JPH0841043A - Epoxy succinic acid derivative - Google Patents

Abstract

(57) [Summary] [Object] To provide an easily synthesized epoxysuccinic acid derivative having a bone resorption inhibitory effect. [Structure] [Chemical 1] [Wherein R ¹ represents a hydrogen atom or an alkyl group having 1 to 9 carbon atoms, which may be substituted with an alkoxy group having 1 to 3 carbon atoms, an alkenyl group, an alkynyl group, or an aralkyl group, and R ² represents R ³ represents an alkyl group having 3 to 4 carbon atoms, and R ³ represents an alkyl group having 2 to 5 carbon atoms. ]
An epoxysuccinic acid derivative represented by or a salt thereof.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a novel epoxysuccinic acid derivative having a bone resorption inhibitory action or a salt thereof.

[0002]

2. Description of the Related Art In normal bone tissue, a phenomenon called remodeling in which old bone is replaced with new bone occurs due to a good balance between bone resorption and bone formation. Bone diseases such as osteoporosis, hypercalcemia, and Paget's disease of bone are conditions caused by an imbalanced state in which bone resorption exceeds bone formation for some reason. For the prevention or treatment of these diseases, estrogen preparations, calcitonin preparations, active vitamin D preparations, calcium preparations, phosphorus preparations, isoflavone preparations, etc. have been used, but the effects are uncertain, the administration method, The target was limited.

On the other hand, an enzyme that decomposes collagen, which is a bone supporting protein in bone resorption, is considered to be a lysosomal cysteine protease, and L- which is known as a specific inhibitor of cysteine protease.
It is known that trans-epoxysuccinyl-L-leucylamide (4-guanidino) butane (hereinafter abbreviated as E-64) suppresses bone resorption (Delais).
se et al., Biochemical and Biophysical Research Communications (Biochem. Bioohys.Res.
Commun), Vol. 125, No. 2, 441-447.
P., 1984).

Therefore, it is considered that the epoxysuccinic acid derivative suppresses bone resorption and is useful as a prophylactic or therapeutic agent for bone diseases, and bone diseases caused by the administration of E-64 or estatin, which is also a kind of epoxysuccinic acid derivative. Has been proposed (Japanese Patent Laid-Open No. 63-284127,
JP-A-2-218610).

[0005]

However, the above-mentioned E
-64 and estatin can be produced by a method of culturing a microorganism (Japanese Patent Publication No. 54-8759, JP-A No. 62-76).
No.) or synthetic method [Agricultural and
Biological Chemistry (Agr.Biol.Chem.), 4th
Volume 2, Issue 3, pages 529-536 (1976), JP-A-62-7
No. 6] is known, but any of the compounds has a low production amount in the culturing method, and in the synthetic method, in the reactions such as the introduction and deprotection of the guanidino group moiety commonly present in both,
There are many problems in yield, purification method, etc., making mass production difficult. Therefore, it is desired to develop a derivative that has a strong bone resorption inhibitory action and is easy to synthesize.

[0006]

Means for Solving the Problems As a result of intensive studies on various epoxysuccinic acid derivatives, the present inventors have found that an epoxysuccinic acid derivative represented by the following formula is easy to synthesize and has a strong bone resorption inhibitory action. The present invention has been completed and the present invention has been completed.

That is, the present invention is

[0008]

[Formula 2]

[Wherein R ¹ is a hydrogen atom or an alkyl group having 1 to 9 carbon atoms, which may be substituted with an alkoxy group having 1 to 3 carbon atoms, an alkenyl group, an alkynyl group,
Alternatively, R ² is an alkyl group having 3 to 4 carbon atoms, and R ³ is an alkyl group having 2 to 5 carbon atoms. ] The epoxy succinic acid derivative represented by these, or its salt.

In the present invention, the alkoxy group is, for example, a methoxy group, an ethoxy group, etc., and the alkyl group is, for example, a straight chain or branched chain such as methyl, ethyl, isopropyl, t-butyl, cyclopentyl or cyclopropylmethyl,
Cyclic alkyl groups, alkenyl groups such as vinyl, 2-methyl-1-propenyl group, 2-cyclohexenyl group and other straight-chain, branched, cyclic alkenyl groups, alkynyl groups such as 2-propynyl group, A 3-butynyl group and the like are referred to as an aralkyl group, for example, a benzyl group, 3-
A phenylpropyl group and the like are shown.

