U.S. patent application number 11/045394 was filed with the patent office on 2005-06-30 for selective preventing and therapeutic agents for progressive lesion after organic damage.
Invention is credited to Ishibashi, Michio, Mioskowski, Charles, Sylvain, Catherine, Wagner, Alain.
Application Number | 20050143453 11/045394 |
Document ID | / |
Family ID | 26588576 |
Filed Date | 2005-06-30 |
United States Patent
Application |
20050143453 |
Kind Code |
A1 |
Ishibashi, Michio ; et
al. |
June 30, 2005 |
Selective preventing and therapeutic agents for progressive lesion
after organic damage
Abstract
An object of the present invention is to provide a
pharmaceutical agents for preventing and/or treating the
progressive lesion after the organic damage without inhibiting
organic function or regeneration function thereof, by selectively
suppressing the induction of cytotoxic effector macrophages which
are induced into the damaged organs in response to chemokines and
cytokines which are expressed depending on the type of damaged
organic tissues.
Inventors: |
Ishibashi, Michio;
(Sakai-shi, JP) ; Wagner, Alain; (Strasbourg,
FR) ; Mioskowski, Charles; (Strasbourg, FR) ;
Sylvain, Catherine; (Gevrey-Chambertin, FR) |
Correspondence
Address: |
WENDEROTH, LIND & PONACK, L.L.P.
2033 K STREET N. W.
SUITE 800
WASHINGTON
DC
20006-1021
US
|
Family ID: |
26588576 |
Appl. No.: |
11/045394 |
Filed: |
January 31, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11045394 |
Jan 31, 2005 |
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10239783 |
Dec 12, 2002 |
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6878745 |
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10239783 |
Dec 12, 2002 |
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PCT/JP01/02513 |
Mar 27, 2001 |
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Current U.S.
Class: |
514/473 ;
549/314 |
Current CPC
Class: |
A61P 13/12 20180101;
C07D 307/33 20130101; A61P 43/00 20180101; A61K 31/341 20130101;
A61K 31/365 20130101; A61P 37/06 20180101; A61P 1/18 20180101; A61K
31/343 20130101; C07D 307/68 20130101; G01N 2500/10 20130101 |
Class at
Publication: |
514/473 ;
549/314 |
International
Class: |
A61K 031/365; C07D
305/12; C07D 307/62 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 28, 2000 |
JP |
88990/2000 |
Sep 22, 2000 |
JP |
289458/2000 |
Claims
1-14. (canceled)
15. A kit for screening a compound which is able to prevent,
mitigate or treat glomerular lesion of kidney, lesion of islet of
Langerhans of pancreas or diabetes mellitus and diabetic glomerular
lesion, which comprises (a) human PBMC, (b) lipopolysaccharide, (c)
human AB type serum, (d) RPMI 1640 medium and (e) a plate to which
monolayered autologous erythrocytes are adhered.
16. A kit for screening a compound which is able to prevent,
mitigate or treat progressive tubulointerstitial lesion after renal
damage, lesion of exocrine, acinar or ductal interstitial tissues
of pancreas or pancreatitis and lesion of interstitial tissues of
urinary tubule, which comprises (a) human PBMC, (b)
mitomycin-treated human PBMC, (c) human AB type serum, (d) RPMI
1640 medium and (e) a plate to which monolayered autologous
erythrocytes are is adhered.
17-27. (canceled)
28. A method for prevention and/or therapy of glomerular lesion of
kidney, lesion of islets of Langerhans of pancreas or diabetes
mellitus and diabetic glomerular lesion, which comprises using a
pharmaceutical containing a compound selectively suppressing the
induction of effector macrophages caused by contact of human PBMC
with lipopolysaccharide.
29. A method for prevention and/or therapy of progressive
tubulointerstitial lesion after renal damage, lesion of exocrine,
acinar or ductal interstitial tissues of pancreas or pancreatitis,
and lesion of interstitial tissues of urinary tubule, which
comprises using a pharmaceutical containing a compound selectively
suppressing the induction of effector macrophages caused by contact
of human PBMC with mitomycin-treated human PBMC.
30. A method for screening a compound which is able to prevent,
mitigate or treat glomerular lesion of kidney, lesion of islet of
Langerhans of pancreas or diabetes mellitus and diabetic glomerular
lesion, which comprises measuring a suppressive action of a
compound to be tested against the induction of effector macrophage
caused by contact of human PBMC with lipopolysaccharide.
31. The method for screening according to claim 30, wherein
effector macrophages are induced by incubation of human untreated
PBMC in RPMI 1640 medium in the presence of a compound to be
tested, lipopolysaccharide and human AB type serum, the said
induced effector macrophages are brought into contact with
monolayered autologous erythrocytes, and a compound showing less
production of SPFC as compared with the absence of the said
compound to be tested is screened.
32. A method for screening a compound which is able to prevent,
mitigate or treat progressive tubulointerstitial lesion after renal
damage, lesion of exocrine interstitial tissues of pancreas or
pancreatitis and lesion of interstitial tissues of urinary tubule,
which comprises measuring a suppressive action of a compound to be
tested against the induction of effector macrophages caused by
contact of human PBMC with mitomycin-treated human PBMC.
33. The method for screening according to claim 32, wherein
effector macrophages are induced by a mixed incubation of
mitomycin-treated human PBMC and human untreated PBMC in RPMI 1640
medium in the presence of a compound to be tested and human AB type
serum, the said induced effector macrophages are brought into
contact with monolayered autologous erythrocytes and a compound
showing less production of SPFC as compared with the absence of the
said compound to be tested is screened.
34. A method for the induction of effector macrophages which are a
cause of glomerular lesion of kidney, lesion of islet of Langerhans
of pancreas or diabetes mellitus and diabetic glomerular lesion,
which comprises bringing lipopolysaccharide into contact with human
PBMC.
35. A method for the induction of effector macrophages which are a
cause of progressive tubulointerstitial lesion after renal damage,
lesion of exocrine interstitial tissues of pancreas or pancreatitis
and lesion of interstitial tissues of urinary tubule, which
comprises bringing human PBMC into contact with mitomycin-treated
human PBMC.
36. The method according to claim 28 or 29, wherein the compound
suppressing the induction of effector macrophages is selected from
the group consisting of: (i) an optical isomer .gamma.-lactone
represented by the formula (3): 44or an optical isomer
.gamma.-lactone represented by the formula (4): 45wherein, R.sup.21
is an optionally substituted naphthyl group, R.sup.22 is an
optionally substituted straight or branched hydrocarbon residue
having 1 to 6 carbon atoms, or a mixture of said optical isomers
represented by the formulae (3) and (4); (ii) one of said optical
isomers represented by the formulae (3) and (4), or a mixture of
said optical isomers represented by the formulae (3) and (4)
wherein R.sup.21 is naphthyl and R.sup.22 is methyl; (iii) a
compound represented by the formula (5): 46wherein, (a) R.sup.1 and
R.sup.2 may be the same or different and each is hydrogen, an
open-chain aliphatic hydrocarbon group which may be substituted or
interrupted by an intervening group, an optionally substituted
cyclic aliphatic hydrocarbon group, an optionally substituted aryl
group, an optionally substituted heterocyclic group or an
optionally substituted condensed heterocyclic group; X.sup.2 is O,
S or NR.sup.3 in which R.sup.3 is hydrogen, oxygen, an open-chain
aliphatic hydrocarbon group which may be substituted or interrupted
by an intervening group, an optionally substituted cyclic aliphatic
hydrocarbon group, an optionally substituted aryl group, an
optionally substituted heterocyclic group or an optionally
substituted condensed heterocyclic group; and n is an integer from
1 to 5; or (b) X.sup.2 is O, S or NR.sup.3, R.sup.1, R.sup.2 and
R.sup.3 each is a substituent represented by the formula
R.sup.10-Z-R.sup.11--, wherein R.sup.10 and R.sup.11 may be the
same or different and each is an optionally substituted open-chain
or cyclic hydrocarbon group, an optionally substituted aryl group,
an optionally substituted heterocyclic group or an optionally
substituted condensed heterocyclic group, and Z is an intervening
group; and n is an integer from 1 to 5, or a pharmacologically
acceptable salt thereof; (iv) a compound represented by the formula
(6): 47wherein, R.sup.1, X.sup.2 and n have the same meaning as
defined above, X.sup.1 is halogen, cyano group, an optionally
substituted mercapto group, an optionally substituted sulfo group,
an optionally substituted sulfonyl group, an optionally substituted
hydroxyl group, an optionally substituted amino group or an
optionally substituted phosphoryl group, or a pharmacologically
acceptable salt thereof, and (v) a compound represented by the
formula (7): 48wherein, R.sup.1, X.sup.1 and X.sup.2 have the same
meaning as defined above, R.sup.5 and R.sup.6 may be the same or
different and each is (a) hydrogen, a straight or branched
aliphatic hydrocarbon group which may be substituted or interrupted
by an intervening group, an optionally substituted cyclic aliphatic
hydrocarbon group, an optionally substituted heterocyclic group, or
an optionally substituted condensed heterocyclic group; (b) a
substituent represented by the formula R.sup.10-Z-R.sup.11--,
wherein R.sup.10 and R.sup.11 may be the same or different and each
is an optionally substituted open-chain or cyclic hydrocarbon
group, an optionally substituted aryl group, an optionally
substituted heterocyclic group or an optionally substituted
condensed heterocyclic group; and Z is an intervening group; or (c)
R.sup.5 and R.sup.6 together with the carbon atom to which they are
attached form an optionally substituted aromatic ring; or a
pharmacologically acceptable salt thereof.
37. The method according to claim 36, wherein the compound
suppressing the induction of effector macrophages is selected from
the group consisting of: 49
38. The method according to claim 28 or 29, wherein the compound
suppressing the induction of effector macrophages is a compound
represented by the formula (4-2): 50
39. The method according to claim 36, wherein the compound
suppressing the induction of effector macrophages is a compound
represented by the formula (6-2): 51wherein Bn is benzyl.
40. The method according to claim 28 or 29, wherein the compound
suppressing the induction of effector macrophages is a compound
represented by the formula (7-3): 52wherein Bn is benzyl.
41. The method according to 36, wherein the compound suppressing
the induction of effector macrophages is a compound represented by
the formula (7), wherein R.sup.5 and R.sup.6 together with the
carbon atom to which they are attached form an optionally
substituted aromatic ring, X.sup.2 is O, X.sup.1 is optionally
substituted hydroxyl and R.sup.1 is hydrogen.
Description
TECHNICAL FIELD
[0001] Organs in organism may be damaged, for example, by blood
flow disorder, ischemia reperfusion injury, hypertension,
hyperglycemia, hyperlipemia, pharmaceutical agent or viral
infection. Damaged organ tissues show any of responses of necrosis,
natural death or self-regeneration depending upon the degree of the
damage. Immune system deeply participates in such a response and
some of macrophages which are carriers of such immune participate
in self-regeneration of organ tissues while others participate in
necrosis or natural death of organ tissues. The latter, i.e. that
which shows cytotoxicity in a functional manner among the much
differentiated macrophages, is called effector macrophage and such
an effector macrophage further worsens the damage after the
above-mentioned damage and causes a progressive lesion after the
organic damage.
[0002] The present invention relates to a pharmaceutical in which
induction of effector macrophage which is a cause of progressive
lesion noted after such an organic damage is selectively suppressed
whereby the said progressive lesion is prevented and/or treated
without inhibiting the function and regeneration process of the
organ, and also relates to a therapeutic method using the same. The
present invention further relates to a screening method for
compounds which can be the said pharmaceutical. The present
invention still further relates to novel .gamma.-lactone
derivatives useful as the said pharmaceutical.
[0003] With regard to one of the progressive lesions noted after
the above-mentioned organic damages, fibrosis of tissue cells may
be exemplified. Such a fibrosis of tissue cells is caused by
effector macrophage showing cytotoxic property the same as above.
The novel .gamma.-lactone derivatives according to the present
invention are able to selectively suppress the induction of the
said effector macrophage. As a result of utilization of such an
action, the present invention also relates to a pharmaceutical as a
fibrosis inhibitor containing the said novel .gamma.-lactone
derivative.
[0004] Rejection, particularly chronic rejection, upon
transplantation of allogenic or xenogenic organ cells is caused by
the same mechanism in the progressive lesion after organic damage
or, at least similar, mechanism to that. The novel .gamma.-lactone
derivatives according to the present invention selectively suppress
the induction of effector macrophage causing the induction of
allograft rejection, particularly chronic rejection, upon allogenic
or xenogenic cell, tissue or organ transplantation whereby they
show an immunosuppressive action only in the organ tissues showing
the rejection. As a result of utilization of such an action, the
present invention also relates to a pharmaceutical as an
immunosuppressant containing the said novel .gamma.-lactone
derivative.
BACKGROUND OF THE INVENTION
[0005] When an organ in organisms is damaged, for example, by blood
flow disorder, ischemia reperfusion injury, hypertension,
hyperglycemia, hyperlipemia, pharmaceutical agent or viral
infection, there works an organism defense mechanism by, for
example, T lymphocytes, macrophages, NK cells, fibroblasts, B
lymphocytes and antibodies, complements, etc. Particularly, T cells
and macrophages have an important participation in the said
organism defense mechanism.
[0006] Damaged organ tissues show any of the responses of necrosis,
natural death or self-regeneration depending upon the degree of the
damage. At that time, chemokines, cytokines, etc. are expressed
from the said tissues to promote self-proliferation and
regeneration and, at the same time, organism defense mechanism of
the host is activated as well. It has been presumed that the
outcome whether the damaged tissues result in necrosis or natural
death by way of atrophy and fibrosis or they result in regeneration
of tissues is the result of the interaction of both.
[0007] For example, there are several reports for the studies that,
in the damaged renal tissue lesions, the damaged renal tissue cells
express tissue-specific chemokine, cytokine or adhesion factor
depending upon the damaged area, etc. whereby the response is
exhibited.
[0008] Wada, et al. reported that, in human crescentic
glomerulonephritis, MIP-1.alpha. of chemokine is expressed in
glomerular cellular crescents in an acute stage while, in
interstitial tissues of the cases of fibrous crescents in a chronic
stage, MCP-1 is expressed (Wada T., Furuichi K., Segawa-Takeda C.,
Shimizu M., Sakai N., Takeda S. I., Takasawa K., Kida H., Kobayashi
K. I., Mukaida N., Ohmoto Y., Matsushima K., Yokoyama H.:
MIP-1.alpha. and MCP-1 contribute to crescents and interstitial
lesions in human crescentic glomerulonephritis. Kidney Int., 56:
995-1003, 1999.
[0009] Matsuda, et al. reported that, in model rats suffering from
crescentic glomerulonephritis, P-secretin and L-secretin are
expressed in a discriminated manner in glomerular endothelial cells
and in interstitial tissues from urinary tubule, respectively
(Michihiro Matsuda, Kenichi Shikata, Daisuke Ogawa, Shinichi Okada,
Yasushi Shikata, Atsushi Wada and Hiroshi Makino: Mechanism of
Induction of Infiltration of Leucocytes to Renal Tissues by
Secretin, Nippon Jinzo Gakkaishi, 42: 213, 2000).
[0010] Tesch, et al. reported that, when nephrotoxic serum
nephritis model was prepared in MCP-1 knockout mice and expression
of MCP-1 was checked, the expression of MCP-1 in the damaged site
of urinary tubule was weak as compared with a wild type while the
expression of MCP-1 in the glomerular lesion had no difference from
the wild type. They also reported that, in the knockout mice where
expression of MCP-1 was weak, infiltration of macrophage decreased
whereby MCP-1 showed the result that it participated in migration
of macrophage while, in terms of degree of proteinuria in an acute
stage, there is no difference between both and there was no
relation between tissue damage and macrophage infiltration in an
acute stage (Tesch G. H., Schwarting A., Kinoshita K., Rolins B.
J., Kelly V. R.: Monocyte chemoattractant protein-1 promotes
macrophage-mediated tubular injury, but not glomerular injury, in
nephritic serum nephritis, J. Clin. Invest. 1999, 103: 73-80.).
[0011] Not only in kidney but also in exocrine gland and islet of
Langerhans of pancreas, there is noted a difference in
chemokine-productive response concerning the lesion after the organ
tissue damage corresponding to the damaged tissue.
[0012] Cameron, et al. reported that, in the mice where nonobese
type I diabetes was spontaneously occurred, promotion of the
expression of MIP-1.alpha. and lowering of the expression of MCP-1
were noted among chemokines in islets of Langerhans and that there
was a correlation between damage of islets of Langerhans and onset
of diabetes (Cameron M. J., Arreaza G. A., Grattan M., Meagher C.,
Sharif S., Burdick M. D., Strieter R. M., Cook D. N., Delovitch T.
L.: Differential expression of CC chemokines and the CCR.sup.5
receptor in the pancreas is associated with progression to type I
diabetes, J. Immunol., 2000, 165: 1102-10).
[0013] In the meanwhile, Andoh, et al. investigated the expression
of chemokines in pancreatitis tissues of human acute pancreatitis
cases. They reported that, in acute pancreatitis, expression of
MIP-1.alpha. was not noted but expression of MCP-1, IL-8 and RANTES
was noted in exocrine, acinar and ductal interstitial pancreas
tissues (Andoh A., Takaya H., Saotome T., Shimada M., Hata K.,
Araki Y., Nakamura F., Shintani Y., Fujiyama Y., Bamba T.: Cytokine
regulation of chemokine (IL-8, MCP-1, and RANTES) gene expression
in human pancreatic periacinar myofibroblasts, Gastroenterology,
2000, 119: 211-9).
[0014] As mentioned above, it was suggested in human clinical
studies and experimental study models using animals that, in
progressive lesion noted after the organic damage, effector
macrophage participated therein corresponding to the damaged
tissues.
[0015] Up to now, therapy of steroids, etc. has been carried out
for progressive lesion after organic damage. However, since
steroidal preparations non-selectively suppress the macrophage,
they also suppress the response of even the macrophage
participating in the reaction for tissue regeneration at the same
time whereby organism defensive mechanism including the
regeneration is lessened. In addition, new tissue damage is induced
resulting in lesion and, as a result, there is a problem that
effector macrophage mediated by the expression of chemokines and
cytokines is actively induced whereby the inherent lesion is
further worsened. As such, the conventional therapy of steroids has
no selectivity in the action, and administration of high dose is
necessary for the therapy of the lesion whereby the side effect is
remarkable. In addition, there is a difficulty that a continuous
therapy by steroids for a long period is accompanied by a severe
side effect.
[0016] On the other hand, like the progressive lesion noted after
the above-mentioned organic damage, organism defense mechanism by T
cells, macrophages, etc. participates in rejection in the
transplantation of organ, skin or the like. Until now, there have
been known many compounds having an immunosuppressive action and,
for example, compounds represented by the formula (8) 1
[0017] (in which R.sup.1 is an optionally substituted phenyl group;
R.sup.2 is an optionally esterified carboxyl group; and X is oxygen
atom or optionally oxidized sulfur atom) have been known to be
useful as a .gamma.-lactone immunosuppressant (Japanese Patent
Laid-Open No. 04/338,331). However, it is to be still improved so
as to show a selective action to target organs or tissues or so as
to show stronger immunosuppressive action.
DISCLOSURE OF THE INVENTION
[0018] Effector macrophage expresses chemokine receptors or O.sub.2
integrin receptors, etc. corresponding to the lesion inherent to
the tissues after the organic damage via either
T-lymphocyte-independent or T-lymphocyte-dependent response and is
selectively induced to and activated in lesion being mediated by
them whereby said effector macrophage causes a progressive lesion
after the tissue damage. An object of the present invention is to
provide a pharmaceutical in which expression and function of
chemokine receptors such as CCR.sup.2, CCR.sup.3 or CCR.sup.8,
.beta.2 integrin receptors such as CD11b/CD18 or other receptors
are suppressed to selectively suppress the induction of the
above-mentioned effector macrophages whereby progressive lesion
after organic damage is prevented and/or treated without inhibiting
the function of the organ and the regeneration function and also to
provide a therapeutic method using the same. Since the
pharmaceutical according to the present invention is able to
suppress the induction of effector macrophages causing the
progressive lesion after organic damage in a more
lesion-selectively manner, it is possible to use in a high dose
and, due to the selectivity of the said pharmaceutical, undesirable
side effect can be avoided even when it is used in a high dose.
Therapy for a long period using the said pharmaceutical is now
possible as well. Further, when the lesions are different resulting
in progressive lesion after the organic damage in plural tissues,
it is possible to carry out more selective and more effective
therapy by combination use of the above pharmaceuticals showing
different selectivity. That is an advantage which is not noted in
the conventional steroidal agents and known .gamma.-lactone
immunosuppressants having no selectivity. The present invention
also aims to provide useful and novel .gamma.-lactone
derivatives.
[0019] Another object of the present invention is to provide a
method for screening the compounds which do not substantially
suppress the macrophage participating in tissue regeneration and
cause the progressive lesion after the above-mentioned organic
damage.
[0020] Still another object of the present invention is to provide
a method for inducing the effector macrophage which causes the
progressive lesion after the above-mentioned organic damage.
[0021] Further object of the present invention is to provide a
pharmaceutical useful as a fibrosis inhibitor preventing from the
fibrosis which is one of lesions inherent to the tissues after the
organic damage or, to be more specific, to provide a fibrosis
inhibitor containing the above-mentioned novel .gamma.-lactone
derivative.
