JPH08109165A - Amino acid derivative having inhibitory action on nitrogen monoxide synthetase - Google Patents

Abstract

PURPOSE: To provide the subject new compound having nitrogen monoxide synthetase inhibiting action and useful for the treatment of cerebrovascular diseases, especially the symptom of the acute stage of occlusive cerebrovascular disease and the hypotension in sepsis. CONSTITUTION: This amino acid derivative is a compound expressed by formula I [R1 is H, a (substituted) 1-6C alkoxycarbonyl or a (substituted) 1-9C acyl; R2 is H or a (substituted 1-6C alkyl; R3 is a (substituted) 1-6C alkyl or a (substituted) 2-6C alkeny; R4 is H, R3 and R4 are each a (substituted) 5-7 membered ring; (n) is an integer of 2-4; excluding the case that R3 is methyl and (n) is 3] or its pharmaceutically permissible salt, e.g. δ-(S-(2-fluoroethyl) isothioureido)-L-ylvaline. The compound of formula I can be produced e.g. by reacting a compound of formula II [R1 is a (substituted) 1-6C alkoxycarbonyl, etc.; R2 is a (substituted) 1-6C alkyl] with a compound of formula III (R3 is not methyl when (n) in the formula II is 3; X is a halogen) in a solvent such as acetone at a temperature between room temperature and the boiling point of the reaction mixture.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an amino acid derivative,
More specifically, nitric oxide synthase (Nitric O
xide Synthase (hereinafter abbreviated as NOS) inhibitory action, nitric oxide (Nitric Oxide,
(Hereinafter abbreviated as NO), cerebrovascular disorder in which NO or a NO metabolite is considered to be involved,
In particular, a compound represented by the general formula (I) or a pharmaceutically acceptable salt thereof, which is useful for the pathophysiology of acute phase of obstructive cerebrovascular disorder and hypotension in sepsis, and containing these as active ingredients The present invention relates to a prophylactic and / or therapeutic agent characterized by the above.

[0002]

2. Description of the Related Art In a region where 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, resulting in exacerbation of cerebral disorder, which determines the extent of subsequent infarct lesions. . 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 I do not have much expectation for effective therapeutic effect ( . 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.

[0003] We, ^N G -nitro-L-arginine, which is known as a NOS inhibitor (L-NN
A) shows cerebral edema and cerebral infarction (Naga) that occur after cerebral ischemia
fuji et al. , Neurosci. Let
t. , 147, 159-162, 1992), neuronal necrosis (Nagafuji et al., Eur. J. et al.
Pharmacol. Env. Tox. , 248, 32
5-328, 1993). However, it has been reported that relatively high doses of NOS inhibitors are not only ineffective but sometimes exacerbated against ischemic brain injury (I
adecola et al. , J. et al. Cereb. Bl
good Flow Metab. , 14,175-19
2, 1994).

At present, the theory that there are at least three types of isoforms of NOS is predominant from the analysis of gene DNA. That is, N-cNOS permanently present in nerve cells and E- permanently present in vascular endothelial cells.
Induced and synthesized by cytokines and in vivo trace toxins in cNOS, macrophages and many other cells i
It is NOS. Among them, N-cNOS and E-cNOS are calcium-dependent, and iNOS is calcium-independent (Nathan et al., FASEB.
J. 16, 3051-3064, 1992).

As a possible mechanism of cerebral 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 for abnormal activation of 0,883-908, 1984; Choi, Trend.
s Neurosci. n, J. Cereb. Blood Flow Meta
b. 9, 127-140, 1989). As mentioned above,
Since N-cNOS is calcium-dependent, inhibiting the abnormal activation of this type of NOS isoform is thought to exert the neuroprotective effect of NOS inhibitors (Dawson). et a
l. , Annals Neurol. 32,297-3
11, 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 gFactor; EDRF), when E-cNOS is unnecessarily inhibited, cerebral blood flow decreases and systemic blood pressure increases, and microcirculatory dynamics deteriorates.
Eventually, the ischemic lesion may expand.

Accordingly, in the case of a therapeutic agent for ischemic brain disease, E-
A NOS inhibitor that has a weak inhibitory effect on cNOS and strongly inhibits N-cNOS is considered to be desirable (Nowicki et al., Eur. J. Pha.
rmacol. , 204, 339-340, 1991;
Dawson et al. , Proc. Natl. A
cad. Sci. USA, 88, 6368-6371,
1991). Is a known NOS inhibitor,
As described above, in comparison with the inhibitory effect on E-cNOS, there is no report that it shows selectivity for N-cNOS.

[0008] In addition, iNOS is induced and synthesized in immunocompetent cells such as macrophages and glial cells and other cells by certain kinds of cytokines and trace toxins in vivo, and as a result, a large amount of generated NO causes fatal decrease in blood pressure. Live Therefore, it is considered that an inhibitor selective for iNOS is effective for hypotension and the like in sepsis (Kilbourn and Griffith, J. et al.
Natl. Cancer Inst. 84,827-83
1, 1992; Cobb et al. , Crit. C
are Med. 21,1261-1263,199
3; Lorente et al. , Crit. Care
Med. 21, 1287-1295, 1993).

To date, the only Aminoguanid
It has been reported that ine (AG) exhibits selectivity for i-NOS in comparison with its inhibitory effect on E-cNOS (Griffith et al., B.
r. J. Pharmacol. 110,963-96
8, 1993), but the inhibitory action itself is not sufficient as a useful therapeutic agent for hypotension in sepsis.