The compound of the formula (1) in which R ¹ is a hydrogen atom can form a salt, and the salt is a pharmaceutically acceptable salt with an alkali metal such as sodium or potassium, calcium or magnesium. And the like, salts with alkaline earth metals such as, salts with organic amines such as triethylamine and pyridine, ammonium salts, salts with basic amino acids such as lysine, arginine, and ornithine.

The compound of the present invention can be produced, for example, by the following method.

[Manufacturing scheme]

[0014]

Embedded image

(In Chemical Formula 3, R ² and R ³ are synonymous with the above, R ⁴ represents a protecting group for an amino group that is commonly used in the field of peptide synthesis chemistry, and R ⁵ represents in the field of peptide synthesis chemistry. Shows a commonly used protective group for a carboxyl group.) R
^{In 4} , the amino-protecting group usually used in the field of synthetic peptide chemistry includes, for example, t-butoxycarbonyl group, benzyloxycarbonyl group, p-nitrobenzyloxycarbonyl group, p-methoxybenzyloxycarbonyl group, 9- Fluorenylmethyloxycarbonyl group and the like.

In R ⁵ , a carboxyl group usually used in the field of peptide synthesis chemistry includes, for example, benzyl group, paramethoxybenzyl group, paranitrobenzyl group, t-butyl group, benzhydryl group, methyl group,
For example, an ethyl group.

The manufacturing method of the present invention will be described below for each step.

Step a: An amino acid derivative represented by the formula (2), which is synthesized from valine, leucine, isoleucine, norvaline, norleucine, etc., using the method and conditions usually used in the field of peptide synthesis chemistry, and the formula (2) 3)
1.0 to 5.0 molar equivalents of the amine represented by N, N'-dicyclohexylcarbodiimide (DCC), N-ethyl-N 'in a solvent such as chloroform, dichloromethane, ethyl acetate, N, N-dimethylformamide. -(3
-Dimethylaminopropyl) carbodiimide hydrochloride (E
DC.HCl), benzotriazol-N-hydroxytrisdimethylaminophosphonium hexafluorophosphate (BOP reagent), carbonyldiimidazole (C
DI), a method using a condensing agent, a mixed acid anhydride method, an acid halide method, an acid azide method, an active ester method, and the like, which are usually used in the field of peptide synthesis chemistry, and condensed using a condition (4) A compound represented by is obtained.

Step b: The protecting group for the amino group of the compound represented by the formula (4) is treated with a catalyst such as palladium carbon or palladium black in a solvent such as methanol, ethanol, ethyl acetate, dioxane or N, N-dimethylformamide. , Catalytic transfer hydrogenation (CTH) method, method using acid such as trifluoroacetic acid, methanesulfonic acid, hydrobromic acid, hydrochloric acid, or method using base such as piperidine or piperazine The compound represented by the formula (5) is obtained by removing the compound according to a method or condition usually used in the field of synthetic peptide chemistry.

Step c: Chemical Pharmaceutical Bulletin (Chem.Pharm.Bull.), Volume 35, pp. 1098 to 1104 (1987), which can be produced according to the formula ( The epoxysuccinic acid derivative represented by 6) and the compound represented by the formula (5) (1.0 to 2.0 molar equivalents) are mixed with chloroform, dichloromethane, ethyl acetate, N,
The compound represented by the formula (1) in which R ¹ is R ⁵ is reacted in a solvent such as N-dimethylformamide and the formula (1-
A compound represented by 1) is obtained.

Step d: The protecting group of the carboxylic acid of the compound represented by the formula (1-1) is treated with methanol, ethanol, N,
Catalytic transfer hydrogenation (CT) using a catalyst such as palladium carbon or palladium black in a solvent such as N-dimethylformamide.
H) method or a hydrolysis method using an acid such as trifluoroacetic acid, methanesulfonic acid, hydrobromic acid, hydrochloric acid, or an alkali such as sodium hydroxide or potassium hydroxide, and the like, which are usually used in the field of peptide synthesis chemistry. Method,
R of the compound represented by the formula (1) is removed under certain conditions.
A compound of formula (1-2) in which ¹ is a hydrogen atom is obtained.

