U.S. patent application number 12/576829 was filed with the patent office on 2010-02-04 for method of treating dermatitis comprising administering a chymase inhibitor.
This patent application is currently assigned to Asubio Pharma Co., Ltd.. Invention is credited to Harukazu Fukami, Yoshiaki Fukuda, Yoshiaki Tomimori, Naohiro Watanabe.
Application Number | 20100029695 12/576829 |
Document ID | / |
Family ID | 18572276 |
Filed Date | 2010-02-04 |
United States Patent
Application |
20100029695 |
Kind Code |
A1 |
Fukami; Harukazu ; et
al. |
February 4, 2010 |
METHOD OF TREATING DERMATITIS COMPRISING ADMINISTERING A CHYMASE
INHIBITOR
Abstract
A medicament safe and free from side effects, for the prevention
or treatment of dermatitis exhibiting a biphasic inflammation
reaction or dermatitis induced by repeated exposure to an antigen,
which suppresses the progress of the condition and improves the
quality of life of the patient, which medicament treats dermatitis
exhibiting a biphasic inflammation reaction or dermatitis induced
by repeated exposure to an antigen by including a quiazoline
derivative having the formula (I) or (II) or its pharmaceutically
acceptable salt as an effective ingredient: ##STR00001##
Inventors: |
Fukami; Harukazu;
(Kyoto-shi, JP) ; Tomimori; Yoshiaki; (Suita-shi,
JP) ; Fukuda; Yoshiaki; (Ibaraki-shi, JP) ;
Watanabe; Naohiro; (Bunkyo-ku, JP) |
Correspondence
Address: |
DRINKER BIDDLE & REATH (DC)
1500 K STREET, N.W., SUITE 1100
WASHINGTON
DC
20005-1209
US
|
Assignee: |
Asubio Pharma Co., Ltd.
Tokyo
JP
|
Family ID: |
18572276 |
Appl. No.: |
12/576829 |
Filed: |
October 9, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09959252 |
Oct 22, 2001 |
7618977 |
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PCT/JP2001/001323 |
Feb 22, 2001 |
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12576829 |
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Current U.S.
Class: |
514/266.3 |
Current CPC
Class: |
A61P 17/00 20180101;
A61K 31/517 20130101; A61P 17/02 20180101; C07D 239/96 20130101;
A61K 31/00 20130101; A61P 17/04 20180101 |
Class at
Publication: |
514/266.3 |
International
Class: |
A61K 31/517 20060101
A61K031/517; A61P 37/06 20060101 A61P037/06; A61P 17/02 20060101
A61P017/02 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 22, 2000 |
JP |
2000-50504 |
Claims
1-16. (canceled)
17. A method for prevention or treatment of dermatitis exhibiting a
biphasic skin reaction comprising administering to a patient in
need of such treatment a chymase inhibitor in an amount effective
for alleviating late-phase reaction.
18. A method according to claim 17, wherein the dermatitis
exhibiting a biphasic skin reaction is atopic dermatitis.
19. A method for alleviation of late-phase reaction of dermatitis
exhibiting biphasic skin reaction comprising administering a
chymase inhibitor to a patient in need of such treatment in an
amount effective for alleviating late-phase reaction of dermatitis
exhibiting biphasic skin reaction.
20. A method according to claim 19, wherein said dermatitis
exhibiting a biphasic skin reaction is atopic dermatitis.
21. A method for the prevention or treatment of dermatitis induced
by repeated exposure to an antigen comprising administering a
chymase inhibitor to a patient in need of such treatment in an
amount effective for prevention or treatment of said
dermatitis.
22. A method according to claim 21, wherein said dermatitis induced
by repeated exposure to an antigen is atopic dermatitis.
Description
TECHNICAL FIELD
[0001] The present invention relates to medical applications of a
chymase inhibitor, more particularly relates to a medicament for
the prevention or treatment of dermatitis, which has a chymase
inhibitor as an effective ingredient.
BACKGROUND ART
[0002] Inflammatory reactions are caused by bacteria, viruses, and
other pathogens and by trauma, foreign matter, etc. They are
immunoreactions where granulocytes, monocytes, lymphocytes, and
other immune cells expel pathogens or injured tissues or foreign
materials. Dermatitis is an acute or chronic inflammation of the
skin, and in particular, there has been a remarkable rise in atopic
dermatitis in recent year, which is becoming a major problem.
[0003] Atopic dermatitis is a chronic disease in which eczema with
itch is the major condition, with exacerbation and remission
observed by turns. In most cases, the patient or his or her family
has a history of cnidosis or allergic rhinitis and bronchial asthma
and other allergic ailments (J. Allergy Clin. Immunol. 104, S123,
1999). The symptoms of atopic dermatitis are diverse and the causes
are still unclear, but it is believed that the disease is elicted
mainly by various natural substances including ticks, hair,
feathers, bacteria and mycetes, or foods including eggs, milk, or
synthetic products including chemical fibers and detergents, etc.
It is also pointed out that the disorder in the barrier function of
the skin due to dry skin plays an important role in atopic
dermatitis.
[0004] The mechanism of onset of atopic dermatitis is still not
clear. It has been thought that the type I allergic reaction
(immediate allergic reaction), in which IgE and mast cells are
involved, plays an important role in atopic dermatitis, since this
disease is one of hypersensitivity reactions to a series of
antigens, the patients or their family sometimes have other
allergic disorders, and an increase in the serum IgE level is
observed in many cases. However, anti-allergic agents that inhibit
type I allergic reaction are ineffective or exhibit no therapeutic
effect in atopic dermatitis, showing that the involvement of the
type I reaction in the pathogenesis of this disease is only
partial.
[0005] It has been reported, on the other hand, that the patients
with atopic dermatitis show biphasic skin reaction, when they are
exposed to the allergens (J. Allergy Clin. Immunol. 101, 222,
1998). This biphasic skin reaction is, for example, observed in the
case of intradermal administration of an antigen such as Ascalis
extract to animals sensitized to the same antigen (J. Immunol. 131,
1096, 1983). The first reaction, termed early-phase reaction, peaks
1 hr after the antigen challenge. The second reaction, late-phase
reaction, is known to show the maximal response after 8 to 24 hours
(Biol. Pharm. Bull. 18, 239, 1995). Early-phase reaction is
suppressed by antagonists to histamine acceptors, suggesting that
the reaction is induced by IgE and mast cells. In contrast, the
mechanism of late-phase reaction is not necessarily clear, but is
characterized by a remarkable infiltration of eosinophils in the
skin (Int. Arch. Allergy Immunol. 113, 196, 1997), which is the
typical histological feature observed in the patients with atopic
dermatitis (J. Am. Acad. Dermatol. 24, 1101, 1991). Further, the
severity of atopic dermatitis patients is known to be correlated
with the serum level of ECP (eosinophil cationic proteins), and the
number of peripheral eosinophils (Medicina 34, 220, 1997). Further,
in recent years, it has been pointed out that the clinical symptoms
of atopic dermatitis are extremely similar to the symptoms of
contact dermatitis classified as a type IV allergic reaction
(Medicina 34, 220, 1997). These findings suggest the possibility
that a type IV allergic reaction is also involved in the mechanism
of pathogenesis of the disease.
[0006] It is generally known that contact hypersensitivity
reaction, the representative type IV allergic reaction, is induced
by applying hapten such as DNFB (dinitrofluorobenzene) to the mice
that had been sensitized once with the same hapten, but it has been
recently reported that a type I allergic reaction is induced in
addition to a type IV allergic reaction when repeatedly applying
such a hapten to the skin (J. Invest. Dermatol. 105, 749, 1995).
For example, by repeatedly applying hapten, the IgE level in the
blood rises and the time-course of the reaction shifts to that of
type I allergic reaction, by repeating the hapten challenge. In
such an animal model, not only the transit response to hapten
challenge, but also the baseline of skin thickness, the thickness
before the hapten challenge, gradually increases, which seems to be
a feature of chronic dermatitis. These findings suggest that the
dermatitis induced by repeating application of hapten is thought to
be useful as an animal model of atopic dermatitis (Anitex 10, 23,
1998).
[0007] Recently, it has been reported that NC/Nga mice
spontaneously develop dermatitis similar to atopic dermatitis (Int.
Immunol. 9, 461, 1997). NC/Nga mice that are maintained in
conventional, non specific pathogen free environment begin to
exhibit remarkable scratching behavior and erythema after about
seven to eight weeks of age, then exhibit hemorrhaging or sores or
ulceration of the skin with aging. Further, they exhibit symptoms
resembling the clinical observations of atopic dermatitis in humans
such as drying or thickening of the skin. Other strains of mice
such as BALB/c do not suffer from similar dermatitis even if made
to cohabitate with NC/Nga mice, suggesting that this dermatitis is
considered specific to NC/Nga mice (Saishin Igaku (Latest
Medicine), 53, 2848, 1998). Further, when raising these mice under
specific paphogen free (SPF) environment, no skin abnormalities are
observed at all, raising the possibility that some sort of
environmental factors are involved in the onset of dermatitis in
these mice. When NC/Nga mice raised under an SPF environment are
repeatedly painted with hapten, only the delayed type
hypersensitivity reaction called contact dermatitis is caused in
the initial period of sensitization, but with the increase in
sensitization, conditions similar to atopic dermatitis are observed
(CRJ Letters 11, 1, 1998). Therefore, while the natural stimulant
for the spontaneous dermatitis in these mice is still not clear, it
is clear that repeated exposure to some sort of antigen under the
natural environment is an important factor. Thus, these mice are
extremely useful as a model for atopic dermatitis spontaneously
caused by repeated exposure to an allergen that would be present in
the air.
[0008] The most effective medicament for the treatment of atopic
dermatitis is a steroid ointment (J. Allergy Clin. Immunol. 104,
S123, 1999). Use of such steroid ointment, however, requires
careful selection of the medicament used according to the location
of application and timing. If the method of use is not appropriate,
no effect will be manifested or the condition will conversely
deteriorate. Further, when a steroid ointment is used over a long
period, side effects such as atrophy and rosacea occur. Further, if
stopping use of this medicament mid way, the phenomenon of rebound,
that is, remarkable deterioration of the skin symptoms, is
sometimes observed.
[0009] In addition to steroid ointment, histamine antagonists and
anti-allergic agents have been used for treatment of atopic
dermatitis. Histamine antagonists are effective in the sense of
eliminating ichiness, but do not lead to a cure of this disease.
Anti-allergic agents such as tranilast, ketotifen, oxatomide, and
azelastine hydrochloride are ineffective against conditions of
atopic dermatitis, or the effect is little, if any. This is
believed to be due to the fact that these drugs have a suppressive
action on a type I allergic reaction, but exhibit almost no effect
on the actions of eosinophils or type IV allergic reaction (Jap. J.
Pharmacol. 63, 73, 1993, Jap. J. Pharmacol. 51, 93, 1989). Recently
ointment of tacrolimus, an immunosuppressive, has been developed as
a medicament for treatment of atopic dermatitis (J. Allergy Clin.
Immunol. 104, S126, 1999), but various side effects due to
suppression of the immunoreaction by use of this medicament cannot
be avoided. Taken together, it is difficult to say that any of the
existing medicaments are sufficiently satisfactory in respect to
efficacy and side effects, and development of a medicament superior
in efficacy and safety is desirable.
[0010] On the other hand, chymase is a serine protease stored in
mast cell granules, and widely present in tissue such as the skin,
heart, vascular walls, intestines, etc. (Mast Cell Proteases in
Immunology and Biology; Caughey, G. H., Ed; Marcel Dekker, Inc.;
New York, 1995). It has been reported long ago that chymase acts on
rat peritoneal mast cells and causes degranulation (J. Immunol.
136, 3812, 1986) and that a chymase inhibitor suppresses the Ig-E
demiated mast cell degranulation (Biochem. Int. 10, 863, 1985) and
has been pointed out that chymase is involved in the function of
mast cells. Recently, it has been reported that administration of
human chymase induces infiltation of leukocytes including
eosinophils in mice as well as guinea pigs (Br. J. Pharmacol, 125,
1491, 1998), that human chymase acts on the precursor of IL-1.beta.
(Interleukin 1.beta.) and converts it to active type IL-1.beta. (J.
Exp. Med. 174, 821, 1991), and that human chymase has the action of
partially digesting membrane-bound stem cell factor (SCF) and
converting it to soluble SCF (Proc. Natl. Acad. Sci. U.S.A. 94,
9017, 1997), etc. These findings suggest the possibility that
chymase has some sort of role in allergic diseases such as atopic
dermatitis. However, it is difficult to say that the
pathophysiological role of chymase has been elucidated by these
studies. At the present time, an energetic search is going on for
substances which can inhibit the activity of chymase in vivo with
the aim of clarifying the role of chymase in various diseases and
the possibility of chymase inhibitors as pharmaceuticals.
[0011] There are chymase inhibitors such as low molecular weight
chymase inhibitors such as shown in textbooks (Protease Inhibitors;
Barrett et al., Eds; Elssevier Science B. V.; Amsterdam, 1996),
.alpha.-keto acid derivatives reported as peptide type inhibitors
(WO93-25574, Proc. Natl. Acad. Sci. USA, 1995, 92, 6738),
.alpha.-difluoro-.beta.-keto acid derivatives (Japanese Unexamined
Patent Publication (Kokai) No. 9-124691), tripeptide inhibitors
(WO93-03625), phosphoric acid derivatives (Oleksyszyn et al.,
Biochemistry 30, 485, 1991), peptide like inhibitors such as
trifluoromethylketone derivatives (WO96-33974, Japanese Unexamined
Patent Publication (Kokai) No. 10-53579) and acetoamide derivatives
(Japanese Unexamined Patent Publication (Kokai) No. 10-7661,
Japanese Unexamined Patent Publication (Kokai) No. 10-53579,
Japanese Unexamined Patent Publication (Kokai) No. 11-246437,
WO99-41277, WO98-18794, WO96-39373), non-peptide type inhibitors
such as triazine derivatives (Japanese Unexamined Patent
Publication (Kokai) No. 8-208654 and Japanese Unexamined Patent
Publication (Kokai) No. 10-245384), phenol ester derivatives
(Japanese Unexamined Patent Publication (Kokai) No. 10-87567),
cephem derivatives (Japanese Unexamined Patent Publication (Kokai)
No. 10-87493), isoxazole derivatives (Japanese Unexamined Patent
Publication (Kokai) No. 11-1479), imidazolidine derivatives
(WO96-04248), hydantoin derivatives (Japanese Unexamined Patent
Publication (Kokai) No. 9-31061), quinazoline derivatives
(WO97-11941), etc. have been reported, but no satisfactory
medicament or treatment method using inhibition of the activity of
chymase as a strategy for treatment has yet been established.
DISCLOSURE OF THE INVENTION
[0012] The object of the present invention is to provide a side
effect-free, safe medicament for prevention or treatment of
dermatitis such as atopic dermatitis, which exhibits biphasic skin
reaction or is induced by repeated exposure to an antigen, which
suppresses the progress of the condition and improves the quality
of life of the patient.
[0013] The present inventors engaged in intensive studies taking
note of the fact that atopic dermatitis exhibits biphasic skin
reaction and that late-phase reaction plays an important role in
the condition. As a result, the present inventors discovered that a
chymase inhibitor acts to alleviate the late-phase reaction in the
biphasic skin reaction of dermatitis and that it is effective even
against dermatitis caused by repeated exposure to an antigen, and
thereby completed the present invention.
[0014] That is, according to the present invention, there is
provided a medicament for prevention or treatment of dermatitis
exhibiting biphasic skin reaction containing a chymase inhibitor as
its effective ingredient.
[0015] According to the present invention, further, there is
provided a medicament for alleviation of late-phase reaction of
dermatitis exhibiting biphasic skin reaction containing a chymase
inhibitor as its effective ingredient.
[0016] According to the present invention, further, there is
provided a medicament for the prevention or treatment of dermatitis
induced by repeated exposure to an antigen containing a chymase
inhibitor as its effective ingredient.
[0017] According to the present invention, there is provided a
pharmaceutical composition for the prevention or treatment of
dermatitis exhibiting biphasic skin reaction containing a chymase
inhibitor in an amount alleviating the late-phase reaction and a
pharmaceutically acceptable vehicle.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] FIG. 1 is a graph showing the time-course of the skin
reaction in Ascaris-induced mouse biphasic skin reaction in Example
2.
[0019] FIGS. 2A, 2B, and 2C are graphs showing the effects of
chymase inhibitor (FIG. 2A) and control drugs, prednisolone (FIG.
2B) and diphenhydramine (FIG. 2C), in Ascaris-induced biphasic skin
reaction in Example 3.
[0020] FIGS. 3A and 3B are graphs showing the time-course of the
skin reaction when the human chymase (FIG. 3A) or histamine (FIG.
3B) was administered intradermally to mice in Example 4.
[0021] FIGS. 4A and 4B are graphs showing dose-dependency of
chymase in the skin reaction when human chymase was administered
intradermally to mice in Example 4 (FIG. 4A, 1 hour after the
chymase administration, FIG. 4B after 16 hours after the chymase
administration).
[0022] FIG. 5 is a graph showing the effect of heat treatment on
the action of human chymase on inducing dermatitis in Example
5.
[0023] FIGS. 6A to 6E are photos showing the histologically
analyzing the dermatitis induced by intradermal injection of human
chymase in Example 6, wherein FIG. 6A, normal mice; FIG. 6B,
dermatitis induced by Ascaris extract (after 1 hour); FIG. 6C,
dermatitis induced by Ascaris extract (after 24 hours), FIG. 6D,
dermatitis induced by human chymase (after 1 hour); FIG. 6E
dermatitis induced by human chymase (after 24 hours).
[0024] FIGS. 7A and 7B are graphs showing the skin reaction when
human chymase administered intradermally to mast cell-deficient
mice in Example 7 (FIG. 7A, the reaction after 1 hour; FIG. 7B the
reaction after 16 hour).
[0025] FIG. 8A is a view showing the concentration dependency of
the effect of human chymase on migration of human polymorphonuclear
leukocytes (PMN) in vitro in Example 8. FIG. 8B is a graph showing
the effect of a chymase inhibitor on the chymase-induced PMN
migration in Example 8.
[0026] FIG. 9 is a graph showing the time-course of the increase in
ear thickness in the dermatitis induced by repeated application of
hapten in Example 9.
[0027] FIG. 10 is a graph showing the change in chymase-like
activity in the ear in the dermatitis induced by repeated
application of hapten in Example 9.
[0028] FIGS. 11A to 11D are views showing the effects of chymase
inhibitor in the dermatitis induced by repeated application of
hapten in Example 10 (FIG. 11A, control drug, prednisolone, FIG.
11B to FIG. 11D, Compound 35, Compound 34, and Compound 18,
respectively).
[0029] FIG. 12 is a graph showing the effect of a chymase inhibitor
on the increase in the number of eosinophils in the dermatitis
induced by repeated application of hapten in Example 11.
[0030] FIG. 13 is a graph showing the effect of a chymase inhibitor
on the increase in the number of mast cells in the dermatitis
induced by repeated application of hapten in Example 12.
[0031] FIGS. 14A, 14B, and 14C are representative photos showing
the effect of a chymase inhibitor on the increase in the number of
eosinophils in the dermatitis induced by repeated application of
hapten in Example 12.
[0032] FIG. 15 is a graph showing the time-course of skin thickness
when human chymase was repeatedly injected intradermally to mice in
Example 13.
[0033] FIG. 16 is a graph showing the time-course of the number of
eosinophils in the skin when human chymase was repeatedly injected
intradermally to mice in Example 4.
[0034] FIG. 17 is a graph showing the time-course of histamine
content in the skin when human chymase was repeatedly injected
intradermally to mice in Example 15.
