JP2003252754A - Ast inhibitor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an AST (Airway Specific Trypsin-like Protease) inhibitor which is clinically applicable as a prophylactic and/or therapeutic agent for chronic respiratory tract diseases. <P>SOLUTION: The AST inhibitor comprises a guanidine derivative represented by a general formula (I) (wherein A is methylene group, ethylene group or vinylene group; R<SP>1</SP>and R<SP>2</SP>are same or different and they are each hydrogen atom or a 1-3C alkyl group), its pharmaceutically acceptable salt or its solvation product. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a human airway trypsin-like enzyme (hereinafter referred to as “airway-specific trypsin-like enzyme” or “AST (Airway Specific Try).
"Psin-like Protease)") inhibitors.

[0002]

2. Description of the Related Art In recent years, AST has been purified from sputum of human chronic airway inflammation patients (Japanese Patent Laid-Open No. 7-067640, Yasuka S. et al., Am. JR).
espir. Cell Mol. Biol. , 16: p
300-308, 1997), and its amino acid sequence and cDNA sequence have been clarified (JP-A-8-892).
46, US5804410, EP699
763, Yamaoka K .; et al. ，
J. Biol. Chem. , 273 (19): 1189.
5-11901, 1998).

Regarding the activity of this enzyme, in v
Some studies have been done in vitro. Since AST has an effect on mucociliary motility, and has an action of enhancing the production of cytokines such as IL-8 and GM-CSF from human bronchial epithelial cell lines (Terao Noriko et al., 1998.
(The Japanese Society of Respiratory Sciences), the possible involvement of airway inflammation in the pathology is considered. Furthermore, AST is a fibrinogen degrading activity (Yoshinaga S. et a.
l. J. Med. Invest. , 45: 77-8
6, 1998), and plasminogen activator (prourokinase) activating action, etc. (Yoshinaga Junko et al., 1998, Protease and Inhibitors Study Group in Disease State and Treatment (Co
nference on Proteases and
Inhibitors in Pathophysi
logic and Therapeutics)),
It acts anti-inflammatory through fibrin formation on the mucosal surface of the respiratory tract, chronic airway disease (chronic obstructive pulmonary disease (including chronic bronchitis, emphysema, diffuse panbronchiolitis, bronchiectasis), bronchial asthma, In sinus bronchial syndrome, pulmonary fibrosis, airway hypersensitivity, and pulmonary hypertension), it may be deeply involved in the pathophysiology, and may be involved in cancer growth or metastasis. ing.

[0004] By the way, in chronic airway diseases, as a pathological condition that has become a problem along with persistent inflammation, there is an exacerbation of the pathological condition due to an excessive increase in mucus secretion (Jeffe.
ry, P.I. K. et al. , Am. J. Respi
r. Crit. Dare. Med. , 150: S6-
13, 1994). The effects of macrolide drugs such as erythromycin on DPB (diffuse panbronchiolitis) belonging to the above-mentioned diseases are known in Japan (Nagai, H. et al., Respira).
tion, 58 (3-4): 145-149), and is not used favorably in Europe and America because it is an antibiotic.

Further, although there are various theories regarding target molecules that are the main cause of overproduction of mucus and hyperplasia of secretory cells (Christian, P. et al.
et al. J. Clin. Invest. , 85: 6
82-689, 1990), there is still no effective therapeutic drug among the drugs created based on them. Therefore, COPD (Chronic Obstructi
In chronic airway diseases represented by ve Pulmonary Disease, an effective drug that suppresses excessive secretion and production enhancement of mucus is desired worldwide.

On the other hand, a compound that inhibits AST is AST
That the production of mucus glycoproteins caused by AST can be suppressed, and that the expression of Muc5AC gene caused by AST can be suppressed, and proEGF and pro on the cell membrane surface caused by AST can be suppressed.
The fact that the cleavage of HB-EGF is suppressed is related to international application J
It has been clarified in P01 / 07349, indicating that the AST inhibitor may suppress the excessive secretion and production enhancement of mucus.

