DESCRIPTION]
[Title of Invention] COMPOSITION FOR MAINTAINING FUNCTION
OF PLATELETS
[Technical Field]
The present invention relates to a composition fo5 r
maintaining a function of platelets, a method for preparing
platelets, a blood product comprising platelets, and a
method for maintaining a function of platelets in a blood
product, which utilize an N-hydroxyformamide derivative.
10 [Background Art]
For the treatment of blood-related diseases typified
by leukemia, it is extremely important to stably amplify
and supply blood cells in an amount necessary for such
treatment. Thus, to date, many researchers have attempted
15 to efficiently amplify hematopoietic stem cells or
hematopoietic progenitor cells.
Among blood cells, megakaryocytes are platelet
progenitor cells, i.e., cells producing platelets and are
known to form a proplatelet structure (cytoplasmic
20 formation) to produce platelets and play an important role
in therapeutic applications. Platelets are essential for
blood coagulation (hemostasis). Accordingly, the demand
for platelets is extremely high for leukemia, bone marrow
transplantation, anticancer therapy, and so forth.
25 Platelet production has been attempted by
administration of thrombopoietin (TPO) and in the way of
3
differentiating umbilical cord blood or bone marrow cells
into megakaryocytes, other than a method of collecting
blood from blood donors so far. Recently, a method also
has been attempted in which hematopoietic progenitor cells
are amplified ex vivo to prepare platelets from suc5 h
progenitor cells. For example, the present inventors have
reported: a method for producing platelets in a relatively
large amount efficiently from a sac-like structure
enclosing hematopoietic progenitor cells, after the
10 sac-like structure was prepared from human embryonic stem
cells (ES cells) (PTL 1); a method for efficiently preparing
mature megakaryocytes and platelets from induced
pluripotent stem cells (iPS cells) in an in-vitro culture
system (PTL 2); and so forth.
15 As described above, researches on the method for
preparing platelets per se exogenously have been advanced.
However, the function of prepared platelets is poorly
stable. For this reason, it is necessary to develop a
method that enables preparation of a large amount of
20 functional platelets capable of being stored for an
extended period of time.
Binding between platelets and an extracellular matrix
is an important process for inducing blood coagulation
(hemostasis, thrombus formation, and the like). It is
25 believed that this process is initiated when a platelet
receptor GPIb binds to von Willebrand factor (VWF) via an
4
α-subunit (GPIbα). However, it has been reported that
under a condition of around 37ºC, a metalloproteinase
ADAM17 (a disintegrin and metallopeptidase domain 17) sheds
an extracellular region of GPIbα (release by cleavage),
thereby inhibiting the association between GPIbα and VWF5 ,
and the platelets lose blood coagulating ability (NPLs 1
and 2).
Hence, it is anticipated that at least the activity
of ADAM17 needs to be suppressed in order to keep the function
10 of platelets prepared in vitro. Actually, the present
inventors have reported that the function of platelets
prepared in vitro can be kept by adding at an appropriate
timing an inhibitor (such as GM6001) directly inhibiting
a metalloproteinase activity or a p38 MAP kinase inhibitor
15 indirectly inhibiting activation of the activity of
metalloproteinase such as ADAM17 (PTL 3).
Nevertheless, inhibitors such as GM6001 inhibit the
activities of not only ADAM17 but also other
metalloproteinases (particularly MMP9 and MMP14 essential
20 for hematopoietic function). Hence, when such inhibitors
are utilized to prepare platelets in vitro, the inhibitors
need to be added at a certain period when platelet production
is observed most abundantly. Since such a complex task
as getting right timing is required in cell culturing that
25 is poor in consistency and reproducibility, the method for
preparing platelets by utilizing these inhibitors has not
5
been satisfactory yet in preparing a large amount of
functional platelets and particularly in establishing a
plant for platelet production system.
Furthermore, non-selective metalloproteinase
inhibitors inhibit the activity of all o5 f
metalloproteinases such as membrane type
metalloproteinases, secreted metalloproteinases and
ADAMs. Accordingly, the in vivo use is believed to bring
about a risk of various side effects (adverse influences
10 caused by the inhibition of essential MMPs or ADAMs acting
on different organs). Actually, since non-selective
metalloproteinase inhibitors cause a severe side effect
called musculoskeletal syndrome, the many developments of
these inhibitors have been terminated (NPL 3). Moreover,
15 it is suggested that an inhibitor with a hydroxamic acid
structure, such as GM6001, should have mutagenicity (NPL
4). Thus, platelets obtained by a method using foregoing
inhibitor are not satisfactory even in safety yet.
Meanwhile, at present, there is no effective method
20 for storing platelets prepared from living donors, other
than a method in which platelets are stored with agitating
at 20ºC to 24ºC. Accordingly, it seems effective to prepare
platelets under room temperature conditions (20ºC to 24ºC)
even when the method for suppressing the metalloproteinase
25 activity of ADAM17 is utilized to keep the function of
platelets. Nevertheless, no verification has been made
6
under the room temperature condition at all so far whether
or not it is possible to get umbilical cord blood or bone
marrow cells differentiated into megakaryocytes, and
whether or not it is possible to produce platelets from
hematopoietic progenitor cells derived from ES cells o5 r
the like.
As described above, a compound practically usable
as an active ingredient of a composition for maintaining
a function of platelets has not been discovered yet. Hence,
10 a method for obtaining functionally stable platelets in
vitro and particularly a method suitable for mass
production of highly safe platelets have not been
established at present.
[Citation List]
15 [Patent Literatures]
[PTL 1] International Publication No. WO2008/041370
[PTL 2] International Publication No. WO2009/122747
[PTL 3] International Publication No. WO2009/119105
[Non Patent Literatures]
20 [NPL 1] Bergmeier et al., Circulation Research, 2004,
vol. 95, pp. 660 677 to 670683
[NPL 2] Bergmeier et al., Blood, 2003, vol. 102, pp.
4229 to 4235
[NPL 3] Peterson et al., Cardiovasc. Res., 2006, vol.
25 69, pp. 677 to 687
[NPL 4] Skipper et al., Cancer Res., 1980, vol. 40, pp.
7
4704 to 4708
[Summary of Invention]
[Technical Problem]
The present invention has been made in view of the
above-described problems of the conventional techniques5 .
An object of the present invention is to identify a compound
capable of maintaining a function of platelets by
specifically inhibiting a metalloproteinase activity of
ADAM17 to suppress GPIbα shedding. Another object of the
10 present invention is to provide a composition for
maintaining a function of platelets, to efficiently produce
platelets, and to improve the quality maintenance of a blood
product, all of which are achieved by utilizing the
identified compound.
15 [Solution to Problem]
The present inventors have earnestly studied in order
to achieve the above objects. As a result, the inventors
have revealed that culturing under room temperature
condition of 25º fails to differentiate hematopoietic
20 progenitor cells derived from ES cells, iPS cells, or the
like into megakaryocytes, or to produce platelets from the
megakaryocytes, but that a culture condition around 37ºC
is preferable. Hence, the present inventors next searched
for a compound capable of specifically inhibiting
25 ADAM17-mediated GPIbα shedding in culturing at around 37ºC.
As a result, the followings were found out.
8
N-hydroxyformamide derivatives with a particular
structure have an inhibitory action specific to ADAM17.
When the N-hydroxyformamide derivatives were added to a
culture system for producing platelets or to human
peripheral blood-derived platelets, GPIbα shedding wa5 s
suppressed, and the function of the platelets was also
maintained under the temperature condition of around 37ºC.
The present inventors have found out that from such actions
of the identified N-hydroxyformamide derivatives, the
10 derivatives are extremely useful for efficient platelet
production, improvement in the quality maintenance of a
blood product comprising platelets, and so on. These
discoveries have led to the completion of the present
invention.
15 The present invention more specifically provides the
following inventions.
(1) A composition for maintaining a function of platelets,
the composition comprising, as an active ingredient, a
compound represented by the following general formula (I)
20 or a salt thereof, or a solvate thereof:
[Chem. 1]
wherein
X represents a phenylene group;
9
Y represents any one of a hydrogen atom and -(CH2)mR1;
wherein
m represents an integer of any one of 0 to 4;
and
R1 is any one o5 f
[Chem. 2]
wherein R2 represents any one of a C1 to C6 alkyl
group, which may be substituted, an aryl group, which may
10 be substituted, and a C1 to C6 alkoxy group;
R3 and R4 each independently represent any one
of a hydrogen atom and a C1 to C6 alkyl group, or R3 and
R4 together with an adjacent nitrogen atom may form a
nitrogen-containing heterocycle; and
R5 15 represents any one of a hydrogen atom, a C1
to C6 alkyl group, and a C1 to C6 alkylsulfonyl group; and
Z represents any one of a hydrogen atom and a C1 to
C6 alkyl group.
(2) The composition according to (1), wherein the
20 compound represented by the general formula (I) is any one
of
N-[2-(4-but-2-ynyloxybenzenesulfonyl)-1-(4-dieth
ylaminomethylphenyl)ethyl]-N-hydroxyformamide and
10
N-{4-[2-(4-but-2-ynyloxybenzenesulfonyl)-1-(form
ylhydroxyamino)ethyl]benzyl}methanesulfonamide.
(3) The composition according to any one of (1) and (2)
in a form of any one of a reagent for maintaining a function
of platelets and an additive to a blood product comprisin5 g
platelets.
(4) A method for preparing platelets, wherein the method
comprises adding, to a culture system for differentiating
megakaryocytes from cells capable of differentiating into
10 megakaryocytes and producing platelets from the
megakaryocytes, a compound represented by the following
general formula (I) or a salt thereof, or a solvate thereof:
[Chem. 3]
15 wherein
X represents a phenylene group;
Y represents any one of a hydrogen atom and -(CH2)mR1;
wherein
m represents an integer of any one of 0 to 4;
20 and
R1 is any one of
[Chem. 4]
11
wherein R2 represents any one of a C1 to C6 alkyl
group, which may be substituted, an aryl group, which may
be substituted, and a C1 to C6 alkoxy group;
R3 and R4 each independently represent any on5 e
of a hydrogen atom and a C1 to C6 alkyl group, or R3 and
R4 together with an adjacent nitrogen atom may form a
nitrogen-containing heterocycle; and
R5 represents any one of a hydrogen atom, a C1
10 to C6 alkyl group, and a C1 to C6 alkylsulfonyl group; and
Z represents any one of a hydrogen atom and a C1 to
C6 alkyl group.
(5) The method according to (4), wherein the compound
represented by the general formula (I) is any one of
15 N-[2-(4-but-2-ynyloxybenzenesulfonyl)-1-(4-dieth
ylaminomethylphenyl)ethyl]-N-hydroxyformamide and
N-{4-[2-(4-but-2-ynyloxybenzenesulfonyl)-1-(form
ylhydroxyamino)ethyl]benzyl}methanesulfonamide.
(6) The method according to any one of (4) and (5), wherein
20 a culture temperature in the culture system is 35 to 38ºC.
(7) A culture which is a culture system for
differentiating megakaryocytes from cells capable of
differentiating into megakaryocytes and for producing
12
platelets from the megakaryocytes, and is added to the
system with a compound represented by the following general
formula (I) or a salt thereof, or a solvate thereof:
[Chem. 5]
5
wherein
X represents a phenylene group;
Y represents any one of a hydrogen atom and -(CH2)mR1;
wherein
10 m represents an integer of any one of 0 to 4;
and
R1 is any one of
[Chem. 6]
wherein R2 15 represents any one of a C1 to C6 alkyl
group, which may be substituted, an aryl group, which may
be substituted, and a C1 to C6 alkoxy group;
R3 and R4 each independently represent any one
of a hydrogen atom and a C1 to C6 alkyl group, or R3 and
R4 20 together with an adjacent nitrogen atom may form a
nitrogen-containing heterocycle; and
13
R5 represents any one of a hydrogen atom, a C1
to C6 alkyl group, and a C1 to C6 alkylsulfonyl group; and
Z represents any one of a hydrogen atom and a C1 to
C6 alkyl group.
(8) The culture according to (7), wherein the compoun5 d
represented by the general formula (I) is any one of
N-[2-(4-but-2-ynyloxybenzenesulfonyl)-1-(4-dieth
ylaminomethylphenyl)ethyl]-N-hydroxyformamide and
N-{4-[2-(4-but-2-ynyloxybenzenesulfonyl)-1-(form
10 ylhydroxyamino)ethyl]benzyl}methanesulfonamide.
(9) A blood product comprising platelets and a compound
represented by the following general formula (I) or a salt
thereof, or a solvate thereof:
[Chem. 7]
15
wherein
X represents a phenylene group;
Y represents any one of a hydrogen atom and -(CH2)mR1;
wherein
20 m represents an integer of any one of 0 to 4;
and
R1 is any one of
[Chem. 8]
14
wherein R2 represents any one of a C1 to C6 alkyl
group, which may be substituted, an aryl group, which may
be substituted, and a C1 to C6 alkoxy group;
R3 and R4 each independently represent any on5 e
of a hydrogen atom and a C1 to C6 alkyl group, or R3 and
R4 together with an adjacent nitrogen atom may form a
nitrogen-containing heterocycle; and
R5 represents any one of a hydrogen atom, a C1
10 to C6 alkyl group, and a C1 to C6 alkylsulfonyl group; and
Z represents any one of a hydrogen atom and a C1 to
C6 alkyl group.
(10) The blood product according to (9), wherein the
compound represented by the general formula (I) is any one
15 of
N-[2-(4-but-2-ynyloxybenzenesulfonyl)-1-(4-dieth
ylaminomethylphenyl)ethyl]-N-hydroxyformamide and
N-{4-[2-(4-but-2-ynyloxybenzenesulfonyl)-1-(form
ylhydroxyamino)ethyl]benzyl}methanesulfonamide.
20 (11) A method for maintaining a function of platelets in
a blood product, wherein the method comprises adding, to
a blood product comprising platelets, a compound
represented by the following general formula (I) or a salt
15
thereof, or a solvate thereof:
[Chem. 9]
wherein
X represents a phenylene group5 ;
Y represents any one of a hydrogen atom and -(CH2)mR1;
wherein
m represents an integer of any one of 0 to 4;
and
R1 10 is any one of
[Chem. 10]
wherein R2 represents any one of a C1 to C6 alkyl
group, which may be substituted, an aryl group, which may
15 be substituted, and a C1 to C6 alkoxy group;
R3 and R4 each independently represent any one
of a hydrogen atom and a C1 to C6 alkyl group, or R3 and
R4 together with an adjacent nitrogen atom may form a
nitrogen-containing heterocycle; and
R5 20 represents any one of a hydrogen atom, a C1
to C6 alkyl group, and a C1 to C6 alkylsulfonyl group; and
16
Z represents any one of a hydrogen atom and a C1 to
C6 alkyl group.
(12) The method according to (11), wherein the compound
represented by the general formula (I) is any one of
N-[2-(4-but-2-ynyloxybenzenesulfonyl)-1-(4-diet5 h
ylaminomethylphenyl)ethyl]-N-hydroxyformamide and
N-{4-[2-(4-but-2-ynyloxybenzenesulfonyl)-1-(form
ylhydroxyamino)ethyl]benzyl}methanesulfonamide.
[Advantageous Effects of Invention]
10 The N-hydroxyformamide derivatives discovered in the
present invention are capable of maintaining a function
of platelets even under a temperature condition of around
37ºC by specifically inhibiting a metalloproteinase
activity of ADAM17 to suppress GPIbα shedding. Thus, the
15 derivatives make it possible to produce a large amount of
functionally stable platelets in vitro, and to keep the
quality of a blood product comprising platelets.
[Brief Description of Drawings]
[Fig. 1] Fig. 1 is a schematic drawing for illustrating
20 a method for inducing megakaryocytes/platelets, under room
temperature conditions, from hematopoietic progenitor
cells derived from ES cells or the like.
[Fig. 2] Fig. 2 is a graph showing the number of
megakaryocytes produced from ES cell-derived
25 hematopoietic progenitor cells by culturing under room
temperature conditions. The unit of the vertical axis is
17
cell count/well.
[Fig. 3] Fig. 3 is a graph showing a total number of
platelets produced from the ES cell-derived hematopoietic
progenitor cells by culturing under room temperature
conditions. The unit of the vertical axis is cel5 l
count/well.
[Fig. 4] Fig. 4 is a graph showing the number of
megakaryocytes produced from umbilical cord blood-derived
CD34(+) cells by culturing under room temperature
10 condition. The unit of the vertical axis is cell
count/well.
[Fig. 5] Fig. 5 is a graph showing a total number of
platelets produced from the umbilical cord blood-derived
CD34(+) cells by culturing under room temperature
15 condition. The unit of the vertical axis is cell
count/well.
[Fig. 6] illustrates application timing of
a compound (S-45282 or S-45457) according to the present
invention in an induction process from ES cell- or iPS
20 cell-derived hematopoietic progenitor cells into
megakaryocytes/platelets. illustrates that
washed platelets were prepared from human peripheral blood,
and the compound (S-45282 or S-45457) according to the
present invention was added thereto.
25 [Fig. 7] Fig. 7 is a graph for illustrating the results
of verification of the function-maintaining effects of
18
GM6001 and S-45282 on ES cell-derived platelets when each
compound was added to a culture system for 2 days (2 days).
The vertical axis represents percentages (%) of CD42b
(GPIbα)(+) and CD42b (GPIbα)(-) where a total number of
platelets (CD41(+)) is taken as 1005 .
[Fig. 8] Fig. 8 is a graph showing a total number of the
ES cell-derived platelets obtained when GM6001 or S-45282
was added to the culture system for 2 days (2 days). The
unit of the vertical axis is cell count/well.