[0022] Still further object of the present invention is to provide
a pharmaceutical which are useful as an immunosuppressant or, to be
more specific, to provide an immunosuppressant which is an
immunosuppressant containing the above-mentioned novel
.gamma.-lactone derivative and shows a selective immunosuppressive
action to target organs or tissues unlike the known .gamma.-lactone
immunosuppressants.
[0023] As mentioned above, damaged tissue cells express chemokine,
cytokine or adhesion molecule inherent to the tissue. Depending
upon type or degree (amount) of the expression or upon defense
mechanism of organism, the expressed chemokine, cytokine, etc.
either promote self-proliferation or regeneration of the damaged
tissues or subject the damaged tissue to necrosis, natural death or
degeneration. Here, effector macrophage showing cytotoxic property
is selectively induced corresponding to type or expressed amount of
the said chemokine and cytokine so that the damaged tissues are
subjected to necrosis, natural death or degeneration. To be more
specific, effector macrophage is selectively induced to the damaged
organ tissues (lesion tissues) by chemokine receptors corresponding
to the said chemokine expressed in the damaged organ tissues
(lesion tissue) or by .beta.2 integrin, etc. corresponding to a
ligand expressed on the said organ tissues (lesion tissues). The
induced effector macrophage recognizes the damaged tissues, acts on
the said tissues in a cytotoxic manner and induces the progressive
lesion after the organic damage. On the other hand, the effector
macrophage does not recognize the normal tissue wherein chemokine
and cytokine which stimulate for inducing and activating the said
effector macrophage are not expressed and, as a result, the normal
tissue is not damaged.
[0024] Accordingly, if induction of effector macrophage in the
damaged organ tissues can be selectively suppressed by way of
suppression of expression and function of chemokine receptor such
as CCR.sup.2, CCR.sup.3 and CCR.sup.8, .beta.2 integrin receptors
such as CD11b/CD18 and other receptors, action of the effector
macrophage in a cytotoxic manner to the damaged organ tissues can
be prevented and, in addition, induction of the macrophage
participating in the tissue regeneration is not suppressed so that
lowering of the defense ability of the organism is not noted
whereby it has been found that progressive lesion after organic
damage can be prevented, mitigated or treated.
[0025] When a compound which is able to selectively suppress the
induction of effector macrophage as such is used as a
pharmaceutical, there is reduced the side effect such as that
defense of organism is unnecessarily lowered or new tissue damage
is induced. In addition, it is possible to result in recovery and
regeneration of the tissue without an unnecessary lowering of
defense of organism and, therefore, it is now possible to
continuously carry out the therapy for a long period.
[0026] Ishibashi who is one of the inventors of the present
invention has carried out a further investigation for the
progressive lesion in kidney on the basis of the above finding and
has obtained an unexpected finding that a compound which suppresses
the induction of effector macrophage caused by contact of human
peripheral blood mononuclear cells abbreviated as PBMC with
lipopolysaccharide suppresses the progress of glomerular lesion of
kidney and that a compound which suppresses the induction of
effector macrophage caused by contact of human PBMC with
mitomycin-treated human PBMC suppresses the progress of progressive
lesion of tubulointerstitial tissue after renal damage.
[0027] Ishibashi who is one of the inventors of the present
invention has also carried out an investigation for progressive
lesion in pancreas and obtained an unexpected finding that a
compound which suppresses the induction of effector macrophage
caused by contact of human PBMC with lipopolysaccharide suppresses
the progress of lesion in islets of Langerhans of pancreas and that
a compound which suppresses the induction of effector macrophage
caused by contact of human PBMC with mitomycin-treated human PBMC
suppresses the progress of lesion of exocrine, acinar and ductal
interstitial tissues of pancreas.
[0028] Here, when lesion of islets of Langerhans of pancreas
progresses, diabetes mellitus is able to occur. When diabetes
mellitus occurs, diabetic nephropathy which is a glomerular lesion
is able to occur as a complication thereof. Ishibashi who is one of
the inventors of the present invention has further obtained an
unexpected finding that a compound which suppresses the induction
of effector macrophage caused by contact of human PBMC with
lipopolysaccharide is also able to prevent and/or treat the onset
of diabetic glomerular lesion (another name: diabetic nephropathy)
together with the onset of diabetes mellitus.
[0029] Ishibashi who is one of the inventors of the present
invention has furthermore obtained an unexpected finding that a
compound which suppresses the induction of effector macrophage
caused by contact of human PBMC with mitomycin-treated human PBMC
is also able to prevent and/or treat the onset of lesion of
tubulointerstitial tissues which is a complication of pancreatitis
together with onset of pancreatitis which is lesion of exocrine,
acinar and ductal interstitial tissues of pancreas.
[0030] The present inventors have carried out an investigation or
the compounds which are able to selectively suppress the induction
of effector macrophage which is caused corresponding to lesion
inherent to tissues after the organic damage and, as a result, they
have found that novel .gamma.-lactone derivatives represented by
the following formulae of from (1) to (7) have such an action.
[0031] The present inventors have further found that, since
fibrosis of tissues is one of the progressive lesions after the
organic damage, the novel .gamma.-lactone derivatives represented
by the following formulae of from (1) to (7) are also useful as
inhibitors for fibrosis.
[0032] With regard to the rejection at the transplantation of organ
cells of allogeneic or xenogenic type, there are acute rejection
and chronic rejection and it has been known that, particularly in
the chronic rejection, not only immunological factors but also
non-immunological factors such as damage by pharmaceuticals,
ischemia reperfusion injury, viral infection, blood flow disorder
and exclusion of cells can be a cause. On the other hand, the
above-mentioned progressive lesion after organic damage also
results from the organic damage, which is a trigger, such as damage
by pharmaceuticals, ischemia reperfusion injury, viral infection,
blood flow disorder and exclusion of cells. Accordingly, chronic
rejection is caused by a mechanism which is the same as or at least
similar to the progressive lesion after the organic damage.
Therefore, it has been found that novel .gamma.-lactone derivatives
represented by the following formulae of from (1) to (7) which are
able to selectively suppress the induction of effector macrophage
induced being correspondent to the lesion inherent to the tissues
after organic damage are useful as an immunosuppressant for the
prevention or the therapy of rejection upon of allogeneic or
xenogenic cell, tissue or organ transplantation, particularly as an
immunosuppressant to chronic rejection.
[0033] Thus, the present invention relates to the followings.
[0034] (1) A pharmaceutical composition, which comprises a compound
suppressing the induction of effector macrophages.
[0035] (2) A preventive and therapeutic pharmaceutical selectively
to progressive lesion of organic damages which comprises a compound
suppressing the induction of effector macrophages.
[0036] (3) A pharmaceutical for prevention and/or therapy of
glomerular lesion of kidney, which comprises a compound suppressing
the induction of effector macrophages caused by contact of human
PBMC with lipopolysaccharide.
[0037] (4) A pharmaceutical for prevention and/or therapy of
progressive tubulointerstitial lesion after renal damage, which
comprises a compound suppressing the induction of effector
macrophages caused by contact of human PBMC with mitomycin-treated
human PBMC.
[0038] (5) A pharmaceutical for prevention and/or therapy of lesion
of islets of Langerhans of pancreas, which comprises a compound
suppressing the induction of effector macrophages caused by contact
of human PBMC with lipopolysaccharide.
[0039] (6) A pharmaceutical for prevention and/or therapy of lesion
of exocrine, acinar and ductal interstitial tissues of pancreas,
which comprises a compound suppressing the induction of effector
macrophages caused by contact of human PBMC with mitomycin-treated
human PBMC.
[0040] (7) A pharmaceutical for prevention and/or therapy of
diabetes mellitus and diabetic glomerular lesion, which comprises a
compound suppressing the induction of effector macrophages caused
by contact of human PBMC with lipopolysaccharide.
[0041] (8) A pharmaceutical for prevention and/or therapy of
pancreatitis and lesion of interstitial tissues of urinary tubule
which is a complication of pancreatitis, which comprises a compound
suppressing the induction of effector macrophages caused by contact
of human PBMC with mitomycin-treated human PBMC.
[0042] (9) A method for prevention and/or therapy of glomerular
lesion of kidney, lesion of islets of Langerhans of pancreas or
diabetes mellitus and diabetic glomerular lesion, which comprises
using a pharmaceutical containing a compound suppressing the
induction of effector macrophages caused by contact of human PBMC
with lipopolysaccharide.
[0043] (10) A method for prevention and/or therapy of progressive
tubulointerstitial lesion after renal damage, lesion of exocrine,
acinar and ductal interstitial tissues of pancreas or pancreatitis,
and lesion of interstitial tissues of urinary tubule, which
comprises using a pharmaceutical containing a compound suppressing
the induction of effector macrophages caused by contact of human
PBMC with mitomycin-treated human PBMC.
[0044] (11) A method for screening a compound which is able to
prevent, mitigate or treat glomerular lesion of kidney, lesion of
islet of Langerhans of pancreas or diabetes mellitus and diabetic
glomerular lesion, which comprises measuring a suppressive action
of a compound to be tested against the induction of effector
macrophage caused by contact of human PBMC with
lipopolysaccharide.
[0045] (12) The method for screening according to the
above-mentioned (11), wherein effector macrophages are induced by
incubation of human untreated PBMC in RPMI 1640 medium in the
presence of a compound to be tested, lipopolysaccharide and human
AB type serum, the said induced effector macrophages are brought
into contact with monolayered autologous erythrocytes and a
compound showing less production of SPFC, Spontaneous
Plaque-Forming Cell, as compared with the absence of the said
compound to be tested is screened.
[0046] (13) A method for screening a compound which is able to
prevent, mitigate or treat progressive tubulointerstitial lesion
after renal damage, lesion of exocrine acinar and ductal
interstitial tissues of pancreas or pancreatitis and lesion of
interstitial tissues of urinary tubule, which comprises measuring a
suppressive action of a compound to be tested against the induction
of effector macrophages caused by contact of human PBMC with
mitomycin-treated human PBMC.
[0047] (14) The method for screening according to the
above-mentioned (13), wherein effector macrophages are induced by a
mixed incubation of mitomycin-treated human PBMC and human
untreated PBMC in RPMI 1640 medium in the presence of a compound to
be tested and human AB type serum, the said induced effector
macrophages are brought into contact with monolayered autologous
erythrocytes and a compound showing less production of SPFC as
compared with the absence of the said compound to be tested is
screened.
[0048] (15) A kit for screening a compound which is able to
prevent, mitigate or treat glomerular lesion of kidney, lesion of
islet of Langerhans of pancreas or diabetes mellitus and diabetic
glomerular lesion, which comprises (a) human PBMC, (b)
lipopolysaccharide, (c) human AB type serum, (d) RPMI 1640 medium
and (e) a plate to which monolayered autologous erythrocytes are
adhered.
[0049] (16) A kit for screening a compound which is able to
prevent, mitigate or treat progressive tubulointerstitial lesion
after renal damage, lesion of exocrine, acinar and ductal
interstitial tissues of pancreas or pancreatitis and lesion of
interstitial tissues of urinary tubule, which comprises (a) human
PBMC, (b) mitomycin-treated human PBMC, (c) human AB type serum,
(d) RPMI 1640 medium and (e) a plate to which monolayered
autologous erythrocytes are adhered.
[0050] (17) A method for the induction of effector macrophages
which are a cause of glomerular lesion of kidney, lesion of islets
of Langerhans of pancreas or diabetes mellitus and diabetic
glomerular lesion, which comprises bringing lipopolysaccharide into
contact with human PBMC.
[0051] (18) A method for the induction of effector macrophages
which are a cause of progressive tubulointerstitial lesion after
renal damage, lesion of exocrine, acinar and ductal interstitial
tissues of pancreas or pancreatitis and lesion of interstitial
tissues of urinary tubule, which comprises bringing human PBMC into
contact with mitomycin-treated human PBMC.
[0052] (19) The pharmaceutical according to any of the
above-mentioned (1) to (8), which comprises a compound represented
by the formula (1) 2
[0053] or a compound represented by the formula (2). 3
[0054] (20) An optical isomer .gamma.-lactone represented by the
formula (3) 4
[0055] or by the formula (4) 5
[0056] (in the formula, R.sup.21 is an optionally substituted
naphthyl group and R.sup.22 is an optionally substituted straight
or branched hydrocarbon group having 1 to 6 carbon atoms) or a
mixture of the above optical isomers.
[0057] (21) The optical isomer .gamma.-lactone or a mixture of the
optical isomers according to the above-mentioned (20), wherein
R.sup.21 is naphthyl and R.sup.22 is methyl.
[0058] (22) A compound represented by the formula (5) or a
pharmacologically acceptable salt thereof. 6
[0059] (in the formula, (a) R.sup.1 and R.sup.2 may be the same or
different and each is hydrogen, an open-chain aliphatic hydrocarbon
group which may be substituted or interrupted by an intervening
group, an optionally substituted cyclic aliphatic hydrocarbon
group, an optionally substituted aryl group, an optionally
substituted heterocyclic group or an optionally substituted
condensed heterocyclic group; X.sup.2 is O, S or NR.sup.3 in which
R.sup.3 is hydrogen, oxygen, an open-chain aliphatic hydrocarbon
group which may be substituted or interrupted by an intervening
group, an optionally substituted cyclic aliphatic hydrocarbon
group, an optionally substituted aryl group, an optionally
substituted heterocyclic group or an optionally substituted
condensed heterocyclic group; and n is an integer from 1 to 5
or.
[0060] (b) X.sup.2 is O, S or NR.sup.3; R.sup.1, R.sup.2 and
R.sup.3 each is a substituent represented by the formula
R.sup.10-Z-R.sup.11-- (in which R.sup.10 and R.sup.11 may be the
same or different and each is an optionally substituted open-chain
or cyclic hydrocarbon group, an optionally substituted aryl group,
an optionally substituted heterocyclic group or an optionally
substituted condensed heterocyclic group; and Z is an intervening
group); and n is an integer from 1 to 5).
[0061] (23) A compound represented by the formula (6) or a
pharmacologically acceptable salt thereof. 7
[0062] (in the formula, R.sup.1, X.sup.2 and n have the same
meaning as defined in the above-mentioned (4); X.sup.1 is halogen,
cyano group, an optionally substituted mercapto group, an
optionally substituted sulfo group, an optionally substituted
sulfonyl group, an optionally substituted hydroxyl group, an
optionally substituted amino group or an optionally substituted
phosphoryl group).
[0063] (24) A compound represented by the formula (7) or a
pharmacologically acceptable salt thereof. 8
[0064] (in the formula, R.sup.1 and X.sup.2 have the same meaning
as defined in the above-mentioned (4); X.sup.1 has the same meaning
as defined in the above-mentioned (5); R.sup.5 and R.sup.6 may be
the same or different and each is (a) hydrogen, a straight or
branched aliphatic hydrocarbon group which may be substituted or
interrupted by an intervening group, an optionally substituted
cyclic aliphatic hydrocarbon group, or an optionally substituted
heterocyclic group, an optionally substituted condensed
heterocyclic group.
[0065] (b) a substituent represented by the formula
R.sup.10-Z-R.sup.11-(in the formula, R.sup.10 and R.sup.11 may be
the same or different and each is an optionally substituted
open-chain or cyclic hydrocarbon group, an optionally substituted
aryl group, an optionally substituted heterocyclic group or an
optionally substituted condensed heterocyclic group; and Z is an
intervening group.), or.
[0066] (c) R.sup.5 and R.sup.6 together with the carbon atom to
which they are attached form an optionally substituted aromatic
ring).
[0067] (25) The pharmaceutical according to any of the
above-mentioned (1) to (8), which comprises a compound mentioned in
any of the above-mentioned (20) to (24).
[0068] (26) An immunosuppressant or a fibrosis inhibitor which
comprises a compound mentioned in any of the above-mentioned (19)
to (24).
[0069] (27) The pharmaceutical according to any of the
above-mentioned (1) to (8), which comprises ethyl 2-ketoglutarate
or benzyl 2-ketoglutarate.
BEST MODE FOR CARRYING OUT THE INVENTION
[0070] (1) Firstly, the present invention provides a method for the
induction of effector macrophage which is induced and activated
corresponding to the lesion inherent to the tissues after the
organic damage and results in progressive lesion after the organic
damage.
[0071] Thus, the present invention provides (a) a method for the
induction of effector macrophage which is a cause of glomerular
lesion of kidney, lesion of islets of Langerhans of pancreas or
diabetes mellitus and diabetic glomerular lesion, characterized in
that, lipopolysaccharide is brought into contact with human PBMC
(hereinafter, abbreviated as "LPS inducing method") and (b) a
method for the induction of effector macrophage which is a cause of
progressive tubulointerstitial lesion after renal damage, lesion of
exocrine, acinar or ductal interstitial tissues of pancreas or
pancreatitis and tubulointerstitial lesion, characterized in that,
human PBMC are brought into contact with mitomycin-treated human
PBMC (hereinafter, abbreviated as "allo-MLC inducing method").
[0072] Hereunder, an LPS inducing method will be illustrated in
detail.
[0073] With regard to a lipopolysaccharide used for the LPS
inducing method, that which has been known per se may be used. For
example, the lipopolysaccharide derived from Gram-negative bacteria
such as Salmonella and Escherichia coli may be used. It may be
either the so-called rough type or smooth type.
[0074] For the preparation of the lipopolysaccharide, a method
which has been known per se may be used. An example is a method
where it is extracted from microbe and, if desired, a treatment for
removing the toxicity is conducted. Examples of the method for
extracting from microbe are a method of extracting with hot phenol
(Westphal & Jann., Methods Carbohydr. Chem., 5, 83-99 (1965))
and a method where microbe is treated with proteinase K in the
presence of sodium lauryl sulfate. In addition, chemically
synthesized one may be used or the commercially available one may
be appropriately used.
[0075] In the present invention, it is preferred that
lipopolysaccharide is used as a solution and, when it is made into
a solution using an appropriate solvent, preferably, RPMI 1640
liquid, it is preferred to use a solution of a high concentration
of about 60 to 100 .mu.g/ml, more preferably about 70 to 90
.mu.m/ml or still more preferably about 80 .mu.g/ml.
[0076] The human PBMC can be obtained from human peripheral blood
by a method known per se. An example for a method for separating
the mononuclear cells from human peripheral blood is a method by a
centrifugal separation using Ficoll-Paque (registered trademark;
manufactured by Pharmacia Fine Chemicals). To be more specific, the
above-mentioned method comprises (a) a step where Ficoll-Paque is
placed at the bottom of a test tube, (b) a step where a blood
sample as it is or after being diluted is carefully transferred
onto the Ficoll-Paque using a pipette, (c) a step where the blood
preparation prepared by Ficoll-Paque in (b) is centrifuged at about
400.about.500 G for about 30.about.40 minutes so that a blood
component having larger specific gravity than the specific gravity
of Ficoll-Paque comes into Ficoll-Paque or passes Ficoll-Paque and
(d) a step where the mononuclear cell layer separated on the upper
area of Ficoll-Paque is collected.
[0077] A specific mode for the LPS inducing method is a method
where human PBMC is incubated in RPMI 1640 medium in the presence
of lipopolysaccharide and human AB type serum to induce the
effector macrophage and that method is advantageously used in the
present invention.
[0078] At that time, a combination of any two of or all of
lipopolysaccharide, human AB type serum and human PBMC may be
previously mixed and then added to the medium or each of them may
be added to the medium solely. There is no limitation for the order
of adding to the medium. More preferably however, human untreated
PBMC are added to RPMI 1640 medium to which human AB type serum is
previously added and then lipopolysaccharide is added thereto.
[0079] RPMI 1640 medium is mentioned in Goding, J. W. (1980) J.
Immunol. Methods, 39, 185 and JAMA, 199 (1957), 519. Alternatively,
a commercially available product (manufactured by Sigma) may be
used as well.
[0080] More preferred mode for carrying out the LPS inducing method
is as follows.
[0081] Human untreated PBMC are dissolved in a concentration of
about 2.times.10.sup.6 cells/ml to RPMI 1640 medium to which
gentamicin, L-glutamine and human AB type serum are added to
concentrations of about 5 .mu.g/ml, about 2 mM and about 10% by
weight, respectively and then lipopolysaccharide is added thereto
so as to make its final concentration about 80 .mu.g/ml to prepare
a culture liquid. The said culture liquid is incubated at about
37.degree. C. for about six days with about 5% of CO.sub.2.
[0082] A method for the induction of allo-MLC will be mentioned in
detail as follows.
[0083] The mitomycin-treated human PBMC used in an allo-MLC
inducing method can be prepared, for example, by adding mitomycin
to the human PBMC obtained by the above-mentioned known method to
make the final concentration of mitomycin about 40 .mu.g/ml
followed by subjecting to a heating treatment at about 37.degree.
C. for about 30 minutes.
[0084] A specific mode for an allo-MLC inducing method is a method
where mitomycin-treated human PBMC and normal human untreated PBMC
are subjected to a mixed culture in RPMI 1640 medium in the
presence of human AB type serum to induce the effector macrophage
and that method is advantageously used in the present
invention.
[0085] At that time, a combination of any two of or all of human AB
type serum, mitomycin-treated human PBMC and human PBMC may be
previously mixed and then added to the medium or each of them may
be added to the medium solely. There is no limitation for the order
of adding to the medium. It is preferred however that normal human
untreated PBMC are added to RPMI 1640 medium to which human AB type
serum is previously added and then mitomycin-treated human PBMC are
added thereto.
