JPS6238353B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention is based on the general formula (In the formula, R ¹ is

[expression] or

[Formula] (In the formula, R ² means a hydrogen atom, an aromatic acyl group or an aryl group (Aryl).), X is a methylene group, an ethylene group, an oxygen atom or a sulfur atom, and Y is a hydroxyl group, a lower It means an alkoxy group, an aralkoxy group, or an amino group substituted with an unsubstituted or lower alkyl group, a hydroxy lower alkyl group, a lower alkoxy lower alkyl group, or a cycloalkyl group. ) 2-Azetidinone-4-carboxylic acid derivatives and salts thereof. A further explanation of the object compound of the present invention [] is as follows. The aromatic acyl group represented by R ² is an unsubstituted or substituted benzoyl group or benzenesulfonyl group, and substituents include methyl, ethyl, propyl, isopropyl, butyl, pentyl, etc. It means a straight chain or branched alkyl group having 1 to 5 carbon atoms. The aryl group (Aryl) meant by R ² is as follows:
It is an unsubstituted or substituted phenyl group. A nitro group can be mentioned as a substituent for the phenyl group. The number of this substituent may be 1 to 3. The lower alkoxy group represented by Y is a straight chain or branched group having 1 to 5 carbon atoms such as methoxy group, ethoxy group, propoxy group, isopropoxy group, butoxy group, tert-butoxy group, pentyloxy group, etc. It is a lower alkoxy group. The aralkoxy group represented by Y includes benzyloxy group, phenethyloxy group, 3-phenylpropyloxy group, α-methylphenethyloxy group

Means a phenyl lower alkoxy group such as [Formula]. Among the substituents in the substituted amino group represented by Y, the lower alkyl group is a linear or branched group having 1 to 5 carbon atoms, such as a methyl group, ethyl group, propyl group, isopropyl group, butyl group, or pentyl group. Examples of the branched alkyl group include a 2-hydroxyethyl group, a 2-hydroxy lower alkyl group, and a 2-hydroxyethyl group.
hydroxypropyl group

[Formula] A lower alkyl group having 1 to 5 carbon atoms substituted with a hydroxyl group such as a 4-hydroxybutyl group (-CH ₂ CH ₂ CH ₂ CH ₂ OH), and a lower alkoxy lower alkyl group as the above hydroxy lower alkyl group. The hydrogen atom in the hydroxyl group of the group is substituted with the above-mentioned lower alkyl group having 1 to 5 carbon atoms, and the cycloalkyl group has a carbon number that may be bridged such as a cyclopentyl group, cyclohexyl group, adamantyl group, etc. It means 5 to 10 cycloalkyl groups. The object compound of the present invention [ ] has at least three asymmetric carbon atoms, and stereoisomers based on this exist. The target compounds of the present invention include separated and mixtures of these isomers. Further, the object compound of the present invention [] forms a salt with an acid or a base. Salts encompassed by this invention include salts with non-toxic acids (e.g., inorganic acid salts such as hydrochlorides, sulfates, and organic acid salts such as citrates, acetates, tartrates); salts with bases (for example, salts with inorganic bases such as sodium salts and potassium salts, and salts with organic bases such as ammonium salts and triethylamine salts). The object compound of the present invention [] can be prepared as it is or mixed with appropriate pharmacologically acceptable carriers, excipients, and diluents to form powders, granules, tablets, capsules, etc.
It can be administered orally or parenterally in the form of injections (intravenously, subcutaneously, intramuscularly), suppositories, and the like. The dosage of the compound [ ] of the present invention varies depending on the type of compound [ ], age, body weight, symptoms, route of administration, etc., but for example, in the case of injection, approximately
0.001 to 10 mg, preferably 0.01 to 0.1 mg (single dose), and for oral administration about 0.05 to 500 mg, preferably 0.1 to 10 mg (single dose). Compounds related to the object compound [ ] of the present invention are also known as "thyrotropin-releasing hormone".
(TRH) L-pyroglutamyl-L-histidyl-L-prolinamide (pGlu-His-Pro
−NH ₂ ) is known. The existence of TRH has been known since the 1960s, but its structure was confirmed in 1970 [Endocrinology 86 , 1143 (1970)]. TRH was originally thought to be a hormone that regulates the release of thyrotropin (TSH) in the pituitary gland of mammals. However, subsequent studies have shown that the biological functions of this tripeptide TRH are not limited to the regulation of TSH release, but also to the central nervous system (CNS).