Step e: The compound of formula (1-2) is converted to the compound of formula R ⁶
A method using an alcohol represented by OH (R ⁶ represents R ¹ containing R ⁵ other than a hydrogen atom) and using an acid catalyst such as sulfuric acid, hydrochloric acid or p-toluenesulfonic acid, dichloromethane,
In a solvent such as ethyl acetate, N, N'-dicyclohexylcarbodiimide (DCC), N-ethyl-N '-(3-
Dimethylaminopropyl) carbodiimide hydrochloride (ED
C.HCl) and the like are esterified by a method using dimethylaminopyridine and the like to obtain a compound represented by the formula (1-3) in which R ¹ is not a hydrogen atom among the compounds represented by the formula (1).

Step f: When R ^{6 of the} compound represented by the formula (1-3) is not equal to R ⁵ , the alcohol represented by the formula R ⁶ OH is used for the compound represented by the formula (1-1). , An acid catalyst such as sulfuric acid, hydrochloric acid, or p-toluenesulfonic acid can be used for transesterification to directly convert the compound represented by the formula (1-3).

The amine compound represented by the formula (3) and the amino group-free compound represented by the formula (5) may be salts with hydrochloric acid, sulfuric acid, p-toluenesulfonic acid and the like. In addition, the reaction can be carried out in the presence of a base such as triethylamine, N, N-diisopropylethylamine, N-methylmorpholine and pyridine.

When the compound represented by formula (1) or a salt thereof is used as a prophylactic and therapeutic agent for bone diseases, tablets, pills, capsules, granules, powders, emulsions, suspensions,
It is administered orally or parenterally in a dosage form such as an injection or a suppository.

Each of the above-mentioned preparations is produced by a technique usually used in the field of preparation, and usual additives such as a bulking agent, a binder, a disintegrating agent, a pH adjusting agent and a dissolving agent can be added.

The dose of the compound represented by the formula (1) or a salt thereof varies depending on the age, body weight, type of disease and condition of the patient, etc., but is usually 10 to 1000 mg per day.
Can be administered in 1 to several divided doses.

[0028]

[Effects of the Invention] As described above, all the compounds represented by the formula (1) obtained by the simple synthetic method are described in Test Example 1,
As shown in 2, it has a strong inhibitory effect on bone resorption and is useful as a preventive and therapeutic agent for bone diseases.

[0029]

EXAMPLES The present invention will be specifically described below with reference to examples and test examples.

Example 1 t-Butoxycarbonyl-L-isoleucine diethyla
Preparation of amide t-butoxycarbonyl-L-isoleucine 23.13
g, diethylamine 8.05 g and benzotriazol-N-hydroxytrisdimethylaminophosphonium hexafluorophosphate (BOP reagent) 44.23 g
A solution of 25.85 g of diisopropylethylamine in 20 ml of dichloromethane was added to a solution of 80 ml of dichloromethane in 20 ml of ice-cooling. After stirring at room temperature for 3 hours, the reaction solution was concentrated under reduced pressure. Add 1000 ml of ethyl acetate to the residue, and add 10
% Citric acid aqueous solution, water, saturated sodium hydrogen carbonate solution, water, saturated saline solution, and the organic layer is dried over anhydrous magnesium sulfate.
It was filtered and concentrated under reduced pressure. The residue was subjected to silica gel column chromatography (eluent; ethyl acetate: n-hexane = 1: 1).
Purification in 4) yielded 25.35 g of the title compound as an oil.

NMR (DMSO-d ₆ ) δ (ppm); 0.77 (3H, d, J = 6.8
Hz), 0.82 (3H, t, J = 7.2Hz), 1.01 (3H, t, J = 7.1Hz), 1.13 (3H,
t, J = 7.1Hz), 1.36〜1.18 (3H, m), 1.36 (9H, s), 3.00〜3.53
(4H, m), 4.14 (1H, dd, J = 9.0,9.0Hz), 6.75 (1H, d, J = 9.0Hz) .IR ^neat ν _max (cm ^-1 ); 3298,2970,2935,2877, 1706,163
5,1521,1504,1463,1384,1366,1313,1288,1247,1221,117
5,1044,1019.

