JPH08104670A - Delta-(s-methylisothioureido)-l-norvaline and cerebrovascular lesion therapeutic agent having nitrogen monoxide synthase-inhibitory action - Google Patents

Abstract

PURPOSE: To obtain the subject new compound excellent in the inhibitory action on a nitrogen monoxide synthase found in intracerebral neurocytes and in the selectivity of the enzyme, and useful as a therapeutic agent for cerebrovascular lesions such as cerebral infarction, cerebral hemorrhage, subarachnoid hemorrhage and cerebral edema. CONSTITUTION: This compound (its salt) is expressed by formula I, and is obtained by either hydrolysis or deprotection through direct treatment with a deprotecting agent (e.g. trifluoroacetic acid, hydrochloric acid) of a compound of formula II [R1 is a (substituted) lower alkoxycarbonyl or (substituted) lower acyl; R2 is a (substituted) l-6C (branched) alkyl] [e.g. α-N-tbutoxycarbonyl-β-(S- methylisothioureido)-L-norvaline-t-butyl ester]. It is preferable that the deprotection is conducted at room temperature in the absence of water by using trifluoroacetic acid.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to amino acid derivatives, and more particularly to nitric oxide synthase.
de synthase (NOS) inhibitory effect and suppresses nitric oxide (nitride oxide, NO) production, thereby contributing to pathological conditions associated with cerebrovascular disorders in which excess NO or NO metabolites are considered to be involved. The present invention relates to a useful δ- (S-methylisothioureido) -L-norvaline or a pharmaceutically acceptable salt thereof and a prophylactic and therapeutic agent comprising these as active ingredients.

[0002]

2. Description of the Related Art Recently, NO is one of the relaxation factors derived from vascular endothelium (Na
Nature, 327, 524-526, 1987; Nat
ure, 333, 664-666, 1988), vasorelaxant, platelet aggregation / adhesion inhibitory action, and it has been clarified that it is involved in the maintenance of biological functions as an information transmitter between cells (TrendsPharmaco).
l. 10, 428-431, 1989; Trends
Neurosci. 14, 29-39, 1987; Tr
ends Biochem. 16, 81-83, 199
1; Trends Neurosci. 13, 1-6,
192; Trends Pharmacol. 12, 1
30-131, 1991; Tends Pharmac.
ol. 12, 87-88, 1991; Trends P.
armacol. 12,125-128,199
1).

Further, NO is not limited to vascular endothelial cells,
It has been clarified that it is also produced by nerve cells, glial cells, macrophages, etc., and the gene encoding each NOS has been isolated (Nature, 351, 714).
-718, 1991; Proc. Natl Acad.
Sci. USA, 89, 11141-11145, 19
92; Proc. Natl. Acad. Sci. US
A, 89, 6348-6352, 1992; FEBS.
Lett. J., 307, 287-293, 1992;
Clin. Inv. , 90,2092-2096,19
92; Science, 256, 225-228, 19
92; Biol. Chem. , 267, 6370-
6374, 1992;

Proc. Natl. Acad. Sci.
USA, 90, 11419-11423, 1993; B.
iochem. , 32, 11600-11605, 19
93; Proc. Natl. Acad. Sci. US
A, 90, 3491-3495, 1993; FEBS
Lett. , 316, 175-180, 1993; Bi
ochem. Biophys. Res. Commu
n. , 191, 89-94, 1993; Proc. Na
tl. Acad. Sci. USA, 89, 6711-6.
715, 1992; Biochem. Biophys.
Res. Commun. , 191,767-774,1
993; Proc. Natl. Acad. Sci. US
A, 90, 9730-9734, 1993).

Furthermore, the pathophysiological role of NO is rapidly elucidated (Pharmacol. Rev.,
43, 109-142, 1991; Neurol. Pr
og. , 32, 197-311, 1992; FASEB.
J. , 6,3051-3064, 1992).

[0006] and is one of the NOS inhibitor ^{N G} -
Nitro-L-arginine (L-NNA)
It was found to have an improving effect on ischemic cerebral infarction and ischemic cerebral edema (Eur. J. Pharmaco.
l. 204, 339-340, 1991; Neuros.
ci. Lett. , 147, 159-162, 199
From the above, 2) has opened the way for the application of substances having NOS inhibitory activity to therapeutic agents for cerebrovascular disorders.