[0010]

SUMMARY OF THE INVENTION The object of the present invention is to selectively inhibit specific NOS isoforms and to eliminate excess NOS.
Another object is to provide a compound useful for diseases in which involvement of NO metabolite is suggested. That is, E-cNOS
The present invention provides a NOS inhibitor useful as a cerebrovascular disorder therapeutic agent, which has a weak inhibitory effect on cerebral vascular disease and strongly inhibits N-cNOS. Further, it has a selective inhibitory effect on iNOS and is useful for hypotension in sepsis.
The present invention provides an OS inhibitor.

[0011]

Means for Solving the Problems The inventors of the present invention have conducted extensive studies to solve such problems, and as a result, in the compound represented by the general formula (I), particularly R ₃ is substituted with a halogen atom. A straight-chain or branched-chain alkyl group having 1 to 6 carbon atoms or a salt thereof has a remarkably superior intracerebral N-cNOS selective inhibitory action as compared with existing NOS inhibitors, and cerebrovascular They have found that they are useful as therapeutic agents for disorders, and have completed the present invention. Further, among compounds represented by the general formula (I), iNOS in which R ₃ is an allyl group or a salt thereof is significantly superior to existing NOS inhibitors.
They found that they have a selective inhibitory action and are useful as a therapeutic agent for sepsis, and have completed the present invention.

That is, the present invention provides the following (1)-
The present invention provides the compound according to (6) or a pharmaceutically acceptable salt thereof.

(1) General formula (I)

Embedded image (In the formula, R ₁ is a hydrogen atom, a linear or branched alkoxycarbonyl group having 1 to 6 carbon atoms in which the alkyl portion may have a substituent, or a straight chain optionally having a substituent. Represents a chain- or branched-chain acyl group having 1 to 9 carbon atoms; R ₂
Represents a hydrogen atom, a linear or branched alkyl group having 1 to 6 carbon atoms which may have a substituent; R ₃ represents a linear or branched chain which may have a substituent. Carbon number 1
~ 6 alkyl group, represents a linear or branched alkenyl group having 2 to 6 carbon atoms which may have a substituent; R ₄
Represents a hydrogen atom; or, R ₃ and R ₄ may be taken together to form a 5 to 7 membered ring which may have a substituent; n is an integer of 2 to 4; Represent Provided that R ₃ is a methyl group and n is 3 at the same time) or a pharmaceutically acceptable salt thereof.

(2) General formula (I)

[Chemical 6] (In the formula, R ₁ is a hydrogen atom, a linear or branched alkoxycarbonyl group having 1 to 6 carbon atoms in which the alkyl portion may have a substituent, or a straight chain optionally having a substituent. Represents a chain- or branched-chain acyl group having 1 to 9 carbon atoms; R ₂
Represents a hydrogen atom or a linear or branched alkyl group having 1 to 6 carbon atoms which may have a substituent; R ₃ represents a linear or branched carbon atom substituted with a halogen atom. Number 1
Represents 6 alkyl group; _{R 4} represents a hydrogen atom; n
Represents an integer of 2 to 4. (1) or a pharmaceutically acceptable salt thereof.

(3) General formula (I)

[Chemical 7] (In the formula, R ₁ is a hydrogen atom, a linear or branched alkoxycarbonyl group having 1 to 6 carbon atoms in which the alkyl portion may have a substituent, or a straight chain optionally having a substituent. Represents a chain- or branched-chain acyl group having 1 to 9 carbon atoms; R ₂
Represents a hydrogen atom or a linear or branched alkyl group having 1 to 6 carbon atoms which may have a substituent; R ₃ represents 2
Represents a fluoroethyl group; R ₄ represents a hydrogen atom;
n represents an integer of 2 to 4. ) The compound according to (1) or a salt thereof which is illicit as a pharmaceutical.

(4) General formula (1)

Embedded image (In the formula, R ₁ and R ₂ each independently represent a hydrogen atom;
₃ is a linear or branched alkyl group having 1 to 6 carbon atoms which may have a substituent, or a linear or branched alkyl group having 2 to 6 carbon atoms which may have a substituent. Represents an alkenyl group; R ₄ represents a hydrogen atom; or R ₃ and R ₄ may be taken together to form a 5- to 7-membered ring which may have a substituent; n is , 2 to 4 are represented. However, R
When n is 3 at the same time when ₃ is a methyl group), the compound according to (1) or a pharmaceutically acceptable salt thereof.

(5) δ- (S- (2-fluoroethyl)
Isothioureido) -L-norvaline, δ- (S-ethylisothioureido) -L-norvaline, and δ-
(S- (3-chloropropyl) isothioureido) -L
-The compound according to (1) selected from the group of compounds consisting of norvaline, or a pharmaceutically acceptable salt thereof. (6) The compound according to (1), which is δ- (S-allylisothioureido) -L-norvaline, or a pharmaceutically acceptable salt thereof.

In particular, δ- (S- (2-fluoroethyl))
Isothioureido) -L-norvaline and δ- (S-
Ethylisothioureido) -L-norvaline has a selective inhibitory effect on N-cNOS in the brain far superior to existing NOS inhibitors, and has a weak inhibitory effect on E-cNOS, and is a therapeutic agent for cerebrovascular disorders. Is useful as

Further, δ- (S-allylisothioureido) -L-norvaline has an iNOS selective inhibitory action far superior to existing NOS inhibitors, and is useful as a therapeutic agent for sepsis.

