have the potential to produce platelets through formation of
proplatelet processes.
Platelets are anucleate cells derived from megakaryocytes and play an important
role in blood coagulation.
CD41a+ cells are means expressing CD (cluster of differentiation) 41a antigen on
35 the cell surface. Likewise, CD42b+ cells are means expressing CD 42b antigen on the
cell surface. These antigens are markers for megakaryocytes and platelets.
Populations of CD41a+ and CD42b+ cells are enriched with megakaryocytes and
platelets.
In the present invention, differentiation of hematopoietic progenitor cells means
40 conversion of hematopoietic progenitor cells to mature blood cells having specific
functions such as erythrocytes, leukocytes, megakaryocytes and platelets.
The specific compounds to be used in the present invention act on hematopoietic
progenitor cells derived from pluripotent stem cells and have such an activity that they
induce megakaryopoiesis and thrombopoiesis from such hematopoietic progenitor cells
45 cultured ex vivo in the presence of a specific compound. Even when hematopoietic
progenitor cells cannot produce megakaryocytes and platelets efficiently, use of a
specific compound makes it possible to produce megakaryocytes and platelets
efficiently by culturing hematopoietic progenitor cells derived from pluripotent stem cells
14
ex vivo. Specifically speaking, it is possible to produce megakaryocytes and platelets
by culturing hematopoietic progenitor cells in a medium containing a specific compound.
It is also possible to produce megakaryocytes and platelets more efficiently by further
adding various cytokines or growth factors, by coculturing them with feeder cells or by
further adding other low-molecular-weight compounds which act on hematopoieti5 c
progenitor cells.
In the present invention, any pluripotent stem cells may be used as long as they
have both pluripotency an self-renewal ability and can differentiate into platelets. The
pluripotent stem cells may, for example, be ES cells, iPS cells, embryonic germ cells
10 (EG cells), Muse cells or the like, and more preferably ES cells or iPS cells. Examples
of transcription factor genes known to be necessary for imparting pluripotency in
establishment of iPS cells include Nanog, Oct3/4, Sox2, Klf4, c-Myc and Lin28. iPS
can be established by introducing the combination of Oct3/4, Sox2, Klf4 and c-Myc, the
combination of Oct3/4, Sox2, Nanog, and Lin28 or the combination of Oct3/4, Sox2 and
15 Klf4 selected from these genes into somatic cells such as fibroblasts. The iPS cells to
be used in the present invention may be established by any methods, and in addidtion
to those established by introduction of the above-mentioned genes, those established
by introduction of genes other than those mentioned above or those established by
using a protein or a low-molecular-weight compound may be used.
20 For culturing and subculturing pluripotent stem cells, a medium usually used to
maintain pluripotency may be used. For example, Iscove’s Modified Dulbecco’s
medium (IMDM), Dulbecco’s Modified Eagles’s Medium (DMEM), F-12 medium, X-VIVO
10 (Lonza), X-VIVO 15 (Lonza), mTeSR (Stemcell Technologies), TeSR2 (Stemcell
Technologies), StemProhESC SFM (Invitrogen) and the like may be mentioned. The
25 culture medium may be supplemented with proteins such as basic fibroblast growth
factor (bFGF), insulin and transforming growth factor (TGF-), serum, KnockOut SR
(Invitrogen), amino acids such as glutamine or 2-mercaptoethanol, and the culture
vessel may be coated with an extracellular matrix such as laminins-1 to -12, collagen,
fibronectin, vitronectin, Matrigel (Becton, Dickinson and Compnay) or Geltrex
30 (Invitrogen). Pluripotent stem cells may be co-cultured with feeder cells. Any feeder
cells that contribute to proliferation and maintenance of pluripotent cells may be used,
and for example, C3H10T1/2 cell line, OP9 cells, NIH3T3 cells, ST2 cells, PA6 cells,
preferably mouse embryonic fibroblast cells (MEF cells) or SL10 cells may be used. It
is preferred to suppress growth of feeder cells, for example, by treatment with
35 mitomycin C or irradiation before use.
Pluripotent stem cells are cultured usually at a temperature of from 25 to 39 C,
preferably 33 to 39 C, in the atmosphere having a CO2 concentration of from 4 to 10
vol%, preferably from 4 to 6 vol%.
The source of hematopoietic progenitor cells to be used in the present invention
40 may be an embryoid body obtained by culturing iPS cells or ES cells under conditions
suitable to induce differentiation of hematopoietic cells or a sac-like structure, preferably
a sac-like structure. An “embryoid body” is an aggregate of cells having a cystic
structure obtained in suspension culture of iPS cells or ES cells in the absence of
factors for maintaining them in the undifferentiated state and feeder cells (see: Blood,
45 2003, 102, 906-915). A “sac-like structure” is an iPS or ES cell-derived threedimensional
saccular structure (having a cavity inside) formed of a population of
endothelial cells or the like and containing hematopoietic progenitor cells inside. For
the details of sac-like structures, see TAKAYAMA et al., BLOOD 2008, 111: 5298-5306.
15
For preparation of a sac-like structure (hereinafter referred to also as an iPS-sac
or ES-sac), suitable culture conditions may be selected, and the suitable culture
conditions vary depending on the organism as the source of the iPS cells or ES cells to
be used. For example, for human iPS cells or ES cells, IMDM containing fetal bovine
serum (FBS) in a final concentration of 15%, optionally supplemented with insulin5 ,
transferrin, lactoferrin, cholesterol, ethanolamine, sodium selenite, -monothioglycerol,
2-mercaptoethanol, bovine serum albumin, sodium pyruvate, ascorbic acid,
polyethylene glycol, various vitamins, various amino acids and various antibiotics, may
be used as the culture medium. As factors which induce human iPS cells or ES cells
10 to form a sac-like structure, vascular endothelial growth factor (VEGF) and placental
growth factor (PGF) may, for example, be mentioned, and VEGF is preferred. VEGF
may be added at a concentration of about 10 ng/mL to100 ng/mL, preferably at a
concentration of about 20 ng/mL. A human iPS or ES cell culture may be incubated,
for example, in 5% CO2 at 36 to 38 C, preferably at 37 C, though the incubation
15 conditions differ depending on the human iPS or ES cells to be used. Further, it is
possible to produce a sac-like structure more efficiently from human iPS or ES cells by
co-culture with feeder cells. Any feeder cells that contribute to induction of
differentiation of pluripotent stem cells into hematopoietic progenitor cells may be used,
and for example, mouse embryonic fibroblast cells (MEF cells) or SL10 cells, preferably,
20 C3H10T1/2 cell line, OP9 cell line, ST2 cells, NIH3T3 cells, PA6 cells or M15 cells ,
more preferably C3H10T1/2 cell line or OP9 cells may be used. It is preferred to
suppress growth of feeder cells, for example, by treatment with mitomycin C or
irradiation before use. The incubation time until formation of a sac-like structure differs
depending on the human iPS or ES cells used, and, for example, the presence of a sac25
like structure can be confirmed 14 to 17 days after inoculation on feeder cells.
The sac-like structure thus formed has a cystic structure demarcated by septa of
cells positive for a mesodermal cell marker Flk1 (fetal liver kinase 1), CD1, CD34 or
UEA-1 lectin (Ulex europaeus.agglutinin-1). The inside of the sac-like structure is rich
in hematopoietic progenitor cells. Before inducing hematopoietic progenitor cells to
30 differentiate into various blood cells, it is necessary to separate hematopoietic
progenitor cells from the cells from the septal cells. The separation may be attained by
physical means. For example, the septal cells can be separated from the
hematopoietic progenitor cells by breaking a sac-like structure with a pipette or a
syringe and then passing the cells through a sterilized sieve-like tool (such as a cell
35 strainer).
In the present invention, the hematopoietic progenitor cells isolated from a suc-like
structure or the like as mentioned above are differentiated into megakaryocytes and/or
platelets. Differentiation of hematopoietic progenitor cells into platelets means
differentiation of hematopoietic progenitor cells into megakaryocytes and production of
40 platelets from the megakaryocytes. Specifically speaking, hematopoietic progenitor
cells derived from pluripotent stem cells are cultured under conditions suitable for
induction of differentiation of megakaryocytes and/or platelets. To differentiate
hematopoietic progenitor cells into megakaryocytes and/or platelets, ordinary culture
media used for hematopoietic cell culture, such as Iscove’s Modified Dulbecco’s
45 medium (IMDM), Dulbecco’s Modified Eagles’s Medium (DMEM), F-12 medium, X-VIVO
10 (Lonza), X-VIVO 15 (Lonza), McCoy’s 5A medium, Eagle’s MEM, MEM, RPMI1640,
StemPro34 (Invitrogen), StemSpan H3000 (Stemcell Technologies),
StemSpanSFEM(Stemcell Technologies), StemlineII(Sigma-Aldrich) or QBSF-60(Quality
16
Biological), may be used. As supplements to the media, bovine fetal serum, human
serum, horse serum, insulin, transferring, lactoferrin, cholesterol, ethanolamine, sodium
selenite, -monothioglycerol, 2-mercaptoethanol, bovine serum albumin, sodium
pyruvate, ascorbic acid, polyethylene glycol, various vitamins, various amino acids,
various antibiotics, agar, agarose, collagen, methylcellulose and the like may b5 e
mentioned.
“Culturing in the presence of a specific compound” means culturing in a medium
containing a specific compound of the present invention, for example, in a medium
containing a specific compound only or a medium containing a specific compound
10 together with other differentiation inducing factors. As the other differentiation inducing
factors, interleukin-1 (IL-1), IL-3, IL-4, IL-5, IL-6, IL-9, IL-11, erythropoietin (EPO),
granulocyte-macrophage colony-stimulating factor (GM-CSF), stem cell factor (SCF),
granulocyte colony-stimulating factor (G-CSF), flk2/flt3 ligand (FL) or Heparin, or a
combination of two or more of them may be mentioned. For example, differentiation
15 into megakaryocytes and platelets can be induced in a culture containing a specific
compound of the present invention (from 1 ng/mL to 1000 ng/mL, preferably from 10
ng/mL to 200 ng/mL, more preferably from 20 ng/mL to 100ng/mL), optionally
supplemented with SCF (from 10 to 200 ng/mL, preferably about 50 ng/mL) and
Heparin (from 10 to 100 U/mL, preferably about 25 U/mL), within about 7 to 15 days. A
20 specific compound of the present invention may be added directly to the culture medium,
or after dissolved in an appropriate solvent before use. Examples of the appropriate
solvent include dimethyl sulfoxide (DMSO) and various alcohols, but it is not restricted
thereto. A specific compound may be immobilized on the surface of a culture plate or a
carrier.
25 A specific compound may be provided or stored in any forms, for example, in a
solid form as a tablet, a pill, a capsule or a granule, in a liquid form as a solution or
suspension in an appropriate solvent or resolvent, in the form bound to a plate or carrier.
When it is formulated into such a form, additives such as a preservative like phydroxybenzoates;
an excipient like lactose, glucose, sucrose and mannitol; a lubricant
30 like magnesium stearate and talc; a binder like polyvinyl alcohol, hydroxypropylcellulose
and gelatin, a surfactant like fatty acid esters, a plasticizer like glycerin may be added.
The additives are not restricted to those mentioned above and a person skilled in the art
can use any additives of choice.
The culture medium may be supplemented with one or more chemical substances
35 effective in differentiation of hematopoietic progenitor cells into platelets (see:
Schweinfurth et al., Platelets, 21: 648-657 2010, Lordier et al., Blood, 112: 3164-3174
2009). Examples of them include retinoic acid receptor ligands such as all-transretinoic
acid, histone deacetylase inhibitors such as valproic acid, trichostatin A, SAHA
(suberoylanilide hydroxamic acid) and APHA (aroyl-pyrrolyl-hydroxyamide), ROCK
40 (Rho-associated coiled-coil forming kinase/Rho-binding kinase) inhibitors such as (R)-
(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide 2HCl H2O (Y27632),
myosin heavy chain II ATPase such as blebbistatin, myosin light chain kinase inhibitors
such as ML7 and prostaglandin E2 but are not restricted thereto. Before treating cells
with these compounds, a person skilled in the art may determine the optimum
45 concentration of these chemical substances by experiment and may choose appropriate
treating time and method. For example, in the case of blebbistatin as a myosin heavy
chain II ATPase inhibitor, from 0.3 to 15 g/mL, or from 1 to 10 g/mL of blabbistatin
may be added, and the incubation time is, preferably, for example, about from 3 to 10
17
days, particularly, about from 4 to 7 days. Alternatively, Y-27632 as a ROCK inhibitor
may be used at 5 to 15 M, or 8 to 12 M, preferably about 10 M, and valproic acid as
a HDAC inhibitor may be used at 0.1 to 1 mM, or 0.2 to 0.7 mM, preferably about 0.5
mM. The treatment time for Y-27632 and valproic acid is about from 3 to 21 days,
preferably about from 7 to 14 day5 s.
Hematopoietic stem cells and/or hematopoietic progenitor cells are cultured
usually at a temperature of from 25 to 39 C, preferably from 33 to 39 C, in the
atmosphere having a CO2 concentration of from 4 to 10 vol%, preferably from 4 to 6
vol%. Hematopoietic progenitor cells may be co-cultured with feeder cells for induction
10 of megakaryocytes and platelets. Any feeder cells that contribute to contribute to
induction of differentiation of hematopoietic progenitor cells into megakaryocytes or
platelets may be used, and for example, mouse embryonic fibroblast cells (MEF cells)
or SL10 cells, preferably, C3H10T1/2 cell line, OP9 cell line, ST2 cells, NIH3T3 cells,
PA6 cells or M15 cells , more preferably C3H10T1/2 cell line or OP9 cells may be used.
15 It is preferred to suppress growth of feeder cells, for example, by treatment with
mitomycin C or irradiation before use.
Hematopoietic progenitor cells can be cultured in a culture vessel generally used
for animal cell culture such as a Petri dish, a flask, a plastic bag, a Teflon (registered
trademark) bag, optionally after preliminary coating with an extracellular matrix or a cell
20 adhesion molecule. The material for such a coating may be collagens I to XIX,
fibronectin, vitronectin, laminins 1 to 12, nitogen, tenascin, thrombospondin, von
Willebrand factor, osteoponin, fibrinogen, various elastins, various proteoglycans,
various cadherins, desmocolin, desmoglein, various integrins, E-selectin, P-selectin, Lselectin,
immunoglobulin superfamily, Matrigel, poly-D-lysine, poly-L-lysine, chitin,
25 chitosan, Sepharose, alginic acid gel, hydrogel or a fragment thereof. Such a coating
material may be a recombinant material having an artificially modified amino acid
sequence. Hematopoietic progenitor cells may be cultured by using a bioreactor which
can mechanically control the medium composition, pH and the like and obtain high
density culture (Schwartz RM, Proc. Natl. Acad. Sci. U.S.A., 88:6760, 1991; Koller MR,
30 Bone Marrow Transplant, 21:653, 1998; Koller, MR, Blood, 82: 378, 1993; Astori G,
Bone Marrow Transplant, 35: 1101, 2005).
When megakaryocytes and platelets are produced from human iPS cells or ES
cells, since the efficiency of sac-like structure production varies clone to clone,
preliminary choice of iPS or ES cell clones which produce a sac-like structure efficiently
35 makes it possible to produce a large number of megakaryocytes and platelets more
efficiently from the sac-like structures produced by the selected iPS or ES cell clones.
As the iPS or ES cell clones producing a sac-like structure efficiently, clones forming at
least 10, preferably at least 15 sac-like structures per 1×105 cells may be chosen.
In addition, introduction of an oncogene or the like increases the proliferative
40 capability of hematopoietic progenitor cells obtained from pluripotent stem cells (see:
WO/2011/034073). The oncogene may, for example, be a MYC family gene (such as
c-Myc, n-myc or l-myc gene), a SRC family gene, a RAS family gene, a g RAF family
gene, c-kit gene, Abl gene or the like, preferably a gene of the Myc family more
preferably c-Myc gene. The senescence of cells resulting from oncogene introduction
45 can be prevented by simultaneous introduction of a polycomb gene or the like. The
polycomb gene may, for example, be Bmi1 gene, Mel18 gene, Ring1a/b gene, Phc1/2/3,
Cbx2/4/6/7/8 gene, Eed gene, Ezh2 gene or Suz12 gene, preferably Bmi1 gene.
Death of cells can be prevented by introduction of an apoptosis suppressor gene, such
18
as BCL2 (B-cell lymphoma 2) gene or BCLXL (B-cell lymphoma-extra large) gene in the
BCL2 family or Survivin, MCL1(myeloid cell leukemia1), preferably BCL2 gene or
BCLXL gene. Suppression of expression of the tumor suppressor gene p53 is effective
for inducing hemeatopoirtic progenitor cells to differentiate into megakaryocytes .(see:
Fuhrken etal., J. Biol. Chem., 283: 15589-15600 2008). Examples of tumo5 r
suppressor genes include p53 gene, p16 gene, p73 gene, Rb gene, BRCA1(breast
cancer susceptibility gene 1) gene, BRCA2 gene and WT1 gene, and p53 gene is
preferred. In addition, RNA genes which promote thrombopoiesis such as antisense
RNAs, small interfering (si) RNAs, short hairpin (sh) RNAs, decoy RNA, ribozymes are
10 also effective as target genes. These genes and RNAs include those having publicly
known nucleotide sequences and their homologues obtained by homologous
modification of these known sequences by conventional techniques. Hereinafter,
genes introduced into hematopoietic progenitor cells for enhancement of their
proliferative capability are referred to as target genes.
15 Target genes may be introduced into cells at any stage of differentiation from
pluripotent stem cells to megakaryocytes, such as mesodermal cells, hematopoietic
stem cells and hematopoietic progenitor cells. For introduction of target genes into
these cells, techniques generally used for transfection of animal cells, for example,
vector transfection using animal cell vectors of viral origin for gene therapy (see Verma,
20 I.M., Nature, 389: 239, 1997 for vectors for gene therapy) such as retrovirus vectors
represented by mouse stem cell virus (MSCV) and Molonry mouse leukemia virus
(MmoLV), adenovirus vectors, adeno-associated vectors (AAVs), herpes simplex virus
vectors, lentivirus vectors, Sendai virus vectors, the calcium phosphate coprecipitation
method, the DEAE-Dextran method, the electroporation method, the liposome method,
25 the lipofection method and the microinjection method, may be used. Among them,
those which allow target genes to be integrated into the chromosomal DNA of the cells
and expressed constitutively are preferred, and when viruses are used, retrovirus
vectors, adeno-associated virus vectors or lentivirus vectors are preferred.
An adeno-associated virus (AAV) vector may be prepared, for example, as follows.
30 First, 293 cells are transfected with a plasmid vector prepared by inserting a therapeutic
gene between the ITRs (inverted terminal repeats) at both end of wild-type adenoassociated
virus DNA and a helper plasmid for virus protein supplementation and
subsequently with adenovirus as a helper virus or with a plasmid expressing an
adenovirus helper genes to produce virus particles containing the AAV vector, which are
35 used for transfection of hematopoietic progenitor cells. It is preferred to insert an
appropriate promoter, enhancer, insulator or the like upstream of the target gene to
regulate expression of the target gene. A marker gene such as a drug resistance gene
may be introduced together with a target gene for easy selection of cells transfected
with the target gene. The target gene may be a sense gene or an antisense gene.
40 A target gene may be introduced into cells via a mammalian expression vector or
virus vector carrying the target gene functionally ligated downstream of an appropriate
promoter so that the introduced target gene is expressed. Promoters such as CMV
promoter, EF promoter and SV40 promoter may be used for constitutive expression of a
target gene. A target gene may be functionally ligeted downstream of an endogeneous
45 enhancer/promoter which induces gene expression at a certain stage of differentiation,
such as glycoprotein IIb/IIIa enhance/promoter (see: Nat. Biotech 26, 209-211 (2008))
so that expression of the target gene is induced in the course of differentiation into
megakaryocytes. Alternatively, an appropriate promoter may be placed under control
19
of an element whose activity is regulated by a trans factor such as a drug-responsive
element to provide a regulatory system which induces or suppresses expression of the
target gene by addition of a drug or the like. Elements whose activity is controlled by a
drug include, for example, tet operator element (see: Proc. Natl. Acad. Sci.USA 89,
5547-5551 (1992)), GAL4-dingin element (see: Proc. Natl. Acad. Sci.USA 91, 81805 -
8184 (1992)), Lac operator element (Environ. Mol. Mutagen. 28, 447-458 (1996)) and
LexA operator element (see: the EMBO journal 7, 3975-3982 (1988)). Introduction of
an appropriate gene having a ligand-binding domain, a DNA –binding domain and a
transcriptional regulatory domain which activates or represses transcription responsive
10 to a drug such as tetracycline, dexamthasone, tamoxyfen, estradiol, doxycycline or
isopropyl--thiogalactopyranoside (IPTG) permits regulation of expression of a target
gene downstream of the element by a drug. Transcriptional regulation by binding of a
dimeric ligand containing a DNA-binding domain and a transcription regulatory domain
is also possible, and it is possible to make such a dimer responsive to a certain drug by
15 using the variable domain of an antibody, as disclosed in Japanese Patent
Application2009-201504 . For regulated expression of a target gene, for example, the
GeneSwitchTM system of Invitrogen, the LacSwitch II Inducible Mammalian Expression
system of Agilent Technologies and the iDimerizeTM system, the Tet-onTM system and
the Tet-offTM system of Clontech may be used. Further, for a drug-responsive regulation
20 of the amount of the protein expressed from a target gene, a target gene to be
introduced may be fused with the destabilization domain of a mutant FK506 binding
protein by using, for example, the ProtoTunerTM system of Clontech. In addition, it is
possible to introduce a target gene at an appropriate location of a genome by using the
homologous recombination technique (see: Nature 317, 230-234 (1985)). Further, an
25 introduced target gene or an oncogene in a genome can be removed from the genome
or repressed, for example, by using the Cre/Lox system or the FLP/frt system disclosed
in Mammalian Genome 15, 677-685 (2004) singly or in combination. Further, removal
of a target gene may be attained by directly introducing the Cre protein or the FLP
protein at a certain stage of differentiation or by introducing a gene encoding such a
30 protein. A target gene preliminarily introduced into cells can be removed at a certain
stage of differentiation by expressing the Cre protein or the FLP protein under control of
a drug-responsive element, as in the Cre-ER system disclosed in Developmental
Biology 244, 305-318 (2002).