[0035] FIGS. 18A and 18B are photos showing representative photos
of immunohistological analysis for SCF expression when human
chymase was repeatedly injected intradermally to mice in Example 16
(FIG. 18A, normal skin; FIG. 18B, chymase-injected)
[0036] FIG. 19 is a graph showing the effect of human chymase on
SCF expression in human keratinocytes in vitro in Example 17.
[0037] FIGS. 20A and 20B are graphs showing the effect of a chymase
inhibitor on clinical skin scores in NC/Nga mice in Example 18.
FIG. 20A, at the start of the experiment;
[0038] FIG. 20B, 35 days after the start of the chymase
administration.
[0039] FIG. 21 is a graph showing the effect of a chymase inhibitor
on histological scores in NC/Nga mice in Example 18.
[0040] FIGS. 22A and 22B are graphs showing the effect of a chymase
inhibitor on the number of skin mast cells in NC/Nga mice in
Example 18. FIG. 22A, the ear; FIG. 22B, the back.
[0041] FIGS. 23A and 23B are graphs showing the effect of a chymase
inhibitor on the number of skin eosinophils in NC/Nga mice in
Example 18 FIG. 23A, the ear; FIG. 23B, the back.
BEST MODE FOR CARRYING OUT THE INVENTION
[0042] The chymase inhibitor able to be used in the present
invention can be selected as a substance able to exhibit an action
inhibiting the activity of chymase by the use of methods workable
by persons skilled in the art. As the method of selection, for
example, the method of the later explained Example 1 may be
mentioned. The compounds obtained in this way include known
compounds previously reported as chymase inhibitors, for example,
the low molecular weight chymase inhibitors such as shown in
textbooks (Protease Inhibitors; Barrett et al., Eds; Elssevier
Science B. V.; Amsterdam, 1996), .alpha.-keto acid derivatives
reported as peptide type inhibitors (WO93-25574, Proc. Natl. Acad.
Sci. USA, 1995, 92, 6738), .alpha.,.alpha.-difluoro-.beta.-keto
acid derivatives (Japanese Unexamined Patent Publication (Kokai)
No. 9-124691), tripeptide inhibitors (WO93-03625), phosphoric acid
derivatives (Oleksyszyn et al., Biochemistry 30, 485, 1991),
peptide like inhibitors such as trifluoromethylketone derivatives
(WO96-33974, Japanese Unexamined Patent Publication (Kokai) No.
10-53579) and acetoamide derivatives (Japanese Unexamined Patent
Publication (Kokai) No. 10-7661, Japanese Unexamined Patent
Publication (Kokai) No. 10-53579, Japanese Unexamined Patent
Publication (Kokai) No. 11-246437, WO99-41277, WO98-18794,
WO96-39373), non-peptide type inhibitors such as triazine
derivatives (Japanese Unexamined Patent Publication (Kokai) No.
8-208654 and Japanese Unexamined Patent Publication (Kokai) No.
10-245384), phenol ester derivatives (Japanese Unexamined Patent
Publication (Kokai) No. 10-87567), cephem derivatives (Japanese
Unexamined Patent Publication (Kokai) No. 10-87493), isoxazole
derivatives (Japanese Unexamined Patent Publication (Kokai) No.
11-1479), imidazolidine derivatives (WO96-04248), hydantoin
derivatives (Japanese Unexamined Patent Publication (Kokai) No.
9-31061), quinazoline derivatives (WO97-11941), etc., but as a
typical examples of a preferable chymase inhibitor, a compound of
the following formula (I) and its pharmaceutically acceptable salts
may be mentioned.
##STR00002##
wherein, the ring A represents an aryl group;
[0043] R.sup.1 represents a hydroxyl group, an amino group, a
C.sub.1 to C.sub.4 lower alkylamino group which may be substituted
with a carboxylic acid group, a C.sub.7 to C.sub.10 lower
aralkylamino group which may be substituted with a carboxylic acid
group, an amino group acylated with a C.sub.1 to C.sub.4 lower
aliphatic acid which may be substituted with a carboxylic acid
group, an amino group acylated with an aromatic ring carboxylic
acid which may be substituted with a carboxylic acid group, an
amino group acylated with a heteroaromatic ring carboxylic acid
which may be substituted with a carboxylic acid group, an amino
group sulfonylated with a C.sub.1 to C.sub.4 lower alkanesulfonic
acid which may be substituted with a carboxylic acid group, an
amino group sulfonylated with an aromatic ring sulfonic acid which
may be substituted with a carboxylic acid group, an amino group
sulfonylated with a heteroaromatic ring sulfonic acid which may be
substituted with a carboxylic acid group, a C.sub.1 to C.sub.4
lower alkyl group substituted with a carboxylic acid group, or a
C.sub.2 to C.sub.4 lower alkylene group which may be substituted
with a carboxylic acid group;
[0044] R.sup.2 and R.sup.3 may be the same or different and
represent a hydrogen atom, an unsubstituted or substituted C.sub.1
to C.sub.4 lower alkyl group, a halogen atom, a hydroxyl group, a
C.sub.1 to C.sub.4 lower alkoxyl group, an amino group, an
unsubstituted or substituted C.sub.1 to C.sub.4 lower alkylamino
group, an unsubstituted or substituted C.sub.7 to C.sub.10
aralkylamino group, an amino group acylated with a C.sub.1 to
C.sub.4 lower aliphatic acid which may be substituted with a
carboxylic acid group, an amino group acylated with an aromatic
ring carboxylic acid which may be substituted with a carboxylic
acid group, an amino group acylated with a heteroaromatic ring
carboxylic acid which may be substituted with a carboxylic acid
group, an amino group sulfonylated with a C.sub.1 to C.sub.4 lower
alkanesulfonic acid which may be substituted with a carboxylic acid
group, an amino group sulfonylated with an aromatic ring sulfonic
acid which may be substituted with a carboxylic acid group, an
amino group sulfonylated with a heteroaromatic ring sulfonic acid
which may be substituted with a carboxylic acid group, or a
carboxylic acid group or when the ring A is a benzene ring, R.sup.1
and R.sup.2 may form, together with the substituting benzene ring,
a fused heterocyclic ring which may be substituted with a
carboxylic acid and in which the carbon atom in the ring may form a
carbonyl group and R.sup.3 is the same as defined above; and
[0045] X represents a hydrogen atom, a C.sub.1 to C.sub.4 lower
alkyl group, a C.sub.1 to C.sub.4 lower alkoxy group, a halogen
atom, a hydroxyl group, an amino group, or a nitro group.
[0046] In the general formula (I), preferable examples of the aryl
group represented by the ring A are a benzene ring and a
naphthalene ring.
[0047] Preferable examples of the C.sub.1 to C.sub.4 lower
alkylamino group which may be substituted with the carboxylic acid
group and the C.sub.7 to C.sub.12 lower aralkylamino group which
may be substituted with a carboxylic acid group represented by
R.sup.1 are a methylamino group, an ethylamino group, a propylamino
group, a butylamino group, a carboxymethylamino group, a
carboxyethylamino group, a carboxypropylamino group, a
carboxybutylamino group, a benzylamino group, a phenetylamino
group, a phenylpropylamino group, a phenylbutylamino group, a
carboxybenzylamino group, a carboxyphenetylamino group, a
carboxyphenylpropylamino group, a carboxyphenylbutylamino group,
etc.
[0048] Preferable examples of the amino group acylated with a
C.sub.1 to C.sub.4 lower aliphatic acid which may be substituted
with a carboxylic acid group, the amino group acylated with an
aromatic ring carboxylic acid which may be substituted with a
carboxylic acid group, and the amino group acylated with a
heteroaromatic ring carboxylic acid which may be substituted with a
carboxylic acid group represented by R.sup.1 are a formylamino
group, an acetylamino group, a propionylamino group, a butyrylamino
group, a benzoylamino group, a naphthoylamino group, a
pyridinecarbonylamino group, a pyrrolecarbonylamino group, a
carboxyacetylamino group, a carboxypropionylamino group, a
carboxybutyrylamino group, a carboxybenzoylamino group, a
carboxynaphthoylamino group, a carboxypyridinecarbonylamino group,
a carboxypyrrolecarbonylamino group, etc.
[0049] Preferable examples of the amino group sulfonylated with a
C.sub.1 to C.sub.4 lower alkanesulfonic acid which may be
substituted with a carboxylic acid group, the amino group
sulfonylated with an aromatic ring sulfonic acid which may be
substituted with a carboxylic acid group, and the amino group
sulfonylated with a heteroaromatic ring sulfonic acid which may be
substituted with a carboxylic acid group represented by R.sup.1 are
a methanesulfonylamino group, an ethanesulfonylamino group, a
propanesulfonylamino group, a butanesulfonylamino group, a
benzenesulfonylamino group, a naphthalenesulfonylamino group, a
pyridinesulfonylamino group, a pyrrolesulfonylamino group, a
carboxymethanesulfonylamino group, a carboxyethanesulfonylamino
group, a carboxypropanesulfonylamino group, a
carboxybutane-sulfonylamino group, a carboxybenzenesulfonylamino
group, a carboxynaphthalenesulfonylamino group, a
carboxypyridinesulfonylamino group, a carboxypyrrolesulfonylamino
group, etc.
[0050] Preferable examples of the C.sub.1 to C.sub.4 lower alkyl
group substituted with a carboxylic acid group represented by
R.sup.1 are an acetic acid group, a propionic acid group, a butyric
acid group, a valeric acid group, etc.
[0051] Preferable examples of the C.sub.2 to C.sub.4 lower alkylene
group substituted with a carboxylic acid group represented by
R.sup.1 are an acrylic acid group, a crotonic acid group, etc.
[0052] Preferable examples of the unsubstituted or substituted
C.sub.1 to C.sub.4 lower alkyl group represented by R.sup.2 or
R.sup.3 are a straight-chain alkyl group such as a methyl group, an
ethyl group, a n-propyl group, and a n-butyl group and a branched
alkyl group such as an isopropyl group, a sec-butyl group, and a
t-butyl group.
[0053] Preferable examples of the substituent group of the C.sub.1
to C.sub.4 lower alkyl group are a carboxylic acid group, a halogen
atom such as a fluorine atom and a chlorine atom, a C.sub.1 to
C.sub.4 lower alkoxy group, an amino group, a methylamino group, a
dimethylamino group, a carboxymethylamino group, a
carboxyethylamino group, etc.
[0054] Preferable examples of the halogen atom represented by
R.sup.2 or R.sup.3 are a fluorine atom, a chlorine atom, a bromine
atom and an iodine atom.
[0055] Preferable examples of the C.sub.1 to C.sub.4 lower alkoxyl
group represented by R.sup.2 or R.sup.3 are a straight-chain
alkyloxy group such as a methoxy group, an ethoxy group, a
n-propyloxy group, and a n-butoxy group and a branched alkyloxy
group such as an isopropyloxy group, a sec-butoxy group, and a
t-butoxy group.
[0056] Preferable examples of the unsubstituted or substituted
C.sub.1 to C.sub.4 lower alkylamino group represented by R.sup.2 or
R.sup.3 are a methylamino group, an ethylamino group, a propylamino
group, a butylamino group, etc.
[0057] Preferable examples of the substituent group of the C.sub.1
to C.sub.4 lower alkylamino group are a carboxylic acid group, a
halogen atom such as a fluorine atom and a chlorine atom, a C.sub.1
to C.sub.4 lower alkoxyl group, etc.
[0058] Preferable examples of the unsubstituted or substituted
C.sub.7 to C.sub.12 lower aralkylamino group represented by R.sup.2
or R.sup.3 are a benzylamino group, a phenetylamino group, a
phenylpropylamino group, a phenylbutylamino group, etc.
[0059] Preferable examples of the substituent group of the
aralkylamino group are a carboxylic acid group, a halogen atom such
as a fluorine atom and a chlorine atom, a C.sub.1 to C.sub.4 lower
alkoxyl group, etc.
[0060] Preferable examples of the amino group acylated with a
C.sub.1 to C.sub.4 lower aliphatic acid which may be substituted
with a carboxylic acid group, the amino group acylated with an
aromatic ring carboxylic acid which may be substituted with a
carboxylic acid group, and the amino group acylated with a
heteroaromatic ring carboxylic acid which may be substituted with a
carboxylic acid group represented by R.sup.2 or R.sup.3 are a
formylamino group, an acetylamino group, a propionylamino group, a
butyrylamino group, a benzoylamino group, a naphthoylamino group, a
pyridinecarbonylamino group, a pyrrolecarbonylamino group, a
carboxyacetylamino group, a carboxypropionylamino group, a
carboxybutyrylamino group, a carboxybenzoylamino group, a
carboxynaphthoylamino group, a carboxypyridinecarbonylamino group,
a carboxypyrrolecarbonylamino group, etc.
[0061] Preferable examples of the amino group sulfonylated with a
C.sub.1 to C.sub.4 lower alkanesulfonic acid which may be
substituted with a carboxylic acid group, the amino group
sulfonylated with an aromatic ring sulfonic acid which may be
substituted with a carboxylic acid group, and the amino group
sulfonylated with a heteroaromatic ring sulfonic acid which may be
substituted with a carboxylic acid group represented by R.sup.2 or
R.sup.3 are a methanesulfonylamino group, an ethanesulfonylamino
group, a propanesulfonylamino group, a benzenesulfbnylamino group,
a naphthalenesulfonylamino group, a pyridinesulfonylamino group, a
pyrrolesulfonylamino group, a carboxymethanesulfonylamino group, a
carboxyethanesulfonylamino group, a carboxypropanesulfonylamino
group, a carboxybenzenesulfonylamino group, a
carboxynaphthalenesulfonylamino group, a
carboxypyridine-sulfonylamino group, a carboxypyrrolesulfonylamino
group, etc.
[0062] Preferable examples of the fused heterocyclic ring which may
be substituted with a carboxylic acid and in which the carbon atom
in the ring may form a carbonyl group which R.sup.1 and R.sup.2
form together with the substituting benzene ring when the ring A is
a benzene ring, are a tetrahydroquinoline ring and a benzoxazine
ring, for example, a tetrahydroquinoline, a benzoxazine, a
quinoxaline, a benzodioxane, a carboxytetrahydroquinoline, a
carboxybenzoxazine, a carboxyquinoxaline, a carboxybenzodioxane,
etc.
[0063] Preferable examples of the C.sub.1 to C.sub.4 lower alkyl
group represented by X are a straight-chain alkyl group such as a
methyl group, an ethyl group, a n-propyl group, and a n-butyl group
and a branched alkyl group such as an isopropyl group, a sec-butyl
group, and a t-butyl group.
[0064] Preferable examples of the C.sub.1 to C.sub.4 lower alkoxyl
group represented by X are a straight-chain alkyloxy group such as
a methoxy group, an ethoxy group, a n-propyloxy group, and a
n-butoxy group and a branched alkyloxy group such as an
isopropyloxy group, a sec-butoxy group, and a t-butoxy group.
[0065] Preferable examples of the halogen atom represented by X,
are a fluorine atom, a chlorine atom, a bromine atom and an iodine
atom.
[0066] Further, examples of a pharmaceutically acceptable salts are
an acid salt such as a hydrochloric acid salt, a methanesulfonic
acid salt, and a trifluoroacetic acid salt and an alkali metal salt
such as a sodium salt and a potassium salt.
[0067] The other typical examples of the preferable chymase
inhibitor are a quinazoline derivative having the formula (II) and
its pharmaceutically acceptable salts:
##STR00003##
wherein, the ring B represents a benzene ring, a pyridine ring, a
pyrrole ring, or a pyrazole ring, m represents 0, 1, or 2,
[0068] Y represents a hydroxy group, a nitro group, a halogen atom,
a C.sub.1 to C.sub.4 lower alkyl group which may be substituted
with a halogen atom, a C.sub.1 to C.sub.4 lower alkoxy group which
may be substituted with a halogen atom, or a C.sub.7 to C.sub.12
aralkyloxy group, or Y represents a group forming a naphthalene
ring or a quinoline ring together with the benzene ring which is
shown as substituted with said Y,
[0069] R.sup.5 and R.sup.6 are the same or different and represent
a hydrogen atom, a halogen atom, a C.sub.1 to C.sub.4 lower alkyl
group which may be substituted with a halogen atom, a nitro group,
a cyano group, a pyrazolyl group, a tetrazolyl group, a carboxyl
group which may be esterified with a C.sub.1 to C.sub.4 lower alkyl
group or an allyl group, or a C.sub.1 to C.sub.4 lower alkoxy group
which may be substituted with one or more substituent groups
selected from the group consisting of a halogen atom, a morpholino
group, a phenylpiperazinyl group, and a carboxyl group which may be
esterified with a C.sub.1 to C.sub.4 lower alkyl group or an allyl
group, or, when the ring B represents a benzene ring, R.sup.5 and
R.sup.6 represent a group forming a naphthalene ring or a quinoline
ring together with the benzene ring which is shown as substituted
with said R.sup.5 and R.sup.6, and
[0070] Z represents a hydrogen atom, a C.sub.1 to C.sub.4 lower
alkyl group which may be substituted with a halogen atom, a C.sub.2
to C.sub.5 alkenyl group, an unsubstituted or substituted aralkyl
group, an unsubstituted or substituted aromatic heterocyclic alkyl
group, a carboxylmethyl group which may be esterified with a
C.sub.1 to C.sub.4 lower alkyl group or an allyl group, a
carbonylmethyl group which is amidated with a primary or secondary
or cyclic amine, an unsubstituted or substituted arylcarbonylmethyl
group, or an unsubstituted or substituted aralkyloxymethyl
group.
[0071] In the general formula (II), the preferable examples of the
halogen atom for Y are fluorine, chlorine, bromine, or iodine. The
examples of the lower alkyl group of the C.sub.1 to C.sub.4 lower
alkyl group for Y, which is substituted with a halogen atom, are
straight chain alkyl groups such as a methyl group, ethyl group,
n-propyl group and n-butyl group, and branched alkyl groups such as
an isopropyl group, sec-butyl group and t-butyl group, and, while
the examples of the halogen atom of the C.sub.1 to C.sub.4 lower
alkyl group for Y which is substituted with a halogen atom, are
fluorine, chlorine, bromine and iodine. The examples of the lower
alkoxy group of the C.sub.1 to C.sub.4 lower alkoxy group for Y,
which is substituted with a halogen atom, are straight chain alkoxy
groups such as a methoxy group, ethoxy group, n-propoxy group and
n-butoxy group, and branched alkoxy groups such as an isopropoxy
group, sec-butoxy group and t-butoxy group, while the examples of
the halogen atom of the C.sub.1 to C.sub.4 lower alkoxy group for
Y, which is substituted with a halogen atom, are fluorine,
chlorine, bromine, and iodine. The examples of the C.sub.7 to
C.sub.12 aralkyloxy group for Y are a benzyloxy group, phenethyloxy
group, phenylpropoxy group and naphthylethyloxy group, etc,
preferably the benzyloxy group.