This discovery opened a new field of research [Science
178 , 417 (1972), Lancet 2 , 999 (1972)]. Thus, in addition to its TSH-releasing activity, TRH also plays a role in reducing sleep duration induced by barbiturates and alcohol, suppressing hypothermia symptoms stimulated by various drugs, increasing locomotor activity, and decreasing sleep duration induced by barbiturates and alcohol. It is known to have effects on the CNS, such as preventing systemic tonicity caused by schizophrenia, enhancing memory, improving symptoms of schizophrenia, and antidepressant effects. Furthermore, TRH has been found to be an ameliorating and therapeutic agent for disorders of consciousness caused by functional or organic disorders in the brain, such as head trauma, brain surgery, cerebrovascular disorders, brain tumors, etc., especially acute or subacute disorders of consciousness. (Unexamined Japanese Patent Publication No. 51-118841)
issue). In terms of clinical treatment, TSH release activity is weaker than that of TRH, but it does not show much, and moreover,
There has been a demand for a TRH derivative that has an effect equal to or greater than that of TRH. Various TRH derivatives have been synthesized for such purposes, and their effects on the CNS have also been further expanded. Compounds synthesized for this purpose include, for example, their TSH release activity is weaker than that of TRH, but they have anesthetic antagonism, increase locomotor activity, or dopamine-like effects, leading to sleep drug addiction, consciousness disorders, hyperactive children, and schizophrenia in humans. , TRH derivatives that are said to be useful for improving treatment of depression and Parkinson's disease (Japanese Patent Application Laid-open No.
No. 116465), has an effect on consciousness disorder after head trauma, has the effect of reducing sleep duration due to hexobarbital, patients with consciousness disorder caused by organic or functional disorders in the brain, patients with senility or mental fatigue. TRH derivatives (Japanese Patent Application Laid-Open No. 1983-1999) are said to be useful for the treatment of depression and depression.
No. 59714) is known. The target compound of the present invention is characterized by its chemical structure in that it is a TRH derivative in which the pyroglutamyl (pGlu) structural part of TRH is converted into an azetidinone structure (β-lactam structure), which has not been employed at all in the past. Moreover, in terms of pharmacological action, it is significantly more powerful than the above-mentioned TRH and conventionally known TRH derivatives.
It has CNS effects and is useful as a medicine. For example, it is useful as an agent for improving consciousness disorders in schizophrenia, depression, sequelae of cerebrovascular accidents, head trauma, senile dementia, epilepsy, etc., or as an agent for improving motivation, depression, memory loss, etc. Experimental Example 1 (Effect on hypothermia caused by pentobarbital) Tests are conducted on groups of 9 male mice each weighing between 18 g and 22 g for each dose of the test compound. Pentobarbital sodium salt 55mg/Kg
is administered intraperitoneally, and 10 minutes after administration, TRH or the test compound is administered intravenously to the animal. The rectal temperature of the animals was recorded 30 minutes after administration of the test compound, and the results were compared with a control group that received intravenous saline alone.
Expressed as the dose that increases rectal temperature by 1.5°C (ED1.5°C). Experimental Example 2 (Effect on disturbance of consciousness due to head trauma) Tests are conducted on groups of 9 male mice each with body weights ranging from 18 g to 22 g per dose of test compound. 18 acrylic cylinders with a diameter of 19 mm and a weight of 20.5 g.
When a shock is applied by dropping a mouse perpendicularly onto the top of the animal's head from a height of cm, the mouse falls over and loses consciousness. The time from this state of unconsciousness to the start of spontaneous movements was compared between a group in which the test compound was administered intravenously 10 minutes before the shock was applied, and a control group in which only physiological saline was administered intravenously. Comparing the results with the control group, the spontaneous movement appearance time was reduced by 50%.
Expressed as shortened drug dose (DE ₅₀ %). The results are shown in Table 1.

[Table] Example 3 (Acute toxicity) Test compound N-(S)-2-azetidinone-4
-carbonyl)-L-histidyl-L-prolinamide 1493 mg/Kg in physiological saline solution (9
Example) was administered intravenously to adult male mice and observed for up to 24 hours, but not a single mouse was observed to die. In other words, the LD ₅₀ (iv) of this compound is 1493mg/Kg
That's all. On the other hand, using TRH mice
LD ₅₀ (iv) was 751 mg/Kg (iv). According to the present invention, the target compound is produced by the following route.