In an area where the cerebral blood flow is interrupted, cellular cerebral edema occurs first, followed by vascular cerebral edema. Vascular cerebral edema appears several hours after cerebral ischemia and continues to progress until one week after the onset. After that, cerebral edema gradually decreases, and is fixed as an infarct lesion within 1 to 3 months depending on the extent of the infarct lesion. Brain edema causes an increase in brain capacity because the brain is covered by a hard skull. When cerebral edema exceeds a certain level, it causes a rapid increase in tissue pressure and intracranial pressure, often leading to fatal hernia,
As a result, brain damage is exacerbated and the extent of subsequent infarct lesions is determined (J. Neurosurg. 77, 16).
9-184, 1992). As described above, the treatment of cerebral edema, which affects the prognosis of a patient, is a clinically extremely important issue. At present, the main treatment methods for cerebral edema are hypertonic fluids, cerebrospinal fluid drainage, and the use of hypertonic fluids and steroids, but these effects are almost transient, and Has no great expectation for effective therapeutic effect (J. Neurosurg. 77, 337-3).
54, 1992). Therefore, it is desired to develop a drug having a completely new mechanism of action different from the conventional one as a therapeutic method for ischemic cerebrovascular disorder.

The present inventors have found that L-NNA causes cerebral edema and cerebral infarction (Neurosci. Let) which occur after cerebral ischemia.
t. , 147, 159-162, 1992), neuronal necrosis (Eur. J. Pharmacol. Env. To.
x. , 248, 325-328, 1993). However, it has been reported that relatively high doses of NOS inhibitors are not only ineffective but also exacerbated in some cases against ischemic brain injury (J. Cereb. BloodFlow).
Metab. , 14, 175-192, 1994).

The theory that there are at least three types of isoforms of NOS is currently dominant from DNA analysis. That is, neu which is constitutively present in nerve cells
local constitutive NOS (N-
cNOS), end which is constitutively present in vascular endothelial cells
othelly constitutive NOS
(E-cNOS), and inducible NOS (iNOS) that is induced and synthesized by cytokines and lipopolysaccharides in macrophages and many other cells
Is. Among them, N-cNOS and E-cNOS are calcium-dependent, and iNOS is calcium-independent (FASEB J. 16, 3051-3064).
1992).

As a possible mechanism of brain tissue damage associated with cerebral ischemia, elevation of extracellular glutamate concentration, excessive activation of glutamate receptors existing at postsynaptic sites, elevation of intracellular calcium concentration, calcium-dependent enzyme A series of pathways has been proposed for the abnormal activation of J. Cere (J. Cere
b. Blood Flow Metab. 1,155-
185, 1981; Neurosurg. 60,8
83-908, 1984; Trends Neuros.
ci. 11, 465-469, 1988; Cere
b. Blood Flow Metab. 9,127-
140, 1989). As described above, since N-cNOS is calcium-dependent, it is considered that inhibition of abnormal activation of this type of NOS isoform exerts the neuroprotective action of NOS inhibitors. (Annals Neurol. 32, 29
7-311, 1992).

On the other hand, NO is a vascular endothelium-derived relaxing factor (en
dothelium-delivered relaxin
g factor (EDRF), which is one of the main bodies of EDRF), when E-cNOS is inhibited to a certain extent or more, cerebral blood flow decreases and systemic blood pressure rises, microcirculatory dynamics deteriorate, and eventually It is possible that the blood lesion will expand. Therefore, in the case of a therapeutic agent for ischemic brain disease, E-cN
A NOS inhibitor that weakly inhibits OS and strongly inhibits N-cNOS is considered desirable (Eu).
r. J. Pharmacol. , 204, 339-34
0, 1991; Proc. Natl. Acad. Sc
i. USA, 88, 6368-6371, 1991).

Recently, a patent application for the substance of the present invention (filing date:
(February 25, 1994), the compound of the present invention δ-
(S-methylisothioureido) -L-norvaline
It was reported to have a concentration-dependent inhibitory effect on both N-cNOS and iNOS (J. Med. Che.
m. 37,885-887,1994 [Issue date: 199
April 1, 4th, Abstract (Advance ACS Abs
issue date: March 1, 1994]). The article describes bNO equivalent to iNOS and N-cNOS.
Although the inhibition rate for S is described, E-cNO
No mention is made of the action on S, and no mention is made of its potential as a therapeutic agent for cerebrovascular disorders.