[0072] The preferable examples of the halogen atom for R.sup.5 or
R.sup.6 are fluorine, chlorine, bromine, or iodine. The examples of
the lower alkyl group of the C.sub.1 to C.sub.4 lower alkyl group
for R.sup.5 or R.sup.6, which is substituted with a halogen atom,
are straight chain alkyl groups such as a methyl group, ethyl
group, n-propyl group and n-butyl group, and branched alkyl groups
such as an isopropyl group, sec-butyl group and t-butyl group,
while the examples of the halogen atom of the C.sub.1 to C.sub.4
lower alkyl group for R.sup.5 or R.sup.6, which is substituted with
a halogen atom, are fluorine, chlorine, bromine, or iodine. The
preferable examples of the C.sub.1 to C.sub.4 lower alkyl group of
the carboxyl group for R.sup.5 or R.sup.6, which may be esterified
with the C.sub.1 to C.sub.4 lower alkyl group or an allyl group,
are straight chain alkyl groups such as a methyl group, ethyl
group, n-propyl group and n-butyl group, and branched alkyl groups
such as an isopropyl group, sec-butyl group and t-butyl group. The
examples of the alkoxy group of the C.sub.1 to C.sub.4 lower alkoxy
group for R.sup.5 or R.sup.6, which is substituted with one or more
substituent groups selected from the group consisting of a halogen
atom, a morpholino group, a phenylpiperazinyl group, and a carboxyl
group which may be esterified with a C.sub.1 to C.sub.4 lower alkyl
group or an allyl group, are straight chain alkoxy groups such as a
methoxy group, ethoxy group, n-propoxy group and n-butoxy group,
and branched alkoxy groups such as an isopropoxy group, sec-butoxy
group and t-butoxy group. The examples of the halogen atom shown as
the above substituent group, are fluorine, chlorine, bromine, or
iodine and the preferable examples of the C.sub.1 to C.sub.4 lower
alkyl group of the carboxyl group which is esterified with a
C.sub.1 to C.sub.4 lower alkyl group or an allyl group shown as the
above substituent group, are a methyl group, ethyl group, n-propyl
group, n-butyl group, and other straight chain alkyl groups and an
isopropyl group, sec-butyl group, t-butyl group, and other branched
alkyl groups.
[0073] The examples of the lower alkyl group of the C.sub.1 to
C.sub.4 lower alkyl group shown as Z which may be substituted with
halogen, are straight chain alkyl groups such as a methyl group,
ethyl group, n-propyl group and n-butyl group, and branched alkyl
groups such as an isopropyl group, sec-butyl group and t-butyl
group, while the examples of the halogen atom of the C.sub.1 to
C.sub.4 lower alkyl group which may be substituted with the halogen
atom are fluorine, chlorine, bromine, or iodine. The examples of
the C.sub.2 to C.sub.5 alkenyl group for Z are an allyl group,
propenyl group, isopropenyl group, butenyl group, etc.
[0074] The examples of the aralkyl group of an unsubstituted or
substituted aralkyl group shown as Z are a C.sub.7 to C.sub.12
aralkyl group, preferably a benzyl group, phenethyl group,
phenylpropyl group, or naphthylethyl group. The preferable examples
of the substituent group of an unsubstituted or substituted aralkyl
group are a carboxyl group which may be esterified with a C.sub.1
to C.sub.4 lower alkyl group or an allyl group, a cyano group, a
nitro group, a carbonyl group amidized with primary amine, an amine
group which may be amidized with a carboxylic acid or an amino
acid, and a guanidino group which may be substituted with a lower
alkoxycarbonyl group. The examples of the lower alkyl group of the
carboxyl group, which may be esterified with a C.sub.1 to C.sub.4
lower alkyl group or an allyl group are straight alkyl groups such
as a methyl group, ethyl group, n-propyl group and n-butyl group,
and branched alkyl groups such as an isopropyl group, sec-butyl
group and t-butyl group. The examples of the primary amine of the
carbonyl group amidized with primary amine are a chain C.sub.1 to
C.sub.4 lower alkylamine or those which may be substituted with
carboxyl group, such as, preferably, methylamine, ethylamine,
isopropylamine and carboxylmethylamine; amines having monocyclic or
polycyclic aromatic hydrocarbon group such as aniline and
naphthylamine; amines having aromatic heterocyclic group such as
aminopyridine, amihopyrrole, and other. The examples of the
carboxylic acid of the amine group which may be amidized with a
carboxylic acid or an amino acid are preferably C.sub.2 to C.sub.5
aliphatic monocarboxylic acids or aliphatic dicarboxylic acids such
as pivalic acid and succinic acid, while the examples of the amino
acid are amino acids, of which carboxyl group may be esterified or
of which amine group may be amidized, such as L-aspartic acid,
.alpha.-O-t-butyl-N-t-butoxycarbonyl-L-aspartic acid and other. The
examples of the guanidino group which may be substituted with a
lower alkoxycarbonyl group are preferably a guadinino group which
may be substituted with a C.sub.2 to C.sub.1 lower alkoxycarbonyl
group such as a guanidino group and
2,3-bis-t-butoxycarbonylguanidino group.
[0075] The examples of the aromatic heterocyclic alkyl group of an
unsubstituted or substituted aromatic heterocyclic alkyl group
shown as Z are thienylalkyl groups such as a 2-thenyl group and a
2-thienylethyl group, furylalkyl groups such as a 2-furfuryl group
and a 2-furylethyl group, pyridylalkyl groups such as a
2-pyridylmethyl group, 3-pyridylmethyl group, 4-pyridylmethyl group
and 4-pyridylethyl group, pyrimidinylalkyl groups such as a
5-pyrimidinylmethyl group, pyrazinylalkyl groups such as a
2-pyrazinylmethyl group, pyridazinylalkyl groups such as a
3-pyridazinylmethyl group tetrazolylalkyl groups such as a
5-tetrazolylmethyl group, isothiazolylalkyl groups such as a
4-isothiazolylmethyl group and a 5-isothiazolylmethyl group,
thiazolylalkyl groups such as a 5-thiazolylmethyl group,
oxazolylalkyl groups such as a 5-oxazolylmethyl group, and
isoxazolylalkyl groups such as a 4-isooxazolylmethyl group and
5-isoxazolylmethyl group. The preferable examples of the
substituent group of an unsubstituted or substituted heterocyclic
alkyl group, are C.sub.1 to C.sub.4 lower alkyl groups such as a
methyl group and ethyl group, and C.sub.1 to C.sub.4 carboxyl lower
alkyl groups such as a carboxylmethyl group and carboxylethyl
group.
[0076] The examples of the lower alkyl group of the carboxymethyl
group which may be esterified with a C.sub.1 to C.sub.4 lower alkyl
group or an allyl group shown as Z are straight chain alkyl groups
such as a methyl group, ethyl group, n-propyl group and n-butyl
group, and branched alkyl groups such as an isopropyl group,
sec-butyl group and t-butyl group.
[0077] The examples of the primary amine of the carbonylmethyl
group which may be amidized with primary or secondary or cyclic
amine shown as Z are chain C.sub.1 to C.sub.4 lower alkylamines or
those which may be substituted with a carboxyl group such as
preferably methylamine, ethylamine, isopropylamine and
carboxylmethylamine, and amines having monocyclic saturated
hydrocarbon group such as a cyclohexylamine, and amines having
monocyclic or polycyclic aromatic hydrocarbon group such as
aniline, a benzylamine and a naphthylamine, and amines having
aromatic heterocyclic group such as an aminopyridine, an
aminomethylpyridine, an aminopyrrole, an aminopyrimidine, an
aminoindole and aminoquinoline, wherein the amines having aromatic
hydrocarbon group or aromatic heterocyclic group may have on its
ring one or more substituent such as
[0078] 1) hydroxy group,
[0079] 2) --OPO (OH).sub.2,
[0080] 3) amino group,
[0081] 4) oxo group,
[0082] 5) halogen atom,
[0083] 6) carboxyl group, which may be esterified with C.sub.1 to
C.sub.4 lower alkyl group such as a methyl group, an ethyl group,
an isopropyl group and a t-butyl group, or an allyl group,
[0084] 7) straight chain or branched C.sub.1 to C.sub.4 lower
alkoxy group such as a methoxy group, an ethoxy group, a n-propoxy
group and t-butoxy group, which may be substituted with a carboxyl
group, which may be esterified with a C.sub.1 to C.sub.4 lower
alkyl group such as a methyl group, an ethyl group, an isopropyl
group and a t-butyl group, or an allyl group,
[0085] 8) straight chain or branched C.sub.1 to C.sub.4 lower alkyl
groups such as a methyl group, an ethyl group, a n-propyl group, a
n-butyl group, an isopropyl group, a sec-butyl group, and t-butyl
group, which may be substituted.
[0086] Further, the preferable examples of the substituent of the
C.sub.1 to C.sub.4 lower alkyl group, which may be substituted, of
the above 8),
[0087] a) a carboxyl group, which may be esterified with C.sub.1 to
C.sub.4 lower alkyl group such as a methyl group, an ethyl group,
an isopropyl group and a t-butyl group, or an allyl group,
[0088] b) piperadinyl group, which may be N-substituted with
carboxy group which is esterified with C.sub.1 to C.sub.4 lower
alkyl group such as a methyl group, an ethyl group, an isopropyl
group and a t-butyl group, or an allyl group,
[0089] c) morpholino group, and
[0090] d) amino group which may be amidized with carboxylic acid or
amino acid
[0091] The examples of the carboxylic acid of amino group of the
above d), which may be amidized with carboxylic acid or amino acid,
are preferably C.sub.2 to C.sub.5 aliphatic mono- or di-carboxylic
acids such as pivalic acid and succinic acid, while the examples of
the amino acid are amino acids of which carboxyl group may be
esterified or of which amino group may be amidized, such as a
L-aspartic acid, an .alpha.-O-t-butyl-N-t-butoxycarbonyl-L-aspartic
acid, and a .beta.-O-t-butyl-N-t-butoxycarbonyl-L-aspartic acid.
Further, the amine having aromatic heterocyclic group may have the
nitrogen atom on its ring, which may be substituted with C.sub.1 to
C.sub.4 lower alkyl group such as a methyl group, and an ethyl
group, or carboxy lower alkyl group, which may be esterified, such
as a carboxylmethyl group and a t-butoxy carbonylmethyl group.
[0092] The examples of the secondary amine of the carbonylmethyl
group shown as Z, which is amidized with primary or secondary or
cyclic amine are di-lower alkylamines such as a dimethylamine and
diethylamine. The examples of the cyclic amine of the
carbonylmethyl group shown as Z, which is amidized with primary or
secondary or cyclic amine are pyrrolidine and piperidine.
[0093] The examples of the arylcarbonylmethyl group of the
unsubstituted or substituted arylcarbonylmethyl group shown as Z
are a phenylcarbonylmethyl group and a naphthylcarbonylmethyl
group, while the preferable examples of the substituent group are a
hydroxy group, a nitro group, halogen atoms such as fluorine,
chlorine, bromine and iodine, straight or branched C.sub.1 to
C.sub.4 lower alkyl groups, which may be substituted with halogen
atom such as methyl group, ethyl group, n-propyl group, n-butyl
group, isopropyl group, sec-butyl group and t-butyl group, straight
or branched C.sub.1 to C.sub.4 lower alkoxy groups, which may be
substituted with halogen atom, such as methoxy group, ethoxy group,
n-propoxy group, n-butoxy group, isopropoxy group, sec-butoxy group
and t-butoxy group.
[0094] The examples of the aralkyloxymethyl group of the
unsubstituted or substituted aralkyloxymethyl group shown as Z, are
preferably C.sub.8 to C.sub.13 aralkyloxymethyl groups such as a
benzyloxy methyl group, phenethyloxymethyl group and
naphthylethyloxymethyl group, while the preferable examples of the
substituent group are a hydroxy group, a nitro group, halogen atoms
such as fluorine, chlorine, bromine and iodine, straight or
branched C.sub.1 to C.sub.4 lower alkyl groups which may be
substituted with halogen atom, such as methyl group, ethyl group,
n-propyl group, n-butyl group, isopropyl group, sec-butyl group and
t-butyl group, straight or branched C.sub.1 to C.sub.4 lower alkoxy
groups, which may be substituted with halogen atom, such as methoxy
group, ethoxy group, n-propoxy group, n-butoxy group, isopropoxy
group, sec-butoxy group and t-butoxy group.
[0095] Further, the examples of a pharmaceutically acceptable salt
are acid salts such as a chlorate and nitrate and alkali metal
salts such as a sodium salt, potassium salt.
[0096] The quinazoline derivative having the formula (I) according
to the present invention may, for example, be synthesized by the
following Synthesis Method (A) or (B).
[0097] Synthesis Method (A)
[0098] A compound having the formula (I-1):
##STR00004##
wherein the ring A is the same as defined above and R.sup.1,
R.sup.2' and R.sup.3' represent R.sup.1, R.sup.2 and R.sup.3, which
may be protected with a protecting group, respectively, and
R.sup.1, R.sup.2 and R.sup.3 represent the same as defined
above
[0099] is reacted with an anthranilic acid derivative having the
formula (I-2):
##STR00005##
wherein X' represents X, which may be protected with a protecting
group, and X represents the same as defined above
[0100] using the method described, for example, in JP-A-6-199839 to
obtain a sulfonylurea derivative having the formula (I-3):
##STR00006##
wherein the ring A, R.sup.1', R.sup.2', R.sup.3' and X' represent
the same as defined above,
[0101] then, a condensing agent for example,
1,1'-carbonyldiimidazole (hereinafter referred to as CDI) is used
to obtain the quinazoline ring, and if necessary, the protecting
groups of R.sup.1, R.sup.2, R.sup.3 and X are deprotected.
[0102] In this reaction, when R.sup.1, R.sup.2 or R.sup.3
represents a group containing a hydroxyl group, an amino group, or
a carboxylic acid group, R.sup.1, R.sup.2 or R.sup.3 may be
optionally protected by a protecting group such as a
benzyloxycarbonyl group, a t-butoxycarbonyl group, a benzyl group,
an allyl group, a t-butyl group, etc. When X represents a hydroxyl
group or an amino group, X may be optionally protected with a
protecting group such as a benzyloxycarbonyl group, a
t-butoxycarbonyl group, a benzyl group, an allyl group, a t-butyl
group, etc.
[0103] The compound having the formula (I-1) used in this reaction
includes a commercially available or known compound or a compound
which can be synthesized by a known method may be used. For
example, using the synthesis method described in the specification
of European Patent No. 0269141, it is possible to use a compound
which can be synthesized from the corresponding sulfonamide
derivative using chlorosulfonyl isocyanate. For example, it is
possible to use 3-allyloxycarbonyl-methylbenzenesulfonyl
isocyanate, 4-allyloxycarbonyl-methylbenzenesulfonyl isocyanate,
4-allyloxybenzenesulfonyl isocyanate, etc.
[0104] As the anthranilic acid derivative having the formula (I-2)
used for this reaction, a commercially available or known compound
or a compound which can be synthesized by a known method may be
used. For example, anthranilic acid, 4-chloroanthranilic acid,
4-methoxyanthranilic acid, 5-chloroanthranilic acid,
4-hydroxyanthranilic acid, etc. may be used.
[0105] The reaction to obtain the quinazoline ring from the
sulfonylurea derivative having the formula (I-3) may be carried out
using an aprotonic solvent such as, for example, an ether solvent
such as tetrahydrofuran and dioxane, a halogen-containing solvent
such as methylene chloride, or dimethylformamide etc. at a
temperature of -50.degree. C. to 50.degree. C., preferably
-20.degree. C. to room temperature. Further, for the cyclization
reaction, it is possible to use an ordinary condensing agent which
includes, for example, CDI, dicyclohexylcarbodiimide, and similar
carbodiimide compounds, mixed anhydrides, etc. The deprotecting
reaction can be carried out by an ordinary method using hydrolysis
with an acid or alkali, reduction or oxidation etc.
[0106] Synthesis Method (B)
[0107] A compound having the formula (I-4):
##STR00007##
wherein the ring A, R.sup.1', R.sup.2' and R.sup.3' represent the
same as defined above
[0108] is condensed with an anthranilic acid derivative having the
formula (I-5):
##STR00008##
wherein X' represents the same as defined above, Ph represents a
phenyl group, and R.sup.4 represents a protecting group of the
carboxyl group, which is specifically a group capable of being
released by hydrolysis or hydrogenolysis, such as, for example, a
methyl group, an ethyl group, or a benzyl group
[0109] using, for example, 1,8-diazabicyclo[5,4,0]-7-undecene
(hereinafter referred to as DBU) to form a sulfonylurea derivative
having the formula (I-6):
##STR00009##
wherein the ring A, R.sup.1', R.sup.2', R.sup.3', R.sup.4 and X'
are the same as defined above,
[0110] which is then hydrolyzed with an alkali or hydrogenolyzed to
derive a corresponding carboxylic acid represented by the formula
(I-3), then the quinazoline ring is obtained and optionally the
protecting groups of R.sup.1, R.sup.2, R.sup.3 and x are
deprotected, in the same way as in Synthesis Method (A). In this
reaction, when R.sup.1, R.sup.2 or R.sup.3 represents a group
containing a hydroxyl group, an amino group, or a carboxylic acid
group, R.sup.1, R.sup.2 or R.sup.3 may be optionally protected by a
protecting group such as a benzyloxycarbonyl group, a
t-butoxycarbonyl group, a benzyl group, an allyl group, a t-butyl
group, etc. When X represents a hydroxyl group or an amino group, X
may be optionally protected with a protecting group such as a
benzyloxycarbonyl group, a t-butoxycarbonyl group, a benzyl group,
an allyl group, a t-butyl group, etc.
[0111] As the compound having the formula (I-4) used in the
reaction, a commercially available or known compound or a compound
which can be synthesized by a known method may be used. For
example, 3-hydroxybenzenesulfonamide, 2-aminobenzenesulfonamide,
3-aminobenzenesulfonamide, 4-aminobenzenesulfonamide,
(.+-.)-2-(4-aminosulfonylphenyl)butyric acid,
3-benzyloxycarbonylamino-4-chlorobenzenesulfonamide,
4-benzyloxycarbonylamino-3-chlorobenzenesulfonamide,
4-amino-3,5-dichlorobenzenesulfonamide,
3-benzyloxycarbonylamino-4-methylbenzenesulfonamide,
4-t-butoxycarbonyl-3-hydroxybenzenesulfonamide,
3-benzyloxycarbonylamino-4-t-butoxycarbonylbenzenesulfonamide,
4-t-butoxycarbonyl-3-hydroxybenzenesulfonamide,
3-t-butoxycarbonyl-4-hydroxybenzenesulfonamide,
3-acetamide-4-methoxybenzenesulfonamide,
3-(3-aminosulfonyl)phenylacrylic acid t-butylester,
3-amino-4-methoxybenzenesulfonamide,
4-methoxy-3-methylsulfonylaminobenzenesulfonamide,
3-carboxy-4-hydroxy-2-naphthalenesulfonamide,
4-benzyloxycarbonylamino-3-t-butoxycarbonylbenzenesulfonamide,
(.+-.)-3-t-butoxycarbonyl-2-oxo-1H,3H-quinoline-7-sulfonamide,
(.+-.)-2-t-butoxycarbonyl-3-oxo-1,4-benzoxazine-6-sulfonamide, etc.
may be used.
[0112] As the anthranilic acid derivative having the formula (I-5)
used in this reaction, a commercially available or known compound
or a compound which can be synthesized by a known method may be
used. For example, methyl 4-chloro-2-N-phenoxycarbonylanthranilate,
ethyl 4-chloro-2-N-phenoxycarbonylanthranilate, benzyl
4-chloro-2-N-phenoxycarbonylanthranilate, methyl
5-chloro-2-N-phenoxycarbonylanthranilate, ethyl
5-chloro-2-N-phenoxycarbonylanthranilate, benzyl
5-chloro-2-N-phenoxycarbonylanthranilate, methyl
4-methoxy-2-N-phenoxycarbonylanthranilate, ethyl
4-methoxy-2-N-phenoxycarbonylanthranilate, benzyl
4-methoxy-2-N-phenoxycarbonylanthranilate, methyl
4-hydroxy-2-N-phenoxycarbonylanthranilate, ethyl
4-hydroxy-2-N-phenoxycarbonylanthranilate, benzyl
4-hydroxy-2-N-phenoxycarbonylanthranilate, etc. may be used.