【table】

[Table] That is, according to the present invention, the target compound [] is produced by (a) reacting compound [] with compound [] to produce compound [], and then producing this compound [].
and the compound [], or (b) reacting the compound [] with the compound [] to form the compound [], and then reacting the obtained compound [] with the compound []. It can be manufactured by folding. Further, the target compound [ ₁ ] thus obtained can also be led to another target compound [ ₂ ] by converting the substituents Y and ^R2 . The reaction for producing the compound [] employed in (a) or (b) above is a peptide synthesis reaction, and a method known per se is used. Commonly used methods include a method using dicyclohexylcarbodiimide as a condensing agent, an azide method, an acid chloride method, an acid anhydride method, and an active ester method. In order to carry out these methods, it is usually necessary to protect functional groups such as amino groups, imino groups, and carboxyl groups that do not participate in the reaction of the raw material compound prior to the peptide formation reaction in each step. The amino group, imino group or carboxyl group involved in the reaction is activated if necessary. A compound in which an amino group, an imino group, or a carboxyl group is activated, such as an active ester, may be once isolated and then subjected to a peptide synthesis reaction, or may be subjected to a peptide synthesis reaction without being isolated. Examples of protecting groups for amino groups include benzyloxycarbonyl group, t-butyloxycarbonyl group, p-methoxybenzyloxycarbonyl group, phthaloyl group, and trifluoroacetyl group. Examples of protecting groups for imino groups include, for example. tosyl group, benzyloxycarbonyl group, p-methoxybenzyloxycarbonyl group, benzyl group,
Examples include 2,4-dinitrophenyl group. As protective groups for carboxyl groups, methyl ester, ethyl ester, benzyl ester, p-
Ester forms such as nitrobenzyl ester and t-butyl ester are used. The groups involved in the reaction can be activated by using the phosphazo method using phosphorus trichloride, the isocyanate method using phosgene, or the phosphite method when the group is an amino group or an imino group, or the activation of a carboxyl group. When , active ester (2,4-dinitrophenol ester, N
-hydroxysuccinimide ester, etc.), azide, carboxylic acid anhydride, etc. Among these, the azide method or the method using dicyclohexylcarbodiimide as a condensing agent is preferred for synthesizing compounds [] and []. It is also possible to use a method of directly preparing a peptide using an N-carboxylic anhydride of an amino acid without using a protecting group. Next, the peptide-forming reaction is carried out in an inert solvent by a conventional method at room temperature or at elevated temperatures. Suitable solvents include dimethylformamide (DMF), ethyl acetate, dichloromethane (methylene chloride), and tetrahydrofuran. When it is necessary to remove protecting groups from the reaction product, for example by catalytic reduction in the case of benzyl esters, anhydrous hydrogen fluoride, HOBT, or hydrogen fluoride-pyridine complex in the case of p-toluenesulfonyl groups. When the protecting group is an alkyl ester, the protecting group can be converted into a benzyloxycarbonyl group,
When the protecting group is p-methoxybenzyloxycarbonyl, it can be easily removed by catalytic reduction or hydrobromic acid-acetic acid treatment, and when the protecting group is t-butyloxycarbonyl group, it can be easily removed by acid decomposition. Next, for the reaction of converting the substituent Y of the target compound [ ] of the present invention to lead to another target compound, appropriate reaction conditions are adopted depending on the properties of the compounds involved in the reaction. These details will be explained by way of examples. The present invention will be further explained below with reference to Examples. Note that a reference example shows a method for producing a raw material compound that is commonly used in a plurality of Examples. The abbreviations used in Examples and Reference Examples have the following meanings. NMR Nuclear magnetic resonance spectrum IR Infrared absorption spectrum Mass Mass spectrometry spectrum Z Benzyloxycarbonyl Bn Benzyl His Histidine Pro Proline DNP 2,4-dinitrophenyl Ts Tosyl BOC t-Butyloxycarbonyl DMF Dimethylformamide HOBT 1-Hydroxy-1,2,3 -Benzotriazole DCC Dicyclohexylcarbodiimide THR Tetrahydrofuran HOSu N-Hydroxysuccinimide Compounds containing the symbol N ^im in the compound name are π nitrogen atoms substituted for the imidazole ring of histidine.