Examples 2-11 Based on the procedure and reaction conditions disclosed in Example 1, t
Instead of butoxycarbonyl-L-isoleucine,
T-butoxycarbonyl-L-leucine and t-, respectively.
Butoxycarbonyl-L-valine, t-butoxycarbonyl-L-norvaline, t-butoxycarbonyl-L-
By using norleucine and di-n-propylamine and diisoamylamine, respectively, in place of diethylamine, compounds shown in the following Tables 1 to 3 represented by the general formula shown in Chemical formula 4 were obtained.

[0033]

[Chemical 4]

[0034]

[Table 1]

[0035]

[Table 2]

[0036]

[Table 3]

Example 12 N- (L-3-trans-ethoxycarbonyloxyla
2-carbonyl) -L-isoleucine diethylami
The compound 25.35g obtained in Example 1 of de 4N hydrochloric acid - dioxane solution 200 ml, and stirred at room temperature for 3 hours. The reaction solution was dried under reduced pressure to give 19.60 g of crude crystals of L-isoleucine diethylamide hydrochloride. This was mixed with L-trans-epoxysuccinic acid ethyl p-nitrophenyl ester 27.22 g and chloroform 25 ml.
50 ml of a chloroform solution of 8.90 g of triethylamine was added under ice cooling.

After stirring at room temperature for 6 hours, the reaction solution was concentrated under reduced pressure. 1000 ml of ethyl acetate was added to the residue, and the mixture was washed successively with 1N aqueous ammonia, water, 5% aqueous hydrochloric acid, water and saturated brine, the organic layer was dried over anhydrous magnesium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent; ethyl acetate: n-hexane = 2: 3) to obtain 23.24 g of the title compound as colorless crystals.

NMR (DMSO-d ₆ ) δ (ppm); 0.76-0.92 (6H,
m), 1.02 (3H, t, J = 7.1Hz), 1.05-1.28 (1H, m), 1.13 (3H, t, J =
7.1Hz), 1.22 (3H, t, J = 7.1Hz), 1.35-1.60 (1H, m), 1.65-1.9
0 (1H, m), 2.95-3.20 (1H, m), 3.30-3.55 (3H, m), 3.60 (1H, d,
J = 1.8Hz), 3.78 (1H, d, J = 1.8Hz), 4.18 (2H, dd, J = 1.3,7.1H
z), 4.57 (1H, dd, J = 9.0,9.0Hz), 8.69 (1H, d, J = 9.0Hz) .IR ^KBr ν _max (cm ^-1 ); 3275,2972,1751,1695,1622,154
4,1493,1465,1370,1343,1252,1214,1196,1095,1021,90
1. mp; 48-51 ° C.

Examples 13-32 Based on the procedures and reaction conditions disclosed in Example 12,
Instead of the compound obtained in Example 1, the compounds obtained in Examples 2 to 11, respectively, and instead of L-trans-epoxysuccinic acid ethyl p-nitrophenyl ester, respectively, L-trans-epoxysuccinic acid were obtained. acid
Methyl p-nitrophenyl ester, L-trans-
Epoxy succinic acid benzyl p-nitrophenyl ester represented by the following general formula shown in Chemical formula 5 shown in Tables 4 to 8 below.
The compound shown in was obtained.

[0041]

Embedded image

[0042]

[Table 4]

[0043]

[Table 5]

[0044]

[Table 6]

[0045]

[Table 7]

[0046]

[Table 8]

Example 33 N- (L-3-trans-carboxyoxirane-2-
Carbonyl) -L-isoleucine diethylamide 1.64 g of the compound obtained in Example 12 was added to ethanol 5
Dissolve in 1 ml of 1N aqueous solution of sodium hydroxide under ice-cooling 5m
1 was added. After further stirring for 2 hours under ice cooling, the reaction solution was concentrated under reduced pressure and 50 ml of water was added. After washing with ethyl acetate, the aqueous layer was adjusted to pH 2 with 6N hydrochloric acid, and the mixture was diluted with 50 ml of ethyl acetate to 1
Once, extracted twice with 25 ml. The organic layers were combined, washed with saturated brine, dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure to obtain 1.42 g of the title compound as a white powder.