[0113] The reaction for obtaining the compound having the formula
(I-4) and the anthranilic acid derivative having the formula (I-5)
condense to obtain a sulfonylurea derivative having the formula
(I-6), may be carried out using an aprotic solvent, for example, an
ether solvent such as tetrahydrofuran or dioxane, a
halogen-containing solvent such as methylene chloride, or
dimethylformamide etc. at a temperature of -50.degree. C. to
50.degree. C., preferably -20.degree. C. to room temperature.
Further, as the usable for the condensation reaction, an organic
strong base such as DBU, inorganic bases such as potassium
carbonate, sodium carbonate, potassium hydroxide, and sodium
hydroxide, or metal bases such as sodium hydride may be used.
[0114] In the reaction for alkali hydrolysis or hydrogenolysis of
the sulfonylurea derivative having the formula (I-6) thus obtained
to obtain the sulfonylurea derivative having the formula (I-3),
ordinary hydrolysis conditions or hydrogenolysis conditions for
esters may be used.
[0115] Note that the above reaction may be carried out while
protecting the functional groups not involved in the reaction.
According to the type of the protecting group, the protection is
removed by chemical reduction or other ordinary protection-removing
reactions. For example, when the protecting group is a t-butyl
group or t-butoxycarbonyl group, trifluoroacetic acid may be used,
while when it is an allyl group, palladium catalysts such as
tetrakis(triphenylphosphine)palladium (0) may be used.
[0116] The compound having the formula (I), wherein R.sup.1
represents an amino group acylated with a C.sub.1 to C.sub.4 lower
aliphatic acid which may be substituted with a carboxylic acid, an
amino group acylated with an aromatic ring carboxylic acid which
may be substituted with a carboxylic acid and an amino group
acylated with an heteroaromatic ring carboxylic acid which may be
substituted with a carboxylic acid, can be obtained from the
compound having the formula (I), wherein R.sup.1 represents an
amino group, by acylating the same with carboxylic acid, carboxylic
acid chloride, carboxylic acid anhydride using an ordinary
method.
[0117] The compound having the formula (I), wherein R.sup.1
represents an amino group sulfonylated with a C.sub.1 to C.sub.4
lower alkane sulfonic acid which may be substituted with a
carboxylic acid, an amino group sulfonylated with an aromatic ring
sulfonic acid which may be substituted with a carboxylic acid and
an amino group sulfonylated with an heteroaromatic ring sulfonic
acid which may be substituted with a carboxylic acid, can be
obtained from the compound having the formula (I), wherein R.sup.1
represents an amino group, by sulfonylating the same with sulfonic
acid or sulfonic acid chloride using an ordinary method.
[0118] A compound of formula (II) of the present invention may be
obtained by a similar method to the above and further is as
described in more detail in International Publication
WO97/11941.
[0119] A compound of the formula (I) or (II) obtained may be
purified by a conventional method such as recrystallization or
column chromatography.
[0120] Further, as necessary, the compound of the formula (I) or
(II) obtained by the above process may be converted into a salt by
causing it to react with various types of acids or bases. As the
acid able to be used to convert the compound of formula (I) or (II)
to a salt, an inorganic acid such as hydrochloric acid, hydrobromic
acid, nitric acid, sulfuric acid, or phosphoric acid and an organic
acid such as methanesulfonic acid, benzenesulfonic acid, p-toluene
sulfonic acid, trifluoroacetic acid, citric acid, lactic acid,
maleic acid, fumaric acid, tartaric acid, acetic acid, adipic acid,
palmitic acid, and tannic acid may be mentioned.
[0121] As the base able to be used for converting the compound of
formula (I) or (II) into a salt, sodium hydroxide, lithium
hydroxide, and potassium hydroxide may be mentioned.
[0122] The compounds having the formula (I) or (II) include ones
containing asymmetric centers. It is possible to isolate a single
optically active substance from the racemic mixture by one or more
methods. For example,
[0123] (1) the method of using an optically active column
[0124] (2) the method of conversion to a salt by an optically
active acid or base and then recrystallization
[0125] (3) the method combining the above (1) and (2) may be
used.
[0126] These compounds may be evaluated for action in preventing or
alleviating conditions of dermatitis exhibiting biphasic skin
reaction and dermatitis induced, by repeated exposure to an antigen
by the later explained methods of Examples 3, 8, 10, 11, 12, and
18.
[0127] When using a compound according to the present invention as
a medicament for the prevention or treatment of dermatitis
exhibiting biphasic skin reaction, a medicament for the alleviation
of the late-phase reaction of dermatitis exhibiting biphasic skin
reaction, or a medicament for prevention or treatment of dermatitis
induced by repeated exposure to an antigen, for example it is
possible to use one type or a mixture of two or more types of the
compound of the present invention to make a preparation of a form
suitable for the method of administration according to an ordinary
method. For example, examples of preparation forms for oral
administration include capsules, tablets, granules, fine granules,
syrups, dry syrups, and other preparations, while examples of
preparation forms for non-oral administration include injections
and also suppositories such as rectal suppositories and vaginal
suppositories, transnasal preparations such as sprays and
ointments, and transdermal preparations such as tapes for
transdermal absorption.
[0128] The clinical dose of the compound according to the present
invention varies according to the symptoms, severity, age, presence
of complications, etc. and also varies according to the form of
preparation. In the case of oral administration, however, it may be
dosed usually, in terms of effective ingredients, as 1 to 1000 mg
per adult per day. In the case of non-oral administration, it is
sufficient to administer 1/10 to 1/2 the amount of the case of oral
administration. These dosages can be suitably adjusted according to
the age, symptoms, etc. of the patient.
[0129] In the present invention, the chymase inhibitor can be
administered alone as it is without being mixed with another
effective ingredient, but considering the disease in question, the
symptoms, complications, etc., it may also administered as a
medicinal preparation containing other effective ingredients.
Further, it may also be combined with these other effective
ingredients. The amounts of the other effective ingredients used
are not particularly limited, but are determined considering the
minimum amounts for expression of their effects alone, the
occurrence of side effects, etc.
[0130] In treatment, the form of preparation and the method of
combined treatment including preparations containing the chymase
inhibitor alone as an effective ingredient and preparations also
containing other effective ingredients are suitably selected by a
physician in accordance with the age of the patient, the symptoms,
etc.
[0131] The toxicity of the compound according to the present
invention is low. The acute toxicity value LD.sub.50 at 24 hours
after oral administration to 5-week old male mice was 1 g/kg or
more. This value is more than 50 times the anticipated clinical
dosage. The compound is therefore judged to be high in safety.
EXAMPLES
[0132] The present invention will now be further explained by, but
is by no means limited to, the following Examples, but the scope of
the invention is not limited to these Examples needless to say.
[0133] In Example 2 and Example 3, a mouse model of dermatitis
exhibiting biphasic skin reaction was used to show the usefulness
of a chymase inhibitor by showing the effect of suppression of the
chymase inhibitor. Further, in Example 4 to Example 6, as further
proof supporting the involvement of chymase in dermatitis
exhibiting biphasic skin reaction, the fact that dermatitis
exhibiting biphasic skin reaction is induced by intradermal
inoculation of human chymase into the ear of mice is presented. In
Example 7 and Example 8, the results of analysis of the mechanism
of action of chymase in such biphasic skin reaction are shown.
[0134] On the other hand, in Example 9 to Example 12, the
usefulness of a chymase inhibitor is demonstrated by using
dermatitis induced by repeated application of hapten as a model for
dermatitis induced by repeated exposure to an antigen and analyzing
the model and showing the effect of a chymase inhibitor in this
model. Further, since it is conceivable that mast cells undergo
repeated degranulation reactions due to repeated exposure to
allergens in patients suffering from atopic dermatitis etc., in
Example 13 to Example 15, dermatitis induced by repeated
inoculation of human chymase into the ears was analyzed for the
purpose of studying the effects of a repeated degranulation
reaction on the skin. Further, in Examples 16 to 17, the effects of
chymase on the expression of SCF, the major cytokine for mast
cells, are shown as one of the mechanisms of chymase-induced
dermatitis. Further, in Example 18, the effects of chymase
inhibitor on dermatitis was examined using NC/Nga mice as the
second model induced by repeated exposure to antigen.
Preparation Example 1
Synthesis of
7-chloro-3-(3-hydroxybenzenesulfonyl)-2,4(1H,3H)-quinazolinedione
(Compound 1)
[0135] Following the Synthesis Method (B), 938 mg (5.42 mmol) of
3-hydroxybenzenesulfonamide was dissolved in 40 ml of
tetrahydrofuran, then 892 .mu.l (5.96 mmol) of
1,8-diazabicyclo[5,4,0]-7-undecene (hereinafter referred to as DBU)
was added dropwise. The reaction solution was stirred at room
temperature for 15 minutes, then 1.66 g (5.42 mmol) of methyl
4-chloro-2-N-phenoxycarbonylanthranilate was added and the mixture
was stirred at room temperature overnight. An excess amount of
water was poured into the reaction solution, then the mixture was
made acidic with hydrochloric acid and extracted with ethyl
acetate. The organic layer was washed with water and saturated
saline, dried over anhydrous magnesium sulfate, and concentrated.
The crude product thus obtained was purified by silica gel column
chromatography (0% to 5% methanol/dichloromethane) to obtain 1.23 g
(yield 59%) of methyl
4-chloro-2-{[(3-hydroxybenzenesulfonylamino)carbonyl]amino}
benzoate. Properties: colorless amorphous, PMR (.delta. ppm,
DMSO-d.sub.6): 3.91 (3H, s), 7.02 (1H, m), 7.09 (1H, m), 7.34 (1H,
t), 7.57 (2H, m), 7.89 (1H, d), 8.38 (1H, d), 10.94 (1H, s). Next,
the 1.23 g (3.2 mmol) of the compound thus obtained was dissolved
in 20 ml of methanol, then 10 ml of 2N sodium hydroxide aqueous
solution was added dropwise. The reaction solution was stirred at
room temperature for 15 minutes, then an excess amount of water was
added and the mixture was made acidic with hydrochloric acid. This
was then stirred to cause crystals to precipitate which were then
obtained by filtration and dried to obtain carboxylic acid. The
product thus obtained was dissolved in 50 ml of tetrahydrofuran
(hereinafter referred to as THF), then 434 mg (2.68 mmol) of CDI
was added under ice cooling and the mixture was stirred for 30
minutes. The reaction solution was diluted with ethyl acetate,
washed with water and saturated saline, and dried over anhydrous
magnesium sulfate, then concentrated to obtain a crude product. The
crude product was purified by silica gel column chromatography
(ethyl acetate:n-hexane=1:2) to obtain 230 mg (yield 20%: 2 steps)
of the above-identified compound. Properties: colorless crystal,
Melting point: >200.degree. C. (decomposition), PMR (.delta.
ppm, DMSO-d.sub.6): 7.12 (2H, s), 7.24 (1H, d), 7.48 (1H, t), 7.58
(2H, s), 7.85 (1H, d), 10.28 (1H, s), 11.63 (1H, s).
Preparation Example 2
Synthesis of
3-(2-aminobenzenesulfonyl)-7-chloro-2,4(1H,3H)-quinazolinedione
(Compound 2)
[0136] 2.7 g (15.7 mmol) of 2-aminobenzenesulfonamide and 4.8 g
(15.7 mmol) of methyl 4-chloro-2-N-phenoxycarbonylanthranilate were
treated in the same way as Preparation Example 1 to obtain 3.2 g
(yield 58%: 3 steps) of the above-identified compound. Properties:
colorless crystal, Melting point: >200.degree. C.
(decomposition), PMR (.delta. ppm, DMSO-d.sub.6): 6.46 (2H, s),
6.65 (1H, t), 6.81 (1H, d), 7.12 (1H, s), 7.23 (1H, d), 7.34 (1H,
t), 7.76 (1H, d), 7.86 (1H, d).
Preparation Example 3
Synthesis of
7-chloro-3-(2-methylsulfonylaminobenzenesulfonyl)-2,4(1H,3H)-quinazolined-
ione (Compound 3)
[0137] 22 mg (0.06 mmol) of Compound 2 was dissolved in 200 .mu.l
of pyridine, 11.6 .mu.l (0.15 mmol) of methanesulfonyl chloride was
added dropwise, then the resultant mixture was stirred at room
temperature overnight. An excess amount of water was added to the
reaction solution and the mixture was extracted with ethyl acetate.
The organic layer was washed with 1N aqueous hydrochloric acid
solution and saturated saline, then dried over anhydrous magnesium
sulfate and concentrated to obtain a crude product. The crude
product was crystallized from diethyl ether to obtain 16 mg (0.04
mmol) of the above-identified compound. Properties: colorless
crystal, Melting point: >200.degree. C. (decomposition), PMR
(.delta. ppm, DMSO-d.sub.6): 3.61 (3H, s), 7.10 (1H, d), 7.20 (1H,
d), 7.74 (1H, d), 7.82-7.90 (4H, m), 8.34 (1H, d), 11.70 (1H,
s).
Preparation Example 4
Synthesis of
3-(4-aminobenzenesulfonyl)-7-chloro-2,4(1H,3H)-quinazolinedione
(Compound 4)
[0138] 2.7 g (15.7 mmol) of 4-aminobenzenesulfonamide and 4.8 g
(15.7 mmol) of methyl 4-chloro-2-N-phenoxycarbonylanthranilate were
treated in the same way as Preparation Example 1 to obtain 7.9 g
(yield 94%) of methyl
2-{[(4-aminobenzenesulfonylamino)carbonyl]amino}-4-chlorobenzoate.
Properties: colorless amorphous, PMR (.delta. ppm, DMSO-d.sub.6):
3.59 (3H, s), 5.37 (2H, s), 6.45 (2H, d), 6.83 (1H, dd), 7.41 (2H,
d), 7.81 (1H, d), 8.66 (1H, d), 9.64 (1H, s).
[0139] Then, from the resultant 7.9 g (14.8 mmol) of sulfonylurea
product, in the same way, 4.3 g (yield 83%: 2 steps) of the
above-identified compound was obtained. Properties: colorless
crystal, Melting point: >200.degree. C. (decomposition), PMR
(.delta. ppm, DMSO-d.sub.6): 6.39 (2H, s), 6.63 (2H, d), 7.09 (1H,
s), 7.22 (1H, d), 7.76 (2H, d), 7.83 (1H, d), 11.51 (1H, s).
Preparation Example 5
Synthesis of
3-(3-carboxymethyl-benzenesulfonyl)-7-chloro-2,4(1H,3H)-quinazolinedione
(Compound 5)
[0140] Following the Synthesis Method (A), 3.27 g (11.6 mmol) of
3-allyloxycarbonylmethylbenzenesulfonyl isocyanate was dissolved in
100 ml of anhydrous THF, then 1.98 g (11.5 mmol) of
4-chloroanthranilic acid was added and the mixture was stirred at
room temperature for 2 hours. The reaction solution was cooled with
ice water, then 1.87 g (11.5 mmol) of CDI was added and the
resultant mixture was stirred under ice cooling for 30 minutes. An
excess amount of water was poured into the reaction solution, then
the mixture was extracted with ethyl acetate. The organic layer was
washed, dried, and concentrated to obtain a crude product. This was
crystallized with a small amount of ethyl acetate to obtain 2.0 g
(yield 40%) of
3-(3-allyloxy-carbonylmethylbenzenesulfonyl)-7-chloro-2,4(1H,3H)-quinazol-
inedione. The allyl product thus obtained was dissolved in 100 ml
of a formic acid-THF (1:9) mixture and 700 mg of triphenylphosphine
was added. The reactor was shaded from light and under nitrogen
atmosphere, then 700 mg of tetrakis(triphenylphosphine)palladium
(0) was added and the resultant mixture was stirred while shaded at
room temperature overnight. The reaction solution was concentrated
in vacuo and the solid obtained was washed with methylene chloride
to obtain 1.47 g (yield 81%) of the above-identified compound.
Properties: colorless crystal, Melting point: >200.degree. C.
(decomposition), PMR (.delta. ppm, DMSO-d.sub.6): 3.76 (2H, s),
7.13 (1H, s), 7.24 (1H, d), 7.61-7.69 (2H, m), 7.86 (1H, d), 8.05
(2H, s), 12.50 (1H, br).
Preparation Example 6
Synthesis of
3-(4-carboxymethyl-benzenesulfonyl)-7-chloro-2,4(1H,3H)-quinazolinedione
(Compound 6)
[0141] 1.10 g (3.95 mmol) of
4-allyloxycarbonylmethyl-benzenesulfonyl isocyanate and 678 mg
(3.95 mmol) of 4-chloroanthranilic acid were treated in the same
way as in Preparation Example 5 to obtain 657 mg (yield 38%) of
3-(4-allyloxycarbonylbenzenesulfonyl)-7-chloro-2,4(1H,3H)-quinazolinedion-
e. 538 mg (1.24 mmol) thereof was treated in the same way to obtain
342 mg of the above-identified compound (yield 70%). Properties:
colorless crystal, Melting point: >200.degree. C.
(decomposition), PMR (.delta. ppm, DMSO-d.sub.6): 3.75 (2H, s),
7.13 (1H, s), 7.23 (1H, d), 7.61-7.69 (2H, m), 7.86 (1H, d), 8.05
(2H, s), 12.07 (2H, br).
Preparation Example 7
Synthesis of
(.+-.)-2-{4-[(7-chloro-2,4(1H,3H)-quinazolin-3-yl)sulfonyl]phenyl}butyric
Acid (Compound 7)
[0142] 1.02 g (3.41 mmol) of t-butyl
(.+-.)-2-(4-amino-sulfonylphenyl)butyrate acid and 1.04 g (3.41
mmol) of methyl 4-chloro-2-N-phenoxycarbonylanthranilate were
treated in the same way as Preparation Example 1 to obtain 1.46 g
(yield 84%) of methyl
2-[({4-[1-(t-butoxycarbonyl)propyl]benzenesulfonylamino}carbonyl)amino]-4-
-chlorobenzoate. Properties: colorless amorphous, PMR (.delta. ppm,
CDCl.sub.3): 0.89 (3H, t), 1.38 (9H, s), 1.69-1.76 (1H, m),
2.03-2.10 (1H, m), 3.42 (1H, t), 3.94 (3H, s), 7.04 (1H, d), 7.47
(2H, d), 7.93 (1H, d), 8.01 (2H, d), 8.45 (1H, br), 11.04 (1H,
br).
[0143] Next, 4.3 ml (8.6 mmol) of 2N sodium hydroxide aqueous
solution was used to similarly form carboxylic acid in an amount of
1.43 g and 463 mg (2.86 mmol) of CDI was used to obtain 970 mg
(yield 71%: 2 steps) of t-butyl
(.+-.)-2-{4-[(7-chloro-2,4(1H,3H)-quinazolin-3-yl)sulfonyl]phenyl-
}butyrate.
[0144] Further, the t-butylester thus obtained was dissolved in 5
ml of dichloromethane, then 5 ml of trifluoroacetic acid was added
and the resultant mixture was stirred at room temperature for 40
minutes. The reaction solution was concentrated in vacuo and the
resultant crude product was washed with a small amount of diethyl
ether to obtain 820 mg of the above-identified compound (yield
96%). Properties: colorless crystal, Melting point: >200.degree.
C. (decomposition), PMR (.delta. ppm, DMSO-d.sub.6): 0.84 (3H, t),
1.67-1.75 (1H, m), 1.98-2.05 (1H, m), 3.62 (1H, t), 7.11 (1H, s),
7.24 (1H, d), 7.61 (2H, d), 7.86 (1H, d), 8.13 (2H, d), 11.62 (1H,
s).