[0020]

BEST MODE FOR CARRYING OUT THE INVENTION The compound of the present invention represented by the general formula (I) is a novel compound not described in the literature, and can be produced, for example, as follows.

[0021]

[Chemical 9] General formula (II) (In formula, R < ₁ > is a linear or branched C1-C6 whose alkyl part may have a substituent.
Represents an alkoxycarbonyl group, which may have a substituent, and represents a linear or branched acyl group having 1 to 9 carbon atoms;
R ₂ represents a linear or branched alkyl group having 1 to 6 carbon atoms which may have a substituent; and n represents an integer of 2 to 4. A compound represented by the general formula (III) (wherein
R ₃ is a linear or branched C 1-6 alkyl group which may have a substituent, or a linear or branched C 2-6 carbon group which may have a substituent. Represents an alkenyl group. However, in the general formula (II), when n is 3,
R ₃ is not a methyl group; X represents a halogen atom. ) And a compound represented by () in a solvent such as acetonitrile, acetone, 1,4-dioxane, methanol and ethanol at a temperature from room temperature to the boiling point of the reaction mixture. It is preferably carried out by heating the reaction mixture to reflux in acetonitrile. Then, by hydrolyzing or directly treating with a deprotecting agent for deprotection, the compound of the general formula (IV) (in the formula, R ₁ and R ₂ represent a hydrogen atom; n represents an integer of 2 to 4). A compound represented by or a salt thereof is obtained.

Further, in the general formula (II) (in the formula, R ₁ is a linear or branched alkoxycarbonyl group having 1 to 6 carbon atoms in which the alkyl portion may have a substituent, or a substituent A linear or branched chain carbon number which may have 1 to 9
It represents an acyl group; R ₂ represents a straight-chain may have a substituent group or branched alkyl group having 1 to 6 carbon atoms;
n represents an integer of 2 to 4. A compound represented by general formula (V) (wherein R ₅ represents an alkylene group having 2 to 4 carbon atoms which may have a substituent; X represents a halogen atom; Y represents a halogen atom) Or represents an alkoxycarbonyl group having 1 to 6 carbon atoms, by reacting the compound represented by with a solvent such as acetonitrile, acetone, 1,4-dioxane, methanol, ethanol at a temperature from room temperature to the boiling point of the reaction mixture. It is preferably carried out by heating the reaction mixture to reflux in acetonitrile.
By deprotecting by hydrolyzing or directly treating with a deprotecting agent, general formula (VI) (in the formula, R ₁ and R ₂ represent a hydrogen atom; R ₅ is a carbon which may have a substituent). Number 2
~ 4 represents an alkylene group or a carbonyl group bonded to the end of the alkylene group; n represents an integer of 2 to 4. ) Or a compound represented by the general formula (VII)
(In the formula, R ₁ , R ₂ , R ₅ and n are the same as the above.) Or a salt thereof is obtained.

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, ethyl acetate and methanol are used at room temperature. And a catalytic reduction reaction in the presence of palladium-carbon in a solvent such as ethanol.
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, 1,4
-It is preferable to use a deprotecting agent such as trifluoroacetic acid, hydrochloric acid, sulfuric acid or methanesulfonic acid under conditions of 0 ° C to room temperature in dioxane, water or no solvent, and especially trifluoroacetic acid under anhydrous conditions at room temperature. It is preferably used.

General formulas (I), (II), (III),
In the definitions of (IV), (V), (VI), and (VII), the linear or branched C1-C6 alkoxycarbonyl group in which the alkyl moiety may have a substituent, Examples thereof include a benzyloxycarbonyl group, a t-butoxycarbonyl group, a 9-fluorenylmethyloxycarbonyl group, and the like, which may have a straight chain or branched chain acyl group having 1 to 9 carbon atoms. Examples of acetyl include an acetyl group and the like. As R ₁ , a t-butoxycarbonyl group is particularly preferable. The linear or branched alkyl group having 1 to 6 carbon atoms in the linear or branched alkyl group having 1 to 6 carbon atoms which may have a substituent is a methyl group, an ethyl group, t A butyl group and the like. Linear or branched chain carbon number 1 to 1 which may have a substituent
Examples of the substituent in the alkyl group of 6 and the linear or branched alkenyl group of 2 to 6 carbon atoms which may have a substituent include a halogen, an alkyl group, an aryl group, and the like. Atoms, cyclopropyl groups and the like are preferred. As R ₂ , a t-butyl group is particularly preferable. R
Examples of ₃ include a 2-fluoroethyl group, a cyclopropylmethyl group, a phenyl group and the like, and a 2-fluoroethyl group is particularly preferable. Examples of the linear or branched alkenyl group having 2 to 6 carbon atoms which may have a substituent include 1-propenyl group. As X, a chlorine atom, a bromine atom or an iodine atom is preferable.
Y is preferably a chlorine atom, a bromine atom, an iodine atom, a methoxycarbonyl group or an ethoxycarbonyl group. Examples of the alkylene group having 2 to 4 carbon atoms which may have a substituent include an ethylene group and a propylene group. R ₅ is preferably an ethylene group or a propylene group.

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 production of the compound of the present invention will be described in more detail based on the following examples, but the invention is not intended to be limited thereto. In addition, in order to show the usefulness of the present invention, selective inhibitory effects of the compounds represented by the general formula (I) on various NOSs are shown in Test Examples.