Hematopoietic progenitor cells with enhanced proliferative capability and/or
35 differentiative capability by regulation of expression of a target gene by genetic
manipulation may be used for production of megakaryocytes and platelets by the
method of the present invention.
Further, the present invention covers a kit for producing platelets as one
embodiment. The kit contains a medium for cell culture, serum, a specific compound
40 of the present invention, supplements such as growth factors (such as TPO, SCF,
Heparin, IL-6 and IL-11), antibiotics and the like. In addition, it may contain an antibody
to detect the marker on sac-like structures (such as antibodies against Flk1, CD31,
CD34 and UEA-I lectin). The reagents and antibodies and the like in the kit are
supplied in any type of vessel in which components effectively sustain their activity over
45 a long period without being adsorbed or denatured by the material of the vessel, such
as a sealed glass ampoule containing a buffer gassed with a neutral inert gas such as
nitrogen gas and an ampoule of an organic polymer such as glass, polycarbonate and
polystyrene, ceramics, a metal or other appropriate materials usually used to retain a
20
reagent or the like.
Since platelets are effective for preventing and alleviating a decrease in platelets
due to leukemia, bone marrow transplantation and anticancer treatment, human platets
obtained by the method of the present invention can be stably supplied in the form of a
platelet preparation. A platelet preparation can be prepared from platelets produced b5 y
the method of the present invention by recovering a fraction of a liquid culture rich in
platelets released from magakaryocytes (for example, in the case of human platelets,
an approximately 22- to 28 day culture of iPS cells or ES cells) and separating platelets
from other components by removing megakaryocytes and other blood cells by using a
10 leukocyte reduction filter (available, for example, from TERUMO and Asahi Kasei
Medical) or the like, or by precipitating non-platelet components by centrifugation at 100
to 150g for 10 to 15 minutes. A platelet preparation may contain other components
which stabilize platelets in view of the storage instability of platelets. As conditions for
platelet stabilization, a method well known to experts in this technical field may be
15 selected. More specifically, platelets obtained (for example, washed platelets derived
from human iPS cells or human ES cells) may be converted to a platelet preparation as
follows.
ACD-A solution and FFP (fresh frozen plasma; prepared from whole blood
obtained by blood donation, which contains all the other blood components other than
20 blood cells such as albumin and a coagulation factor) are mixed at a ratio of 1:10,
irradiated with 15-50 Gy radiation and stored with shaking at 20 to 24 C. The ACD-A
solution is prepared by dissolving 22 g of sodium citrate/8 g of citric acid/22 g of glucose
with water for injection to a total volume of 1 L.
When the above-mentioned method is used, the platelet density is set desirably at
25 1×109 platelets/mL, for example.
Addition of GM6001(a broad-range hydroxamic acid-based metalloprotease
inhibitor) (Calbiochem, La Jolla, CA, USA) prevents platelet deactivation during
cryopreservation and storage at room temperature caused by cleavage of the molecules
essential for platelet function such as GPIb-V-IX and GPVI. The present inventors
30 confirmed that inactivation of platelets derived from mouse ES cells can be prevented
by this method. For the mechanisms of inactivation of human platelets by this method,
see Bergmeier, W et al., Cir Res 95: 677-683, 2004 and Gardiner, EE et al., J
Thrombosis and Haemostasis, 5:1530-1537, 2007.
For a container of a preparation containing platelets, materials which activate
35 platelets such as glass are preferably avoided.
Platelets produced by the method of the present invention may be used for
treatment of diseases accompanied by a decrease in platelets and effective for
treatment of various diseases.
As specific examples, lysosomal storage disease such as Gaucher’s disease and
40 mucopolysaccharidosis, adrenoleukodystrophy, various kinds of cancers and tumors,
especially blood cancers such as acute or chronic leukemia, Fanconi syndrome,
aplastic anemia, granulocytopenia, lymphopenia, thrombocytopenia, idiopathic
thrombocytopenic purpura, thrombotic thrombocytopenic purpura, Kasabach-Merritt
syndrome, malignant lymphoma, Hodgkin’s disease, multiple myeloma, chronic
45 hepatopathy, renal failure, massive blood transfusion of bank blood or during operation,
hepatitis B, hepatitis C, severe infections, systemic lupus erythematodes, articular
rheumatism, xerodermosteosis, systemic sclerosis, polymyositis, dermatomyositis,
mixed connective tissue disease, polyarteritis nodosa, Hashimoto’s disease, Basedow’s
21
disease, myasthenia gravis, insulin dependent diabetes mellitus, autoimmune hemolytic
anemia, snake bite, hemolytic uremic syndrome, hypersplenism, bleeding, Bernard-
Soulier syndrome, Glanzmann’s thrombasthenia, uremia, myelodysplastic syndrome,
polycythemia rubra vera, erythremia, myeloproliferative disease, and the like may be
mentioned. Now, the specific compound to be used in the present invention will b5 e
described in terms of the definitions of terms used for it and its best mode.
In the compound to be used in the present invention, “n” denotes normal, “i”
denotes iso, “s” denotes secondary, “t” denotes tertiary, “c” denotes cyclo, “o” denotes
ortho, “m” denotes meta, and “p” denotes para.
10 First, the terms in the respective substituents R1 to R20 and V1 to V4 will be
explained.
As a halogen atom, a fluorine atom, a chlorine atom, a bromine atom or an iodine
atom may be mentioned.
A C1-3 alkyl group may be linear, branched or a C3 cycloalkyl group, and methyl,
15 ethyl, n-propyl, i-propyl and c-propyl and the like may be mentioned.
A C1-6 alkyl group may be linear, branched or a C3-6 cycloalkyl group, and as
specific examples, in addition to those mentioned above, n-butyl, i-butyl, s-butyl, t-butyl,
c-butyl, 1-methyl-c-propyl, 2-methyl-c-propyl, n-pentyl, 1-methyl-n-butyl, 2-methyl-nbutyl,
3-methyl-n-butyl, 1,1-dimethyl-n-propyl, 1,2-dimethyl-n-propyl, 2,2-dimethyl-n20
propyl, 1-ethyl-n-propyl, c-pentyl, 1-methyl-c-butyl, 2-methyl-c-butyl, 3-methyl-c-butyl,
1,2-dimethyl-c-propyl, 2,3-dimethyl-c-propyl, 1-ethyl-c-propyl, 2-ethyl-c-propyl, n-hexyl,
1-methyl-n-pentyl, 2-methyl-n-pentyl, 3-methyl-n-pentyl, 4-methyl-n-pentyl, 1,1-dimethyln-
butyl, 1,2-dimethyl-n-butyl, 1,3-dimethyl-n-butyl, 2,2-dimethyl-n-butyl, 2,3-dimethyl-nbutyl,
3,3-dimethyl-n-butyl, 1-ethyl-n-butyl, 2-ethyl-n-butyl, 1,1,2-trimethyl-n-propyl,
25 1,2,2-trimethyl-n-propyl, 1-ethyl-1-methyl-n-propyl, 1-ethyl-2-methyl-n-propyl, c-hexyl, 1-
methyl-c-pentyl, 2-methyl-c-pentyl, 3-methyl-c-pentyl, 1-ethyl-c-butyl, 2-ethyl-c-butyl, 3-
ethyl-c-butyl, 1,2-dimethyl-c-butyl, 1,3-dimethyl-c-butyl, 2,2-dimethyl-c-butyl, 2,3-
dimethyl-c-butyl, 2,4-dimethyl-c-butyl, 3,3-dimethyl-c-butyl, 1-n-propyl-c-propyl, 2-npropyl-
c-propyl, 1-i-propyl-c-propyl, 2-i-propyl-c-propyl, 1,2,2-trimethyl-c-propyl, 1,2,3-
30 trimethyl-c-propyl, 2,2,3-trimethyl-c-propyl, 1-ethyl-2-methyl-c-propyl, 2-ethyl-1-methyl-cpropyl,
2-ethyl-2-methyl-c-propyl, 2-ethyl-3-methyl-c-propyl and the like may be
mentioned.
A C1-10 alkyl group may be linear, branched or a C3-10 cycloalkyl group, and as
specific examples, in addition to those mentioned above, 1-methyl-1-ethyl-n-pentyl, 1-
35 heptyl, 2-heptyl, 1-ethyl-1,2-dimethyl-n-propyl, 1-ethyl-2,2-dimethyl-n-propyl, 1-octyl, 3-
octyl, 4-methyl-3-n-heptyl, 6-methyl-2-n-heptyl, 2-propyl-1-n-heptyl, 2,4,4-trimethyl-1-npentyl,
1-nonyl, 2-nonyl, 2,6-dimethyl-4-n-heptyl, 3-ethyl-2,2-dimethyl-3-n-pentyl, 3,5,5-
trimethyl-1-n-hexyl, 1-decyl, 2-decyl, 4-decyl, 3,7-dimethyl-1-n-octyl, 3,7-dimethyl-3-noctyl
and the like may be mentioned.
40 A C2-6 alkenyl group may be linear, branched or a C3-6 cycloalkenyl group, and as
specific examples, ethenyl, 1-propenyl, 2-propenyl, 1-methyl-1-ethenyl, 1-butenyl, 2-
butenyl, 3-butenyl, 2-methyl-1-propenyl, 2-methyl-2-propenyl, 1-ethylethenyl, 1-methyl-
1-propenyl, 1-methyl-2-propenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 1-npropylethenyl,
1-methyl-1-butenyl, 1-methyl-2-butenyl, 1-methyl-3-butenyl, 2-ethyl-2-
45 propenyl, 2-methyl-1-butenyl, 2-methyl-2-butenyl, 2-methyl-3-butenyl, 3-methyl-1-
butenyl, 3-methyl-2-butenyl, 3-methyl-3-butenyl, 1,1-dimethyl-2-propenyl, 1-ipropylethenyl,
1,2-dimethyl-1-propenyl, 1,2-dimethyl-2-propenyl, 1-c-pentenyl, 2-cpentenyl,
3-c-pentenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexenyl, 1-
22
methyl-1-pentenyl, 1-methyl-2-pentenyl, 1-methyl-3-pentenyl, 1-methyl-4-pentenyl, 1-nbutylethenyl,
2-methyl-1-pentenyl, 2-methyl-2-pentenyl, 2-methyl-3-pentenyl, 2-methyl-
4-pentenyl, 2-n-propyl-2-propenyl, 3-methyl-1-pentenyl, 3-methyl-2-pentenyl, 3-methyl-
3-pentenyl, 3-methyl-4-pentenyl, 3-ethyl-3-butenyl, 4-methyl-1-pentenyl, 4-methyl-2-
pentenyl, 4-methyl-3-pentenyl, 4-methyl-4-pentenyl, 1,1-dimethyl-2-butenyl, 1,15 -
dimethyl-3-butenyl, 1,2-dimethyl-1-butenyl, 1,2-dimethyl-2-butenyl, 1,2-dimethyl-3-
butenyl, 1-methyl-2-ethyl-2-propenyl, 1-s-butylethenyl, 1,3-dimethyl-1-butenyl, 1,3-
dimethyl-2-butenyl, 1,3-dimethyl-3-butenyl, 1-i-butylethenyl, 2,2-dimethyl-3-butenyl, 2,3-
dimethyl-1-butenyl, 2,3-dimethyl-2-butenyl, 2,3-dimethyl-3-butenyl, 2-i-propyl-2-propenyl,
10 3,3-dimethyl-1-butenyl, 1-ethyl-1-butenyl, 1-ethyl-2-butenyl, 1-ethyl-3-butenyl, 1-npropyl-
1-propenyl, 1-n-propyl-2-propenyl, 2-ethyl-1-butenyl, 2-ethyl-2-butenyl, 2-ethyl-3-
butenyl, 1,1,2-trimethyl-2-propenyl, 1-t-butylethenyl, 1-methyl-1-ethyl-2-propenyl, 1-
ethyl-2-methyl-1-propenyl, 1-ethyl-2-methyl-2-propenyl, 1-i-propyl-1-propenyl, 1-i-propyl-
2-propenyl, 1-methyl-2-c-pentenyl, 1-methyl-3-c-pentenyl, 2-methyl-1-c-pentenyl, 2-
15 methyl-2-c-pentenyl, 2-methyl-3-c-pentenyl, 2-methyl-4-c-pentenyl, 2-methyl-5-cpentenyl,
2-methylene-c-pentyl, 3-methyl-1-c-pentenyl, 3-methyl-2-c-pentenyl, 3-methyl-
3-c-pentenyl, 3-methyl-4-c-pentenyl, 3-methyl-5-c-pentenyl, 3-methylene-c-pentyl, 1-chexenyl,
2-c-hexenyl, 3-c-hexenyl and the like may be mentioned.
As a C2-6 alkynyl group, ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 3-
20 butynyl, 1-methyl-2-propynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl, 4-pentynyl, 1-methyl-2-
butynyl, 1-methyl-3-butynyl, 2-methyl-3-butynyl, 3-methyl-1-butynyl, 1,1-dimethyl-2-
propynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl, 5-hexynyl, 1-methyl-2-pentynyl, 1-
methyl-3-pentynyl, 1-methyl-4-pentynyl, 2-methyl-3-pentynyl, 2-methyl-4-pentynyl, 3-
methyl-1-pentynyl, 3-methyl-4-pentynyl, 4-methyl-1-pentynyl, 4-methyl-2-pentynyl, 1,1-
25 dimethyl-2-butynyl, 1,1-dimethyl-3-butynyl, 1,2-dimethyl-3-butynyl, 2,2-dimethyl-3-
butynyl, 3,3-dimethyl-1-butynyl, 1-ethyl-2-butynyl, 1-ethyl-3-butynyl, 1-n-propyl-2-
propynyl, 2-ethyl-3-butynyl, 1-methyl-1-ethyl-2-propynyl, 1-i-propyl-2-propynyl and the
like may be mentioned.
A C2-14 aryl group may be a C6-14 aryl group containing no hetero atoms as ring
30 constituting atoms, a C2-9 heteroaryl group or a C2-14 fused polycyclic group.
As a C6-14 aryl group containing no hetero atoms, a phenyl group, a 1-indenyl
group, a 2-indenyl group, a 3-indenyl group, a 4-indenyl group, a 5-indenyl group, a 6-
indenyl group, a 7-indenyl group, an -naphthyl group, a -naphthyl group, an obiphenylyl
group, a m-biphenylyl group, a p-biphenylyl group, a 1-anthryl group, a 2-
35 anthryl group, a 9-anthryl group, a 1-phenanthryl group, a 2-phenanthryl group, a 3-
phenanthryl group, a 4-phenanthryl group, a 9-phenanthryl group,
or the like may be mentioned.
A C2-9 heteroaryl group may be a 5 to 7-membered C2-6 heteromonocyclic group or
40 8 to 10-membered C5-9 fused heterobicyclic group containing from 1 to 3 oxygen atoms,
23
nitrogen atoms or sulfur atoms singly or in combination.
As a 5 to 7-membered C2-6 heteromonocyclic group, a 2-thienyl group, a 3-thienyl
group, a 2-furyl group, a 3-furyl group, a 2-pyranyl group, a 3-pyranyl group, a 4-pyranyl
group, a 1-pyrrolyl group, a 2-pyrrolyl group, a 3-pyrrolyl group, a 1-imidazolyl group, a
2-imidazolyl group, a 4-imidazolyl group, a 1-pyrazolyl group, a 3-pyrazolyl group, a 5 4-
pyrazolyl group, a 2-thiazolyl group, a 4-thiazolyl group, a 5-thiazolyl group, a 3-
isothiazolyl group, a 4-isothiazolyl group, a 5-isothiazolyl group, a 1-1,2,4-triazole group,
a 3-1,2,4-triazole group, a 5-1,2,4-triazole group, a 1-1,2,3-triazole group, a 4-1,2,3-
triazole group, a 5-1,2,3-triazole group, a 2-oxazolyl group, a 4-oxazolyl group, a 5-
10 oxazolyl group, a 3-isoxazolyl group, a 4-isoxazolyl group, a 5-isoxazolyl group, a 2-
pyridyl group, a 3-pyridyl group, a 4-pyridyl group, a 2-pyrazinyl group, a 2-pyrimidinyl
group, a 4-pyrimidinyl group, a 5-pyrimidinyl group, a 3-pyridazinyl group, a 4-
pyridazinyl group, a 2-1,3,4-oxadiazolyl group, a 2-1,3,4-thiadiazolyl group, a 3-1,2,4-
oxadiazolyl group, a 5-1,2,4-oxadiazolyl group, a 3-1,2,4-thiadiazolyl group, a 5-1,2,4-
15 thiadiazolyl group, a 3-1,2,5-oxadiazolyl group, a 3-1,2,5-thiadiazolyl group or the like
may be mentioned.
As a 8 to 10-membered C5-9 fused heterocyclic group, a 2-benzofuranyl group, a
3-benzofuranyl group, a 4-benzofuranyl group, a 5-benzofuranyl group, a 6-
benzofuranyl group, a 7-benzofuranyl group, a 1-isobenzofuranyl group, a 4-
20 isobenzofuranyl group, a 5-isobenzofuranyl group, a 2-benzothienyl group, a 3-
benzothienyl group, a 4-benzothienyl group, a 5-benzothienyl group, a 6-benzothienyl
group, a 7-benzothienyl group, a 1-isobenzothienyl group, a 4-isobenzothienyl group, a
5-isobenzothienyl group, a 2-chromenyl group, a 3-chromenyl group, a 4-chromenyl
group, a 5-chromenyl group, a 6-chromenyl group, a 7-chromenyl group, a 8-chromenyl
25 group, a 1-indolizinyl group, a 2-indolizinyl group, a 3-indolizinyl group, a 5-indolizinyl
group, a 6-indolizinyl group, a 7-indolizinyl group, a 8-indolizinyl group, a 1-isoindolyl
group, a 2-isoindolyl group, a 4-isoindolyl group, a 5-isoindolyl group, a 1-indolyl group,
a 2-indolyl group, a 3-indolyl group, a 4-indolyl group, a 5-indolyl group, a 6-indolyl
group, a 7-indolyl group, 1-indazolyl group, a 2-indazolyl group, a 3-indazolyl group, a 4-
30 indazolyl group, a 5-indazolyl group, a 6-indazolyl group, a 7-indazolyl group, a 1-purinyl
group, a 2-purinyl group, a 3-purinyl group, a 6-purinyl group, a 7-purinyl group, a 8-
purinyl group, a 2-quinolyl group, a 3-quinolyl group, a 4-quinolyl group, a 5-quinolyl
group, a 6-quinolyl group, a 7-quinolyl group, a 8-quinolyl group, a 1-isoquinolyl group, a
3-isoquinolyl group, a 4-isoquinolyl group, a 5-isoquinolyl group, a 6-isoquinolyl group, a
35 7-isoquinolyl group, a 8-isoquinolyl group, a 1-phthalazinyl group, a 5-phthalazinyl group,
a 6-phthalazinyl group, a 1-2,7-naphthyridinyl group, a 3-2,7-naphthyridinyl group, a 4-
2,7-naphthyridinyl group, a 1-2,6-naphthyridinyl group, a 3-2,6-naphthyridinyl group, a
4-2,6-naphthyridinyl group, a 2-1,8-naphthyridinyl group, a 3-1,8-naphthyridinyl group, a
4-1,8-naphthyridinyl group, a 2-1,7-naphthyridinyl group, a 3-1,7-naphthyridinyl group, a
40 4-1,7-naphthyridinyl group, a 5-1,7-naphthyridinyl group, a 6-1,7-naphthyridinyl group, a
8-1,7-naphthyridinyl group, 2-1,6-naphthyridinyl group, a 3-1,6-naphthyridinyl group, a
4-1,6-naphthyridinyl group, a 5-1,6-naphthyridinyl group, a 7-1,6-naphthyridinyl group, a
8-1,6-naphthyridinyl group, a 2-1,5-naphthyridinyl group, a 3-1,5-naphthyridinyl group, a
4-1,5-naphthyridinyl group, a 6-1,5-naphthyridinyl group, a 7-1,5-naphthyridinyl group, a
45 8-1,5-naphthyridinyl group, a 2-quinoxalinyl group, a 5-quinoxalinyl group, a 6-
quinoxalinyl group, a 2-quinazolinyl group, a 4-quinazolinyl group, a 5-quinazolinyl
group, a 6-quinazolinyl group, a 7-quinazolinyl group, a 8-quinazolinyl group, a 3-
cinnolinyl group, a 4-cinnolinyl group, a 5-cinnolinyl group, a 6-cinnolinyl group, a 7-
24
cinnolinyl group, a 8-cinnolinyl group, a 2-pteridinyl group, a 4-pteridinyl group, a 6-
pteridinyl group, a 7-pteridinyl group or the like may be mentioned.