Preparation Example 8
Synthesis of
3-(3-amino-4-chlorobenzenesulfonyl)-7-chloro-2,4(1H,3H)-quinazolinedione
(Compound 8)
[0145] 1.0 g (2.93 mmol) of
3-benzyloxycarbonylamino-4-chlorobenzenesulfonamide and 1.18 g
(2.93 mmol) of benzyl 4-chloro-2-N-phenoxycarbonylanthranilate were
treated in the same way as Preparation Example 1 to obtain 1.43 g
(yield 78%) of benzyl 2-{[(3-benzyloxycarbonylamino-4-chlorobenzene
sulfonylamino)carbonyl]amino}-4-chlorobenzoate. Properties:
colorless amorphous, PMR (.delta. ppm, DMSO-d.sub.6): 5.19 (2H, s),
5.36 (2H, s), 7.21 (1H, dd), 7.34-7.48 (10H, m), 7.72-7.76 (2H, m),
7.97 (1H, d), 8.25 (1H, d), 8.30 (1H, d), 9.53 (1H, s), 10.30 (1H,
s). 1.38 g (2.20 mmol) thereof was dissolved in 50 ml of THF, then
200 mg of palladium-carbon (10%) was added and the mixture was
stirred under a hydrogen flow for 2 hours. The reaction mixture was
filtered with Celite to remove the palladium-carbon, then the
filtrate was concentrated in vacuo to obtain a carboxylic acid. The
product obtained was suspended in 50 ml of THF, then 356 mg (2.20
mmol) of CDI was added under ice cooling and the resultant mixture
was treated in the same way as Preparation Example 1 to obtain 560
mg (yield 66%: 2 steps) of the above-identified compound.
Properties: colorless crystal, Melting point: >200.degree. C.
(decomposition), PMR (.delta. ppm, DMSO-d.sub.6): 6.00 (2H, s),
7.12 (1H, s), 7.26 (2H, t), 7.48 (1H, d), 7.66 (1H, s), 7.86 (1H,
d), 11.76 (1H, br).
Preparation Example 9
Synthesis of
3-(4-amino-3,5-dichlorobenzenesulfonyl)-7-chloro-2,4(1H,3H)-quinazolinedi-
one (Compound 9)
[0146] 1.06 g (4.40 mmol) of
4-amino-3,5-dichloro-benzenesulfonamide and 1.34 g (4.40 mmol) of
methyl 4-chloro-2-N-phenoxycarbonylanthranilate were treated in the
same way as Preparation Example 1 to obtain 905 mg (yield 44%) of
methyl
2-{[(4-amino-3,5-dichlorobenzenesulfonylamino)carbonyl]amino}-4-chloroben-
zoate. Properties: colorless amorphous, PMR (.delta. ppm,
DMSO-d.sub.6): 3.87 (3H, s), 6.59 (2H, br), 7.22 (1H, dd), 7.72
(2H, s), 7.93 (1H, d), 8.24 (1H, d), 10.17 (1H, s).
[0147] Then, from 905 mg (2.0 mmol) of the resultant sulfonylurea
product, in the same way, 660 mg (yield 82%: 2 steps) of the
above-identified compound was obtained. Properties: colorless
crystal, Melting point: >200.degree. C. (decomposition), PMR (6
ppm, DMSO-d.sub.6): 6.80 (2H, s), 7.12 (1H, s), 7.24 (1H, d), 7.86
(1H, d), 7.92 (2H, s), 11.63 (1H, br).
Preparation Example 10
Synthesis of
3-(3-amino-4-methylbenzenesulfonyl)-7-chloro-2,4(1H,3H)-quinazolinedione
(Compound 10)
[0148] 960 mg (3.00 mmol) of
3-benzyloxycarbonylamino-4-methylbenzenesulfonamide and 1.14 g
(3.00 mmol) of benzyl 4-chloro-2-N-phenoxycarbonylanthranilate were
treated in the same way as in Preparation Example 8 to obtain 1.14
g (yield 62% of benzyl
2-{[(3-benzyloxycarbonylamino-4-methylbenzenesulfonylamino)carbony-
l]amino}-4-chlorobenzoate. Properties: colorless amorphous, PMR
(.delta. ppm, DMSO-d.sub.6): 2.30 (3H, s), 5.17 (2H, s), 5.36 (2H,
s), 7.20 (1H, dd), 7.33-7.48 (11H, m), 7.63 (1H, d), 7.97 (1H, d),
8.11 (1H, s), 8.25 (1H, s), 9.27 (1H, s), 10.30 (1H, s), 12.20 (1H,
br).
[0149] Then, from 1.14 g (1.87 mmol) of the resultant sulfonylurea
product, in the same way, 190 mg (yield 27%: 2 steps) of the
above-identified compound was obtained. Properties: colorless
crystal, Melting point: >200.degree. C. (decomposition), PMR
(.delta. ppm, DMSO-d.sub.6): 2.12 (3H, s), 5.47 (2H, s), 7.12 (1H,
s), 7.16-7.25 (3H, m), 7.38 (1H, s), 7.85 (1H, d), 11.58 (1H,
s).
Preparation Example 11
Synthesis of
3-[(3-carboxymethylaminophenyl)sulfonyl]-7-chloro-2,4(1H,3H)-quinazolined-
ione (Compound 11)
[0150] 1.62 g (5.65 mmol) of
3-t-butoxycarbonyl-methylaminobenzenesulfonamide and 1.73 g (5.65
mmol) of methyl 4-chloro-2-N-phenoxycarbonylanthranilate were
treated in the same way as in Preparation Example 7 to obtain 209
mg (yield 9%: 4 steps) of the above-identified compound.
Properties: colorless crystal, Melting point: >200.degree. C.
(decomposition), PMR (.delta. ppm, DMSO-d.sub.6): 3.86 (2H, s),
6.88 (1H, s), 7.12 (1H, s), 7.24 (1H, d), 7.30-7.38 (3H, m), 7.86
(1H, d), 11.61 (1H, br).
Preparation Example 12
Synthesis of
3-(3-aminobenzenesulfonyl)-7-chloro-2,4(1H,3H)-quinazolinedione
(Compound 12)
[0151] 3.5 g (12.9 mmol) of
3-t-butoxycarbonylamino-benzenesulfonamide and 3.9 g (12.8 mmol) of
methyl 4-chloro-2-N-phenoxycarbonylanthranilate were treated in the
same way as in Preparation Example 7 to obtain 2.2 g (yield 49%: 4
steps) of the above-identified compound. Properties: colorless
crystal, Melting point: >200.degree. C. (decomposition), PMR
(.delta. ppm, DMSO-d.sub.6): 5.72 (2H, s), 6.87 (1H, d), 7.12 (1H,
s), 7.23-7.27 (2H, m), 7.33 (1H, s), 7.86 (1H, d), 11.61 (1H,
s).
Preparation Example 13
Synthesis of
2-{3-[(7-chloro-2,4(1H,3H)-quinazolinedion-3-yl)sulfonyl]phenylaminocarbo-
nyl}propionic Acid (Compound 13)
[0152] 100 mg (0.28 mmol) of Compound 12 was dissolved in 5 ml of
THF, 100 mg (1.0 mmol) of succinic anhydride was added, and the
resultant mixture was heated and refluxed for 3 hours. The reaction
solution was concentrated in vacuo and the crude product thus
obtained was crystallized with ethyl acetate-diethyl ether to
obtain 120 mg (yield 96%) of the above-identified compound.
Properties: colorless crystal, Melting point: 187-188.degree. C.,
PMR (.delta. ppm, DMSO-d.sub.6): 2.54 (2H, d), 2.59 (2H, d), 7.12
(1H, s), 7.24 (1H, d), 7.59 (1H, t), 7.80 (1H, d), 7.86 (1H, d),
7.96 (1H, d), 8.41 (1H, s), 10.40 (1H, s), 11.63 (1H, br), 12.10
(1H, br).
Preparation Example 14
Synthesis of
3-{3-[(7-chloro-2,4(1H,3H)-quinazolinedion-3-yl)sulfonyl]phenyl}acrylic
acid (Compound 14)
[0153] 1.54 g (5.44 mmol) of t-butyl
3-(3-aminosulfonyl)phenylacrylate and 1.66 g (5.44 mmol) of methyl
4-chloro-2-N-phenoxycarbonylanthranilate were treated in the same
way as in Preparation Example 7 to obtain 2.18 g (yield 81%) of
methyl
2-({[3-(3-t-butoxy-3-oxo-1-propenyl)benzenesulfonylamino]carbonyl}amino)--
4-chlorobenzoate. Properties: colorless amorphous, PMR (.delta.
ppm, CDCl.sub.3): 1.53 (9H, s), 3.95 (3H, s), 6.46 (1H, d), 7.05
(1H, d), 7.55 (1H, m), 7.57 (1H, d), 7.72 (1H, m), 7.93 (1H, m),
8.04 (1H, m), 8.27 (1H, s), 8.46 (1H, d), 11.05 (1H, br).
[0154] Then, from 2.18 g (4.4 mmol) of the resultant sulfonylurea
product, in the same way, 698 mg (yield 37%: 3 steps) of the
above-identified compound was obtained. Properties: colorless
crystal, Melting point: >200.degree. C. (decomposition), PMR
(.delta. ppm, DMSO-d.sub.6): 6.65 (1H, d), 7.12 (1H, s), 7.25 (1H,
d), 7.69 (1H, d), 7.72 (1H, t), 7.87 (1H, d), 8.12 (2H, q), 8.37
(1H, s), 11.64 (1H, s).
Preparation Example 15
Synthesis of
4-[(7-chloro-2,4(1H,3H)-quinazolinedion-3-yl)sulfonyl]salicylic
acid (Compound 15)
[0155] 1.0 g (3.66 mmol) of
4-t-butoxycarbonyl-3-hydroxybenzenesulfonamide and 1.12 g (3.66
mmol) of methyl 4-chloro-2-N-phenoxycarbonylanthranilate were
treated in the same way as in Preparation Example 7 to obtain 1.79
g (yield 100%) of methyl
2-{[(4-t-butoxycarbonyl-3-hydroxybenzenesulfonylamino)carbonyl]amino}-4-c-
hlorobenzoate. Properties: colorless amorphous, PMR (.delta. ppm,
DMSO-d.sub.6): 1.57 (9H, s), 3.87 (3H, s), 7.14 (1H, d), 7.40-7.45
(2H, m), 7.85 (1H, d), 7.92 (1H, d), 8.32 (1H, d), 10.13 (1H, s),
10.82 (1H, s).
[0156] Then, from 1.78 g (3.66 mmol) of the resultant sulfonylurea
product, in the same way, 370 mg (yield 25%: 3 steps) of the
above-identified compound was obtained. Properties: colorless
crystal, Melting point: >200.degree. C. (decomposition), PMR
(.delta. ppm, DMSO-d.sub.6): 7.13 (1H, s), 7.26 (1H, d), 7.69 (1H,
d), 7.87 (1H, d), 8.01 (1H, d), 11.67 (1H, s).
Preparation Example 16
Synthesis of
4-[(7-chloro-2,4(1H,3H)-quinazolinedion-3-yl)sulfonyl]salicylic
Acid Monosodium Salt (Compound 16)
[0157] 50 mg (0.13 mmol) of Compound 15 was suspended in
approximately 1 ml of THF, then 126 .mu.l of 1N sodium hydroxide
aqueous solution was added dropwise. The solution was confirmed to
have become uniform, then 30 ml of water was added and the mixture
freeze-dried to quantitatively obtain the above-identified compound
in an amorphous state in an amount of 52 mg. Properties: colorless
amorphous, PMR (.delta. ppm, CD.sub.3OD): 7.11 (1H, s), 7.19 (1H,
d), 7.58 (1H, d), 7.63 (1H, s), 7.92 (1H, d), 8.03 (1H, d).
Preparation Example 17
Synthesis of
4-[(7-chloro-1-2,4(1H,3H-quinazolinedion-3-yl)sulfonyl]anthranilic
Acid (Compound 17)
[0158] 2.84 g (6.99 mmol) of
3-benzyloxycarbonylamino-4-t-butoxycarbonylbenzenesulfonamide and
2.67 g (6.99 mmol) of benzyl
4-chloro-2-N-phenoxycarbonylanthranilate were treated in the same
way as in Preparation Example 8 to obtain 3.74 g (yield 77%) of
benzyl
2-{[(3-benzyloxycarbonylamino-4-t-butoxycarbonylbenzenesulfonylamino)carb-
onyl]amino}-4-chlorobenzoate. Properties: colorless amorphous, PMR
(.delta. ppm, DMSO-d.sub.6): 1.54 (9H, s), 5.19 (2H, s), 5.34 (2H,
s), 7.05 (1H, m), 7.34-7.58 (10H, m), 7.60 (1H, d), 7.90 (1H, d),
7.98 (1H, d), 8.50 (1H, br), 8.62 (1H, s), 10.00 (1H, br), 10.41
(1H, s).
[0159] Then, from 3.74 g (5.39 mmol) of the resultant sulfonylurea,
in the same way, 690 mg (yield 30%: 2 steps) of t-butyl
4-[(7-chloro-2,4(1H,3H)-quinazolinedion-3-yl)sulfonyl]anthranilate
was obtained, then this was subjected to a similar debutylation
reaction to obtain 503 mg (yield 84%) of the above-identified
compound. Properties: colorless crystal, Melting point:
>200.degree. C. (decomposition), PMR (.delta. ppm,
DMSO-d.sub.6): 7.14 (1H, s), 7.18 (1H, d), 7.25 (1H, d), 7.59 (1H,
s), 7.87 (1H, d), 7.89 (1H, d), 11.62 (1H, s).
Preparation Example 18
Synthesis of
4-[(7-chloro-2,4(1H,3H)-quinazolinedion-3-yl)sulfonyl]anthranilic
Acid Monosodium Salt (Compound 18)
[0160] 50 mg (0.13 mmol) of Compound 17 was suspended in
approximately 1 ml of THF, then 126 .mu.l of 1N sodium hydroxide
aqueous solution was added dropwise. The solution was confirmed to
have become uniform, then 30 ml of water was added and the mixture
was freeze-dried to quantitatively obtain the above-identified
compound in an amorphous state in an amount of 52 mg. Properties:
colorless amorphous, PMR (.delta. ppm, DMSO-d.sub.6): 7.11-7.22
(3H, m), 7.37 (1H, s), 7.83 (1H, d), 7.91 (1H, d).
Preparation Example 19
Synthesis of
3-(4-hydroxybenzenesulfonyl)-7-chloro-2,4(1H,3H)-quinazolinedione
(Compound 19)
[0161] 1.50 g (7.03 mmol) of 4-allyloxybenzenesulfonyl isocyanate
and 1.2 g (7.03 mmol) of 4-chloroanthranilic acid were treated in
the same way as in Preparation Example 5 to obtain 1.5 g (yield
53%) of
3-(4-allyloxybenzenesulfonyl)-7-chloro-2,4(1H,3H)-quinazolinedione.
500 mg (1.27 mmol) thereof was similarly treated to obtain 405 mg
of the above-identified compound (yield 90%). Properties: colorless
crystal, Melting point: >200.degree. C. (decomposition), PMR
(.delta. ppm, DMSO-d.sub.6): 6.98 (2H, d), 7.11 (1H, s), 7.23 (1H,
d), 7.85 (1H, d), 8.00 (2H, d), 11.25 (1H, br).
Preparation Example 20
Synthesis of 4-[(2,4(1H,3H)-quinazolinedion-3-yl)sulfonyl]salicylic
Acid (Compound 20)
[0162] 618 mg (2.26 mmol) of
4-t-butoxycarbonyl-3-hydroxybenzenesulfonamide and 613 mg (2.26
mmol) of methyl 2-N-phenoxycarbonylanthranilate were treated in the
same way as in Preparation Example 17 to obtain 792 mg (yield 78%)
of methyl
2-{[(4-t-butoxycarbonyl-3-hydroxybenzene-sulfonylamino)carbonyl]amino}ben-
zoate. Properties: colorless amorphous, PMR (.delta. ppm,
CDCl.sub.3): 1.60 (9H, s), 3.97 (3H, s), 7.09 (1H, t), 7.49-7.52
(2H, m), 7.65 (1H, d), 7.90 (1H, d), 8.01 (1H, dd), 8.33 (1H, d),
10.98 (1H, s), 11.18 (1H, s).
[0163] Then, from 790 mg (1.75 mmol) of the resultant sulfonylurea
product, in the same way, 100 mg (yield 8%: 3 steps) of the
above-identified compound was obtained. Properties: colorless
crystal, Melting point: >200.degree. C. (decomposition), PMR
(.delta. ppm, DMSO-d.sub.6): 7.13 (1H, d), 7.22 (1H, t), 7.63-7.69
(3H, m), 7.87 (1H, d), 8.01 (1H, d), 11.57 (1H, s).
Preparation Example 21
Synthesis of
5-[(7-chloro-2,4(1H,3H)-quinazolinedion-3-yl)sulfonyl]salicylic
Acid (Compound 21)
[0164] 320 mg (1.17 mmol) of
3-t-butoxycarbonyl-4-hydroxybenzenesulfonamide and 447 mg (1.17
mmol) of benzyl 4-chloro-2-N-phenoxycarbonylanthranilate were
treated in the same way as in Preparation Example 17 to obtain 611
mg (yield 93%) of benzyl
2-{[(3-t-butoxycarbonyl-4-hydroxybenzenesulfonylamino)carbonyl]amino}-4-c-
hlorobenzoate. Properties: colorless amorphous, PMR (.delta. ppm,
CDCl.sub.3): 1.62 (9H, s), 5.35 (2H, s), 7.01-7.05 (2H, m),
7.37-7.41 (5H, m), 7.96 (1H, d), 8.10 (1H, dd), 8.46-8.48 (2H, m),
10.99 (1H, s), 11.66 (1H, s).
[0165] Then, from 611 mg (1.09 mmol) of the resultant sulfonylurea
product, in the same way, 114 mg (yield 33%: 3 steps) of the
above-identified compound was obtained. Properties: colorless
crystal, Melting point: >200.degree. C. (decomposition), PMR
(.delta. ppm, DMSO-d.sub.6): 7.11 (1H, s), 7.19 (1H, d), 7.24 (1H,
d), 7.86 (1H, d), 8.20 (1H, d), 8.56 (1H, s), 11.57 (1H, s).
Preparation Example 22
Synthesis of
3-(3-acetamide-4-methoxybenzenesulfonyl)-7-chloro-2,4(1H,
3H)-quinazolinedione (Compound 22)
[0166] 500 mg (2.19 mmol) of
3-acetamide-4-methoxybenzenesulfonamide and 836 mg (2.19 mmol) of
benzyl 4-chloro-2-N-phenoxycarbonylanthranilate were treated in the
same way as in Preparation Example 8 to obtain 812 mg (yield 70%)
of benzyl
2-{[(3-acetylamino-4-methoxybenzenesulfonylamino)carbonyl]amino}-4-chloro-
benzoate. Properties: colorless amorphous, PMR (.delta. ppm,
DMSO-d.sub.6): 2.12 (3H, s), 3.93 (3H, s), 5.36 (2H, s), 7.20 (1H,
d), 7.24 (1H, d), 7.36-7.48 (5H, m), 7.69 (1H, d), 7.96 (1H, d),
8.24 (1H, s), 8.67 (1H, s), 9.39 (1H, s), 10.25 (1H, s), 12.11 (1H,
br).
[0167] Then, from 611 mg (1.09 mmol) of the resultant sulfonylurea
product, in the same way, 250 mg (yield 39%: 2 steps) of the
above-identified compound was obtained. Properties: colorless
crystal, Melting point: >200.degree. C. (decomposition), PMR
(.delta. ppm, DMSO-d.sub.6): 2.12 (3H, s), 3.95 (3H, s), 7.12 (1H,
s), 7.23 (1H, d), 7.30 (1H, d), 7.85 (1H, d), 7.89 (1H, d), 8.80
(1H, s), 9.42 (1H, s), 11.59 (1H, br).