[0013]

Means for Solving the Problems In view of these problems, the present inventors have earnestly studied, and as a result, the following formula (I)

Embedded image Δ- (S-methylisothioureido) -L-
It was found that norvaline or a salt thereof has an inhibitory effect on N-cNOS superior to existing NOS inhibitors such as L-NNA described above, and is useful as a therapeutic agent for cerebrovascular disorders, and completed the present invention. It was That is, the present invention provides a novel cerebrovascular disorder therapeutic agent having a mechanism of action (selective inhibitory action on N-cNOS present in nerve cells in the brain) that is completely different from existing therapeutic agents for cerebrovascular disorder. It is a thing.

The compound of the present invention was prepared according to the method described in the above literature (J. Med.
Chem. 37, 885-887, 1994), but can also be manufactured, for example, as follows.

That is, the general formula (II)

Embedded image (In the formula, R ₁ represents a lower alkoxycarbonyl group whose alkyl part may have a substituent or a lower acyl group which may have a substituent. R ₂ has a substituent. Which may be a linear or branched alkyl group having 1 to 6 carbon atoms) is directly treated with a hydrolysis or deprotecting agent to be deprotected to obtain δ- (represented by the formula (I). S-Methylisothioureido) -L-norvaline or a salt thereof is obtained.

In the definition of the general formula (II), the lower alkoxycarbonyl group whose alkyl part may have a substituent includes, for example, a benzyloxycarbonyl group, a 9-fluorenylmethyloxycarbonyl group, and t-butoxy. Examples thereof include a carbonyl group and the like, and examples of the lower acyl group which may have a substituent include an acetyl group. As R ₁ , a t-butoxycarbonyl group is particularly preferable. Linear or branched carbon number 1 which may have a substituent
Examples of the alkyl group of to 6 include a methyl group, an ethyl group, a benzyl group, a t-butyl group and the like, and a t-butyl group is particularly preferable.

The deprotection reaction is carried out under the conditions usually used depending on the kind of the protecting group. For example, when R ₁ is a benzyloxycarbonyl group and R ₂ is a benzyl group, at room temperature, ethyl acetate, A catalytic reduction reaction in the presence of palladium-carbon in a solvent such as methanol or ethanol can be mentioned.
Particularly when R ₁ is a t-butoxycarbonyl group and R ₂ is a t-butyl group, a solvent that does not adversely influence the reaction, for example, methylene chloride, ethyl acetate, methanol, ethanol, dioxane, in water or without a solvent, is used at 0 ° C. to room temperature. It is preferable to use a deprotecting agent such as trifluoroacetic acid, hydrochloric acid, sulfuric acid, or methanesulfonic acid under the conditions described above, and it is particularly preferable to use trifluoroacetic acid under anhydrous conditions at room temperature.

The pharmaceutically acceptable salt of the compound (I) of the present invention is, for example, acetate, maleate, tartrate, methanesulfonate, benzenesulfonate, formate, p.
-Organic acid salts such as toluenesulfonic acid salts and trifluoroacetic acid salts, for example, inorganic acid salts such as hydrochlorides, sulfates, hydrobromides and phosphates, and dihydrochlorides are preferable.

The salt is not particularly limited as long as it is a pharmaceutically acceptable salt. For example, hydrochloric acid, sulfuric acid, nitric acid, hydrobromic acid, inorganic acid salts with phosphoric acid, acetic acid, oxalic acid,
Organic acid salts with tartaric acid, maleic acid, methanesulfonic acid, benzenesulfonic acid, formic acid, p-toluenesulfonic acid, trifluoroacetic acid, etc., alkali metal salts with sodium, potassium, etc., alkaline earth metals with calcium, magnesium, etc. Examples thereof include metal salts and the like, and dihydrochloride is preferable.

The compounds of the present invention or salts thereof are suitable excipients, auxiliaries, lubricants, preservatives, disintegrating agents, buffers, binders, stabilizers, wetting agents, emulsifiers, coloring agents, scent agents. Or, it may be added orally or parenterally in the form of tablets, granules, fine granules, powders, capsules, syrups, elixirs, suspensions / emulsions, injections, etc. by adding an fragrance or the like. Can be administered intramuscularly or intravenously.