10 [Fig. 9] Fig. 9 is a graph for illustrating the result
of verifying effects of GM6001 and S-45282 maintaining a
function of ES cell-derived platelets when each compound
was added to a culture system for 8 days (8 days). The
vertical axis represents percentages (%) of CD42b
15 (GPIbα)(+) and CD42b (GPIbα)(-) where a total number of
platelets (CD41(+)) is taken as 100.
[Fig. 10] Fig. 10 is a graph showing a total number of
the ES cell-derived platelets obtained when GM6001 or
S-45282 were added to the culture system for 8 days (8 days).
20 The unit of the vertical axis is cell count/well.
[Fig. 11] Fig. 11 is a graph showing the number of ES
cell-derived platelets (the number of CD41(+)CD42b(+) and
CD41(+)CD42b(-)) obtained when GM6001, S-45282, or S-45457
was added to a culture system for 8 days. The unit of the
25 vertical axis is cell count/well.
[Fig. 12] Fig. 12 is a graph for illustrating the result
19
of verification of function-maintaining effects of GM6001,
S-45282, and S-45457 on the ES cell-derived platelets when
each compound was added to the culture system for 8 days.
The vertical axis represents a percentage (%) of CD42b
(GPIbα)(+) where a total number of platelets (CD41(+)) i5 s
taken as 100.
[Fig. 13] Fig. 13 is a graph showing a total number of
ES cell-derived platelets obtained when GM6001 or S-45457
was added to a culture system for 8 days. The unit of the
10 vertical axis is cell count/well. The value is average
+ standard error (n = 4).
[Fig. 14] Fig. 14 is a graph for illustrating a
concentration-dependence of function-maintaining effect
of S-45457 on the ES cell-derived platelets. The vertical
15 axis represents a percentage (%) of CD42b (GPIbα)(+) where
a total number of platelets is taken as 100. The value
is average + standard error (n = 4). # (sharp sign)
indicates p < 0.1.
[Fig. 15] Fig. 15 is a graph showing a total number of
20 iPS cell-derived platelets obtained when GM6001 or S-45457
was added to a culture system for 8 days. The unit of the
vertical axis is cell count/well. The value is average
+ standard error (n = 4).
[Fig. 16] Fig. 16 is a graph for illustrating a
25 concentration-dependence of function-maintaining effect
of S-45457 on the iPS cell-derived platelets. The vertical
20
axis represents a percentage (%) of CD42b (GPIbα)(+) where
a total number of platelets is taken as 100. * (asterisk)
and ** (two asterisks) indicate p < 0.05 and p < 0.01,
respectively.
[Fig. 17] Fig. 17 is a graph for illustrating 5 a
concentration-dependence of function-maintaining effect
of S-45457 on peripheral blood-derived platelets. The
vertical axis represents a percentage of CD41(+)CD42b
(GPIbα)(+) where a total number of platelets is taken as
10 1.00. The value is average value + standard error (n =
4). ** (two asterisks) indicates p < 0.01.
[Fig. 18] Fig. 18 shows dot plot charts for illustrating
function-maintaining effects of S-45457 and p38 inhibitors
on peripheral blood-derived platelets.
15 [Fig. 19] Fig. 19 is a graph for illustrating
function-maintaining effects of GM6001, S-45457, and the
p38 inhibitors on peripheral blood-derived platelets. The
vertical axis represents a percentage of
CD41(+)CD42b(GPIbα)(+) to a total number of platelets which
20 is taken as 1.00. The value is average value + standard
error (n = 4).
[Fig. 20] Fig. 20 is a graph showing a total number of
ES cell-derived platelets obtained when GM6001, S-45457,
or the p38 inhibitors was added to a culture system for
25 8 days. The unit of the vertical axis is cell count/well.
[Fig. 21] Fig. 21 is a graph for illustrating the result
21
of verification of function-maintaining effects of GM6001,
S-45457, and the p38 inhibitors on the ES cell-derived
platelets when each compound was added to the culture system
for 8 days. The vertical axis represents a percentage (%)
of CD42b (GPIbα)(+) where a total number of platelets i5 s
taken as 100.
[Fig. 22] Fig. 22 is a graph for illustrating the result
of verification of function-maintaining effects of GM6001,
S-45457, and S-45282 on peripheral blood-derived
10 platelets. The vertical axis represents a percentage (%)
of CD42b (GPIbα)(+) where a total number of platelets is
taken as 100.
[Fig. 23] Fig. 23 shows graphs for illustrating the result
of verification of function-maintaining effects of GM6001,
15 S-45457, S-45282, and the p38 inhibitors on peripheral
blood-derived PRP. The vertical axis represents
percentages (%) of CD41(+)CD42b(+) and CD41(+)CD42b(-)
where a total number of platelets (CD41(+)) is taken as
100.
20 [Fig. 24] Fig. 24 shows dot plot charts for illustrating
function-maintaining effects of GM6001, S-45457, S-45282,
and a p38 inhibitor on peripheral blood-derived platelets.
[Fig. 25] Fig. 25 shows dot plot charts for illustrating
a concentration-dependence of function-maintaining effect
25 of S-45457 on peripheral blood-derived platelets.
[Fig. 26] Fig. 26 is a graph for illustrating the result
22
of chronological evaluation of the adhesion ability of
platelets prepared in the presence of S-45457.
[Fig. 27] Fig. 27 is a graph for illustrating the result
of evaluation of the adhesion ability of the platelets
prepared in the presence of S-454575 .
[Fig. 28] Fig. 28 shows microphotographs at the time of
laser irradiation (0 seconds) and 20 seconds thereafter,
showing thrombus formation in mice into which platelets
prepared in the presence of S-45457 were injected.
10 [Fig. 29] Fig. 29 is a graph for illustrating the
contribution of the platelets prepared in the presence of
S-45457 to the thrombus formation. The vertical axis is
a percentage (%) of the number of human platelets to a total
number of platelets (mouse + human) involved in the thrombus
15 formation, which was obtained by observing obstructive
thrombi induced in mensenteric capillaries of mice
transfused with each sample. The value is the average of
the percentages + standard error obtained by observing 20
blood vessels.
20 [Description of Embodiments]
A composition of the present invention is a
composition for maintaining a function of platelets, the
composition comprising, as an active ingredient, a compound
represented by the following general formula (I) or a salt
25 thereof, or a solvate thereof:
[Chem. 11]
23
wherein
X represents a phenylene group;
Y represents any one of a hydrogen atom and -(CH2)mR1;
wherei5 n
m represents an integer of any one of 0 to 4;
and
R1 is any one of
[Chem. 12]
10
wherein R2 represents any one of a C1 to C6 alkyl
group, which may be substituted, an aryl group, which may
be substituted, and a C1 to C6 alkoxy group;
R3 and R4 each independently represent any one
of a hydrogen atom and a C1 to C6 alkyl group, or R3 15 and
R4 together with an adjacent nitrogen atom may form a
nitrogen-containing heterocycle; and
R5 represents any one of a hydrogen atom, a C1
to C6 alkyl group, and a C1 to C6 alkylsulfonyl group; and
20 Z represents any one of a hydrogen atom and a C1 to
C6 alkyl group.
24
In the compound represented by the above-mentioned
general formula (I), "C1 to C6" and "C6 to C14" each mean
that the carbon number falls within a range of from 1 to
6, and from 6 to 14, respectively.
"C1 to C6 alkyl group" of "C1 to C6 alkyl group, whic5 h
may be substituted" in R2, R3, R4, R5 and Z means a linear
or branched C1 to C6 alkyl group, and its specific examples
include a methyl group, an ethyl group, an n-propyl group,
an isopropyl group, an n-butyl group, an isobutyl group,
10 a tert-butyl group, a sec-butyl group, an n-pentyl group,
a tert-amyl group, a 3-methylbutyl group, a neopentyl group,
an n-hexyl group, etc.
The substituent in the above-mentioned "C1 to C6 alkyl
group, which may be substituted" includes a hydroxyl group,
15 a halogen atom, a cyano group, a nitro group, a C1 to C6
alkoxy group, a carboxyl group, a C1 to C6 alkoxycarbonyl
group, etc. At least one or more of these may be substituted
in any and every substitutable position. In case where
the compound has multiple substituents, the substituents
20 may be the same or different, and may be substituted on
the same carbon atom or on different carbon atoms.
"Halogen atom" means a fluorine atom, a chlorine atom,
a bromine atom, an iodine atom.
"C1 to C6 alkoxy group" means an alkoxy group in which
25 the alkyl moiety is has the same meaning as that of the
above-mentioned "C1 to C6 alkyl group", for which, for
25
example, there is mentioned a linear or branched alkoxy
group such as a methoxy group, an ethoxy group, an n-propoxy
group, an isopropoxy group, an n-butoxy group, an isobutoxy
group, a tert-butoxy group, a sec-butoxy group, an
n-pentyloxy group, a tert-amyloxy group, a 3-methylbutox5 y
group, a neopentyloxy group, an n-hexyloxy group, etc.
"C1 to C6 alkoxycarbonyl group" means one in which
the alkyl moiety excluding the oxycarbonyl moiety therein
is a linear or branched C1 to C6 alkyl group, including,
10 for example, a methoxycarbonyl group, an ethoxycarbonyl
group, an n-propoxycarbonyl group, an isopropoxycarbonyl
group, an n-butoxycarbonyl group, an isobutoxycarbonyl
group, a tert-butoxycarbonyl group, a sec-butoxycarbonyl
group, an n-pentyloxycarbonyl group, a
15 tert-amyloxycarbonyl group, a 3-methylbutoxycarbonyl
group, a neopentyloxycarbonyl group, an
n-hexyloxycarbonyl group, etc.
"Nitrogen-containing heterocycle" which R3 and R4
together with an adjacent nitrogen atom form includes, for
20 example, a 5- to 7-membered nitrogen-containing
heterocycle which contains at least one nitrogen atom in
addition to the carbon atom as the cycle-constituting atom
and may further contain one or two hetero atoms selected
from an oxygen atom, a sulfur atom and a nitrogen atom.
25 Preferred examples of the nitrogen-containing heterocycle
include a piperidine ring, a piperazine ring, a morpholine
26
ring, a thiomorpholine ring, a pyrrolidine ring, an
imidazolidine ring, etc.
"Aryl group" of " aryl group, which may be substituted
" in R2 means an aromatic carbocycle, preferably a C6 to
C14 aromatic carbocycle, and includes, for example, 5 a
phenyl group, a naphthyl group, etc.
The substituent on the aromatic ring of the
above-mentioned " aryl group, which may be substituted "
includes a hydroxyl group, a halogen atom, a cyano group,
10 a nitro group, a trifluoromethyl group, a C1 to C6 alkyl
group which may be substituted, a C1 to C6 alkoxy group,
a carboxyl group, a C1 to C6 alkoxycarbonyl group, etc.
At least one or more of these may be substituted in any
and every substitutable position. In case where the
15 compound has multiple substituents, the substituents may
be the same or different, and may be substituted on the
same carbon atom or on different carbon atoms. In this,
"halogen atom", "C1 to C6 alkyl group, which may be
substituted", "C1 to C6 alkoxy group" and "C1 to C6
20 alkoxycarbonyl group" have the same meanings as above.
"C1 to C6 alkylsulfonyl group" in R5 means an
alkylsulfonyl group in which the alkyl moiety has the same
meaning as that of the above-mentioned "C1 to C6 alkyl group",
including, for example, a methanesulfonyl group, an
25 ethanesulfonyl group, etc.
In case where the compound represented by the general
27
formula (I) has an asymmetric carbon, racemates and
diastereomers thereof and also individual optical active
forms of the compound are all included in the invention.
In case where the compound has a geometric isomer, the (E)
form and the (Z) form thereof and also the mixture thereo5 f
are all included in the invention.
Not specifically defined, the salt of the compound
represented by the general formula (I) may be any
pharmaceutically-acceptable salt thereof, including, for
10 example, salts with an inorganic base, salts with an organic
base, salts with an organic acid, salts with an inorganic
acid, salts with an amino acid, etc. Examples of the salts
with an inorganic base include alkali metal salts and
alkaline earth metal salts such as lithium salts, sodium
15 salts, potassium salts, calcium salts, magnesium salts,
etc. Examples of the salts with an organic base include
triethylamine salts, pyridine salts, ethanolamine salts,
cyclohexylamine salts, dicyclohexylamine salts,
dibenzylethanolamine salts, etc. Examples of the salts
20 with an organic acid include formates, acetates, tartrates,
maleates, succinates, lactates, malates, ascorbates,
oxalates, glycolates, phenylacetates, methanesulfonates,
etc. Examples of the salts with an inorganic acid include
hydrochlorides, hydrobromides, phosphates, sulfamates,
25 nitrates, etc. Examples of the salts with an amino acid
include glycine salts, alanine salts, arginine salts,
28
glutamates, aspartates, etc.
The compound represented by the general formula (I)
may have a form of prodrug. Examples of prodrug include
methyl ester, ethyl ester and aminoalkyl ester derivatives
at the carboxyl group of the compound of the general formul5 a
(I), acetate, formate and benzoate derivatives at the
hydroxyl group and the amine functional group of the
compound of the general formula (I), etc., to which, however,
the invention is not limited.
10 The compound represented by the above-mentioned
general formula (I) can be produced according to various
methods but may be efficiently produced according to the
method mentioned below. Note that,Specific examples of
the "protective group" for use in the production method
15 mentioned below include a tert-butyl group, a benzyl group,
an o-methylbenzyl group, a p-nitrobenzyl group, a
p-methoxybenzyl group, an o-chlorobenzyl group, a
2,4-dichlorobenzyl group, a p-bromobenzyl group, an allyl
group, a tert-butoxycarbonyl group, a benzyloxycarbonyl
20 group, an o-methylbenzyloxycarbonyl group, a
p-nitrobenzyloxycarbonyl group, a
p-methoxybenyloxycarbonyl group, an
o-chlorobenzyloxycarbonyl group, a
2,4-dichlorobenzyloxycarbonyl group, a
25 p-bromobenzyloxycarbonyl group, an allyloxycarbonyl group,
a tert-butyldimethylsilyl group, a
29
tert-butyldiphenylsilyl group, a triethylsilyl group, a
trimethylsilyl group, a triisopropylsilyl group, a
methoxymethyl group, a tetrahydropyranyl group, carbonyl
protective groups (for example, protective groups with
ethanediol, propanediol, mercaptoethanol5 ,
mercaptopropanol, ethanedithiol, propanedithiol, etc.),
etc.
The compound represented by the general formula (I)
can be produced, for example, through the reaction of the
10 following step 1 and step 2.
[Chem. 13]
Scheme 1:
(In the formulae, X, Y and Z have the same meanings as
15 mentioned above.)

In the step 1, hydroxylamine or its salt is added
30
to the compound (II) to produce the compound represented
by the general formula (III). In case where hydroxylamine
is a salt thereof (hydrochloride, acetate, etc.), the
addition reaction is attained in the presence of an
inorganic base such as potassium carbonate, sodiu5 m
carbonate, sodium hydrogen carbonate, sodium hydroxide,
potassium hydroxide, lithium hydroxide, etc. Not
specifically defined, the reaction solvent may be any
solvent not significantly interfering with the reaction,
10 but is preferably water, tetrahydrofuran, cyclopentyl
methyl ether, acetonitrile, 1,4-dioxane, diethyl ether or
their mixed solvent, etc.
Not specifically defined, the reaction temperature
may be generally from 0 to 100 C, and the reaction time
15 is preferably from 2 hours to 1 week.

In the step 2, the compound (III) obtained in the
step 1 is condensed with the intermediate represented by
the general formula (IV) to produce the compound
20 represented by the general formula (I). The intermediate
(IV) is a reactive intermediate to be obtained from a mixed
acid anhydride with formic acid (mixed acid anhydride of
formic acid and acetic acid, etc.), pentafluorophenyl
formate, or formic acid and a carbodiimide
25 (dicyclohexylcarbodiimide, diisopropylcarbodiimide or
water-soluble carbodiimide). For smoothly attaining the
31
reaction, an organic base such as triethylamine,
diisopropylethylamine, pyridine, lutidine, collidine,
dimethylaminopyridine or the like may be made to coexist
in the system. Adding 1-hydroxybenzotriazole and/or
4-dimethylaminopyridine to some of these cases (especiall5 y
where the reactive intermediate is obtained from
carbodiimide) could promote the reaction. Not
specifically defined, the reaction solvent may be any
solvent not significantly interfering with the reaction,
10 but is preferably chloroform, methylene chloride,
tetrahydrofuran, acetonitrile, cyclopentyl methyl ether,
1,4-dioxane, dimethylformamide, dimethyl sulfoxide,
pyridine, etc. Not specifically defined, the reaction
temperature may be generally from 0 to 100 C, and the
15 reaction time is preferably from 1 to 24 hours. In this
step, a CHO group may be added to also to the hydroxyl group
of the hydroxylamino group, depending on the chemical
properties of the starting materials; but in such a case,
the product may be processed with a lower alcohol in an
20 acidic, basic or neutral condition to be converted into
the intended product, compound (I). The lower alcohol is
preferably methanol, ethanol, propanol, etc. An auxiliary
solvent may be used here, and when used, the auxiliary
solvent is not specifically defined.
25 Needless to say, depending on the properties of X,
Y and Z, it is necessary to previously use the corresponding
32
protective group in the reaction of the above-mentioned
step 1 and step 2 and to remove the protective group after
the reaction. In case where the group is not protected,
the yield in the next step and further in the next step
after that next step may lower and the intermediate ma5 y
be difficult to handle.
The above-mentioned compound (II) may be produced
according to the process of the step 3 to step 5, as mentioned
below.