[0027]

【Example】

Example 1 α-Nt-butoxycarbonyl-δ- (S-ethyl group
Sothioureido) -L-norvaline-t-butyl ester
Synthesis of hydroiodide salt Paul L. et al. Felderman's method (Tetra
hedron Lett. , 32, 7, 875-87
8, 1991). Α-Nt-butoxycarbonyl-δ-thioureido-L-norvaline-t-
Ethyl iodide (0.17 ml) was added to a mixture of butyl ester (0.48 g) and acetonitrile (5 ml), and the mixture was heated under reflux for 2 hours. After the reaction solution was concentrated under reduced pressure, the residue was purified by silica gel column chromatography (developing solution, methylene chloride: methanol = 95: 5), and α-Nt-butoxycarbonyl-δ- (S-ethylisothiothiol was used. 0.67 g of raid) -L-norvaline-t-butyl ester hydroiodide was obtained. ¹ H = NMR (CDCl ₃ ) δ: 1.39-1.47 (12 H, m), 1.48 (9
H, s), 1.63-1.97 (4H, m), 3.36.
-3.62 (4H, m), 4.07-4.19 (1H,
m), 5.22-5.41 (1H, m) FAB-MS (m / z) 376 (M ⁺ +1).

[0028]

Example 2 δ- (S-ethylisothioureido) -L-norvaline
Synthesis of α-Nt-butoxycarbonyl-δ- (S-ethylisothioureido) -L-norvaline-t-butyl ester hydroiodide (0.67 g) and trifluoroacetic acid (5 ml) After stirring at room temperature for 2 hours, the mixture was concentrated under reduced pressure. The residue was dissolved in ethanol, a 1,4-dioxane solution of hydrogen chloride (4N, 1 ml) was added at room temperature, and the reaction solution was concentrated under reduced pressure. The residue was subjected to ion exchange chromatography (DOWEX, 50W-X8, H
⁺ Form) to obtain 0.25 g of δ- (S-ethylisothioureido) -L-norvaline. ¹ H-NMR (D ₂ O) δ: 1.30-1.35 (3H, m), 1.56-1.
89 (4H, m), 3.03-3.10 (2H, m),
3.35-3.38 (2H, m), 3.43-3.50
(1H, m) FAB-MS (m / z) 220 (M ⁺ +1)

[0029]

Example 3 α-Nt-butoxycarbonyl-δ- (S- (2- phenyl
Luoroethyl) isothioureido) -L-norvaline-
Synthesis of t-butyl ester hydrobromide As in Example 1, the reaction was carried out using 2-fluoroethane bromide as a reactant, and α-Nt-butoxycarbonyl-δ- (S- ( 0.29 g of 2-fluoroethyl) isothioureido) -L-norvaline-t-butyl ester hydrobromide was obtained. ¹ H-NMR (CDCl ₃ ) δ: 1.44 (9H, s), 1.48 (9H, s),
1.62-1.96 (4H, m), 3.38-3.61
(2H, m), 3.77 (2H, destroyed)
dt, J = 25.1 Hz, 4.95-5.28 Hz),
4.03-4.19 (1H, m), 4.75 (2H, d
estorted dt, J = 46.9 Hz, 4.95
-5.28 Hz), 5.24-5.37 (1 H, m) FAB-MS (m / z) 394 (M ⁺ +1).

[0030]

Example 4 δ- (S- (2-Fluoroethyl) isothioureido)
Synthesis of -L-norvaline dihydrochloride The compound obtained in Example 3 and trifluoroacetic acid (10 m
The mixture of l) was stirred at room temperature for 2 hours and then concentrated under reduced pressure. The residue was dissolved in ethanol (5 ml), hydrogen chloride in 1,4-dioxane (4N, 0.6 ml) was added at room temperature, and the reaction mixture was concentrated under reduced pressure. The residue was dissolved in water, freeze-dried and then δ- (S- (2-fluoroethyl) isothioureido) -L-norvaline dihydrochloride 0.17.
g was obtained. ¹ H-NMR (D ₂ O) δ: 1.76-2.06 (4H, m), 3.43-3.
56 (4H, m), 4.05-4.09 (1H, m),
4.80 (2H, destroyed dt, J = 4
6.5 Hz, 4.95-5.61 Hz) FAB-MS (m / z) 238 (M ⁺ +1)

[0031]

Example 5 α-Nt-butoxycarbonyl-δ- (S- (2-p-
Lopenyl) isothioureido) -L-norvaline-t-
Synthesis of Butyl Ester / Hydrobromide The reaction was carried out using allyl bromide as a reactant in the same manner as in Example 1, and α-Nt-butoxycarbonyl-δ- (S-
0.52-g of (2-propenyl) isothioureido) -L-norvaline-t-butyl ester hydrobromide was obtained. ¹ H-NMR (CDCl ₃ ) δ: 1.44 (9H, s), 1.47 (9H, s),
1.67-1.99 (4H, m), 3.32-3.63
(4H, m), 3.81-3.91 (1H, m), 3.
99-4.18 (2H, m), 5.35-5.44 (1
H, m) FAB-MS (m / z) 388 (M ⁺ +1)

[0032]

Example 6 δ- (S- (2-propenyl) isothioureido) -L
-Synthesis of norvaline dihydrochloride Using the compound obtained in Example 5 as a starting material, a deprotection reaction was carried out in the same manner as in Example 4 to obtain δ- (S- (2-propenyl)).
Isothioureido) -L-norvaline dihydrochloride 0.3
3 g were obtained. ¹ H-NMR (D ₂ O) δ: 1.72-2.09 (4H, m), 3.41-3.
46 (2H, m), 3.78 (2H, d, J = 6.8H
z), 4.07-4.12 (1H, m), 5.26-
5.40 (2H, m), 5.85-5.99 (1H,
m) FAB-MS (m / z) 232 (M ⁺ +1)