A C2-9 nitrogen-containing heteroaryl group is a C2-9 heteroaryl group containing
one to three nitrogen atoms.
A C2-14 fused polycyclic group is a fused bicyclic or fused tricyclic group consistin5 g
of a C6-14 aryl group containing no hetero atoms and at most 12 carbon atoms as
mentioned above or a C2-9 heteroaryl group fused with a C2-9 heterocyclyl group, and:
may be mentioned specifically.
10 A C1-10 thioalkyl group may linear, branched or a C3-10 cyclothioalkyl group, and as
specific examples, methylthio, ethylthio, n-propylthio, i-propylthio, c-propylthio, nbutylthio,
i-butylthio, s-butylthio, t-butylthio, c-butylthio, 1-methyl-c-propylthio, 2-methylc-
propylthio, n-pentylthio, 1-methyl-n-butylthio, 2-methyl-n-butylthio, 3-methyl-n-butylthio,
1,1-dimethyl-n-propylthio, 1,2-dimethyl-n-propylthio, 2,2-dimethyl-n-propylthio, 1-ethyl-n15
propylthio, c-pentylthio, 1-methyl-c-butylthio, 2-methyl-c-butylthio, 3-methyl-c-butylthio,
1,2-dimethyl-c-propylthio, 2,3-dimethyl-c-propylthio, 1-ethyl-c-propylthio, 2-ethyl-cpropylthio,
n-hexylthio, 1-methyl-n-pentylthio, 2-methyl-n-pentylthio, 3-methyl-npentylthio,
4-methyl-n-pentylthio, 1,1-dimethyl-n-butylthio, 1,2-dimethyl-n-butylthio, 1,3-
dimethyl-n-butylthio, 2,2-dimethyl-n-butylthio, 2,3-dimethyl-n-butylthio, 3,3-dimethyl-n20
butylthio, 1-ethyl-n-butylthio, 2-ethyl-n-butylthio, 1,1,2-trimethyl-n-propylthio, 1,2,2-
trimethyl-n-propylthio, 1-ethyl-1-methyl-n-propylthio, 1-ethyl-2-methyl-n-propylthio, chexylthio,
1-methyl-c-pentylthio, 2-methyl-c-pentylthio, 3-methyl-c-pentylthio, 1-ethyl-cbutylthio,
2-ethyl-c-butylthio, 3-ethyl-c-butylthio, 1,2-dimethyl-c-butylthio, 1,3-dimethyl-cbutylthio,
2,2-dimethyl-c-butylthio, 2,3-dimethyl-c-butylthio, 2,4-dimethyl-c-butylthio, 3,3-
25 dimethyl-c-butylthio, 1-n-propyl-c-propylthio, 2-n-propyl-c-propylthio, 1-i-propyl-cpropylthio,
2-i-propyl-c-propylthio, 1,2,2-trimethyl-c-propylthio, 1,2,3-trimethyl-cpropylthio,
2,2,3-trimethyl-c-propylthio, 1-ethyl-2-methyl-c-propylthio, 2-ethyl-1-methyl-cpropylthio,
2-ethyl-2-methyl-c-propylthio, 2-ethyl-3-methyl-c-propylthio, 1-methyl-1-ethyln-
pentylthio, 1-heptylthio, 2-heptylthio, 1-ethyl-1,2-dimethyl-n-propylthio, 1-ethyl-2,2-
30 dimethyl-n-propylthio, 1-octylthio, 3-octylthio, 4-methyl-3-n-heptylthio, 6-methyl-2-nheptylthio,
2-propyl-1-n-heptylthio, 2,4,4-trimethyl-1-n-pentylthio, 1-nonylthio, 2-
nonylthio, 2,6-dimethyl-4-n-heptylthio, 3-ethyl-2,2-dimethyl-3-n-pentylthio, 3,5,5-
trimethyl-1-n-hexylthio, 1-decylthio, 2-decylthio, 4-decylthio, 3,7-dimethyl-1-n-octylthio,
3,7-dimethyl-3-n-octylthio or the like may be mentioned.
35 A C1-3 alkylsulfonyl group may be linear, branched or a C3 cycloalkylsulfonyl group,
25
and as specific examples, methylsulfonyl, ethylsulfonyl, n-propylsulfonyl, i-propylsulfonyl,
c-propylsulfonyl or the like may be mentioned.
A C1-10 alkylsulfonyl group may be linear, branched or a C3-10 cycloalkylsulfonyl
group, and as specific examples, in addition to those mentioned above, n-butylsulfonyl,
i-butylsulfonyl, s-butylsulfonyl, t-butylsulfonyl, c-butylsulfonyl, 1-methyl-c-propylsulfonyl5 ,
2-methyl-c-propylsulfonyl, n-pentylsulfonyl, 1-methyl-n-butylsulfonyl, 2-methyl-nbutylsulfonyl,
3-methyl-n-butylsulfonyl, 1,1-dimethyl-n-propylsulfonyl, 1,2-dimethyl-npropylsulfonyl,
2,2-dimethyl-n-propylsulfonyl, 1-ethyl-n-propylsulfonyl, c-pentylsulfonyl,
1-methyl-c-butylsulfonyl, 2-methyl-c-butylsulfonyl, 3-methyl-c-butylsulfonyl, 1,2-dimethyl10
c-propylsulfonyl, 2,3-dimethyl-c-propylsulfonyl, 1-ethyl-c-propylsulfonyl, 2-ethyl-cpropylsulfonyl,
n-hexylsulfonyl, 1-methyl-n-pentylsulfonyl, 2-methyl-n-pentylsulfonyl, 3-
methyl-n-pentylsulfonyl, 4-methyl-n-pentylsulfonyl, 1,1-dimethyl-n-butylsulfonyl, 1,2-
dimethyl-n-butylsulfonyl, 1,3-dimethyl-n-butylsulfonyl, 2,2-dimethyl-n-butylsulfonyl, 2,3-
dimethyl-n-butylsulfonyl, 3,3-dimethyl-n-butylsulfonyl, 1-ethyl-n-butylsulfonyl, 2-ethyl-n15
butylsulfonyl, 1,1,2-trimethyl-n-propylsulfonyl, 1,2,2-trimethyl-n-propylsulfonyl, 1-ethyl-1-
methyl-n-propylsulfonyl, 1-ethyl-2-methyl-n-propylsulfonyl, c-hexylsulfonyl, 1-methyl-cpentylsulfonyl,
2-methyl-c-pentylsulfonyl, 3-methyl-c-pentylsulfonyl, 1-ethyl-cbutylsulfonyl,
2-ethyl-c-butylsulfonyl, 3-ethyl-c-butylsulfonyl, 1,2-dimethyl-c-butylsulfonyl,
1,3-dimethyl-c-butylsulfonyl, 2,2-dimethyl-c-butylsulfonyl, 2,3-dimethyl-c-butylsulfonyl,
20 2,4-dimethyl-c-butylsulfonyl, 3,3-dimethyl-c-butylsulfonyl, 1-n-propyl-c-propylsulfonyl, 2-
n-propyl-c-propylsulfonyl, 1-i-propyl-c-propylsulfonyl, 2-i-propyl-c-propylsulfonyl, 1,2,2-
trimethyl-c-propylsulfonyl, 1,2,3-trimethyl-c-propylsulfonyl, 2,2,3-trimethyl-cpropylsulfonyl,
1-ethyl-2-methyl-c-propylsulfonyl, 2-ethyl-1-methyl-c-propylsulfonyl, 2-
ethyl-2-methyl-c-propylsulfonyl, 2-ethyl-3-methyl-c-propylsulfonyl, 1-methyl-1-ethyl-n25
pentylsulfonyl, 1-heptylsulfonyl, 2-heptylsulfonyl, 1-ethyl-1,2-dimethyl-n-propylsulfonyl,
1-ethyl-2,2-dimethyl-n-propylsulfonyl, 1-octylsulfonyl, 3-octylsulfonyl, 4-methyl-3-nheptylsulfonyl,
6-methyl-2-n-heptylsulfonyl, 2-propyl-1-n-heptylsulfonyl, 2,4,4-trimethyl-
1-n-pentylsulfonyl, 1-nonylsulfonyl, 2-nonylsulfonyl, 2,6-dimetyl-4-n-heptylsulfonyl, 3-
ethyl-2,2-dimethyl-3-n-pentylsulfonyl, 3,5,5-trimethyl-1-n-hexylsulfonyl, 1-decylsulfonyl,
30 2-decylsulfonyl, 4-decylsulfonyl, 3,7-dimethyl-1-n-octylsulfonyl, 3,7-dimethyl-3-noctylsulfonyl
or the like may be mentioned.
A C1-10 alkylsulfonylamino group may be linear, branched or a C3-10
cycloalkylsulfonylamino group, and as specific examples, methylsulfonylamino,
ethylsulfonylamino, n-propylsulfonylamino, i-propylsulfonylamino, c-propylsulfonylamino,
35 n-butylsulfonylamino, i-butylsulfonylamino, s-butylsulfonylamino, t-butylsulfonylamino, cbutylsulfonylamino,
1-methyl-c-propylsulfonylamino, 2-methyl-c-propylsulfonylamino, npentylsulfonylamino,
1-methyl-n-butylsulfonylamino, 2-methyl-n-butylsulfonylamino, 3-
methyl-n-butylsulfonylamino, 1,1-dimethyl-n-propylsulfonylamino, 1,2-dimethyl-npropylsulfonylamino,
2,2-dimethyl-n-propylsulfonylamino, 1-ethyl-n-propylsulfonylamino,
40 c-pentylsulfonylamino, 1-methyl-c-butylsulfonylamino, 2-methyl-c-butylsulfonylamino, 3-
methyl-c-butylsulfonylamino, 1,2-dimethyl-c-propylsulfonylamino, 2,3-dimethyl-cpropylsulfonylamino,
1-ethyl-c-propylsulfonylamino, 2-ethyl-c-propylsulfonylamino, nhexylsulfonylamino,
1-methyl-n-pentylsulfonylamino, 2-methyl-n-pentylsulfonylamino, 3-
methyl-n-pentylsulfonylamino, 4-methyl-n-pentylsulfonylamino, 1,1-dimethyl-n45
butylsulfonylamino, 1,2-dimethyl-n-butylsulfonylamino, 1,3-dimethyl-nbutylsulfonylamino,
2,2-dimethyl-n-butylsulfonylamino, 2,3-dimethyl-nbutylsulfonylamino,
3,3-dimethyl-n-butylsulfonylamino, 1-ethyl-n-butylsulfonylamino, 2-
ethyl-n-butylsulfonylamino, 1,1,2-trimethyl-n-propylsulfonylamino, 1,2,2-trimethyl-n26
propylsulfonylamino, 1-ethyl-1-methyl-n-propylsulfonylamino, 1-ethyl-2-methyl-npropylsulfonylamino,
c-hexylsulfonylamino, 1-methyl-c-pentylsulfonylamino, 2-methyl-cpentylsulfonylamino,
3-methyl-c-pentylsulfonylamino, 1-ethyl-c-butylsulfonylamino, 2-
ethyl-c-butylsulfonylamino, 3-ethyl-c-butylsulfonylamino, 1,2-dimethyl-cbutylsulfonylamino,
1,3-dimethyl-c-butylsulfonylamino, 2,2-dimethyl-5 cbutylsulfonylamino,
2,3-dimethyl-c-butylsulfonylamino, 2,4-dimethyl-cbutylsulfonylamino,
3,3-dimethyl-c-butylsulfonylamino, 1-n-propyl-c-propylsulfonylamino,
2-n-propyl-c-propylsulfonylamino, 1-i-propyl-c-propylsulfonylamino, 2-i-propyl-cpropylsulfonylamino,
1,2,2-trimethyl-c-propylsulfonylamino, 1,2,3-trimethyl-c10
propylsulfonylamino, 2,2,3-trimethyl-c-propylsulfonylamino, 1-ethyl-2-methyl-cpropylsulfonylamino,
2-ethyl-1-methyl-c-propylsulfonylamino, 2-ethyl-2-methyl-cpropylsulfonylamino,
2-ethyl-3-methyl-c-propylsulfonylamino, 1-methyl-1-ethyl-npentylsulfonylamino,
1-heptylsulfonylamino, 2-heptylsulfonylamino, 1-ethyl-1,2-dimethyln-
propylsulfonylamino, 1-ethyl-2,2-dimethyl-n-propylsulfonylamino, 1-octylsulfonylamino,
15 3-octylsulfonylamino, 4-methyl-3-n-heptylsulfonylamino, 6-methyl-2-nheptylsulfonylamino,
2-propyl-1-n-heptylsulfonylamino, 2,4,4-trimethyl-1-npentylsulfonylamino,
1-nonylsulfonylamino, 2-nonylsulfonylamino, 2,6-dimethyl-4-nheptylsulfonylamino,
3-ethyl-2,2-dimethyl-3-n-pentylsulfonylamino, 3,5,5-trimethyl-1-nhexylsulfonylamino,
1-decylsulfonylamino, 2-decylsulfonylamino, 4-decylsulfonylamino,
20 3,7-dimethyl-1-n-octylsulfonylamino, 3,7-dimethyl-3-n-octylsulfonylamino, cheptylsulfonylamino,
c-octylsulfonylamino, 1-methyl-c-hexylsulfonylamino, 2-methyl-chexylsulfonylamino,
3-methyl-c-hexylsulfonylamino, 1,2-dimethyl-c-hexylsulfonylamino,
1-ethyl-c-hexylsulfonylamino, 1-methyl-c-pentylsulfonylamino, 2-methyl-cpentylsulfonylamino,
3-methyl-c-pentylsulfonylamino or the like may be mentioned.
25 A C1-3 alkoxy group may be linear, branched or a C3 cycloalkoxy group, and as
specific examples, methoxy, ethoxy, n-propoxy, i-propoxy, c-propoxy or the like may be
mentioned.
A C1-6 alkoxy group may be linear, branched or a C3-6 cycloalkoxy group, and as
specific examples, in addition to those mentioned above, n-butoxy, i-butoxy, s-butoxy, t30
butoxy, c-butoxy, 1-methyl-c-propoxy, 2-methyl-c-propoxy, n-pentyloxy, 1-methyl-nbutoxy,
2-methyl-n-butoxy, 3-methyl-n-butoxy, 1,1-dimethyl-n-propoxy, 1,2-dimethyl-npropoxy,
2,2-dimethyl-n-propoxy, 1-ethyl-n-propoxy, c-pentyloxy, 1-methyl-c-butoxy, 2-
methyl-c-butoxy, 3-methyl-c-butoxy, 1,2-dimethyl-c-propoxy, 2,3-dimethyl-c-propoxy, 1-
ethyl-c-propoxy, 2-ethyl-c-propoxy, n-hexyloxy, 1-methyl-n-pentyloxy, 2-methyl-n35
pentyloxy, 3-methyl-n-pentyloxy, 4-methyl-n-pentyloxy, 1,1-dimethyl-n-butoxy, 1,2-
dimethyl-n-butoxy, 1,3-dimethyl-n-butoxy, 2,2-dimethyl-n-butoxy, 2,3-dimethyl-n-butoxy,
3,3-dimethyl-n-butoxy, 1-ethyl-n-butoxy, 2-ethyl-n-butoxy, 1,1,2-trimethyl-n-propoxy,
1,2,2-trimethyl-n-propoxy, 1-ethyl-1-methyl-n-propoxy, 1-ethyl-2-methyl-n-propoxy, chexyloxy,
1-methyl-c-pentyloxy, 2-methyl-c-pentyloxy, 3-methyl-c-pentyloxy, 1-ethyl-c40
butoxy, 2-ethyl-c-butoxy, 3-ethyl-c-butoxy, 1,2-dimethyl-c-butoxy, 1,3-dimethyl-c-butoxy,
2,2-dimethyl-c-butoxy, 2,3-dimethyl-c-butoxy, 2,4-dimethyl-c-butoxy, 3,3-dimethyl-cbutoxy,
1-n-propyl-c-propoxy, 2-n-propyl-c-propoxy, 1-i-propyl-c-propoxy, 2-i-propyl-cpropoxy,
1,2,2-trimethyl-c-propoxy, 1,2,3-trimethyl-c-propoxy, 2,2,3-trimethyl-c-propoxy,
1-ethyl-2-methyl-c-propoxy, 2-ethyl-1-methyl-c-propoxy, 2-ethyl-2-methyl-c-propoxy, 2-
45 ethyl-3-methyl-c-propoxy or the like may be mentioned.
A C1-10 alkoxy group may be linear, branched or a C3-10 cycloalkoxy group, and as
specific examples, in addition to those mentioned above, 1-methyl-1-ethyl-n-pentyloxy,
1-heptyloxy, 2-heptyloxy, 1-ethyl-1,2-dimethyl-n-propyloxy, 1-ethyl-2,2-dimethyl-n27
propyloxy, 1-octyloxy, 3-octyloxy, 4-methyl-3-n-heptyloxy, 6-methyl-2-n-heptyloxy, 2-
propyl-1-n-heptyloxy, 2,4,4-trimethyl-1-n-pentyloxy, 1-nonyloxy, 2-nonyloxy, 2,6-
dimethyl-4-n-heptyloxy, 3-ethyl-2,2-dimethyl-3-n-pentyloxy, 3,5,5-trimethyl-1-n-hexyloxy,
1-decyloxy, 2-decyloxy, 4-decyloxy, 3,7-dimethyl-1-n-octyloxy, 3,7-dimethyl-3-n-octyloxy
or the like may be mentioned5 .