10
33
[Chem. 14]
Scheme 2:
(In the formulae, X, Y and Z have the same meanings as above;
E represents a releasing functional group such as a C15 to
C6 alkoxy group, a halogen atom, an
N,O-dimethylhydroxyamino group or the like; M1 represents
Li, CeCl2, NaBH3, LiBH3, LiBEt3, KBEt3, LiB[CH(CH3)C2H5]3,
KB[CH(CH3)C2H5]3, Al[CH(CH3)C2H5]2 or the like; Et
10 represents an ethyl group.)

In the step 3, the compound represented by the general
formula (V) is converted into an anion with a base, and
then reacted with the compound represented by the general
15 formula (VI) to produce the compound (VII). The base to
be used includes lithium diisopropylamide, lithium
(bistrimethylsilyl)amide, lithium tetramethylpiperazide,
34
sodium (bistrimethylsilyl)amide, potassium
(bistrimethylsilyl)amide, n-butyllithium,
sec-butyllithium, tert-butyllithium, etc. One alone or,
as the case may be, two or more of these may be used either
singly or as combined. Not specifically defined, th5 e
reaction solvent may be any one not significantly
interfering with the reaction, but is preferably
tetrahydrofuran, cyclopentyl methyl ether,
tetrahydrofuran, diethyl ether, tert-butyl methyl ether,
10 or their mixed solvent, etc.
The reaction temperature may be generally from -100
to 40 C and the reaction time is preferably from 1 to 12
hours. In this step, the compound (II) may be produced
depending on the chemical properties of the compound (VI),
15 which, however, causes no problem in consideration of the
intended production object.

In the step 4, the compound (VII) obtained in the
step 3 is reacted with the compound represented by the
20 general formula (VIII) to produce the compound represented
by the general formula (IX). The reaction solvent is, when
the compound (VIII) is sodium borohydride or lithium
borohydride, preferably methanol, ethanol, isopropanol,
tetrahydrofuran, cyclopentyl methyl ether,
25 dichloromethane, chloroform or their mixture, etc.; but
when the compound (VIII) is any other than those two, the
35
reaction solvent is preferably tetrahydrofuran,
cyclopentyl methyl ether, tetrahydropyran, diethyl ether,
tert-butyl methyl ether or their mixed solvent, etc. The
reaction temperature may be generally from -100 to 30 C
and the reaction time is preferably from 1 to 12 hours5 .
During or after the reaction of the step 4, the hydroxyl
group may be spontaneously eliminated from the formed
compound (IX) whereby the compound may be partly or wholly
converted into the compound (II). In the case of partial
10 conversion, the step 5 may be carried out without separating
the converted compound; and in the case of complete
conversion, the step 5 may be omitted.

In the step 5, the compound (IX) obtained in the step
15 4 may be dehydrated to produce the compound (II). The
dehydration reaction is attained by a combination of a
hydroxyl group activator and an organic base. The hydroxyl
group activator includes methanesulfonyl chloride,
p-toluenesulfonyl chloride, benzenesulfonyl chloride,
20 methanesulfonyl chloride, thionyl chloride, surfuryl
chloride, phosphorus pentachloride, etc. The organic base
includes triethylamine, diisopropylethylamine,
diazabicycloundecene, diazabicyclononene, pyridine,
dimethylaminopyridine, lutidine, collidine, etc.
25 Preferred is a combination of methanesulfonyl chloride and
triethylamine. As other dehydration reagents, there may
36
be mentioned triphenylphosphine-diethyl azodicarboxylate,
triphenylphosphine-diisopropyl azocarboxylate,
tri-n-butylphosphine-diethyl azodicarboxylate,
tri-n-butylphosphine-diisopropyl azocarboxylate, etc.
The reaction solvent may be any one not significantl5 y
interfering with the reaction, but is preferably chloroform,
methylene chloride, tetrahydrofuran, cyclopentyl methyl
ether, acetonitrile, 1,4-dioxane, dimethyl formamide, etc.
Not specifically defined, the reaction temperature may be
10 generally from 0 to 100 C, and the reaction time is
preferably from 1 to 24 hours.
Needless to say, depending on the properties of X,
Y and Z, it is necessary to previously use the corresponding
protective group in the reaction of the above-mentioned
15 step 3 to step 5 and to remove the protective group after
the reaction.
The compound (V) may be produced according to the
step 6 mentioned below.
37
[Chem. 15]
Scheme 3:
(In the formulae, Z had the same meaning as above; J1
represents a halogen atom, a methanesulfonyloxy group, 5 a
p-toluenesulfonyloxy group, a benzenesulfonyloxy group,
a trifluoromethanesulfonyloxy group or a hydroxyl group.)

In the step 6, the compound represented by the general
10 formula (X) or its salt is condensed with the compound
represented by the general formula (XI) in the presence
of an inorganic base to produce the compound (V). Preferred
inorganic bases include sodium hydroxide, potassium
hydroxide, lithium hydroxide, sodium carbonate, potassium
15 carbonate, cesium carbonate, calcium carbonate, etc.
However, when J1 is a hydroxyl group, the hydroxyl group
of the compound represented by the general formula (X) or
its salt is activated with a reagent and then the resulting
compound is condensed with the compound represented by the
20 general formula (XI) to produce the compound (V). As the
reagent suitable for activating the hydroxyl group, there
may be mentioned diethyl azodicarboxylate
38
(DEAD)-triphenylphosphine, diisopropyl
azodicarboxylate-triphenylphosphine, cyanomethylene
tributylphosphorane, cyanomethylene
trimethylphosphorane,
butyllithium-chlorodiphenylphosphine, etc. In case o5 f
activating the group with
butyllithium-chlorodiphenylphosphine, a quinone compound
such as 2,6-dimethyl-1,4-benzoquinone,
tetrafluoro-1,4-benzoquinone or the like is added to the
10 system.
Not specifically defined, the reaction solvent may
be any one not significantly interfering with the reaction,
but is preferably water, methanol, ethanol, tert-butanol,
tetrahydrofuran, cyclopentyl methyl ether, acetonitrile,
15 diethyl ether, dimethyl ether, dichloromethane,
1,4-dioxane, 2-methoxyethanol, N,N-dimethylformamide, or
their mixed solvent, etc. Not specifically defined, the
reaction temperature may be generally from -80 to 120 C,
and the reaction time is preferably from 1 to 24 hours.
20 The above-mentioned compound (II) may also be
produced through the reaction of the following step 7 to
step 11, as mentioned below.
[Chem. 16]
Scheme 4:
39
(In the formulae, X, Y, Z, E, M1 and J1 are the same as
mentioned above; and P1 represents a hydroxyl-protective
group.)

In the step 7, the compound represented by the general
formula (XII) is converted into an anion with a base, and
then reacted with the compound (VI) to produce the compound
(XIII), like in the step 3.
10
In the step 8, the compound represented by the general
formula (XIII) is reacted with the compound represented
by the general formula (VIII) to produce the compound (XIV),
like in the step 4.
15 Needless to say, depending on the properties of X
and Y, it is necessary to previously use the corresponding
protective group in the reaction of the above-mentioned
40
step 7 and step 8 and to remove the protective group after
the reaction.

In the step 9, the compound (XIV) is dehydrated to
produce the compound (XV), like in the step 55 .
In the step 9, the protective group P1 may be
spontaneously removed from the formed compound whereby the
compound may be partly or wholly converted into the compound
(XVI). In the case of partial conversion, the step 10 may
10 be carried out without separating the converted compound;
and in the case of complete conversion, the step 10 may
be omitted.

In the step 10, the compound represented by the general
15 formula (XV) is deprotected according to any known method
depending on the type of the protective group P1 therein,
thereby producing the compound represented by the general
formula (XVI).

20 In the step 11, the compound (XVI) is condensed with
the compound represented by the general formula (X) or its
salt to produce the compound (II), like in the step 6.
The N-hydroxyformamide derivative of the invention,
thus produced according to the above-mentioned method, may
25 be isolated and purified as a free compound thereof, or
as its salt, its hydrate or its various types of solvates
41
such as an ethanolate thereof, or as a polymorphic form
thereof. The pharmaceutically-acceptable salt of the
compound of the general formula (I) can be produced
according to conventional salt-forming reaction. The
isolation and purification may be attained by chemica5 l
operation of extractive fractionation, crystallization,
various types of fractionation chromatography, etc. An
optical isomer may be obtained as stereochemically pure
isomer by selecting suitable starting materials or by
10 optical resolution of racemic compounds.
The composition of the present invention is a
composition for maintaining a function of platelets, the
composition comprising, as an active ingredient, the
above-described compound or a salt thereof, or a solvate
15 thereof (hereinafter also referred to as "the compound
represented by the general formula (I) or the like"). In
the present invention, the phrase "maintaining a function
of platelets" mainly means maintaining a function related
to blood coagulation such as hemostasis and thrombus
20 formation by platelets. The composition of the present
invention is capable of achieving the function by
suppressing ADAM17-mediated GPIbα shedding (release by
cleavage).
"Maintaining a function of platelets" can be
25 evaluated, for example, by employing a method in which the
adhesion ability of platelets to VWF is evaluated using
42
a flow chamber system as described in International
Publication No. WO2009/119105 and Test Example 15 described
later, a method in which the in vivo lifespan of platelets
is evaluated using thrombocytopenic mouse, a method in
which the shape of platelets is observed on 5 a
fibrinogen-coated cover glass in the presence of thrombin,
a biomolecular imaging technique as described in Test
Example 16 later in which a thrombus formation model animal
is prepared using laser irradiation together with
10 hematoporphyrin and so forth to observe the kinetics of
platelets at an individual level in the model animal, or
other approaches.
ADAM17 (a disintegrin and metallopeptidase domain
17) is a protein having a structure containing a disintegrin
15 domain and has a metalloproteinase activity. ADAM17 is
a protein also called TACE (TNF-alpha converting enzyme)
because ADAM17 sheds, other than GPIbα, membrane-bound
TNF-α (tumor necrosis factor-alpha) to produce free TNF-α.
Typically, human-derived ADAM17 is a protein (gene)
20 specified under ACCESSION No. NP_003174.3 (No.
NM_003183.4).
GPIbα (glycoprotein Ib alpha) is a membrane protein
of platelets, and functions as a receptor for von Willebrand
factor (VWF). In addition, since GPIbα is expressed only
25 on platelets in a human body, GPIbα is also called CD42b
antigen and used as a surface marker for platelets.
43
Typically, human-derived GPIbα is a protein (gene)
specified under ACCESSION No. NP_000164.5 (No.
NM_000173.5).
Although reference sequences registered in GenBank
are exemplified as typical examples of ADAM17 and GPIbα5 ,
the amino acid sequences of the proteins may be mutated
naturally (i.e., non-artificially). Thus, it should be
understood that ADAM17 and GPIbα involved in the action
mechanism of the composition of the present invention
10 include such naturally-occurring mutants.
In the present invention, the phrase "suppressing
ADAM17-mediated GPIbα shedding" includes both complete
suppression (inhibition) and partial suppression of
shedding by ADAM17. Further, as described in Examples
15 later, it is possible to evaluate the composition of the
present invention as suppressing ADAM17-mediated GPIbα
shedding when a percentage of the number of CD42b (GPIbα)(+)
platelets to a total number of platelets in the presence
of the composition of the present invention is calculated
20 to be a large value in comparison with a percentage obtained
in the absence of the composition of the present invention.
As the degree of GPIbα shedding suppressed by the
composition of the present invention, the percentage of
the number of CD42b(+) platelets in the presence of the
25 composition of the present invention is preferably 80% or
higher, and a higher percentage is more preferable (for
44
example, 85% or higher, 90% or higher, 95% or higher).
Note that when an ability of the composition of the
present invention to suppress GPIbα shedding is evaluated
using a method described in Test Example 3 later, the 50%
inhibitory concentration (IC50) is preferably 0.01 nmol/5 L
to 100 nmol/L.
Examples of the compound represented by the general
formula (I) as an active ingredient of the composition of
the present invention include
10 N-[2-(4-but-2-ynyloxybenzenesulfonyl)-1-(4-diethylamin
omethylphenyl)ethyl]-N-hydroxyformamide,
N-[2-(4-but-2-ynyloxybenzenesulfonyl)-1-(4-dimet
hylaminomethylphenyl)ethyl]-N-hydroxyformamide,
N-{4-[2-(4-but-2-ynyloxybenzenesulfonyl)-1-(form
15 ylhydroxyamino)ethyl]benzyl}-2-methoxyacetamide,
N-{4-[2-(4-but-2-ynyloxybenzenesulfonyl)-1-(form
ylhydroxyamino)ethyl]benzyl}methanesulfonamide,
N-{4-[2-(4-but-2-ynyloxybenzenesulfonyl)-1-(form
ylhydroxy-amino)ethyl]benzyl}benzamide,
20 N-[2-(4-but-2-ynyloxybenzenesulfonyl)-1-(4-morph
olin-4-ylmethylphenyl)ethyl]-N-hydroxyformamide,
N-hydroxy-N-[1-(4-morpholin-4-ylmethylphenyl)-2-
(4-pent-2-ynyloxybenzenesulfonyl)ethyl]formamide,
N-[2-(4-but-2-ynyloxybenzenesulfonyl)-1-(4-dimet
25 hylaminophenyl)ethyl]-N-hydroxyformamide,
N-[2-(4-but-2-ynyloxybenzenesulfonyl)-1-(3-dimet
45
hylaminophenyl)ethyl]-N-hydroxyformamide,
N-[2-(4-but-2-ynyloxybenzenesulfonyl)-1-(2-dimet
hylaminophenyl)ethyl]-N-hydroxyformamide,
N-[2-(4-but-2-ynyloxybenzenesulfonyl)-1-(4-piper
idin-1-ylmethylphenyl)ethyl]-N-hydroxyformamide5 ,
N-[2-(4-but-2-ynyloxybenzenesulfonyl)-1-(3-piper
idin-1-ylmethylphenyl)ethyl]hydroxyformamide,
N-[2-(4-but-2-ynyloxybenzenesulfonyl)-1-(3-morph
olin-4-ylmethylphenyl)ethyl]-N-hydroxyformamide,
10 N-[2-(4-but-2-ynyloxybenzenesulfonyl)-1-{4-[(eth
ylmethylamino)methyl]phenyl]ethyl}-N-hydroxyformamide,
N-(2-(4-but-2-ynyloxybenzenesulfonyl)-1-{3-[(eth
ylmethylamino)methyl]phenyl}ethyl)-N-hydroxyformamide,
N-{4-[2-(4-but-2-ynyloxybenzenesulfonyl)-1-(form
15 ylhydroxyamino)ethyl]benzyl}-N-methylmethanesulfonamid
e,
N-{4-[2-(4-but-2-ynyloxybenzenesulfonyl)-1-(form
ylhydroxyamino)ethyl]benzyl}-4-methylbenzenesulfonamid
e,
20 N-{4-[2-(4-but-2-ynyloxybenzenesulfonyl)-1-(form
ylhydroxyamino)ethyl]benzyl}-4,N-dimethylbenzenesulfon
amide,
N-{4-[2-(4-but-2-ynyloxybenzenesulfonyl)-1-(form
ylhydroxyamino)ethyl]benzyl}-N-methylsulfonylmethanesu
25 lfonamide,
N-{2-(4-but-2-ynyloxybenzenesulfonyl)-1-[4-(2-di
46
methylaminoethyl)phenyl]ethyl}-N-hydroxyformamide,
N-{2-(4-but-2-ynyloxybenzenesulfonyl)-1-[4-(2-mo
rpholin-4-ylethyl)phenyl]ethyl}-N-hydroxyformamide,
N-(2-{4-[2-(4-but-2-ynyloxybenzenesulfonyl)-1-(f
ormylhydroxyamino)ethyl]phenyl}ethyl)methanesulfonami5 d
e,
N-{2-(4-but-2-ynyloxybenzenesulfonyl)-1-[4-(3-di
methylaminopropyl)phenyl]ethyl}-N-hydroxyformamide,
N-{2-(4-but-2-ynyloxybenzenesulfonyl)-1-[4-(3-di
10 ethylaminopropyl)phenyl]ethyl}-N-hydroxyformamide,
N-{2-(4-but-2-ynyloxybenzenesulfonyl)-1-[4-(3-mo
rpholin-4-ylpropyl)phenyl]ethyl}-N-hydroxyformamide,
N-{2-(4-but-2-ynyloxybenzenesulfonyl)-1-[4-(4-mo
rpholin-4-ylbutyl)-phenyl]-ethyl}-N-hydroxyformamide,
15 N-{4-[1-(formylhydroxyamino)-2-(4-pent-2-ynyloxy
benzenesulfonyl)ethyl]benzyl}methanesulfonamide, and
N-{4-[1-(formylhydroxyamino)-2-(4-oct-2-ynyloxyb
enzenesulfonyl)ethyl]benzyl}methanesulfonamide. As
described in Test Example 4 later, from the viewpoint of
20 having a high specificity to ADAM17,
N-[2-(4-but-2-ynyloxybenzenesulfonyl)-1-(4-diethylamin
omethylphenyl)ethyl]-N-hydroxyformamide or
N-{4-[2-(4-but-2-ynyloxybenzenesulfonyl)-1-(formylhydr
oxyamino)ethyl]benzyl}methanesulfonamide is preferable.
25 Note that
N-[2-(4-but-2-ynyloxybenzenesulfonyl)-1-(4-diethylamin
47
omethylphenyl)ethyl]-N-hydroxyformamide is "I-1" or
"S-45282" in Examples described later, and
N-{4-[2-(4-but-2-ynyloxybenzenesulfonyl)-1-(formylhydr
oxyamino)ethyl]benzyl}methanesulfonamide is "I-4" or
"S-45457" in Examples described later5 .