[Formula] Compound and τ nitrogen atom position exchange

[Formula] is a mixture of compounds. In addition, in this specification, when the N-terminus is a free form, hydrogen is expressed as a method for expressing peptides and amino acids (for example, H-His-Pro-NH ₂ ).
and if hydrogen is not displayed (for example, His−Pro
-NH ₂ ), but both have the same meaning. Reference example 1 (S)-4-benzyloxycarbonyl-2-
Azetidinone 1 (3.46 g) was mixed with methanol (350
10% palladium-carbon (350 mg) dissolved in
Hydrogenate using as a catalyst. When the catalyst was removed and the liquid was concentrated to dryness, (S)-2-azetidinone-4-
Carboxylic acid 2 (1.94 g) is obtained. NMR (DMSO-d ₆ ) δppm: 8.26 (s, 1H),
4.02 (dd, 1H, J=3.4Hz, 6.9Hz), 3.21
(dd, 1H, J = 6.9Hz, 16.0Hz), 2.82 (dd,
1H, J = 3.4Hz, 16.0Hz), IR (KBr) cm ^-1 : 3320, 1740, 1720 Mass: 116 (M ⁺ +1) Reference example 2 N-benzyloxycarbonyl-L-histidine hydrazide 6 (10.01 g) was dissolved in a 1N aqueous hydrochloric acid solution (99 ml). Add ethyl acetate (132ml) to this
and an aqueous solution (8.25 ml) of sodium nitrite (2.313 g) while stirring vigorously under ice-cooling. After reacting this mixture at 0° C. for 5 minutes, a 50% aqueous potassium carbonate solution (39.6 ml) was added to the mixture while cooling on ice to make the liquid alkaline. Transfer this to a separatory funnel and remove the organic layer. Further, the aqueous layer is extracted with ethyl acetate (20 ml), combined with the organic layer, and dried over anhydrous sodium sulfate for 10 minutes under ice cooling. After removing the anhydrous sodium sulfate, an ethyl acetate solution (152 ml) of N-benzyloxycarbonyl-L-histidine azide 7 was obtained. This was cooled on ice and L-proline benzyl ester 8 (5.703
Add an ethyl acetate solution (20 ml) of g). This mixture is allowed to react overnight at 0°C. After concentrating the reaction solution to dryness, chloroform-methanol (10:1)
Dissolve in 22 ml and subject to silica gel column chromatography. Chloroform-methanol (95:
The eluate obtained in step 5) was concentrated to dryness to obtain N-benzyloxycarbonyl-L-histidyl-L-proline benzyl ester 9b (6.602 g). NMR (CDCl ₃ ) δppm: 7.45 (1H), 7.14 (s,
5H), 7.10 (s, 5H), 6.82 (1H), 5.70 (d,
1H, J=8.5Hz) 5.20 (s, 2H), 5.06 (s,
2H), 4.4-4.8 (m, 2H), 2.8-3.9 (m,
2H), 3.09 (d, 2H, J=5.7Hz), 1.6-2.5
(m, 4H) Mass: 476 (M ⁺ ), 396, 325, 244, 91, 70 Reference example 3 Cool 25% hydrobromic acid monoacetic acid solution (20 ml) on ice,
Compound 9b (1.91 g) was added to this and reacted at 5° to 10°C for 2 hours. The reaction mixture was added to dry ether (200 ml), the resulting precipitate was quickly removed, and the mixture was poured into a desiccator containing potassium hydroxide for 1 hour.
After drying overnight, L-histidyl-L-proline benzyl ester dihydrobromide 10b (1.99 g) is obtained. NMR (CDCl ₃ + CD ₃ OD) δppm: 8.75 (1H),
7.57 (1H), 7.35 (s, 5H), 5.2 (2H) Example 1 L-Histidyl-L-prolinamide dihydrobromide 10a (826 mg) is dissolved in dimethylformamide (DMF) (13 ml), and a DMF solution (2 ml) of triethylamine (405 mg) is added under ice cooling.
After reacting for 30 minutes under ice-cooling and removing the precipitate, L-histidyl-L-prolinamide 11a
is obtained. This is immediately used for the next synthesis reaction.