[0086] A more preferred mode of carrying out the allo-MLC inducing
method will be as follows.
[0087] Human untreated PBMC are dissolved in a concentration of
about 2.times.10.sup.6 cells/ml to RPMI 1640 medium to which
gentamicin, L-glutamine and human AB type serum are added to
concentrations of about 5 .mu.g/ml, about 2 mM and about 10% by
weight, respectively and then mitomycin-treated human PBMC are
added thereto so as to make the final concentration of about
2.times.10.sup.6 cells/ml to prepare a culture liquid. The said
culture liquid is incubated at about 37.degree. C. for about six
days with about 5% of CO.sub.2.
[0088] (2) The present invention then provides a method for
screening a compound which is able to prevent, mitigate or treat
the progressive lesion after organic damage by a selective
suppression of induction of effector macrophage corresponding to
lesion inherent to the tissues after organic damage.
[0089] Thus, the present invention provides a method for screening
a compound which is able to prevent, mitigate or treat glomerular
lesion of kidney, lesion of islets of Langerhans of pancreas or
diabetes mellitus and diabetic glomerular lesion, characterized in
that, a suppressive action of a compounded to be tested to the
induction of effector macrophage caused by contact of human PBMC
with lipopolysaccharide is measured.
[0090] Preferred embodiment of the above-mentioned screening method
according to the present invention will be as follows.
[0091] First, human PBMC are brought into contact with
lipopolysaccharide in the presence of the compound to be tested to
induce effector macrophage. Then, measurement is carried out to
check whether the compounded to be tested shows a suppressive
action to induction of effector macrophage.
[0092] It is preferred that the above-mentioned induction of
effector macrophage is carried out in the same manner as in the
above-mentioned LPS inducing method except that, when
lipopolysaccharide is brought into contact with human PBMC, the
compound to be tested is further present.
[0093] It is preferred that the measurement of the above-mentioned
suppressive action to the induction of effector macrophage is
carried out by measuring the numbers of spontaneous plaque-forming
cells (hereinafter, referred to as SPFC) which are produced by
bringing the induced effector macrophage into contact with
monolayered autologous erythrocytes. Thus, when the numbers of the
produced SPFC are less than the case where no test compound to be
tested is present, it is judged that induction of effector
macrophage is suppressed.
[0094] More preferred embodiment will be as follows.
[0095] Human untreated PBMC are dissolved in a concentration of
about 2.times.10.sup.6 cells/ml to RPMI 1640 medium to which
gentamicin, L-glutamine and human AB type serum are added to
concentrations of about 5 .mu.g/ml, about 2 mM and about 10% by
weight, respectively and then lipopolysaccharide is added to the
solution so as to make its final concentration of about 80
.mu.g/ml.
[0096] A compound to be tested is dissolved in an appropriate
solvent, preferably dimethyl sulfoxide (DMSO) of about 0.001% by
weight and the resulting test solution is added to above-mentioned
medium. At that time, there are prepared various media where
concentrations of the compound to be tested are different. It is
preferred that the concentrations of the compound to be tested are
about 1 .mu.M to 0.001 .mu.M.
[0097] The mixture is incubated at about 37.degree. C. for about
six days in the presence of about 5% Of CO.sub.2 to induce effector
macrophage. The effector macrophage generated in the cultured PBMC
induced from the culture liquid is recovered. In the recovering,
known methods may be used and, for example, there is a method where
the adhered thing is recovered using a rubber-policeman (spatula
made of rubber). After that, washing may be carried out. For the
washing, it is preferred to use a Hanks solution to which
gentamicin is added so as to make its concentration of about 5
.mu.g/ml.
[0098] In the meanwhile, monolayered autologous erythrocytes are
prepared. The monolayered autologous erythrocytes may be
manufactured by methods known per se but the following method is
preferred.
[0099] Autologous erythrocytes are made about 4% by weight
concentration by a Hanks solution without serum supplementation. It
is preferred to use the autologous erythrocytes which are prepared
by the above-mentioned known method and preserved at about
4.degree. C. in a phosphate-buffered physiological saline
(hereinafter, referred to as PBS) with addition of 0.1% of AB
serum. Poly-L-lysine is added to a Terasaki plate, treated at about
37.degree. C. for about 20 minutes and washed with PBS, the
above-mentioned autologous erythrocytes are added immediately
thereafter and allowed to stand at about 37.degree. C. for about 30
minutes and the erythrocytes which are not adhered are removed to
give a Terasaki plate to which monolayered autologous erythrocytes
are adhered.
[0100] The induced effector macrophage which is recovered
hereinabove is dissolved/suspended in the Hanks solution with 5
.mu.g/ml of gentamicin so as to make about 2.times.10.sup.6
cells/ml. To the above Terasaki plate to which the monolayered
autologous erythrocytes are adhered is added 1 to 10 .mu.l of the
Hanks solution, the above dissolved/suspended liquid is added
thereto in the same amount and the mixture is allowed to stand at
about 37.degree. C. for about 2 hours. After completion of the
reaction, it is preferred to fix by formalin.
[0101] Numbers of the produced SPFC can be easily measured by a
phase-contrast microscope.
[0102] With regard to an index for the suppression of induction of
effector macrophage, it is preferred to use an IC.sub.50
concentration. The IC.sub.50 can be calculated as follows.
[0103] When plural experiments are carried out under the same
conditions, a mean value of the SPFC production numbers measured as
above is determined. Then, SPFC production numbers per
1.times.10.sup.6 of induced macrophages are calculated and, from
the recovered induced macrophage numbers, the produced SPFC numbers
are determined (this value will be called S1). Effector macrophage
is induced as above without addition of the compound to be tested
and SPFC production numbers are measured by the same manner (this
value will be called S2). Concentration of the substance to be
tested when S1 becomes one half of S2 is defined as IC.sub.50.
[0104] With regard to the compound which is able to prevent,
mitigate or cure the progressive lesion after organic damage found
by the screening method according to the present invention, the
compound where IC.sub.50 is 1 .mu.M or less is preferred.
[0105] The present invention further provides a method for
screening a compound which is able to prevent, mitigate or treat
progressive tubulointerstitial lesion after renal damage, lesion of
exocrine, acinar or ductal interstitial tissues of pancreas or
pancreatitis, and lesion of tubulointerstitial tissues associated
with pancreatitis, characterized in that, a suppressive action of a
compounded to be tested to the induction of effector macrophage
caused by contact of human PBMC with mitomycin-treated human PBMC
is measured.
[0106] A preferred embodiment of the above-mentioned screening
method according to the present invention is as follows. Firstly,
human PBMC are brought into contact with mitomycin-treated human
PBMC in the presence of a compound to be tested to induce effector
macrophage. After that, a measurement is carried out to check
whether the compound to be tested shows a suppressive action to the
induction of effector macrophage.
[0107] It is preferred to induce the above effector macrophage in
the same manner as in the case of inducing method for the above
allo-MLC except that the compound to be tested is further present
in bringing the human PBMC into contact with mitomycin-treated
human PBMC. Concentration of the compound to be tested is
preferably in about 1 .mu.M to 0.001 .mu.M the same as in the above
screening method and it is preferred to conduct the induction of
effector macrophage by adding the compounded to be tested in
various concentrations.
[0108] It is preferred that the measurement of the suppressive
action of the compound to be tested to induction of the
above-mentioned effector macrophage is carried out by the same
manner as in the above-mentioned screening method.
[0109] The above-mentioned compound to be tested may be anything
and may be, for example, peptide, protein, non-peptidic compound,
synthetic compound, fermented product, cell extract, vegetable
extract, animal tissue extract and plasma. It may be either a known
compound or a novel compound.
[0110] It is also possible to combine known methods in such a
manner that plural compounds to be tested are screened at the same
time and, only when a suppressive action to the induction of
effector macrophage is detected, suppressive action for each
compound is measured by the above-mentioned method and the compound
having a suppressive action is identified, etc.
[0111] The present invention further provides a screening kit for
carrying out the above-mentioned screening method. There is no
particular limitation for the form of the said screening kit
although the forms which have been known per se may be used.
[0112] For example, a preferred embodiment of a screening kit for
screening a compound which is able to prevent, mitigate or treat
glomerular lesion of kidney, lesion of islets of Langerhans of
pancreas or diabetes mellitus and diabetic glomerular lesion is a
kit which comprises (a) human PBMC, (b) lipopolysaccharide, (c)
human AB type serum, (d) RPMI 1640 medium and (e) a plate to which
monolayered autologous erythrocytes are adhered.
[0113] Preferred one is such a kit containing Hanks solution to
which (a) a solution prepared by adding human PBMC and
lipopolysaccharide are added to an extent of about
2.times.10.sup.6/ml and about 80 .mu.g/ml, respectively to RPMI
1640 containing about 5 .mu.g/ml of gentamicin, about 2 mM of
L-glutamine and about 10% by weight of human AB type serum, (b) a
plate to which monolayered erythrocytes are adhered and (c) about 5
.mu.g/ml of gentamicin are added.
[0114] A preferred embodiment of a screening kit for screening a
compound which is able to prevent, mitigate or treat progressive
tubulointerstitial lesion after renal damage, or lesion of
exocrine, acinar or ductal interstitial tissues of pancreas is a
kit comprising (a) human PBMC, (b) mitomycin-treated human PBMC,
(c) human AB type serum, (d) RPMI 1640 medium and (e) a plate to
which monolayered autologous erythrocytes are adhered.
[0115] Preferred one is the said kit containing a Hanks solution to
which (a) a solution prepared by adding human PBMC and
mitomycin-treated human PBMC to an extent of about
2.times.10.sup.6/ml each to RPMI 1640 containing about 5 .mu.g/ml
of gentamicin, about 2 mM of L-glutamine and about 10% by weight of
human AB type serum, (b) a plate to which monolayered erythrocytes
are adhered and (c) about 5 .mu.g/ml of gentamicin are added.
[0116] (3) The present invention provides a pharmaceutical for
prevention or therapy of progressive lesion after organic damage by
a selective suppression of induction of effector macrophage
corresponding to the lesion inherent to the tissues after the
organic damage and also provides a therapeutic method using the
said pharmaceutical.
[0117] Thus, the present invention provides a pharmaceutical for
prevention and/or therapy of glomerular lesion of kidney, lesion of
islets of Langerhans of pancreas or diabetes mellitus or diabetic
glomerular lesion, characterized in that, the pharmaceutical
contains a compound which suppresses the induction of effector
macrophage caused by contact of human PBMC with
lipopolysaccharide.
[0118] The present invention also provides a pharmaceutical for
prevention and/or therapy of progressive tubulointerstitial lesion
after renal damage, lesion of exocrine, acinar or ductal
interstitial tissues of pancreas and pancreatitis and lesion of
tubulointerstitial tissues which is a complication of pancreatitis,
characterized in that, the pharmaceutical contains a compound which
suppresses the induction of effector macrophage caused by contact
of human PBMC with mitomycin-treated human PBMC.
[0119] With regard to (a) a compound which suppresses the induction
of effector macrophage caused by contact of human PBMC with
lipopolysaccharide and (b) a compound which suppresses the
induction of effector macrophage caused by contact of human PBMC
with mitomycin-treated human PBMC, the compounds which show the
suppression to the induction of effector macrophage by the
above-mentioned screening methods may be exemplified.
[0120] When a compound which shows activity as a result of the
screening is acidic or basic, a salt of such a compound may be used
as the pharmaceutical as well. Salt of the said compound is a salt
with a physiologically acceptable acid (such as inorganic acid and
organic acid) or base (such as alkaline metal). To be more
specific, there may be used inorganic acid salt such as a salt with
hydrochloric acid, phosphoric acid, hydrobromic acid and sulfuric
acid; organic acid salt such as a salt with acetic acid, formic
acid, propionic acid, fumaric acid, maleic acid, succinic acid,
tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid,
methanesulfonic acid and benzenesulfonic acid; inorganic base salt
such as sodium salt, potassium salt and ammonium salt; and organic
base salt such as dimethylamine salt and cyclohexylamine salt.
[0121] The compound which is used for a pharmaceutical or a
therapeutic method according to the present invention may be a
prodrug or a derivative of the above-mentioned compound as
well.
[0122] With regard to the compound used as a pharmaceutical
according to the present invention, the following compounds may be
specifically exemplified.
[0123] Specific examples of the compound which is able to prevent,
mitigate or cure glomerular lesion of kidney, lesion of islet of
Langerhans of pancreas or diabetes mellitus or diabetic glomerular
lesion by suppressing the induction of effector macrophage caused
by contact of human PBMC with lipopolysaccharide are the following
compounds (9-1), (7-3), (7-5) and (4-2).
[0124] Specific examples of the compounds which is able to prevent,
mitigate or cure progressive tubulointerstitial lesion after renal
damage, lesion of exocrine, acinar or ductal interstitial tissues
of pancreas and pancreatitis and lesion of tubulointerstitial
tissues which is a complication of pancreatitis by suppressing the
induction of effector macrophage caused by contact of human PBMC
with mitomycin-treated human PBMC are the following compounds
(6-1), (6-2) and (3-2).
[0125] With regard to the above-mentioned compounds, there are a
compound represented by the formula (1) 9
[0126] and a compound represented by the formula (2). 10
[0127] There is also an optical isomer .gamma.-lactone represented
by the formula (3) 11
[0128] or by the formula (4) 12
[0129] (in the formulae, R.sup.21 is an optionally substituted
naphthyl and R.sup.22 is an optionally substituted open-chain
hydrocarbon group having 1 to 6 carbons) and a mixture of such
optical isomers.
[0130] Among them, the compound where R.sup.21 is naphthyl group
and R.sup.22 is methyl group is preferred.
[0131] There is also a compound represented by the formula (5) and
a pharmacologically acceptable salt thereof. 13
[0132] (in the formula,
[0133] (a) R.sup.1 and R.sup.2 may be the same or different and
each is hydrogen, an open-chain aliphatic hydrocarbon group which
may be substituted or interrupted by an intervening group, an
optionally substituted cyclic aliphatic hydrocarbon group, an
optionally substituted aryl group, an optionally substituted
heterocyclic group or an optionally substituted condensed
heterocyclic group; X.sup.2 is O, S or NR.sup.3 in which R.sup.3 is
hydrogen, oxygen, an open-chain aliphatic hydrocarbon group which
may be substituted or interrupted by an intervening group., an
optionally substituted cyclic aliphatic hydrocarbon group, an
optionally substituted aryl group, an optionally substituted
heterocyclic group or an optionally substituted condensed
heterocyclic group; and n is an integer from 1 to 5 or
[0134] (b) X.sup.2 is O, S or NR.sup.3; R.sup.1, R.sup.2 and
R.sup.3 each is a substituent represented by the formula
R.sup.10-Z-R.sup.11-- (in which R.sup.10 and R.sup.11 may be the
same or different and each is an optionally substituted open-chain
or cyclic hydrocarbon group, an optionally substituted aryl group,
an optionally substituted heterocyclic group or an optionally
substituted condensed heterocyclic group; and Z is an intervening
group); and n is an integer from 1 to 5).
[0135] There is further a compound represented by the formula (6)
or a pharmacologically acceptable salt thereof. 14
[0136] (in the formula, R.sup.1, X.sup.2 and n have the same
meaning as defined in the above-mentioned item (4); X.sup.1 is
halogen, cyano group, an optionally substituted mercapto group, an
optionally substituted sulfo group, an optionally substituted
sulfonyl group, an optionally substituted hydroxyl group, an
optionally substituted amino group or an optionally substituted
phosphoryl group).
[0137] There is still further a compound represented by the formula
(7) or a pharmacologically acceptable salt thereof. 15
[0138] (in the formula, R.sup.1 and X.sup.2 have the same meaning
as defined in the above-mentioned item (4); X.sup.1 has the same
meaning as defined in the above-mentioned item(S); R.sup.5 and
R.sup.6 may be the same or different and each is.
[0139] (a) hydrogen, an open-chain aliphatic hydrocarbon group
which may be substituted or interrupted by an intervening group, an
optionally substituted cyclic aliphatic hydrocarbon group, an
optionally substituted heterocyclic group or an optionally
substituted condensed heterocyclic group,
[0140] (b) a substituent represented by the formula
R.sup.10-Z-R.sup.11-- (in the formula, R.sup.10 and R.sup.11 may be
the same or different and each is an optionally substituted
open-chain or cyclic hydrocarbon group, an optionally substituted
aryl group, an optionally substituted heterocyclic group or an
optionally substituted condensed heterocyclic group; and Z is an
intervening group.), or
[0141] (c) R.sup.5 and R.sup.6 is an optionally substituted
aromatic ring together with the carbon atom to which they are
attached).
[0142] In the compound represented by the formula (3) or (4),
R.sup.21 is an optionally substituted naphthyl group.
[0143] Examples of the substituent are halogen (preferably,
fluorine, chlorine and bromine), an oxo group, an alkanoyl group
(preferably C.sub.1-8), an alkanoyloxy group (preferably
C.sub.1-8), an alkanoylamino group (preferably C.sub.1-8), carboxyl
group, an alkoxycarbonyl group (preferably C.sub.2-8), a
haloalkylcarbonyl group (preferably C.sub.2-8), an alkoxy group
(preferably C.sub.1-8), a haloalkoxy group (preferably C.sub.1-8),
amino group, an alkylamino group (preferably C.sub.1-8), a
dialkylamino group (preferably C.sub.2-16), a cyclic amino group,
an alkylaminocarbonyl group (preferably C.sub.2-8), carbamoyl
group, hydroxyl group, nitro group, cyano group, mercapto group, an
alkylthio group (preferably C.sub.1-8), an alkylsulfonyloxy group
(preferably C.sub.1-8), an alkylsulfonylamino group (preferably
C.sub.1-8) and phenyl group.
[0144] Further, the naphtyl group may be substituted by such
group(s) at one or more position(s).
[0145] The naphthyl group represented by a substituent R.sup.21 may
also be substituted with open-chain or cyclic hydrocarbon group
which will be mentioned later in detail. Carbon number(s) of the
hydrocarbon group is/are preferably 1 to 8. The said open-chain or
cyclic hydrocarbon group may be substituted, for example, with
halogen, hydroxyl group, amino group, nitro group, cyano group,
mercapto group, carbamoyl group, alkanoyl group, alkanoyloxy group
or alkanoylamino group.
[0146] The open-chain hydrocarbon group as a substituent for
naphthyl group may be interrupted by an intervening group such as
--O--, --CO--, --COO--, --S--, --SO--, --SO.sub.2--, --NH--,
--NR.sup.3--, --NH--CO--, --NR.sup.3--CO--, --NH--SO.sub.2--,
--NR.sup.3--SO.sub.2--, --Si-- or phosphoryl group.
[0147] The substituent R.sup.3 is
[0148] (a) hydrogen, oxygen, an open-chain hydrocarbon residue
which may be substituted or interrupted by an intervening group, an
optionally substituted cyclic aliphatic hydrocarbon group, an
optionally substituted aryl group, an optionally substituted
heterocyclic group or an optionally substituted condensed
heterocyclic group or
[0149] (b) a substituent represented by the formula
R.sup.10-Z-R.sup.11-- (in which R.sup.10 and R.sup.11 may be the
same or different and each is an optionally substituted open-chain
or cyclic hydrocarbon group, an optionally substituted aryl group,
an optionally substituted heterocyclic group or an optionally
substituted condensed heterocyclic group; and Z is an intervening
group).
[0150] The naphthyl group represented by the substituent R.sup.21
may be substituted with a substituent represented by
R.sup.10-Z-R.sup.11-- (in the formula, R.sup.10 and R.sup.11 may be
the same or different and each is an optionally substituted
open-chain or cyclic hydrocarbon group, an optionally substituted
aryl group, an optionally substituted heterocyclic group or an
optionally substituted condensed heterocyclic group; and Z is an
intervening group).
[0151] In the compound represented by the formula (3) or (4),
R.sup.22 is an optionally substituted open-chain hydrocarbon group
having 1 to 6 carbon(s).
[0152] The term "open-chain hydrocarbon group having 1 to 6
carbon(s)" means that carbon number(s) therein is/are 1 to 6 and it
may be either straight or branched and either saturated or
unsaturated.
[0153] Its examples are methyl group, ethyl group, propyl group,
isopropyl group, n-butyl group, isobutyl group, tert-butyl group,
n-pentyl group, isopentyl group, tert-pentyl group and n-hexyl
group.
[0154] Examples of the substituent in the open-chain hydrocarbon
group are halogen, hydroxyl group, amino group, nitro group, cyano
group, mercapto group, carbamoyl group, alkanoyl group, alkanoyloxy
group and alkanoylamino group.
[0155] In the compounds represented by the formulae (5) to (7), the
substituents R.sup.1 and R.sup.2 may be the same or different and
each is
[0156] (a) hydrogen, an open-chain aliphatic hydrocarbon group
which may be substituted or interrupted by an intervening group, an
optionally substituted cyclic aliphatic hydrocarbon group, an
optionally substituted aryl group, an optionally substituted
heterocyclic group or an optionally substituted condensed
heterocyclic group or
[0157] (b) a substituent represented by the formula
R.sup.10-Z-R.sup.11-- (in which R.sup.10 and R.sup.11 may be the
same or different and each is an optionally substituted open-chain
or cyclic hydrocarbon group, an optionally substituted aryl group,
an optionally substituted heterocyclic group or an optionally
substituted condensed heterocyclic group; and Z is an intervening
group).