The dose of the compound of the present invention or a salt thereof can be appropriately selected according to the patient's body type, age, physical condition, degree of disease, elapsed time after onset, etc., but is preferably 0.6 per day. ~ 60 mg / body.

In the case of formulating as an internal preparation, for example, as a carrier for the formulation, a starch or starch derivative such as lactose, sucrose, sorbitol, mannitol, potato starch or corn starch, a starch derivative such as a cellulose derivative or gelatin is usually used. Auxiliary agents that can be used are suitable, and at the same time, a lubricant such as magnesium stearate, carbowax or polyethylene glycol can be added, and a mixture of these is made into granules, tablets, capsules and the like by a conventional method. can do.

When formulated as an aqueous preparation, for example, an effective amount of the main component is dissolved in distilled water for injection, and if necessary, an antioxidant, a stabilizer, a solubilizing agent, a buffer, a preservative and the like are added. In addition, an injection can be prepared by completely dissolving, then filtering, filling and sealing by a conventional method, and sterilizing by a high pressure steam sterilization method, a dry heat sterilization method and the like.

When formulated as a freeze-drying agent, an aqueous solution in which the main component is dissolved in distilled water for injection may be freeze-dried by a conventional method. , Mannitol, inositol, lactose, maltose, sucrose and the like, sugars or sugar alcohols, glycine and the like can be added and lyophilized in a conventional manner to prepare.

The production of the compound of the present invention will be described in more detail in the following examples, but the present invention is not limited to these examples. Further, in order to demonstrate the usefulness of the present invention, δ- (S-methylisothioureido) -L-norvaline has an excellent selective inhibitory effect on N-cNOS in the brain, and human ischemic cerebral edema and cerebral infarction. The test results regarding the anti-occlusive cerebrovascular disorder action using a pathological model animal similar to the above are shown in Test Examples.

[0026]

【Example】

Embodiment 1

Δ = (S-methylisothioureido) -L
-Synthesis of norvaline dihydrochloride Paul L. et al . Feldman's method (Tetrahhe
dron Lett. , 32,7,875-878,1
991) and a mixture of α-Nt-butoxycarbonyl-δ- (S-methylisothioureido) -L-norvaline-t-butyl ester (1.03 g) and trifluoroacetic acid (10 ml) at room temperature. After stirring for 2 hours, the mixture was concentrated under reduced pressure. The residue is ethanol (5m
After dissolving in 1), a solution of hydrogen chloride in dioxane (4N, 2 ml) was added at room temperature, and the reaction solution was evaporated under reduced pressure.
The residue was dissolved in water and freeze-dried to obtain δ- (S-methylisothioureido) -L-norvaline dihydrochloride at 0.6.
0 g was obtained.

Embedded image

¹ H-NMR (D ₂ O) δ: 1.78-2.36 (4 H, m), 2.63 (3
H, s), 3.47 (2H, t, J = 6.60 Hz),
4.17 (1H, t, J = 5.97Hz) FAB-MS (m / z) 206 (M ⁺⁺ 1)

[0029]

[Test example]

[Test Example 1] N-cNOS and E-c of the compound of Example 1
The inhibitory effect on NOS was examined by comparison with existing NOS inhibitors. As a crude enzyme fraction of each NOS isoform, N-cNOS was a soluble fraction of rat cerebral cortex, and
E-cNOS is a bovine pulmonary artery vascular endothelial cell line (CPAE)
Was used.

The following NOS inhibitors were used as control compounds. ^{L-NNA, N G -nitro-} L-arg
inine methyl ester (L-NAM
E), N ^G -amino-L-arginine (L
-AA), N ^G -iminoethyl-ornith
ine (L-NIO), N ^G -monomethyl-
L-arginine (L-NMMA), ^NG- all
yl-L-arginine (L-ALA), 7-ni
Troindazole (7-NI) aminoguanidine (AG).

A crude enzyme preparation of N-cNOS was prepared by the following procedure. Normal untreated male Sprague Daw
Ley (SD) rats (weight 300-400 g) were decapitated, the whole brain was quickly removed, and the cerebral cortex was separated on ice. Then 5 volumes of 50 mM Tris-HCl, 1
A mM DTT (pH 7.4) solution was added, and the mixture was homogenized for 3 minutes and then centrifuged at 1,000 xg for 10 minutes.
The obtained supernatant was centrifuged at 100,000 xg for 60 minutes, and the soluble cytoplasmic fraction of the finally obtained supernatant was added to Nc.
A crude enzyme preparation of NOS was used.