Compound 2 (230 mg) was dissolved in DMF (10 ml), and 1-hydroxy-1,2,3-benzotriazole (HOBT) (351 mg) and dicyclohexylcarbodiimide (DCC) (453 mg) were added under ice-cooling.
After stirring for 1 minute, the mixture was allowed to react at room temperature for 15 minutes. This is cooled on ice again, and a DMF solution (15 ml) of the above compound 11a is added. The reaction mixture is allowed to react overnight at 0°C. After removing the precipitate and concentrating the liquid to dryness, it was dissolved in 10 ml of chloroform-methanol (4:1) and subjected to silica gel chromatography. The eluate with chloroform-methanol (7:3) was collected and concentrated to dryness to give N-[(S)-2-azetidinone-4-carbonyl]-L-histidyl-L.
- A crude product (509 mg) of prolinamide 3a is obtained. This was again subjected to silica gel chromatography and eluted with chloroform-methanol-ammonia water (40:10:1).
mg) is obtained. NMR (CD ₃ OD) δppm: 7.59 (s, 1H), 6.98
(s, 1H), 4.41 (dd, 1H), 4.11 (dd, 1H,
J = 3.1Hz, 5.9Hz), 3.36-3.96 (m, 2H),
3.05 (dd, 1H, J=5.9Hz, 14.9Hz) 2.80
(dd, 1H, J=3.1Hz, 14.9Hz) 17.2~2.20
(m, 4H) Mass: 348 (M ⁺ ), 234, 207, 154, 82, 70 [α] ²³ _D = -75.8° (C = 0.6, methanol) When this compound 3a is tritiated with a small amount of methanol, it crystallizes. do. mp 183～185゜(decomposition) Elemental analysis value C ₁₅ H ₂₀ N ₆ O ₄・1/2H ₂ O Calculated value (%)
C: 50.41, H: 5.92, N: 23.52 Experimental value (%)
C: 50.35, H: 6.00, N: 23.64 When compound 3a is recrystallized from methanol, different crystal forms can be obtained depending on the crystallization conditions. For example, mp 145-149℃, 154-157℃, 154-163℃,
181.5-183.5℃, 187-189℃, etc. are obtained, and these have infrared absorption spectra (KBr tablets),
It has been confirmed that it is a crystal polymorph by powder X-ray diffraction, differential scanning calorimetry, etc. The difference in melting points is due to the mixture of different crystal forms. Their physical properties (NMR, optical rotation, etc.) in the solution state are the same. Example 2 L-Histidyl-L-proline benzyl ester dihydrobromide 10b (1.99 g) was dissolved in dichloromethane (45 ml), cooled to -20°, and dichloromethane solution (5 ml) of triethylamine (900 mg) was dissolved in dichloromethane (45 ml).
ml). After reacting at -10° to -20° for 1 hour, the resulting precipitate is removed to obtain a solution containing L-histidyl-L-proline benzyl ester 11b. This is used for the next synthesis reaction immediately after generation. Compound 2 (455 mg) was suspended in dichloromethane (30 ml), HOBT (801 mg), DCC
(1.059 g), stirred for 15 minutes, and reacted for 15 minutes at room temperature. This was cooled on ice again to form the above compound.
Add a dichloromethane solution (50 ml) of 11b. This mixture is allowed to react on ice for 1 hour and then overnight at room temperature. After removing the precipitate and concentrating the liquid to dryness, it was dissolved in 20 ml of water-methanol (4:1).
Subject to HP-20 column chromatography. water-
The eluate with methanol (1:4) was concentrated to dryness to yield a crude product (1.135 g) of N-[(S)-2-azetidinone-4-carbonyl]-L-histidyl-L-proline benzyl ester 3b. can get. This was again subjected to silica gel chromatography and eluted with chloroform-methanol-ammonia water (40:10:1), yielding pure 3b (827 mg).