[0158] The term "open-chain aliphatic hydrocarbon group" in the
substituents R.sup.1 and R.sup.2 may be straight or branched and
may be saturated or unsaturated.
[0159] To be more specific, there may be exemplified an alkyl group
such as methyl group, ethyl group, n-propyl group, isopropyl group,
n-butyl group, isobutyl group, tert-butyl group, n-pentyl group,
isopentyl group, tert-pentyl group, n-hexyl group,
1,1-dimethylpropyl group and 3-methyl-3-butenyl group; an alkenyl
group such as vinyl group, allyl group, 1-propenyl group,
isopropenyl group, 2-butenyl group, 1,3-butadienyl group and
2-pentenyl group; and an alkynyl group such as ethynyl group,
2-propynyl group, 1-butynyl group and 2-butynyl group.
[0160] It is also possible that both double bond and triple bond
are present in one substituent such as 2-penten-4-ynyl.
[0161] Carbon number(s) is/are preferably 1 to 8. Especially for
the substituent R.sup.2, ethynyl group or 2-propynyl group are
preferred.
[0162] The "cyclic aliphatic hydrocarbon group" in the substituent
R.sup.1 and R.sup.2 may be saturated or unsaturated or may be
cross-linked.
[0163] To be more specific, there may be exemplified a cycloalkyl
group such as cyclopropyl group, cyclobutyl group, cyclopentyl
group, cyclohexyl group, cycloheptyl group, adamantyl group and
bicyclo[2.2.1]heptyl group; and a cycloalkenyl group such as
2-cyclopenten-1-yl group and 2,4-cyclopentadien-1-yl group.
[0164] Carbon numbers of the cyclic aliphatic hydrocarbon group are
preferably 3 to 12.
[0165] The open-chain or cyclic aliphatic hydrocarbon group in the
substituents R.sup.1 and R.sup.2 may be substituted with the
substituent which will be mentioned later. With regard to the
position of such a substituent, there is no particular limitation
so far as it is chemically allowed.
[0166] The open-chain hydrocarbon group as a substituent in the
substituents R.sup.1 and R.sup.2 may be interrupted by an
intervening group such as --O--, --CO--, --COO--, --S--, --SO--,
--SO.sub.2--, --NH--, --NR.sup.3--, --NH--CO--, --NR.sup.3--CO--,
--NH--SO.sub.2--, --NR.sup.3--SO.sub.2--, --Si-- or phosphoryl
group. R.sup.3 is
[0167] (a) hydrogen, oxygen, an open-chain hydrocarbon residue
which may be substituted or interrupted by an intervening group, an
optionally substituted cyclic aliphatic hydrocarbon group, an
optionally substituted aryl group, an optionally substituted
heterocyclic group or an optionally substituted condensed
heterocyclic group or
[0168] (b) a substituent represented by the formula
R.sup.10-Z-R.sup.11-- (in which R.sup.10 and R.sup.11 may be the
same or different and each is an optionally substituted open-chain
or cyclic hydrocarbon group, an optionally substituted aryl group,
an optionally substituted heterocyclic group or an optionally
substituted condensed heterocyclic group; and Z is an intervening
group).
[0169] The "aryl group" in the substituents R.sup.1 and R.sup.2 is
an aromatic hydrocarbon group which may be partially saturated and
there may be exemplified phenyl group, benzyl group, biphenyl
group, indenyl group and naphthyl group or a partially saturated
group thereof such as 2,3-dihydroindenyl group and
1,2,3,4-tetrahydronaphthyl group.
[0170] Preferred carbon numbers of the aryl group are 6 to 20.
[0171] Such an aryl group may be substituted with the substituent
which will be mentioned later and, with regard to the position of
the linkage and the position of the substituent, there is no
particular limitation so far as they are chemically allowed.
[0172] With regard to the substituent R.sup.1 and R.sup.2, benzyl
group is preferred and, with regard to the substituent R.sup.2, it
is also preferred that 4-position is substituted with fluorine.
[0173] With regard to the "heterocyclic group" in the substituent
R.sup.1 and R.sup.2, there may be exemplified five- to six-membered
saturated or unsaturated ring containing 1 to 3 hetero atom(s)
selected from nitrogen atom, oxygen atom and sulfur atoms in a
ring.
[0174] Examples of such a heterocyclic group are an aromatic
heterocyclic group such as pyrrolyl group, furyl group, thienyl
group, imidazolyl group, oxazolyl group, thiazolyl group, pyrazolyl
group, isoxazolyl group, isothiazolyl group, oxadiazolyl group,
triazolyl group, indolyl group, benzofuryl group, benzothienyl
group, benzimidazolyl group, benzoxazolyl group, benzothiazolyl
group, pyridyl group, pyrimidinyl group, pyrazinyl group,
pyridazinyl group, triazolyl group, tetrazolyl group, quinolyl
group and isoquinolyl group; a partially saturated heterocyclic
group such as pyranyl group, 1,2-dihydroquinolyl group,
1,2,3,4-tetrahydroquinolyl group, 1,2-dihydroisoquinolyl group,
1,2,3,4-tetrahydroisoquinolyl group, dihydrofuryl group and
dihydrothienyl group; and a saturated heterocyclic group such as
pyrrolidinyl group, piperidinyl group, piperazinyl group,
morpholinyl group, tetrahydrofuryl group and tetrahydrothienyl
group.
[0175] Such a heterocyclic group may be substituted with the
substituent which will be mentioned later and, with regard to the
position of the linkage and the position of the substituent, there
is no particular limitation so far as they are chemically
allowed.
[0176] With regard to the "condensed heterocyclic group" in the
substituent R.sup.1 and R.sup.2, there may be exemplified a case
where a five- to six-membered saturated, partially saturated or
unsaturated ring containing 1 to 3 hetero atom(s) selected from
nitrogen atom, oxygen atom and sulfur atom in the ring is condensed
with a benzene ring or other heterocyclic ring.
[0177] With regard to the condensed heterocyclic ring, there may be
exemplified indole, 3H-indole, isoindole, benzofuran,
benzothiophene, 1H-indazole, benzimidazole, benzoxazole,
benzothiazole, benzisodxazole, benzisothiazole, quinoline,
isoquinoline, quinazoline, 1,2-dihydroquinoline,
1,2,3,4-tetrahydroquinoline, 1,2-dihydroisoquinoline and
1,2,3,4-tetrahydroisoquinoline.
[0178] Such a condensed heterocyclic group may be substituted with
the substituent which will be mentioned later. With regard to the
position of the linkage and the position of the substituent in case
substituent is present, there is no particular limitation so far as
they are chemically allowed.
[0179] The substituents R.sup.1 and R.sup.2 may be a substituent
represented by the formula R.sup.10-Z-R.sup.11--.
[0180] R.sup.10 and R.sup.11 may be the same or different and each
is an optionally substituted open-chain or cyclic hydrocarbon
group, an optionally substituted aryl group, an optionally
substituted heterocyclic group or an optionally substituted
condensed hetero group. With regard to the substituent, there may
be exemplified a substituent which will be mentioned later.
[0181] With regard to the "substituent" in the open-chain or cyclic
aliphatic hydrocarbon group, the aryl group, the heterocyclic group
or the condensed heterocyclic group, a substituent which is
conventionally used in the field of pharmaceuticals may be
used.
[0182] Examples of the substituent are halogen (preferably,
fluorine, chlorine and bromine), an oxo group, an alkanoyl group
(preferably C.sub.1.about.8), an alkanoyloxy group (preferably
C.sub.1.about.8), an alkanoylamino group (preferably
C.sub.1.about.8), carboxyl group, an alkoxycarbonyl group
(preferably C.sub.2.about.8), a haloalkylcarbonyl group (preferably
C.sub.2.about.8), an alkoxy group (preferably C.sub.1.about.8), a
haloalkoxy group (preferably C.sub.1.about.8), an alkyl group
(preferably C.sub.1.about.20), amino group, an alkylamino group
(preferably C.sub.1.about.8), a dialkylamino group (preferably
C.sub.2.about.16), a cyclic amino group, an alkylaminocarbonyl
group (preferably C.sub.2.about.8), carbamoyl group, hydroxyl
group, nitro group, cyano group, mercapto group, an alkylthio group
(preferably C.sub.1.about.8), an alkylsulfonyloxy group (preferably
C.sub.1.about.8), an alkylsulfonylamino group (preferably
C.sub.1.about.8) and phenyl group.
[0183] Further, the group may be substituted by such group(s) at
one or more position(s).
[0184] The substituent X.sup.2 is O, S, NH or NR.sup.3. Here, the
substituent R.sup.3 has the same meaning as defined above.
[0185] n is an integer of 1 to 5 and, preferably, it is 1.
[0186] The substituent X.sup.1 is (a) halogen, (b) cyano group, (c)
an optionally substituted mercapto group, an optionally substituted
sulfo group or an optionally substituted sulfonyl group, (d) an
optionally substituted hydroxyl group, (e) an optionally
substituted amino group or (f) an optionally substituted phosphoryl
group.
[0187] Examples of the substituent are halogen (preferably,
fluorine, chlorine and bromine), an oxo group, an alkanoyl group
(preferably C.sub.1.about.8), an alkanoyloxy group (preferably
C.sub.1.about.8), an alkanoylamino group (preferably
C.sub.1.about.8), carboxyl group, an alkoxycarbonyl group
(preferably C.sub.2.about.8), a haloalkylcarbonyl group (preferably
C.sub.2.about.8), an alkoxy group (preferably C.sub.1.about.8), a
haloalkoxy group (preferably C.sub.1.about.8), an alkyl group
(preferably C.sub.1.about.20), amino group, an alkylamino group
(preferably C.sub.1.about.8), a dialkylamino group (preferably
C.sub.2.about.16), a cyclic amino group, an alkylaminocarbonyl
group (preferably C.sub.2.about.8), carbamoyl group, hydroxyl
group, nitro group, cyano group, mercapto group, an alkylthio group
(preferably C.sub.1.about.8), an alkylsulfonyloxy group (preferably
C.sub.1.about.8), an alkylsulfonylamino group (preferably
C.sub.1.about.8) and phenyl group.
[0188] Further, the group may be substituted by such group(s) at
one or more position(s), if chemically acceptable.
[0189] With regard to the above-mentioned optionally substituted
mercapto group, optionally substituted sulfo group or optionally
substituted sulfonyl group, there may be exemplified benzoylthio
group, tosyl group, phenylsulfo group and phenylsulfinyl group.
[0190] With regard to the above-mentioned optionally substituted
hydroxyl group, there may be exemplified methoxy, ethoxy,
propionyloxy, allyloxy, benzoxy and naphthoxy.
[0191] With regard to the above-mentioned optionally substituted
amino group, there may be exemplified methylamino group, ethylamino
group, anilino group and anisidino group.
[0192] With regard to the above-mentioned optionally substituted
phosphoryl group, a substituent represented by the formula (9)
16
[0193] or by the formula (10) 17
[0194] (in the formulae, substituents R.sup.7 and R.sup.8 may be
the same or different and each is an optionally substituted
open-chain or cyclic hydrocarbon group, an optionally substituted
aryl group, an optionally substituted heterocyclic group or an
optionally substituted condensed hetero group) is preferred. There
may be exemplified methylphosphoryl group, dimethylphosphoryl group
and methylethylphosphoryl group. Carbon number(s) of the said
phosphoryl group is/are preferably 1 to 20.
[0195] In the compound (7), the substituents R.sup.5 and R.sup.6
each is (a) hydrogen, an open-chain hydrocarbon residue which may
be substituted or interrupted by an intervening group, an
optionally substituted cyclic aliphatic hydrocarbon group, an
optionally substituted aryl group, an optionally substituted
heterocyclic group or an optionally substituted condensed
heterocyclic group or is (b) a substituent represented by the
formula R.sup.10-Z-R.sup.11-- (in which R.sup.10 and R.sup.11 may
be the same or different and each is an optionally substituted
open-chain or cyclic hydrocarbon group, an optionally substituted
aryl group, an optionally substituted heterocyclic group or an
optionally substituted condensed heterocyclic group; and Z is an
intervening group).
[0196] To be specific, the above is the same description as that
for the substituents R.sup.1 and R.sup.2.
[0197] The substituents R.sup.5 and R.sup.6 may be the same or
different.
[0198] The substituents R.sup.5 and R.sup.6 may also form an
aromatic ring together with a carbon to which they are bonded.
There are exemplified the cases where benzene, naphthalene or
indene is formed.
[0199] The said aromatic ring may be substituted. In that case, one
or more position(s) may be substituted. Examples of the substituent
are halogen (preferably, fluorine, chlorine and bromine), an oxo
group, an alkanoyl group (preferably C.sub.1.about.8), an
alkanoyloxy group (preferably C.sub.1.about.8), an alkanoylamino
group (preferably C.sub.1.about.8), carboxyl group, an
alkoxycarbonyl group (preferably C.sub.2.about.8), a
haloalkylcarbonyl group (preferably C.sub.2.about.8), an alkoxy
group (preferably C.sub.1.about.8), a haloalkoxy group (preferably
C.sub.1.about.8), an alkyl group (preferably C.sub.1.about.20),
amino group, an alkylamino group (preferably C.sub.1.about.8), a
dialkylamino group (preferably C.sub.2.about.16), a cyclic amino
group, an alkylaminocarbonyl group (preferably C.sub.2.about.8),
carbamoyl group, hydroxyl group, nitro group, cyano group, mercapto
group, an alkylthio group (preferably C.sub.1.about.8), an
alkylsulfonyloxy group (preferably C.sub.1.about.8), an
alkylsulfonylamino group (preferably C.sub.1.about.8) and phenyl
group.
[0200] Further, the said aromatic ring may be substituted with the
above-mentioned optionally substituted open-chain or cyclic
hydrocarbon group. Preferably, carbon number(s) of the hydrocarbon
group is/are 1 to 8. The said open-chain hydrocarbon group may be
interrupted by an intervening group such as --O--, --CO--, --COO--,
--S--, --SO--, --SO.sub.2--, --NH--, --NR.sup.3--, --NH--CO--,
--NR.sup.3--CO--, --NH--SO.sub.2--, --NR.sup.3--SO.sub.2--, --Si--
or phosphoryl group (where R.sup.3 has the same meaning as the
above-mentioned definition).
[0201] Still further, the said aromatic ring may be substituted
with a substituent represented by the formula R.sup.10-Z-R.sup.11--
(in the formula, R.sup.10 and R.sup.11 may be the same or different
and each is an optionally substituted open-chain or cyclic
hydrocarbon group, an optionally substituted aryl group, an
optionally substituted heterocyclic group or an optionally
substituted condensed heterocycylic group; and Z is an intervening
group).
[0202] The compounds represented by the formulae (5) to (7) have an
asymmetric carbon atom and, therefore, two optical isomers can
exist. Accordingly, the pharmaceutical according to the present
invention may contain one of the optical isomers only or may
contain a racemate.
[0203] There is no particular limitation for the pharmacologically
acceptable salts of the compounds represented by the formulae (5)
to (7) and, to be more specific, there may be exemplified inorganic
acid salt such as a salt with hydrochloric acid, phosphoric acid,
hydrobromic acid and sulfuric acid; organic acid salt such as a
salt with acetic acid, formic acid, propionic acid, fumaric acid,
maleic acid, succinic acid, tartaric acid, citric acid, malic acid,
oxalic acid, benzoic acid, methanesulfonic acid and benzenesulfonic
acid; inorganic base salt such as sodium salt, potassium salt and
ammonium salt; and organic base salt such as dimethylamine salt and
cyclohexylamine salt.
[0204] With regard to the specific examples of the above-mentioned
substituents in the present invention, the following substituents
may be exemplified.
[0205] With regard to the above "alkanoyl group", there may be
exemplified formyl group, acetyl group, propionyl group, butyryl
group and pivaloyl group.
[0206] With regard to the above "alkanoyloxy group", there may be
exemplified formyloxy group, acetoxy group, propionyloxy group,
butyryloxy group and pivaloyloxy group.
[0207] With regard to the above "alkanoylamino group", there may be
exemplified acetylamino group, propionylamino group, butyrylamino
group and pivaloylamino group.
[0208] With regard to the above "alkoxycarbonyl group", there may
be exemplified methoxycarbonyl group, ethoxycarbonyl group,
propoxycarbonyl group, isopropoxycarbonyl group, butoxycarbonyl
group, isobutoxycarbonyl group, tert-butoxycarbonyl group and
pentyloxycarbonyl group.
[0209] With regard to the above "haloalkylcarbonyl group", there
may be exemplified fluoroacetyl group, difluoroacetyl group,
trifluoroacetyl group, chloroacetyl group, dichloroacetyl group,
trichloroacetyl group, bromoacetyl group, dibromoacetyl group,
tribromoacetyl group, 3-chloropropionyl group and 4-chlorobutyryl
group.
[0210] The above "alkoxy group" means a straight or branched alkoxy
group and there may be exemplified methoxy group, ethoxy group,
propoxy group, isopropoxy group, butoxy group, tert-butoxy group,
pentyloxy group, tert-pentyloxy group and hexyloxy group.
[0211] The above "haloalkoxy group" means a group where halogen
atom is substituted in the above "alkoxy group" and there may be
exemplified fluoromethoxy group, difluoromethoxy group,
trifluoromethoxy group, chloromethoxy group, dichloromethoxy group,
trichloromethoxy group, bromomethoxy group, dibromomethoxy group,
tribromomethoxy group, iodomethoxy group, diiodomethoxy group,
triiodomethoxy group, 2-fluoroethoxy group, 2,2-difluoroethoxy
group, 2,2,2-trifluoroethoxy group, 2-chloroethoxy group,
2,2-dichloroethoxy group, 2,2,2-trichloroethoxy group,
2-bromoethoxy group, 2,2-dibromoethoxy group, 2,2,2-tribromoethoxy
group, 3-chloropropoxy group and 4-chlorobutoxy group.
[0212] The above "alkylamino group" means a group where an amino
group is substituted with an alkyl group and there may be
exemplified methylamino group, propylamino group, isopropylamino
group, butylamino group, isobutylamino group, tert-butylamino
group, pentylamino group, isopentylamino group, tert-pentylamino
group and hexylamine group.
[0213] The above "dialkylamino group" means a group where an amino
group is disubstituted with an alkyl group in which the types of
the alkyl groups may be the same or different and there may be
exemplified dimethylamino group, ethylmethylamino group,
diethylamino group, methylpropylamino group, ethylpropylamino
group, dipropylamino group, diisopropylamino group, dibutylamino
group, diisobutylamino group, di-tert-butylamino group,
dipentylamino group, diisopentylamino group, di-tert-pentylamino
group and dihexylamino group.
[0214] The above "cyclic amino group" means a group where an amino
group is in a cyclic form where four- to eight-membered cyclic
amino groups are preferred and there may be exemplified azetidinyl
group, pyrrolidinyl group and piperidino group as well as those
having oxygen atom, sulfur atom or nitrogen atom as a hetero atom
such as morpholino group, thiomorpholino group and piperazinyl
group. The nitrogen atom of 4-position of the piperazinyl group may
bear a substituent such as a lower alkyl group or an aryl
group.
[0215] The above "alkylaminocarbonyl group" means a group where the
"alkylamino" moiety is the already-mentioned "alkylamino group" and
there may be exemplified methylaminocarbonyl group,
ethylaminocarbonyl group, propylaminocarbonyl group,
isopropylaminocarbonyl group, butylaminocarbonyl group,
isobutylaminocarbonyl group, tert-butylaminocarbonyl group,
pentylaminocarbonyl group, isopentylaminocarbonyl group,
tert-pentylaminocarbonyl group and hexylaminocarbonyl group.
[0216] The above "alkylthio group" means a straight or branched
alkylthio group and there may be exemplified methylthio group,
ethylthio group, propylthio group, isopropylthio group, butylthio
group, tert-butylthio group, pentylthio group, tert-pentylthio
group and hexylthio group.
[0217] The above "alkylsulfonyloxy group" means a straight or
branched alylsulfonyloxy group, and there may be exemplified
methylsulfonyloxy group, ethylsulfonyloxy group, propylsulfonyloxy
group, isopropylsulfonyloxy group, butylsulfonyloxy group,
tert-butylsulfonyloxy group, pentylsulfonyloxy group,
tert-pentylsulfonyloxy group and hexylsulfonyloxy group.
[0218] The above "alkylsulfonylamino group" means a group where an
amino group is substituted with a straight or branched
alkylsulfonyl group and there may be exemplified
methylsulfonylamino group, ethylsulfonylamino group,
propylsulfonylamino group, isopropylsulfonylamino group,
butylsulfonylamino group, tert-butylsulfonylamino group,
pentylsulfonylamino group, tert-pentylsulfonylamino group and
hexylsulfonylamino group.
[0219] In a substituent represented by the formula
R.sup.10-Z-R.sup.11-- (in the formula, R.sup.10 and R.sup.11 may be
the same or different and each is an optionally substituted
open-chain or cyclic hydrocarbon group, aryl group, heterocyclic
group or condensed heterocyclic group; and Z is an intervening
group), examples of the intervening group are --O--, --CO--,
--COO--, --S--, --SO--, --SO.sub.2--, --NH--, --NR.sup.3--,
--NH--CO--, --NR.sup.3--CO--, --NH--SO.sub.2--,
--NR.sup.3--SO.sub.2--, --Si-- are phosphoryl group where R.sup.3
has the same meaning as the above-mentioned definition.
[0220] With regard to the above-mentioned substituent, specific
examples thereof are the following substituents.