NMR (DMSO-d ₆ ) δ (ppm); 0.76-0.92 (6H,
m), 1.02 (3H, t, J = 7.0Hz), 1.05-1.27 (1H, m), 1.12 (3H, t, J =
7.0Hz), 1.35-1.58 (1H, m), 1.67-1.90 (1H, m), 2.98-3.19 (1
H, m), 3.25-3.58 (3H, m), 3.47 (1H, dJ = 1.8Hz), 3.73 (1H, d,
J = 1.8Hz), 4.57 (1H, dd, J = 9.0,9.0Hz), 8.65 (1H, d, J = 9.0),
13.00-13.85 (1H, broad) .IR ^KBr ν _max (cm ^-1 ); 3401,2972,1752,1620,1546,146
3,1385,1363,1215,1099,896.

Examples 34-41 Based on the procedures and reaction conditions disclosed in Example 33,
Instead of the compound obtained in Example 12, the compounds obtained in Examples 14, 15 and 18 to 23 were used to obtain the compounds shown in the following Tables 9 and 10 represented by the general formula shown in Chemical formula 5. It was

[0050]

[Table 9]

[0051]

[Table 10]

Example 42 N- (L-3-trans-isopropoxycarbonylo
Xylan-2-carbonyl) -L-isoleucine diet
Preparation of luamide 769 mg of the compound obtained in Example 33, 312 mg of isopropyl alcohol and 35 mg of 4-dimethylaminopyridine were dissolved in 25 ml of dichloromethane, and N-ethyl-N ′-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC. HCl) 591 mg was added under ice cooling. After stirring under ice cooling for 2 hours and further at room temperature for 2 hours,
The reaction solution was concentrated under reduced pressure.

100 ml of ethyl acetate was added to the residue, and 5% was added.
The organic layer was washed successively with hydrochloric acid, water, saturated aqueous sodium hydrogen carbonate, water and saturated brine, dried over anhydrous magnesium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent; ethyl acetate: n-hexane = 1: 1) to obtain 770 mg of the title compound as colorless crystals.

NMR (DMSO-d ₆ ) δ (ppm); 0.82 (3H, d, J = 6.6
Hz), 0.83 (3H, d, J = 7.1Hz), 1.02 (3H, t, J = 7.1Hz), 1.13 (3H,
t, J = 7.1Hz), 1.16-1.32 (1H, m), 1.22 (3H, d, J = 6.3Hz), 1.23
(3H, d, J = 6.3Hz), 1.36-1.61 (1H, m), 1.64-1.95 (1H, m), 2.9
5-3.20 (1H, m), 3.26-3.63 (3H, m), 3.55 (1H, d, J = 1.8Hz), 3.
78 (1H, d, J = 1.8Hz), 4.57 (1H, dd, J = 8.9,8.9Hz), 4.98 (1H, q
q, d = 6.3,6.3Hz), 8.69 (1H, d, J = 8.9Hz) .IR ^KBr ν _max (cm ^-1 ); 3272,2981,2940,2877,1748,169
6,1625,1547,1467,1377,1361,1328,1252,1214,1198,110
5,906.mp; 61-63 ° C.

Examples 43-65 Based on the procedures and reaction conditions disclosed in Example 42,
Instead of isopropyl alcohol, the corresponding alcohol was used to obtain the compounds represented by the general formula shown in Chemical formula 6 and shown in Tables 11 to 16 below.

[0056]

[Chemical 6]

[0057]

[Table 11]

[0058]

[Table 12]

[0059]

[Table 13]

[0060]

[Table 14]

[0061]

[Table 15]

[0062]

[Table 16]

Example 66 N- (L-3-trans-cyclopentoxycarbonyl
Oxirane-2-carbonyl) -L-isoleucine diet
Preparation of tilamide 1.013 g of the compound obtained in Example 12 was dissolved in 8 ml of cyclopentanol, 1 drop of concentrated sulfuric acid was added, and 100 ° C was added.
And stirred for 4 hours. 100 ml of ethyl acetate was added to the reaction solution, and the mixture was washed successively with saturated aqueous sodium hydrogen carbonate, water and saturated brine, the organic layer was dried over anhydrous magnesium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent; ethyl acetate: n-hexane = 1: 1),
980 mg of the title compound as colorless crystals were obtained.