is obtained. NMR ( _CD3OD ) δppm: 7.58 (1H), 7.34
(s, 5H), 6.88 (1H), 5.15 (s, 2H), 4.50
(dd, 1H), 4.08 (dd, 1H, J=3.3Hz, 5.9
Hz), 3.40-3.92 (m, 2H), 3.04 (dd, 1H,
J=5.9Hz, 14.9Hz), 2.76(dd, 1H, J=
3.3H, 14.9Hz), 1.65-2.20 (m, 4H) Mass: 439 (M ⁺ ), 325 Example 3 N-[(S)-2-azetidinone-4-carbonyl]-L-histidyl-L-proline benzyl ester 3b (782 mg) was dissolved in methanol (150 ml) and 10% paldium on carbon (156 mg) was used as a catalyst. Hydrogenate for 2 hours at room temperature. remove the catalyst,
Concentrating the liquid yields N-[(S)-2-azetidinone-4-carbonyl]-L-histidyl-L-proline 3c (620 mg). NMR ( _CD3OD ) δppm: 8.44 (1H), 7.28
(1H), 4.94 (1H), 4.44 (dd, 1H), 4.14
(dd, 1H, J=3.1Hz, 5.9Hz), 3.3~4.0 (m,
2H), 3.25 (dd, 1H, 5.9Hz, 14.9Hz), 2.86
(dd, 1H, J=3.1Hz, 14.9Hz), 1.7~2.4
(m, 4H) Mass (diazomethane treatment, as dimethyl form): 377 (M ⁺ ), 263, 221, 96, 70 Example 4 Compound 3c (277 mg) was dissolved in DMF (8 ml),
Add HOBT (168 mg) and DCC (330 mg) under ice cooling, react for 1 hour, and then react for 2 hours at room temperature. This was cooled on ice again, a DMF solution (2 ml) of monoethanolamine (80 mg) was added, and the mixture was reacted for 1 hour and then overnight at room temperature. After removing the precipitate and concentrating the liquid to dryness, it was dissolved in 10 ml of chloroform-methanol-ammonia water (40:10:1) and subjected to silica gel chromatography. When eluted with the same solvent, N-[(S)-2-azetidinone-4-carbonyl]-L-histidyl-N-
(2-hydroxyethyl)-L-prolinamide
3d (106mg) is obtained. NMR ( _CD3OD ) δppm: 7.74 (1H), 7.00
(1H), 4.41 (1H, d, d), 4.12 (d, d, J
= 2.9Hz, 5.5Hz), 3.082H, t, J = 7.1Hz),
3.62 (2H, t, J = 7.1Hz), 2.81 (d, d, J
= 2.9Hz, 15.7Hz) Mass: 392 (M ⁺ ), 279, 207 When this compound 3d is tritiated with ether, it crystallizes. Then, it is recrystallized from ethanol. mp 239-241° (decomposition) [α] ²⁴ _D = -87.3° (C = 0.13, methanol) IR (KBr) cm ^-1 ; 3280, 3180, 2950, 1760,
1650, 1635, 1550 Elemental analysis Calculated value (%) as C ₁₇ H ₂₄ N ₆ O ₅
C: 52.03, H: 6.16, N: 21.42 Experimental value (%)
C: 51.90, H: 6.16, N: 21.23 Example 5 3-[N ^im -(2,4-dinitrophenyl)-L
-Histidyl]-L-thiazolidine-4-carboxylic acid amide dihydrochloride 12 (876 mg) was dissolved in dichloromethane (30 ml), and a dichloromethane solution (2 ml) of triethylamine (388 mg) was added under ice cooling.
When reacted at 0℃ for 30 minutes, 3-[N ^im -(2・4-
A solution of dinitrophenyl)-L-histidyl]-L-thiazolidine-4-carboxylic acid amide 13 is obtained. This is used for the next synthesis reaction immediately after generation. Compound 2 (223 mg) was dissolved in 6 ml of DMF, and HOBT (389 mg) and DCC (517 mg) were added under ice cooling.