[0221] (a) With regard to a substituent where the intervening group
is --O--, there may be exemplified methoxymethyl group,
ethoxymethyl group, ethoxyethyl group, propoxymethyl group,
propoxyethyl group, isopropoxymethyl group, butoxymethyl group,
butoxyethyl group, butoxypropyl group, tert-butoxymethyl group,
tert-butoxyethyl group, pentyloxymethyl group, pentyloxyethyl
group, pentyloxypropyl group, pentyloxybutyl group,
tert-pentyloxymethyl group, tert-pentyloxyethyl group,
hexyloxymethyl group, hexyloxyethyl group, hexyloxypropyl group,
hexyloxypropyl group, benzyloxymethyl group and phenoxymethyl
group.
[0222] Preferred carbon number(s) of the said substituent is/are 1
to 10.
[0223] (b) With regard to a substituent where the intervening group
is --CO--, there may be exemplified acetylmethyl group, acetylethyl
group, acetylpropyl group, acetylbutyl group, acetylpentyl group,
acetylhexyl group, propionylmethyl group, butyrylmethyl group,
isobutyrylmethyl group, valerylmethyl group, isovalerylmethyl
group, hexanoylmethyl group and phenylacetylmethyl group.
[0224] Preferred carbon number(s) of the said substituent is/are 1
to 10.
[0225] (c) With regard to a substituent where the intervening group
is --COO--, there may be exemplified acetoxymethyl group,
acetoxyethyl group, acetoxypropyl group, acetoxybutyl group,
acetoxypentyl group, acetoxyhexyl group, propionyloxymethyl group,
tert-butyloxycarbonylmethyl group, 1-isobutyryloxyethyl group,
1-cyclohexyloxycarbonylethyl group, benzyloxycarbonylmethyl group,
phenoxycarbonylmethyl group and pivaloyloxymethyl group.
[0226] Preferred carbon number(s) of the said substituent is/are 1
to 10.
[0227] (d) With regard to a substituent where the intervening group
is --S--, there may be exemplified methylthiomethyl group,
methylthioethyl group, methylthiopropyl group, methylthiobutyl
group, methylthioheptyl group, methylthiohexyl group,
methylthioisobutyl group, ethylthiomethyl group, propylthiomethyl
group, butylthiomethyl group, heptylthiomethyl group,
hexylthiomethyl group, benzylthiomethyl group and phenylthiomethyl
group.
[0228] Preferred carbon number(s) of the said substituent is/are 1
to 10.
[0229] (e) With regard to a substituent where the intervening group
is --SO.sub.2--, there may be exemplified methylsulfonylmethyl
group, methylsulfonylethyl group, methylsulfonylpropyl group,
methylsulfonylbutyl group, methylosulfonylheptyl group,
methylsulfonylhexyl group, methylsulfonylisobutyl group,
ethylsulfonylmethyl group, propylsulfonylmethyl group,
butylsulfonylmethyl group, heptylsulfonylmethyl group,
hexylsulfonylmethyl group, benzylsulfonylmethyl group and
phenylsulfonylmethyl group.
[0230] Preferred carbon number(s) of the said substituent is/are 1
to 10.
[0231] (f) With regard to a substituent where the intervening group
is --SO--, there may be exemplified methylsulfinylmethyl group,
methylsulfinylethyl group, methylsulfinylpropyl group,
methylsulfinylbutyl group, methylsulfinylheptyl group,
methylsulfinylhexyl group, methylsulfinylisobutyl group,
ethylsulfinylmethyl group, propylsulfinylmethyl group,
butylsulfinylmethyl group, heptylsulfinylmethyl group,
hexylsulfinylmerhyl group, benzylsulfinylmethyl group and
phenylsulfinylmethyl group.
[0232] Preferred carbon number(s) of the said substituent is/are 1
to 10.
[0233] (g) The substituent where the intervening group is --NH-- is
a compound represented by the formula R.sup.10--NH--R.sup.11--
where R.sup.11 is an optionally substituted open-chain or cyclic
hydrocarbon group, an optionally substituted aryl group, an
optionally substituted heterocyclic group and an optionally
substituted condensed heterocyclic group as mentioned already.
Examples of R.sup.10--NH-- are methylamino group, ethylamino group,
propylamino group, isopropylamino group, butylamino group,
isobutylamino group, tert-butylamino group, pentylamino group,
isopentylamino group, tert-pentylamino group, hexylamino group,
anilino group and benzylamino group.
[0234] Preferred carbon number(s) of the said substituent is/are 1
to 10.
[0235] (h) The substituent where the intervening group is
--NR.sup.3-- is a compound represented by the formula
R.sup.10--NR.sup.3--R.sup.11-- where R.sup.11 is an optionally
substituted open-chain or cyclic hydrocarbon group, an optionally
substituted aryl group, an optionally substituted heterocyclic
group and an optionally substituted condensed heterocyclic group as
mentioned already.
[0236] Examples of R.sup.10--NR.sup.3-- are dimethylamino group,
ethylmethylamino group, diethylamino group, methylpropylamino
group, ethylpropylamino group, dipropylamino group,
diisopropylamino group, dibutylamino group, diisobutylamino group,
di-tert-butylamino group, dipentylamino group, diisopentylamino
group, di-tert-pentylamino group, dihexylamino group, dibenzylamino
group and methylbenzylamino group.
[0237] The substituent R.sup.3 has the same meaning as the
above-mentioned definition.
[0238] Preferred carbon number(s) of the said substituent is/are 1
to 10.
[0239] (i) With regard to the substituent where the intervening
group is --NH--CO--, --NR.sup.3--CO--, NH--SO.sub.2-- or
--NR.sup.3--SO.sub.2--, there may be exemplified a compound where
an intervening group is changed to the above-mentioned one in the
above-mentioned compound where the intervening group Z is --NH-- or
--NR.sup.3--.
[0240] (j) With regard to the substituent where the intervening
group Z is --Si--, there may be exemplified methylsilylmethyl
group, methylsilylethyl group, methylsilylpropyl group,
methylsilylbutyl group, methylsilylheptyl group, methylsilylhexyl
group, methylsilylisobutyl group, ethylsilylmethyl group,
propylsilylmethyl group, butylsilylmethyl group, heptylsilylmethyl
group, hexylsilylmethyl group, benzylsilylmethyl group and
phenylsilylmethyl group.
[0241] (k) The substituent where the intervening group Z is a
phosphoryl group is represented by the formulae; 18
[0242] (in the formulae, the substituents R.sup.10, R.sup.11 and
R.sup.12 may be the same or different and each is an optionally
substituted open-chain or cyclic hydrocarbon group, an optionally
substituted aryl group, an optionally substituted heterocyclic
group or an optionally substituted condensed hetero cyclic group).
There may be exemplified methylphosphoryl group, dimethylphosphoryl
group and methylethylphosphoryl group. Carbon number(s) of such a
substituent is/are preferably 1 to 20.
[0243] All of the above-mentioned compounds according to the
present invention may be manufactured by known methods or methods
similar thereto. Methods for the manufacture of the compounds of
the present invention will be exemplified as follows.
[0244] In the case of the compound represented by the formula (5)
where R.sup.1 is not hydrogen, it can be manufactured by
cyclization of a compound, for example, represented by the formula
(11) 19
[0245] (in the formula, R.sup.1, R.sup.2 and X.sup.2 are the same
as those defined already; and W is a leaving group) by means of an
intramolecular ring closing reaction.
[0246] Here, preferred leaving groups are the leaving groups which
are known per se such as halogen, ester group, mercapto group,
etc.
[0247] As to the condition for the intramolecular ring closure
reaction, known conditions may be used. Thus, for example, heating
is carried out in an organic solvent, preferably in toluene, at
about 30 to 100.degree. C. or, preferably, about 50 to 70.degree.
C. for about 5 to 20 hours or, preferably, about 8 to 15 hours.
[0248] After that, the solvent is usually removed. Removal of the
solvent may be carried out under reduced pressure or in vacuo
depending upon necessity.
[0249] If desired, purification may be carried out by known methods
such as chromatography or filtration to give the compound
represented by the formula (5).
[0250] In the case of the compound represented by the formula (5)
where R.sup.1 is hydrogen, a starting material where R.sup.1 is a
protective group such as benzyl group is firstly prepared and then
it is hydrolyzed by a known method or by a method similar
thereto.
[0251] Depending upon the type of the protective group represented
by R.sup.1, catalytic reduction may be carried out instead of
hydrolysis. Condition for the catalytic reduction may follow the
known reaction condition and there is exemplified a method where
the starting material is brought into contact with hydrogen gas
under ordinary or high pressure in the presence of a catalyst such
as palladium-carbon, palladium hydroxide-carbon, platinum oxide or
palladium black in an amount of about 3 to 20% by weight or,
preferably, about 5 to 15% by weight.
[0252] If desired, purification may be carried out by known methods
such as chromatography or filtration to give the compound
represented by the formula (5).
[0253] In the case of the compound (6) where R.sup.1 is not
hydrogen and the substituent X.sup.2 is O, the aimed compound is
manufactured by adding a nucleophilic agent containing X.sup.1
(definition of X.sup.1 is the same as defined above) such as
(COCl).sub.2 when X.sup.1 is Cl or by adding a cyanide when X.sup.1
is CN to a compound represented by the formula (12) 20
[0254] (in the formula, R.sup.1 has the same meaning as defined
already) in an organic solvent, preferably ether.
[0255] This reaction is believed to proceed in such a mechanism
that an intramolecular ketalization firstly takes place and a
nucleophilic agent attacks the resulting hydroxyl group.
[0256] After that, there is carried out an after-treatment such as
removal of the solvent. Removal of the solvent may be carried out
either under reduced pressure or in vacuo upon necessity.
[0257] If desired, purification may be carried out by known methods
such as chromatography or filtration to give the compound
represented by the formula (6).
[0258] In the case of the compound (7) where R.sup.1 is not
hydrogen, the substituent X.sup.2 is O and R.sup.5 and R.sup.6 form
a benzene ring together with the carbon atoms bonded thereto, a
compound where X.sup.1 is OH is synthesized by, for example,
addition of 1,2-isochroman-1,3,4-tr- ione to benzyl alcohol and
pyridine.
[0259] After that, a nucleophilic agent containing X.sup.1
(definition of X.sup.1 is the same as defined above) to be
manufactured such as (COCl).sub.2 is added when X.sup.1 is Cl or,
when X.sup.1 is CN, a cyanide is added to substitute the OH group
with the substituent X.sup.2 to manufacture the aimed compound.
[0260] Such a reaction may be carried out according to conventional
means.
[0261] The compounds represented by the above-mentioned formulae
(1) to (7) may be metabolized in vivo, for example, as follows.
Such metabolites may be a pharmaceutical according to the present
invention as well. 21
[0262] (in the formulae, Bn is benzyl group and Et is ethyl
group).
[0263] The following compound (7-2), (7-3), (7-5) or (7-6) may be
metabolized to a compound represented by the formula (7-1). In some
cases, the following compound (7-2), (7-3) or (7-5) may also be
metabolized to a compound where an ester group is converted to a
carboxyl group. 22
[0264] The pharmaceutical in accordance with the present invention
may be in a dosage form such as tablets which are, if desired,
sugar-coated or film-coated, capsules, elixirs or microcapsules and
may be administered orally. Further, the pharmaceutical according
to the present invention may be a parenteral preparation
represented by injection such as an aseptic solution or suspension
preparation with water or other pharmaceutically acceptable
liquid.
[0265] The above-mentioned preparations may be manufactured by a
method which is known per se.
[0266] The pharmaceutical according to the present invention may
further contain other pharmacologically active component which is
effective to progressive lesion after organic damage.
[0267] The pharmaceutical according to the present invention may
still further contain additives which have been used in the related
art such as binders, disintegrating agents, fillers, antiseptic
agents, stabilizers and flavors.
[0268] With regard to the additive which may be mixed with tablets
or capsules, there may be exemplified those which have been used in
the related art such as binders, disintegrating agents, fillers,
antiseptic agents, stabilizers and flavors. To be more specific,
there may be used binders such as hydroxypropyl cellulose,
hydroxypropyl methyl cellulose, Macrogol, gelatin, corn starch,
tragacanth and gum arabic; disintegrating agents such as starch and
carboxymethyl cellulose calcium; fillers such as lactose, starch
and crystalline cellulose; swelling agents such as corn starch,
gelatin and alginic acid; lubricants such as magnesium stearate and
talc; sweeteners such as sucrose, lactose and saccharine; and
flavors such as peppermint, oil derived from Gaultheria ovatifolia
ssp.Adenothrix and cherry. When the dosage form is a capsule, it is
also possible to add a liquid carrier such as fat/oil in addition
to the above additives.
[0269] With regard to an aqueous solution for injection, it is
possible to use a physiological saline and other isotonic solution
containing glucose and other excipients such as D-sorbitol,
D-mannitol and sodium chloride. In that case, it is further
possible to jointly use the appropriate solubilization aids such as
alcohol (e.g., ethanol), polyalcohol (e.g., propylene glycol and
polyethylene glycol) and nonionic surface-active agent (e.g.,
Polysorbate 80.TM. and HCO-50). In the case of an oily liquid for
injection, sesame oil, soybean oil, etc. may be used. Solubilizing
aids such as benzyl benzoate and benzyl alcohol may be also used
together therewith. It is also possible to compound with buffer
such as phosphate buffer and sodium acetate buffer; anesthetizing
agent such as benzalkonium chloride and procaine hydrochloride;
stabilizer (such as human serum albumin and polyethylene glycol;
preserving agent (antiseptic) such as chlorobutanol, methyl
p-hydroxybenzoate, propyl p-hydroxybenzoate, benzyl alcohol and
phenol; antioxidant; etc. The pharmaceutical preparation such as
injection prepared as such is usually filled in appropriate
ampoules.
[0270] Daily dose of the pharmaceutical of the present invention
may vary depending upon the type of the effective ingredient,
diseases to be treated, route for administration, dosage form, etc.
and is not definitely decided. Preferably however, it is about 0.1
to 100 mg/kg, more preferably about 1 to 50 mg/kg and it is also
possible to be about 0.5 to 50 mg/kg. Those compounds are of low
toxicity and are able to be administered either orally or
parenterally.
[0271] The compounds of the present invention represented by the
above-mentioned formulae (1) to (7) have immunosuppressive or
fibrosis inhibiting action. To be more specific, the compound
according to the present invention exhibits an effect of
selectively suppressing the effector macrophage expressed in the
damaged tissue caused by organic damager or immune disease whereby
it is able to selectively exhibit an immunosuppressive action
selectively to specific tissues. Further, it is able to effectively
inhibit the progress or worsening of the disease by the said
effector macrophage whereby it specifically exhibits a fibrosis
inhibiting action selectively to damaged tissues.
[0272] Accordingly, the pharmaceutical which contains the compound
of the present invention represented by the above-mentioned
formulae (1) to (7) is able to be used as an immunosuppressant or a
fibrosis inhibitor. To be more specific, the pharmaceutical can be
used for prevention of onset or progress of rejection upon
allogeneic or xenogeneic cell, tissue or organ transplantation,
acute or chronic glomerular nephritis, interstitial nephritis or
diabetes mellitus; therapy and/or prevention of complications such
as diabetic nephropathy, diabetic retinopathy and diabetic
neuropathy; therapy and/or prevention of chronic pancreatitis,
arteriosclerosis, arteriosclerotic restenosis, pulmonary fibrosis,
dialytic amyloidosis, chronic hepatitis, cerebrospinal degenerative
disease, asthma, rheumatic arthritis, chronic pigmentary
dermatitis, psoriasis, autoimmune chronic organic tissue damage,
endotoxin shock reaction by bacterial toxin, systemic intravascular
coagulation and cancer or metastasis thereof; and prevention and
therapy of aids virus infection. It may also be used as a
substitute for steroidal therapeutic agents.
[0273] Daily dose of the above-mentioned pharmaceutical according
to the present invention used as an immunosuppressants or a
fibrosis inhibitor may vary depending upon the type of the
effective ingredient, the disease to be treated, route of
administration, dosage form, etc. and is not definitely decided.
Preferably however, it is about 0.1 to 100 mg/kg, more preferably
about 1 to 50 mg/kg and it is also possible to be about 0.5 to 50
mg/kg in terms of the compound represented by the formulae (1) to
(7). Those compounds are of low toxicity and are able to be
administered either orally or parenterally.
[0274] As mentioned above, the pharmaceutical according to the
present invention which is used as an immunosuppressant or a
fibrosis inhibitor may contain other pharmacological components
showing an immunosuppressive action or a fibrosis inhibiting action
as mentioned above. Further, it may be in various dosage forms as
mentioned above and, still further, it may contain known additives
depending upon the above-mentioned dosage form.
[0275] In the following Examples, Bn means a benzyl group.
EXAMPLE 1
Manufacture of (RS)-(-)-.alpha.-methyl-2-naphthalene-methyl
2-(4-fluorophenoxy)-5-oxotetrahydrofuran-2-carboxylate
[0276] 23
[0277] To a solution prepared by dissolving 164 mg (0.6 mmol, 1
equivalent) of
2-(4-fluorophenoxy)-5-oxotetrahydrofuran-2-carboxylic acid
manufactured by a method mentioned in paragraph [0018], page 5 of
Japanese Patent Laid-Open No. 04/338,331 in 2.2 ml of ether at
0.degree. C. were added 240 .mu.l (2.7 mmol, 4 equivalents) of
(COCl).sub.2 gradually and then two drops of dimethylformamide
(DMF) were added thereto. At that time, discharge of gas was
observed.
[0278] The reaction solution was allowed to stand for 1 hour with
stirring at 0.degree. C., ether was removed in vacuo and the
product was dried in vacuo.
[0279] The product was dissolved in 2.5 ml of ether, then 130 mg
(0.75 mmol, 1.1 equivalents) of
(S)-(-)-.alpha.-methyl-2-naphthalenemethanol and 8 mg (0.07 mmol,
0.1 equivalent) of dimethylaminopyrrolidone (hereinafter,
abbreviated as DMAP) were gradually added and, at the same time,
140 .mu.l (1 mmol, 1.5 equivalents) of triethylamine were added
thereto as well.
[0280] After the reaction, the product is dissolved in 20 ml of
ether, the solution was washed with an aqueous solution of
NaHCO.sub.3 and the organic phase was dried over MgSO.sub.4.
[0281] The solvent was removed in vacuo and, as a result of the
first purification by silica gel, 175 mg (yield: 74%) of the
product were obtained. By means of purification using silica gel
for several times thereafter, an optical isomer was separated from
a mixture of two diastereomers.
[0282] About the compound of the formula (3), i.e.
(S)-(-)-.alpha.-methyl-- 2-naphthalenemethyl
2-(4-fluorophenoxy)-5-oxotetrahydrofuran-2-carboxylate- :
[0283] Thin-layer chromatography: R.sub.f=0.56 (hexane/ether, 1:1
(v/v))
[0284] T.sub.f=97-98.degree. C.
[0285] [.alpha.].sup.23.sub.D=-113 (c=3 in CHCl.sub.3)
[0286] RMN .sup.1H (300 MHz; CDCl.sub.3) d ppm: 1.44 (3H, d, J=6.7
Hz) 2.47-2.8 (4H, m); 6.03 (1H, q, J=6.7 Hz); 6.82-6.88 (2H, m);
7.03-7.09 (2H, m); 7.34-7.35 (1H, m); 7.49-7.51 (2H, m) 7.73 (1H,
s); 7.8-7.9 (3H, m).
[0287] RMN .sup.13C (75 MHz; CDCl.sub.3) dppm: 21.54; 27.4; 33.1;
75.5; 105.3; 116 (d, .sup.2J.sub.CF=23 Hz); 120.5 (d,
.sup.3J.sub.CF=7.2 Hz); 123.8; 125.6; 126.6; 127.8; 128.2; 128.7;
133.1; 133.3; 137.2; 150.4; 159.3 (d, .sup.1J.sub.CF=244 Hz);
166.0; 174.0.
[0288] IR (CsI) .nu.: 3423; 2981; 1797; 1758; 1504; 1290; 1194;
1165; 1082; 1044; 914; 857; 822; 751 cm.sup.-1.
[0289] About the compound of the formula (4), i.e.
(R)-(-)-.alpha.-methyl-- 2-naphthalenemethyl
2-(4-fluorophenoxy)-5-oxotetrahydrofuran-2-carboxylate- :
[0290] Thin-layer chromatography: R.sub.f=0.51 (hexane/ether, 1:1
(v/v))
[0291] T.sub.f=117-118.degree. C.
[0292] [.alpha.].sup.23.sub.D=-20 (c=3 in CHCl.sub.3)
[0293] RMN .sup.1H (300 MHz; CDCl.sub.3) d ppm: 1.62 (3H, d, J=6.7
Hz) 2.49-2.87 (4H, m); 6.05 (1H, q, J=6.7 Hz); 6.76-6.81 (2H, m);
6.82-7.1 (2H, m); 7.17 (1H, m); 7.5-7.6 (2H, m); 7.65 (1H, s);
7.7-7.8 (3H, m).
[0294] RMN .sup.13C (75 MHz; CDCl.sub.3) d ppm: 21.5; 27.41; 33.2;
75.6; 116 (d, .sup.2J.sub.CF=23 Hz); 120.4 (d, .sup.3J.sub.CF=8.6
Hz); 123.8; 125.8; 126.6; 127.7; 128.1; 128.6; 133.0; 136.9; 150.3;
159.5 (d, .sup.1J.sub.CF=255 Hz); 166.3; 174.0.