NMR (DMSO-d ₆ ) δ (ppm); 0.82 (3H, d, J = 6.6
Hz), 0.83 (3H, t, J = 7.2Hz), 0.96-1.28 (1H, m), 1.02 (3H, t, J
= 7.0Hz), 1.12 (3H, t, J = 7.0Hz), 1.34-2.06 (10H, m), 2.94-
3.19 (1H, m), 3.23-3.61 (3H, m), 3.54 (1H, d, J = 1.8Hz), 3.77
(1H, d, J = 1.8Hz), 4.57 (1H, dd, J = 8.9,8.9Hz), 5.07-5.24 (1
H, m), 8.70 (1H, d, J = 8.9Hz) .IR ^KBr ν _max (cm ^-1 ); 3267,2970,2875,1746,1696,162
4,1551,1466,1338,1252,1215,1195,1163,1091,904.mp; 81-82 ° C.

Test examples are shown below.

Test Example 1 Evaluation of In Vitro Bone Resorption Inhibitory Action Evaluation was carried out by inhibition of resorption pit formation.

That is, a femur and a tibia were excised from a 5-day-old rabbit, and the pieces were minced with scissors and stirred in α-minimum essential medium (α-MEM) for 30 seconds.
After standing for 3 minutes, the supernatant is taken and the cell suspension is made to a thickness of 150
4 in a 96-well plate containing ivory pieces sliced to μm
The seeds were seeded at × 10 ⁵ cells / 250 μl / well. After culturing at 37 ° C. in a 5% incubator for 2 hours, the medium was removed, and the medium was removed with dimethyl sulfoxide to 10 ^−7.
Alternatively, 100 μl / well of α-MEM containing 5% FBS containing the compound of the present invention prepared to a concentration of 10 ⁻⁸ M was added. After culturing in a 10% incubator for 24 hours, the medium and cells on the ivory pieces were removed, and the resorption pits formed on the ivory pieces were stained with acid hematoxylin stock solution. The number of stained resorption pits was measured under a microscope, and the specific activity (%) of the compound-added group was calculated when the number of resorption pits formed in the control group containing no compound was 100. The results are shown in Table 17.
Shown in

[0068]

[Table 17]

Test Example 2 Evaluation of In Vivo Bone Resorption Inhibitory Action The method of Delaisse et al. (Biochemical and Biophysical Research Communications (Biochem.
oohys. Res. Commun), Vol. 125, No. 2, 441
Pp.-447, 1984), it was evaluated by the lowering effect of blood calcium concentration which was increased by hyperparathyroidism caused by administration of a calcium deficient diet.

That is, 4-week-old male Wistar rats (Charles River Japan) were bred for 13 days on a low calcium diet for rats (CLEA Japan, Inc.) to prepare model bone resorption-promoting rats. The compound of the present invention was suspended in 5% acacia so as to have a dose of 1 ml per 100 g body weight, and was orally administered by force using a sonde. The control group was administered with a solvent, and blood was collected from the tail artery under ether anesthesia at 3, 9 and 24 hours after the administration, and the serum calcium concentration was determined by a kit using the octocresolphthalein complexone (OCPC) method (calcium C test Wako ). The results are shown in Table 18.

[0071]

[Table 18]

Significant difference from drug-untreated group *: P <
0.05 **: P <0.01

Claims (1)

[Claims] Claims: [Wherein R ¹ represents a hydrogen atom or an alkyl group having 1 to 9 carbon atoms, which may be substituted with an alkoxy group having 1 to 3 carbon atoms, an alkenyl group, an alkynyl group, or an aralkyl group, and R ² represents R ³ represents an alkyl group having 3 to 4 carbon atoms, and R ³ represents an alkyl group having 2 to 5 carbon atoms. ]
An epoxysuccinic acid derivative represented by or a salt thereof.