Preparation Example 23
Synthesis of
3-(3-amino-4-methoxybenzenesulfonyl)-7-chloro-2,4(1H,3H)-quinazolinedione
(Compound 23)
[0168] 400 mg (1.40 mmol) of
3-t-butoxycarbonylamino-4-methoxybenzenesulfonamide and 533 mg
(1.40 mmol) of benzyl 4-chloro-2-N-phenoxycarbonylanthranilate were
treated in the same way as in Preparation Example 17 to obtain 86
mg (yield 16%: 4 steps) of the above-identified compound.
Properties: colorless crystal, Melting point: >200.degree. C.
(decomposition), PMR (.delta. ppm, DMSO-d.sub.6): 3.81 (3H, s),
7.26-7.37 (5H, m), 7.77 (1H, s), 7.90 (1H, d), 7.94 (1H, d), 11.73
(1H, s).
Preparation Example 24
Synthesis of
7-chloro-3-(4-methoxy-3-methylsulfonylaminobenzenesulfonyl)-2,4(1H,3H)-qu-
inazolinedione (Compound 24)
[0169] 500 mg (1.89 mmol) of
4-methoxy-3-methylsulfonylaminobenzenesulfonamide and 722 mg (1.89
mmol) of benzyl 4-chloro-2-N-phenoxycarbonylanthranilate were
treated in the same way as in Preparation Example 8 to obtain 888
mg (yield 83%) of benzyl
2-({[(4-methoxy-3-methylsulfonylamino)benzene
sulfonylamino]carbonyl}amino)-4-chlorobenzoate. Properties:
colorless amorphous, PMR (.delta. ppm, DMSO-d.sub.6): 2.12 (3H, s),
3.93 (3H, s), 5.36 (2H, s), 7.20 (1H, d), 7.24 (1H, d), 7.36-7.48
(5H, m), 7.69 (1H, d), 7.96 (1H, d), 8.24 (1H, s), 8.67 (1H, s),
9.39 (1H,'s), 10.25 (1H, s), 12.11 (1H, br).
[0170] Then, from 880 mg (1.55 mmol) of the resultant sulfonylurea
product, in the same way, 620 mg (yield 85%: 2 steps) of the
above-identified compound was obtained. Properties: colorless
crystal, Melting point: >200.degree. C. (decomposition), PMR
(.delta. ppm, DMSO-d.sub.6): 3.04 (3H, s), 3.94 (3H, s), 7.11 (1H,
s), 7.23 (1H, d), 7.34 (1H, d), 7.86 (1H, d), 7.99 (1H, d), 8.10
(1H, s).
Preparation Example 25
Synthesis of 4-[(7-chloro-2,4(1H,3H)-quinazolinedion-3-yl
sulfonyl]-1-hydroxy-naphthalene-2-carboxylic Acid (Compound 25)
[0171] 323 mg (1.00 mmol) of
3-t-butoxycarbonyl-4-hydroxy-1-naphthalenesulfonamide and 381 mg
(1.00 mmol) of benzyl 4-chloro-2-N-phenoxycarbonylanthranilate were
treated in the same way as in Preparation Example 17 to obtain 447
mg (yield 73%) of
4-({[(2-benzyloxycarbonyl-5-chloroanilino)carbonyl]amino}sulfonyl)-1-hydr-
oxy-2-naphthalenecarboxylic acid t-butyl ester. Properties:
colorless amorphous, PMR (.delta. ppm, DMSO-d.sub.6): 1.66 (9H, s),
5.34 (3H, s), 6.98 (1H, d), 7.35-7.48 (5H, m), 7.66 (1H, m), 7.81
(1H, m), 7.89 (1H, d), 8.37 (2H, m), 8.44 (1H, s), 8.71 (1H, d),
10.02 (1H, br), 12.52 (1H, br).
[0172] Then, from 445 mg (0.72 mmol) of the resultant sulfonylurea
product, in the same way, 56 mg (yield 18%: 3 steps) of the
above-identified compound was obtained. Properties: colorless
crystal, Melting point: >200.degree. C. (decomposition), PMR
(.delta. ppm, DMSO-d.sub.6): 7.08 (1H, s), 7.20 (1H, d), 7.63 (1H,
t), 7.77 (1H, t), 7.84 (1H, d), 8.42 (1H, d), 8.51 (1H, d), 8.75
(1H, s), 11.57 (1H, s).
Preparation Example 26
Synthesis of
5-[(7-chloro-2.4(1H,3H)-quinazolinedion-3-yl)sulfonyl]anthranilic
Acid (Compound 26)
[0173] 834 mg (2.05 mmol) of
4-benzyloxycarbonylamino-3-t-butoxycarbonylbenzenesulfonamide and
783 mg (2.05 mmol) of benzyl
4-chloro-2-N-phenoxycarbonylanthranilate were treated in the same
way as in Preparation Example 17 to obtain 1.18 g (yield 83%) of
benzyl
2-{[(4-benzyloxycarbonylamino-3-t-butoxycarbonylbenzenesulfonylamino)carb-
onyl]amino}-4-chlorobenzoate. Properties: colorless amorphous, PMR
(.delta. ppm, CDCl.sub.3): 1.56 (9H, s), 5.22 (2H, s), 5.37 (2H,
s), 7.04 (1H, dd), 7.33-7.42 (10H, m), 7.97 (1H, d), 8.14 (1H, d),
8.45 (1H, d), 8.60 (1H, d), 8.65 (1H, d), 11.01 (1H, s), 11.11 (1H,
s).
[0174] Then, from 1.17 g (1.69 mmol) of the resultant sulfonylurea
product, in the same way, 404 mg (yield 60%: 3 steps) of the
above-identified compound was obtained. Properties: colorless
crystal, Melting point: >200.degree. C. (decomposition), PMR
(.delta. ppm, DMSO-d.sub.6): 6.89 (1H, d), 7.11 (1H, s), 7.23 (1H,
d), 7.85 (1H, d), 7.98 (1H, d), 8.51 (1H, s), 11.51 (1H, s).
Preparation Example 27
Synthesis of
4-[(7-methoxy-2,4(1H,3H)-quinazolinedion-3-yl)sulfonyl]anthranilic
Acid (Compound 27)
[0175] 500 mg (1.23 mmol) of
3-benzyloxycarbonylamino-4-t-butoxycarbonylbenzenesulfonamide and
460 mg (1.22 mmol) of benzyl
4-methoxy-2-N-phenoxycarbonylanthranilate were treated in the same
way as in Preparation Example 17 to obtain 15 mg (yield 3.1%: 4
steps) of the above-identified compound. Properties: colorless
crystal, Melting point: >200.degree. C. (decomposition), PMR
(.delta. ppm, DMSO-d.sub.6): 3.82 (3H, s), 6.58 (1H, s), 6.80 (1H,
d), 7.16 (1H, d), 7.56 (1H, s), 7.80 (1H, d), 7.90 (1H, d), 11.49
(1H, s).
Preparation Example 28
Synthesis of
(.+-.)-7-[(7-chloro-2,4(1H,3H)-quinazolinedion-3-yl)sulfonyl]-2-oxo-1H,3H-
-quinoline-3-carboxylic acid (Compound 28)
[0176] 400 mg (1.23 mmol) of
(.+-.)-3-t-butoxycarbonyl-2-oxo-1H,3H-quinoline-7-sulfonamide and
468 mg (1.23 mmol) of benzyl
4-chloro-2-N-phenoxycarbonylanthranilate were treated in the same
way as in Preparation Example 17 to obtain 649 mg (yield 86%) of
8-({[(2-benzyloxycarbonyl-5-chloroanilino)carbonyl]amino}sulfonyl)-2-oxo--
1,2,3,4-tetrahydro-3-quinoline carboxylic acid t-butyl ester.
Properties: colorless amorphous, PMR (6 ppm, CDCl.sub.3): 1.32 (9H,
s), 3.18-3.30 (2H, m), 3.54 (1H, m), 5.35 (2H, s), 6.85 (1H, m),
7.00 (1H, m), 7.35-7.39 (5H, m), 7.87-7.96 (3H, m), 8.47 (1H, m),
8.78 (1H, br), 10.92 (1H, br).
[0177] Then, from 640 mg (1.04 mmol) of the resultant sulfonylurea
product, in the same way, 258 mg (yield 55%: 3 steps) of the
above-identified compound was obtained. Properties: colorless
crystal, Melting point: >200.degree. C. (decomposition), PMR (6
ppm, DMSO-d.sub.6): 3.23-3.31 (2H, m), 3.59 (1H, t), 7.07 (1H, d),
7.12 (1H, s), 7.25 (1H, d), 7.86 (1H, d), 7.96 (1H, d), 7.98 (1H,
d), 10.84 (1H, s), 11.60 (1H, s).
Preparation Example 29
Synthesis of
(.+-.)-6-[(7-chloro-2,4(1H,3H)-quinazolinedion-3-yl)sulfonyl]-3-oxo-1,4-b-
enzoxazine-2-carboxylic Acid (Compound 29)
[0178] 300 mg (0.91 mmol) of
(.+-.)-2-t-butoxycarbonyl-3-oxo-1,4-benzoxazin-6-sulfonamide and
349 mg (0.91 mmol) of benzyl
4-chloro-2-N-phenoxycarbonylanthranilate were treated in the same
way as in Preparation Example 17 to obtain 417 mg (yield 74%) of
5-({[(2-benzyloxycarbonyl-5-chloroanilino)carbonyl]amino}sulfonyl)-3-oxo--
3,4-dihydro-2H-1,4-benzoxazine-2-carboxylic acid t-butyl ester.
Properties: colorless amorphous, PMR (6 ppm, DMSO-d.sub.6): 1.29
(9H, s), 5.37 (2H, s), 5.42 (2H, s), 7.19-7.26 (2H, m), 7.37-7.57
(7H, m), 7.97 (1H, d), 8.25 (1H, d), 10.27 (1H, s), 11.25 (1H, s),
12.22 (1H, br).
[0179] Then, from 417 mg (0.68 mmol) of the resultant sulfonylurea
product, in the same way, 100 mg (yield 32%: 3 steps) of the
above-identified compound was obtained. Properties: colorless
crystal, Melting point: >200.degree. C. (decomposition), PMR (6
ppm, DMSO-d.sub.6): 5.47 (1H, s), 7.11 (1H, s), 7.24 (1H, d), 7.29
(1H, d), 7.76 (1H, s), 7.78 (1H, d), 7.86 (1H, d), 11.25 (1H, s),
11.62 (1H, s).
Preparation Example 30
Synthesis of
4-[(7-hydroxy-2,4(1H,3H)-quinazolinedion-3-yl)sulfonyl]anthranilic
Acid (Compound 30)
[0180] 620 mg (1.53 mmol) of
3-benzyloxycarbonylamino-4-t-butoxycarbonylbenzenesulfonamide and
550 mg (1.51 mmol) of benzyl
4-hydroxy-2-N-phenoxycarbonylanthranilate were treated in the same
way as in Preparation Example 17 to obtain 25 mg (yield 4%: 4
steps) of the above-identified compound. Properties: colorless
crystal, Melting point: >200.degree. C. (decomposition), PMR
(.delta. ppm, DMSO-d.sub.6): 6.48 (1H, s), 6.61 (1H, d), 7.14 (1H,
d), 7.51 (1H, s), 7.70 (1H, d), 7.90 (1H, d), 10.80 (1H, s), 11.39
(1H, s).
Preparation Example 31
Synthesis of
4-[(7-chloro-2,4(1H,3H)-quinazolinedion-3-yl)sulfonyl]-2-N-propionylanthr-
anilic Acid (Compound 31)
[0181] 840 mg (1.86 mmol) of Compound 17 was dissolved in 8 ml of
1,4-dioxane, 240 .mu.l (2.79 mmol) of propionyl chloride was added
dropwise, then the resultant mixture was stirred overnight at
60.degree. C. An excess of water was added to the reaction solution
and the mixture was extracted with ethyl acetate. The organic layer
thus obtained was washed, dried, and concentrated to obtain a crude
product of t-butyl
4-[(7-chloro-2,4(1H,3H)-quinazolinedion-3-yl)sulfonyl]-2-N-propionylanthr-
anilate. The obtained crude product was stirred at room temperature
in 3 ml of trifluoroacetic acid for 1 hour, then the reaction
solution was concentrated in vacuo to obtain a crude product. This
was washed by diethyl ether to obtain 400 mg (yield 48%: 2 steps)
of the above-identified compound. Properties: colorless crystal,
Melting point: >200.degree. C. (decomposition), PMR (.delta.
ppm, DMSO-d.sub.6): 1.10 (3H, t), 2.45 (2H, dd), 7.11 (1H, s), 7.24
(1H, d), 7.85 (1H, d), 7.88 (1H, d), 8.17 (1H, d), 9.18 (1H, s),
11.07 (1H, s), 11.63 (1H, s).
Preparation Example 32
Synthesis of
4-[(6-chloro-2,4(1H,3H)-quinazolinedion-3-yl)sulfonyl]anthranilic
Acid (Compound 32)
[0182] 300 mg (0.74 mmol) of
3-benzyloxycarbonylamino-4-t-butoxycarbonylbenzenesulfonamide and
310 mg (0.81 mmol) of benzyl
5-chloro-2-N-phenoxycarbonylanthranilate were treated in the same
way as in Preparation Example 17 to obtain 75 mg (yield 26%: 4
steps) of the above-identified compound. Properties: colorless
crystal, Melting point: >200.degree. C. (decomposition), PMR
(.delta. ppm, DMSO-d.sub.6): 7.13-7.20 (2H, m), 7.56 (1H, s), 7.72
(1H, d), 7.82 (1H, s), 7.90 (1H, d), 11.68 (1H, s).
Preparation Example 33
Synthesis of
4-[(7-chloro-2,4(1H,3H)-quinazolinedion-3-yl)sulfonyl]-2-N-methanesulfony-
lanthranilic Acid (Compound 33)
[0183] 200 mg (0.44 mmol) of Compound 17 was treated in the same
way as in Preparation Example 3 to obtain 81 mg of t-butyl
4-[(7-chloro-2,4(1H,3H)-quinazolinedion-3-yl)sulfonyl]-2-N-methanesulfony-
lanthranilate. This was used to perform the same debutylation
reaction to obtain 53 mg (yield 25%: 2 steps) of the
above-identified compound. Properties: colorless crystal, Melting
point: >200.degree. C. (decomposition), PMR (.delta. ppm,
DMSO-d.sub.6): 3.24 (3H, s), 7.11 (1H, s), 7.25 (1H, d), 7.85-7.91
(2H, m), 8.23 (1H, d), 8.39 (1H, s), 11.05 (1H, br), 11.70 (1H,
s).
Preparation Example 34
Synthesis of
3-(3-aminobenzenesulfonyl)-7-chloro-2,4-(1H,3H)quinazolinedion
Methanesulfonic Acid Salt (Compound 34) 2.15 g (6.10 mmol) of
compound 12 was dissolved in
[0184] 65 ml of THF and 0.4 ml of methanesulfonic acid was added
dropwise. To this solution, 200 ml of ether was added and the
resultant precipate was filtered to obtain 2.59 g (yield 95%) of
the above-identified compound. Properties: colorless amorphous, PMR
(.delta. ppm, DMSO-d.sub.6): 2.35 (3H, s), 6.98 (1H, d), 7.12 (1H,
m), 7.25 (1H, m), 7.34 (2H, s), 7.43 (1H, m), 7.86 (1H, s), 11.64
(1H, s).
Preparation Example 35
Synthesis of
7-chloro-3-[4-(pyrazol-3-yl)benzenesulfonyl]-2,4(1H,3H)-quinazolinedione
hydrochloride (Compound 35)
[0185] Following the Synthesis Method (B), 5.65 g (25.34 mmol) of
4-(pyrazol-3-yl)beneznesulfonamide was dissolved in 60 ml of THF,
then 7.8 ml (52.16 mmol) of DBU was added dropwise. The reaction
solution was stirred at room temperature for 10 minutes, then added
with 8.5 g (27.86 mmol) of methyl
4-chloro-2-phenoxycarbonyl-aminobenzoate and stirred at room
temperature for 3 hours. The reaction solution was further added
with 400 mg (0.131 mmol) of methyl
4-chloro-2-phenoxycarbonylaminobenzoate and then stirred at room
temperature for 2 hours. An excess of an aqueous solution of citric
acid was added to the reaction solution, then extraction was
performed using ethyl acetate. The organic layer was washed by
water and saturated saline, then dried over anhydrous sodium
sulfate and condensed. Methanol was added to the condensed residue,
then the mixture was stirred and the resultant crystals were
obtained by filtration to obtain 10.49 g of a crude product.
[0186] 10.49 g of the crude product obtained was suspended in 45 ml
of methanol, then 90 ml of a 1N sodium hydroxide aqueous solution
was added. The reaction solution was stirred at 60.degree. C. for
40 minutes, then the precipitate was removed by filtration. The
filtrate was concentrated in vacuo and the methanol distilled off,
then the obtained aqueous mixture was washed by ethyl acetate. The
aqueous layer was made acidic by hydrochloric acid to cause the
precipitation of crystals. These were then obtained by filtration.
The filtrate was extracted by ethyl acetate, the organic layer was
washed by saturated saline, and the result was dried and condensed
over anhydrous sodium sulfate. The condensed residue and the
crystals obtained by filtration above were combined and
recrystallized from THF-ethyl acetate-hexane to obtain 7.70 g
(yield of 72% in two steps) of
N-[4-(pyrazol-3-yl)benzenesulfonyl]-N'-(2-carboxyl-5-chlorophenyl)urea
(properties: colorless crystal, melting point: 129 to 132.degree.
C., PMR (.delta. ppm, DMSO-d.sub.6): 6.81 (1H, d), 7.02 (1H, dd),
7.78 (1H, s), 7.89-7.92 (3H, m), 7.96 (2H, d), 8.24 (1H, s), 10.57
(1H, br).
[0187] 3.0 g (7.14 mmol) of the urea derivative obtained above was
dissolved in 60 ml of THF. 1.2 g (7.40 mmol) of CDI was then added
under ice cooling and the result stirred for 2 hours. The reaction
solution was diluted by ethyl acetate, then successively washed by
a citric acid aqueous solution, saturated saline, a 0.5M sodium
hydrogencarbonate aqueous solution, and saturated saline. The
organic layer was dried over anhydrous sodium sulfate, then
condensed to obtain a crude product. The crude product was
recrystallized from ethyl acetate to obtain 1.93 g (yield: 67%) of
7-chloro-3-[4-(pyrazol-3-yl)
benzenesulfonyl]-2,4(1H,3H)-quinazolinedione (properties: colorless
crystal, melting point: 124 to 126.degree. C. (decomposition), PMR
(.delta. ppm, CDCl.sub.3-CD.sub.3OD): 6.73 (1H, s), 7.09 (1H, s),
7.16 (2H, d), 7.48 (1H, s), 7.66 (1H, s), 7.9-8.1 (3H, m), 8.32
(2H, d).
[0188] 545 mg (1.35 mmol) of the quinazoline derivative obtained
above was dissolved in 35 ml of THF, then 0.4 ml of a 1,4-dioxane
solution of 4M hydrochloric acid was added dropwise. 20 ml of ether
was added to this solution, then the precipitated crystal was
obtained by filtration to obtain 572 mg (yield: 96%) of the
above-referenced compound. Properties: colorless crystal, melting
point: >200.degree. C. (decomposition), PMR (.delta. ppm,
DMSO-d.sub.6): 6.91 (1H, d), 7.15 (1H, d), 7.24 (1H, dd), 7.84 (1H,
d), 7.86 (1H, d), 8.01 (2H, d), 8.17 (2H, d), 11.7 (1H, s).