[0295] IR (CsI) .nu.: 3423; 2981; 1797; 1758; 1504; 1290; 1194;
1165; 1082; 1044; 914; 857; 822; 751 cm.sup.-1.
EXAMPLE 2
Manufacture of
(.+-.)-2-(4-fluorophenoxy)-5-oxotetrahydrofuran-2-carboxyli- c
Acid
(a) Manufacture of
(-)-2-(4-fluorophenoxy)-5-oxotetrahydrofuran-2-carboxyl- ic
Acid
[0296] 24
[0297] C.sub.11H.sub.9FO.sub.5
[0298] Molecular weight 240.1
[0299] White solid
[0300] T.sub.f=128.degree. C.
[0301] To a solution of 98 mg (0.24 mmol, 1 equivalent) of
(S)-(-)-.alpha.-methyl-2-naphthalenemethyl
2-(4-fluorophenoxy)-5-oxotetra- hydrofuran-2-carboxylate
represented by the formula (3-1) manufactured in Example 1
dissolved in 4 ml of ethyl acetate and 8 drops of ethanol was added
a Pd/C catalyst in an amount of 10 parts by weight to 100 parts by
weight of the above solution. The resulting solution was washed
with water several times, stirred at room temperature and subjected
to a catalytic reduction using hydrogen for 4 hours. The product
was dissolved in ethyl acetate, the catalyst was removed by
filtering through Celite (manufactured by Johns Manville Sales Co.)
and the solvent was removed in vacuo. The product was purified by
means of a reverse phase silica gel chromatography (RP 18) (eluent:
acetonitrile/water, 1:1 (v/v)). Acetonitrile was removed in vacuo
and an aqueous phase was freeze-dried. The present compound was
white powder and 45 mg (yield: 78%) were prepared.
(b) (+)-2-(4-fluorophenoxy)-5-oxotetrahydrofuran-3carboxylic
Acid
[0302] 25
[0303] C.sub.11H.sub.9FO.sub.5
[0304] Molecular weight=240.1
[0305] White solid
[0306] T.sub.f=128.degree. C.
[0307] (R)-(-)-.alpha.-methyl-2-naphthalenemethyl
2-(4-fluoro-phenoxy)-5-o- xotetrahydrofuran-2-carboxylate
represented by the formula (4-1) manufactured in Example 1 was
subjected to a catalytic reduction with hydrogen in the same manner
as above whereupon the present compound was selectively
manufactured. The present compound was white powder and 16 mg
(yield: 58%) were obtained.
[0308] Compound (1): [a].sup.23.sub.D=-101 (c=0.6 in MeOH)
[0309] Compound (2): [a].sup.23.sub.D=+116 (c=0.3 in MeOH)
[0310] RMN .sup.1H (300 MHz; CDCl.sub.3) d ppm: 2.63-2.84 (4H, m)
7.08-7.14 (4H, m).
[0311] RMN .sup.13C (75 MHz; CDCl.sub.3) d ppm: 26.7; 32.6; 105.7;
115.4 (d, .sup.2J.sub.CF=23 Hz); 120.5 (d, 3J.sub.CF=8.6 Hz);
150.9; 159.2 (d, .sup.1J.sub.CF=235 Hz); 168.4; 175.1
[0312] IR (CsI) .nu.: 3082; 1775; 1507; 1253; 1199; 1041; 978; 830;
708 cm.sup.-1
EXAMPLE 3
Manufacture of Benzyl
2-(4-fluorobenzyl)-5-oxotetrahydrofuran-2-carboxylat- e
[0313] 26
[0314] C.sub.19H.sub.17FO.sub.4
[0315] Molecular weight=328.3
[0316] White crystals
[0317] T.sub.f=70.degree. C.
[0318] In the presence of Amberyst resin 15 (manufactured by Rohm
& Haas Co.), a solution where 192 mg (0.4 mmol, 1 equivalent)
of dibenzyl 2-(4-fluorobenzyl)-2-hydroxypentane-1,5-dicarboxylate
were dissolved in 6 ml of anhydrous toluene was heated at
60.degree. C. for 10 hours. This was returned to room temperature
and filtered through cotton to remove the Amberyst resin 15. The
solvent was removed in vacuo and the product was separated by
chromatography (eluent: hexane/ethyl acetate, 8.5:2.5 (v/v)). After
purification by recrystallization from ether-hexane twice, the
present compound was obtained as thin flaky crystals in an amount
of 99 mg (yield: 76%).
[0319] Thin-layer chromatography: R.sub.f=0.24 (hexane/ethyl
acetate, 7:3 (v/v))
[0320] RMN .sup.1H (300 MHz; CDCl.sub.3) d ppm: 2.15-2.52 (4H, m);
3.1 (1H, d. J=14.4 Hz); 3.3 (1H, d, J=14.4 Hz); 5.14 (1H, d, J=12
Hz); 5.18 (1H, d, J=12 Hz); 6.92 (2H, t, J=8.6 Hz) 7.1 (2H, t,
J=8.6 Hz); 7.25-7.36 (5H, m).
[0321] RMN .sup.13C (75 MHz; CDCl.sub.3) d ppm: 27.8; 30.4; 41.6;
67.6; 86.1; 115.2 (d, .sup.2J.sub.CF=22 Hz); 128.3; 128.6; 129.5;
131.9 (d, .sup.3J.sub.CF=6.8 Hz); 134.7; 162.3 (d,
.sup.1J.sub.CF=244 Hz); 170.8; 175.3.
[0322] IR (CsI) .nu.: 1784; 1736%; 1508; 1223; 1189; 1097; 1056;
973; 910; 840; 700; 607 cm.sup.-1.
[0323] SM (IC/NH.sub.3) m/z (intensite relative): 346=100%
(MNH.sub.4.sup.+); 347=23%; 674=19% (2xM+NH.sub.4.sup.+).
EXAMPLE 4
Manufacture of
2-(4-fluorobenzyl)-5-oxo-tetrahydrofuran-2-carboxylic Acid
[0324] 27
[0325] C.sub.12H.sub.11FO.sub.4
[0326] Molecular weight=238.2
[0327] White crystals
[0328] T.sub.f=93-94.degree. C.
[0329] To a solution prepared by dissolving 71 mg (0.2 mmol, 1
equivalent) of benzyl
2-(4-fluorobenzyl)-5-oxo-tetrahydrofuran-2-carboxylate represented
by the formula (5-1), manufactured by the above Example 3, in a
mixture of 1.5 ml of ethyl acetate and 3 drops of ethanol was added
a Pd/C catalyst in an amount of 10 parts by weight to 100 parts by
weight of the above solution. The reaction solution was washed with
water several times and subjected to a catalytic reduciton with
hydrogen for 6 hours with stirring. Completion of the reaction was
confirmed by means of a thin-layer chromatography.
[0330] The product was filtered through Celite (manufactured by
Johns Manville Sales Co.) and the solvent was evaporated in vacuo.
As a result, the present compound in white powder was obtained in
an amount of 46 mg.
[0331] Thin-layer chromatography R.sub.f=0.23 (ethyl acetate/acetic
acid, 98:2 (v/v))
[0332] RMN .sup.1H (300 MHz; CDCl.sub.3) d ppm: 2-2.55 (4H, m);
3.12 (H, d, J=14 Hz); 3.37 (1H, d, J=14 Hz); 7.01 (2H, t, J=8 Hz);
7.25 (2H, t, J=8 Hz); 8.01 (1H, s).
[0333] RMN .sup.13C (75 MHz; CDCl.sub.3) d ppm: 28.8; 31.9; 42.6;
88.0; 115.7 (d, .sup.2J.sub.CF=21 Hz); 132.2; 133.3 (d,
.sup.3J.sub.CF=7.2 Hz); 163.5 (d, .sup.1J.sub.CF=242 Hz); 174.2;
178.3.
[0334] IR (CsI) .nu.: 3124; 1769; 1511; 1409; 1270; 1244; 1223;
1178; 1037; 961; 933; 848; 773; 696; 658 cm.sup.-1.
[0335] SM (IC/NH.sub.3) m/z (relative intensity): 256=100%
(MNH.sub.4.sup.+); 257=14%; 494=14% (2x M+NH.sub.4.sup.+).
EXAMPLE 5
Manufacture of benzyl
2-prop-2-ynyl-5-oxo-tetrahydrofuran-2-carboxylate
[0336] 28
[0337] C.sub.15H.sub.14O.sub.4
[0338] Molecular weight=258.2
[0339] Colorless liquid
[0340] In the presence of Amberyst resin 15 (manufactured by Rohm
& Haas Co.), a solution of 160 mg (0.43 mmol, 1 equivalent) of
dibenzyl 2-hydroxy-2-(2-prop-2-ynyl)pentane-1,5-dicarboxylate in 5
ml of anhydrous toluene was stirred overnight at 60.degree. C.
[0341] When the reaction finished, the reaction solution was
returned to ordinary temperature and spherical Amberyst resin was
removed by filtering through cotton. The solvent was removed in
vacuo.
[0342] The product was purified by a silica type chromatography
(eluent: hexane/ethyl acetate, 7:3 (v/v)). The present compound was
colorless liquid and was obtained in an amount of 85 mg (yield:
77%).
[0343] Thin-layer chromatography: R.sub.f=0.23 (hexane/ethyl
acetate, 7:3 (v/v))
[0344] RMN .sup.1H (300 MHz; CDCl.sub.3) d ppm: 2.08 (1H, t, J=2.6
Hz) 2.44-2.7 (4H, m); 2.89 (1H, dd, J=17.3 Hz, J=2.6 Hz); 2.95 (1H,
dd, J=17.3 Hz, J=2.6 Hz); 5.22 (1H, d, J=12 Hz); 5.28 (1H, d, J=12
Hz); 7.38 (5H, ml).
[0345] RMN .sup.13C (75 MHz; CDCl.sub.3) d ppm: 27.1; 28.1; 29.5;
67.8; 72.2; 77.0; 83.9; 128.1; 128.6; 134.6; 169.8; 175.2. IR (CsI)
.nu.: 3286; 1793; 1746; 1456; 1419; 1339; 1261; 1170; 1068; 933;
753; 699 cm.sup.-1.
[0346] SM (IC/NH.sub.3) m/z (intensite relative): 276=100%
(MNH.sub.4.sup.+) 277=19%; 534=16% (2xM+NH.sub.4.sup.+).
EXAMPLE 6
Manufacture of Benzyl
2-benzyl-5-oxo-tetrahydrofuran-2-carboxylate
[0347] 29
[0348] C.sub.19H.sub.18O.sub.4
[0349] Molecular weight=310.3
[0350] White crystals
[0351] T.sub.f=93-94.degree. C.
[0352] In the presence of Amberyst resin 15 (manufactured by Rohm
& Haas Co.), a solution of 180 mg (0.43 mmol, 1 equivalent) of
dibenzyl 2-benzyl-2-hydroxypentane-1,5-dicarboxylate in 6 ml of
anhydrous toluene was stirred overnight at 60.degree. C.
[0353] When the reaction finished, the reaction solution was
returned to ordinary temperature and spherical Amberyst resin was
removed by filtering through cotton. The solvent was removed in
vacuo.
[0354] The product was purified by a silica type chromatography
(eluent: hexane/ethyl acetate, 8:2 (v/v)). The present compound was
a white solid and was obtained in an amount of 84 mg (yield:
63%).
[0355] Thin-layer chromatography: R.sub.f=0.27 (hexane/ethyl
acetate, 7:3 (v/v))
[0356] RMN .sup.1H (300 MHz; CDCl.sub.3) d ppm: 2.1-2.5 (4H, m);
3.15 (1H, d, J=14.3 Hz); 3.38 (1H, d, J=14.3 Hz); 5.2 (2H, s),
7.2-7.38 (10H, m).
[0357] RMN .sup.13C (75 MHz; CDCl.sub.3) d ppm: 27.9; 30.1; 42.3;
67.7; 86.2; 127.4; 128.3; 128.5; 128.6; 128.7; 130.4; 171;
175.5.
[0358] IR (CsI) .nu.: 1780; 1744; 1457; 1432; 1269; 1174; 1082;
1040; 914 857; 758; 701; 604 cm.sup.-1.
[0359] SM (IC/NH.sub.3) m/z (relative intensity): 328=100%
(MNH.sub.4.sup.+); 329=34.
EXAMPLE 7
Manufacture of 2-benzyl-5-oxotetrahydrofuran-2-carboxylic Acid
[0360] 30
[0361] C.sub.12H12O4
[0362] Molecular weight=220.2
[0363] White crystals
[0364] T.sub.f=110-111.degree. C.
[0365] According to the manufacturing method for
2-(3-fluorobenzyl)-5-oxot- etrahydrofuran-2-carboxylic acid
(Example 4), the present compound in white solid was obtained in an
amount of 34 mg (yield: 90%) from benzyl
2-benzyl-5-oxotetrahydrofuran-2-carboylate represented by the
formula (5-4) manufactured in the above Example 6.
[0366] Thin-layer chromatography: R.sub.f=0.19 (ethyl
acetate/hexane/acetic acid, 6:4:0.2 (v/v/v)).
[0367] RMN .sup.1H (300 MHz; CDCl.sub.3) d ppm: 2.07-2.7 (6H, m);
3.16 (1H, d, J=14.3 Hz); 3.39 (1H, d, J=14.3 Hz); 7.19-7.35 (5H,
m); 7.88 (1H, sl).
[0368] RMN .sup.13C (75 MHz; CDCl.sub.3) d ppm: 27.9; 29.9; 42.2;
86.0; 127.5; 128.5; 130.5; 133.5; 175.5; 176.1.
[0369] IR (CsI) .nu.: 3032; 2938; 1782; 1712; 1185; 1082; 1044;
930; 746; 698 cm.sup.-1.
[0370] SM (IC/NH.sub.3) m/z (relative intensity): 238=100%
(MNH.sub.4.sup.+); 239=15.
EXAMPLE 8
Manufacture of Benzyl
2-chloro-5-oxotetrahydrofuran-2-carboxylate
[0371] 31
[0372] C.sub.12H.sub.11ClO.sub.4
[0373] Molecular weight=254.6
[0374] Yellow oil
[0375] To a solution of 7.5 g (31.7 mmol, 1 equivalent) of benzyl
2-oxohemiglutarate dissolved in 105 ml of ether at 0.degree. C.
were added 8.3 ml (98 mmol, 3 equivalents) of (COCl).sub.2 and then
DMF (400 .mu.l) was added as well. At that time, discharge of gas
was observed.
[0376] The mixture was stirred at 0.degree. C. for 1 hour and then
stirred at ordinary temperature for 2 hours. The temperature was
set at 0.degree. C. and the product was gradually neutralized with
an aqueous solution of K.sub.2CO.sub.3. The product was extracted
from ether (2.times.150 ml). The extracted organic phase was washed
with water. After that, the reaction solution was dried over
MgSO.sub.4 and the solvent was removed in vacuo. The residue was
added to a column filled with silica gel and purified by passing an
eluent of hexane/ethyl (7:3 (v/v)) through the column to give 8 g
of the present invention product as yellowish oil.
[0377] Thin-layer chromatography: R.sub.f=0.4 (hexane/ethyl
acetate, 7:3 (v/v))
[0378] RMN .sup.1H (200 MHz; CDCl.sub.3) d ppm: 2.48-2.89 (4H, m);
5.23 (2H, s); 7.25-7.35 (5H, m).
[0379] RMN .sup.13C (50 MHz; CDCl.sub.3) d ppm: 26.6; 35.9; 68.6;
96.4; 128.1; 128.6; 134.1; 164.6; 172.5.
[0380] IR (CsI) .nu.: 1817; 1762; 1271; 1166; 1088 cm.sup.-1.
[0381] SM (IC/NH.sub.3) m/z (relative intensity): 255=13%
(MH.sup.+) 272=100% (MNH.sub.4.sup.+); 273=15%.
EXAMPLE 9
Manufacture of Benzyl
2-fluoro-5-oxotetrahydrofuran-2-carboxylate
[0382] 32
[0383] C.sub.12H.sub.11FO.sub.4
[0384] Molecular weight=238.2
[0385] White crystals
[0386] T.sub.f=57-58.degree. C.
[0387] To a solution of 1 g (4.23 mmol, 1 equivalent) of benzyl
2-oxohemiglutarate dissolved in 10 ml of methylene chloride was
added a solution of 670 .mu.l (5 mmol, 1.2 equivalents) of
diethylaminotrifluorosulfonic acid (hereinafter abbrivated as DAST)
in 4 ml of methylene chloride at 0.degree. C. After mixing, the
reaction solution becomes dark red and was placed in a darkroom of
4.degree. C. for 48 hours. The product was adsorbed on silica gel
and separated by a silica type chromatography (eluent: hexane/ethyl
acetate, 8.5:1.5 (v/v)). The present compound was white crystalline
and obtained in an amount of 725 mg (yield: 72%).
[0388] Thin-layer chromatography: R.sub.f=0.3 (hexane/ethyl
acetate, 8:2 (v/v)).
[0389] RMN .sup.1H (300 MHz; CDCl.sub.3) d ppm: 2.48-2.91 (4H, m);
5.3 (1H, d, J=12 Hz); 5.39 (1H, d, J=12 Hz); 7.44 (5H, sl).
[0390] RMN .sup.13C (75 MHz; CDCl.sub.3) d ppm: 26.1; 30.6 (d,
.sup.2J.sub.CF=26 Hz); 68.8; 11.2 (d, .sup.1J.sub.CF=238 Hz);
128.6; 128.9; 129.0; 134.2; 164.3 (d, .sup.2J.sub.CF=36 Hz);
173.0.
[0391] IR (CsI) .nu.: 1815; 1764; 1339; 1313; 1199; 1177; 1103;
1058; 973; 908; 776; 751; 699 cm.sup.-1.
[0392] SM (IC/NH.sub.3) m/z (relative intensity): 256=100%
(MNH.sub.4.sup.+); 257=12%.
[0393] Incidentally, the compound represented by the formula (6-5)
33
[0394] was able to be manufactured according to the method of
Example 4 from benzyl 2-fluoro-5-oxotetrahydrofuran-2-carboxylate
manufactured in this Example.
EXAMPLE 10
Manufacture of Benzyl
2-cyano-5-oxotetrahydrofuran-2-carboxylate
[0395] 34
[0396] C.sub.12H.sub.11FO.sub.4
[0397] Molecular weight=238.2
[0398] White crystals
[0399] T.sub.f=57-58.degree. C.
[0400] To a solution where 200 mg (0.78 mmol, 1 equivalent) of
benzyl 2-chloro-5-oxotetrahydrofuran-2-carboxylate manufactured in
Example 8 was dissolved in 1.2 ml of tetrahydrofuran (THF) was
added at -78.degree. C. a solution of 209 mg (0.78 mmol, 1
equivalent) of cyanotetrabutylammonium dissolved in 1.5 ml of THF.
The reaction solution soon became red. This was stirred at
-78.degree. C. for 15 minutes. The reaction solution was returned
to ordinary temperature and gradually mixed with a mixture of 5 ml
of ether and 5 ml of water. After that, water was removed by ether
(2.times.15 ml) and an organic phase was collected. The organic
layer was washed with water and dried over Na.sub.2SO.sub.4. After
removal of the solvent, the residue was added to a column filled
with silica gel and purified by adding an eluent hexane/ethyl
acetate (7:3 (v/v)). The brown oil obtained at that time was
further purified. The present compound was a white solid and
obtained in an amount of 177 mg (yield: 93%)
[0401] Thin-layer chromatography: R.sub.f=0.3 (hexane/ethyl
acetate, 7:3 (v//v))
[0402] RMN .sup.1H (300 MHz; CDCl.sub.3) dppm: 2.64-2.85 (4H, m);
5.35 (2H, s); 7.4 (5H, ml).
[0403] RMN .sup.13C (75 MHz; CDCl.sub.3) dppm: 25.9; 32; 69.6;
74.6; 114.6; 128.3; 128.7; 129.0; 133.4; 163.8; 172.4.
[0404] IR (CsI) .nu.: 3035; 1815; 1764; 1498; 1456; 1417; 1379;
1271; 1158; 1072; 1048; 961; 895; 754; 698 cm.sup.-1.
[0405] SM (IC/NH.sub.3) m/z (relative intensity): 263=100%
(MNH.sub.4.sup.+); 264=12%; 280=24%.
EXAMPLE 12
Manufacture of Benzyl
1-hydroxy-3-oxo-1,3-dihydroisobenzofuran-1-carboxyla- te
[0406] 35
[0407] C.sub.16H.sub.12O.sub.5
[0408] Molecular weight=284.2
[0409] White solid
[0410] T.sub.f=77.degree. C.
[0411] To a solution of 500 mg (2.84 mmol, 1 equivalent) of
1,2-isochroman-1,3,4-trione dissolved in 6.5 ml of THF were added
292 .mu.l (2.84 mmol,1 equivalent) of benzyl alcohol and 230 .mu.l
(2.84 mmol, 1 equivaleent) of pyridine. The reaction solution which
was yellow at first became transparent after 15 minutes. The
reaction solution was stirred at ordinary temperature for 2
hours.