A crude enzyme preparation of E-cNOS was prepared by the following procedure. A bovine pulmonary artery vascular endothelial cell line (CPAE) was cultured in MEM medium containing 20% FBS. A few days later,
This was peeled from the flask with a 0.25% tripsin, 1 mM EDTA solution and diluted with physiological saline to 0.1.
M phosphate buffer solution (phosphate-buffer)
ed saline, PBS) (pH 7.4),
It was centrifuged at 1,000 rpm for 5 minutes. Then discard the supernatant,
The cells were washed by adding an appropriate amount of PBS and centrifuging at 1,000 rpm for 5 minutes. Furthermore, 50 mM T
The cells were washed by performing the same operation once again with ris-HCl and 1 mM DTT (pH 7.4). Appropriate amount of 50 mM Tris-HCl, 1 mM DTT to the cells for precipitation
(PH 7.4) solution was added and homogenized for 3 minutes to obtain a crude enzyme preparation of E-cNOS.

The measurement of NOS activity basically follows the method reported by the present inventors and is one of the substrates, L- [ ³ H].
L-, which is one of the reaction products from arginine
It was determined by quantifying the amount of conversion to [ ³ H] citrullline (Brain Edema IX (I
to et al. eds. ) 60, pp. 285-2
88, 1994, Acta Neurochir. , S
printer-Verlag).

The reaction solution was 100 nM L- [ ³ H] ar.
Gine, N-cNOS or E-cNOS crude enzyme preparation (6-20 μg / ml protein), 1.25 mM C
aCl ₂ , 1 mM EDTA, 10 μg / ml cal
modulin, 1 mM NADPH, 100 μM t
etrahydrobiopterine, 10 μMF
AD, 10 μM FMN, 50 mM Tris-HCl
(PH 7.4), to which the compound of the present invention,
Alternatively, a control compound was added.

The reaction was started by adding L- [ ³ H] arginine, and after incubating at 37 ° C. for 10 minutes, 50 mM Tris-HCl (pH 5.5), 1 m.
2 ml of M EDTA was added and the reaction was stopped by placing on ice. The reaction solution is a cation exchange resin column (Dowex
AG50WX-8, Na ⁺ form, 3.2 ml), and left unreacted substrate L- [ ³ H] argin.
ine and the reaction product L- [ ³ H] citrulli
ne was separated. This eluate and the eluate obtained by further passing 3 ml of distilled water through the column were put in a mini vial, and L-
[ ³ H] citrulline was collected. Then 5
ml scintillator was added, the radioactivity was measured with a liquid scintillation counter, and L- [ ³ H] citrul was measured.
The line was quantified. The protein concentration in the crude enzyme preparation was determined using a micro assay kit manufactured by Bio-Rad.

Table 1 shows the IC ₅₀ values (concentration required for 50% activity inhibition) of both test compounds for both NOS isoforms and the IC ₅₀ values of E ₅₀ for Ec-NOS for N-cNOS as an index showing selectivity. The percentage of IC ₅₀ value is shown. All the values in the table are shown as mean ± standard error, and the number of cases is shown in parentheses. From Table 1, 1. The compound of the present invention is stronger than existing NOS inhibitors in N-
It has a cNOS inhibitory action. 2. Regarding the selectivity for N-cNOS and E-cNOS, the compound of the present invention has extremely high selectivity as compared with existing NOS inhibitors (ratio of IC ₅₀ values (IC ₅₀ [E ₅₀
-NOS] / IC ₅₀ [N-cNOS]) 42.
5). It is clear.

[0037]

Test Example 2 The compound of the present invention was used to examine the action on a rat model of regional cerebral ischemia. Rat left middle cerebral artery (M)
A regional cerebral ischemia model due to (CA) occlusion was prepared according to the method reported by the present inventors (Neurosci. Le.
tt. 147, 159-162, 1992; Brain
Edema IX (Ito et al. Eds.)
60, pp. 285-288, 1994, Acta N
eurochir. , Springer-Verla
g).