Examples of the form of the composition of the present
invention include a drug additive and a reagent used for
purposes of research and development (for example, in vitro
and in vivo research and development).
10 The composition of the present invention has an action
of maintaining a function of platelets by suppressing
ADAM17-mediated GPIbα shedding. Accordingly, the
composition of the present invention can be suitably used
as a reagent for maintaining a function of platelets (for
15 example, a reagent used in a culture system for
differentiating megakaryocytes from cells capable of
differentiating into megakaryocytes and then producing
platelets from the megakaryocytes, or a reagent for
maintaining a function of produced platelets by adding the
20 regent) or as a drug additive added to a blood product
comprising platelets from donation and the like. When used
as the above-described additive or reagents, the
composition of the present invention may comprise, for
example, a stabilizer, a solvent, or the like, in addition
25 to the compound represented by the general formula (I) or
the like as the active ingredient.
48
A product (for example, reagent, drug additive) of
the composition of the present invention or a protocol
thereof may be labelled to indicate that the use is to
maintain a function of platelets. Herein, the phrase "a
product or a protocol is labelled" means that the body o5 f
the product, a container or a package therefor, or the like
is labelled, or that a protocol, an attachment document,
an advertisement, other prints, or the like disclosing
information on the product is labelled. The label
10 indicating that the use is to maintain a function of
platelets may include information on a mechanism of how
the compound represented by the general formula (I) or the
like as the active ingredient of the composition of the
present invention demonstrates an effect of maintaining
15 a function of platelets. An example of the information
on the mechanism includes information that a function of
platelets is maintained by suppressing ADAM17-mediated
GPIbα shedding. Moreover, the label indicating that the
use is to maintain a function of platelets may include
20 information that the use is to produce or store platelets
or for other purposes.
The compound represented by the general formula (I)
or the like serves as an active ingredient for manufacturing
the composition of the present invention. Thus, the
25 present invention also provides: the use of the compound
represented by the general formula (I) or the like for
49
manufacturing the composition of the present invention;
and a method for manufactureing the composition of the
present invention comprising the compound represented by
the general formula (I) or the like.
Further, the present invention provides a cultur5 e
which is a culture system for differentiating
megakaryocytes from cells capable of differentiating into
megakaryocytes and for producing platelets from the
megakaryocytes, and is added to the system with the compound
10 represented by the general formula (I) or the like.
Furthermore, the present invention provides a method for
preparing platelets, wherein the method comprises adding,
to a culture system for differentiating megakaryocytes from
cells capable of differentiating into megakaryocytes and
15 producing platelets from the megakaryocytes, the compound
represented by the general formula (I) or the like.
In the present invention, "megakaryocytes" are cells
also called platelet progenitor cells or megakaryocytic
cells, and they divide the cytoplasm to produce platelets.
20 In the present invention, "cells capable of
differentiating into megakaryocytes" are not particularly
limited, as long as the cells are capable of differentiating
into megakaryocytes. Examples thereof include fertilized
eggs, pluripotent stem cells such as embryonic stem cells
25 (ES cells), induced pluripotent stem cells (iPS cells),
embryonic germ cells (EG cells), hematopoietic stem cells,
50
hematopoietic progenitor cells, megakaryoblasts, and
adipocytes. The hematopoietic stem cells and the
hematopoietic progenitor cells may be collected from bone
marrow, umbilical cord blood, or the like. Further, among
these, iPS cells are preferable from the viewpoints tha5 t
there is no ethical problem because an embryo is not
destroyed, and that it is easy to match the human leukocyte
antigen (HLA) type of a patient transfused with platelets
prepared according to the present invention. Moreover,
10 iPS cells established using 4 factors (Oct3/4, Sox2, Klf4
and c-Myc) are more preferable from the viewpoints that
megakaryocytes derived from the iPS cells can continue to
increase until Days 25 to 35 after culturing of the iPS
cells is started, and that the number of platelets produced
15 from the megakaryocytes is 2 to 10 times larger than the
number of platelets from iPS cells established using 3
factors (Oct3/4, Sox2 and Klf4). Note that the present
inventors have confirmed that such increase in the number
of megakaryocytes and platelets produced are caused by
20 re-activation of the c-Myc gene.
Further, an animal species from which the
above-described cells used in the method of the present
invention are derived is not particularly limited.
Examples thereof include human, mice, rats, dogs, cats,
25 cattle, horses, sheep, and the like. Preferable are mice,
rats, and human, more preferable are mice and human, and
51
particularly preferable is human.
Examples of the "culture system for differentiating
megakaryocytes from cells capable of differentiating into
megakaryocytes and producing platelets from the
megakaryocytes" in the present invention include a cultur5 e
system for forming embryoid bodies (cell population
containing differentiation-induced, undifferentiated
mesodermal cells) in the middle of platelet production (Eto
et al., Proc. Natl. Acad. Sci. USA, 2002, vol. 99, pp.
10 12819-12824, and so forth), a culture system for forming
a sac-like structure (sac structure) enclosing
hematopoietic progenitor cells (see PTLs 1 to 3 and so forth),
a culture system for producing platelets from umbilical
cord blood-derived hematopoietic progenitor cells (see
15 Robert et al., "Glycoprotein Ibalpha receptor instability
is associated with loss of quality of platelets produced
in culture.", Stem Cells and Development, published online
on May 26, 2010, and so forth), and the like (for others,
see Fujimoto et al., Blood, 2003, vol. 102, pp. 4044-4051:
20 Hiroyama et al., Exp. Hematol., 2006, vol. 34, pp. 760-769:
Gaur et al., J Thromb Haemost., 2005, vol. 4, pp. 436-442;
and so forth). Among these, a culture system for forming
a sac-like structure is preferable because hematopoietic
progenitor cells are condensed in the sac-like structure,
25 enabling efficient ex vivo production of megakaryocytes,
platelets, and the like.
52
Culture conditions of the culture system according
to the present invention are not particularly limited, as
long as the conditions are suitable for culturing of
megakaryocytes and platelet production. The culture
temperature is preferably 35 to 38ºC, more preferably 37ºC5 .
If the culture temperature is below the lower limit, the
numbers of megakaryocytes and platelets produced tend to
be small. If the culture temperature exceeds the upper
limit, cell culturing and maintenance tend to be difficult.
10 Furthermore, when the "cells capable of
differentiating into megakaryocytes" according to the
present invention are cultured (particularly, in the
process of inducing differentiation to megakaryocytes),
the cells are preferably co-cultured with feeder cells.
15 The "feeder cells" used herein are not particularly limited,
as long as the feeder cells contribute to the
differentiation induction of the "cells capable of
differentiating into megakaryocytes." For example, it is
possible to use mouse embryonic fibroblasts, preferably
20 a 10T1/2 cell line, OP9 cells, and the like. An example
of the cells other than immortalized cell lines includes
human bone marrow-derived mesenchymal stem cells (cultured
cells directly prepared from human bone marrow). Even when
the mesenchymal stem cells are used as the feeder cells,
25 megakaryocyte maturation and platelet production from
human ES cells or iPS cells are possible. Platelets can
53
be induced by culturing on an extracellular substrate such
as Matrigel also, but the efficiency is low. When the
"feeder cells" are used, it is preferable to suppress the
growth of cells through radiation exposure or the like.
An amount (concentration) of the compound represente5 d
by the general formula (I) or the like added to the culture
system is preferably 0.01 to 100 μM. If the amount added
is below the lower limit, it tends to be difficult to suppress
the GPIbα cleavage on platelets. If the amount exceeds
10 the upper limit, cell growth tends to be suppressed.
Additionally, the adding period of the compound represented
by the general formula (I) or the like to the culture system
is preferably throughout the entire production period from
hematopoietic progenitor cells to platelets, from the
15 viewpoints of ensuring uniformity during the culture
process and ensuring product uniformity, regardless of
non-uniform situation for blood cell differentiation.
This is greatly different from adding period of GM6001 which
is limited to a production period from megakaryocytes to
20 platelets.
Hereinafter, the method for preparing platelets of
the present invention will be more specifically described
by taking an example of a method for forming a sac-like
structure suitably used for the culture system of the
25 present invention.
First, description will be given of the method for
54
forming a sac-like structure (sac structure). Culture
conditions suitable for preparing a sac-like structure vary
depending on ES cells or iPS cells used. However, for
example, Iscove's Modified Dulbecco's Medium (IMDM)
supplemented with FBS to a final concentration of 15% i5 s
used as a medium. Meanwhile, even in a case of a serum-free
medium, a growth factor, a supplement, or the like may be
added thereto as appropriate for use. Further, in order
to form a sac-like structure efficiently, a vascular
10 endothelial growth factor (VEGF) should be added at
approximately 0 to 100 ng/ml, 0 to 300 ng/ml, more preferably
approximately 20 ng/ml, 200 ng/ml. The culture
environment varies depending on the type of ES cells or
iPS cells used. However, the conditions are preferably
15 5% CO2 at 35 to 38ºC, more preferably 37ºC. The culturing
period until a sac-like structure is formed varies
depending on the type of ES cells or iPS cells, but the
presence can be observed on approximately Day 15 (14 to
16 days later) after seeding on feeder cells.
20 The formed sac-like structure has a follicular
structure. In the structure, hematopoietic progenitor
cells, particularly CD34-positive cells, are present in
a concentrated state. The hematopoietic progenitor cells
present inside the sac-like structure can be separated by
25 physical means, for example, by passing the cells through
55
a sterilized sieve-like tool (for example, cell strainer
or the like).
Next, description will be given of a method for
preparing platelets from the hematopoietic progenitor
cells separated from the sac-like structure. Th5 e
hematopoietic progenitor cells obtained by the separation
are seeded on feeder cells, and cultured under conditions
suitable for producing megakaryocytes and platelets.
Herein, examples of the "conditions suitable for producing
10 megakaryocytes and platelets" include culturing for
approximately 7 to 15 days in the presence of thrombopoietin
(TPO, approximately 10 to 200 ng/mL, preferably 100 ng/mL),
or in the presence of a stem cell factor (SCF, approximately
10 to 200 ng/mL, preferably 50 ng/mL), heparin
15 (approximately 10 to 100 U/mL, preferably 25 U/ml), and
TPO (approximately 10 to 200 ng/mL, preferably 100 ng/mL).
As the culture environment, the conditions are preferably
5% CO2 at 35 to 38ºC, more preferably 37ºC.
Moreover, in such a culture system, the timing of
20 adding the compound represented by the general formula (I)
or the like to this culture system is preferably when the
hematopoietic progenitor cells are reseeded on the feeder
cells. It is more preferable to add the compound
represented by the general formula (I) or the like on
25 approximately Day 22 after the culturing is started (Days
20 to 23, or Days 6 to 10 after the sac-like structure is
56
reseeded).
Furthermore, in the method of the present invention,
platelets can be prepared by: collecting a culture solution
fraction (for example, in the method for forming a sac-like
structure, a fraction present on approximately Days 22 t5 o
28 after human iPS cells or ES cells are cultured) in which
platelets released from megakaryocytes are abundant; and
then removing components other than platelets (i.e.,
megakaryocytes and other blood cells) using a leukocyte
10 reduction filter (available from, for example, Terumo
Corporation, Asahi Kasei Medical Co., Ltd., and so forth)
or the like.
As described above, the compound represented by the
general formula (I) or the like suppresses ADAM17-mediated
15 GPIbα shedding and has an action of maintaining a function
of platelets. Thus, the present invention also provides
a blood product comprising platelets and the compound
represented by the general formula (I) or the like; it also
provides a method for maintaining a function of platelets
20 in a blood product, wherein the method comprises adding,
to a blood product comprising platelets, the compound
represented by the general formula (I) or the like.
Further, the compound represented by the general
formula (I) or the like serves as an active ingredient for
25 producing a blood product comprising platelets. Thus, the
present invention also provides the compound represented
57
by the general formula (I) or the like as a drug additive
used to produce the blood product of the present invention.
The platelets comprised in the blood product of the
present invention is not particularly limited, and may be
platelets obtained by the above-described method fo5 r
preparing platelets of the present invention or may be
platelets derived from peripheral blood or the like
obtained by collecting blood. When such a blood product
is prepared, the blood product may also comprise other
10 ingredients used to stabilize platelets by taking the
storage instability of platelets and the like into
consideration. Conditions for stabilizing platelets can
be selected from among methods well known to those skilled
in the art. For example, the product can be prepared by
15 suspending platelets in a solution necessary to keep a
function of platelets (for example, an ACD-A solution (a
solution prepared from sodium citrate/citric
acid/glucose) and the like; in some cases, frozen plasma
or the like may be added as appropriate) at an appropriate
concentration (for example, approximately 1 × 108 20 to 1 ×
1010 platelets/mL, preferably approximately 1 × 109
platelets/mL). Note that, as a container for storing the
product comprising platelets, it is preferable to avoid
using a material that activates platelets, such as glass.
25 An amount (concentration) added of the compound represented
by the general formula (I) or the like is preferably 0.01
58
to 100 μmol/L. If the amount added is below the lower limit,
platelets tend not to keep the adhesion function. Even if
the amount exceeds the upper limit, the effects are at a
plateau.
[Examples5 ]
Hereinafter, the present invention will be described
more specifically based on Examples and Test Examples.
However, the present invention is not limited to the
following Examples. Note that megakaryocytes and platelets
10 used in Test Examples described later were produced and
analyzed by employing methods described below.

As human ES cells (hES cells), a KhES cell line (KhES-3)
15 established by and provided from Institute for Frontier
Medical Sciences, Kyoto University was used.
As human iPS cells (hiPS cells), an iPS cell line (TkDA3-4)
established at the University of Tokyo by introducing Oct4,
Klf4, Sox2, and c-Myc into human-derived skin cells was
20 also used.with 0.1 mM non-essential amino acid
(manufactured by Invitrogen Corp.)Further, the TkDA3-4
cells were cultured on radiation-exposed mouse fibroblasts
in a medium mixture of Dulbecco's modified Eagle's medium
and Ham's F-12 medium (manufactured by Sigma-Aldrich Co.)
25 (mixing ratio 1:1) supplemented with 0.1 mM non-essential
amino acid (manufactured by Invitrogen Corp.), 2 mM
59
L-glutamine (manufactured by Invitrogen Corp.), 20%
knockout serum replacement additive (KSR, manufactured by
Invitrogen Corp.), 0.1 mM 2-mercaptoethanol, and 5 ng/ml
basic fibroblast growth factor (bFGF, manufactured by
Upstate). The cells were subcultured for every 3 days5 ,
and those keeping an undifferentiated state were used.
In addition, mouse embryo-derived fibroblasts, a
C3H10T1/2 cell line (hereinafter also referred to as
"10T1/2 cells"), purchased from RIKEN BioResource Center
10 were cultured in basal medium Eagle (BME, manufactured by
Invitrogen Corp.) supplemented with 10% fetal bovine serum
(FBS) and 2 mM L-glutamine. Then, the 10T1/2 cells were
adjusted to a cell count of 8 × 106/10-cm dish, and exposed
to 50-Gy radiation for use as feeder cells.
15
The human ES cells or the human iPS cells were seeded
on the feeder cells such that the cell count was 5 × 104
to 1 × 105/10-cm dish. The cells were cultured in Iscove's
20 Modified Dulbecco's Medium (IMDM, manufactured by
Invitrogen Corp/GIBCO) supplemented with 15% FBS (product
name: CELLect(TM) GOLD, manufactured by ICN Biomedicals
Inc.), 2 mM L-glutamine (manufactured by Invitrogen Corp.),
ITS supplement (10 μg/ml insulin, 5.5 mg/ml human
25 transferrin, 5 ng/ml sodium selenite) (manufactured by
Sigma-Aldrich Co.), 50 μg/ml ascorbic acid (Sigma-Aldrich
60
Co.), 0.45 mM α-monothioglycerol (MTG, manufactured by
Sigma-Aldrich Co.), and 20 ng/ml vascular endothelial
growth factor (VEGF, manufactured by R&D Systems, Inc.).
Further, this culturing was carried out at 37ºC in 5% CO2
and 21% O2 (atmospheric oxygen) using a CO2 incubato5 r
(product name: HERA CELL 150i, manufactured by Thermo
Fisher Scientific K. K.). Then, the medium was replaced
every 3 days, and the culturing was continued under the
conditions until Day 15 when a large number of sac structures
10 (sac-like structures) containing hematopoietic progenitor
cells therein were observed.
Next, after detached from the dish, the sac structures
were disrupted and suspended in a 3% FBS-containing
phosphate buffer solution (PBS). Then, the suspension was
15 passed through a 40-μm cell strainer (manufactured by BD).
The solution thus passed through was centrifuged at 440
× g for 10 minutes to collect hematopoietic progenitor cells,
and the cells was counted. The hematopoietic progenitor
cells were seeded on 10T1/2 cells newly prepared in a 6-well
plate (6 × 105 20 cells/plate) exposed to 50-Gy radiation such
that the number of the hematopoietic progenitor cells was
2 to 3 × 104/well. The hematopoietic progenitor cells were
further cultured for 8 days in IMDM (manufactured by
Invitrogen Corp/GIBCO) supplemented with 15% FBS (product
25 name: CELLect(TM) GOLD, manufactured by ICN Biomedicals
Inc.), 2 mM L-glutamine (manufactured by Invitrogen Corp.),
61
ITS supplement (10 μg/ml insulin, 5.5 mg/ml transferrin,
5 ng/ml sodium selenite) (manufactured by Sigma-Aldrich
Co.), 50 μg/ml ascorbic acid (manufactured by Sigma-Aldrich
Co.), 0.45 mM MTG (manufactured by Sigma-Aldrich Co.), 100
ng/ml human thrombopoietin (human TPO, manufactured b5 y
PeproTech, Inc.), 50 ng/ml human stem cell factor (SCF,
manufactured by PeproTech, Inc.), and 25 U/ml heparin.