Therefore, the AST inhibitor can be a new preventive and / or therapeutic agent for chronic airway diseases and cancers due to the direct and indirect actions of AST.

On the other hand, compounds of the general formula (I), for example 4
The mesylate salt of N, N-dimethylcarbamoylmethyl ester of-(4-guanidinobenzoyloxy) phenylacetic acid is a known compound and is described in US Pat. No. 402147.
No. 2 describes that it has a plasmin inhibitory action and a trypsin inhibitory action and is used for treating hemorrhagic diseases and acute pancreatitis. However, it has never been known that the compound of the general formula (I) inhibits AST.

[0009]

An object of the present invention is to provide a clinically applicable AST inhibitor as a preventive and / or therapeutic drug for chronic airway diseases and the like.

[0010]

[Means for Solving the Problems] Under the above-mentioned problems, the inventors of the present invention have conducted extensive studies to find compounds that inhibit AST, and as a result, compounds of the following general formula (I) Surprisingly, they discovered that they have AST inhibitory activity and completed the invention. That is, the present invention provides the following formula (I)

[0011]

[Chemical 2]

(In the formula, A is a methylene group, an ethylene group, or a vinylene group, and R ¹ and R ² may be the same or different and each represents a hydrogen atom or a C1-C3 alkyl group). The AST inhibitor is characterized by containing the indicated guanidine derivative, a pharmaceutically acceptable salt thereof, or a solvate thereof (hereinafter sometimes referred to as "guanidine derivative etc.").

The present invention also provides a guanidine derivative in which A is a methylene group in the general formula (I) and R ¹ and R ² are each a methyl group, a pharmaceutically acceptable salt thereof, or a solvate thereof. It is an AST inhibitor characterized by containing a substance.

The present invention further comprises a methanesulfonate salt of a guanidine derivative in which A is a methylene group in the general formula (I) and R ¹ and R ² are each a methyl group, or a solvate thereof. Is an AST inhibitor.

Further, the present invention is a preventive and / or therapeutic agent for chronic airway diseases containing an AST inhibitor.

[0016]

BEST MODE FOR CARRYING OUT THE INVENTION An object of the present invention is to administer a guanidine derivative represented by the general formula (I), or a prodrug thereof, to inhibit AST, prevent chronic airway disease and / or Provide a therapeutic agent.

[0017]

[Chemical 3]

(In the formula, A is a methylene group, an ethylene group, or a vinylene group, and R ¹ and R ² may be the same or different and each represents a hydrogen atom or a C1-C3 alkyl group.) In the formula (I), A is a methylene group, an ethylene group or a vinylene group, preferably a methylene group.

R ¹ and R ^{2 in} the general formula (I) may be the same or different and each is a hydrogen atom or a C _{1 to} C ₃ alkyl group. R ¹ and R ² are preferably the same substituent. The “C ₁ -C ₃ alkyl group” has ₁ to ₃ carbon atoms.
Of straight-chain and branched-chain alkyl, and represents, for example, methyl, ethyl or n-propyl, preferably a methyl group.

The "pharmaceutically acceptable salt" of the present invention is, for example, an inorganic acid salt such as hydrochloric acid, sulfuric acid, nitric acid or phosphoric acid,
Alternatively, an organic acid salt such as paratoluenesulfonic acid, methanesulfonic acid, oxalic acid or citric acid can be mentioned, and methanesulfonic acid is preferable.

The compound of the present invention may form a solvate by coordinating with a suitable organic solvent and / or water, and examples thereof include a hydrate.

Examples of the compound represented by the formula (I) include 4- (4-guanidinobenzoyloxy) phenylacetic acid carbamoylmethyl ester, 4- (4-guanidinobenzoyloxy) phenylacetic acid N-methylcarbamoylmethyl ester, 4- (4-guanidinobenzoyloxy) phenylacetic acid N, N-dimethylcarbamoylmethyl ester, 4- (4-guanidinobenzoyloxy) phenylpropionic acid carbamoylmethyl ester, 4- (4-guanidinobenzoyloxy) phenylpropionic acid N- Methyl carbamoyl methyl ester,
4- (4-guanidinobenzoyloxy) phenylpropionic acid N, N-dimethylcarbamoylmethyl ester and the like can be mentioned.