[0412] After that, the solvent was removed in vacuo. The product
was dissolved again in a mixture of chloroform and water and, after
that, an aqueous phase was extracted with chloroform (3.times.20
ml). The organic phase was washed with 5% by volume of hydrochloric
acid and with water. The organic phase was dried over
Na.sub.2SO.sub.4 and the solvent was removed in vacuo. The product
was purified by chromatography using silica gel (eluent:
hexane/ethyl acetate, 6:4 (v/v)). The present compound was a white
solid and obtained in an amount of 605 mg (yield: 75%).
[0413] Thin-layer chromatography: R.sub.f=0.23 (hexane/ethyl
acetate, 6:4 (v/v)
[0414] RMN .sup.1H (300 MHz; CDCl.sub.3) d ppm: 5.24 (2H, s);
7.15-7.33 (1H, d, J=7.5 Hz); 7.64-7.76 (2H, m); 7.9 (1H, d, J=6.7
Hz).
[0415] RMN .sup.13C (75 MHz; CDCl.sub.3) d ppm 68.9; 122.8; 125.7;
126.8; 127.7; 128.5; 128.6; 131.3; 133.9; 134.7; 144.6; 167.4;
167.8.
[0416] IR (CsI) .nu.: 3384; 1746; 1467; 1277; 1237; 1202; 1157;
1105; 1083; 907; 750; 694 cm.sup.-1.
[0417] SM (IC/NH.sub.3) m/z (relative intensity): 302=100%
(MNH.sub.4.sup.+) 303=22%.
EXAMPLE 13
Manufacture of Benzyl
1-fluoro-3-oxo-1,3-dihydro-isobenzofuran-1-carboxyla- te
[0418] 36
[0419] C.sub.6H.sub.11O.sub.4
[0420] Molecular weight=302.7
[0421] White solid
[0422] T.sub.f=78.degree. C.
[0423] To a solution where 300 mg (1.05 mmol, 1 equivalent) of
benzyl 1-hydroxy-3-oxo-1,3-dihydroisobenzofuran-1-carboxylate
represented by the formula (7-1) manufactured in the above Example
12 were dissolved in 2 ml of methylene chloride was added at
0.degree. C. a solution of 167 .mu.l (1.22 mmole, 1 equivalent) of
DAST dissolved in 1.5 ml of methylene chloride. After addition, the
reaction solution was stirred at 4.degree. C. for 20 hours. The
product was dissolved in methylene chloride, directly adsorbed on
silica gel and then purified by silica gel chromatography (eluent:
hexane/ethyl acetate, 8:2 (v/v)). The present compound was
colorless oil and obtained in an amount of 213 mg (yield: 71%).
[0424] Thin-layer chromatography: R.sub.f=0.3 (hexane/ethyl
acetate, 8:2 (v/v))
[0425] RMN .sup.1H (300 MHz; CDCl.sub.3) d ppm: 5.2 (1H, d, J=12
Hz); 5.3 (1H, d, J=12 Hz); 7.29-7.38 (5H, m); 7.68-7.81 (3H, m);
7.95-7.98 (1H, dd, J=6.7 Hz, J=1.8 Hz).
[0426] RMN .sup.13C (75 MHz; CDCl.sub.3) d ppm: 68.7; 107.2 (d,
.sup.1J.sub.CF=238 Hz); 123.4; 126.1; 132.6; 135.4; 125.4; 128.2;
128.6; 128.8; 142 (d, .sup.2J.sub.CF=20 Hz); 163.3 (d,
.sup.2J.sub.CF=38.9 Hz); 165.7.
[0427] IR (CsI) .nu.: 3035; 1811; 1766; 1605; 1498; 1468; 1456;
1380; 1341; 1296; 1256; 1200; 1163; 1129; 1107; 1083; 1046; 965;
905; 749; 688; 594 cm.sup.-1.
[0428] SM (IC/NH.sub.3) m/z (relative intensity): 320=100%
(MNH.sub.4.sup.+); 305=25%.
EXAMPLE 14
Manufacture of Benzyl
1-chloro-3-oxo-1,3-dihydro-isobenzofuran-1-carboxyla- te
[0429] 37
[0430] C.sub.16H.sub.11O.sub.4Cl
[0431] Molecular weight=302.7
[0432] White solid
[0433] T.sub.f=78.degree. C.
[0434] Into 0.5 ml of ether were dissolved 50 mg (0.17 mmol, 1
equivalent) of benzyl
1-hydrodxy-3-oxo-1,3-dihydro-isobenzofuran-1-carboxylate
represented by the formula (7-1) manufactured in the above Example
12. The solution was cooled at 0.degree. C. and 80 .mu.l (0.8 mmol,
5 equivalents) of (COCl).sub.2 and 3 drops of DMF were added
thereto. At that time, discharge of gas and precipitate of white
turbidity were observed. The reaction solution was returned to
ordinary temperature and stirred for 3 hours. After the reaction,
the product was dissolved in an aqueous solution of
K.sub.2CO.sub.3, and the aqueous phase was extracted with ether
(3.times.10 ml). The organic phase was washed with water and the
solution was dried over Na.sub.2SO.sub.4. The solvent was removed
in vacuo. There is no need to purify the present compound whereupon
51 mg of white solid were obtained.
[0435] Thin-layer chromatography: R.sub.f=0.4 (hexane/ethyl
acetate, 8:2 (v/v))
[0436] RMN .sup.1H (300 MHz; CDCl.sub.3) d ppm: 5.32 (2H, s);
7.35-7.38 (5H, m); 7.66-7.95 (4H, m).
[0437] RMN .sup.13C (75 MHz; CDCl.sub.3) d ppm: 69.2; 91.9; 124;
124.3; 125.8; 131.8; 135.5; 128.2; 128.7; 128.8; 134; 14.6.3;
163.8; 166.
[0438] IR (CsI) .nu.: 3034; 1804; 1764; 1603; 1468; 1456; 1288;
1237; 1188; 1114; 1042; 985; 960; 905; 746; 697 cm.sup.-1.
[0439] SM (IC/NH.sub.3) m/z (relative intensity) 286=100%; 320 =95%
(MNH.sub.4.sup.+); 321=22%
EXAMPLE 15
Manufacture of Benzyl
3-oxo-1,3-dihydroisobenzofuran-1-carboxylate
[0440] 38
[0441] C.sub.16H.sub.12O.sub.4
[0442] Molecular weight=268.2
[0443] White solid
[0444] T.sub.f=101.degree. C.
[0445] To a solution where 133 mg (0.43 mmol, 1 equivalent) of
benzyl 1-chloro-3-oxo-1,3-dihydroisobenzofuran-1-carboxylate
represented by the formula (7-3) manufactured in the above Example
14 were dissolved in 6 ml of benzene were gradually added 122 .mu.l
(0.46 mmol, 1.05 equivalents) of Bu.sub.3SnH and 4 mg (0.02 mmol,
0.05 equivalent) of .alpha.,.alpha.'-azoisobutyronitrile (AIBN).
The reaction solution was stirred at 70.degree. C. for 5 hours and
then stirred overnight at ordinary temperature. After the reaction,
the solvent was removed in vacuo. The product was placed in ether
and washed with a 10% by volume of KF solution. The organic phase
was dried over Na.sub.2SO.sub.4 and the solvent was removed in
vacuo. The residue was purified by a silica gel chromatography
(eluent: hexane/ether, 7:3 (v/v)). The present compound was a white
solid and obtained in an amount of 26 mg (yield: 23%).
[0446] Thin-layer chromatography: R.sub.f=0.32 (hexane/ether, 6:4
(v/v))
[0447] RMN .sup.1H (300 MHz; CDCl.sub.3) d ppm: 5.17 (1H, d, J=12
Hz) 5.23 (1H, d, J=12 Hz); 5.86 (1H, s); 7.3 (5H, m); 7.65-7.51
(3H, m); 7.83 (1H, d, J=7 Hz).
[0448] RMN .sup.13C (75 MHz; CDCl.sub.3) dppm: 68.0; 77.2; 122.7;
126.0; 130.2; 125.0; 128.4; 128.7; 128.8; 134.5; 143.9; 166.5;
169.3.
[0449] IR (CsI) .nu.: 3493; 3069; 2966; 1780; 1758; 1601; 1468;
1455; 1378; 1320; 1281; 1256; 1215; 1197; 1058; 1040; 949; 895;
755; 734; 697 cm.sup.-1.
[0450] SM (IC/NH.sub.3) m/z (relative intensity) 286=100%
(MNH.sub.4.sup.+); 287=227%, 554=17% (2x M+NH.sub.4.sup.+).
EXAMPLE 16
Manufacture of Benzyl
1-(4-fluorophenoxy)-3-oxo-1,3-dihydroisobenzofuran-1-
-carboxylate
[0451] 39
[0452] C.sub.22H.sub.15O.sub.5
[0453] Molecular weight=378.34
[0454] White solid
[0455] T.sub.f=75.degree. C.
[0456] A solution where 450 mg (1.5 mmol, 1 equivalent) of benzyl
1-hydroxy-3-oxo-1,3-dihydrobenzofuran-1-carboxylate represented by
the formula (7-1) manufactured in the above Example 12 and 532 mg
(4.75 mmol, 3 equivalents) of 4-fluorophenol were dissolved in 2 ml
of methylene chloride was heated at 65.degree. C. for 5 minutes.
After heating, it was gradually added drop-by-drop to a solution
where 355 mg (1.7 mmol, 1 equivalent) of
N,N-dicyclohexylcarbodiimide (DCC) were dissolved in 0.8 ml of
methylene chloride. After addition, the reaction solution was
heated to reflux for 2.5 hours and the reaction container was
returned to ordinary temperature followed by diluting with 10 ml of
methylene chloride. The precipitate was removed by filtration and
the solvent was removed in vacuo. The product was purified by
chromatography using silica gel (CH.sub.2Cl.sub.2/hexane, 6:4
(v/v)). The present compound was a white solid and obtained in an
amount of 227 mg (yield; 40%).
[0457] Thin-layer chromatography: R.sub.f=0.27
(CH.sub.2Cl.sub.2/hexane, 6:4 (v/v)).
[0458] RMN .sup.1H (300 MHz; CDCl.sub.3) d ppm: 5.1 (1H, d, J=12
Hz); 5.2 (1H, d, J=12 Hz); 6.85-7.17 (4H, m); 7.28-7.34 (5H, m);
7.64-7.92 (4H, m).
[0459] RMN .sup.13C (75 MHz; CDCl.sub.3) d ppm: 68.5; 103.2; 115.9
(d, .sup.2J.sub.CF=23 Hz); 121.5 (d, .sup.3J.sub.CF=8 Hz) 123.9;
125.8; 128.6; 131.9; 126.2; 128.2; 128.5; 134; 134.9; 143.6; 149.4;
159.4 (d, .sup.1J.sub.CF=242 Hz); 164.9; 166.9.
[0460] IR (CsI) .nu.: 1790; 1747; 1603; 1506; 1465; 1272; 1258;
1205; 1097; 1040; 996; 959; 830; 784; 741; 697 cm.sup.-1.
[0461] SM (IC/NH.sub.3) m/z (relative intensity): 396=100%
(MNH.sub.4.sup.+) 397=33%.
EXAMPLE 17
Manufacture of
1-(4-fluorophenoxy)-3-oxo-1,3-dihydro-isobenzofuran-1-carbo- xylic
Acid
[0462] 40
[0463] C.sub.15H.sub.9O.sub.5
[0464] Molecular weight=288.2
[0465] White solid
[0466] T.sub.f=143-147.degree. C.
[0467] To a solution where 40 mg (0.1 mmol, 1 equivalent) of benzyl
1-(4-fluorophenoxy)-3-oxo-1,3-dihydroisobenzofuran-1-carboxylate
represented by the formula (7-5) manufactured in the above Example
16 were dissolved in a mixture of 1.2 ml of ethyl acetate and 2
drops of ethanol was added a Pd/C catalyst in an amount of 10 parts
by weight to 100 parts by weight of the said solution. After the
reaction solution was degassed several times, it was subjected to a
catalytic reduction with hydrogen with stirring at ordinary
temperature for 1 hour. The catalyst was removed by filtering
through Celite (manufactured by Johns Manville Sales Co.). The
solvent was removed in vacuo. The compound was a white solid and
obtained in an amount of 28 mg.
[0468] Thin-layer chromatography: R.sub.f=0.16 (ethyl
acetate/methanol/acetic acid, 95:0.5:0.1 (v/v/v)).
[0469] RMN .sup.1H (300 MHz; CDCl.sub.3) d ppm: 6.97-7.19 (4H, m)
7.70-7.88 (4H, m).
[0470] RMN .sup.13C (75 MHz; CDCl.sub.3) d ppm: 105; 116.7 (d,
.sup.2J.sub.CF=23 Hz); 123.3 (d, .sup.3J.sub.CF=8.6 Hz); 125.3;
126.4; 133.1; 136.3; 127.5; 145.5; 151.2; 160.9 (d,
.sup.1J.sub.CF=239 Hz); 168.0; 168.2.
[0471] IR (CsI) .nu.: 3448; 1797; 1734; 1501; 1466; 1257; 1188;
1099; 1032; 960; 851; 783; 727 cm.sup.-1.
[0472] SM (IC/NH.sub.3) m/z (relative intensity): 306=100%
(MNH.sub.4.sup.+) 307=24% 594=23% (2x M+18).
EXAMPLE 18
Manufacture of the Compound of the Following Formula
[0473] 41
[0474] C.sub.9H.sub.6O.sub.5
[0475] Molecular weight=194.1
[0476] White solid
[0477] T.sub.f=130-140.degree. C.
[0478] Isochroman-1,3,4-trione (64 mg, 0.36 mmol, 1 equivalent) was
dissolved in 0.5 ml of water followed by stirring overnight at
ordinary temperature. After that, the reaction solution was frozen
and then freeze-dried. No purification was necessary and the
present compound was obtained white solid in an amount of 69
mg.
[0479] RMN .sup.1H (300 MHz; CDCl.sub.3) d ppm: 7.61-7.91 (4H,
m).
[0480] RMN .sup.13C (75 MHz; CDCl.sub.3) d ppm: 123.1 (sl); 125.1
(sl) 131.8; 134.6 (sl); 168.8 (sl).
[0481] IR (CsI) .nu.: 3494; 3067; 1777; 1745; 1467; 1386; 1285;
1232; 1197; 1163; 1104; 1079; 1001; 930; 769; 706 cm.sup.-1.
[0482] n SM (IC/NH.sub.3) m/z (relative intensity): 212=100%
(MNH.sub.4.sup.+); 229=85%.
EXAMPLE 19
Manufacture of (RS)-(+)-.alpha.-methyl-2-naphthalene-methyl
2-(4-fluorophenoxy)-5-oxotetrahydrofuran-2-carboxylate
[0483] 42
[0484] The above two compounds were manufactured by the same manner
as in Example 1.
[0485] The above .gamma.-lactone derivatives may be classified into
the following groups A-D when the conventional gamma-lactone
immunosuppressive compound (a racemate of ethyl
2-(4-fluorophenoxy)-5-oxo- -2-tetrahydrofurancarboxylate mentioned
in Japanese Patent Laid-Open No. 04/338,331) is taken as a lead
compound.
[0486] Group A: compounds where the conventional compound which is
racemic is separated into single compounds which are optical
isomers.
[0487] Group B: compounds where oxocyclic oxygen of the lead
compound is substituted with carbon which are more stable to an
enzymatic hydrolysis and show an activity similar to the lead
compound.
[0488] Group C: compounds where 4-fluorophenoxy group of the lead
compound is substituted with an unstable substituent such as
halogen, alkoxy group, benzoxy group, nitrile group, etc.
Incidentally, such compounds may also be grasped as prodrugs of
ketoglutaric acid having activity.
[0489] Group D: compounds where an aromatic ring is added to the
lead compound for stability of the compound and, in addition,
4-fluorophenoxy group or halogen is introduced for affinity to
lipid. Such compounds may also be grasped as a compound represented
by the formula (7-1) having activity and prodrugs thereof. 43
[0490] (1) Evaluation of effector macrophage suppressive action
showing a selective suppressive action to target cell damage in
vitro.
[0491] A suppressive effect of the test compound to effector
macrophage induction was investigated in vitro by an induction and
production system of spontaneous plaque-forming cell (SPEC)
mentioned in M. Ishibashi, S. Jiang, Y. Kokado, S. Takahara, T.
Sonoda: Immunopharmacologic effects of immunosuppressive agents
explored by a new effector generation assay. Transplant Proc., 21:
1854-1858, 1989.
TEST EXAMPLE 1
Step 1: Separation of Human PBMC
[0492] Heparin-added peripheral blood (40 ml) was collected from a
healthy person and the same amount of EDTA-added physiological
saline was added. PBMC were obtained by a specific gravity
centrifugal method using Ficoll-Hypaque (manufactured by Pharmacia
Fine Chemicals). Self-plasma was added thereto and allowed to stand
at 37.degree. C. for 10 minutes and platelets were removed by a
low-speed centrifugal separation. RPMI 1640 liquid containing 15
.mu.g/ml of gentamicin and 2 mM L-glutamine was used for washing
the PBMC. Concentration of the PBMC was adjusted to
2.times.10.sup.6 cells/ml using the same liquid.
Step 2: Addition of an Agonist and a Substance to be Tested
[0493] Each 200 .mu.l of a solution containing 2.times.10.sup.6
PBMC/ml prepared in the step 1 were added to each well of a micro
test plate, lipopolysaccharide (hereinafter, abbreviated as LPS)
was diluted with the RPMI 1640 liquid to make the final
concentration 80 .mu.g/ml and the said LPS solution was added to
each well of the 200 .mu.l-micro test plate. Further, each 50 .mu.l
of human serum of type AB were added so as to make the
concentration 10% by weight. Two such micro test plates were
prepared.
[0494] After that, to the above two micro test plates was added a
solution where the test compound was diluted with 1% by weight of
DMSO so as to make the concentration of the test compound 1 .mu.M.
Similarly were prepared each two micro test plates where the final
concentrations of the test compound were 0.1 .mu.M, 0.01 .mu.M and
0.001 .mu.M.
[0495] The culture liquid prepared as such was incubated for 6 days
at 37.degree. C. in 5% CO.sub.2.
Step 3: Recovery of the Incubated PBMC
[0496] The effector macrophage induced after the incubation was all
recovered using a rubber-policeman (spatula made of rubber). A
Hanks solution containing 5 .mu.g/ml of gentamicin was used for
washing and, using the said solution, there was prepared a solution
where the concentration of the induced effector macrophage was
2.times.10.sup.6 per ml.
Step 4: Preparation of Monolayered Autologous Erythrocyte-Adhered
Plate
[0497] With regard to autologous erythrocytes, those which were
preserved at 4.degree. C. in a phosphate physiological saline
(hereinafter, abbreviated as PBS) to which 0.1% by weight of AB
serum and autologous erythrocytes obtained from PBMC separation
were used were used. The preserved autologous erythrocytes were
washed for three times with a Hanks solution to which no serum was
added and made into a solution of 4% by weight concentration using
the said Hanks solution. Poly-L-lysine was added to a Terasaki
plate and washed with PBS every 20 minutes at 37.degree. C., the
above-mentioned autologous erythrocyte solution was added thereto
immediately, the mixture was allowed to stand at 37.degree. C. for
30 minutes and the erythrocytes which were not adhered to the plate
were removed to prepare a Terasaki plate to which self-erythrocytes
in a monolayer were adhered.
[0498] A Hanks solution (4 .mu.l) was added to the said Terasaki
plate, 4 .mu.l of a solution containing the induced effector
macrophage obtained in the step 3 were added thereto and the
mixture was allowed to stand at 37.degree. C. for 2 hours. After
completion of the reaction, it was fixed by formalin. Numbers of
the produced SPFC which were adhered or phagocytized were
measured.
Step 5: Calculation of Total Amount of SPFC Produced
[0499] A mean SPFC production number was calculated from the
measured values of SPFC production when the concentration of the
test compound was the same. SPFC production number per
1.times.10.sup.6 of the induced effector macrophage was calculated
and, from the recovered induced effector macrophage numbers, total
SPFC production amount (S2) was calculated.
Step 6: Judgment of Positive Compound Having the Activity
[0500] As a control, no test compound was added and the same
operation as above was carried out to calculate the total SPFC
production amount (S1). The concentration of the test compound in
which the total SPFC production amount when test compound was added
(S2) was one half of that when no test compound was added (SI) was
defined as IC.sub.50. The test compound when its IC.sub.50 was 1
.mu.M or less was judged to be positive.
TEST EXAMPLE 2
[0501] The same operation as in Test Example 1 was carried out
except that, in the step 2, mitomycin-treated human PBMC (allogenic
MLC; hereinafter, abbreviated as allo-MLC) was added to each well
of the micro test plate so as to make its concentration
2.times.10.sup.6 per ml.
[0502] Here, the allo-MLC used was prepared in such a manner that
mitomycin was added to PBMC which was prepared by the same manner
as in the step 1 of Test Example 1 so as to make the concentration
of mitomycin 40 .mu.g/ml followed by allowing to stand at
37.degree. C. for 30 minutes.
[0503] The result is shown in the following Table. The term
"structure characteristic" in the Table means the above-mentioned
groups A-D. With regard to a functional classification of the
compound, the compound which selectively suppresses the SPFC
production under the conditions of allo-MLC stimulation or, in
other words, the compound which selectively suppresses the
induction of effector macrophage in the presence of allo-MLC is
classified as group I while the compound which selectively
suppresses the SPFC production under the conditions of LPS
stimulation or, in other words, the compound which selectively
suppresses the induction of effector macrophage in the presence of
LPS is classified as group II. Further, the compound which
selectively suppresses the SPFC production under the conditions of
both allo-MLC stimulation and LPS stimulation or, in other words,
the compound which non-selectively suppresses the induction of
effector macrophage is classified as group III. Each of the
"compound" in the Table is the compound bearing the above-mentioned
number.