Thereby, megakaryocytes/platelets were induced.
Subsequently, the induced megakaryocytes/platelets
10 were collected with an anticoagulant solution (Acid Citrate
Dextrose, ACD), followed by centrifugation at 900 rpm for
10 minutes without a break. The supernatant obtained by
the centrifugation was further centrifuged at 1500 rpm for
10 minutes without a break to remove the supernatant.
15 Thereafter, the numbers of megakaryocytes and platelets
in the precipitate thus obtained were analyzed by flow
cytometry using antibodies for staining.
Note that, in the flow cytometry, FACS Aria
manufactured by Becton, Dickinson and Company was used.
20 As the antibodies used to stain the megakaryocytes and the
platelets, used were a Phycoerythrin(PE)-modified
anti-human CD42a (GPIX) antibody, a PE-modified anti-human
CD42b (GPIbα) antibody, and an allophycocyanin
(APC)-labelled anti-CD41a (integrin αIIbbeta3 complex,
25 HIP8 clone) antibody (manufactured by BD Bioscience).
Additionally, in order to accurately measure the absolute
62
numbers of the megakaryocytes and the platelets, the cells
were stained with these antibodies, and the analysis was
conducted by flow cytometry using TruCount beads
(manufactured by BD Bioscience), also.
(Test Example 1) Induction to Megakaryocytes/Platelet5 s
from ES Cell-Derived Hematopoietic Progenitor Cells under
Room Temperature Condition
In order to examine whether or not it is possible
to induce megakaryocytes/platelets by culturing under room
10 temperature condition (culturing at 25ºC), the induction
of megakaryocytes/platelets from the ES cell–derived
hematopoietic progenitor cells obtained as described above
was attempted under conditions illustrated in Fig. 1.
Specifically, in the above-described 8-day culture process
15 for inducing hematopoietic progenitor cells to
megakaryocytes/platelets, each culturing was conducted
under the following conditions:
Control: culturing at 37ºC for 8 days
Room-temperature culturing for 2 days: culturing at 37ºC
20 for 6 days, followed by culturing at 25ºC for 2 days, and
Room-temperature culturing: culturing at 25ºC for 8 days.
Then, the numbers of megakaryocytes and platelets collected
as described above were counted by flow cytometry. Figs.
2 and 3 show the obtained results.
25 As apparent from the results shown in Figs. 2 and
3, megakaryocytes and platelets were hardly obtained by
63
the culturing at 25ºC for 8 days. Additionally, by the
culturing at 25ºC for 2 days before the flow cytometry
analysis, megakaryocytes and platelets were obtained, but
the amounts were only approximately half of those of the
control5 .
(Test Example 2) Induction to Megakaryocytes/Platelets
from Umbilical Cord Blood-Derived CD34(+) Cells under Room
Temperature Condition
Whether or not it is possible to induce platelets
10 from umbilical cord blood–derived CD34(+) cells by
culturing under room temperature condition (culturing at
24 to 25ºC) was examined.
First, 106 CD45low CD34+ cells were selected and
isolated from umbilical cord blood (CB) using a bead column.
15 Of the obtained umbilical cord blood-derived CD34(+) cells,
4 × 104 cells were seeded on the feeder cells described
above and cultured for 4 days in a medium A (IMDM
(manufactured by Invitrogen Corp/GIBCO) supplemented with
15% FBS (product name: CELLect(TM) GOLD, manufactured by
20 ICN Biomedicals Inc.), 2 mM L-glutamine (manufactured by
Invitrogen Corp.), ITS supplement (10 μg/ml insulin, 5.5
mg/ml transferrin, 5 ng/ml sodium selenite) (manufactured
by Sigma-Aldrich Co.), 50 μg/ml ascorbic acid (manufactured
by Sigma-Aldrich Co.), 0.45 mM MTG (manufactured by
25 Sigma-Aldrich Co.), and a cytokine cocktail (2 ng/ml TPO
(manufactured by PeproTech, Inc.), 7.5 ng/ml SCF
64
(manufactured by PeproTech, Inc.), 11 ng/ml FLT-3)). On
Day 4 after the culturing, to the umbilical cord
blood-derived CD34(+) cells, a medium B was added in the
same amount as that of the medium A. Note that the medium
B is different from the medium A in that the medium B wa5 s
supplemented with 30 ng/ml TPO, 1 ng/ml SCF, 7.5 ng/ml IL-6,
and 13.5 ng/ml IL-9 as a cytokine cocktail. Meanwhile,
the medium A and the medium B have the same composition,
except that the supplemented cytokine cocktails are
10 different.
Then, the umbilical cord blood-derived CD34(+) cells
on Day 7 thus cultured were adjusted to a cell count of
4 × 105/mL in the medium B. The cells were further cultured
for 7 days at 37ºC (control), or further cultured for 7
15 days at 24 to 25ºC (room-temperature culturing). On Day
14 after the culturing, the numbers of megakaryocytes and
platelets were counted by flow cytometry. Figs. 4 and 5
show the obtained results.
As apparent from the results shown in Figs. 4 and
20 5, megakaryocytes and platelets were hardly obtained by
the culturing at approximately 25ºC for 7 days as in the
case of the ES cell-derived hematopoietic progenitor cells
described above. Thus, the results shown in Test Examples
1 and 2 reveal that, in a culture system for differentiating
25 megakaryocytes from cells capable of differentiating into
megakaryocytes, such as ES cells and umbilical cord
65
blood-derived CD34(+) cells, to produce platelets from the
megakaryocytes, the culture temperature is preferably
around 37ºC, while megakaryocytes and platelets cannot be
produced by culturing under room temperature condition of
around 25º5 C.
However, if the culture temperature is around 37ºC
in the culture system for producing platelets, ADAM17 sheds
GPIbα, which is an important factor for blood coagulation
located on the surface of platelets. This brings about
10 a problem that it is hard to obtain platelets not
demonstrating a hemostatic function. Meanwhile,
inhibitors (for example, GM6001) targeting
metalloproteinases including ADAM17 may be added to a
culture system in order to suppress GPIbα shedding. In
15 such a case, however, metalloproteinases essential for
hematopoietic function (MMP-9, MMP-14) other than ADAM17
are also inhibited. Hence, such inhibitors need to be added
only at a certain period when platelet production is
observed most abundantly, and are not preferable in
20 establishing a plant or the like for a culture system
producing platelets. Furthermore, there is a concern
about safety when non-specific metalloproteinase
inhibitors such as GM6001 are administered in vivo. Thus,
it is not preferable to use platelets mixed with such
25 inhibitors as a blood product, either.
For these reasons, compounds specifically inhibiting
66
ADAM17 described in Examples below were designed and
synthesized. Note that 1H-NMR spectra described below were
measured with an ECA400 spectrometer (400 MHz, manufactured
by JEOL Ltd.) using deuterated chloroform (CDCl3) or
deuterated dimethyl sulfoxide (DMSO-d6) as a solvent, an5 d
tetramethylsilane (TMS) as the internal standard. In the
measurement result of chemical shifts, δ values were
expressed in ppm, and coupling constant J values were
expressed in Hz. The abbreviation s means singlet; d,
10 doublet; t, triplet; q, quartet; dd, doublet doublet; m,
multiplet; and br, broad. For low-resolution mass
spectrum (fast atom bombardment: FAB-MS) measurement, JEOL
Ltd. JMS-HX-110A model was used, and for mass spectrum
(electrospray ionization: ESI-MS) measurement, Exactive
15 manufactured by Thermo Fisher Scientific K. K. was used.
(Example 1)
N-[2-(4-But-2-ynyloxybenzenesulfonyl)-1-(4-dieht
ylaminomethylphenyl)ethyl]-N-hydroxyformamide (I-1)
67
[Chem. 17]
(1-1): 1-But-2-ynyloxy-4-methanesulfonylbenzene
(V-1)
2.88 g (15.9 mmol) of 4-methylsulfonylphenol wa5 s
added to and dissolved in a dimethylsulfoxide solution (30
mL) of 2.12 g (15.9 mmol) of 1-bromo-2-butyne, and then
2.64 g (19.1 mmol) of potassium carbonate was added thereto.
After stirred for 6 hours, brine was added thereto and
10 extracted with ethyl acetate. The organic layer was washed
with brine, and dried over anhydrous magnesium sulfate.
3.39 g (15.11 mmol) of
1-but-2-ynyloxy-4-methanesulfonylbenzene (V-1) was
obtained as a roughly-purified product (yield 95%). Its
15 physical properties are shown below.
MS (FAB) m/z: 225 (M+H)+.
1H-NMR(CDCl3): 7.88 (2H, m), 7.10 (2H, m), 4.73 (2H, m),
3.04 (3H, s), 1.87 (3H, t, J = 2.3 Hz).
(1-2): tert-Butyl
20 {4-[2-(4-but-2-ynyloxybenzenesulfonyl)acetyl]benzyl}ca
rbamate (VII-1)
In an argon atmosphere at -78 C, 3.65 mL (7.29 mmol)
68
of a hexane-heptane-ethylbenzene solution of 2.0 M lithium
diisopropylamide was added to a tetrahydrofuran (70 mL)
solution of 1.36 g (6.08 mmol) of the compound (V-1) obtained
in the above (1-1), stirred for 30 minutes, and then 12.16
mL (12.16 mmol) of a tetrahydrofuran solution of 1.0 5 M
lithium hexamethyldisilazide and 5 mL of a tetrahydrofuran
solution of 1.61 g (6.08 mmol) of methyl
4-(tert-butoxycarbonylaminomethyl)benzoate were added
thereto. After stirred at -78 C for 5 minutes, this was
10 gradually heated up to room temperature, and stirred for
1 hour. After brine was added thereto, this was extracted
with ethyl acetate, and the organic layer was washed with
brine. After dried over anhydrous magnesium sulfate, the
solvent was evaporated away under reduced pressure.
15 Purified by silica gel column chromatography (hexane/ethyl
acetate = 2/1 1/1), this gave 2.02 g (4.41 mmol) of
tert-butyl
{4-[2-(4-but-2-ynyloxybenzenesulfonyl)acetyl]benzyl}ca
rbamate (VII-1) as a colorless molten caramel-like
20 substance (yield 72%). Its physical properties are shown
below.
MS (FAB) m/z: 480 (M+Na)+.
1H-NMR (CDCl3): 7.93 (2H, br d, J = 8.2 Hz), 7.81 (2H,
m), 7.40 (2H, br d, J = 8.2 Hz), 7.07 (2H, m), 4.72 (2H,
25 m), 4.70 (2H, s), 4.39 (2H, m), 1.87 (3H, t, J = 2.3 Hz),
1.57 (9H, s).
69
(1-3): tert-Butyl
{4-[2-(4-but-2-ynyloxybenzenesulfonyl)-1-hydroxyethyl]
benzyl}carbamate (IX-1)
At 0 C, 167 mg (4.41 mmol) of sodium borohydride was
added to a methanol (50 mL) solution of 2.02 g (4.41 mmol5 )
of the compound (VII-1) obtained in the above (1-2). After
stirred for 2 hours and 30 minutes, brine and aqueous
saturated ammonium chloride solution were added thereto.
Methanol was evaporated away under reduced pressure, then
10 the residue was extracted with ethyl acetate, and the
organic layer was washed with brine, and dried over
anhydrous magnesium sulfate. The solvent was evaporated
away under reduced pressure to give 2.04 g (4.41 mmol) of
tert-butyl
15 {4-[2-(4-but-2-ynyloxybenzenesulfonyl)-1-hydroxyethyl]
benzyl}carbamate (IX-1), as a roughly-purified amorphous
solid substance (yield 99%). Its physical properties are
shown below.
MS (FAB) m/z: 482 (M+Na)+.
20 (1-4): tert-Butyl
{4-[2-(4-but-2-ynyloxybenzenesulfonyl)vinyl]benzyl}car
bamate (II-1a)
At 0 C, 0.7 mL (8.8 mmol) of methanesulfonyl chloride
was added to a dichloromethane (45 mL) solution of 2.04
25 g (4.41 mmol) of the compound (IX-1) obtained in the above
(1-3) and 3.1 mL (22.1 mmol) of triethylamine. After
70
stirred for 3 hours and 30 minutes, brine was added thereto,
and extracted with chloroform. The organic layer was
washed with brine, and dried over anhydrous magnesium
sulfate. The solvent was evaporated away under reduced
pressure, and the residue was purified by silica gel colum5 n
chromatography (chloroform/ethyl acetate = 3/1) to give
1.77 g (4.00 mmol) of tert-butyl
{4-[2-(4-but-2-ynyloxybenzenesulfonyl)vinyl]benzyl}car
bamate (II-1a) as a colorless amorphous solid (yield 91%).
10 Its physical properties are shown below.
1H-NMR (CDCl3): 7.87 (2H, d, J = 8.7 Hz), 7.61 (1H, d,
J = 15 Hz), 7.43 (2H, J = 8.2 Hz), 7.30 (2H, d, J = 8.2
Hz), 7.10 (2H, d, J = 8.7 Hz), 6.82 (1H, d, J = 15 Hz),
4.88 (1H, m), 4.71 (2H, m), 4.33 (2H, m), 1.86 (3H, m),
15 1.45 (9H, s).
(1-5):
4-[(E)-2-(4-But-2-ynyloxybenzenesulfonyl)vinyl]benzyla
mine hydrochloride (II-1b)
At 0 C, 2 mL of 4 M-hydrochloric acid/dioxane was added
20 to a methanol (5 mL) solution of 295 mg (0.67 mmol) of the
compound (II-1a) obtained in the above (1-4), stirred for
10 minutes, heated up to room temperature, and stirred for
2 hours. The solvent was evaporated away under reduced
pressure, then 20 mL of methanol was added thereto, and
25 the solvent was again evaporated away under reduced
pressure to give
71
4-[(E)-2-(4-but-2-ynyloxybenzenesulfonyl)vinyl]benzyla
mine hydrochloride (II-1b) as a colorless solid. Its
physical properties are shown below.
1H-NMR (DMSO-d6): 7.85 (2H, d, J = 8.7 Hz), 7.78 (2H,
d, J = 8.2 Hz), 7.52 (2H, d, J = 8.2 Hz), 7.20 (2H, d, 5 J
= 8.7 Hz), 4.87 (2H, m), 4.05 (2H, m), 1.83 (3H, m).
(1-6):
{4-[(E)-2-(4-But-2-ynyloxybenzenesulfonyl)vinyl]benzyl
}diethylamine (II-1c)
10 At 0 C, 1 mL of acetaldehyde, 212 mg (1.00 mmol) of
sodium triacetoxyhydroborate and 3 drops of acetic acid
were added to a methanol (6 mL) solution of the compound
(II-1b) obtained in the above (1-5), and then stirred at
room temperature for 1 hour and 30 minutes. After brine
15 and saturated aqueous sodium bicarbonate solution were
added thereto, and the solvent was evaporated away under
reduced pressure. This was extracted with chloroform, the
organic layer was washed with brine and dried over anhydrous
magnesium sulfate. The solvent was evaporated away under
20 reduced pressure, and the residue was purified by silica
gel column chromatography (chloroform/methanol = 20/1
10/1) to give 111.7 mg (0.28 mmol) of
{4-[(E)-2-(4-but-2-ynyloxybenzenesulfonyl)vinyl]benzyl
}diethylamine (II-1c) as a pale yellow amorphous solid (two
25 steps yield 42%). Its physical properties are shown below.
MS (FAB) m/z: 398 (M+H)+.
72
(1-7):
N-[2-(4-But-2-ynyloxybenzenesulfonyl)-1-(4-diethylamin
omethylphenyl)ethyl]hydroxylamine (III-1)
50% hydroxylamine solution (3 mL) was added to a
tetrahydrofuran (8 mL) solution of 108 mg (0.27 mmol) o5 f
the compound (II-1c) obtained in the above (1-6), and
stirred at room temperature for 25 hours. The reaction
solution was evaporated under reduced pressure, and then
water was added thereto and extracted with chloroform. The
10 organic layer was washed with brine, dried over anhydrous
magnesium sulfate, and concentrated under reduced pressure.
92.9 mg (0.22 mmol) of
N-[2-(4-but-2-ynyloxybenzenesulfonyl)-1-(4-diethylamin
omethylphenyl)ethyl]hydroxylamine (III-1) was obtained as
15 a pale yellow amorphous solid (yield 81%). Its physical
properties are shown below.
1H-NMR (CDCl3): 7.84 (2H, m), 7.27 (2H, m), 7.20 (2H,
m), 7.07 (2H, m), 4.72 (2H, m), 3.75 (1H, m), 3.51 (2H,
s), 3.33 (1H, m), 2.49 (4H, m), 1.87 (3H, s), 1.03 (6H,
20 m).
(1-8):
N-[2-(4-But-2-ynyloxybenzenesulfonyl)-1-(4-diethylamin
omethylphenyl)ethyl]-N-hydroxyformamide (I-1)
1 mL of formic acid was cooled at 0 C, then 0.3 mL
25 of acetic anhydride was dropwise added thereto and stirred
for 30 minutes to prepare a formic acid/acetic acid mixed
73
acid anhydride solution. At 0 C, 0.6 ml of the formic
acid/acetic acid mixed acid anhydride solution prepared
previously was added to a tetrahydrofuran (3 mL) solution
of 92 mg (0.21 mmol) of the compound (III-1) obtained in
the above (1-7), and stirred at room temperature for 4 hours5 .