That is, 2% of 10-week-old male SD rats
Anesthesia was induced by inhalation of halothane (diluted with 70% N ₂ O and 30% O ₂ ) and maintained by inhalation of 1% halothane, and fixed in a lateral decubitus position on the operating table. A linear skin incision was made midway between the left external ear canal and the lateral canth, and along the anterior edge of the temporalis muscle, the incision was made down to the zygomatic arch. The temporal muscle, intra-orbital adipose tissue, and eyeball were removed and immersed in physiological saline containing antibiotics. The back of the orbit was cauterized using a bipolar coagulator to prevent bleeding. Under a surgical microscope, a small hole with a diameter of 3 mm was made with a dental electric drill in the middle of the foramen ovale and the orbital fissure where the third branch of the trigeminal nerve running inside the temporal muscle was found deep in the floor of the temporal bone. Opened One layer of thin bone was left and it was removed using a microhook and microneedle holder. Using a 27G needle and a microhook, the dura and arachnoid are cut slightly, and the lens nucleus striatal artery (lentic
The MCA trunks were occluded for 48 hours with mini clips at the position of the bifurcation with the urostrate artery (LSA), or were reperfused by removing the clips for 2 hours after 2 hours of occlusion. During the operation, the temperature of the right temporal muscle and the temperature in the rectum were monitored and kept constant at around 37 ° C.

In the 48-hour MCA occlusion model, a control solvent (physiological saline, 2 ml / kg) or the compound of the present invention (0.1, 0.3, 1 mg / kg) was intraperitoneally administered immediately after MCA occlusion. Administration or 24.2 μg / kg was administered immediately after MCA occlusion through a catheter attached to the right femoral vein, and then continuously administered at a flow rate of 84.2 ng / kg / min. The water content in the brain was measured by the dry weight method (Neurosci. Lett. 147, 159-1).
62, 1992). The rat was decapitated 48 hours after MCA occlusion, and the wet weight of the left cerebral hemisphere taken out within 60 seconds was
Each measurement was made within 90 seconds on an analytical balance. Then 1
After drying in an oven at 05 ° C for 3 days, the dry weight was measured. The water content in the brain was calculated by "(wet weight-dry weight) / (wet weight) x 100%" and displayed in Figs.

[0040]

[Figure 1]

[0041]

FIG. 2

In a model in which MCA was occluded for 2 hours and then reperfused for 2 hours, a control solvent (physiological saline, 2 ml / kg) was used.
Alternatively, the compound of the present invention (0.1, 0.3, 1 mg / k)
g) was intraperitoneally administered immediately after MCA occlusion and immediately after reperfusion. 2 hours after reperfusion, 10 IU / m under ether anesthesia
perfuse the brain transcardially with 1 heparin-containing saline,
Subsequently, it was fixed with a 4% formalin solution. Coronal serial sections at 1 mm intervals from the frontal lobe prepared by a conventional method were stained with hematoxylin and eosin (Neurosci. L).
ett. , 147, 159-162, 1992; Eu.
r. J. Pharmacol. Env. Tox. , 24
8, 325-328, 1993). The area of the infarct lesion was determined by "image area of infarct lesion on affected side" = "area of normal region on healthy side"-"area of normal region on affected side" using an image analyzer and displayed in FIG.

[0043]

FIG. 3

All the values in the figure are shown as the average value ± standard error, and the number of animals is shown in parentheses. Statistical analysis is D
Unnett's multiple comparison test was performed, and a risk factor (P value) of less than 0.05 was considered to be statistically significant.

From FIG. 1, the water content in the left cerebral hemisphere increased by cerebral ischemia due to MCA occlusion for 48 hours was dose-dependently improved by a single intraperitoneal administration of the compound of the present invention,
The effect was significant in the 1 mg / kg dose group. Further, from FIG. 2, cerebral edema 48 hours after MCA was significantly reduced by continuous intravenous administration of the compound of the present invention.
Furthermore, as shown in FIG. 3, the infarct lesion that occurs when MCA is occluded for 2 hours and reperfused for 2 hours is dose-dependently improved by two intraperitoneal administrations of the compound of the present invention, and its action is 1 m.
It was significant in the g / kg administration group.

[0046]

EFFECT OF THE INVENTION δ- (S-Methylisothioureido)-
L-norvaline has a significantly superior effect to existing NOS inhibitors in (1) N-cNOS inhibitory action and (2) selectivity for N-cNOS and E-cNOS, and as a cerebrovascular disorder therapeutic agent. It is useful.

[0047]

[Brief description of drawings]

1 shows δ- (S-methylisothioureido) -L
FIG. 6 is a view showing a cerebral edema improving effect after permanent occlusion of rat MCA by a single intraperitoneal administration of norvaline.