Example 1
Measurement of Chymase Inhibitory Activity
[0189] Human heart chymase was purified according to the method of
Urata et al. (J. Biol. Chem., 1990, 265, 22348). The inhibitory
activity of the quinazoline derivatives of the present invention
with respect to chymase was measured in the following manner. That
is, the purified enzyme solution was diluted to a suitable
concentration with 0.1M tris-hydrochloride buffer (pH=7.5), 1M
sodium chloride, and 0.01% TritonX-100 to obtain an enzyme
solution. A 10 mM dimethyl sulfoxide (hereinafter referred to as
DMSO) solution of Suc-Ala-Ala-Pro-Phe-MCA (Peptide Institute) was
diluted 20-fold at the time of use by 0.1M tris-hydrochlorate, 1M
sodium chloride, and 0.01% TritonX-100 to obtain the substrate
solution.
[0190] 75 .mu.l of the enzyme solution warmed to 30.degree. C. was
mixed with 5 .mu.l of DMSO solution of the test sample. The mixture
was preincubated at 30.degree. C. for 10 minutes. Next, 20 .mu.l of
a substrate solution warmed to 30.degree. C. was mixed with the
test sample-enzyme mixture and incubated at 30.degree. C. After 10
minutes, 50 .mu.l of 30% acetic acid was added to stop the
enzymatic reaction. The amount of the AMC produced was quantified
using a fluorescent photometer. At the same time, a blind test was
carried out by adding, instead of the test sample solution, 5 .mu.l
of DMSO and performing the same reaction. The chymase inhibitory
activity was expressed by a rate of inhibition, that is, the 50%
inhibition concentration (IC.sub.50), based on the blind test
value.
[0191] The quinazoline derivatives of the present invention all
strongly inhibited human chymase at concentrations of 100 .mu.M.
The IC.sub.5, values for typical compounds are shown in Table
I.
TABLE-US-00001 TABLE 1 Example No. IC.sub.50 value (.mu.M) 1 0.36 2
0.14 8 0.035 10 0.17 12 0.44 13 0.3 16 0.84 17 0.14 18 0.14 21 0.34
22 0.3 24 0.32 27 4.0 29 1.7 32 1.5 34 0.36
Example 2
Time-Course of Skin Reaction in Ascaris-Induced Mouse Biphasic
Dermatitis Model
[0192] Ascaris-induced biphasic dermatitis was induced according to
the method described previously (Folia Pharmacol. Jap. 112, 221,
1998). That is, 8-week old BALB/c mice (Charles River Japan) were
sensitized by intraperitoneal injection of 0.5 ml of a 1:1 mixture
of ascaris extract (800 .mu.g/ml, Cosmo Bio Co., Ltd.) and an alum
saline suspension (16 mg/ml in saline). Two weeks after the
sensitization, 10 .mu.l of ascaris extract (1 mg/ml) was injected
intradermally to the right ear of mice. The edema induced at the
ear were evaluated immediately before intradermal injection of the
ascaris extract (n=3) and 1 hour after (n=4), 2 hours after (n=4),
4 hours after (n=4), 6 hours after (n=4), 16 hours after (n=4), and
24 hours after injection (n=4), by weighing ear biopsy prepared
with a punch (a diameter of 6 mm, Fukui Kiko Shokai) and measuring
their weights. The edema (mg) was expressed as the difference in
the weight of the ear punch biopsy between the right and the left
ears of the same mouse.
[0193] Biphasic dermatitis was induced by intradermal
administration of ascaris extract to the ears of mice sensitized by
the same antigen (FIG. 1). The first reaction reached its peak
after 1 hour, while the second reaction reached its peak after 16
hours.
Example 3
Effects of Chymase Inhibitor in Ascaris-Induced Mouse Biphasic
Dermatitis Model
[0194] Dermatitis was induced in accordance with the method
described in Example 2 and the ear edema was measured in the same
way as in Example 2, 1 hour (n=6) and 16 hours (n=8) after the
intradermal administration of ascaris extract to the ears to
investigate the effects of the test substance on dermatitis. As the
chymase inhibitor, Compound 34 was used. As the control drug,
diphenhydrazine (antihistamine, Sigma) and prednisolone (steroid,
Nakarai Tesc Co.) were used. Each drug under study was suspended in
saline containing 0.5% hydroxypropyl cellulose and administered
intraperiotoneally 60 minutes before intradermal administration of
ascaris extract. A group of mice sensitized with ascaris extract
and challenged by intradermal injection of saline was used as a
control (n=3).
[0195] Results
[0196] As a result of the intraperiotoneal administration of the
chymase inhibitor (Compound 34), the reaction after 1 hour
(early-phase reaction) and reaction after 16 hours (late-phase
reaction) of the biphasic dermatitis induced by ascaris extract
were both suppressed in a dose-dependent manner. A statistically
significant difference was observed in the dosage of 50 mg/kg (FIG.
2A). The rate of suppression at 50 mg/kg was about 41% for the
early-phase reaction and about 45% for the late-phase reaction
(both p<0.01, Dunnett's test). Prednisolone, which is effective
against atopic dermatitis, was substantially ineffective against
the early-phase reaction, but strongly inhibited the late-phase
reaction in a dosage of 30 mg/kg (rate of suppression: 67%) (FIG.
2B). On the other hand, diphenhydrazine significantly suppressed
the early-phase reaction (rate of suppression: 79%), but exhibited
almost no effect against the late-phase reaction (FIG. 2C).
[0197] The fact that a chymase inhibitor exhibits a suppressive
action in an allergic dermatitis model exhibiting biphasic skin
reaction shows the involvement of chymase in allergic biphasic
dermatitis and usefulness of a chymase inhibitor for such
dermatitis. In particular, the finding that a chymase inhibitor,
like a steroid, significantly suppresses late-phase reaction, in
which antihistamines and anti-allergic agents exhibit little
effect, shows the usefulness of a chymase inhibitor in atopic
dermatitis. In the following Examples 4 to 6, the skin conditions
induced by inoculation of human chymase into the ears of mice were
analyzed for the purpose of further confirming the importance of
chymase in a biphasic skin reaction.
Example 4
Ability of Single Administration of Human Chymase to Induce
Dermatitis
[0198] Recombinant human chymase was used in this Example.
[0199] Recombinant human chymase was obtained by expression and
purification in accordance with the already reported method of
production of serine protease (Biochem. Biophys. Acta 1350, 11,
1997). That is, first, cDNA (79-756) encoding mature human chymase
(J. Biol. Chem. 266, 17173, 1991) was amplified by the PCR method.
The PCR product was cloned to the pDE vector along with the signal
sequence of human trypsin II and the region including the cleaved
site of enterokinase (23 amino acid). The constructed human chymase
expression plasmid was transfected to CHOdhfr.sup.- cells, and the
transfectants were selected by an already reported method (Arch.
Biochem. Biophys. 307, 133, 1993). The fused protein of the human
chymase and trypsin secreted in the culture supernatent of the
obtained cells was concentrated using a HiTrap Heparin column
(Amersham Pharmacia Biotech), then cleaved with enterokinase
(Invitrogen) to produce human mature chymase. The human mature
chymase was purified using a heparin 5PW column (Tosoh Corp.) In
SDS-polyacrylamide gel electrophoresis analysis, the purified
chymase showed a 33-36 kDa broad band. Further, chymase activity
was measured in a 0.1M Tris/HCl buffer (pH8.0) by using 1 mM
Suc-Ala-Ala-Pro-Phe-MCA (Peplide Institute) as a substrate and
measuring the intensity of fluorescense of the free MCA. As a
result, it was confirmed that the purified chymase certainly has
the enzymatic activity.
[0200] Next, 20 .mu.l of the above recombinant human chymase
(hereinafter called human chymase) (0.1 mg/ml) was administered
intradermally to an ear of BALB/c mice (Japan Charles River) and
the time-course of the edematous reaction of the ears measured by
the method described in Example 2 for the purpose of investigating
the role of chymase in dermatitis (n=3 to 4). Further, histamine,
an inflammation mediator of mast cells, was similarly administered
intradermally and the time-course was compared with the case of
administration of human chymase. The histamine (Sigma-Aldrich) was
injected by dissolving in saline (0.25 mg/ml).
[0201] As shown in FIG. 3A, by administering human chymase (2.0
.mu.g/ear) to the ears of mice, a biphasic edematous reaction
resembling the allergic skin reaction shown in the case of Example
2 was induced. That is, the first skin reaction was immediately
induced after administration of chymase and reached a peak after 30
minutes to 1 hour. Further, the second skin reaction peaked after 6
hours and continued for at least 24 hours. On the other hand, an
immediate edematous reaction was induced even when inoculating
histamine, but this skin reaction completely disappeared 20 hours
after the inoculation in contrast to the case of inoculation of
chymase (FIG. 3B). The analysis of the dose-dependency in the
chymase-induced dermatitis revealed that early-phase reaction
(after 1 hour) is dose-dependent and that the maximum reaction is
observed at 2.0 .mu.g/ear amount the dose used in the experiment
(FIG. 4A). On the other hand, in the second (after 16 hours)
reaction, while the reactions at 0.5 .mu.g/ear and 1.0 .mu.g/ear
were about the same in level, the maximum response was obtained at
2.0 .mu.g/ear in the same way as in the first reaction (FIG.
4B).
[0202] As shown above, it was shown that intradermal administration
of human chymase in mice induces dermatitis, and that its
time-course resembles that of antigen-induced biphasic dermatitis,
an acute model of dermatitis, showing the involvement of chymase in
biphasic dermatitis.
Example 5
Involvement of Chymase Activity in Dermatitis Induced by Single
Administration of Chymase
[0203] The ability of heat-treated human chymase to induced
dermatitis was investigated for the purpose of studying whether the
enzymatic activity of chymase is involved in dermatitis induced by
human chymase shown. The human chymase was inactivated by
incubating a 0.1 mg/ml human chymase solution at 50.degree. C. for
2 hours, then boiling it at 100.degree. C. for 5 minutes. This
inactivated human chymase (2.0 .mu.g/ear) was administered to the
ears of mice by the method described in Example 4, and the
dermatitis was evaluated 1 hour after the administration.
[0204] As a result of the heat treatment of the human chymase, the
edema reaction induced by the human chymase completely disappeared
(p<0.01 vs. untreated chymase administration group, Student's
t-test, N=4) (FIG. 5). This result shows that chymase activity is
essential for inducing dermatitis.
Example 6
Histological Analysis of Dermatitis Induced by Single
Administration of Chymase
[0205] A pathohistological analysis of dermatitis induced by
chymase was conducted and a comparison was performed with the
biphasic dermatitis shown in Example 2 for the purpose of
investigating in further detail the involvement of chymase in
biphasic dermatitis. These types of dermatitis were induced in
accordance with the methods described in Example 2 and Example 4
(dosage of human chymase was 2.0 .mu.g/ear). The ears were fixed in
formalin and paraffin sections were prepared in accordance with an
ordinary method 1 hour and 24 hours after administration in both
models. The sections were stained with hematoxylin and eosin, then
observed under-microscope and photographed. Further, sections of
ears of normal BALB/c mice were used as negative controls.
[0206] In the section 1 hour after elicitation of chymase
dermatitis (FIG. 6D), remarkable thickening was observed compared
with the sections of ears of normal mice (FIG. 6A), but no
difference could be observed between the two in respect to
infiltration of leukocytes. As shown in FIG. 6E, however,
remarkable cellular infiltration was observed in sections 24 hours
after chymase injection. The pattern of the histological change
shown by chymase dermatitis resembled that of the ascaris-induced
biphasic dermatitis model (FIGS. 6B and 6C). That is, there was
remarkable thickening of the tissue at 1 hour, but cellular
infiltration was observed only in sections after 24 hours. In
summary, it was shown that the dermatitis induced by chymase
resembles the biphasic dermatitis induced by an antigen even in a
pathohistological analysis.
[0207] In the following Example 7 and Example 8, an analysis was
conducted on the mechanism of dermatitis induced by chymase to
investigate the role of chymase in biphasic dermatitis.
Example 7
Ability of Human Chymase to Induce Dermatitis in Mast
Cell-Deficient Mice
[0208] Since it has been reported that chymase induces a
degranulation in rat peritoneal mast cells (J. Immunol. 136, 3812,
1986), it was considered possible that dermatitis induced by
chymase is induced through the release of the inflammation mediator
from the mast cells. Thus, next the involvement of mast cells in
chymase-induced dermatitis was studied using mast-cell deficient
mice (Blood 52, 447-425, 1978). Mast cell-deficient
(WBB6F1-W/W.sup.v) mice and their control mice (WBB6F1-+/+) were
obtained from SLC Japan. Chymase dermatitis was induced by
intradermal administration of human chymase (2.0 .mu.g/ear) and the
edema reaction was evaluated after 1 hour and 16 hours using the
method described in Example 4.
[0209] As shown in FIGS. 7A and 7B, a skin reaction of a similar
extent as the control mice (WBB6F1-+/+) was observed even in mast
cell deficient (WBB6F1-W/W.sup.v) mice after 1 hour (FIG. 7A) and
after 16 hours (FIG. 7B). This result indicates that chymase
induces a biphasic phase skin reaction regardless of the existence
of mast cells.
Example 8
Ability of Human Chymase to Promote Migration of Polymorphonuclear
Leukocytes
[0210] In Example 6, it was shown that a remarkable infiltration of
leukocytes is observed in late-phase reaction of human
chymase-induced dermatitis. The effect of human chymase on the
migration of polymorphonuclear leukocytes (PMN) in vitro was
investigated for the purpose of investigating the mechanism of
chymase-induced leukocyte infiltration. PMN was isolated by adding
a 1/5th volume of 6% dextran solution to heparinized whole blood
from normal healthy subject and allowing it to stand at 37.degree.
C. for 1 hour, then layering the supernatent on Ficoll-Paque
(Amersham Pharmacia Biotech) and centrifuging it. Further, the
migration of PMN was measured by using a 48-well chemotaxis chamber
(NeuroProbe Co.) by the textbook method (Seibutsu Yakkagaku Jikken
Koza (Biopharmacology Experiment Lectures) 12, 315, Hirokawa
Shoten). That is, a medium containing human chymase (200 to 800 nM)
or fMLP (N-formyl-L-methionyl-L-leucyl-L-phenylalanine,
Sigma-Aldrich Co.) (10 nM) was placed in the lower well of the
chamber. The upper well and the lower well were separated by a
polycarbonate filter (pore size 5 .mu.m) (NeuroProbe Co.) PMN
(1.times.10.sup.6/ml) was added to the upper well and cultured at
37.degree. C. for 1 hour, then the filter was fixed, stained and
the number of cells in the membrane were counted under a microscope
(400.times.) (Seibutsu Yakkagaku Jikken Koza (Biopharmacology
Experiment Lectures) 12, 315). For the cell staining, a hemacolor
solution (Merck) was used. In this test, when investigating the
effect of the chymase inhibitor, a chymase inhibitor (Compound 18
or Compound 34) was dissolved in dimethyl sulfoxide and added to
the lower well of the chamber just before the addition of the human
chymase. The concentration of the compound was adjusted so that the
final concentration of the dimethyl sulfoxide became 1%.
[0211] As shown in FIG. 8A, human chymase exhibited an activity
promoting migration of human PMN in a concentration-dependent
manner, with a statistical significance observed at .gtoreq.2400 nM
(p<0.05, Dunnett's test). From the fact that human chymase
exhibits activity promoting migration to the same extent as 10 nM
fMLP in a concentration of 200 to 400 nM, it is deduced to have an
activity of about 1/30th that of fMLP. The action of human chymase
in promoting the migration of human PMN was significantly
suppressed by chymase inhibitors, Compound 18 and Compound 34 (FIG.
8B). These results suggest that chymase acts directly on
polymorphonuclear leukocytes to promote their migration, and show
the involvement of enzymatic activity of chymase in that
action.
[0212] Taken together, since chymase released by mast cells upon
antigen stimuli induces biphasic skin reaction when injected
intradermally to mice ear (Example 4 to 6), it is clear that
chymase plays an important role in a skin reaction exhibiting
biphasic reaction. The data of Examples 7 and 8 suggest a mechanism
of involvement of chymase in biphasic skin reaction. In addition,
it was also shown that a chymase inhibitor suppresses
ascaris-induced dermatitis, a biphasic skin reaction (Example 2 and
Example 3).
[0213] Next, a mouse dermatitis model induced by repeated
application of hapten was analyzed as a model of dermatitis induced
by repeated exposure to an antigen and the effects of a chymase
inhibitor in this model is shown.
Example 9
Time-Course of Increase in Ear Thickness in Mouse Dermatitis Model
Induced by Repeated Application of Hapten
[0214] Dermatitis was induced in accordance with an already
reported method (J. Pharmacol. Exp. Ther. 283, 321, 1997) using
dinitrofluorobenzene (DNFB) as hapten. That is, the right ears of
eight-week old female C3H/HeN mice (Nippon Clare) (n=7) were
painted with 0.15% DNFB (25 .mu.l) dissolved in an acetone/olive
oil (3:1) every seven days six times. When applying the hapten each
time, the ear thickness was measured by a micrometer (Digimatic
Indicator, Mitsutoyo Co.) immediately before the application and 1
hour, 6 hours, 24 hours, and 48 hours after the application so as
to find the amount of increase from the ear thickness before the
first application of hapten. Further, a group similarly treated
with an acetone/olive oil solution (3:1) not containing DNFB was
used as a control. Further, in a test separate from the above, the
ears were cut off immediately before the third application of
hapten, the chymase-like activity of the skin was measured in
accordance with an already reported method (n=3), and this was
compared with the activity of mice treated with an acetone/olive
oil solution (3:1) not containing DNFB (n=2).
[0215] A transient skin reaction was induced by the application of
DNFB. This transient skin reaction gradually became larger with
each instance of application (FIG. 9). By the repeated application
of DNFB, in addition to an increase in the response to DNFB, the
ear thickness before application (baseline value) gradually
increased. For example, at the fifth week after the start of
application, the thickness of the ear immediately before
application increased by about 240 .mu.m compared with the ear
before the first application (FIG. 9). In the control group treated
with only a solvent not containing DNFB, almost no thickening of
the ear was detected at any point of time. The chymase activity of
the ear immediately before the third application was significantly
higher as compared with the control group treated with only a
solvent not containing DNFB (p<0.05, Student's t-test) (FIG.
10). In summary, it was shown that repeated application of hapten
DNFB to the ears of mice induces a sustained ear thickness, in
addition to an increase in the chymase activity and a remarkable
transient skin reaction.
Example 10
Effect of Chymase Inhibitor in Mouse Dermatitis Model Induced by
Repeated Application of Hapten
[0216] The effect of a test substance on dermatitis was
investigated by inducing dermatitis in accordance with the method
described in Example 9 (n=7) and measuring the ear thickness in the
same way as in Example 9. As the test substance, three chymase
inhibitors (Compound 18, Compound 34, and Compound 35) were used,
while as the control, the steroid prednisolone (Nakarai Tesc Inc.)
was used. Further, the test substance was suspended in saline
containing 0.5% hydroxypropyl cellulose in the same way as in
Example 3 (HPC/saline) and intraperitoneally administered in
dosages of 10 mg/kg or 50 mg/kg once a day for five consecutive
days a week until the end of the test; the first administration was
performed immediately before the start of the hapten-application.
Further, a group similarly treated with DNFB and administered
HPC/saline instead of the test substance was used as a control
group.