Among the compounds of the general formula (I), particularly preferably, the mesylate salt of 4- (4-guanidinobenzoyloxy) phenylacetic acid N, N-dimethylcarbamoylmethyl ester represented by the following formula (X) (general formula: Name: camostat mesylate).

[0024]

[Chemical 4]

The preparation of the AST inhibitor compound of the present invention represented by the general formula (I) is carried out according to the above-mentioned US Pat. No. 402147.
It can be carried out by the method described in No. 2. That is, guanidinobenzoic acid halide (II) and the general formula (I
It can be obtained by reacting the compound represented by II) in the presence of a dehydrohalogenating agent.

[0026]

[Chemical 5]

(In the above reaction formula, X represents halogen,
A, R ¹ and R ² have the same definitions as in formula (I). In order to proceed the above reaction, the raw material (II) and the raw material (III) may be heated and refluxed in an organic solvent such as acetonitrile in the presence of a dehydrohalogenating agent such as triethylamine.

The starting material guanidinobenzoic acid halide (II) can be obtained from p-guanidinobenzoic acid by a conventional method. For example, thionyl chloride and p-guanidinobenzoic acid can be heated to obtain the corresponding acid chloride. You can In addition, the raw material (III) can be synthesized by the reaction shown in the following formula.

[0029]

[Chemical 6]

(In the above reaction formula, X'represents halogen,
A, R ¹ and R ² have the same definitions as in formula (I). That is, the raw material (VI) and the raw material (V) are mixed in an organic solvent such as acetonitrile, THF and benzene in the presence of an organic base such as triethylamine for 2 to 20 hours at 60 ° C to 150 ° C.
The desired raw material (III) can be obtained by heating at ° C.

The desired product compound (I) is obtained as a salt with hydrogen halide, and the carbonate of the general formula (I) can be obtained by adding an aqueous sodium bicarbonate solution to the reaction solution. By using this carbonate, it can be converted into an inorganic acid salt such as hydrochloric acid, sulfuric acid, phosphoric acid or the like, or an organic acid salt such as acetic acid, methanesulfonic acid or the like.

The activity of the AST inhibitor containing the guanidine derivative represented by the general formula (I) and the like can be measured by the following method. That is, 2 recombinant AST
ng / mL, as a stabilizer for recombinant AST such as Tw
50 mM Tris-HCl buffer solution (pH 8.00) containing 0.01% of een20 (trade name, manufactured by SIGMA).
6) DMS containing a test compound against 0.05 mL
2 μL of O (dimethyl sulfoxide) solution, and the synthetic substrate Boc-Phe-Ser-Arg-MCA (MC
50 m containing 30 μM of A: methylcoumarinamide)
M Tris-HCl buffer (pH 8.6) 0.05m
Add L and incubate at 24 ° C. for 6 minutes. The amount of AMC produced over time was measured by fluorescence measurement (fluorescence 46
0 nm, excitation light 380 nm), and the ability of the compound to inhibit the enzyme activity is measured. By this method, it is possible to determine the IC50 (molar concentration showing 50% inhibitory activity) as the inhibitory activity value by changing the concentration of the test compound.

The IC50 value of the guanidine derivative represented by the general formula (I) is 0.1 to 100 μM, preferably 0.1 to 10 μM, more preferably 0.1 to 1 μM.
It is M.

From this fact, the guanidine derivative represented by the general formula (I) can be used as an AST inhibitor having AST inhibitory activity, and further, it can be used as a preventive agent for chronic airway disease or cancer and / or Alternatively, it can be clinically applied as a therapeutic agent.

The administration methods include oral administration and parenteral administration. The dose can be appropriately increased or decreased depending on the patient's symptoms, age, body weight, and degree of disease symptoms. For example, in the case of oral administration, 1 to 3000 mg per adult
/ Day, preferably 10 to 500 mg / day is suitable.
Examples of dosage forms include tablets, capsules, powders and granules. These can be produced by known formulation techniques together with usual excipients, lubricants, additives such as binders and the like. In the case of parenteral administration, 0.01 to 300 mg / day, preferably 0.1 mg to 50 mg / day, per adult is administered by respiratory tract, subcutaneous administration, intravenous administration, intravenous drip infusion, suppository, etc. It can be applied by rectal administration or the like.