A C1-10 alkoxycarbonyl group may be linear, branched or a C3-10
cycloalkoxycarbonyl group, and as specific examples, methoxycarbonyl, ethoxycarbonyl,
n-propoxycarbonyl, i-propoxycarbonyl, c-propoxycarbonyl, n-butoxycarbonyl, ibutoxycarbonyl,
s-butoxycarbonyl, t-butoxycarbonyl, c-butoxycarbonyl, 1-methyl-c10
propoxycarbonyl, 2-methyl-c-propoxycarbonyl, n-pentyloxycarbonyl, 1-methyl-nbutoxycarbonyl,
2-methyl-n-butoxycarbonyl, 3-methyl-n-butoxycarbonyl, 1,1-dimethyl-npropoxycarbonyl,
1,2-dimethyl-n-propoxycarbonyl, 2,2-dimethyl-n-propoxycarbonyl, 1-
ethyl-n-propoxycarbonyl, c-pentyloxycarbonyl, 1-methyl-c-butoxycarbonyl, 2-methyl-cbutoxycarbonyl,
3-methyl-c-butoxycarbonyl, 1,2-dimethyl-c-propoxycarbonyl, 2,3-
15 dimethyl-c-propoxycarbonyl, 1-ethyl-c-propoxycarbonyl, 2-ethyl-c-propoxycarbonyl, nhexyloxycarbonyl,
1-methyl-n-pentyloxycarbonyl, 2-methyl-n-pentyloxycarbonyl, 3-
methyl-n-pentyloxycarbonyl, 4-methyl-n-pentyloxycarbonyl, 1,1-dimethyl-nbutoxycarbonyl,
1,2-dimethyl-n-butoxycarbonyl, 1,3-dimethyl-n-butoxycarbonyl, 2,2-
dimethyl-n-butoxycarbonyl, 2,3-dimethyl-n-butoxycarbonyl, 3,3-dimethyl-n20
butoxycarbonyl, 1-ethyl-n-butoxycarbonyl, 2-ethyl-n-butoxycarbonyl, 1,1,2-trimethyl-npropoxycarbonyl,
1,2,2-trimethyl-n-propoxycarbonyl, 1-ethyl-1-methyl-npropoxycarbonyl,
1-ethyl-2-methyl-n-propoxycarbonyl, c-hexyloxycarbonyl, 1-methyl-cpentyloxycarbonyl,
2-methyl-c-pentyloxycarbonyl, 3-methyl-c-pentyloxycarbonyl, 1-
ethyl-c-butoxycarbonyl, 2-ethyl-c-butoxycarbonyl, 3-ethyl-c-butoxycarbonyl, 1,2-
25 dimethyl-c-butoxycarbonyl, 1,3-dimethyl-c-butoxycarbonyl, 2,2-dimethyl-cbutoxycarbonyl,
2,3-dimethyl-c-butoxycarbonyl, 2,4-dimethyl-c-butoxycarbonyl, 3,3-
dimethyl-c-butoxycarbonyl, 1-n-propyl-c-propoxycarbonyl, 2-n-propyl-c-propoxycarbonyl,
1-i-propyl-c-propoxycarbonyl, 2-i-propyl-c-propoxycarbonyl, 1,2,2-trimethyl-cpropoxycarbonyl,
1,2,3-trimethyl-c-propoxycarbonyl, 2,2,3-trimethyl-c-propoxycarbonyl,
30 1-ethyl-2-methyl-c-propoxycarbonyl, 2-ethyl-1-methyl-c-propoxycarbonyl, 2-ethyl-2-
methyl-c-propoxycarbonyl, 2-ethyl-3-methyl-c-propoxycarbonyl, 1-methyl-1-ethyl-npentyloxycarbonyl,
1-heptyloxycarbonyl, 2-heptyloxycarbonyl, 1-ethyl-1,2-dimethyl-npropyloxycarbonyl,
1-ethyl-2,2-dimethyl-n-propyloxycarbonyl, 1-octyloxycarbonyl, 3-
octyloxycarbonyl, 4-methyl-3-n-heptyloxycarbonyl, 6-methyl-2-n-heptyloxycarbonyl, 2-
35 propyl-1-n-heptyloxycarbonyl, 2,4,4-trimethyl-1-n-pentyloxycarbonyl, 1-
nonyloxycarbonyl, 2-nonyloxycarbonyl, 2,6-dimethyl-4-n-heptyloxycarbonyl, 3-ethyl-2,2-
dimethyl-3-n-pentyloxycarbonyl, 3,5,5-trimethyl-1-n-hexyloxycarbonyl, 1-
decyloxycarbonyl, 2-decyloxycarbonyl, 4-decyloxycarbonyl, 3,7-dimethyl-1-noctyloxycarbonyl,
3,7-dimethyl-3-n-octyloxycarbonyl or the like may be mentioned.
40 A C1-3 alkylcarbonyl group may linear, branched or a C3 cycloalkylcarbonyl group,
and as specific examples, methylcarbonyl, ethylcarbonyl, n-propylcarbonyl, ipropylcarbonyl,
c-propylcarbonyl or the like may be mentioned.
A C1-10 alkylcarbonyl group may linear, branched or a C3-10 cycloalkylcarbonyl
group, and as specific examples, in addition to those mentioned above, n-butylcarbonyl,
45 i-butylcarbonyl, s-butylcarbonyl, t-butylcarbonyl, c-butylcarbonyl, 1-methyl-cpropylcarbonyl,
2-methyl-c-propylcarbonyl, n-pentylcarbonyl, 1-methyl-n-butylcarbonyl,
2-methyl-n-butylcarbonyl, 3-methyl-n-butylcarbonyl, 1,1-dimethyl-n-propylcarbonyl, 1,2-
dimethyl-n-propylcarbonyl, 2,2-dimethyl-n-propylcarbonyl, 1-ethyl-n-propylcarbonyl, c28
pentylcarbonyl, 1-methyl-c-butylcarbonyl, 2-methyl-c-butylcarbonyl, 3-methyl-cbutylcarbonyl,
1,2-dimethyl-c-propylcarbonyl, 2,3-dimethyl-c-propylcarbonyl, 1-ethyl-cpropylcarbonyl,
2-ethyl-c-propylcarbonyl, n-hexylcarbonyl, 1-methyl-n-pentylcarbonyl, 2-
methyl-n-pentylcarbonyl, 3-methyl-n-pentylcarbonyl, 4-methyl-n-pentylcarbonyl, 1,1-
dimethyl-n-butylcarbonyl, 1,2-dimethyl-n-butylcarbonyl, 1,3-dimethyl-n-butylcarbonyl5 ,
2,2-dimethyl-n-butylcarbonyl, 2,3-dimethyl-n-butylcarbonyl, 3,3-dimethyl-n-butylcarbonyl,
1-ethyl-n-butylcarbonyl, 2-ethyl-n-butylcarbonyl, 1,1,2-trimethyl-n-propylcarbonyl, 1,2,2-
trimethyl-n-propylcarbonyl, 1-ethyl-1-methyl-n-propylcarbonyl, 1-ethyl-2-methyl-npropylcarbonyl,
c-hexylcarbonyl, 1-methyl-c-pentylcarbonyl, 2-methyl-c-pentylcarbonyl,
10 3-methyl-c-pentylcarbonyl, 1-ethyl-c-butylcarbonyl, 2-ethyl-c-butylcarbonyl, 3-ethyl-cbutylcarbonyl,
1,2-dimethyl-c-butylcarbonyl, 1,3-dimethyl-c-butylcarbonyl, 2,2-dimethylc-
butylcarbonyl, 2,3-dimethyl-c-butylcarbonyl, 2,4-dimethyl-c-butylcarbonyl, 3,3-
dimethyl-c-butylcarbonyl, 1-n-propyl-c-propylcarbonyl, 2-n-propyl-c-propylcarbonyl, 1-ipropyl-
c-propylcarbonyl, 2-i-propyl-c-propylcarbonyl, 1,2,2-trimethyl-c-propylcarbonyl,
15 1,2,3-trimethyl-c-propylcarbonyl, 2,2,3-trimethyl-c-propylcarbonyl, 1-ethyl-2-methyl-cpropylcarbonyl,
2-ethyl-1-methyl-c-propylcarbonyl, 2-ethyl-2-methyl-c-propylcarbonyl, 2-
ethyl-3-methyl-c-propylcarbonyl, 1-methyl-1-ethyl-n-pentylcarbonyl, 1-heptylcarbonyl, 2-
heptylcarbonyl, 1-ethyl-1,2-dimethyl-n-propylcarbonyl, 1-ethyl-2,2-dimethyl-npropylcarbonyl,
1-octylcarbonyl, 3-octylcarbonyl, 4-methyl-3-n-heptylcarbonyl, 6-methyl-
20 2-n-heptylcarbonyl, 2-propyl-1-n-heptylcarbonyl, 2,4,4-trimethyl-1-n-pentylcarbonyl, 1-
nonylcarbonyl, 2-nonylcarbonyl, 2,6-dimethyl-4-n-heptylcarbonyl, 3-ethyl-2,2-dimethyl-3-
n-pentylcarbonyl, 3,5,5-trimethyl-1-n-hexylcarbonyl, 1-decylcarbonyl, 2-decylcarbonyl,
4-decylcarbonyl, 3,7-dimethyl-1-n-octylcarbonyl, 3,7-dimethyl-3-n-octylcarbonyl or the
like may be mentioned.
25 A C1-10 alkylcarbonyloxy group may be linear, branched or a C3-10
cycloalkylcarbonyloxy group, and as specific examples, in addition to those mentioned
above, n-butylcarbonyloxy, i-butylcarbonyloxy, s-butylcarbonyloxy, t-butylcarbonyloxy, cbutylcarbonyloxy,
1-methyl-c-propylcarbonyloxy, 2-methyl-c-propylcarbonyloxy, npentylcarbonyloxy,
1-methyl-n-butylcarbonyloxy, 2-methyl-n-butylcarbonyloxy, 3-methyl30
n-butylcarbonyloxy, 1,1-dimethyl-n-propylcarbonyloxy, 1,2-dimethyl-n-propylcarbonyloxy,
2,2-dimethyl-n-propylcarbonyloxy, 1-ethyl-n-propylcarbonyloxy, c-pentylcarbonyloxy, 1-
methyl-c-butylcarbonyloxy, 2-methyl-c-butylcarbonyloxy, 3-methyl-c-butylcarbonyloxy,
1,2-dimethyl-c-propylcarbonyloxy, 2,3-dimethyl-c-propylcarbonyloxy, 1-ethyl-cpropylcarbonyloxy,
2-ethyl-c-propylcarbonyloxy, n-hexylcarbonyloxy, 1-methyl-n35
pentylcarbonyloxy, 2-methyl-n-pentylcarbonyloxy, 3-methyl-n-pentylcarbonyloxy, 4-
methyl-n-pentylcarbonyloxy, 1,1-dimethyl-n-butylcarbonyloxy, 1,2-dimethyl-nbutylcarbonyloxy,
1,3-dimethyl-n-butylcarbonyloxy, 2,2-dimethyl-n-butylcarbonyloxy, 2,3-
dimethyl-n-butylcarbonyloxy, 3,3-dimethyl-n-butylcarbonyloxy, 1-ethyl-nbutylcarbonyloxy,
2-ethyl-n-butylcarbonyloxy, 1,1,2-trimethyl-n-propylcarbonyloxy, 1,2,2-
40 trimethyl-n-propylcarbonyloxy, 1-ethyl-1-methyl-n-propylcarbonyloxy, 1-ethyl-2-methyl-npropylcarbonyloxy,
c-hexylcarbonyloxy, 1-methyl-c-pentylcarbonyloxy, 2-methyl-cpentylcarbonyloxy,
3-methyl-c-pentylcarbonyloxy, 1-ethyl-c-butylcarbonyloxy, 2-ethyl-cbutylcarbonyloxy,
3-ethyl-c-butylcarbonyloxy, 1,2-dimethyl-c-butylcarbonyloxy, 1,3-
dimethyl-c-butylcarbonyloxy, 2,2-dimethyl-c-butylcarbonylxoy, 2,3-dimethyl-c45
butylcarbonyloxy, 2,4-dimethyl-c-butylcarbonyloxy, 3,3-dimethyl-c-butylcarbonyloxy, 1-npropyl-
c-propylcarbonyloxy, 2-n-propyl-c-propylcarbonyloxy, 1-i-propyl-cpropylcarbonyloxy,
2-i-propyl-c-propylcarbonyloxy, 1,2,2-trimethyl-c-propylcarbonyloxy,
1,2,3-trimethyl-c-propylcarbonyloxy, 2,2,3-trimethyl-c-propylcarbonyloxy, 1-ethyl-2-
29
methyl-c-propylcarbonyloxy, 2-ethyl-1-methyl-c-propylcarbonyloxy, 2-ethyl-2-methyl-cpropylcarbonyloxy,
2-ethyl-3-methyl-c-propylcarbonyloxy, 1-methyl-1-ethyl-npentylcarbonyloxy,
1-heptylcarbonyloxy, 2-heptylcarbonyloxy, 1-ethyl-1,2-dimethyl-npropylcarbonyloxy,
1-ethyl-2,2-dimethyl-n-propylcarbonyloxy, 1-octylcarbonyloxy, 3-
octylcarbonyloxy, 4-methyl-3-n-heptylcarbonyloxy, 6-methyl-2-n-heptylcarbonyloxy, 5 2-
propyl-1-n-heptylcarbonyloxy, 2,4,4-trimethyl-1-n-pentylcarbonyloxy, 1-nonylcarbonyloxy,
2-nonylcarbonyloxy, 2,6-dimethyl-4-n-heptylcarbonyloxy, 3-ethyl-2,2-dimethyl-3-npentylcarbonyloxy,
3,5,5-trimethyl-1-n-hexylcarbonyloxy, 1-decylcarbonyloxy, 2-
decylcarbonyloxy, 4-decylcarbonyloxy, 3,7-dimethyl-1-n-octylcarbonyloxy, 3,7-dimethyl-
10 3-n-octylcarbonyloxy or the like may be mentioned.
A C1-10 alkylcarbonylamino group may be linear, branched or a C3-10
cycloalkylcarbonylamino group, and as specific examples, methylcarbonylamino,
ethylcarbonylamino, n-propylcarbonylamino, i-propylcarbonylamino, cpropylcarbonylamino,
n-butylcarbonylamino, i-butylcarbonylamino, s15
butylcarbonylamino, t-butylcarbonylamino, c-butylcarbonylamino, 1-methyl-cpropylcarbonylamino,
2-methyl-c-propylcarbonylamino, n-pentylcarbonylamino, 1-
methyl-n-butylcarbonylamino, 2-methyl-n-butylcarbonylamino, 3-methyl-nbutylcarbonylamino,
1,1-dimethyl-n-propylcarbonylamino, 1,2-dimethyl-npropylcarbonylamino,
2,2-dimethyl-n-propylcarbonylamino, 1-ethyl-n20
propylcarbonylamino, c-pentylcarbonylamino, 1-methyl-c-butylcarbonylamino, 2-methylc-
butylcarbonylamino, 3-methyl-c-butylcarbonylamino, 1,2-dimethyl-cpropylcarbonylamino,
2,3-dimethyl-c-propylcarbonylamino, 1-ethyl-cpropylcarbonylamino,
2-ethyl-c-propylcarbonylamino, n-hexylcarbonylamino, 1-methyln-
pentylcarbonylamino, 2-methyl-n-pentylcarbonylamino, 3-methyl-n25
pentylcarbonylamino, 4-methyl-n-pentylcarbonylamino, 1,1-dimethyl-nbutylcarbonylamino,
1,2-dimethyl-n-butylcarbonylamino, 1,3-dimethyl-nbutylcarbonylamino,
2,2-dimethyl-n-butylcarbonylamino, 2,3-dimethyl-nbutylcarbonylamino,
3,3-dimethyl-n-butylcarbonylamino, 1-ethyl-n-butylcarbonylamino,
2-ethyl-n-butylcarbonylamino, 1,1,2-trimethyl-n-propylcarbonylamino, 1,2,2-trimethyl-n30
propylcarbonylamino, 1-ethyl-1-methyl-n-propylcarbonylamino, 1-ethyl-2-methyl-npropylcarbonylamino,
c-hexylcarbonylamino, 1-methyl-c-pentylcarbonylamino, 2-methylc-
pentylcarbonylamino, 3-methyl-c-pentylcarbonylamino, 1-ethyl-c-butylcarbonylamino,
2-ethyl-c-butylcarbonylamino, 3-ethyl-c-butylcarbonylamino, 1,2-dimethyl-cbutylcarbonylamino,
1,3-dimethyl-c-butylcarbonylamino, 2,2-dimethyl-c35
butylcarbonylamino, 2,3-dimethyl-c-butylcarbonylamino, 2,4-dimethyl-cbutylcarbonylamino,
3,3-dimethyl-c-butylcarbonylamino, 1-n-propyl-cpropylcarbonylamino,
2-n-propyl-c-propylcarbonylamino, 1-i-propyl-cpropylcarbonylamino,
2-i-propyl-c-propylcarbonylamino, 1,2,2-trimethyl-c-propylcarbonylamino,
1,2,3-trimethyl-c-propylcarbonylamino, 2,2,3-trimethyl-c40
propylcarbonylamino, 1-ethyl-2-methyl-c-propylcarbonylamino, 2-ethyl-1-methyl-cpropylcarbonylamino,
2-ethyl-2-methyl-c-propylcarbonylamino, 2-ethyl-3-methyl-cpropylcarbonylamino,
1-methyl-1-ethyl-n-pentylcarbonylamino, 1-heptylcarbonylamino,
2-heptylcarbonylamino, 1-ethyl-1,2-dimethyl-n-propylcarbonylamino, 1-ethyl-2,2-
dimethyl-n-propylcarbonylamino, 1-octylcarbonylamino, 3-octylcarbonylamino, 4-methyl-
45 3-n-heptylcarbonylamino, 6-methyl-2-n-heptylcarbonylamino, 2-propyl-1-nheptylcarbonylamino,
2,4,4-trimethyl-1-n-pentylcarbonylamino, 1-nonylcarbonylamino,
2-nonylcarbonylamino, 2,6-dimethyl-4-n-heptylcarbonylamino, 3-ethyl-2,2-dimethyl-3-npentylcarbonylamino,
3,5,5-trimethyl-1-n-hexylcarbonylamino, 1-decylcarbonylamino, 2-
30
decylcarbonylamino, 4-decylcarbonylamino, 3,7-dimethyl-1-n-octylcarbonylamino, 3,7-
dimethyl-3-n-octylcarbonylamino or the like may be mentioned.
A C1-10 monoalkylamino group may be linear, branched or a C3-10 cycloalkylamino
group, and specific examples, methylamino, ethylamino, n-propylamino, i-propylamino,
c-propylamino, n-butylamino, i-butylamino, s-butylamino, t-butylamino, c-butylamino, 5 1-
methyl-c-propylamino, 2-methyl-c-propylamino, n-pentylamino, 1-methyl-n-butylamino,
2-methyl-n-butylamino, 3-methyl-n-butylamino, 1,1-dimethyl-n-propylamino, 1,2-
dimethyl-n-propylamino, 2,2-dimethyl-n-propylamino, 1-ethyl-n-propylamino, cpentylamino,
1-methyl-c-butylamino, 2-methyl-c-butylamino, 3-methyl-c-butylamino, 1,2-
10 dimethyl-c-propylamino, 2,3-dimethyl-c-propylamino, 1-ethyl-c-propylamino, 2-ethyl-cpropylamino,
n-hexylamino, 1-methyl-n-pentylamino, 2-methyl-n-pentylamino, 3-methyln-
pentylamino, 4-methyl-n-pentylamino, 1,1-dimethyl-n-butylamino, 1,2-dimethyl-nbutylamino,
1,3-dimethyl-n-butylamino, 2,2-dimethyl-n-butylamino, 2,3-dimethyl-nbutylamino,
3,3-dimethyl-n-butylamino, 1-ethyl-n-butylamino, 2-ethyl-n-butylamino,
15 1,1,2-trimethyl-n-propylamino, 1,2,2-trimethyl-n-propylamino, 1-ethyl-1-methyl-npropylamino,
1-ethyl-2-methyl-n-propylamino, c-hexylamino, 1-methyl-c-pentylamino, 2-
methyl-c-pentylamino, 3-methyl-c-pentylamino, 1-ethyl-c-butylamino, 2-ethyl-cbutylamino,
3-ethyl-c-butylamino, 1,2-dimethyl-c-butylamino, 1,3-dimethyl-c-butylamino,
2,2-dimethyl-c-butylamino, 2,3-dimethyl-c-butylamino, 2,4-dimethyl-c-butylamino, 3,3-
20 dimethyl-c-butylamino, 1-n-propyl-c-propylamino, 2-n-propyl-c-propylamino, 1-i-propyl-cpropylamino,
2-i-propyl-c-propylamino, 1,2,2-trimethyl-c-propylamino, 1,2,3-trimethyl-cpropylamino,
2,2,3-trimethyl-c-propylamino, 1-ethyl-2-methyl-c-propylamino, 2-ethyl-1-
methyl-c-propylamino, 2-ethyl-2-methyl-c-propylamino, 2-ethyl-3-methyl-c-propylamino,
1-methyl-1-ethyl-n-pentylamino, 1-heptylamino, 2-heptylamino, 1-ethyl-1,2-dimethyl-n25
propylamino, 1-ethyl-2,2-dimethyl-n-propylamino, 1-octylamino, 3-octylamino, 4-methyl-
3-n-heptylamino, 6-methyl-2-n-heptylamino, 2-propyl-1-n-heptylamino, 2,4,4-trimethyl-1-
n-pentylamino, 1-nonylamino, 2-nonylamino, 2,6-dimethyl-4-n-heptylamino, 3-ethyl-2,2-
dimethyl-3-n-pentylamino, 3,5,5-trimethyl-1-n-hexylamino, 1-decylamino, 2-decylamino,
4-decylamino, 3,7-dimethyl-1-n-octylamino, 3,7-dimethyl-3-n-octylamino or the like may
30 be mentioned.