[0033]

Example 7 α-Nt-butoxycarbonyl-δ- (2,3-dehydroxy)
Dorothiazolin-2-yl) amino-L-norvaline-
Synthesis of t-butyl ester / hydrobromide The reaction was conducted in the same manner as in Example 1 except that 1,2-dibromoethane was used as the reactant, and α-Nt-butoxycarbonyl-δ- (2,3- Dehydrothiazolin-2-yl) amino-L-norvaline-t-butyl ester / hydrobromide (0.23 g) was obtained. ¹ H-NMR (CDCl ₃ ) δ: 1.44 (9H, s), 1.47 (9H, s),
1.67-1.99 (4H, m), 3.32-3.63
(4H, m), 3.81-3.91 (1H, m), 3.
99-4.18 (2H, m), 5.35-5.44 (1
H, m) FAB-MS (m / z) 374 (M ⁺ +1)

[0034]

Example 8 δ- (2,3-dehydrothiazolin-2-yl) amino
Synthesis of -L-norvaline dihydrochloride Using the compound obtained in Example 7 as a starting material, a deprotection reaction was carried out in the same manner as in Example 4 to obtain δ- (2,3-dehydrothiazolin-2-yl) amino-L. -0.11 g of norvaline dihydrochloride was obtained. ¹ H-NMR (D ₂ O) δ: 1.68-1.89 (4H, m), 3.28-3.
49 (4H, m), 3.98-4.04 (1H, m),
4.69-4.78 (2H, m) FAB-MS (m / z) 218 (M ⁺⁺ 1)

[0035]

Example 9 α-Nt-butoxycarbonyl-ε- (S-methyl group
Sothioureido) -L-norleucine-t-butyleth
Synthesis of ter hydroiodide K. Narayanan's method (J. Med. Che
m. , 37,885-887, 1994), and α-Nt-butoxycarbonyl-ε-thioureido-L-norleucine-t-butyl ester (0.19).
g) was used as a reactant and methyl iodide was used as a reaction agent.
0.29 g of-(S-methylisothioureido) -L-norleucine-t-butyl ester hydroiodide was obtained. ¹ H-NMR (CDCl ₃ ) δ: 1.44 (9H, s), 1.47 (9H, s),
1.67-1.85 (6H, m), 2.81 (3H,
s), 3.32-3.53 (2H, m), 4.05-
4.18 (1H, m), 5.16-5.27 (1H,
m) FAB-MS (m / z) 376 (M ⁺ +1)

[0036]

Example 10 ε- (S-methylisothioureido) -L-norleucine
The synthesis of ε- (S-methylisothioureido) -L-norleucine dihydrochloride was carried out in the same manner as in Example 4 except that the compound obtained in Example 9 was used as a starting material. 15 g was obtained. ¹ H-NMR (D ₂ O) δ: 1.42-1.64 (2H, m), 1.68-1.
81 (2H, m) 1.90-2.10 (2H, m),
2.60 (3H, s), 3.38-3.43 (2H,
m), 4.09 (1H, t, J = 6.3 Hz) FAB-MS (m / z) 220 (M ⁺ +1)

[0037]

Example 11 α-Nt-butoxycarbonyl-δ- (S- (n-p
Ropyl) isothioureido) -L-norvaline-t-bu
Synthesis of tyl ester / hydroiodide The reaction was carried out in the same manner as in Example 1 using n-propyl iodide as a reactant to obtain α-Nt-butoxycarbonyl-δ.
-(S- (n-propyl) isothioureido) -L-norvaline-t-butyl ester / hydroiodide 0.7
5 g was obtained. ¹ H-NMR (CDCl ₃ ) δ: 1.09 (3H, t, J = 7.3 Hz) 1.44
(9H, s), 1.48 (9H, s), I.S. 77-1.
92 (6H, m), 3.33-3.38 (2H, m),
3.39-3.72 (2H, m), 4.05-4.17
(1H, m), 5.20-5.52 (1H, m)

[0038]

Example 12 δ- (S- (n-propyl) isothioureido) -L-
Synthesis of norvaline Using the compound obtained in Example 11 as a starting material, Example 2
In the same manner as described above, 0.31 g of δ- (S- (n-propyl) isothioureido) -L-norvaline was obtained. ¹ H-NMR (DMSO-d ₆ ) δ: 0.95 (3H, t, J = 7.3 Hz), 1.54.
−1.67 (6H, m), 1.92 to 2.02 (2H,
m), 3.13-3.30 (2H, m), 4.17-
4.27 (1H, m) FAB-MS (m / z) 234 (M ⁺ +1)

[0039]

Example 13 α-Nt-butoxycarbonyl-δ- (4H-5,6
-Dihydro-1,3-thiazin-2-yl) amino-L
-Norvaline-t-butyl ester / hydrobromide combination
The reaction was carried out using 1,3-dibromopropane as a reactant in the same manner as in Synthesis Example 1, and α-Nt-butoxycarbonyl-δ- (4H-5,6-dihydro-1,3-thiazine-2 was used. -Yl) Amino-L-norvaline-t-butyl ester / hydrobromide 0.19 g was obtained. ¹ H-NMR (CDCl ₃ ) δ: 1.44 (9H, s), 1.46 (9H, s),
1.55-1.91 (4H, m), 2.06-2.18
(2H, m), 2.96-3.00 (2H, m), 3.
29 (2H, t, J = 5.6Hz), 4.11-4.2
3 (1H, m), 5.39-5.48 (1H, m) FAB-MS (m / z) 388 (M ⁺ +1)