AST inhibitors are described in International Application JP01 / 07
As disclosed in 349, it is expected to be used as a preventive agent and / or a therapeutic agent for a disease associated with a condition associated with AST. That is, AST has secretory cell-promoting action or mucus production-promoting action, action in coagulation / fibrinolysis system, action on airway remodeling, action on airway inflammation, fibroblast / epithelial cell / hair cell / smooth muscle cell・ Suppress or modify physiological effects such as effects on goblet cell proliferation and damage, effects on cancer growth and metastasis, effects on mucociliary movement, or antiviral infection,
It can be expected to be used for improvement and treatment of pathological conditions.

Further, as the AST inhibitor, AST is PAR
By activating (protease activated receptor),
Secretion promoting action on secretory cells of PAR (mucus gland cells, serous gland cells, goblet cells promoting mucus production, nerve cells / neuroendocrine cells promoting secretion, chloride ion secretion promoting action on airway epithelial cells, etc.), epithelial cells Or, relaxation action of blood vessels, respiratory tracts, intestinal tract, etc. via endothelial cells, contraction action to smooth muscle, inflammatory cytokine production induction and enhancement action of fibroblasts and epithelial cells, fibroblast / epithelial cells (cilia cells, Expected to be used for the improvement and treatment of pathological conditions by suppressing or modifying the effects on cell proliferation and disorders of goblet cells, basal cells), smooth muscle cells, secretory gland cells, etc., and physiological effects such as enhancing hypersensitivity to nerve cells. it can.

[0038] As the AST inhibitor, AST is EGFR
Route (Epidermal Growth Fact)
r Receptor pathway)
Secretory cells (mucous gland cells, serous gland cells, goblet cells, squamous cells, cancer cells, etc.) as an effect of EGFR activation
Secretion-enhancing action and cytokine production-enhancing action on fibroblasts, endothelial cells, epithelial cells, smooth muscle cells, and other inflammatory cytokine production-inducing and enhancing actions on various cells having EGFR pathway, fibroblasts, endothelial cells, Epithelial cells, hair cells, goblet cells, basal cells, squamous cells, cancer cells, smooth muscle cells, secretory gland cells, etc. EGFR
It can be expected to suppress or modify physiological effects such as cell proliferation effect on various cells having a pathway and to be used for improvement and treatment in pathological conditions.

[0039]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited thereto.

[Reference Example] Preparation of AST JP-A-8-89246, US5804410, EP699733, and Yamaok
a K. et al. J. Biol. Chem. , 2
73 (19): 11895-11901, 1998, a recombinant baculovirus vector was prepared based on the cDNA sequence disclosed, and recombinant AST was produced by infecting insect cells, and then recombinant from the cell fraction. AST was purified.

That is, insect cells (Tn-5 cells) were grown in a monolayer to a density of 5 × 10 ⁶ cells / mL, the medium was removed, and then MOI (Multiplicity) per cell.
Serum-free medium containing recombinant AST-expressing baculovirus is added so that yof Infection = 0.2-0.5 to infect insect cells (Tn5 cell line).
The cells were cultured for one day to express AST. The expressed protein was confirmed by Western blotting using an SDS-PAGE and an anti-AST peptide antibody (Anal. Bioche.
m. , 112, 195-203, 1981).