A di-C1-10 alkylamino group may be symmetric or asymmetric. A symmetric di-C1-
10 alkylamino group may be linear, branched or a C3-10 cycloalkylamino group, and as
specific examples, dimethylamino, diethylamino, di-n-propylamino, di-i-propylamino, dic-
propylamino, di-n-butylamino, di-i-butylamino, di-s-butylamino, di-t-butylamino, di-c35
butylamino, di-(1-methyl-c-propyl)amino, di-(2-methyl-c-propyl)amino, di-n-pentylamino,
di-(1-methyl-n-butyl)amino, di-(2-methyl-n-butyl)amino, di-(3-methyl-n-butyl)amino, di-
(1,1-dimethyl-n-propyl)amino, di-(1,2-dimethyl-n-propyl)amino, di-(2,2-dimethyl-npropyl)
amino, di-(1-ethyl-n-propyl)amino, di-c-pentylamino, di-(1-methyl-c-butyl)amino,
di-(2-methyl-c-butyl)amino, di-(3-methyl-c-butyl)amino, di-(1,2-dimethyl-c-propyl)amino,
40 di-(2,3-dimethyl-c-propyl)amino, di-(1-ethyl-c-propyl)amino, di-(2-ethyl-c-propyl)amino,
di-n-hexylamino, di-(1-methyl-n-pentyl)amino, di-(2-methyl-n-pentyl)amino, di-(3-methyln-
pentyl)amino, di-(4-methyl-n-pentyl)amino, di-(1,1-dimethyl-n-butyl)amino, di-(1,2-
dimethyl-n-butyl)amino, di-(1,3-dimethyl-n-butyl)amino, di-(2,2-dimethyl-n-butyl)amino,
di-(2,3-dimethyl-n-butyl)amino, di-(3,3-dimethyl-n-butyl)amino, di-(1-ethyl-n-butyl)amino,
45 di-(2-ethyl-n-butyl)amino, di-(1,1,2-trimethyl-n-propyl)amino, di-(1,2,2-trimethyl-npropyl)
amino, di-(1-ethyl-1-methyl-n-propyl)amino, di-(1-ethyl-2-methyl-n-propyl)amino,
di-c-hexylamino, di-(1-methyl-c-pentyl)amino, di-(2-methyl-c-pentyl)amino, di-(3-methylc-
pentyl)amino, di-(1-ethyl-c-butyl)amino, di-(2-ethyl-c-butyl)amino, di-(3-ethyl-c31
butyl)amino, di-(1,2-dimethyl-c-butyl)amino, di-(1,3-dimethyl-c-butyl)amino, di-(2,2-
dimethyl-c-butyl)amino, di-(2,3-dimethyl-c-butyl)amino, di-(2,4-dimethyl-c-butyl)amino,
di-(3,3-dimethyl-c-butyl)amino, di-(1-n-propyl-c-propyl)amino, di-(2-n-propyl-cpropyl)
amino, di-(1-i-propyl-c-propyl)amino, di-(2-i-propyl-c-propyl)amino, di-(1,2,2-
trimethyl-c-propyl)amino, di-(1,2,3-trimethyl-c-propyl)amino, di-(2,2,3-trimethyl-5 cpropyl)
amino, di-(1-ethyl-2-methyl-c-propyl)amino, di-(2-ethyl-1-methyl-c-propyl)amino,
di-(2-ethyl-2-methyl-c-propyl)amino, di-(2-ethyl-3-methyl-c-propyl)amino, di-(1-methyl-1-
ethyl-n-pentyl)amino, di-(1-heptyl)amino, di-(2-heptyl)amino, di-(1-ethyl-1,2-dimethyl-npropyl)
amino, di-(1-ethyl-2,2-dimethyl-n-propyl)amino, di-(1-octyl)amino, di-(3-
10 octyl)amino, di-(4-methyl-3-n-heptyl)amino, di-(6-methyl-2-n-heptyl)amino, di-(2-propyl-
1-n-heptyl)amino, di-(2,4,4-trimethyl-1-n-pentyl)amino, di-(1-nonyl)amino, di-(2-
nonyl)amino, di-(2,6-dimethyl-4-n-heptyl)amino, di-(3-ethyl-2,2-dimethyl-3-npentyl)
amino, di-(3,5,5-trimethyl-1-n-hexyl)amino, di-(1-decyl)amino, di-(2-decyl)amino,
di-(4-decyl)amino, di-(3,7-dimethyl-1-n-octyl)amino, di-(3,7-dimethyl-3-n-octyl)amino or
15 the like may be mentioned.
An asymmetric di-C1-10 alkylamino group may be linear, branched or a C3-10
cycloalkylamino group, and as specific examples, (methyl, ethyl)amino, (methyl, npropyl)
amino, (methyl, i-propyl)amino, (methyl, c-propyl)amino, (methyl, n-butyl)amino,
(methyl, i-butyl)amino, (methyl, s-butyl)amino, (methyl, t-butyl)amino, (methyl, n20
pentyl)amino, (methyl, c-pentyl)amino, (methyl, n-hexyl)amino, (methyl, c-hexyl)amino,
(ethyl, n-propyl)amino, (ethyl, i-propyl)amino, (ethyl, c-propyl)amino, (ethyl, nbutyl)
amino, (ethyl, i-butyl)amino, (ethyl, s-butyl)amino, (ethyl, t-butyl)amino, (ethyl, npentyl)
amino, (ethyl, c-pentyl)amino, (ethyl, n-hexyl)amino, (ethyl, c-hexyl)amino, (npropyl,
i-propyl)amino, (n-propyl, c-propyl)amino, (n-propyl, n-butyl)amino, (n-propyl, i25
butyl)amino, (n-propyl, s-butyl)amino, (n-propyl, t-butyl)amino, (n-propyl, n-pentyl)amino,
(n-propyl, c-pentyl)amino, (n-propyl, n-hexyl)amino, (n-propyl, c-hexyl)amino, (i-propyl,
c-propyl)amino, (i-propyl, n-butyl)amino, (i-propyl, i-butyl)amino, (i-propyl, s-butyl)amino,
(i-propyl, t-butyl)amino, (i-propyl, n-pentyl)amino, (i-propyl, c-pentyl)amino, (i-propyl, nhexyl)
amino, (i-propyl, c-hexyl)amino, (c-propyl, n-butyl)amino, (c-propyl, i-butyl)amino,
30 (c-propyl, s-butyl)amino, (c-propyl, t-butyl)amino, (c-propyl, n-pentyl)amino, (c-propyl, cpentyl)
amino, (c-propyl, n-hexyl)amino, (c-propyl, c-hexyl)amino, (n-butyl, i-butyl)amino,
(n-butyl, s-butyl)amino, (n-butyl, t-butyl)amino, (n-butyl, n-pentyl)amino, (n-butyl, cpentyl)
amino, (n-butyl, n-hexyl)amino, (n-butyl, c-hexyl)amino, (i-butyl, s-butyl)amino, (ibutyl,
t-butyl)amino, (i-butyl, n-pentyl)amino, (i-butyl, c-pentyl)amino, (i-butyl, n35
hexyl)amino, (i-butyl, c-hexyl)amino, (s-butyl, t-butyl)amino, (s-butyl, n-pentyl)amino, (sbutyl,
c-pentyl)amino, (s-butyl, n-hexyl)amino, (s-butyl, c-hexyl)amino, (t-butyl, npentyl)
amino, (t-butyl, c-pentyl)amino, (t-butyl, n-hexyl)amino, (t-butyl, c-hexyl)amino,
(n-pentyl, c-pentyl)amino, (n-pentyl, n-hexyl)amino, (n-pentyl, c-hexyl)amino, (c-pentyl,
n-hexyl)amino, (c-pentyl, c-hexyl)amino, (n-hexyl, c-hexyl)amino, (methyl, n40
heptyl)amino, (methyl, n-octyl)amino, (methyl, n-nonanyl)amino, (methyl, n-decyl)amino,
(ethyl, n-heptyl)amino, (ethyl, n-octyl)amino, (ethyl, n-nonanyl)amino, (ethyl, ndecyl)
amino or the like may be mentioned.
A C1-10 alkylaminocarbonyl group may be linear, branched or a C1-10
cycloalkylaminocarbonyl group and may be a di-C1-10 alkylaminocarbonyl group, and as
45 specific examples, methylaminocarbonyl, ethylaminocarbonyl, n-propylaminocarbonyl, ipropylaminocarbonyl,
c-propylaminocarbonyl, n-butylaminocarbonyl, ibutylaminocarbonyl,
s-butylaminocarbonyl, t-butylaminocarbonyl, c-butylaminocarbonyl,
1-methyl-c-propylaminocarbonyl, 2-methyl-c-propylaminocarbonyl, n32
pentylaminocarbonyl, 1-methyl-n-butylaminocarbonyl, 2-methyl-n-butylaminocarbonyl,
3-methyl-n-butylaminocarbonyl, 1,1-dimethyl-n-propylaminocarbonyl, 1,2-dimethyl-npropylaminocarbonyl,
2,2-dimethyl-n-propylaminocarbonyl, 1-ethyl-npropylaminocarbonyl,
c-pentylaminocarbonyl, 1-methyl-c-butylaminocarbonyl, 2-methylc-
butylaminocarbonyl, 3-methyl-c-butylaminocarbonyl, 1,2-dimethyl-5 cpropylaminocarbonyl,
2,3-dimethyl-c-propylaminocarbonyl, 1-ethyl-cpropylaminocarbonyl,
2-ethyl-c-propylaminocarbonyl, n-hexylaminocarbonyl, 1-methyln-
pentylaminocarbonyl, 2-methyl-n-pentylaminocarbonyl, 3-methyl-npentylaminocarbonyl,
4-methyl-n-pentylaminocarbonyl, 1,1-dimethyl-n10
butylaminocarbonyl, 1,2-dimethyl-n-butylaminocarbonyl, 1,3-dimethyl-nbutylaminocarbonyl,
2,2-dimethyl-n-butylaminocarbonyl, 2,3-dimethyl-nbutylaminocarbonyl,
3,3-dimethyl-n-butylaminocarbonyl, 1-ethyl-n-butylaminocarbonyl,
2-ethyl-n-butylaminocarbonyl, 1,1,2-trimethyl-n-propylaminocarbonyl, 1,2,2-trimethyl-npropylaminocarbonyl,
1-ethyl-1-methyl-n-propylaminocarbonyl, 1-ethyl-2-methyl-n15
propylaminocarbonyl, c-hexylaminocarbonyl, 1-methyl-c-pentylaminocarbonyl, 2-methylc-
pentylaminocarbonyl, 3-methyl-c-pentylaminocarbonyl, 1-ethyl-c-butylaminocarbonyl,
2-ethyl-c-butylaminocarbonyl, 3-ethyl-c-butylaminocarbonyl, 1,2-dimethyl-cbutylaminocarbonyl,
1,3-dimethyl-c-butylaminocarbonyl, 2,2-dimethyl-cbutylaminocarbonyl,
2,3-dimethyl-c-butylaminocarbonyl, 2,4-dimethyl-c20
butylaminocarbonyl, 3,3-dimethyl-c-butylaminocarbonyl, 1-n-propyl-cpropylaminocarbonyl,
2-n-propyl-c-propylaminocarbonyl, 1-i-propyl-cpropylaminocarbonyl,
2-i-propyl-c-propylaminocarbonyl, 1,2,2-trimethyl-cpropylaminocarbonyl,
1,2,3-trimethyl-c-propylaminocarbonyl, 2,2,3-trimethyl-cpropylaminocarbonyl,
1-ethyl-2-methyl-c-propylaminocarbonyl, 2-ethyl-1-methyl-c25
propylaminocarbonyl, 2-ethyl-2-methyl-c-propylaminocarbonyl, 2-ethyl-3-methyl-cpropylaminocarbonyl,
1-methyl-1-ethyl-n-pentylaminocarbonyl, 1-heptylaminocarbonyl,
2-heptylaminocarbonyl, 1-ethyl-1,2-dimethyl-n-propylaminocarbonyl, 1-ethyl-2,2-
dimethyl-n-propylaminocarbonyl, 1-octylaminocarbonyl, 3-octylaminocarbonyl, 4-methyl-
3-n-heptylaminocarbonyl, 6-methyl-2-n-heptylaminocarbonyl, 2-propyl-1-n30
heptylaminocarbonyl, 2,4,4-trimethyl-1-n-pentylaminocarbonyl, 1-nonylaminocarbonyl,
2-nonylaminocarbonyl, 2,6-dimethyl-4-n-heptylaminocarbonyl, 3-ethyl-2,2-dimethyl-3-npentylaminocarbonyl,
3,5,5-trimethyl-1-n-hexylaminocarbonyl, 1-decylaminocarbonyl, 2-
decylaminocarbonyl, 4-decylaminocarbonyl, 3,7-dimethyl-1-n-octylaminocarbonyl, 3,7-
dimethyl-3-n-octylaminocarbonyl or the like may be mentioned.
35 A di-C1-10 alkylaminocarbonyl group may be symmetric or asymmetric. A
symmetric di-C1-10 alkylaminocarbonyl group may be linear, branched or a C3-10
cycloalkylaminocarbonyl group, and as specific examples, dimethylaminocarbonyl,
diethylaminocarbonyl, di-n-propylaminocarbonyl, di-i-propylaminocarbonyl, di-cpropylaminocarbonyl,
di-n-butylaminocarbonyl, di-i-butylaminocarbonyl, di-s40
butylaminocarbonyl, di-t-butylaminocarbonyl, di-c-butylaminocarbonyl, di-(1-methyl-cpropyl)
aminocarbonyl, di-(2-methyl-c-propyl)aminocarbonyl, di-n-pentylaminocarbonyl,
di-(1-methyl-n-butyl)aminocarbonyl, di-(2-methyl-n-butyl)aminocarbonyl, di-(3-methyl-nbutyl)
aminocarbonyl, di-(1,1-dimethyl-n-propyl)aminocarbonyl, di-(1,2-dimethyl-npropyl)
aminocarbonyl, di-(2,2-dimethyl-n-propyl)aminocarbonyl, di-(1-ethyl-n45
propyl)aminocarbonyl, di-c-pentylaminocarbonyl, di-(1-methyl-c-butyl)aminocarbonyl, di-
(2-methyl-c-butyl)aminocarbonyl, di-(3-methyl-c-butyl)aminocarbonyl, di-(1,2-dimethyl-cpropyl)
aminocarbonyl, di-(2,3-dimethyl-c-propyl)aminocarbonyl, di-(1-ethyl-cpropyl)
aminocarbonyl, di-(2-ethyl-c-propyl)aminocarbonyl, di-n-hexylaminocarbonyl, di33
(1-methyl-n-pentyl)aminocarbonyl, di-(2-methyl-n-pentyl)aminocarbonyl, di-(3-methyl-npentyl)
aminocarbonyl, di-(4-methyl-n-pentyl)aminocarbonyl, di-(1,1-dimethyl-nbutyl)
aminocarbonyl, di-(1,2-dimethyl-n-butyl)aminocarbonyl, di-(1,3-dimethyl-nbutyl)
aminocarbonyl, di-(2,2-dimethyl-n-butyl)aminocarbonyl, di-(2,3-dimethyl-nbutyl)
aminocarbonyl, di-(3,3-dimethyl-n-butyl)aminocarbonyl, di-(1-ethyl-5 nbutyl)
aminocarbonyl, di-(2-ethyl-n-butyl)aminocarbonyl, di-(1,1,2-trimethyl-npropyl)
aminocarbonyl, di-(1,2,2-trimethyl-n-propyl)aminocarbonyl, di-(1-ethyl-1-methyln-
propyl)aminocarbonyl, di-(1-ethyl-2-methyl-n-propyl)aminocarbonyl, di-chexylaminocarbonyl,
di-(1-methyl-c-pentyl)aminocarbonyl, di-(2-methyl-c10
pentyl)aminocarbonyl, di-(3-methyl-c-pentyl)aminocarbonyl, di-(1-ethyl-cbutyl)
aminocarbonyl, di-(2-ethyl-c-butyl)aminocarbonyl, di-(3-ethyl-cbutyl)
aminocarbonyl, di-(1,2-dimethyl-c-butyl)aminocarbonyl, di-(1,3-dimethyl-cbutyl)
aminocarbonyl, di-(2,2-dimethyl-c-butyl)aminocarbonyl, di-(2,3-dimethyl-cbutyl)
aminocarbonyl, di-(2,4-dimethyl-c-butyl)aminocarbonyl, di-(3,3-dimethyl-c15
butyl)aminocarbonyl, di-(1-n-propyl-c-propyl)aminocarbonyl, di-(2-n-propyl-cpropyl)
aminocarbonyl, di-(1-i-propyl-c-propyl)aminocarbonyl, di-(2-i-propyl-cpropyl)
aminocarbonyl, di-(1,2,2-trimethyl-c-propyl)aminocarbonyl, di-(1,2,3-trimethyl-cpropyl)
aminocarbonyl, di-(2,2,3-trimethyl-c-propyl)aminocarbonyl, di-(1-ethyl-2-methyl-cpropyl)
aminocarbonyl, di-(2-ethyl-1-methyl-c-propyl)aminocarbonyl, di-(2-ethyl-2-methyl20
c-propyl)aminocarbonyl, di-(2-ethyl-3-methyl-c-propyl)aminocarbonyl, di-(1-methyl-1-
ethyl-n-pentyl)aminocarbonyl, di-(1-heptyl)aminocarbonyl, di-(2-heptyl)aminocarbonyl,
di-(1-ethyl-1,2-dimethyl-n-propyl)aminocarbonyl, di-(1-ethyl-2,2-dimethyl-npropyl)
aminocarbonyl, di-(1-octyl)aminocarbonyl, di-(3-octyl)aminocarbonyl, di-(4-
methyl-3-n-heptyl)aminocarbonyl, di-(6-methyl-2-n-heptyl)aminocarbonyl, di-(2-propyl-1-
25 n-heptyl)aminocarbonyl, di-(2,4,4-trimethyl-1-n-pentyl)aminocarbonyl, di-(1-
nonyl)aminocarbonyl, di-(2-nonyl)aminocarbonyl, di-(2,6-dimethyl-4-nheptyl)
aminocarbonyl, di-(3-ethyl-2,2-dimethyl-3-n-pentyl)aminocarbonyl, di-(3,5,5-
trimethyl-1-n-hexyl)aminocarbonyl, di-(1-decyl)aminocarbonyl, di-(2-
decyl)aminocarbonyl, di-(4-decyl)aminocarbonyl, di-(3,7-dimethyl-1-n30
octyl)aminocarbonyl, di-(3,7-dimethyl-3-n-octyl)aminocarbonyl or the like may be
mentioned.
An asymmetric C1-10 dialkylaminocarbonyl group may be linear, branched or a C3-
10 cycloalkylaminocarbonyl group, and as specific examples, (methyl,
ethyl)aminocarbonyl, (methyl, n-propyl)aminocarbonyl, (methyl, i-propyl)aminocarbonyl,
35 (methyl, c-propyl)aminocarbonyl, (methyl, n-butyl)aminocarbonyl, (methyl, ibutyl)
aminocarbonyl, (methyl, s-butyl)aminocarbonyl, (methyl, t-butyl)aminocarbonyl,
(methyl, n-pentyl)aminocarbonyl, (methyl, c-pentyl)aminocarbonyl, (methyl, nhexyl)
aminocarbonyl, (methyl, c-hexyl)aminocarbonyl, (ethyl, n-propyl)aminocarbonyl,
(ethyl, i-propyl)aminocarbonyl, (ethyl, c-propyl)aminocarbonyl, (ethyl, n40
butyl)aminocarbonyl, (ethyl, i-butyl)aminocarbonyl, (ethyl, s-butyl)aminocarbonyl, (ethyl,
t-butyl)aminocarbonyl, (ethyl, n-pentyl)aminocarbonyl, (ethyl, c-pentyl)aminocarbonyl,
(ethyl, n-hexyl)aminocarbonyl, (ethyl, c-hexyl)aminocarbonyl, (n-propyl, ipropyl)
aminocarbonyl, (n-propyl, c-propyl)aminocarbonyl, (n-propyl, nbutyl)
aminocarbonyl, (n-propyl, i-butyl)aminocarbonyl, (n-propyl, s-butyl)aminocarbonyl,
45 (n-propyl, t-butyl)aminocarbonyl, (n-propyl, n-pentyl)aminocarbonyl, (n-propyl, cpentyl)
aminocarbonyl, (n-propyl, n-hexyl)aminocarbonyl, (n-propyl, chexyl)
aminocarbonyl, (i-propyl, c-propyl)aminocarbonyl, (i-propyl, n-butyl)aminocarbonyl,
(i-propyl, i-butyl)aminocarbonyl, (i-propyl, s-butyl)aminocarbonyl, (i-propyl, t34
butyl)aminocarbonyl, (i-propyl, n-pentyl)aminocarbonyl, (i-propyl, cpentyl)
aminocarbonyl, (i-propyl, n-hexyl)aminocarbonyl, (i-propyl, chexyl)
aminocarbonyl, (c-propyl, n-butyl)aminocarbonyl, (c-propyl, i-butyl)aminocarbonyl,
(c-propyl, s-butyl)aminocarbonyl, (c-propyl, t-butyl)aminocarbonyl, (c-propyl, npentyl)
aminocarbonyl, (c-propyl, c-pentyl)aminocarbonyl, (c-propyl, 5 nhexyl)
aminocarbonyl, (c-propyl, c-hexyl)aminocarbonyl, (n-butyl, i-butyl)aminocarbonyl,
(n-butyl, s-butyl)aminocarbonyl, (n-butyl, t-butyl)aminocarbonyl, (n-butyl, npentyl)
aminocarbonyl, (n-butyl, c-pentyl)aminocarbonyl, (n-butyl, n-hexyl)aminocarbonyl,
(n-butyl, c-hexyl)aminocarbonyl, (i-butyl, s-butyl)aminocarbonyl, (i-butyl, t10
butyl)aminocarbonyl, (i-butyl, n-pentyl)aminocarbonyl, (i-butyl, c-pentyl)aminocarbonyl,
(i-butyl, n-hexyl)aminocarbonyl, (i-butyl, c-hexyl)aminocarbonyl, (s-butyl, tbutyl)
aminocarbonyl, (s-butyl, n-pentyl)aminocarbonyl, (s-butyl, c-pentyl)aminocarbonyl,
(s-butyl, n-hexyl)aminocarbonyl, (s-butyl, c-hexyl)aminocarbonyl, (t-butyl, npentyl)
aminocarbonyl, (t-butyl, c-pentyl)aminocarbonyl, (t-butyl, n-hexyl)aminocarbonyl,
15 (t-butyl, c-hexyl)aminocarbonyl, (n-pentyl, c-pentyl)aminocarbonyl, (n-pentyl, nhexyl)
aminocarbonyl, (n-pentyl, c-hexyl)aminocarbonyl, (c-pentyl, nhexyl)
aminocarbonyl, (c-pentyl, c-hexyl)aminocarbonyl, (n-hexyl, c-hexyl)aminocarbonyl,
(methyl, n-heptyl)aminocarbonyl, (methyl, n-octyl)aminocarbonyl, (methyl, nnonanyl)
aminocarbonyl, (methyl, n-decyl)aminocarbonyl, (ethyl, n-heptyl)aminocarbonyl,
20 (ethyl, n-octyl)aminocarbonyl, (ethyl, n-nonanyl)aminocarbonyl, (ethyl, ndecyl)
aminocarbonyl or the like may be mentioned.