The reaction mixture was concentrated under reduced
pressure, and then azeotroped with toluene. The obtained
oily substance was dissolved in 2 mL of chloroform and 10
mL of methanol, and stirred for 12 hours. The solution
10 was concentrated under reduced pressure, and the resulting
oily substance was dissolved in chloroform and neutralized
with saturated aqueous sodium bicarbonate solution added
thereto. After extracted with chloroform, the extract was
washed with brine and dried over anhydrous magnesium
15 sulfate. Purifying by middle-pressure silica gel column
chromatography (chloroform/methanol = 95/5 75/25) gave
53.9 mg (0.11 mmol) of
N-[2-(4-but-2-ynyloxybenzenesulfonyl)-1-(4-diethylamin
omethylphenyl)ethyl]-N-hydroxyformamide (I-1) as a pale
20 yellow amorphous solid (yield 52%). Its physical
properties are shown below.
MS (FAB) m/z: 459(M+H)+.
1H-NMR (CDCl3): 8.32 (0.6H, s), 8.10 (0.4H, s), 7.80-7.86
(2H, m), 7.21-7.30 (4H, m), 7.06-7.14 (2H, m), 5.65 (0.6H,
25 m), 5.36 (0.4H, m), 4.74 (2H, br s), 4.20 (0.4H, m), 4.05
(0.6H, br t, J = 13 Hz), 3.48-3.57 (3H, m), 2.46 (4H, m),
74
1.87 (3H, br s), 0.99 (6H, m).
(Example 2)
N-[2-(4-But-2-ynyloxybenzenesulfonyl)-1-(4-dimet
hylaminomethylphenyl)ethyl]-N-hydroxyformamide (I-2)
[Chem. 185 ]
According to the same operation as in Example 1, the
above-mentioned compound (I-2) was obtained.
MS (FAB) m/z : 431 (M+H)+.
110 H-NMR (CDCl3): 8.28 (0.6H, s), 8.14 (0.4H, s), 7.79-7.91
(2H, m), 7.04-7.32 (6H, m), 5.69 (0.6H, m), 5.35 (0.4H,
m), 4.74 (2H, br s), 4.18 (0.4H, m), 4.07 (0.6H, m), 3.47
(1H, m), 3.33 (2H, m), 2.11 (6H, s), 1.87 (3H, s).
(Example 3)
15 N-{4-[2-(4-But-2-ynyloxybenzenesulfonyl)-1-(form
ylhydroxyamino)ethyl]benzyl}-2-methoxyacetamide (I-3)
75
[Chem. 19]
(3-1):
N-{4-[2-(4-But-2-ynyloxybenzenesulfonyl)vinyl]benzyl}-
2-methoxyacetamide (II-5 3)
At room temperature, 0.2 mL (1.85 mmol) of
methoxyacetyl chloride was dropwise added to a pyridine
solution (5 mL) of 283 mg (0.75 mmol) of the compound (II-1b)
obtained in the above-mentioned Example 1 (1-5). After
10 2 days, brine was added thereto, extracted with ethyl
acetate, and washed with brine. This was dried over
anhydrous magnesium sulfate, the solvent was evaporated
away under reduced pressure, and the residue was purified
by silica gel column chromatography (chloroform/methanol
15 = 20/1 10/1) to give 296 mg (0.71 mmol) of the compound
(II-3) as a pale yellow solid (yield 94%). Its physical
properties are shown below.
MS (FAB) m/z: 414 (M+H)+.
(3-2):
20 N-{4-[2-(4-But-2-ynyloxybenzenesulfonyl)-1-(formylhydr
oxyamino)ethyl]benzyl}-2-methoxyacetamide (I-3)
According to the same process as in the
76
above-mentioned Example 1 (1-7 and 1-8) but using the
compound (II-3) obtained in the above (3-1),
N-{4-[2-(4-but-2-ynyloxybenzenesulfonyl)-1-(formylhydr
oxyamino)ethyl]benzyl}-2-methoxyacetamide (I-3) was
obtained. Its physical properties are shown below5 .
1H-NMR (CDCl3): 8.32 (0.6H, s), 8.09 (0.4H, s), 7.77-7.88
(2H, m), 7.20-7.31 (4H, m), 7.06-7.16 (2H, m), 5.65 (0.6H,
m), 5.37 (0.4H, m), 4.75 (2H, br s), 4.39-4.47 (2H, m),
4.16 (0.4H, m), 4.03 (0.6H, br t), 3.93 (0.8H, br s), 3.90
10 (1.2H, br s), 3.46 (1H, m), 3.40 (3H, br s), 1.87 (3H, br
s).
(Example 4)
N-{4-[2-(4-But-2-ynyloxybenzenesulfonyl)-1-(form
ylhydroxyamino)ethyl]benzyl}methanesulfonamide (I-4)
15 [Chem. 20]
According to the same operation as in Example 3, the
above-mentioned compound (I-4) was obtained.
MS (FAB) m/z : 481 (M+H)+.
120 H-NMR (CDCl3): 8.42 (0.6H, s), 7.87 (0.4H, s), 7.78-7.88
(2H, m), 7.30-7.35 (4H, m), 7.06-7.17 (2H, m), 5.65 (0.6H,
m), 5.39 (0.4H, m), 4.75 (2H, m), 4.28 (2H, m), 4.15 (0.4H,
m), 4.00 (0.6H, br t, J = 12 Hz), 3.45 (1H, m), 2.91 (1.2H,
77
s), 2.89 (1.8H, s), 1.87 (3H, m).
(Example 5)
N-{4-[2-(4-But-2-ynyloxybenzenesulfonyl)-1-(form
ylhydroxyamino)ethyl]benzyl}benzamide (I-5)
[Chem. 215 ]
According to the same operation as in Example 3, the
above-mentioned compound (I-5) was obtained.
MS (FAB) m/z: 507 (M+H)+.
110 H-NMR (CDCl3): 8.31 (0.6H, s), 8.08 (0.4H, s), 7.73-7.87
(4H, m), 7.51 (1H, m), 7.42 (2H m), 7.27-7.33 (3H, m),
7.07-7.14 (2H, m), 5.65 (0.6H, m), 5.37 (0.4H, m), 4.73
(2H, m), 4.54-4.62 (2H, m), 4.15 (0.4H, m), 4.00 (0.6H,
m), 3.45 (1H, m), 1.86 (3H, br s).
15 (Example 6)
N-[2-(4-But-2-ynyloxybenzenesulfonyl)-1-(4-morph
olin-4-ylmethylphenyl)ethyl]-N-hydroxyformamide (I-6)
[Chem. 22]
20 (6-1):
78
tert-Butyl-(4-methanesulfonylphenoxy)dimethylsilane
(XII-6)
9.9 g (145.18 mmol) of imidazole was added to and
dissolved in an N,N-dimethylformamide solution (150 mL)
of 10 g (58.07 mmol) of 4-methylsulfonylphenol, and the5 n
10.5 g (69.7 mmol) of tert-butyldimethylchlorosilane was
added thereto and stirred. After the reaction, brine was
added thereto and extracted with ethyl acetate. The
organic layer was washed three times with brine, and dried
10 over anhydrous magnesium sulfate. Purifying by silica gel
column chromatography (hexane/ethyl acetate = 5/1 3/1)
gave 15.97 g (55.75 mmol) of
tert-butyl-(4-methanesulfonylphenoxy)dimethylsilane
(XII-6) (yield 96%). Its physical properties are shown
15 below.
1H-NMR (CDCl3): 7.83 (1H, m), 7.81 (1H, m), 6.97 (1H,
m), 6.95 (1H, m), 3.04 (1H, s), 1.56 (9H, s), 0.25 (6H,
s).
(6-2):
20 2-[4-(tert-Butyldimethylsilanyloxy)benzenesulfonyl]-1-
(4-morpholin-4-ylmethylphenyl)ethanone (XIII-6)
In an argon atmosphere at -78 C, 4.19 mL (8.37 mmol)
of a hexane-heptane-ethylbenzene solution of 2.0 M lithium
diisopropylamide was added to a tetrahydrofuran solution
25 of 2.6 g (6.98 mmol) of
tert-butyl-(4-methanesulfonylphenoxy)dimethylsilane
79
(XII-6) obtained in the above (6-1), and 6.98 mL (6.98 mmol)
of a tetrahydrofuran solution of 1.0 M lithium
hexamethyldisilazide and 5 mL of a tetrahydrofuran solution
of 1.6 g (6.98 mmol) of methyl
4-morpholin-4-ylmethylbenzoate (IX-6) were added thereto5 .
Subsequently, this was gradually heated up to room
temperature with stirring. After the reaction, brine was
added thereto, extracted with ethyl acetate, and the
organic layer was washed with brine. After dried over
10 anhydrous magnesium sulfate, the solvent was evaporated
away under reduced pressure. 3.74 g of
2-[4-(tert-butyldimethylsilanyloxy)benzenesulfonyl]-1-
(4-morpholin-4-ylmethylphenyl)ethanone (XIII-6) was
obtained as a roughly-purified product.
15 (6-3):
2-[4-(tert-Butyldimethylsilanyloxy)benzenesulfonyl]-1-
(4-morpholin-4-ylmethylphenyl)ethanol (XIV-6)
At 0 C, 264 mg (6.98 mmol) of sodium borohydride was
added to a methanol (50 mL) solution of 3.74 g (6.98 mmol)
20 of the compound (XIII-6) obtained in the above (6-2). After
stirred for 1 hour, brine was added thereto. Methanol was
evaporated away under reduced pressure, and the residue
was extracted with ethyl acetate. The organic layer was
washed with brine, and dried over anhydrous magnesium
25 sulfate. The solvent was evaporated away under reduced
pressure, and the residue was purified by silica gel column
80
chromatography (ethyl acetate ethyl acetate/methanol
= 10/1) to give 2.19 g (4.48 mmol) of
2-[4-(tert-butyldimethylsilanyloxy)benzenesulfonyl]-1-
(4-morpholin-4-ylmethylphenyl)ethanol (XIV-6). Its
physical properties are shown below5 .
1H-NMR (CDCl3): 7.83 (2H, m), 7.25-7.30 (4H, m), 6.98
(2H, m), 5.22 (1H, d, J = 7.7 Hz), 3.68 (4H, m), 3.46 (3H,
m), 3.30 (1H, dd, J= 1.5, 14 Hz), 2.40 (4H, m), 0.99 (9H,
s), 0.25 (6H, s).
10 (6-4):
4-[(E)-2-(4-Morpholin-4-ylmethylphenyl)ethenesulfonyl]
phenol (XVI-6)
3.10 mL of triethylamine was added to a
dichloromethane solution(45 mL) of 2.19 g (4.48 mmol) of
15 2-[4-(tert-butyldimethylsilanyloxy)benzenesulfonyl]-1-
(4-morpholin-4-ylmethylphenyl)ethanol (XIV-6) obtained
in the above (6-3), and stirred at 0 C. 0.61 mL (8.91 mmol)
of methanesulfonyl chloride was added thereto, and stirred
at room temperature for 8 hours. Further, 3.10 mL of
20 triethylamine and 0.61 mL of methanesulfonyl chloride were
added thereto, and stirred for 4 hours. Brine was added
and extracted with chloroform. The organic layer was
washed with brine, and dried over anhydrous magnesium
sulfate. The solvent was evaporated away under reduced
25 pressure, and the residue was purified by silica gel column
chromatography (hexane/ethyl acetate = 1/2 ethyl acetate
81
ethyl acetate/methanol = 10/1) to give 0.757 g (2.11
mmol) of
4-[(E)-2-(4-morpholin-4-ylmethylphenyl)ethenesulfonyl]
phenol (XVI-6) (yield 47%). Its physical properties are
shown below5 .
1H-NMR (CDCl3): 8.01 (1H, d, J = 8.7 Hz), 7.81 (1H, d,
J = 8.7 Hz), 7.61 (1H, d, J = 15 Hz), 7.35-7.47 (5H, m),
6.92 (1H, d, J = 6.9 Hz), 6.81 (1H, d, J = 15 Hz), 3.71
(4H, m), 3.51 (2H, m), 2.44 (4H, m).
10 (6-5):
4-[4-[(E)-2-(4-But-2-ynyloxybenzenesulfonyl)vinyl]benz
yl]morpholine (II-6)
108 mg (0.780 mmol) of potassium carbonate and 0.094
mL (1.04 mmol) of 1-bromo-2-butyne were added to an
15 N,N-dimethylformamide solution of 187 mg (0.520 mmol) of
4-[(E)-2-(4-morpholin-4-ylmethylphenyl)ethenesulfonyl]
phenol (XVI-6) obtained in the above (6-4), and stirred
for 4 hours. Brine was added thereto, and extracted with
ethyl acetate. The organic layer was washed with brine,
20 and dried over anhydrous magnesium sulfate. The solvent
was evaporated away under reduced pressure, and the residue
was purified by silica gel column chromatography (ethyl
acetate/chloroform = 1/1 1/2 ethyl acetate) to give
0.0238 g (0.0578 mmol) of
25 4-[4-[(E)-2-(4-but-2-ynyloxybenzenesulfonyl)vinyl]benz
yl]morpholine (II-6) (yield 11%). Its physical properties
82
are shown below.
1H-NMR (CDCl3): 7.87 (2H, d, J = 8.7 Hz), 7.63 (1H, d,
J = 15.5 Hz), 7.35-7.44 (4H, m), 7.07 (2H, d, J = 8.7 Hz),
6.83 (1H, d, J = 15.5 Hz), 4.71 (2H, m), 3.69 (4H, m), 3.50
(2H, s), 2.43 (4H, m), 1.86 (3H, s)5 .
(6-6):
N-[2-(4-But-2-ynyloxybenzenesulfonyl)-1-(4-morpholin-4
-ylmethylphenyl)ethyl]-N-hydroxyformamide (I-6)
According to the same process as in the above (1-7
10 and 1-8) but using the compound (II-6) obtained in the above
(6-5),
N-[2-(4-but-2-ynyloxybenzenesulfonyl)-1-(4-morpholin-4
-ylmethylphenyl)ethyl]-N-hydroxyformamide (I-6) was
obtained. Its physical properties are shown below.
MS (FAB) m/z: 473 (M+H)+15 .
1H-NMR (CDCl3): 8.45 (0.6H, s), 8.11 (0.4H, s), 7.80-7.88
(2H, m), 7.23-7.32 (4H, m), 7.07-7.16 (2H, m), 5.63 (0.6H,
m), 5.38 (0.4H, m), 4.75 (2H, m), 4.19 (0.4H, m), 4.03 (0.6H,
m), 3.64-3.71 (4H, m), 3.40-3.51 (2H, m), 2.39 (4H, m),
20 1.87 (3H, m).
(Example 7)
N-Hydroxy-N-[1-(4-morpholin-4-ylmethylphenyl)-2-
(4-pent-2-ynyloxybenzenesulfonyl)ethyl]formamide (I-7)
[Chem. 23]
83
According to the same operation as in Example 6, the
above-mentioned compound (I-7) was obtained.
MS (FAB) m/z: 487 (M+H)+.
1H-NMR (CDCl3): 8.44 (0.6H, s), 8.11 (0.4H, s), 7.78-7.85 8
(2H, m), 7.26-7.32 (4H, m), 7.06-7.16 (2H, m), 5.63 (0.6H,
dd, J = 3.6, 12.3 Hz), 5.38 (0.4H, dd, J = 3.0, 10.1 Hz),
4.75 (2H, m), 4.18 (0.4H, dd, J = 10.1, 15.7 Hz), 4.03 (0.6H,
dd, J = 12.3, 14.6 Hz), 3.72 (2H, q, J = 6.9 Hz), 3.64-3.70
10 (4H, m), 3.41-3.48 (3H, m), 2.36-2.43 (4H, m), 1.24 (3H,
t, J = 6.9 Hz).
According to the schemes 1 to 4 and according to the
same process as in Examples 1 to 7, the following compounds
(I-8 to I-28) were obtained.
15 (Example 8)
N-[2-(4-But-2-ynyloxybenzenesulfonyl)-1-(4-dimet
hylaminophenyl)ethyl]-N-hydroxyformamide (I-8)
[Chem. 24]
MS (ESI) m/z: 417(M+H)+20 .
84
1H-NMR (CDCl3): 8.40 (0.5H, s), 8.04 (0.5H, s), 7.73-7.89
(2H, m), 7.02-7.22 (4H, m), 6.62 (2H, d, J = 8.7 Hz),
5.49-5.58 (0.5H, m), 5.22-5.31 (0.5H, m), 4.73 (2H, d, J
= 9.2 Hz), 3.98-4.23 (1H, m), 3.37-3.55 (1H, m), 2.93 (3H,
s), 2.91 (3H, s), 1.87 (3H, t, J = 2.3 Hz)5 .
(Example 9)
N-[2-(4-But-2-ynyloxybenzenesulfonyl)-1-(3-dimet
hylaminophenyl)ethyl]-N-hydroxyformamide (I-9)
[Chem. 25]
10
MS (ESI) m/z: 417(M+H)+.
1H-NMR (CDCl3): 8.45 (0.5H, s), 8.07 (0.5H, s), 7.77-7.89
(2H, m), 7.04-7.21 (4H, m), 6.57-6.68 (2H, m), 5.54-5.62
(0.5H, m), 5.27-5.34 (0.5H, m), 4.69-4.77 (2H, m),
15 4.00-4.25 (1H, m), 3.41-3.54 (1H, m), 2.90-2.95 (6H, m),
1.87 (3H, t, J = 2.3 Hz).