Incubate for 30 minutes at 0°C and 30 minutes at room temperature. This is cooled on ice again, and a dichloroethane solution (32 ml) of the above compound 13 is added. This mixture is allowed to react overnight at 0°C. After removing the precipitate and concentrating the solution to dryness, it was dissolved in 20 ml of chloroform-methanol-ammonia water (40:10:1) and subjected to silica gel chromatography. When eluted with the same solvent, 3
-[N〓-[(S)-2-azetidinone-4-carbonyl]-N ^im -(2,4-dinitrophenyl)-L
-Histidyl]-L-thiazolidine-4-carboxylic acid amide 4 (347 mg) is obtained. NMR (CD ₃ OD) δppm: 8.93 (d, 1H, J=
3.1Hz), 8.63 (dd, 1H, J=3.1Hz, 9.0Hz),
7.92 (d, 1H, J=9.0Hz), 7.87 (s, 1H),
7.26 (s, 1H) 4.43 (d, 2H, J=9.5Hz),
4.14 (dd, 1H, J=3.1Hz, 5.9Hz), 2.86
(dd, 1H, J=3.1Hz, 14.7Hz) Mass: 419, (M ⁺ −C ₄ H ₅ N ₂ O ₂ ), 372, 248, 81 Example 6 Compound 4 (337 mg) is dissolved in DMF (15 ml). Add mercaptoethanol (2 ml) to this solution and react for 30 minutes at room temperature. After the reaction solution was concentrated to dryness, it was dissolved in 20 ml of chloroform-methanol-ammonia (30:10:1) and subjected to silica gel chromatography. When eluted with the same solvent, 3
-[N〓-[(S)-2-azetidinone-4-carbonyl]-L-histidyl]-L-thiazolidine-4
-Carboxylic acid amide 5 (214 mg) is obtained. NMR ( _CD3OD ) δppm: 7.55 (1H), 6.91
(1H), 4.32 (d, 2H, J=9.5Hz), 4.07
(dd, 1H, J=3.1Hz, 5.9Hz) Mass: 367 (M ⁺ +1), 253, 206, 115 Reference example 4 N-t-butyloxycarbonyl-N ^im -tosyl-L-histidyl-DL-pipecolic acid 14
(6.28 g) and N-hydroxysuccinimide (HOSu) (1.39 g) were dried in methylene chloride (90 ml).
Dissolve in water and cool in an ice bath. After adding DCC (2.74 g), the mixture is stirred in an ice bath for 3 hours. After removing the insoluble matter, the liquid is concentrated under reduced pressure. The residue was dissolved in ethyl acetate, aqueous sodium bicarbonate solution, water,
Wash sequentially with saline. After drying the organic layer, the solvent is removed and the resulting syrup is triturated with a 1:1 mixture of ether and petroleum ether. The obtained crystalline compound 15 is used as it is for the next reaction after drying. Compound 15 (2.3 g) was dissolved in 40 ml of tetrahydrofuran (THF), and a solution of ethanolamine (251 mg) in 5 ml of THF was added under ice cooling. The reaction mixture was stirred for 1 hour under ice cooling and the reaction solvent was removed under reduced pressure. The residue is dissolved in chloroform and washed successively with an aqueous sodium bicarbonate solution, water, and brine. The residue obtained by removing the solvent from the organic layer is triturated with ether. Solid N-t-butyloxycarbonyl-N ^im -tosyl-L-histidyl-N-
(2-Hydroxyethyl)-DL-pipecolamide 16 (1.05 g) is obtained. After concentration, the mother liquor is subjected to silica gel column chromatography. Compound 16 (0.66
g) is obtained. IR (KBr) cm ^-1 ; 3360, 2920, 1680, 1640, 1170 NMR ( _CD3OD ) δppm: 7.28-8.4 (6H, imidazole ring hydrogen, benzene ring hydrogen), 2.44
(3H, methyl of tosyl group), 1.0-1.5 (broad, BOC hydrogen) Example 7 Compound 16 (1.71 g) was dissolved in 40 ml of methylene chloride, and 40 ml of trifluoroacetic acid was added under ice cooling. After stirring for 2 hours to react, the mixture was dried under reduced pressure. The residue is azeotropically dehydrated several times using toluene and dried. Trituration with ether then yields the trifluoroacetate of compound 17 in solid form. This is used for the next reaction as it is after drying. Dissolve this trifluoroacetate (1.3 g) in DMF (8 ml),
Add 274 mg of triethylamine while cooling on ice. Adjust the pH to 7-8 with additional triethylamine while checking with pH test paper. The resulting free amine solution was added to a reaction solution prepared from a mixture of Compound 2 (286 mg) and DCC (557 mg) in methylene chloride (8 ml) and DMF (1.5 ml) under ice cooling. Stir the reaction mixture in the refrigerator overnight. After removing the insoluble matter, the liquid is concentrated under reduced pressure. The residue is dissolved in chloroform and washed three times with water. After drying the organic layer and removing the solvent, a syrup-like substance is obtained. This syrup was subjected to column chromatography on silica gel (140 ml). The target N〓[(S)-2-azetidinone-] was obtained by elution with a 7% methanol-chloroform mixture.