A C1-10 alkylaminosulfonyl group may be linear, branched, a C3-10
cycloalkylsulfonylamino group or a di-C1-10 alkylaminosulfonyl group, and as specific
examples, methylaminosulfonyl, ethylaminosulfonyl, n-propylaminosulfonyl, i25
propylaminosulfonyl, c-propylaminosulfonyl, n-butylaminosulfonyl, i-butylaminosulfonyl,
s-butylaminosulfonyl, t-butylaminosulfonyl, c-butylaminosulfonyl, 1-methyl-cpropylaminosulfonyl,
2-methyl-c-propylaminosulfonyl, n-pentylaminosulfonyl, 1-methyln-
butylaminosulfonyl, 2-methyl-n-butylaminosulfonyl, 3-methyl-n-butylaminosulfonyl,
1,1-dimethyl-n-propylaminosulfonyl, 1,2-dimethyl-n-propylaminosulfonyl, 2,2-dimethyl-n30
propylaminosulfonyl, 1-ethyl-n-propylaminosulfonyl, c-pentylaminosulfonyl, 1-methyl-cbutylaminosulfonyl,
2-methyl-c-butylaminosulfonyl, 3-methyl-c-butylaminosulfonyl, 1,2-
dimethyl-c-propylaminosulfonyl, 2,3-dimethyl-c-propylaminosulfonyl, 1-ethyl-cpropylaminosulfonyl,
2-ethyl-c-propylaminosulfonyl, n-hexylaminosulfonyl, 1-methyl-npentylaminosulfonyl,
2-methyl-n-pentylaminosulfonyl, 3-methyl-n-pentylaminosulfonyl,
35 4-methyl-n-pentylaminosulfonyl, 1,1-dimethyl-n-butylaminosufonyl, 1,2-dimethyl-nbutylaminosufonyl,
1,3-dimethyl-n-butylaminosufonyl, 2,2-dimethyl-n-butylaminosufonyl,
2,3-dimethyl-n-butylaminosufonyl, 3,3-dimethyl-n-butylaminosufonyl, 1-ethyl-nbutylaminosufonyl,
2-ethyl-n-butylaminosufonyl, 1,1,2-trimethyl-n-propylaminosulfonyl,
1,2,2-trimethyl-n-propylaminosulfonyl, 1-ethyl-1-methyl-n-propylaminosulfonyl, 1-ethyl-2-
40 methyl-n-propylaminosulfonyl, c-hexylaminosulfonyl, 1-methyl-c-pentylaminosulfonyl, 2-
methyl-c-pentylaminosulfonyl, 3-methyl-c-pentylaminosulfonyl, 1-ethyl-cbutylaminosulfonyl,
2-ethyl-c-butylaminosulfonyl, 3-ethyl-c-butylaminosulfonyl, 1,2-
dimethyl-c-butylaminosulfonyl, 1,3-dimethyl-c-butylaminosulfonyl, 2,2-dimethyl-cbutylaminosulfonyl,
2,3-dimethyl-c-butylaminosulfonyl, 2,4-dimethyl-c45
butylaminosulfonyl, 3,3-dimethyl-c-butylaminosulfonyl, 1-n-propyl-c-propylaminosulfonyl,
2-n-propyl-c-propylaminosulfonyl, 1-i-propyl-c-propylaminosulfonyl, 2-i-propyl-cpropylaminosulfonyl,
1,2,2-trimethyl-c-propylaminosulfonyl, 1,2,3-trimethyl-cpropylaminosulfonyl,
2,2,3-trimethyl-c-propylaminosulfonyl, 1-ethyl-2-methyl-c35
propylaminosulfonyl, 2-ethyl-1-methyl-c-propylaminosulfonyl, 2-ethyl-2-methyl-cpropylaminosulfonyl,
1-methyl-1-ethyl-n-pentylaminosulfonyl, 1-heptylaminosulfonyl, 2-
heptylaminosulfonyl, 1-ethyl-1,2-dimethyl-n-propylaminosulfonyl, 1-ethyl-2,2-dimethyl-npropylaminosulfonyl,
1-octylaminosulfonyl, 3-octylaminosulfonyl, 4-methyl-3-nheptylaminosulfonyl,
6-methyl-2-n-heptylaminosulfonyl, 2-propyl-1-5 nheptylaminosulfonyl,
2,4,4-trimethyl-1-n-pentylaminosulfonyl, 1-nonylaminosulfonyl, 2-
nonylaminosulfonyl, 2,6-dimethyl-4-n-heptylaminosulfonyl, 3-ethyl-2,2-dimethyl-3-npentylaminosulfonyl,
3,5,5-trimethl-1-n-hexylaminosulfonyl, 1-decylaminosulfonyl, 2-
decylaminosulfonyl, 4-decylaminosulfonyl, 3,7-dimetyl-1-n-octylaminosulfonyl, 3,7-
10 dimetyl-3-n-octylaminosulfonyl, c-heptylaminosulfonyl, c-octylaminosulfonyl, 1-methyl-chexylaminosulfonyl,
2-methyl-c-hexylaminosulfonyl, 3-methyl-c-hexylaminosulfonyl, 1,2-
dimethyl-c-hexylaminosulfonyl, 1-ethyl-c-hexylaminosulfonyl, 1-methyl-cpentylaminosulfonyl,
2-methyl-c-pentylaminosulfonyl, 3-methyl-c-pentylaminosulfonyl or
the like may be mentioned.
15 A di-C1-10 alkylaminosulfonyl group may be symmetric or asymmetric. A
symmetric di-C1-10 dialkylaminosulfonyl group may be linear, branched or a C3-10
cycloalkylaminosulfonyl group, and as specific examples, dimethylaminosulfonyl,
diethylaminosulfonyl, di-n-propylaminosulfonyl, di-i-propylaminosulfonyl, di-cpropylaminosulfonyl,
di-n-butylaminosulfonyl, di-i-butylaminosulfonyl, di-s20
butylaminosulfonyl, di-t-butylaminosulfonyl, di-c-butylaminosulfonyl, di-(1-methyl-cpropyl)
aminosulfonyl, di-(2-methyl-c-propyl)aminosulfonyl, di-n-pentylaminosulfonyl, di-
(1-methyl-n-butyl)aminosulfonyl, di-(2-methyl-n-butyl)aminosulfonyl, di-(3-methyl-nbutyl)
aminosulfonyl, di-(1,1-dimethyl-n-propyl)aminosulfonyl, di-(1,2-dimethyl-npropyl)
aminosulfonyl, di-(2,2-dimethyl-n-propyl)aminosulfonyl, di-(1-ethyl-n25
propyl)aminosulfonyl, di-c-pentylaminosulfonyl, di-(1-methyl-c-butyl)aminosulfonyl, di-(2-
methyl-c-butyl)aminosulfonyl, di-(3-methyl-c-butyl)aminosulfonyl, di-(1,2-dimethyl-cpropyl)
aminosulfonyl, di-(2,3-dimethyl-c-propyl)aminosulfonyl, di-(1-ethyl-cpropyl)
aminosulfonyl, di-(2-ethyl-c-propyl)aminosulfonyl, di-n-hexylaminosulfonyl, di-(1-
methyl-n-pentyl)aminosulfonyl, di-(2-methyl-n-pentyl)aminosulfonyl, di-(3-methyl-n30
pentyl)aminosulfonyl, di-(4-methyl-n-pentyl)aminosulfonyl, di-(1,1-dimethyl-nbutyl)
aminosulfonyl, di-(1,2-dimethyl-n-butyl)aminosulfonyl, di-(1,3-dimethyl-nbutyl)
aminosulfonyl, di-(2,2-dimethyl-n-butyl)aminosulfonyl, di-(2,3-dimethyl-nbutyl)
aminosulfonyl, di-(3,3-dimethyl-n-butyl)aminosulfonyl, di-(1-ethyl-nbutyl)
aminosulfonyl, di-(2-ethyl-n-butyl)aminosulfonyl, di-(1,1,2-trimethyl-n35
propyl)aminosulfonyl, di-(1,2,2-trimethyl-n-propyl)aminosulfonyl, di-(1-ethyl-1-methyl-npropyl)
aminosulfonyl, di-(1-ethyl-2-methyl-n-propyl)aminosulfonyl, di-chexylaminosulfonyl,
di-(1-methyl-c-pentyl)aminosulfonyl, di-(2-methyl-cpentyl)
aminosulfonyl, di-(3-methyl-c-pentyl)aminosulfonyl, di-(1-ethyl-cbutyl)
aminosulfonyl, di-(2-ethyl-c-butyl)aminosulfonyl, di-(3-ethyl-c-butyl)aminosulfonyl,
40 di-(1,2-dimethyl-c-butyl)aminosulfonyl, di-(1,3-dimethyl-c-butyl)aminosulfonyl, di-(2,2-
dimethyl-c-butyl)aminosulfonyl, di-(2,3-dimethyl-c-butyl)aminosulfonyl, di-(2,4-dimethylc-
butyl)aminosulfonyl, di-(3,3-dimethyl-c-butyl)aminosulfonyl, di-(1-n-propyl-cpropyl)
aminosulfonyl, di-(2-n-propyl-c-propyl)aminosulfonyl, di-(1-i-propyl-cpropyl)
aminosulfonyl, di-(2-i-propyl-c-propyl)aminosulfonyl, di-(1,2,2-trimethyl-c45
propyl)aminosulfonyl, di-(1,2,3-trimethyl-c-propyl)aminosulfonyl, di-(2,2,3-trimethyl-cpropyl)
aminosulfonyl, di-(1-ethyl-2-methyl-c-propyl)aminosulfonyl, di-(2-ethyl-1-methylc-
propyl)aminosulfonyl, di-(2-ethyl-2-methyl-c-propyl)aminosulfonyl, di-(2-ethyl-3-
methyl-c-propyl)aminosulfonyl, di-(1-methyl-1-ethyl-n-pentyl)aminosulfonyl, di-(1-
36
heptyl)aminosulfonyl, di-(2-heptyl)aminosulfonyl, di-(1-ethyl-1,2-dimethyl-npropyl)
aminosulfonyl, di-(1-ethyl-2,2-dimethyl-n-propyl)aminosulfonyl, di-(1-
octyl)aminosulfonyl, di-(3-octyl)aminosulfonyl, di-(4-methyl-3-n-heptyl)aminosulfonyl, di-
(6-methyl-2-n-heptyl)aminosulfonyl, di-(2-propyl-1-n-heptyl)aminosulfonyl, di-(2,4,4-
trimethyl-1-n-pentyl)aminosulfonyl, di-(1-nonyl)aminosulfonyl, di-(2-nonyl)aminosulfonyl5 ,
di-(2,6-dimethyl-4-n-heptyl)aminosulfonyl, di-(3-ethyl-2,2-dimethyl-3-npentyl)
aminosulfonyl, di-(3,5,5-trimethyl-1-n-hexyl)aminosulfonyl, di-(1-
decyl)aminosulfonyl, di-(2-decyl)aminosulfonyl, di-(4-decyl)aminosulfonyl, di-(3,7-
dimethyl-1-n-octyl)aminosulfonyl, di-(3,7-dimethyl-3-n-octyl)aminosulfonyl or the like
10 may be mentioned.
An asymmetric di-C1-10 alkylaminosulfonyl group may be linear, branched or a
C3-10 cycloalkylaminosulfonyl group, and as specific examples, (methyl,
ethyl)aminosulfonyl, (methyl, n-propyl)aminosulfonyl, (methyl, i-propyl)aminosulfonyl,
(methyl, c-propyl)aminosulfonyl, (methyl, n-butyl)aminosulfonyl, (methyl, i15
butyl)aminosulfonyl, (methyl, s-butyl)aminosulfonyl, (methyl, t-butyl)aminosulfonyl,
(methyl, n-pentyl)aminosulfonyl, (methyl, c-pentyl)aminosulfonyl, (methyl, nhexyl)
aminosulfonyl, (methyl, c-hexyl)aminosulfonyl, (ethyl, n-propyl)aminosulfonyl,
(ethyl, i-propyl)aminosulfonyl, (ethyl, c-propyl)aminosulfonyl, (ethyl, nbutyl)
aminosulfonyl, (ethyl, i-butyl)aminosulfonyl, (ethyl, s-butyl)aminosulfonyl, (ethyl, t20
butyl)aminosulfonyl, (ethyl, n-pentyl)aminosulfonyl, (ethyl, c-pentyl)aminosulfonyl, (ethyl,
n-hexyl)aminosulfonyl, (ethyl, c-hexyl)aminosulfonyl, (n-propyl, i-propyl)aminosulfonyl,
(n-propyl, c-propyl)aminosulfonyl, (n-propyl, n-butyl)aminosulfonyl, (n-propyl, ibutyl)
aminosulfonyl, (n-propyl, s-butyl)aminosulfonyl, (n-propyl, t-butyl)aminosulfonyl,
(n-propyl, n-pentyl)aminosulfonyl, (n-propyl, c-pentyl)aminosulfonyl, (n-propyl, n25
hexyl)aminosulfonyl, (n-propyl, c-hexyl)aminosulfonyl, (i-propyl, c-propyl)aminosulfonyl,
(i-propyl, n-butyl)aminosulfonyl, (i-propyl, i-butyl)aminosulfonyl, (i-propyl, sbutyl)
aminosulfonyl, (i-propyl, t-butyl)aminosulfonyl, (i-propyl, n-pentyl)aminosulfonyl, (ipropyl,
c-pentyl)aminosulfonyl, (i-propyl, n-hexyl)aminosulfonyl, (i-propyl, chexyl)
aminosulfonyl, (c-propyl, n-butyl)aminosulfonyl, (c-propyl, i-butyl)aminosulfonyl,
30 (c-propyl, s-butyl)aminosulfonyl, (c-propyl, t-butyl)aminosulfonyl, (c-propyl, npentyl)
aminosulfonyl, (c-propyl, c-pentyl)aminosulfonyl, (c-propyl, n-hexyl)aminosulfonyl,
(c-propyl, c-hexyl)aminosulfonyl, (n-butyl, i-butyl)aminosulfonyl, (n-butyl, sbutyl)
aminosulfonyl, (n-butyl, t-butyl)aminosulfonyl, (n-butyl, n-pentyl)aminosulfonyl, (nbutyl,
c-pentyl)aminosulfonyl, (n-butyl, n-hexyl)aminosulfonyl, (n-butyl, c35
hexyl)aminosulfonyl, (i-butyl, s-butyl)aminosulfonyl, (i-butyl, t-butyl)aminosulfonyl, (ibutyl,
n-pentyl)aminosulfonyl, (i-butyl, c-pentyl)aminosulfonyl, (i-butyl, nhexyl)
aminosulfonyl, (i-butyl, c-hexyl)aminosulfonyl, (s-butyl, t-butyl)aminosulfonyl, (sbutyl,
n-pentyl)aminosulfonyl, (s-butyl, c-pentyl)aminosulfonyl, (s-butyl, nhexyl)
aminosulfonyl, (s-butyl, c-hexyl)aminosulfonyl, (t-butyl, n-pentyl)aminosulfonyl, (t40
butyl, c-pentyl)aminosulfonyl, (t-butyl, n-hexyl)aminosulfonyl, (t-butyl, chexyl)
aminosulfonyl, (n-pentyl, c-pentyl)aminosulfonyl, (n-pentyl, n-hexyl)aminosulfonyl,
(n-pentyl, c-hexyl)aminosulfonyl, (c-pentyl, n-hexyl)aminosulfonyl, (c-pentyl, chexyl)
aminosulfonyl, (n-hexyl, c-hexyl)aminosulfonyl, (methyl, n-heptyl)aminosulfonyl,
(methyl, n-octyl)aminosulfonyl, (methyl, n-nonanyl)aminosulfonyl, (methyl, n45
decyl)aminosulfonyl, (ethyl, n-heptyl)aminosulfonyl, (ethyl, n-octyl)aminosulfonyl, (ethyl,
n-nonanyl)aminosulfonyl, (ethyl, n-decyl)aminosulfonyl or the like may be mentioned.
A C2-14 arylene group is a bivalent group formed by removing a hydrogen atom
from a ring-constituting atom in a C2-14 aryl group, and as specific examples,
44
or the like may be mentioned.
A C2-9 heterocyclyl group may be a monocyclic or fused bicyclic heterocyclic group
containing at least one atom optionally selected from nitrogen atoms, oxygen atoms an5 d
sulfur atoms and from 2 to 9 carbon atoms, and specifically mentioned are:
45
The protecting group in a protected hydroxy group, a protected amino group, a
protected thiol group or an amino-protecting group may be a C1-4 alkoxymethyl group
(such as MOM: methoxymethyl, MEM: 2-methoxyethoxymethyl, ethoxymethyl, npropoxymethyl,
i-propoxymethyl, n-butoxymethyl, iBM: isobutyloxymethyl, BUM: 5 tbutoxymethyl,
POM: pivaloyloxymethyl, SEM: trimethylsilylethoxymethyl and the like,
preferably a C1-2 alkoxymethyl or the like), an aryloxymethyl (such as BOM:
benzyloxymethyl, PMBM: p-methoxybenzyloxymethyl, P-AOM: p-anisyloxymethyl and
the like, preferably benzyloxymethyl), a C1-4 alkylaminomethyl group (such as
10 dimethylaminomethyl), a substituted acetamidomethyl group (such as Acm:
acetamidomethyl, Tacm: trimethylacetamidomethyl and the like), a substituted
thiomethyl group (such as MTM: methylthiomethyl, PTM: phenylthiomethyl, Btm:
benzylthiomethyl and the like), a carboxyl group, a C1-7 acyl group (such as formyl,
acetyl, fluoroacetyl, difluoroacetyl, trifluoroacetyl, chloroacetyl, dichloroacetyl,
15 trichloroacetyl, propionyl, Pv: pivaloyl, tigloyl and the like), an arylcarbonyl group (such
as benzoyl, p-bromobenzoyl, p-nitrobenzoyl, 2,4-dinitrobenzoyl, benzoylformyl,
benzoylpropionyl, phenylpropionyl and the like), a C1-4 alkoxycarbonyl group (such as
methoxycarbonyl, ethoxycarbonyl, n-propoxycarbonyl, i-propoxycarbonyl, nbutoxycarbonyl,
i-butoxycarbonyl, BOC: t-butoxycarbonyl, AOC: t-amyloxycarbonyl,
20 VOC: vinyloxycarbonyl, AOC: allyloxycarbonyl, Teoc: 2-(trimethylsilyl)ethoxycarbonyl,
Troc: 2,2,2-trichloroethoxycarbonyl and the like, preferably BOC and the like), an
aryloxycarbonyl group (such as Z: benzyloxycarbonyl, p-nitrobenzyloxycarbonyl, MOZ:
p-methoxybenzyloxycarbonyl and the like), a C1-4 alkylaminocarbonyl group (such as
methylcarbamoyl, Ec: ethylcarbamoyl, n-propylcarbamoyl and the like), an
25 arylaminocarbonyl group (such as phenylcarbamoyl and the like), a trialkylsilyl group
(such as TMS: trimethylsilyl, TES: triethylsilyl, TIPS: triisopropylsilyl, DEIPS:
diethylisopropylsilyl, DMIPS: dimethylisopropylsilyl, DTBMS: di-t-butylmethylsilyl,
46
IPDMS: isopropyldimethylsilyl, TBDMS: t-butyldimethylsilyl, TDS: thexyldimethylsilyl and
the like, preferably t-butyldimethylsilyl and the like), a trialkylarylsilyl group (such as
DPMS: diphenylmethylsilyl, TBDPS: t-butyldiphenylsilyl, TBMPS: tbutyldimethoxyphenylsilyl,
TPS: triphenylsilyl and the like), an alkylsulfonyl group, (such
as Ms: methanesulfonyl, ethanesulfonyl and the like) or an arylsulfonyl group (such 5 as
benzenesulfonyl, Ts: p-toluenesulfonyl, p-chlorobenzenesulfonyl, MBS: pmethoxybenzenesulfonyl,
m-nitrobenzenesulfonyl, o-nitrobenzenesulfonyl, pnitrobenzenesulfonyl,
2,4-nitrobenzenesulfonyl, iMds: 2,6-dimethoxy-4-
methylbenzenesulfonyl, Mds: 2,6-dimethyl-4-methoxybenzenesulfonyl, Mtb: 2,4,6-
10 trimethoxybenzenesulfonyl, Mte: 2,3,5,6-tetramethyl-4-methoxybenzenesulfonyl, Mtr:
2,3,6-trimethyl-4-methoxybenzenesulfonyl, Mts: 2,4,6-trimethylbenzenesulfonyl, Pme:
pentamethylbenzenesulfonyl and the like).