[0217] Prednisolone suppressed dermatitis in this model in a
dose-dependent manner (FIG. 11A). On the other hand, a chymase
inhibitor also remarkably suppressed the transient skin reaction
induced by the application of hapten, particularly after the fourth
week (3 weeks after initial application of hapten) (FIG. 11B-D).
For example, Compound 35 significantly suppressed the increase in
ear thickness in the fourth to sixth weeks (3 weeks initial
application of hapten, and thereafter) at 50 mg/kg 1 hour, 6 hours,
24 hours, and 48 hours after hapten application (p<0.05,
Dunnett's test). Compound 34 also significantly suppressed the skin
reaction 1 to 48 hours after the application after the fifth week
(4 weeks after initial application of hapten, and thereafter) at 50
mg/kg and exhibited a significant suppressive effect 1, 24, and 48
hours after the fifth week (after the fifth application of hapten)
and 1 hour after the sixth week (after sixth application of hapten)
in an amount of 10 mg/kg. Further, Compound 18 significantly
suppressed the reaction 1 to 24 hours after the application in the
fourth and fifth weeks (after fourth application and fifth
application of hapten) and the reaction 1 to 48 hours in the sixth
week (after sixth application of hapten) at 50 mg/kg and exhibited
a significant effect 1 to 48 hours after the application in the
sixth week (after sixth application of hapten) even at 10
mg/kg.
[0218] These results show that chymase inhibitor suppresses edema
in mouse dermatitis model induced by repeated application of
hapten.
Example 11
Effect of Chymase Inhibitor on Increase in Eosinophils of Skin in
Mouse Dermatitis Model Induced by Repeated Application of
Hapten
[0219] The effect of a chymase inhibitor on the infiltration of
eosinophils of the skin in the test of Example 10 was studied. That
is, 48 hours after the sixth application of hapten, the ears of the
mice were fixed with formalin and paraffin sections prepared in
accordance with an ordinary method. The sections were stained with
Fast Green (Fluka), known to specifically stain eosinophils
(Current Protocol in Immunology, Wiley Interscience). That is, the
deparaffined sections were fixed with 100% methanol for 1 minute,
then stained for 30 minutes by 0.2% Fast Green dissolved in 70%
ethanol. The eosinophils were measured by randomly selecting 10
fields (X400) under a microscope and counting the number of cells
per area using an ocular grid. As the chymase inhibitor, the
Compound 18 and Compound 34 were used and administered by the
method described in Example 10. Further, a group treated with an
acetone/olive oil solution (3:1) not containing DNFB was used as
the non-induced control group, while a group treated with DNFB and
administered HPC/saline instead of the test substance was used as
the control for the group administered the chymase inhibitor.
[0220] As shown in FIG. 12, the number of eosinophils remarkably
increased by the repeated application of hapten (about 22 times
that of the control group, p<0.01, Student's t-test). As a
result of administration of a chymase inhibitor (Compound 18), the
increase in the eosinophils was significantly suppressed in a
dose-dependent manner. That is, the rates of suppression at 10
mg/kg and 50 mg/kg were 37.1% and 60.5%, respectively. For the
Compound 34 as well, while no statistically significant difference
was recognized, a trend toward suppression dependent on the dosage
was shown. These results suggest that chymase inhibitor suppresses
the increase in eosinophils of the skin in mouse dermatitis model
induced by repeated application of hapten.
Example 12
Effect of Chymase Inhibitor on Increase in Number of Mast Cells of
Skin in Mouse Dermatitis Model Induced by Repeated Application of
Hapten
[0221] The effect of a chymase inhibitor on the increase in mast
cells of skin in the test of Example 10 was studied. In the same
way as Example 11, the ears of the mice were fixed with formalin
and paraffin sections prepared by an ordinary method 48 hours after
the sixth application of hapten. The mast cells of the sections
were stained by the Toluidine Blue method, then the number of mast
cells were counted under a microscope (X400) for 10 fields per
section. The density of mast cells of the skin was measured in
accordance with the method of Kitagaki et al. (J. Invest. Dermatol.
105, 749, 1995). As the chymase inhibitor, use was made of Compound
18 and Compound 34. These were administered by the method described
in Example 10. Further, a group treated with an acetone/olive oil
solution (3:1) not containing DNFB was used as the non-induced
control group, while a group treated with DNFB and administered
HPC/saline instead of the test substance was used as the control
for the group administered the chymase inhibitor.
[0222] As shown in FIG. 13, the density of mast cells of the skin
significantly increased by the application of hapten (about 2.5
times that of the non-induced group), but as a result of
administration of a chymase inhibitor (Compound 18 and Compound
34), the increase in the density of mast cells was significantly
suppressed by both the compounds in all dosages. The rate of
suppression by Compound 18 was about 57% at 10 mg/kg and about 64%
at 50 mg/kg, while the rate of suppression by Compound 34 was about
37% at 10 mg/kg and about 51% at 50 mg/kg. FIG. 14 shows
representative micrographs. FIG. 14A shows the ear of a non-induced
mouse, FIG. 14B shows the ear of a mouse treated with DNFB and
administered HPC/saline (48 hours after the sixth application of
DNFB), and FIG. 14C shows the ear a mouse treated with DNFB and
administered Compound 34 (48 hours after the sixth application of
DNFB). These results suggest that chymase inhibitor suppresses the
increase in the number of mast cells of the skin in mouse
dermatitis model induced by repeated application of hapten.
Example 13
Ability of Repeated Administration of Human Chymase to Induce
Dermatitis
[0223] The role of chymase in dermatitis induced by repeated
exposure to an allergen was investigated by repeatedly
administering intradermally human chymase to the ear of mice. The
human chymase was administered once a week (2.0 .mu.g/ear/shot) in
accordance with the method described in Example 4. The thickness of
the ear was measured by a micrometer (Digimatic Indicator,
Mitsutoyo Co.) immediately before each administration of chymase
and 1 hour, 6 hours, 24 hours, and 48 hours after the
administration to find the increase from the ear thickness before
the first administration of chymase. Further, heat-treated chymase
was prepared by the method described in Example 5, and its effect
was also studied at the same time.
[0224] A transient skin reaction was induced by intradermal
administration of human chymase to the ears of the mice. The
reactivity with respect to chymase was about the same among the
first to third administrations of chymase, but was amplified by the
fourth to fifth administrations (FIG. 15). No transient reaction
was observed when the inactivated chymase was administered
repeatedly. Further, no skin reaction at all was observed when
repeatedly administering this inactivated chymase (FIG. 15). This
shows that the skin reaction observed was due to the enzymatic
activity of chymase and that the amplification of the skin reaction
seen when administering chymase four to five times is not due to
the administration of foreign protein to the mice.
Example 14
Effect of Repeated Administration of Human Chymase on Number of
Eosinophils of Skin
[0225] Human chymase was repeatedly administered to the ears of
mice to investigate the change in the number of eosinophils of the
skin. Human chymase was repeatedly administered to the ears of mice
in accordance with the method described in Example 13. The number
of eosinophils of the skin was measured by the method described in
Example 11. Further, as a control, ear tissue repeatedly
administered saline was used.
[0226] As shown in FIG. 16, in an ear 24 hours after single
administration of 2.0 .mu.g of human chymase, there was an
approximately 7.6 fold increase in the number of eosinophils
compared with an ear administered saline (p<0.01, Student's
t-test). When human chymase was administered further at 1 week
intervals four times in total and the number of eosinophils was
measured 1 week after each administration, the number of
eosinophils further increased to about 21-fold the group
administered saline (p<0.01, Student's t-test). These results
indicate the possibility that chymase increases the number of
eosinophils and that the rate of the increase is dependent on the
frequency of the exposure to chymase.
Example 15
Effect of Repeated Administration of Human Chymase on Number of
Mast Cells of Skin
[0227] Human chymase was repeatedly administered intradermally to
the ears of mice to investigate the change in the number of mast
cells of the skin. Human chymase was administered repeatedly in
accordance with the method described in Example 13 and the change
in the mast cells of the skin was studied 1 week after
administration of the chymase by the method described in Example 12
or the method of measuring the histamine content of the skin. The
skin histamine content was measured by cutting off the ear, then
homogenizing it in a 20 mM Tris-HCl buffer (pH7.5), centrifuging
the extract (10,000 rpm, 10 minutes), then assaying the amount of
histamine in the supernatent using an ELISA kit (Medical Biological
Laboratories). Further, as the control, use was made of ear tissue
repeatedly administered saline.
[0228] The change in the number of mast cells of the skin was
observed in accordance with the method described in Example 12,
whereupon a tendency for an increase in the number of mast cells by
the administration of human chymase was seen. The increase in the
number of cells however was not remarkable. Thus, the histamine
content of the skin, an indicator of the number of mast cells of
tissue, was measured for the purpose of more objectively or
quantitatively evaluating the increase in the number of mast cells
of the skin. As a result, as shown in FIG. 17, the histamine
content of the skin was a significantly higher than the group
administered saline a week after the fourth administration of human
chymase. From these results, the existence of a mechanism where the
chymase derived from mast cells acts on the mast cells in a
positive feedback manner is suggested.
Example 16
Effect of Administration of Human Chymase on Stem Cell Factors
(SCF)
[0229] The expression of stem cell factors (SCF), known as a factor
for differentiation and proliferation of mast cells (Blood 90,
1345, 1997), was analyzed by the immunohistochemical method for the
purpose of clarifying the mechanism of action of the increase in
the number of mast cells of the skin when administering human
chymase. Human chymase (2.0 .mu.g/ear) was administered to mouse
ears by the method described in Example 4. The ears were harvested
after 1 hour, after 6 hours, and after 24 hours and frozen tissue
sections of 5 .mu.m were prepared by an ordinary method. As a
control, sections of ears of normal mice were used. In the
immunohistochemical studies, anti-mouse SCF goat IgG (made by
R&D Systems) was used as the primary antibody for detection by
a PAP kit (DAKO Co.) Note that as a negative control, normal goat
IgG (Vector Laboratories Co.) was used instead of an SCF antibody.
Further, after immunostaining, nucleus was stained using Methyl
Green (Wako Pure Chemicals) in accordance with an ordinary method
(Sensyokuho no Subete (Everything About Dyeing), Ishiyaku
Shuppan).
[0230] In normal mouse ears, a strong immunostaining was observed
around the corneal layer of the epidermis (FIG. 18A). On the other
hand, no immunostaining at all was seen when using normal goat IgG
instead of anti-SCF antibody as the primary antibody. This shows
that the immunostaining in the corneal layer is specific. On the
other hand, in the ears of mice administered chymase, the staining
around the corneal layer becomes weaker in a time-dependent manner.
After 24 hours, almost no formation of color is seen in the ears
(FIG. 18B).
Example 17
Effect of Human Chymase on Expression of SCF in Human
Keratinocytes
[0231] The effect of human chymase on the expression of SCF in
human normal keratinocytes in vitro was studied. Human normal
keratinocytes were obtained from Cell Applications Co. The cultured
keratinocytes were harvested by an enzyme-free cell dissociation
buffer (GIBCO BRL Co.), washed three times by PBS, then adjusted to
a cell concentration of 10.sup.6/50 .mu.l, added with human
chymase, and allowed to react at 37.degree. C. for 10 minutes. The
chymase reaction was stopped by the addition of fetal calf serum
(FCS) to give a final concentration of 10%, then the cells were
removed by centrifugation. The SCF in the supernatent was assayed
by an ELISA kit (PeproTech Co.) The cytotoxicity of chymase to the
keratinocytes was investigated using as an indicator the release of
lactate dehydrogenase (LDH) using a cytotoxicity detection kit
(made by Roche Molecular Biochemicals Co.)
[0232] As shown in FIG. 19, the release of SCF was promoted in a
concentration-dependent manner by incubating human keratinocytes in
the presence of human chymase for 10 minutes. The amount of release
of SCF when incubating it in the absence of human chymase for 10
minutes was about the same as when not incubating it. On the other
hand, the release of LDH in the supernatent did not change all
under these conditions. It was confirmed that the release of SCF
due to human chymase was not by cell damage. Above, it was shown in
vitro that human chymase acts on membrane-bound SCF of human
keratinocytes and causes the release of free SCF.
[0233] SCF in composed of two types of molecules of SCF.sup.248 and
SCF.sup.220 that are generated by the differences in splicing
(Blood 90, 1345, 1997). SCF.sup.246 is first synthesized as a cell
membrane protein, then processed by some sort of protease to become
free SCF which then is released from the cells. On the other hand,
SCF.sup.220 does not have any site digested by an enzyme, so
functions only as a membrane protein (Blood 90, 1345, 1997).
Longley et al. have provided data that the SCF of the skin of
healthy subjects is expressed mainly on the cell membranes of
keratinocytes of the epidermis, but that in dermatitis accompanied
with an increase in skin mast cells, the expression of SCF on the
cells is no longer observed and that SCF is detected in the dermis
and the intercellular spaces of the keratinocytes (N. Engl. J. Med.
328, 1302, 1993). Further, in transgenic mice made to excessively
express both of SCF.sup.220 and SCF.sup.248, an increase in the
number of skin mast cells is observed, but this phenomenon is not
seen in transgenic mice made to excessively express only
SCF.sup.220 (J. Exp. Med. 187, 1565, 1998). These findings suggest
that the patho physiological roles differ between membrane-bound
type and free SCF and in particular that free SCF is closely
related to the increase in number of mast cells of the skin. In
fact, it is known that administration of free SCF to the human skin
causes an increase in the number of mast cells of the skin (J. Exp.
Med. 183, 2681, 1996).
[0234] Longley et al. further report that human chymase cleaves
membrane binding SCF and converts it to free SCF (Proc. Natl. Acad.
Sci. USA, 94, 9017; 1997). The findings obtained from Example 16
are completely novel findings proving the data of Longley et al. in
vivo. The findings of Example 17 are initial data shown using human
cells. Further, the findings of Examples 16 and 17 can be said to
be data explaining the increase in skin mast cells induced by
administration of chymase shown in Example 15 and the mechanism of
action in suppressing the increase in skin mast cells by a chymase
inhibitor shown in Example 12.
[0235] From the fact that skin reaction that is induced by
administrating artificially from the outside chymase released by
mast cells upon antigen stimuli is amplified along with the
increase in the number of the repeated administration, (Example
13), it is clear that chymase plays an important role in dermatitis
induced by repeated exposure to an antigen. Further, from the fact
that a chymase inhibitor suppresses the increase in eosinophills or
mast cells in skin in dermatitis induced by repeated application of
hapten (Examples 11 and 12) and that the administration of chymase
causes an increase in eosinophils or mast cells (Examples 14 to
15), it is clear that chymase controls the number of eosinophils or
mast cells playing an important role in allergic reactions.
Further, it was shown that a chymase inhibitor improves the
condition of dermatitis in the dermatitis model induced by repeated
application of hapten, which is a model of dermatitis induced by
repeated exposure to an antigen (Examples 9 and 10).
[0236] Next, the effect of a chymase inhibitor on natural onset
dermatitis (NC/Nga) mice as a second model of dermatitis induced by
repeated exposure to an antigen will be shown.
Example 18
Effect of Chymase Inhibitor on Natural Onset Dermatitis NC/Nga)
Mice
[0237] NC/Nga mice were bred and raised in accordance with the
methods described in previous reports (Progress in Medicine 19,
1201, 1999). Specifically, 5-week old NC/Nga mice obtained from
Charles River Japan and raised under a specific pathogen free (SPF)
environment were mixed with NC/Nga mice bred by the Faculty of
Applied Biological Science of Hiroshima University starting from 6
weeks of age, kept until 15 weeks of age in a normal non-SPF
environment, then used for the tests. The tests were conducted
under non-SPF, conventional environment. The test substance
(Compound 18) was mixed with the drinking water and administered
for 35 consecutive days at 150 mg/kg/day (n=7), then an evaluation
was conducted in accordance with the method described in previous
reports (Progress in Medicine 19, 1201, 1999). That is, first, 35
days after the start of administration, the five items of (1)
scratching behavior, (2) edema, (3) erythema and hemorrhaging, (4)
depilation and ulceration, and (5) dryness were scored as 0 to 2
and the total found to judge the outer appearance of the skin.
Next, the ears and skin of the back were fixed with formalin,
embedded in paraffin, and histologically analyzed by staining the
ear specimens by the hematoxylin and Eosin method, Toluidine Blue
method, or Congo Red method (Stain Technol. 56, 323, 1981) and by
staining the back skin specimens by the Toluidine Blue method or
Congo Red method. Sections of the ear stained with Hematoxylin and
Eosin were evaluated by judging the change in tissue in four scales
of 0 to 3 for three items of (1) thickness of epidermis, (2)
thickness of dermis, and (3) cellular infiltration and expressing
as the total score. Further, the specimens stained with Toluidine
Blue and stained with Congo Red were used for counting the mast
cells and eosinophils. Specifically, the eosinophils of the back
skin were counted in a X400 field of a microscope, while the other
cells were counted in a total of five X200 fields and the totals
expressed.
[0238] As shown in FIGS. 20A and 20B, at the start of the test (15
weeks age), no difference could be seen in the scores of the skin
conditions between the control group and the chymase inhibitor
(Compound 18) administered group (FIG. 20A), but 35 days after
administration of the chymase inhibitor, the dermatitis score
decreased significantly compared with the control group (p<0.05,
Mann-Whitney test) (FIG. 20B). Similarly, the histological score of
the ears also decreased significantly by the administration of a
chymase inhibitor (FIG. 21, p<0.05, Mann-Whitney test). On the
other hand, the mast cells of the ear skin (FIG. 22A) and back skin
(FIG. 22B) were counted. As a result, a significant suppressive
effect was recognized by the administration of a chymase inhibitor
in both specimens. Further, it was shown that the numbers of
eosinophils of the ear skin (FIG. 23A) and back skin (FIG. 23B)
were significantly suppressed by a chymase inhibitor.
[0239] Above, in a natural onset atopic dermatitis model where
onset is considered to occur due to repeated exposure to an antigen
in the air under a non-SPF environment, a chymase inhibitor
improves the outer appearance of the skin and the histological
change of the skin and suppresses the increase in mast-cells and
infiltration of eosinophils, so the usefulness of a chymase
inhibitor against dermatitis induced by repeated exposure to an
antigen is demonstrated.
Formulation Example 1
Production of Tablets
[0240] 100.0 g of Compound 1 was mixed with 22.5 g of
microcrystalline cellulose and 2.5 g of magnesium stearate and then
tabletized by a single-action type tabletizing machine to produce
tablets of a diameter of 9 mm and a weight of 250 mg each
containing 200 mg of Compound 1.
Formulation Example 2
Production of Granules
[0241] 30 g of Compound 1 was mixed well with 265 g of lactose and
5 g of magnesium stearate. The mixture was press molded, then
pulverized, granulated, and sieved to obtain excellent 10% granules
of 20 to 50 mesh.
Formulation Example 3
Production of Rectal Suppositoru
[0242] Vitepsol H-15 (Dynamite Nobel Co.) was warmed to melt. To
this was added Compound 1 to a concentration of 12.5 mg/ml. This
was homogeneously mixed, then was added in 2 ml amounts to rectal
suppository mold and cooled to obtain rectal suppositories each
containing 25 mg of Compound 1.
INDUSTRIAL APPLICABILITY
[0243] According to the present invention, a chymase inhibitor
alleviates a biphasic skin inflammation reaction or its late-phase
reaction and is effective against skin thickening of dermatitis
induced by repeated application of hapten, one of the animal
disease models of atopic dermatitis, and can effectively prevent
and/or treat conditions of dermatitis exhibiting a biphasic
inflammation reaction or dermatitis induced by repeated exposure to
an antigen.
* * * * *