Next, the culture solution of the recombinant AST-expressing baculovirus-infected insect cells was centrifuged (about 500 × g),
Separated into supernatant and cells. The cell fraction corresponding to 450 mL of the culture solution was added to M-PER (Mammalian Prote
in Extraction Reagent, PIER
It was suspended in 100 mL (manufactured by CE) and incubated at room temperature for 30 minutes. Further sonication for 15 minutes on ice,
Solubilized. The obtained solubilized product was added at 10,000 rpm, 4
Centrifugation was performed at 30 ° C. for 30 minutes, and the supernatant was collected. 1 liter of 50 mM sodium acetate (pH 4) /
After dialysis with 0.01% PEG6000 (polyethylene glycol, trade name, manufactured by Wako Pure Chemical Industries, Ltd.) at room temperature for 2 hours with stirring, the mixture was centrifuged at 4 ° C. and 10,000 rpm for 30 minutes to collect the supernatant.

Next, BSA was added to this sample (final concentration 100 μg / mL), and 2 liters of 50 mM Tris was added.
-HCl (pH 8) /0.5M NaCl / 0.01%
After dialysis twice with PEG6000, 1000 at 4 ℃
The supernatant was recovered by centrifugation at 0 rpm for 30 minutes. The collected supernatant was placed on benzamidine sepharose 6B (trade name, manufactured by Amersham Pharmacia) (10 mL bed volume), and 100 mL of 50 mM Tris-HCl.
(PH 8) /0.5M NaCl / 0.01% PEG6
The column was washed at 000. Further 80mL 10m
After washing the column with M sodium phosphate buffer (pH 7.5), the bound protein was eluted with 10 mM HCl (pH 2).

After elution, the absorbance (280 nm) of each fraction was measured, and the main peaks were collected. And SDS polyacrylamide gel electrophoresis (SDS-P
AGE) to detect a protein having a molecular weight of about 30 kD,
After confirmation, this was cryopreserved as a purified recombinant AST standard.

[Example] Evaluation of AST inhibitory activity of camostat mesylate 2 ng / mL Tw of recombinant AST obtained in Reference Example
50 mM Tris-HCl buffer containing 0.01% of een20 (trade name, manufactured by SIGMA) (pH 8.
6) To 0.05 mL, 2 μL of DMSO solution containing camostat mesylate (manufactured by Wako Pure Chemical Industries, Ltd.), and synthetic substrate Boc-Phe-Ser-Arg-MCA (MC
50 m containing 30 μM of A: methylcoumarinamide)
M Tris-HCl buffer (pH 8.6) 0.05m
L was added and incubated at 24 ° C. for 6 minutes. The amount of AMC produced over time was measured by fluorescence measurement (fluorescence 46
0 nm, excitation light 380 nm), and the ability of the compound to inhibit the enzyme activity was evaluated. The results are shown in Table 1 as IC50 (molar concentration showing 50% inhibitory activity).

[0046]

[Table 1]

[0047]

The compound of formula (I) of the present invention is
It exhibits excellent AST inhibitory activity and is useful as an AST inhibitor. Further, due to this action, the compound represented by the formula (I) of the present invention can be used as a novel prophylactic and / or therapeutic agent for chronic airway diseases and the like.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61P 43/00 105 A61P 43/00 105 111 111 (72) Inventor Hiroshi Eguchi 4-3 Asahigaoka, Hino City, Tokyo No. 2 F-Term in Tokyo Research Center, Teijin Limited (reference) 4C206 AA01 AA02 AA03 GA09 GA22 MA01 MA04 NA14 ZA59 ZB21 ZB26 ZB33 ZC20

Claims (4)

[Claims] 1. The following formula (I): (In the formula, A is a methylene group, an ethylene group, or a vinylene group, and R ¹ and R ² may be the same or different and each represents a hydrogen atom or a C1-C3 alkyl group.)
And the guanidine derivative, a pharmaceutically acceptable salt thereof, or a solvate thereof. 2. A guanidine derivative, wherein A in the formula (I) is a methylene group, and R ¹ and R ² are each a methyl group, a pharmaceutically acceptable salt thereof, or a solvate thereof. The AST inhibitor according to claim 1, wherein 3. In the formula (I), methanesulfonate of a guanidine derivative in which A is a methylene group and R ¹ and R ² are each a methyl group, or a solvate thereof is contained. The AST inhibitor according to claim 1. 4. A according to any one of claims 1 to 3.
A prophylactic and / or therapeutic agent for chronic airway disease containing an ST inhibitor.