In addition, a 1-methyl-1-methoxyethyl group, a 1-ethoxyethyl group, a 2,2,2-
trichloroethyl group, a 2-trimethylsilylethoxy group, a t-butyl group, an allyl group, a
15 benzyl group, a p-methoxybenzyl group, a 2,4-dinitrophenyl group, a p-chlorophenyl
group, a p-methoxyphenyl group, a tetrahydropyranyl group, a tetrahydrofuranyl group
or the like may be mentioned.
Herein, the expression “may be substituted” means that a group may have
substituents in any positions of a group in each of which a substituent may be present,
20 and that each substituent is dependent of one another.
For example, the expression “a C1-3 alkoxy group which may be substituted with
one or more halogen atoms” means an unsubstituted C1-3 alkoxy group or an alkoxy
group with a C1-3 alkyl group in which optional hydrogen atom(s) may be substituted
with halogen atom(s) provided that the number of halogen atoms are 2 or more, each
25 halogen atoms may be identical to or different from one another, such as a
trifluoromethoxy group, a 2,2,2-trifluoroethoxy group or a 1,1-difluoroethoxy group.
The wavy line in the formula of a group indicates a “site of bonding”.
Preferred examples of the substituents in the compounds of the present invention
represented by the formula (I) are given below.
30 Preferred examples of R1 are a hydrogen atom and a C1-6 alkyl group which may
be substituted with one or more halogen atoms, more preferred examples are a
hydrogen atom and a C1-3 alkyl group, and a particularly preferred example is a methyl
group.
Preferred examples of R2, R3, R4 and R6 are a hydrogen atom and a C1-3 alkyl
35 group (the C1-3 alkyl group is unsubstituted or substituted with one or more halogen
atoms), more preferred examples are a hydrogen atom and C1-3 alkyl group (the C1-3
alkyl group is unsubstituted), and a particularly preferred example is a hydrogen atom.
Preferred examples of R5 are a phenyl group which may be substituted with one
or more substituents independently represented by V1 and a C2-9 heteroaryl group which
40 may be substituted with one or more substituents independently represented by V1, and
the C2-9 heteroaryl group is preferably a C2-9 nitrogen-containing heteroaryl group.
Specific examples of the C2-9 heteroaryl group are a 2-thienyl group, a 3-thienyl group, a
2-furyl group, a 3-furyl group, a 2-pyranyl group, a 3-pyranyl group, a 4-pyranyl group, a
1-pyrrolyl group, a 2-pyrrolyl group, a 3-pyrrolyl group, a 1-imidazolyl group, a 2-
45 imidazolyl group, a 4-imidazolyl group, a 1-pyrazolyl group, a 3-pyrazolyl group, a 4-
pyrazolyl group, a 2-thiazolyl group, a 4-thiazolyl group, a 5-thiazolyl group, a 3-
isothiazolyl group, a 4-isothiazolyl group, a 5-isothiazolyl group, a 1-1,2,4-triazole group,
a 3-1,2,4-triazole group, a 5-1,2,4-triazole group, a 1-1,2,3-triazole group, a 4-1,2,3-
47
triazole group, a 5-1,2,3-triazole group, a 2-oxazolyl group, a 4-oxazolyl group, a 5-
oxazolyl group, a 3-isooxazolyl group, a 4-isooxazolyl group, a 5-isooxazolyl group, a 2-
pyridyl group, a 3-pyridyl group, a 4-pyridyl group, a 2-pyrazinyl group, a 2-pyrimidinyl
group, a 4-pyrimidinyl group, a 5-pyrimidinyl group, a 3-pyridazinyl group, a 4-
pyridazinyl group, a 2-1,3,4-oxadiazolyl group, a 2-1,3,4-thiadiazolyl group, a 3-1,2,45 -
oxadiazolyl group, a 5-1,2,4-oxadiazolyl group, a 3-1,2,4-thiadiazolyl group, a 5-1,2,4-
thiadiazolyl group, a 3-1,2,5-oxadiazolyl group and a 3-1,2,5-thiadiazolyl group.
As V1, the formulae (V), (VI), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), (XIV), (XV),
(XVI), (XVII), (XVIII), (XIX), (XX), (XXI) and (XXII) may be mentioned.
10 More preferred examples of R5 are a phenyl group, a 2-thienyl group, a 3-thienyl
group, a 2-pyridyl group, a 3-pyridyl group, a 4-pyridyl group, a 2-pyrazinyl group, a 2-
pyrimidinyl group, a 4-pyrimidinyl group, a 5-pyrimidinyl group, a 3-pyridazinyl group, a
4-pyridazinyl group and groups obtained by substituting these groups with one or more
substituents selected from the formulae (V), (VI), (VII), (VIII), (IX), (X), (XI), (XII), (XIII),
15 (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XXI) and (XXII).
Further more preferred examples of R5 are a phenyl group, 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, a 2-pyrazinyl group and
groups obtained by substituting these groups with one or more substituents selected
20 from the formulae (V), (VI), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), (XIV), (XV), (XVI),
(XVII), (XVIII), (XIX), (XX), (XXI) and (XXII).
Particularly preferred examples of R5 are a 4-pyridyl group, a phenyl group (the
phenyl group is unsubstituted or substituted with one or more substituents selected from
the formulae (V), (VI), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), (XIV), (XV), (XVI), (XVII),
25 (XVIII), (XIX), (XX), (XXI) and (XXII)) and the like.
The most preferred examples of R5 are a 4-pyridyl group and a phenyl group
substituted with one or more substituents selected from the formulae (VII), (VIII), (XI)
and (XII).
A preferred examples of R7 is a C2-14 aryl group (the C2-14 aryl group is
30 unsubstituted or substituted with one or more substituents selected from the group
consisting of C1-10 alkyl groups (the C1-10 alkyl groups are unsubstituted or substituted
with one or more halogen atoms), halogen atoms, C1-10 alkoxy groups and C1-3 alkoxy
groups (the C1-3 alkoxy groups are substituted with one or more halogen atoms)).
A more preferred example of R7 is a phenyl group (the phenyl group is substituted
35 with one or more substituents selected from the group consisting of C1-10 alkyl groups
(the C1-10 alkyl groups are unsubstituted or substituted with one or more halogen atoms),
halogen atoms, C1-10 alkoxy groups and C1-3 alkoxy groups (the C1-3 alkoxy groups are
substituted with one or more halogen atoms), and the formulae (A01), (A02), (A03),
(A04), (A05), (A06), (A07), (A08), (A09), (A10), (A11), (A12), (A13), (A14) and (A15)).
40 Particularly preferred examples of R7 are a phenyl group (the phenyl group is
substituted with one or more substituents selected from the group consisting of C1-6
alkyl groups, C1-3 alkyl groups (the C1-3 alkyl groups are substituted with one or more
halogen atoms), halogen atoms, C1-3 alkoxy groups and C1-3 alkoxy groups (the C1-3
alkoxy groups are substituted with one or more halogen atoms)) and the formulae (A05),
45 (A06), (A08), (A09), (A10), (A11), (A12), (A13), (A14) and (A15).
More specific particular preferred examples are a phenyl group (the phenyl group
is substituted with one or more substituents selected from the group consisting of
methyl groups, t-butyl groups, halogen atoms, methoxy groups, trifluoromethyl groups
48
and trifluoromethoxy groups) and the formulae (A11), (A13) and (A15).
Preferred examples of Ar1are structures represented by the formulae (IV).
A preferred example of X is OH.
A preferred example of Y is an oxygen atom.
A preferred example of Z is an oxygen atom5 .
n is preferably an integer of 1 or 2, more preferably an integer of 1. When n is 1,
it is particularly preferred that R5 is a 4-pyridyl group or a phenyl group substituted with
one or more substituents selected from the formulae (VII), (VIII), (XI) and (XII).
Preferred examples of the compounds of the present invention are compounds
10 wherein Ra, Ar and Q are any of the following combinations shown in Tables 1 to 13,
tautomers or pharmaceutically acceptable salts of the compounds or solvates thereof.
The symbols in Tables 1 to 13 denote the following substituents.
15
S
A1 A2 A3
N
A4 A5
N
62
TABLE 13
A compounds of the present invention represented by the formula (I) may be
converted to a pharmaceutically acceptable salt or may be liberated from the resulting
salt, if necessary. The pharmaceutically acceptable salt of the present invention may
be, for example, a salt with an alkali metal (such as lithium, sodium and potassium), a5 n
alkaline earth metal (such as magnesium and calcium), ammonium, an organic base or
an amino acid. It may be a salt with an inorganic acid (such as hydrochloric acid,
hydrobromic acid, phosphoric acid and sulfuric acid) or an organic acid (such as acetic
acid, citric acid, maleic acid, fumaric acid, benzenesulfonic acid and p-toluenesulfonic
10 acid). A compound of the present invention represented by the formula (I) or a
pharmaceutically acceptable salt thereof may be in the form of arbitrary crystals or an
arbitrary hydrate, depending on the production conditions. The present invention
covers these crystals, hydrates and mixtures. They may be in the form of a solvate
with an organic solvent such as acetone, ethanol and tetrahydrofuran, and the present
15 invention covers any of these forms.
In the present invention, the compounds of the present invention represented by
the formula (I) may be present in the form of tautomers or geometrical isomers
generated by endocyclic or exocyclic isomerization, mixtures of tautomers or geometric
isomers or mixtures of thereof. When the compounds of the present invention has an
20 asymmetric center, whether or not resulting from an isomerization, the compounds of
the present invention may be in the form of resolved optical isomers or in the form of
mixtures containing them in certain ratios.
The compounds which serve as prodrugs are derivatives of the present invention
having chemically or metabolically degradable groups which give pharmacologically
25 active compounds of the present invention upon solvolysis or under physiological
conditions in vivo. Methods for selecting or producing appropriate prodrugs are
disclosed, for example, in Design of Prodrugs (Elsevier, Amsterdam 1985). In the
present invention, when the compound has a hydroxy group, acyloxy derivatives
obtained by reacting the compound with appropriate acyl halides or appropriate acid
Ｎｏ． Ｒａ Ａ Ｑ Ｎｏ． Ｒａ Ａ Ｑ
817 Ｒａ9 Ａ4 Ｑ１ 834 Ｒａ10 Ａ5 Ｑ１
818 Ｒａ9 Ａ4 Ｑ２ 835 Ｒａ10 Ａ5 Ｑ２
819 Ｒａ9 Ａ4 Ｑ３ 836 Ｒａ10 Ａ5 Ｑ３
820 Ｒａ9 Ａ4 Ｑ４ 837 Ｒａ10 Ａ5 Ｑ４
821 Ｒａ9 Ａ4 Ｑ５ 838 Ｒａ10 Ａ5 Ｑ5
822 Ｒａ9 Ａ4 Ｑ６ 839 Ｒａ10 Ａ5 Ｑ6
823 Ｒａ9 Ａ4 Ｑ７ 840 Ｒａ10 Ａ5 Ｑ7
824 Ｒａ9 Ａ4 Ｑ８ 841 Ｒａ10 Ａ5 Ｑ8
825 Ｒａ9 Ａ4 Ｑ９ 842 Ｒａ10 Ａ5 Ｑ9
826 Ｒａ9 Ａ4 Ｑ１０ 843 Ｒａ10 Ａ5 Ｑ10
827 Ｒａ9 Ａ4 Ｑ１１ 844 Ｒａ10 Ａ5 Ｑ11
828 Ｒａ9 Ａ4 Ｑ１２ 845 Ｒａ10 Ａ5 Ｑ12
829 Ｒａ9 Ａ4 Ｑ１３ 846 Ｒａ10 Ａ5 Ｑ13
830 Ｒａ9 Ａ4 Ｑ１４ 847 Ｒａ10 Ａ5 Ｑ14
831 Ｒａ9 Ａ4 Ｑ１５ 848 Ｒａ10 Ａ5 Ｑ15
832 Ｒａ9 Ａ4 Ｑ１６ 849 Ｒａ10 Ａ5 Ｑ16
833 Ｒａ9 Ａ4 Ｑ１７ 850 Ｒａ10 Ａ5 Ｑ17
63
anhydrides may, for example, be mentioned as prodrugs. Acyloxys particularly
preferred as prodrugs include -OCOC2H5, -OCO(t-Bu), -OCOC15H31,
-OCO(m-CO2Na-Ph), -OCOCH2CH2CO2Na, -OCOCH(NH2)CH3, -OCOCH2N(CH3)2 and
the like. When the compounds of the present invention have an amino group, amide
derivatives obtained by reacting the compound having an amino group with appropriat5 e
acid halides or appropriate mixed acid anhydrides may, for example, be mentioned as
prodrugs. Amides particularly preferred as prodrugs include
-NHCO(CH2)20OCH3, -NHCOCH(NH2)CH3 and the like.
The specific compound to used in the method of the present invention can be
10 synthesized chemically by reference to Patent Documents WO2004/108683,
WO2006/064957, WO2007/010954, WO2010/140685 and the like, though there are no
particular restrictions.
EXAMPLES
15 Now, the present invention will be described in further detail with reference to
Examples. However, it should be understood that the present invention is by no
means restricted by these specific Examples.
The CO2 concentration (%) in the CO2 incubator is expressed in the percentage of
the volume of CO2 in the atmosphere. PBS denotes phosphate buffered saline
20 (Sigma-Aldrich Japan), and FBS denotes fetal bovine serum.
(TEST EXAMPLE 1: Preparation of human iPS cell-derived sac-like structures (iPSsacs))
In this Example, an iPS cell line TkDA3-4 (established by Tokyo University by
introducing Oct3/4,Klf4,Sox2 and c-Myc into skin cells: see: WO2009122747) was used.
25 As the feeder cells, a mouse embryo-derived cell line C3H10T1/2, provided by
BIoResource center, Riken Tsukuba Institute, was used. On the day before the
differentiation experiment, C3H10T1/2 cells were irradiated with 50 Gy radiation,
seeded on dishes coated with 0.1% gelatin at a density of from 6 to 8×105 /10cm dish
and used as feeder cells.
30 Human iPS cells were seeded on the C3H10T1/2 cells and cultured in IMDM
(Invitrogen/GIBCO) supplemented with 15% FBS (JRH BIOSCIENCES, U.S.A), 2 mM
L-glutamine (Invitrogen), 100 Unit/mL Penicillin-100g/mL Streptmycin (Sigma), ITS
supplement (10 g/mL insulin, 5.5mg/mL transferrin, 5 ng/mL sodium selenite) (Sigma),
50 g/mL ascorbic acid (Sigma), 0.45 mM MTG (Sigma) and 20 ng/mL VEGF (R&D
35 systems) and incubated in 5% CO2 at 37 C.
After about 14 to 15 days of incubation, a number of sac-like structures (iPS-sacs)
containing blood cell-like cells were observed.
(TEST EXAMPLE 2: Induction of megakaryocytes / platelets from the sac-like structures
(iPS-sacs))
40 Next, the sac-like structures were physically disrupted with a 10mL disposable
pipette, and hematopoietic progenitor cells and sac-like structures were separated by
using a 70 m cell strainer. The hematopoietic progenitor cells were seeded on
irradiated C3H10T1/2 cells (from 6 to 8×105 cells/6-well plate) newly prepared on a 6-
well plate, at a density of 3×104 cells/well and incubated in IMDM (Invitrogen/GIBCO)
45 supplemented with 15% FBS (JRH BIOSCIENCES, U.S.A), 2 mM L-glutamine
(Invitrogen), 100 Unit/mL Penicillin-100g/mL Streptmysin (Sigma), ITS supplement (10
g/mL insulin, 5.5 mg/mL transferrin, 5 ng/mL sodium selenite) (Sigma), 50 g/mL
ascorbic acid (Sigma), 0.45 mM monothioglycerol (MTG, Sigma) and 100 ng/mL human
64
TPO (Peprotec) or one of the following specific compounds (100 ng/mL No.1, 30 ng/mL
No.2, 50 ng/mL No.3, 1000 ng/mL No.4, 100 ng/mL No.5, 100 ng/mL No.6, 300 ng/mL
No.7, 300 ng/mL No.8) to induce megakaryocytes / platelets.
The names and structural formulae of the specific compounds used in this
Example are given below5 .
Specific Compound No.1:(E)-5-(2-{1-[5-(3,4-dichlorophenyl)-4-hydroxythiophen-3-
yl]ethyliden}hydrazinecarbonyl)-N-[4-(2-hydroxyethylcarbamoyl)benzyl]thiophene-2-
carboxamide
Specific Compound No.2:(E)-5-(2-{1-[5-(4-bromophenyl)-4-hydroxythiophen-3-
10 yl]ethylidene}hydrazinecarbonyl)-N-[4-(2- hydroxyethylcarbamoyl)benzyl]thiophene-2-
carboxamide
Specific Compound No.3:(E)-5-(2-{1-[5-(3,4-dichlorophenyl)-4-hydroxythiophen-3-
yl]ethylidene}hydrazinecarbonyl)-N-(pyridin-4-ylmethyl)thiophene-2-carboxamide
Specific Compound No.4:(E)-5-(2-{1-[5-(2,3-dihydro-1H-inden-5-yl)-4-hydroxythiophen-
15 3-yl]ethylidene}hydrazinecarbonyl)thiophene-2-carboxylic acid
Specific Compound No.5: potassium (E)-2-(3,4-dichlorophenyl)-4-[1-(2-{5-[(pyrazin-2-
ylmethyl)carbamoyl]thiophene-2-carbonyl}hydrazono)ethyl]thiophen-3-olate
Specific Compound No.6:(E)-5-(2-{1-[5-(4-bromophenyl)-4-hydroxythiophen-3-
yl]ethylidene}hydrazinecarbonyl)-N-{4-[2-(piperazin-1-
20 yl)ethylcarbamoyl]benzyl}thiophene-2-carboxamide
Specific Compound No.7:(E)-N-[4-(2-amino-2-oxoethyl)benzyl]-5-(2-{1-[5-(3,4-
dichlorophenyl)-4-hydroxythiophen-3-yl]ethylidene}hydrazinecarbonyl)thiophene-2-
carboxamide
Specific Compound No.8:(E)-N-(4-{2-[bis(2-
25 hydroxyethyl)amino]ethylcarbamoyl}benzyl)-5-(2-{1-[5-(4-t-butylphenyl)-4-
hydroxythiophen-3-yl]ethylidene}hydrazinecarbonyl)thiophene-2-carboxamide.
65
These compounds were synthesized by known procedures in accordance with,
WO2007/010954, WO2010/140685, WO2011/049213 and the like.
(TEST EXAMPLE 3: Megakaryocyte / platelet counts in cell cultures)
The nonadherent cells in the 23- to 24-day TkDA3-4 cultures were characterize5 d
by cell surface antigens with a fluocytometer (Becton, Dickinson and Company,
BDFACSAia) after addition of 8.5 mM sodium citrate (Sigma), 6.5 mM citric acid (Sigma),
10.4 mM glucose (Sigma), anti-human CD41a antibody (Becton, Dickinson and
66
Company) and anti-human CD42b antibody (BioLegend) in terms of final concentration.
Platelets were sorted out by size with a flow cytometer and counted by using BD
Trucount tubes (Becton, Dickinson and Company). Megakaryocytes were sorted by
size from platelets by centrifugation (310 g, 5 minutes) and flow cytometry and counted
with a hemocytometer. The megakaryocytes and platelets were positive for the cel5 l
surface antigens specific to megakaryocytes and platelets, human CD41a (integrin IIb)
and human CD42b (GPIb) (Figs. 1 and 2; megakaryocytes, Figs. 3 and 4; platelets).