[0040]

Example 14 δ- (4H-5,6-dihydro-1,3-thiazine-2
Synthesis of -yl) amino-L-norvaline Using the compound obtained in Example 13 as a starting material, Example 2
In the same manner as δ- (4H-5,6-dihydro-1,3-
Thiazin-2-yl) amino-L-norvaline 0.13
g was obtained. ¹ H-NMR (D ₂ O) δ: 1.69-1.81 (6H, m), 2.20-2.
28 (2H, m), 3.17-3.30 (2H, m),
3.55-3.73 (3H, m) FAB-MS (m / z) 232 (M ⁺⁺ 1)

[0041]

Example 15 α-Nt-butoxycarbonyl-δ- (2,3-dehydroxy)
Dro-4-oxothiazolin-2-yl) amino-L-
Synthesis of norvaline-t-butyl ester The reaction was performed using methyl bromoacetate as a reactant in the same manner as in Example 1, and α-Nt-butoxycarbonyl-δ-
(2,3-dehydro-4-oxothiazolin-2-yl) amino-L-norvaline-t-butyl ester 0.
28 g was obtained. ¹ H-NMR (CDCl ₃ ) δ: 1.44 (9H, s), 1.46 (9H, s),
1.61-1.78 (4H, m), 3.35-3.40
(2H, m), 3.86 (2H, s), 4.11-4.
26 (1H, m) FAB-MS (m / z) 388 (M ⁺ +1)

[0042]

Example 16 δ- (2,3-dehydro-4-oxothiazoline-2-
Synthesis of yl) amino-L-norvaline Using the compound obtained in Example 15 as the starting material, Example 2
Similarly to δ- (2,3-dehydro-4-oxothiazolin-2-yl) amino-L-norvaline 0.12 g
I got ¹ H-NMR (D ₂ O) δ: 1.74-1.93 (4H, m), 3.42-3.
57 (2H, m), 3.72-3.76 (1H, m),
4.06 (2H, s) FAB-MS (m / z) 232 (M ⁺ +1)

[0043]

Example 17 α-Nt-butoxycarbonyl-δ- (S- (cyclo
Propylmethyl) isothioureido) -L-norvaline
Synthesis of -t-butyl ester hydrobromide The reaction was carried out using cyclopropylmethyl bromide as a reactant in the same manner as in Example 1, and α-Nt-butoxycarbonyl-δ- (S- (cyclo 0.54 g of propylmethyl) isothioureido) -L-norvaline-t-butyl ester hydrobromide was obtained. ¹ H-NMR (CDCl ₃ ) δ: 0.38-0.49 (2H, m), 0.63-0.
75 (2H, m), 1.00-1.18 (1H, m),
1.48 (9H, s), 1.76 (9H, s), 1.6
2-1.96 (4H, m), 3.34 (2H, d, J =
7.6 Hz), 3.36-3.53 (2H, m), 4.
07-4.21 (1H, m), 5.20-5.33 (1
H, m) FAB-MS (m / z) 402 (M ⁺ +1)

[0044]

Example 18 δ- (S- (Cyclopropylmethyl) isothiourei
De) Synthesis of -L-norvaline dihydrochloride Example 4 using the compound obtained in Example 17 as a starting material
The deprotection reaction was carried out in the same manner as in, to obtain 0.36 g of δ- (S- (cyclopropylmethyl) isothioureido) -L-norvaline dihydrochloride. ¹ H-NMR (D ₂ O) δ: 0.33-0.36 (2H, m), 0.64-0.
66 (2H, m) 1.09-1.14 (2H, m),
1.76-2.07 (4H, m), 3.10 (2H, m)
d, J = 7.3 Hz), 3.42-3.47 (2H,
m), 4.09-4.13 (1H, m) FAB-MS (m / z) 246 (M ⁺ +1).

[0045]

Example 19 α-Nt-butoxycarbonyl-2- (2-S-methyl)
Luisothioureidoethyl) glycine-t-butyleth
Synthesis of Ter-hydroiodide Example 1 using α-Nt-butoxycarbonyl-2- (2-thioureidoethyl) glycine-t-butyl ester as a starting material and methyl iodide as a reactant The reaction is performed in the same manner as in α-Nt-butoxycarbonyl-2
0.32 g of-(2-S-methylisothioureidoethyl) glycine-t-butyl ester / hydroiodide was obtained. ¹ H-NMR (CDCl ₃ ) δ: 1.45 (9H, s), 1.49 (9H, s),
1.77-2.06 (1H, m), 2.21-2.38
(1H, m), 2.79 (3H, s), 3.38-3.
56 (2H, m), 4.06-4.20 (1H, m),
5.30-5.67 (1H, m) FAB-MS (m / z) 348 (M ⁺⁺ 1)

[0046]

Example 20 2- (2-S-Methylisothioureidoethyl) glycy
Example The compound obtained in Example 19 of emissions dihydrochloride as starting material 4
The deprotection reaction was carried out in the same manner as above to obtain 0.18 g of 2- (2-S-methylisothioureidoethyl) glycine dihydrochloride. ¹ H-NMR (D ₂ O) δ: 2.23-2.35 (2H, m), 2.60 (3
H, s), 3.60-3.65 (2H, m), 4.04.
-4.09 (1H, m) FAB-MS (m / z) 192 (M ⁺ +1)