1TABLE 1 SPFC Production SPFC Production Inhibiting Inhibiting
Concentration Structure Concentration by all-MLC by LPS Stimulation
Functional Characteristic Compound Stimulation (.mu.M: IC.sub.50)
(.mu.M: IC.sub.50) Classification A (1) 0.01 >1.0 Group I (2)
>1.0 1 Group II (3-2) >1.0 1 Group II (4-2) 0.01 >1.0
Group I B (5-2) 0.1 0.01 Group II (5-4) 0.01 1 Group I C (6-1)
>1.0 0.001 Group II (6-2) not done 0.001 Group II (6-3) 0.1
0.001 Group II D (7-1) 0.1 1 Group I (7-2) >1.0 0.1 Group II
(7-3) 0.01 >1.0 Group I (7-5) 0.01 >1.0 Group I (7-6) >1.0
0.01 Group II (7-7) >1.0 0.01 Group II Metabolite (9-1) 0.01
>1.0 Group I Conventional Compound 0.1 0.1 Group III
[0504] In the Table, "Conventional Compound" is a racemic ethyl
2-(4-fluorophenoxy)-5-oxo-2-tetrahydrofurancarboxylate.
[0505] The optical isomer according to the present invention
selectively suppressed the SPFC production by allo-MLC stimulation
and that by LPS stimulation. The correlation in the biological
activity depending upon the structure as such was confirmed in the
two types of optical isomers of "(1) and (2)" and "(3-2) and
(4-2)".
[0506] With regard to the compounds (group I) where SPFC production
by allo-MLC stimulation is selectively suppressed, i.e., induction
of effector macrophage in the presence of allo-MLC is selectively
suppressed, the compounds represented by the formulae (1), (4-2),
(5-4), (7-1), (7-3) and (7-5) correspondeded thereto. With regard
to the compounds (group II) where SPFC production by LPS
stimulation is selectively suppressed, i.e., induction of effector
macrophage in the presence of LPS is selectively suppressed, the
compounds represented by the formulae (2), (3-2), (5-2), (6-1),
(6-2), (6-3), (7-2), (7-6) and (7-7) corresponded thereto. There
were some compounds where the SPFC production suppressive activity
was from 10-fold to 100-fold as compared with the conventional
compound.
[0507] (2) Evaluation of immunosuppressive action showing a
selective suppressive effect on target cell damage in animal
experiments.
TEST EXAMPLE 3
[0508] Obstruction Release Model after Complete Obstruction of
Unilateral Ureter for 14 Days
[0509] Experimental models were prepared by a method devised and
established by Ishibashi (Michio Ishibashi, et al.: The Japanese
Journal of Nephrology, 42: 248, 2000) using male SD rats of 8-9
weeks age and about 280 g. Thus, the rat was laparotomized under
anesthetization with ether and ureter was ligated with 7-0 Nylon at
the height of the margin of lower pole of left kidney to close the
abdomen. On the 14th day after obstruction, the obstruction was
released and urinary passage was reconstructed using a cuff. Thus,
after 14 days, the part of ligated obstructed ureter was resected,
a polyethylene tube of 25 gages (manufactured by Nippon Sherwood)
was used as a cuff and inserted into and retained at the lumen from
the cut end of the lower normal ureter, then the cuff was also
retained in the expanded upper ureter and each of them was ligated
and fixed by 7-0 Nylon to reconstruct the urinary passage. At the
same time, the right kidney at the opposite side was excised. After
the release of the obstruction, body weight was measured, blood was
collected on the 2nd day, 5th day and 7th day from the release to
measure serum creatinine, then the rat was sacrificed under
anesthetization and the left obstruction-released kidney was
excised. With regard to the excised kidney, there were carried out
measurement of weight of the kidney and pathological test for the
kidney. When the compound of the present invention was not
administered to the model, destruction of renal structure was
observed in pathological and morphological investigations during
the obstructed period and after release of the obstruction with a
lapse of time causing thickening of glomerular Bowman's capsule
wall, hyperplasia of mesangial cells, glomerular sclerosis,
involution or dilation of urinary tubule, cellular infiltration to
interstitial tissues and fibrosis. Cellular infiltration did not
increase the CD5-positive T cells and CD11b/CD18 (ED8)-positive
macrophage became dominant on the tenth day.
[0510] An in vivo biological test was carried out for the
.gamma.-lactone derivatives according to the present invention
using the above models. With regard to the test compound, bulk
powder of the test compound was dissolved in an aseptic
physiological saline together with gum arabic and the final
concentration of gum arabic was 5%(vol/vol). The concentration of
the test compound 30 mg/ml was suspended in 5% gum arabic-saline
solution. The preparation was subcutaneously injected at the dose
of 30 mg/kg every day throughout the experiment. With regard to the
test compound, there were used (9-1), (4-2) and (7-3) as the
compounds of group I suppressing the SPFC production by allo-MLC
stimulation, i.e., the compounds which selectively suppress the
induction of effector macrophage in the presence of allo-MLC while
there was used (3-2) as the compound of group II suppressing the
SPFC production by LPS stimulation, i.e., the compounds which
selectively suppress the induction of effector macrophage in the
presence of LPS. Further, with regard to the compound of group III
which suppresses the SPFC production by both allo-MLC stimulation
and LPS stimulation, i.e., the compound which non-selectively
suppresses the induction of effector macrophage, there was used a
racemate of ethyl
2-(4-flyuorophenoxy)-5-oxo-2-tetrahydrofurancarboxylate which is
the known compound.
[0511] The result is given in the following Table. In the Table,
"functional classification" of the compound means the above group
I, group II and group III. "Compound" means the compound bearing
each of the above-mentioned compound numbers. The fact whether
glomerular lesion was suppressed was judged by the ameriolation of
the lesions such as thickness of wall of Bowman's capsule,
hyperplasia of mesangial cells and glomerular sclerosis. The fact
whether lesion of tubulointerstitial tissue was suppressed was
judged by the ameriolation of the lesions such as thickness of
basement membrane of tubules, cellular infiltration and fibrosis of
interstitial tissue. In the table, "control" is the result when the
same test as above was carried out using 5% gum arabic only which
is a solvent.
2TABLE 2 Serum Creatinine Rate of Weight of level (mg/dl) on Cases
where Rate of Cases Kidney (g) on the 7th Day from Glomerular where
tubulointerstitial the 7th Day Functional Case Release of Lesion
was Lesion was from Release Classification Compound Numbers
Obstruction Suppressed Suppressed of Obstruction Group I (9-1) n =
3 2.0 .+-. 0.2 0% (0/4) 100% (4/4) 3.01 .+-. 0.56 Control n = 3 2.0
.+-. 0.2 0% (0/3) 33% (1/3) 2.62 .+-. 0.70 (4-2) n = 4 2.7 .+-. 0.3
50% (2/4) 100% (4/4) 3.94 .+-. 0.32 Control n = 4 2.7 .+-. 0.9 0%
(0/4) 0% (0/4) 4.68 .+-. 0.66 (7-3) n = 4 2.0 .+-. 0.6 25% (1/4)
100% (4/4) 2.72 .+-. 0.47 Control n = 4 2.7 .+-. 0.9 25% (1/4) 25%
(1/4) 2.69 .+-. 0.40 Group (3-2) n = 5 2.0 .+-. 0.4 100% (5/5) 0%
(5/5) 4.36 .+-. 0.95 II Control n = 3 2.6 .+-. 0.9 33% (1/3) 0%
(0/3) 5.61 .+-. 1.08 Group Known n = 4 2.2 .+-. 0.2 75% (3/4) 75%
(3/4) 2.73 .+-. 0.41 III Compd Control n = 4 3.1 .+-. 0.8 0% (0/4)
0% (0/4) 2.21 .+-. 0.50
[0512] Both (9-1), (4-2) and (7-3) as the compounds of the group I
which suppress the SPFC production by allo-MLC stimulation, i.e.,
the compounds which selectively suppress the induction of effector
macrophage in the presence of allo-MLC and (3-2) as the compound of
the group I which suppresses the SPFC production by LPS
stimulation, i.e., the compound which selectively suppresses the
induction of effector macrophage in the presence of SPFC showed a
selective suppressive effect. Thus, the group I dominantly
suppressed the lesion of tubulointerstitial tissue. The (3-2) of
the group II did not suppress the lesion of tubulointerstitial
tissue but suppressed the glomerular lesion only. The conventional
compound belongs to the group III and, although it showed
suppression, no selective suppression was observed.
[0513] In order to further confirm whether the preventive and
therapeutic effect of the .gamma.-lactone derivatives of the
present invention to the target cell damage is selective, (7-3) of
the group I and (6-1) of the group II were used as test compounds
and they were subcutaneously injected at the dose of 30 mg/kg every
day and compared with the control group. With regard to the control
group, only 5% gum arabic which is a solvent was used and the same
test as above was carried out.
[0514] The result is shown in the following Table wherefrom it is
apparent that, in all cases combined with group I and group II
compounds, lesion of glomerulus and of tubulointerstitial tissue
were suppressed.
3TABLE 3 Creatinine Rate of Rate of Cases Weight of level (mg/dl)
Cases where where Lesion of Kidney (g) on Combination on 7th Day
Glomerular Tubulointerstitial 7th Day from of Case from Release
Lesion was Tissue was Release of compounds Numbers of Obstruction
Suppressed Suppressed Obstruction Combined n = 3 2.1 .+-. 0.3 100%
(3/3) 100% (3/3) 2.27 .+-. 0.16 use of (7-3) and (6-1) Control n =
5 2.5 .+-. 0.7 0% (0/5) 20% (1/5) 2.55 .+-. 0.45
TEST EXAMPLE 4
[0515] Evaluation Using Puromycin Chronic Nephrosis Model in
Rat
[0516] Male SD rat of 8 weeks age of about 250 g was used and 50
.mu.g/kg of puromycin were intravenously administered once
according to a method of Diamond, et al. (J. R. Diamond, I. Pesek,
S. Ruggeri, M. J. Karnovsky: Essential fatty acid deficiency during
acute puromycin nephrosis ameliorates late renal injury. Am. J.
Physiol, 257: F798.about.F807, 1989). There was induced a chronic
puromycin nephrosis rat where albuminuria increased from about
tenth to twelfth week. After eighth week from administration of
puromycin, each of compound (9-1) and compound (6-1) was
subcutaneously injected at the dose of 30 mg/kg/day everyday until
the 22nd week. Incidentally, as shown in the above Table 1, (9-1)
is a compound which suppresses the SPFC production by allo-MLC
stimulation or, in other words, a compound which selectively
suppresses the induction of effector macrophage in the presence of
allo-MLC (group I) while (6-1) is a compound which suppresses the
SPFC production by LPS stimulation or, in other words, a compound
which selectively suppresses the induction of effector macrophage
in the presence of LPS (group II).
[0517] As a functional evaluation of kidney, albumin in urine was
measured every week using a kit for the measurement of albumin. On
the 22nd week, the rat was killed and anatomized to observe
functional and morphological changes of kidney.
[0518] The result is shown in the following Table. The compound
(1-1) (group I) well suppressed the lesion of tubulointerstitial
tissues while the compound (2-1) (group II) well suppressed the
glomerular lesion. Functional average daily albuminuria was
proportional to the morphological change.
[0519] Accordingly, it was found that the compounds of the group I
which suppressed the induction of effector macrophage by allo-MLC
stimulation selectively suppressed the lesion of tubulointerstitial
tissues while the compounds of the group II which suppressed the
induction of effector macrophage by LPS stimulation selectively
suppressed the glomerular lesion.
4TABLE 4 Rate of Cases where Glomerular Rate of Cases where
Functioal Case Albumin in Urine Lesion was Lesion of
Tubulointerstitial Classification Compound Numbers (mg/day)
Suppressed Tissue was Suppressed Group I (9-1) n = 6 20, 7, 5, 5,
5, 4 50% (3/6) 83% (5/6) Group II (6-1) n = 6 56, 20, 11, 7, 4, 3
67% (4/6) 17% (1/6)
[0520] Correlation between the structural characteristic of
immunosuppressive .gamma.-lactone derivative and biological
activities in vitro and in vivo is given.
5 TABLE 5 Activity in vitro Activity in vivo Inhibiting
Concentration Inhibiting Concentration Suppressive Suppressive
Effect Structure- for SPEC Production by for SPEC Production by
Effect to to Lesion of Characteristic allo-MLC Stimulation LPS
Stimulation Glomerular Tubulointerstitial Experimental of compound
Compound (.mu.M: IC.sub.50) (.mu.M: IC.sub.50) Lesion Tissue Model
of Rat A (3-2) >1.0 1 .smallcircle. x UUO/Release (4-2) 0.01
>1.0 .DELTA. .smallcircle. UUO/Release C (6-1) >1.0 0.001
.smallcircle. x CPN (6-2) not done 0.001 .smallcircle. x
UUO/Release D (7-3) 0.01 0.01 x .smallcircle. UUO/Release
Metabolite (9) 0.01 >1.0 x .smallcircle. UUO/Release
Conventional Compound 0.1 0.1 .smallcircle. .smallcircle.
UUO/Release and CPN .smallcircle.: available .DELTA.: somewhat
available x: not available UUO/Release: ureter was obstructed for
14 days followed by releasing CPN: chronic PAN nephrosis
[0521] As such, the .gamma.-lactone derivatives in accordance with
the present invention are able to selectively suppress the
induction of effector macrophage by allo-MLC stimulation and the
induction of effector macrophage by LPS stimulation. It has been
also proved that, as a result thereof, a suppressive effect
depending upon the lesion is able to be achieved.
[0522] Accordingly, pharmaceutical preparations containing the
.gamma.-lactone derivatives according to the present invention can
be applied depending upon the lesions. When, for example, the
progressive lesion after the renal damage comes to glomerulus in
the case of renal diseases, administration of the pharmaceutical
containing the compound of the group II is effective. On the other
hand, when the progressive lesion after the renal damage comes to
tubulointerstitial tissue, administration of the pharmaceutical
containing the compound of the group I is effective. When the
progressive lesion after the renal damage comes to both glomerulus
and tubulointerstitial tissue, combination use of the
pharmaceutical containing the compounds of the above-mentioned
groups I and II is effective. When the progressive lesion after
pancreatic damage comes to islets of Langerhans in the case of the
pancreatic disease, administration of the pharmaceutical containing
the compound of the group II is effective while, when the
progressive lesion after pancreatic damage comes to exocrine,
acinar and ductal interstitial tissue of pancreas, administration
of the pharmaceutical containing the compound of the group I is
effective. When the progressive lesion after the renal damage comes
to both islets of Langerhans and exocrine, acinar and ductal
interstitial tissue of pancreas, combination use of the
pharmaceutical containing the compounds of the above-mentioned
groups I and II is effective.
[0523] As mentioned above, the .gamma.-lactone derivatives in
accordance with the present invention achieve a selective
suppressive action unlike the conventional compounds. As a result
of such an action, the .gamma.-lactone derivatives in accordance
with the present invention only suppress the activation of
induction of effector macrophage acting in a cytotoxic manner to
damaged organ tissue cells and do not suppress the activation of
induction of macrophage participating in the regeneration of
tissues and, therefore, they are able to more effectively prevent
or cure the progressive lesion after the organic damage without
lowering the defensive ability of organism.
[0524] It has been also found that the compound (9-1), i.e. ethyl
2-ketoglutarate, is one of the active metabolite derived from the
compounds of the group I which selectively suppress the SPFC
production by allo-MLC stimulation. Such an active metabolite shows
an activity for suppressing the lesion of in tubulointerstitial
tissue in vivo. Similarly, the compound (9-2), i.e. benzyl
2-ketoglutarate, is one of the active metabolite of the group II
which selectively suppresses the SPFC production by LPS stimulation
and is able to suppress the lesion of glomerulus in vivo.
TEST EXAMPLE 5
[0525] Acute and Chronic Toxicity Tests
[0526] Toxicity test of benzyl
2-chloro-5-oxotetrahydrofuran-2-carboxylate represented by the
formula (6-1) manufactured in Example 8 and ethyl 2-ketoglutarate
represented by the formula (9-1) was carried out. To be more
specific, the former and the latter were subcutaneously injected at
the doses of 30 mg/kg/day and 90 mg/kg/day, respectively for ten
days and their toxicity was investigated whereupon there was only
noted an increase in weights of the liver and the spleen in a light
degree.
[0527] When both were administered at the dose of 30 mg/kg/day for
consecutive 14 days, neither reduction of body weight nor death was
noted and there was no abnormality at the subcutaneously injected
area whereupon they were found to be of low toxicity.
PREPARATION EXAMPLE 1
[0528]
6 (Tablets) (1) Benzyl 2-chloro-5-oxotetrahydrofuran-2- 10 g
carboxylate (2) Lactose 90 g (3) Corn starch 29 g (4) Magnesium
stearate 1 g 130 g
[0529] The components (1) and (2) and 24 g of the component (3)
were mixed with water to granulate, the resulting granules were
mixed with 5 g of the component (3) and the component (4), and the
mixture was compressed using a compressive tabletting machine to
manufacture 1,000 tablets of 7 mm diameter containing 10 mg of the
component (1) per tablet.
PREPARATION EXAMPLE 2
[0530]
7 (Capsules) (1) Benzyl 2-chloro-5-oxotetrahydrofuran-2- 50 mg
carboxylate (2) Lactose 14 mg (3) Corn starch 29 mg (4)
Hydroxypropyl cellulose 6 mg (5) Magnesium stearate 1 mg 100 mg per
capsule
[0531] The above-mentioned components (1), (2), (3) and (4) were
mixed and granulated according to a conventional method. The
component (5) was added thereto and placed into a gelatin capsule
by a conventional method to give a capsule preparation.
Industrial Applicability
[0532] The present invention is able to provide a method for the
induction of effector macrophage which is induced and activated
corresponding to the lesion inherent to the tissues after organic
lesion and results in a progressive lesion after the organic
damage.
[0533] By utilizing the said method, the present invention is able
to provide a method for screening compounds which are able to
prevent, mitigate or cure the progressive lesion after organic
damage such as glomerular lesion and tubulointerstitial lesion in
the case of kidney and, in the case of pancreas, exocrine
interstitial lesion or pancreatitis and Langerhans islet lesion or
diabetes. The present invention also provides a method for
screening compounds which are able to prevent, mitigate or cure
diabetes and diabetic retinitis or pancreatitis and
tubulointerstitial lesion complicated with pancreatitis at the same
time.
[0534] It is also possible to provide a pharmaceutical for the
prevention or the therapy of the above-mentioned progressive lesion
after the organic damage or a therapeutic method therefor by
utilizing the said screening method. The said pharmaceutical or the
said therapeutic method has little side effect such as an
unnecessary lowering of defense of organism or induction of new
tissue damage and, in addition, it achieves repair and regeneration
of the tissues without unnecessary lowering of the defense of
organism whereby it is now possible to conduct the therapy for a
long period on a continuous basis using the said pharmaceutical or
therapeutic method.
[0535] The novel .gamma.-lactone derivatives according to the
present invention are compounds which are able to selectively
suppress the induction of effector macrophage corresponding to the
lesion inherent to the tissues after the organic damage and are
able to be used as the above-mentioned pharmaceutical.
[0536] The novel .gamma.-lactone derivatives according to the
present invention have a selective induction-suppressive action for
the above effector macrophage and, as a result, they show a
selective immunosuppressive action or a fibrosis inhibiting action
to specific tissues. Accordingly, the novel .gamma.-lactone
derivatives according to the present invention are able to be used
not only as the above-mentioned pharmaceutical but also as an
immunosuppressant or a fibrosis inhibitor. To be more specific, the
pharmaceutical containing the novel .gamma.-lactone derivative of
the present invention is able to selectively suppress the progress
or worsening of the diseases to the target organ or the like and,
since its immunosuppressive action or fibrosis inhibiting action is
strong, it is effective for therapy and/or prevention of rejection
reaction upon of xenogenic or allogeneic cell, tissue or organ
transplantation and endotoxin shock reaction by bacterial toxin,
systemic intravascular coagulation, various inflammatory diseases,
chronic inflammatory diseases and cancer. To be more specific, the
pharmaceutical containing the novel .gamma.-lactone derivative
according to the present invention is able to be used for
prevention of onset or progress of rejection reaction upon
transplantation of allogeneic or xenogenic organs, cells or
tissues, or acute or chronic glomerular nephritis and onset or
progress of interstitial retinitis or diabetes; therapy and/or
prevention of complications such as diabetic nephropathy, diabetic
retinopathy and diabetic neuropathy; therapy and/or prevention of
chronic pancreatitis, arteriosclerosis, arteriosclerotic
restenosis, pulmonary fibrosis, dialytic amyloidosis, chronic
hepatitis, cerebrospinal degeneration, asthma, rheumatic arthritis,
chronic pigmentary skin diseases, psoriasis, autoimmune chronic
organic tissue damage, endotoxin shock reaction by cytotoxin,
systemic intravascular coagulation or cancer or its metastasis; and
prevention and therapy of infection of AIDS virus. It is also able
to be used as a substitute for steroidal therapeutic agent.
* * * * *