(Example 10)
N-[2-(4-But-2-ynyloxybenzenesulfonyl)-1-(2-dimet
hylaminophenyl)ethyl]-N-hydroxyformamide (I-10)
20 [Chem. 26]
85
MS (ESI) m/z: 417(M+H)+.
1H-NMR (CDCl3): 8.31 (0.6H, s), 8.08 (0.4H, s), 7.88 (0.6H,
d, J = 9.2 Hz), 7.83 (0.4H, d, J = 8.7 Hz), 7.07-7.42 (6H,
m), 6.57-6.68 (2H, m), 6.04 (0.4H, dd, J = 2.7, 10 Hz)5 ,
5.88 (0.6H, dd, J = 2.7, 11 Hz), 4.71-4.78 (2H, m), 3.99-4.08
(1H, m), 3.39-3.51 (1H, m), 2.61 (2.4H, s), 2.54 (3.6H,
s), 1.86 (3H, t, J = 2.3 Hz).
(Example 11)
10 N-[2-(4-But-2-ynyloxybenzenesulfonyl)-1-(4-piper
idin-1-ylmethylphenyl)ethyl]-N-hydroxyformamide (I-11)
[Chem. 27]
MS (ESI) m/z: 471(M+H)+.
115 H-NMR (CDCl3): 8.28 (0.6H, s), 8.10 (0.4H, s), 7.77-7.89
(2H, m), 7.04-7.30 (6H, m), 5.61-5.69 (0.6H, m), 5.31-5.39
(0.4H, m), 4.69-4.78 (2H, m), 3.99-4.26 (1H, m), 3.35-3.53
(3H, m), 2.37 (4H, br s), 1.87 (3H, br s), 1.35-1.59 (6H,
m).
20 (Example 12)
86
N-[2-(4-But-2-ynyloxybenzenesulfonyl)-1-(3-piper
idin-1-ylmethylphenyl)ethyl]hydroxyformamide (I-12)
[Chem. 28]
MS (ESI) m/z: 471(M+H)+5 .
1H-NMR (CDCl3): 8.38 (0.5H, s), 8.10 (0.5H, s), 7.77-7.89
(2H, m), 7.04-7.30 (6H, m), 5.70 (0.5H, dd, J = 3.7, 11
Hz), 5.30-5.38 (0.5H, m), 4.69-4.77 (2H, m), 4.01-4.27 (1H,
m), 3.23-3.60 (3H, m), 2.30 (4H, br s), 1.87 (3H, t, J =
10 2.3 Hz), 1.31-1.55 (6H, m).
(Example 13)
N-[2-(4-But-2-ynyloxybenzenesulfonyl)-1-(3-morph
olin-4-ylmethylphenyl)ethyl]-N-hydroxyformamide (I-13)
[Chem. 29]
15
MS (ESI) m/z: 473(M+H)+.
1H-NMR (CDCl3): 8.46 (0.5H, s), 8.10 (0.5H, s), 7.78-7.90
(2H, m), 7.06-7.31 (6H, m), 5.65 (0.5H, dd, J = 3.7, 12
Hz), 5.35-5.42 (0.5H, m), 4.70-4.78 (2H, m), 3.98-4.26 (1H,
20 m), 3.68 (4H, dd, J = 4.6, 4.6 Hz), 3.39-3.54 (3H, m),
87
2.36-2.44 (4H, m), 1.87 (3H, t, J = 2.3 Hz).
(Example 14)
N-[2-(4-But-2-ynyloxybenzenesulfonyl)-1-{4-[(eth
ylmethylamino)methyl]phenyl]ethyl}-N-hydroxyformamide
(I-145 )
[Chem. 30]
MS (ESI) m/z: 445(M+H)+.
1H-NMR (CDCl3): 8.35 (0.5H, s), 8.11 (0.5H, s), 7.78-7.89
10 (2H, m), 7.06-7.28 (6H, m), 5.65 (0.5H, dd, J = 3.7, 12
Hz), 5.32-5.40 (0.5H, m), 4.71-4.77 (2H, m), 3.99-4.24 (1H,
m), 3.38-3.53 (3H, m), 2.38 (3H, q, J = 7.3 Hz), 1.87 (3H,
t, J = 2.3 Hz), 1.05 (3H, t, J = 7.3 Hz).
(Example 15)
15 N-(2-(4-But-2-ynyloxybenzenesulfonyl)-1-{3-[(eth
ylmethylamino)methyl]phenyl}ethyl)-N-hydroxyformamide
(I-15)
[Chem. 31]
MS (ESI) m/z: 445(M+H)+20 .
88
1H-NMR (CDCl3): 8.37 (0.5H, s), 8.11 (0.5H, s), 7.78-7.90
(2H, m), 7.06-7.34 (6H, m), 5.70 (0.5H, dd, J = 3.7, 12
Hz), 5.32-5.40 (0.5H, m), 4.70-4.78(2H, m), 4.00-4.22 (1H,
m), 3.29-3.59 (3H, m), 2.38 (3H, q, J = 6.9 Hz), 1.87 (3H,
t, J = 2.3 Hz), 1.03 (3H, t, J = 6.9 Hz)5 .
(Example 16)
N-{4-[2-(4-But-2-ynyloxybenzenesulfonyl)-1-(form
ylhydroxyamino)ethyl]benzyl}-N-methylmethanesulfonamid
e (I-16)
10 [Chem. 32]
MS (ESI) m/z: 495(M+H)+.
1H-NMR (DMSO-d6): 8.22 (0.5H, br s), 8.11 (0.5H, br s),
7.75-7.86 (2H, m), 7.09-7.42 (4H, m), 7.13 (2H, d, J = 9.2
15 Hz), 5.70 (0.5H, br s), 5.40 (0.5H, br s), 4.83-4.89 (2H,
m), 4.18 (2H, s), 3.86-4.16 (2H, m), 2.94 (3H, s), 2.63
(3H, s), 1.84 (3H, t, J = 2.3 Hz).
(Example 17)
N-{4-[2-(4-But-2-ynyloxybenzenesulfonyl)-1-(form
20 ylhydroxyamino)ethyl]benzyl}-N-methylbenzenesulfonamid
e (I-17)
[Chem. 33]
89
MS (ESI) m/z: 557(M+H)+.
1H-NMR (CDCl3): 8.37 (0.6H, br s), 8.03 (0.4H, br s),
7.69-7.88 (4H, m), 7.04-7.33 (8H, m), 5.61 (0.6H, dd, J
= 3.6, 12 Hz), 5.31-5.39 (0.4H, m), 4.70-4.83 (2H, m)5 ,
3.92-4.17 (3H, m), 3.36-3.51 (1H, m), 2.43 (3H, s), 1.87
(3H, t, J = 2.3 Hz).
(Example 18)
N-{4-[2-(4-But-2-ynyloxybenzenesulfonyl)-1-(form
10 ylhydroxyamino)ethyl]benzyl}-4,N-dimethylbenzenesulfon
amide (I-18)
[Chem. 34]
MS (ESI) m/z: 571(M+H)+.
115 H-NMR (CDCl3): 8.47 (0.6H, br s), 8.11 (0.4H, br s),
7.78-7.89 (2H, m), 7.70(2H, d, J = 8.2 Hz), 7.35 (2H, d,
J = 8.2 Hz), 7.23-7.32 (4H, m), 7.06-7.17 (2H, m), 5.64
(0.6H, dd, J = 3.6, 12 Hz), 5.36-5.43 (0.4H, m), 4.71-4.78
(2H, m), 3.96-4.22 (3H, m), 3.38-3.52 (1H, m), 2.58 (1.2H,
20 s), 2.54 (1.8H, s), 2.45 (3H, s), 1.87 (3H, t, J = 2.3 Hz).
90
(Example 19)
N-{4-[2-(4-But-2-ynyloxybenzenesulfonyl)-1-(form
ylhydroxyamino)ethyl]benzyl}-N-methylsulfonylmethanesu
lfonamide (I-19)
[Chem. 355 ]
MS (ESI) m/z: 559(M+H)+.
1H-NMR(DMSO-d6): 8.12 (0.5H, br s), 8.21 (0.5H, br s),
7.80 (2H, br s), 7.35-7.44 (1H, m), 7.32 (2H, d, J = 9.2
10 Hz), 7.31 (1H, s), 7.14 (2H, d, J = 9.2 Hz), 5.41 (0.5H,
br s), 5.71 (0.5H, br s), 4.87 (2H, q, J = 2.3 Hz), 4.83
(2H, s), 4.00-4.16 (1H, m), 3.84-3.98 (1H, m), 3.25 (6H,
s), 1.84 (3H, t, J = 2.3 Hz).
(Example 20)
15 N-{2-(4-But-2-ynyloxybenzenesulfonyl)-1-[4-(2-di
methylaminoethyl)phenyl]ethyl}-N-hydroxyformamide
(I-20)
[Chem. 36]
91
MS (ESI) m/z: 445(M+H)+.
1H-NMR (CDCl3): 8.30 (0.5H, s), 8.14 (0.5H, s), 7.76-7.89
(2H, m), 7.00-7.24 (6H, m), 5.71 (0.5H, dd, J = 3.7, 11
Hz), 5.29-5.36 (0.5H, m), 4.69-4.76 (2H, m), 4.02-4.21 (1H5 ,
m), 3.42-3.59 (1H, m), 2.43-2.54 (2H, m), 2.10-2.28 (2H,
m), 2.19 (6H, s), 1.87 (3H, t, J = 2.3 Hz).
(Example 21)
N-{2-(4-But-2-ynyloxybenzenesulfonyl)-1-[4-(2-mo
10 rpholin-4-ylethyl)phenyl]ethyl}-N-hydroxyformamide
(I-21)
[Chem. 37]
MS (ESI) m/z: 487(M+H)+.
115 H-NMR (CDCl3): 8.44 (0.5H, s), 8.10 (0.5H, s), 7.77-7.89
(2H, m), 7.05-7.27 (6H, m), 5.62 (0.5H, dd, J = 3.7, 12
Hz), 5.32-5.39 (0.5H, m), 4.69-4.78 (2H, m), 3.96-4.23 (1H,
m), 3.71 (4H, dd, J = 4.1, 4.6 Hz), 3.39-3.53 (1H, m),
92
2.69-2.77 (2H, m), 2.43-2.56 (6H, m), 1.87 (3H, t, J = 2.3
Hz).
(Example 22)
N-(2-{4-[2-(4-But-2-ynyloxybenzenesulfonyl)-1-(f
ormylhydroxyamino)ethyl]phenyl}ethyl)methanesulfonami5 d
e (I-22)
[Chem. 38]
MS (ESI) m/z: 495(M+H)+.
110 H-NMR (CDCl3): 8.39 (0.5H, s), 8.07 (0.5H, s), 7.78-7.89
(2H, m), 7.05-7.30 (6H, m), 5.65 (0.5H, dd, J = 3.7, 12
Hz), 5.33-5.41 (0.5H, m), 4.70-4.78 (2H, m), 3.95-4.21 (1H,
m), 3.31-3.52 (3H, m), 2.80-2.91 (5H, m), 1.87 (3H, t, J
= 2.3 Hz).
15 (Example 23)
N-{2-(4-But-2-ynyloxybenzenesulfonyl)-1-[4-(3-di
methylaminopropyl)phenyl]ethyl}-N-hydroxyformamide
(I-23)
[Chem. 39]
93
MS (ESI) m/z: 459(M+H)+.
1H-NMR (CDCl3): 8.06-8.13 (1H, m), 7.74-7.88 (2H, m),
7.02-7.24 (6H, m), 5.64-5.75 (0.5H, m), 5.22-5.32 (0.5H,
m), 4.68-4.76 (2H, m), 4.02-4.22 (1H, m), 3.40-3.58 (1H5 ,
m), 2.51 (2H, dd, J = 7.3, 7.8 Hz), 2.17 (2H, dd, J = 7.3,
7.8 Hz), 2.08 (3H, s), 2.06 (3H, s), 1.87 (3H, t, J = 2.3
Hz), 1.52-1.66 (2H, m).
(Example 24)
10 N-{2-(4-But-2-ynyloxybenzenesulfonyl)-1-[4-(3-di
ethylaminopropyl)phenyl]ethyl}-N-hydroxyformamide
(I-24)
[Chem. 40]
MS (ESI) m/z: 487(M+H)+15 .
1H-NMR (CDCl3): 8.29 (0.5H, s), 8.07 (0.5H, m), 7.75-7.89
(2H, m), 7.04-7.24 (6H, m), 5.64 (0.5H, dd, J = 3.7, 11
Hz), 5.28-5.37 (0.5H, m), 4.69-4.77 (2H, m), 4.00-4.23 (1H,
m), 3.41-3.55 (1H, m), 2.33-2.60 (6H, m), 1.87 (3H, t, J
20 = 2.3 Hz), 1.61-1.72 (2H, m), 0.91-1.01 (6H, m).
94
(Example 25)
N-{2-(4-But-2-ynyloxybenzenesulfonyl)-1-[4-(3-mo
rpholin-4-ylpropyl)phenyl]ethyl}-N-hydroxyformamide
(I-25)
[Chem. 415 ]
MS (ESI) m/z: 501(M+H)+.
1H-NMR (CDCl3): 8.37 (0.5H, s), 8.08 (0.5H, s), 7.76-7.89
(2H, m), 7.04-7.24 (6H, m), 5.64 (0.5H, dd, J = 3.7, 12
10 Hz), 5.29-5.38 (0.5H, m), 4.68-4.77 (2H, m), 3.98-4.23 (1H,
m), 3.67 (4H, br s), 3.40-3.53 (1H, m), 2.52-2.63 (2H, m),
2.24-2.46 (6H, m), 1.87 (3H, t, J = 2.3 Hz), 1.65-1.76 (2H,
m).
(Example 26)
15 N-{2-(4-But-2-ynyloxybenzenesulfonyl)-1-[4-(4-mo
rpholin-4-ylbutyl)phenyl]ethyl}-N-hydroxyformamide
(I-26)
[Chem. 42]
95
MS (ESI) m/z: 515(M+H)+.
1H-NMR (CDCl3): 8.41 (0.5H, s), 8.10 (0.5H, s), 7.77-7.89
(2H, m), 7.05-7.24 (6H, m), 5.57-5.66 (0.5H, m), 5.31-5.38
(0.5H, m), 4.69-4.77 (2H, m), 3.97-4.23 (1H, m), 3.67 (4H5 ,
dd, J = 4.1, 4.6 Hz), 3.40-3.53 (1H, m), 2.52-2.63 (2H,
m), 2.24-2.46 (6H, m), 1.87 (3H, t, J = 2.3 Hz), 1.39-1.63
(4H, m).
(Example 27)
10 N-{4-[1-(Formylhydroxyamino)-2-(4-pent-2-ynyloxy
benzenesulfonyl)ethyl]benzyl}methanesulfonamide (I-27)
[Chem. 43]
MS (ESI) m/z: 495(M+H)+.
115 H-NMR (CDCl3): 8.29 (0.6H, s), 8.00 (0.4H, s), 7.75-7.88
(2H, m), 7.23-7.35 (4H, m), 7.05-7.16 (2H, m), 5.66 (0.6H,
dd, J = 3.7, 12 Hz), 5.31-5.41 (0.4H, m), 4.71-4.80 (2H,
m), 4.26 (2H, br s), 3.95-4.18 (1H, m), 3.39-3.50 (1H, m),
2.89 (3H, br s), 2.24 (2H, tq, J = 1.8, 7.3 Hz), 1.14 (3H,
96
t, J = 7.3 Hz).
(Example 28)
N-{4-[1-(Formylhydroxyamino)-2-(4-oct-2-ynyloxyb
enzenesulfonyl)ethyl]benzyl}methanesulfonamide (I-28)
[Chem. 445 ]
MS (ESI) m/z: 537(M+H)+.
1H-NMR (CDCl3): 8.35 (0.6H, s), 8.03 (0.4H, s), 7.76-7.88
(2H, m), 7.27-7.36 (4H, m), 7.06-7.17 (2H, m), 5.66 (0.6H,
10 dd, J = 3.7, 12 Hz), 5.34-5.42 (0.4H, m), 4.72-4.82 (2H,
m), 4.23-4.33 (2H, m), 3.94-4.18 (1H, m), 3.38-3.51 (1H,
m), 2.90 (1.2H, s), 2.89 (1.8H, s), 2.22 (2H, tt, J = 2.3,
7.3 Hz), 1.45-1.55 (2H, m), 1.23-1.38 (4H, m), 0.87 (3H,
t, J = 7.3 Hz).
15 (Test Example 3) ADAM17 Inhibition Experiment
The N-hydroxyformamide derivatives obtained in
Examples 1 to 7 were examined to detemine whether they had
an ADAM17 inhibitory action or not.
Specifically, first, since the nucleotide sequence
20 coding for ADAM17 had been reported by Moss et al. (see
Moss et al., Nature, 1997, vol. 385, pp. 733-736), the cDNA
of ADAM-17 was obtained from a human monocytic cell line,
97
THP-1 cells, by a standard method. The cDNA was
incorporated into an expression vector. Then, this vector
was transfected into mammalian cells or insect cells, and
ADAM17 was expressed and obtained.
Next, the activity of ADAM17 was measured, as follow5 s
in the presence or absence of the test substances, in which
obtained ADAM17 was used as an enzyme, and a fluorescent
synthetic substrate Nma (N-methyl
anthranilate)-Leu-Ala-Gln-Ala-Val-Arg-Ser-Ser-Lys-Dnp
10 (dinitrophenyl)-D-Arg-NH2 containing an ADAM17-cleavage
sequence of membrane-bound TNF was used as a substrate.