[0047]

Example 21 α-Nt-butoxycarbonyl-δ- (S- (3-
Lolopropyl) isothioureido) -L-norvaline-
Synthesis of t-Butyl Ester / Hydroiodide The reaction was conducted in the same manner as in Example 1 using 1-chloro-3-iodopropyl as a reactant to obtain α-Nt-butoxycarbonyl-δ- (S -(3-chloropropyl) isothioureido) -L-norvaline-t-butyl ester
0.475 g of hydroiodide was obtained. ¹ H-NMR (CDCl ₃ ) δ: 1.45 (9H, s), 1.49 (9H, s),
1.60-2.03 (4H, m), 2.18-2.32.
(2H, m), 3.35 (2H, t, J = 6.6H
z), 3.46-3.65 (2H, m), 3.71-
3.76 (2H, m), 3.99-4.22 (2H,
m), 5.16 (1H, m)

[0048]

Example 22 δ- (S- (3-chloropropyl) isothioureido)
Synthesis of -L-norvaline dihydrochloride Example 4 using the compound obtained in Example 21 as a starting material
The deprotection reaction was carried out in the same manner as in, to obtain δ- (S- (3-chloropropyl) isothioureido) -L-norvaline dihydrochloride. _{^{1 H-NMR (DMSO-d}} 6) δ: 1.54-1.95 (4H, m), 1.98-2.
13 (2H, m), 3.22-3.65 (4H, m),
3.69-3.78 (2H, m), 3.85-3.98
(1H, m)

[0049]

[Table 1] Tables 1 to 3 show the chemical structural formulas of the example compounds.

[0050]

[Test Example] The inhibitory action of the compounds of the present invention on NOS isoforms was compared and examined with existing NOS inhibitors. N-cNO was used as a crude enzyme fraction of each NOS isoform.
S is a soluble fraction of rat cerebral cortex, E-cNOS is a homogenate of bovine pulmonary artery vascular endothelial cell line (CPAE), iN
For OS, LPS (10 mg / kg, ip) was administered to rats, and the soluble fractions of the lungs extracted 6 hours later were used.

The following NOS inhibitors were used as control compounds. N ^G -nitro-L-arginine
(L-NNA), N ^G -cyclopropyl-L-
arginine (L-CPA), N ^G -nitro-
L-arginine methyl ester (L
-NAME), N ^G -amino-L-arginin
e (L-AA), N ^G -iminoethyl-orn
itine (L-NIO), ^NG- monometh
yl-L-arginine (L- NMMA), N G -
allyl-L-arginine (L-ALA), 7
-Nitroindazole (7-NI), and a
minoguanidine (AG).

A crude enzyme preparation of N-cNOS was prepared by the following procedure. Untreated male SD rats (weight 300-4
00g) was decapitated, the whole brain was quickly removed, and the cerebral cortex was separated on ice. Then 5 volumes of 50 mM Tris
-HCl, 1 mM DTT (pH 7.4) solution was added,
Homogenize for 3 minutes and centrifuge at 1,000 xg for 10 minutes. The resulting supernatant is 6 at 100,000 xg
After centrifugation for 0 minute, the soluble cytoplasmic fraction of the finally obtained supernatant was used as a crude enzyme preparation of N-cNOS.

A crude enzyme preparation of E-cNOS was prepared by the following procedure. 2 bovine pulmonary artery vascular endothelial cell lines (CPAE)
The cells were cultured in MEM medium containing 0% 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
d saline; PBS) (pH 7.4) was added,
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.

A crude enzyme preparation of iNOS was prepared by the following procedure. Lipopolysaccharide
(Challide, LPS) (10 mg / kg) was intraperitoneally administered to a rat, and 6 hours later, it was transcardially perfused with physiological saline containing 10 U / ml heparin, and then the lung was extracted. 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 converted to iNO.
It was used as a crude enzyme preparation of S.

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] citrulline (Nagafuji et al.,
in Brain Edema IX (Ito et
al. eds. ) 60, pp. 285-288,199
4, Acta Neurochir. , Springe
r-Verlag).

The reaction solution was 100 μM L- [ ³ H] ar.
gine, soluble fraction (6-20 μg / ml protein), 1.25 mM CaCl ₂ , 1 mM EDTA,
10 μg / ml calmodulin, 1 mM NA
DPH, 100 μM tetrahydrobiopt
erine, 10 μM FAD, 10 μM FMN, 5
It was composed of 0 mM Tris-HCl (pH 7.4) to which the compound of the present invention or 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 MEDTA was added and placed on ice to stop the reaction. The reaction solution is a cation exchange resin column (Dowex
AG50WX-8, Na ⁺ form, 3.2 ml), and left unreacted substrate L- [ ³ H] argini.
ne and the reaction product L- [ ³ H] citrullin
e was separated. This eluate and the eluate obtained by passing 3 ml of distilled water through the column were put in a mini vial, and L- [
³ H] citrullline was collected. Then 5m
l scintillator was added, and the radioactivity was measured with a liquid scintillation counter, and L- [ ³ H] citrull was measured.
ine was quantified. The protein concentration in the crude enzyme preparation was determined using a micro assay kit manufactured by Bio-Rad.

Table 4 shows IC ₅₀ values (concentration required for 50% activity inhibition) of each test compound for each NOS isoform and E-cNOS of IC ₅₀ values for N-cNOS as an index showing selectivity. IC for iNOS
The ratios of ₅₀ values are shown respectively. All the values in the table are shown as mean ± standard error, and the number of cases is shown in parentheses.