[0048]

FIG. 2 shows δ- (S-methylisothioureido) -L
FIG. 3 is a view showing the cerebral edema improving effect after permanent occlusion of rat MCA by continuous intravenous administration of norvaline.

[0049]

FIG. 3 shows δ- (S-methylisothioureido) -L
FIG. 6 is a view showing a cerebral infarction reducing effect after temporary occlusion of rat MCA by intraperitoneal administration of norvaline twice.

[Table 1]

─────────────────────────────────────────────────── ───

[Procedure amendment]

[Submission date] February 22, 1995

[Procedure Amendment 1]

[Document name to be amended] Statement

[Name of item to be corrected] 0029

[Correction method] Change

[Correction content]

[0029]

[Test example]

[Test Example 1] N-cNOS and E-c of the compound of Example 1
The inhibitory effect on NOS was examined by comparison with existing NOS inhibitors. As a crude enzyme fraction of each NOS isoform, N-cNOS was a rat cerebral cortex soluble fraction, and
E-cNOS is a bovine pulmonary artery vascular endothelial cell line (CPAE)
Was used.

[Submission date] September 19, 1995

[Procedure Amendment 2]

[Document name to be amended] Statement

[Correction target item name] 0039

[Correction method] Change

[Correction content]

In the 48-hour MCA occlusion model, a control solvent (physiological saline, 2 ml / kg) or the compound of the present invention (0.1, 0.3, 1 mg / kg) was intraperitoneally administered immediately after MCA occlusion. Administration or 24.2 μg / kg was administered immediately after MCA occlusion through a catheter attached to the right femoral vein, and then continuously administered at a flow rate of 84.2 ng / kg / min. The water content in the brain was measured by the dry weight method (Neurosci. Lett. 147, 159-1).
62, 1992). Rats were decapitated MCA occlusion 48 hours later, the wet weight of the left cerebral hemisphere was removed within 60 seconds,
It was measured within 90 seconds in the chemical balance. Then, it dried in the oven of 105 degreeC for 3 days, and measured the dry weight. The brain water content was calculated as "(wet weight-dry weight) / (wet weight) x 1
00% "and displayed in FIGS.

[Submission date] September 19, 1995

[Procedure 3]

[Document name to be amended] Statement

[Name of item to be corrected] 0045

[Correction method] Change

[Correction content]

[0045] From FIG. 1, the water content in the 48 hours by Ri increase <br/> much left hemisphere to MCA occlusion is dose-dependently improved by single intraperitoneal dose of the compounds of the present invention, its effect Is 1
It was significant in the mg / kg administration group. Further, from FIG. 2, MC was obtained by continuous intravenous administration of the compound of the present invention.
Brain edema after 48 hours was significantly reduced. Furthermore, as shown in FIG. 3, the infarct lesion that occurs when MCA is occluded for 2 hours and reperfused for 2 hours is dose-dependently improved by two intraperitoneal administrations of the compound of the present invention, and its action is 1 mg / kg. It was significant in the administration group.

Claims (8)

[Claims] 1. δ- (S-Methylisothioureido) -L
-Norvaline or a pharmaceutically acceptable salt thereof. 2. δ- (S-Methylisothioureido) -L
-A therapeutic agent for cerebrovascular disorders, which comprises norvaline or a pharmaceutically acceptable salt thereof as an active ingredient. 3. The therapeutic agent according to claim 2, wherein the type of cerebrovascular disorder is cerebral infarction. 4. The therapeutic agent according to claim 2, wherein the type of cerebrovascular disorder is cerebral hemorrhage. 5. The therapeutic agent according to claim 2, wherein the type of cerebrovascular disorder is subarachnoid hemorrhage. 6. The therapeutic agent according to claim 2, wherein the type of cerebrovascular disorder is cerebral edema. 7. A selective inhibitor of N-cNOS, the selectivity of which is the ratio of IC ₅₀ values (IC ₅₀ [E-NOS] /
A therapeutic agent for cerebrovascular disease, which comprises a compound having an IC ₅₀ [N-cNOS] of 30 or more. 8. The therapeutic agent for cerebrovascular disorders according to claim 7, wherein the compound is δ- (S-methylisothioureido) -L-norvaline or a pharmaceutically acceptable salt thereof.