The specific compounds of the present invention showed higher megakaryopoietic and
thrombopoietic effects than TPO.
10 (TEST EXAMPLE 4: Functional analysis of the platelets)
Next, activation of integrin by platelet activators was examined. From
nonadherent cells in 23- or 24-day culture of TkDA3-4 cells, nucleate cells were
removed, and platelets were separated by centrifugation (400 g, 10 minutes) and
treated with human anti-CD42b antibody (BioLegend), FITC-labeled PAC-1 (Becton,
15 Dickinson and Company) and 500 M a platelet activator, adenosine diphosphate (ADP,
Sigma). 15 minutes later, the binding of PAC-1 as a platelet activation maker to
palatelets was analyzed with a flow cytometer and expressed as the mean fluorescence
intensity (MFI). As a result, the platelets derived from human iPS cells by using the
specific compounds of the present invention showed as much activation of the integrin
20 (binding of PAC-1 to platelets) as platelets from peripheral blood. The results
demonstrate that platelets derived from iPS cells by using a specific compound of the
present invention are as functional as platelets from peripheral blood.
(TEST EXAMPLE 5: Preparation of human ES cell-derived sac-like structures (ESsacs))
25 In this Example, an ES cell line KhES-3 (established by Kyoto University, see:
Biochem Biophys Res Commun. 2006. 345: 926-932) was used. As the feeder cells, a
mouse embryo-derived cell line C3H10T1/2, provided by BIoResource center, Riken
Tsukuba Institute, was used. On the day before the differentiation experiment,
C3H10T1/2 cells were irradiated with 50 Gy radiation, seeded on dishes coated with
30 0.1% gelatin at a density of from 6 to 8×105/10 cm dish and used as feeder cells.
Human ES cells were seeded on the C3H10T1/2 cells and cultured in IMDM
(Invitrogen/GIBCO) supplemented with 15% FBS (JRH BIOSCIENCES, U.S.A), 2 mM
L-glutamine (Invitrogen), 100 Unit/mL Penicillin-100 g/mL Streptmycin (Sigma), ITS
supplement (10 g/mL insulin, 5.5 mg/mL transferrin, 5 ng/mL sodium selenite) (Sigma),
35 50 g/mL ascorbic acid (Sigma), 0.45 mM MTG (Sigma) and 20 ng/mL VEGF (R&D
systems) and incubated in 5% CO2 at 37 C.
After about 14 to 15 days of incubation, a number of sac-like structures (ES-sacs)
containing blood cell-like cells were observed.
(TEST EXAMPLE 6: Induction of megakaryocytes / platelets from the sac-like structures
40 (ES-sacs))
Next, the sac-like structures were mechanically disrupted with a 10 mL disposable
pipette, and hematopoietic progenitor cells and sac-like structures were separated by
using a 70 m cell strainer. The hematopoietic progenitor cells were seeded on
irradiated C3H10T1/2 cells (from 6 to 8×105 cells/6-well plate) newly prepared on a 6-
45 well plate, at a density of 3×104 cells/well and incubated in IMDM (Invitrogen/GIBCO)
supplemented with 15% FBS (JRH BIOSCIENCES, U.S.A), 2 mM L-glutamine
(Invitrogen), 100 Unit/mL Penicillin-100 g/mL Streptmysin (Sigma), ITS supplement (10
g/mL insulin, 5.5 mg/mL transferrin, 5 ng/mL sodium selenite) (Sigma), 50 g/mL
67
ascorbic acid (Sigma), 0.45 mM monothioglycerol (MTG, Sigma) and 100 ng/mL human
TPO (Peprotec) or a specific compound used in Test Example 2 (30 ng/mL No.2, 50
ng/mL No.3) to induce megakaryocytes / platelets.
(TEST EXAMPLE 7: Megakaryocyte / platelet counts in cell cultures)
The nonadherent cells in the 23- to 24-day KhES-3 cultures were characterized b5 y
cell surface antigens after addition of 8.5 mM sodium citrate (Sigma), 6.5 mM citric acid
(Sigma), 10.4 mM glucose (Sigma), human anti-CD41a antibody (Becton, Dickinson
and Company) and human anti-CD42b antibody (BioLegend) in terms of final
concentration. Platelets were sorted out by size with a flow cytometer and counted by
10 using BD Trucount tubes (Becton, Dickinson and Company). Megakaryocytes were
sorted by size from platelets by centrifugation (310 g, 5 minutes) and flow cytometry and
counted with a hemocytometer. The megakaryocytes and platelets were positive for
the cell surface antigens specific to megakaryocytes and platelets, human CD41a
(integrin IIb) and human CD42b (GPIb) (Fig. 6; platelet counts). The specific
15 compound of the present invention showed higher megakaryopoietic and
thrombopoietic effects than TPO did.
(TEST EXAMPLE 8: Preparation of genetically manipulated hematopoietic progenitor
cells)
Hematopoietic progenitor cells were obtained from KhES-3 cell-derived sac-like
20 structures obtained in Test Examples 5 and 6, and cells (Myc-Bmi cell line) showing
enhanced proliferative capability in the presence of estradiol through enhanced
expression of the oncogene c-Myc and the polycomb gene Bmi1 by using a pMX tet off
vector system for regulated gene expression were obtained by using the method
described in WO 2011/034073. In the absence of estradiol in the presence of
25 doxycylcline, Myc-Bmi cells show repressed expression of c-Myc and Bmi1 and produce
functional platelets, but hardly proliferate. From the Myc-Bmi cells, cells (Myc-Bmi-
BCLXL cell line) which show enhanced expression of the apoptosis suppressor gene
BCLXL in the absence of estradiol in the presence of doxycycline and can proliferate
even in the absence of estradiol in the presence doxycycline by using an Ai-Lv tet on g
30 vector system for regulated gene expression (Clontech) were obtained. Further,
expression of p53 gene was suppressed by short hairpin (sh) RNA interference to
promote polyploidization in the course of differentiation into mature megakaryocytes.
Myc-Bmi-BCLXL cells were transfected with a FG12 lenti virus carrying shp53 to obtain
Myc-Bmi-BCLXL cells showing repressed p53 expression (p53KD-Myc-Bmi-BCLXL cell
35 line). The p53KD-Myc-Bmi-BCLXL cells were maintained by culturing on C3H10T1/2
cells inactivated by preliminary treatment with 10 g/mL mitocycin C (Wako Pure
Chemical Industries) for 0.5 to 5 hours in IMDM (Invitrogen/GIBCO) supplemented with
15% FBS (Invitrogen/GIBCO), 2m M L-glutamine- 100 Unit/mL Penicillin-100 g/mL
Streptmysin (Invitrogen/GIBCO), ITS supplement (10 g/mL insulin, 5.5 mg/mL
40 transferrin, 5 ng/mL sodium selenite) (Invitrogen/GIBCO), 50 g/mL ascorbic acid
(Sigma), 0.45 mM monothiglycerol (MTG, Sigma), 10 g/mL doxycycline (Clontech), 50
ng/mL SCF (R&D system) and 100 ng/mL human TPO (R&D system) in 5% CO2 at
39 C.
(TEST EXAMPLE 9: Induction of megakaryocytes / platelets from genetically
45 manipulated hematopoietic progenitor cells)
p53KD-Myc-Bmi-BCLXL cells were seeded on C3H10T1/2 cells inactivated by
preliminary treatment with 10 g/mL mitocycin C (Wako Pure Chemical Industries) for
0.5 to 5 hours (6~8×105 cells/6-well plate) and cultured in IMDM (Invitrogen/GIBCO)
68
supplemented with 15% FBS (Invitrogen/GIBCO), 2 mM L-glutamine-100 Unit/mL
Penicillin-100 g/mL Streptmysin (Invitrogen/GIBCO), ITS suplement (10 g/mL insulin,
5.5 mg/mL transferin, 5 ng/mL sodium selenite) (Invitrogen/GIBCO), 50 g/mL ascorbic
acid (Sigma), 0.45 mM monothioglycerol (MTG, Sigma), 10 g/mL doxycyclin (Clontech),
0.5 mM valproic acid (Sigma), 10 M Y27632 (Wako Pure Chemical Industries), 5 5 M
(S)(-/-)-Blebbistatin (Toronto Research Chemicals), 50 ng/mL SCF (R&D system) and
100 ng/mL human TPO (R&D system) or a specific compound mentioned in Test
Example 2 (100 ng/mL No.2 or 50 ng/mL No.3) in 5% CO2 at 39 C for 7 days to induce
mature megakaryocytes and platelets. The nonadherent cells in the 7-day cultures
10 were characterized by cell surface antigens with a flow cytometer (Becton, Dickinson
and Company, BDFACSAria) after addition of 8.5 mM sodium citrate (Sigma), 6.5 mM
citric acid (Sigma), 10.4 mM glucose (Sigma), anti-human CD41a antigen (Becton,
Dickinson and Company), anti-human CD42b antibody (BioLegend) in terms of final
concentration. Platelets were sorted out by size with a flow cytometer and counted by
15 using BD Trucount tubes (Becton, Dickinson and Company). The platelets were
positive for the cell surface antigens specific to platelets, human CD41a (integrin IIb)
and human CD42b (GPIb) (Fig.7; platelets). The specific compounds of the present
invention showed higher thrombopoietic effect than TPO did.
These results demonstrate that megakaryocytes and platelets are induced
20 efficiently from human iPS cells and ES cells in accordance with the method of the
present invention by using the specific compounds of the present invention.
INDUSTRIAL APPLICABILITY
Megakaryocytes and platelets can be expanded from human pluripotent stem cells
25 more efficiently in the presence of a specific compound of the present invention as an
active ingredient in culture than in its absence or in the presence of TPO. Platelets
produced by using a specific compound are useful for diseases accompanied by a
decrease in platelets such as hematopoietic dysfunction and tumors, and hence their
application to transfusion therapy is expected. According to the present invention, it is
30 possible to provide platelets which can overcome the problem of HLA compatibility.
Therefore, it is possible to supply platelets to patients who require transfusion and solve
the problem of platelet destruction by generation of anti-platelet antibodies.
The entire disclosure of Japanese Patent Application No. 2011-219545 filed on
October 3, 2011 including specification, claims, drawings and summary is incorporated
35 herein by reference in its entirety.
69
CLAIMS
1. A method for producing megakaryocytes and/or platelets, comprising culturing
hematopoietic progenitor cells derived from pluripotent stem cells ex vivo in the
presence of a compound represented by the formula (I), a tautomer, prodrug or
pharmaceutically acceptable salt of the compound or a solvate thereof an5 d
differentiating the hematopoietic progenitor cells into megakaryocytes and/or platelets: ;
wherein W is a substituent represented by the formula (Ia) or a carboxy group:
10 each of R1, R2, R3 and R4 is independently a C1-10 alkyl group which may be substituted
with one or more halogen atoms or a hydrogen atom,
n is an integer of 0, 1, 2 or 3,
R5 is a C2-14 aryl group which may be substituted with one or more substituents
independently represented by V1, provided that when n is 2, R5 is not an unsubstituted
15 pyridyl group,
R6 is a C1-10 alkyl group which may be substituted with one or more halogen atoms or a
hydrogen atom,
R7 is a C2-14 aryl group which may be substituted with one or more substituents
independently represented by V2,
20 Ar1 is a C2-14 arylene group which may be substituted with one or more substituents
independently represented by V3,
X is -OR20,
each of Y and Z is independently an oxygen atom or a sulfur atom,
V1 is -(CH2)m1M1NR8R9, -(CH2)m6NR16R17, -M2NR18(CH2)m7R19 or -C(=O)-(piperazine-
25 1,4-diyl)-U,
each of V2, V3 and V4 is 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 C1-3 alkoxy group which may be
30 substituted with one or more halogen atoms, a C1-10 alkyl group which may be
substituted with one or more halogen atoms, a C2-6 alkenyl group, a C2-6 alkynyl group,
a C1-10 alkylcarbonyloxy group, a C1-10 alkoxycarbonyl group, a C1-10 alkoxy group, a C1-
10 alkylcarbonyl group, a C1-10 alkylcarbonylamino group, a mono- or di-C1-10 alkylamino
70
group, a C1-10 alkylsulfonyl group, a C1-10 alkylaminosulfonyl group, a C1-10
alkylaminocarbonyl group, a C1-10 alkylsulfonylamino group or a C1-10 thioalkyl group,
each of M1 and M2 is independently -(C=O)- or -(SO2)-,
m1 is an integer of 0, 1 or 2,
each of m2, m3, m4, m5, m6 and m7 is independently an integer of 1 or 25 ,
R8 is a hydrogen atom or a C1-3 alkyl group,
each of R9 and U is independently -(CH2)m2OR10 or -(CH2)m4NR11R12, provided that
when m1 is 1 or 2, R9 may be any of those mentioned above or a hydrogen atom,
R10 is a hydrogen atom, a C1-3 alkyl group or -(CH2)m3T,
10 each of R11 and R12 is independently a hydrogen atom or -(CH2)m5Q, or N, R11 and R12
mean, as a whole, a substituent represented by the formula (II):
or a substituent represented by the formula (III):
,
15 T is a hydroxy group, a C1-6 alkoxy group or a C1-6 alkyl group,
Q is a hydroxy group, a C1-3 alkoxy group or -NR13R14,
each of R13 and R14 is independently a hydrogen atom or a C1-3 alkyl group,
R15 is a hydrogen atom, a C1-3 alkyl group or an amino-protecting group,
each of R16 and R17 is independently a hydrogen atom, a C1-3 alkylcarbonyl group or a
20 C1-3 alkylsulfonyl group,
R18 is a hydrogen atom or a C1-3 alkyl group,
R19 is a C2-9 heterocyclyl group or a C2-14 aryl group, and
R20 is a hydrogen atom, a C1-10 alkyl group which may be substituted with one or more
substituents independently represented by V4 or a C1-10 alkylcarbonyl group which may
25 be substituted with one or more substituents independently represented by V4.
2. The method according to Claim 1, wherein W is a substituent represented by the
formula (Ia):
.
3. The method according to Claim 2, wherein R1 is a hydrogen atom or a C1-6 alkyl
30 group which may be substituted with one or more halogen atoms,
each of R2,R3,R4 and R6 is independently a hydrogen atom or a C1-3 alkyl group,
n is an integer of 1 or 2,
Ar1 is represented by the formula (IV):
（IV)
S ,
71
R7 is a phenyl group which may be substituted with one or more substituents selected
from the group consisting of C1-10 alkyl groups which may be substituted with one or
more halogen atoms, C1-10 alkoxy groups, C1-3 alkoxy groups substituted with one or
more halogen atoms and halogen atoms,
X is -OH, an5 d
Y and Z are oxygen atoms.
4. The method according to Claim 3, wherein R2, R3, R4 and R6 are hydrogen atoms.
5. The method according to any one of Claims 2 to 4 , wherein R5 is a phenyl group
which may be substituted with one or more substituents independently represented by
10 V1.
6 The method according to any one of Claims 2 to 4 , wherein R5 is a C2-9 heteroaryl
group which may be substituted with one or more substituents independently
represented by V1.
7. The method according to Claim 6, wherein the C2-9 heteroaryl group is a C2-9
15 nitrogen-containing heteroaryl group.
8. The method according to Claim 7, wherein the C2-9 nitrogen-containing heteroaryl
group is selected from 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.
20 9. The method according to Claim 7, wherein the C2-9 nitrogen-containing heteroaryl
group is a 4-pyridyl group.
10. The method according to any one of Claims 2 to 9, wherein V1 is represented by
any one of the formulae (V) to (XXII):
O
HN
O
O
HN
N O
O
HN
N NH
O
HN
N
OH
OH
O
HN
OH
O
HN
O
OH
O
HN
O
O CH3
（V） （VＩ） （VII）
O
NH2
NH
CH3
O
（VIII） （IX） （X）
（X Ｉ） （X Ｉ Ｉ） （X Ｉ Ｉ Ｉ）
72
.
11. The method according to Claim 3 or 4, wherein R5 is a phenyl group substituted
with a substituent represented by the formula (VIII):
.
12. The method according to Claim 3 or 4, wherein R5 is a 4-pyridyl group5 .
13. The method according to any one of Claims 2 to 12 , wherein n is an integer of 1.
14. The method according to any one of Claims 2 to 13 , wherein R7 is a phenyl group
substituted with one or more substituents selected from methyl groups, t-butyl groups,
halogen atoms, methoxy groups, trifluoromethyl groups and trifluoromethoxy groups.
10 15. The method according to any one of Claims 2 to 13 , wherein R7 is a phenyl group
which may be substituted with one or two halogen atoms.
16. The method according to any one of Claims 2 to 15 , wherein R1 is a methyl group.
17. The method according to Claim 2, wherein the compound represented by the
formula (I) is (E)-5-(2-{1-[5-(3,4-dichlorophenyl)-4-hydroxythiophen-3-
15 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.
20 18. The method according to Claim 1, wherein W is a carboxy group.
19. The method according to Claim 18, wherein R1 is a hydrogen atom or a C1-6 alkyl
group which may be substituted with one or more halogen atoms,
73
R6 is a hydrogen atom or a C1-3 alkyl group which may be substituted with one or more
halogen atoms,
R7 is a C2-14 aryl group
X is -OH,
Y is an oxygen atom or a sulfur atom, an5 d
Ar1 is represented by the formula (IV):
（IV)
S .
20. The method according to Claim 19, wherein R1 is a hydrogen atom or a C1-6 alkyl
group,
10 R6 is a hydrogen atom,
R7 is a substituent represented by any one of the formulae (A01) to (A15):
O
O
(A01) (A02) (A03) (A04)
(A08) (A09) (A10) (A11)
(A12) (A13) (A14) (A15)
(A05) (A06) (A07)
, and
Y is an oxygen atom.
15 21. The method according to Claim 1, wherein the compound represented by the
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 R1 is a hydrogen atom or a C1-6 alkyl
group which may be substituted with one or more halogen atoms,
20 each of R2, R3, R4 and R6 is independently a hydrogen atom or a C1-3 alkyl group,
n is an integer of 1 or 2,
R5 is a phenyl group or a C2-9 heteroaryl group which may be substituted with one or
74
more substituents independently represented by V1,
R7 is a phenyl group which may be substituted with one or more substituents selected
from C1-10 alkyl groups which may be substituted with one or more halogen atoms, C1-10
alkoxy groups, C1-3 alkoxy groups substituted with one or more halogen atoms and
halogen atoms or a substituent represented by any one of the formulae (A01) to (A15)5 :
,
Ar1 is represented by the formula (IV):
（IV)
S ,
X is -OH, and
10 each of Y and Z is independently an oxygen atom or a sulfur atom.
23. The method according to Claim 22, wherein R1 is a hydrogen atom or a C1-6 alkyl
group,
R2, R3, R4 and R6 are hydrogen atoms,
n is an integer of 1,
15 R5 is a pyridyl group, a pyrazinyl group or a phenyl group substituted with a substituent
represented by the formula (VII), (VIII), (XI) or (XII):
75
O
HN
N
OH
OH
O
NH2
（X Ｉ） （X Ｉ Ｉ）
O
HN
OH
（VIII）
O
HN
N NH
（VII）
,
R7 is a phenyl group which may be substituted with one or two halogen atoms or C1-10
alkyl groups or a substituent represented by the formula (A11), (A13) or (A15):
, and
Y and Z are oxygen atom5 s.
24. The method according to Claim 1, wherein the compound represented by the
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-
10 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-
15 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-
20 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 any one of Claims 1 to 24, wherein the pluripotent stem
cells are ES cells or iPS cells.
25 26. The method according to any one of Claims 1 to 25, wherein the hematopoietic
progenitor cells derived from pluripotent stem cells are hematopoietic progenitor cells
obtained from a sac-like structure formed by differentiating pluripotent stem cells into
hematopoietic progenitor cells.
27. The method according to any one of Claims 1 to 26, wherein the hematopoietic
30 progenitor cells derived from pluripotent stem cells have one or more introduced genes
76
selected from oncogenes, polycomlj genes, apoptosis suppressor genes and genes which suppress a tumor suppressor gene and have proliferative and/or diffenentiative capability enhanced by regulation or expression of the introduced genes.
28. The method according to any one of Claims 1 to 27, wherein the hematopoietic
progenitor cells derived from pluripotent stem cells are hematopoietic progenitor cells
which have one or more introduced genes selected from MYC family genes. BniH
genes, BCL2 family genes and genes which suppress the p53 gene expression and
have proliferative andVor differentiatiye capability enhanced by regulation of expression
of the introduced genes.
29. Megakaryocytes andjor platelets obtained by the method as defined in any one of Claims 1 to 23.
30. A blood preparation containing p atelets obtained by the method as defined in any one of Claims 1 to 2S, as an active ingredient.
31. A kit for producing platelets by the method as defined in any one of Claims 1 to 23.