From Table 4, 1. The compounds of Examples 2 and 4 have a stronger N-cNOS inhibitory action than existing NOS inhibitors. 2. The compounds of Examples 2 and 4 were N-cNOS and E-cN.
In terms of selectivity for OS, it shows higher N-cNOS selectivity than existing NOS inhibitors. 3. The compound of Example 2 exhibits significantly higher N-cNOS selectivity than existing NOS inhibitors in terms of selectivity for N-cNOS and iNOS. 4. The compound of Example 6 has a significantly stronger iNOS inhibitory action than existing NOS inhibitors. 5. The compound of Example 6 exhibits extremely high iNOS selectivity in selectivity to N-cNOS and iNOS. It is clear.

[0060]

INDUSTRIAL APPLICABILITY The compound of the present invention has a selective inhibitory effect on N-cNOS in the brain, which is far superior to existing NOS inhibitors, and is a therapeutic agent for cerebrovascular disorders (especially in the acute stage of obstructive cerebrovascular disorder). It is useful as a therapeutic agent. In particular, Example 2,
The compound of 4 has a strong intracerebral N-cNOS inhibitory action, and at the same time, has a significantly high intracerebral N-cNOS selectivity in selectivity for N-cNOS and E-cNOS.
It is useful as a therapeutic agent for cerebrovascular disorders (particularly a therapeutic agent in the acute phase of obstructive cerebrovascular disorder). In addition, it has a significantly superior iNOS selective inhibitory action compared to existing NOS inhibitors,
It is useful as a therapeutic agent for hypotension in sepsis. In particular,
The compound of Example 6 has a strong iNOS inhibitory action and, at the same time, has a high iNOS selectivity with respect to N-cNOS and iNOS, and is useful as a therapeutic agent for hypotension in sepsis.

[Table 2]

[Table 3]

[Table 4]

Continuation of the front page (51) Int.Cl. ⁶ Identification number Office reference number FI technical display location A61K 31/425 31/54 ABV 31/55

Claims (6)

[Claims] 1. A compound represented by the general formula (I): (In the formula, R ₁ is a hydrogen atom, a linear or branched alkoxycarbonyl group having 1 to 6 carbon atoms in which the alkyl portion may have a substituent, or a straight chain optionally having a substituent. Represents a chain- or branched-chain acyl group having 1 to 9 carbon atoms; R ₂
Represents a hydrogen atom, a linear or branched alkyl group having 1 to 6 carbon atoms which may have a substituent; R ₃ represents a linear or branched chain which may have a substituent. Carbon number 1
~ 6 alkyl group, represents a linear or branched alkenyl group having 2 to 6 carbon atoms which may have a substituent; R ₄
Represents a hydrogen atom; or, R ₃ and R ₄ may be taken together to form a 5 to 7 membered ring which may have a substituent; n is an integer of 2 to 4; Represent Provided that R ₃ is a methyl group and n is 3 at the same time) or a pharmaceutically acceptable salt thereof. 2. A compound represented by the general formula (I): (In the formula, R ₁ is a hydrogen atom, a linear or branched alkoxycarbonyl group having 1 to 6 carbon atoms in which the alkyl portion may have a substituent, or a straight chain optionally having a substituent. Represents a chain- or branched-chain acyl group having 1 to 9 carbon atoms; R ₂
Represents a hydrogen atom or a linear or branched alkyl group having 1 to 6 carbon atoms which may have a substituent; R ₃ represents a linear or branched carbon atom substituted with a halogen atom. Number 1
Represents 6 alkyl group; _{R 4} represents a hydrogen atom; n
Represents an integer of 2 to 4. ] The compound of Claim 1 represented by these, or its pharmaceutically acceptable salt. 3. A compound represented by the general formula (I): (In the formula, R ₁ is a hydrogen atom, a linear or branched alkoxycarbonyl group having 1 to 6 carbon atoms in which the alkyl portion may have a substituent, or a straight chain optionally having a substituent. Represents a chain- or branched-chain acyl group having 1 to 9 carbon atoms; R ₂
Represents a hydrogen atom or a linear or branched alkyl group having 1 to 6 carbon atoms which may have a substituent; R ₃ represents 2
Represents a fluoroethyl group; R ₄ represents a hydrogen atom;
n represents an integer of 2 to 4. ] The compound of Claim 1 represented by these, or its pharmaceutically acceptable salt. 4. General formula (I): (In the formula, R ₁ and R ₂ each independently represent a hydrogen atom;
₃ is a linear or branched alkyl group having 1 to 6 carbon atoms which may have a substituent, or a linear or branched alkyl group having 2 to 6 carbon atoms which may have a substituent. Represents an alkenyl group; R ₄ represents a hydrogen atom; or R ₃ and R ₄ may be taken together to form a 5- to 7-membered ring which may have a substituent; n is , 2 to 4 are represented. However, R
The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein n is 3 at the same time when ₃ is a methyl group. 5. δ- (S- (2-Fluoroethyl) isothioureido) -L-norvaline, δ- (S-ethylisothioureido) -L-norvaline, and δ- (S-
The compound according to claim 1, which is selected from the group consisting of (3-chloropropyl) isothioureido) -L-norvaline, or a pharmaceutically acceptable salt thereof. 6. δ- (S-allylisothioureido) -L
-The compound according to claim 1, which is norvaline, or a pharmaceutically acceptable salt thereof.