Specifically, 90 μL of 14 units of an enzyme solution
(1 unit is defined as an amount of enzyme degrading 1 pmol
of the substrate in 1 minute at 25ºC) prepared with Assay
15 Buffer A (50 mmol/L Tris-HCl buffer (pH 7.5) containing
200 mmol/L sodium chloride, 5 mmol/L calcium chloride, 10
μmol/L zinc sulfate, and 2 mg/mL bovine serum albumin) was
mixed with 90 μL of the fluorescent synthetic substrate
adjusted to be 20 μmol/L with Assay Buffer B (50 mmol/L
20 Tris-HCl buffer (pH 7.5) containing 200 mmol/L sodium
chloride, 5 mmol/L calcium chloride, 10 μmol/L zinc
sulfate, and 0.05% PLURONIC F-68), and allowed for reaction
at 37ºC for 1.5 hours. Thereafter, the enzyme activity
was determined by measurement using a fluorometer
25 (Fluoroskan Ascent) under conditions of an excitation
wavelength of 355 nm and a measurement wavelength of 460
98
nm. Then, an inhibition rate was obtained based on the
enzyme activities in the presence and absence of the test
compounds, and the 50% inhibitory concentration (IC50) was
calculated. Table 1 show the obtained result.
[Table 15 ]
As apparent from the result shown in Table 1, any
of the compounds I-1 to I-7 (N-hydroxyformamide
derivatives) according to the present invention was
10 confirmed to inhibit the ADAM17-enzyme activity at low
concentrations.
(Test Example 4) Various Evaluation experiments
Next, compounds I-1 (hereinafter also referred to
as "S-45282") and I-4 (hereinafter also referred to as
15 "S-45457") having a strong inhibitory effect on ADAM17
among the N-hydroxyformamide derivatives obtained in
Examples 1 to 7 were examined for the specificity as an
inhibitor and the TACE-inhibitory activity in cells by the
following methods. For comparison, a non-selective
99
metalloproteinase inhibitor GM6001 (EMD/Calbiochem,
product number 364205) was also evaluated.

Enzymes used in this test are as follow:
Matrix metalloproteinase 1 (MMP-1) (manufactured b5 y
Calbiochem, product number: 444208)
Matrix metalloproteinase 2 (MMP-2) (manufactured by
Calbiochem, product number: 444213)
Matrix metalloproteinase 3 (MMP-3) (manufactured by
10 Calbiochem, product number: 444217)
Matrix metalloproteinase 8 (MMP-8) (manufactured by
Calbiochem, product number: 444229)
Matrix metalloproteinase 9 (MMP-9) (manufactured by
Calbiochem, product number: 444231)
15 Matrix metalloproteinase 13 (MMP-13) (manufactured
by Chemicon, product number: CC068)
Matrix metalloproteinase 14 (MMP-14) (manufactured
by Calbiochem, product number: 475935)
Matrix metalloproteinase 17 (MMP-17) (manufactured
20 by Calbiochem, product number: 475940)
A disintegrin and metalloproteinase 10 (ADAM10)
(manufactured by Funakoshi Co., Ltd., product code:
936-AD-020) and
A disintegrin and metalloproteinase 17 (ADAM17, TACE)
25 (used was one prepared at Kaken Pharmaceutical Co., Ltd.
by employing the genetic recombination in the same way as
100
in Test Example 3. Note that, the same as in Test Example
3, 1 unit is defined as an amount of enzyme degrading 1
pmol of substrate in 1 minute at 25ºC).
In addition, substrates used in this test are as
follows5 :
444221 substrate
(DNP-Pro-Cha-Gly-Cys(Me)-His-Ala-Lys(Nma)-NH2, custom
synthesis): excitation wavelength at 355 nm, emission
wavelength at 460 nm
10 TACE substrate (the same as one used in Test Example
3): excitation wavelength at 355 nm, emission wavelength
at 460 nm
3168 substrate
(MOCAc-Arg-Pro-Lys-Pro-Val-Glu-Nva-Trp-Arg-Lys(Dnp)-NH
15 2, manufactured by Peptide Institute, Inc.): excitation
wavelength at 320 nm, emission wavelength at 405 nm, and
3163 substrate
(MOCAc-Pro-Leu-Gly-Leu-A2pr(Dnp)-Ala-Arg-NH2,
manufactured by Peptide Institute, Inc.): excitation
20 wavelength at 320 nm, emission wavelength at 405 nm.
The enzyme reaction was carried out using the enzymes
and substrates. Specifically, if necessary, the enzyme
was activated in advance with APMA (final concentration
1 mmol/L, p-aminophenylmercuric acetate, manufactured by
25 Sigma-Aldrich Co., A9563) according to information from
the manufacturer. Then, in a 96-well plate for
101
fluorescence measurement, 90 μL of an enzyme solution
prepared with Assay Buffer A (50 mmol/L Tris-HCl buffer
(pH 7.5) containing 200 mmol/L sodium chloride, 5 mmol/L
calcium chloride, 10 μmol/L zinc sulfate, and 2 mg/mL bovine
serum albumin) was mixed with 90 μL of the substrate adjuste5 d
to be 20 μmol/L with Assay Buffer B (50 mmol/L Tris-HCl
buffer (pH 7.5) containing 200 mmol/L sodium chloride, 5
mmol/L calcium chloride, 10 μmol/L zinc sulfate, and 0.05%
PLURONIC F-68), and incubated for reaction at 25ºC or 37ºC
10 for several hours. Moreover, the fluorescence intensities
before and after the reaction were measured using a
fluorometer to determine the enzyme activity. An
inhibition rate was obtained based on the enzyme activities
in the presence and absence of the test substances, and
15 the 50% inhibitory concentration (IC50) was calculated.
Table 2 shows the reaction conditions for each enzyme.
102
[Table 2]
103

THP-1 cells (human acute monocytic leukemia–derived
cells, ATCC catalog number: TIB202) were suspended in 10
v/v% FBS-containing RPMI 1640, dispensed in a volume of
200 μL/well (1 × 105 cells) into a 96-well culture plate5 ,
treated with PMA (final concentration 50 nmol/L) and
cultured at 37ºC in 5% CO2 for 40 to 48 hours, so that the
THP-1 cells differentiated into macrophage-like cells.
After the differentiation, the medium was discarded, and
10 the cells were washed with THP-1 washing buffer (RPMI 1640,
20 mM HEPES-NaOH (pH 7.4)). After the washing of the cells,
a THP-1 assay buffer (RPMI 1640, 20 mmol/L HEPES-NaOH (pH
7.4), 0.01 w/v% PF-68, 10 v/v% FBS) was added thereto at
100 μL/well. Then, a THP-1 assay buffer containing the
15 test substance (I-1 or I-4) was further added at 50 μL/
well and mixed by tapping. After incubation in a CO2
incubator for 30 to 45 minutes, a THP-11 assay buffer
containing LPS was added at 50 μL/well (the final
concentration of LPS was 1 μg/mL). Further, the plate was
20 tapped for mixing, followed by incubation in the CO2
incubator for 6 hours. After the incubation was finished,
human TNF-α concentration in the culture supernatant was
measured with a commercially available kit (Human TNF-α
CytoSet (manufactured by BIOSOURCE) or Human TNF-α
25 Ready-Set-Go! (manufactured by eBioscience, Inc.)).
Table 3 show the result obtained by these experimental
104
methods.
[Table 3]
105
As apparent from the result shown in Table 3, both
of the compounds I-1 and I-4 according to the present
invention were confirmed to have a high specificity to
ADAM17 in comparison with metalloproteinases other tha5 n
ADAM17. Their inhibitory activity was confirmed on TACE
expressed on the macrophage-like cells, also. Moreover,
the two compounds suppressed ADAM10, as well, although the
degree of suppression was somewhat low in comparison with
10 ADAM17. ADAM10 is an enzyme that can cleave the same
substrate as ADAM17 (see Caescu et al., Biochemical J.,
2009, vol. 424, pp. 79 to 88). It is suggested that the
inhibition is useful for storing the platelet function (see
Bender et al., "Differentially regulated GPVI ectodomain
15 shedding by multiple platelet-expressed proteinases.,"
Blood, July 19, 2010, online publication). From the above,
the compounds I-1 and I-4 according to the present invention
are ADAM17-specific inhibitors, suggesting that the other
compounds having the same basic structure (for example,
20 I-2, I-3, I-5, I-6) according to the present invention be
also ADAM17-specific inhibitors. Meanwhile, GM6001
having a hydroxamic acid structure non-specifically and
strongly inhibited metalloproteinases as has been known
conventionally, but the inhibitory activity on ADAM17 was
25 weak.
(Test Example 5) Verification of Function-Maintaining
106
Effects of GM6001 and S-45282 on ES Cell-Derived Platelets
Under conditions (Protocol 1) illustrated in Fig.
6, platelets were induced from ES cell-derived
hematopoietic progenitor cells obtained as described
above. Specifically, in the above-described 8-day cultur5 e
process for inducing hematopoietic progenitor cells to
megakaryocytes/platelets, culturing was conducted under
the following conditions:
2 days: culturing while GM6001, S-45282, or DMSO was added
10 2 days before the flow cytometry analysis (on "d22" shown
in Fig. 6), or
8 days: culturing for 8 days while GM6001 (50 μM), S-45282
(5 μM or 50 μM), or DMSO was added after ES cell-derived
hematopoietic progenitor cells were reseeded (after "d15"
15 shown in Fig. 6).
After collection as described above, the number of
CD41(+)CD42b(+), the number of CD41(+)CD42b(-), and a total
number of platelets were counted by flow cytometry. Figs.
7 to 10 show the obtained results. Note that since CD41
20 (integrin αIIb) is a surface marker for platelets, CD41(+)
indicates platelets. Moreover, CD42b (GPIbα)(+)
indicates that platelets have GPIbα on the surface, while
CD42b (GPIbα)(-) indicates that platelets of which GPIbα
was shed by ADAM17. In addition, in the graphs, "GM" shows
25 the result of adding 50 μM GM6001 (solvent: DMSO), and
107
"45282" shows the result of adding S-45282 (solvent: DMSO)
at each concentration.
As apparent from the results shown in Figs. 7 and
9, it was confirmed that each of GM6001 and S-4

[CLAIMS]
[Claim 1]
A composition for maintaining a function of platelets,
the composition comprising, as an active ingredient, a
compound represented by the following general formula (5 I)
or a salt thereof, or a solvate thereof:
[Chem. 1]
wherein
10 X represents a phenylene group;
Y represents any one of a hydrogen atom and -(CH2)mR1;
wherein
m represents an integer of any one of 0 to 4;
and
R1 15 represents any one of
[Chem. 2]
wherein R2 represents any one of a C1 to C6 alkyl
group, which may be substituted, an aryl group, which may
20 be substituted, and a C1 to C6 alkoxy group;
R3 and R4 each independently represent any one
126
of a hydrogen atom and a C1 to C6 alkyl group, or R3 and
R4 together with an adjacent nitrogen atom may form a
nitrogen-containing heterocycle; and
R5 represents any one of a hydrogen atom, a C1
to C6 alkyl group, and a C1 to C6 alkylsulfonyl group; an5 d
Z represents any one of a hydrogen atom and a C1 to
C6 alkyl group.
[Claim 2]
The composition according to claim 1, wherein the
10 compound represented by the general formula (I) is any one
of
N-[2-(4-but-2-ynyloxybenzenesulfonyl)-1-(4-dieth
ylaminomethylphenyl)ethyl]-N-hydroxyformamide and
N-{4-[2-(4-but-2-ynyloxybenzenesulfonyl)-1-(form
15 ylhydroxyamino)ethyl]benzyl}methanesulfonamide.
[Claim 3]
The composition according to any one of claims 1 and
2 in a form of any one of a reagent for maintaining a function
of platelets and an additive to a blood product comprising
20 platelets.
[Claim 4]
A method for preparing platelets, wherein the method
comprises adding, to a culture system for differentiating
megakaryocytes from cells capable of differentiating into
25 megakaryocytes and producing platelets from the
megakaryocytes, a compound represented by the following
127
general formula (I) or a salt thereof, or a solvate thereof:
[Chem. 3]
wherein
X represents a phenylene group5 ;
Y represents any one of a hydrogen atom and -(CH2)mR1;
wherein
m represents an integer of any one of 0 to 4;
and
R1 10 is any one of
[Chem. 4]
wherein R2 represents any one of a C1 to C6 alkyl
group, which may be substituted, an aryl group, which may
15 be substituted, and a C1 to C6 alkoxy group;
R3 and R4 each independently represent any one
of a hydrogen atom and a C1 to C6 alkyl group, or R3 and
R4 together with an adjacent nitrogen atom may form a
nitrogen-containing heterocycle; and
R5 20 represents any one of a hydrogen atom, a C1
to C6 alkyl group, and a C1 to C6 alkylsulfonyl group; and
128
Z represents any one of a hydrogen atom and a C1 to
C6 alkyl group.
[Claim 5]
The method according to claim 4, wherein the compound
represented by the general formula (I) is any one o5 f
N-[2-(4-but-2-ynyloxybenzenesulfonyl)-1-(4-dieth
ylaminomethylphenyl)ethyl]-N-hydroxyformamide and
N-{4-[2-(4-but-2-ynyloxybenzenesulfonyl)-1-(form
ylhydroxyamino)ethyl]benzyl}methanesulfonamide.
10 [Claim 6]
The method according to any one of claims 4 and 5,
wherein a culture temperature in the culture system is 35
to 38ºC.
[Claim 7]
15 A culture which is a culture system for
differentiating megakaryocytes from cells capable of
differentiating into megakaryocytes and for producing
platelets from the megakaryocytes, and is added to the
system with a compound represented by the following general
20 formula (I) or a salt thereof, or a solvate thereof:
[Chem. 5]
wherein
X represents a phenylene group;
129
Y represents any one of a hydrogen atom and -(CH2)mR1;
wherein
m represents an integer of any one of 0 to 4;
and
R1 is any one o5 f
[Chem. 6]
wherein R2 represents any one of a C1 to C6 alkyl
group, which may be substituted, an aryl group, which may
10 be substituted, and a C1 to C6 alkoxy group;
R3 and R4 each independently represent any one
of a hydrogen atom and a C1 to C6 alkyl group, or R3 and
R4 together with an adjacent nitrogen atom may form a
nitrogen-containing heterocycle; and
R5 15 represents any one of a hydrogen atom, a C1
to C6 alkyl group, and a C1 to C6 alkylsulfonyl group; and
Z represents any one of a hydrogen atom and a C1 to
C6 alkyl group.
[Claim 8]
20 The culture according to claim 7, wherein the compound
represented by the general formula (I) is any one of
N-[2-(4-but-2-ynyloxybenzenesulfonyl)-1-(4-dieth
ylaminomethylphenyl)ethyl]-N-hydroxyformamide and
130
N-{4-[2-(4-but-2-ynyloxybenzenesulfonyl)-1-(form
ylhydroxyamino)ethyl]benzyl}methanesulfonamide.
[Claim 9]
A blood product comprising platelets and a compound
represented by the following general formula (I) or a sal5 t
thereof, or a solvate thereof:
[Chem. 7]
wherein
10 X represents a phenylene group;
Y represents any one of a hydrogen atom and -(CH2)mR1;
wherein
m represents an integer of any one of 0 to 4;
and
R1 15 is any one of
[Chem. 8]
wherein R2 represents any one of a C1 to C6 alkyl
group, which may be substituted, an aryl group, which may
20 be substituted, and a C1 to C6 alkoxy group;
R3 and R4 each independently represent any one
131
of a hydrogen atom and a C1 to C6 alkyl group, or R3 and
R4 together with an adjacent nitrogen atom may form a
nitrogen-containing heterocycle; and
R5 represents any one of a hydrogen atom, a C1
to C6 alkyl group, and a C1 to C6 alkylsulfonyl group; an5 d
Z represents any one of a hydrogen atom and a C1 to
C6 alkyl group.
[Claim 10]
The blood product according to claim 9, wherein the
10 compound represented by the general formula (I) is any one
of
N-[2-(4-but-2-ynyloxybenzenesulfonyl)-1-(4-dieth
ylaminomethylphenyl)ethyl]-N-hydroxyformamide and
N-{4-[2-(4-but-2-ynyloxybenzenesulfonyl)-1-(form
15 ylhydroxyamino)ethyl]benzyl}methanesulfonamide.
[Claim 11]
A method for maintaining a function of platelets in
a blood product, wherein the method comprises adding, to
a blood product comprising platelets, a compound
20 represented by the following general formula (I) or a salt
thereof, or a solvate thereof:
[Chem. 9]
wherein
132
X represents a phenylene group;
Y represents any one of a hydrogen atom and -(CH2)mR1;
wherein
m represents an integer of any one of 0 to 4;
5 and
R1 is any one of
[Chem. 10]
wherein R2 represents any one of a C1 to C6 alkyl
10 group, which may be substituted, an aryl group, which may
be substituted, and a C1 to C6 alkoxy group;
R3 and R4 each independently represent any one
of a hydrogen atom and a C1 to C6 alkyl group, or R3 and
R4 together with an adjacent nitrogen atom may form a
15 nitrogen-containing heterocycle; and
R5 represents any one of a hydrogen atom, a C1
to C6 alkyl group, and a C1 to C6 alkylsulfonyl group; and
Z represents any one of a hydrogen atom and a C1 to
C6 alkyl group.
20 [Claim 12]
The method according to claim 11, wherein the compound
represented by the general formula (I) is any one of
N-[2-(4-but-2-ynyloxybenzenesulfonyl)-1-(4-dieth
133
ylaminomethylphenyl)ethyl]-N-hydroxyformamide and
N-{4-[2-(4-but-2-ynyloxybenzenesulfonyl)-1-(form
ylhydroxyamino)ethyl]benzyl}methanesulfonamide.