537 mg of 4-carbonyl]-N ^im -tosyl-L-histidyl-N-(2-hydroxyethyl)-DL-pipecolamide 18 are obtained. NMR (CDCl ₃ ) δppm; 7.0 to 8.3 (9H, imidazole hydrogen, benzene ring hydrogen, NH) 5.08 centers (2H, α-methine hydrogen of histidine, α-methine hydrogen of pipecolic acid), 4.08 (1H, azetidinone ring 4 position hydrogen), 2.44 (3H, methyl of tosyl group) Mass m/e; 559 (M-1) 472, 402, 388 Example 8 Compound 18 (250 mg) and HOBT (73 mg) are dissolved in dry methylene chloride (15 ml) and reacted by stirring at room temperature for 5 hours. Insoluble matter precipitates. The residue obtained by removing the solvent from the reaction mixture was subjected to column chromatography on silica gel (100 ml). Chloroform-methanol-ammonia water (80:
By elution with a mixture of 20:2), the target product N-[(S)-2-azetidinone-4-carbonyl]-L-histidyl-N-(2-hydroxyethyl)-DL-pipecolamide 19 (116 mg ). This is a mixture of diastereomers
Two spots are shown on TLC. IR (Measurement of sample after freeze-drying using KBr tablet) cm
^-1 ; 3250 (NH, OH), 1750 (4-membered ring lactam),
1630 center (broad, amide) NMR (CD ₃ OD), δppm; 7.65 (1H, imidazole ring hydrogen), 6.92 (1H, imidazole ring hydrogen), 4.14 (1H, d, d, azetidinone ring 4-position hydrogen), 1.2 ~1.8 (6H, methylene hydrogen of piperidine ring) Mass m/e; 406 (M ⁺ ), 388, 345, 318 Reference example 5 Compound 15 (2.8 g) synthesized from Compound 14 in the same manner as Reference Example 4 and 1-aminoadamantane (755
mg) in quantitative yield from N-t-butyloxycarbonyl-N ^im -tosyl-L-histidyl-N.
-(1-adamantyl)-DL-pipecolamide 20
get. NMR (CD ₃ OD) δppm; 7.1-8.3 (6H, imidazole ring hydrogen, benzene ring hydrogen), 2.44 (3H,
methyl of tosyl group) Mass m/e; 653 (M ⁺ ), 502, 475, 419 Example 9 The BOC group is removed using Compound 20 in the same manner as in Example 7 above. The resulting trifluoroacetate of 21 is similarly made into the free base using triethylamine. A coupling reaction is performed using this free amine (2.15 mM) and Compound 2 (290 mg) in the same manner as described above. After the reaction, insoluble materials are removed and the liquid is concentrated under reduced pressure. The residue was dissolved in ethyl acetate and washed three times with water. After drying the organic layer, remove the solvent and subject it to column chromatography on silica gel (220 ml). By eluting with a mixture of 3% methanol and chloroform, the target product N
-[(S)-2-azetidinone-4-carbonyl]-
598 mg of N ^im -tosyl-L-histidyl-N-(1-adamantyl)-DL-pipecolamide 22 is obtained as a powder. NMR (CD ₃ OD) δppm; 7.2-8.24 (6H, imidazole ring hydrogen, benzene ring hydrogen), 4.04 center (1H, azetidinone ring 4-position hydrogen), 2.44 (3H,
methyl of tosyl group), 1.2-2.3 (21H, methylene hydrogen of piperidine ring, hydrogen of adamantyl group) IR (KBr) cm ^-1 ; 3280, 2900, 1760, 1640 center (broad) Mass m/e; 650 (M ⁺ ), 472, 402, 360 Example 10 Compound 22 (510 mg) and HOBT (130 mg) were dissolved in 10 ml of methylene chloride and stirred at room temperature for 7 hours. After removing the solvent, the residue is subjected to column chromatography on silica gel. The two diastereomers are separated by elution with a mixture of chloroform-methanol-aqueous ammonia (80:20:2). On the silica gel column, the less polar compound N-[2-azetidinone-4-carbonyl]-L-histidyl-N-(1-adamantyl)pipecolamide 23a (98 mg) was eluted first, followed by 23a and its Mixture of stereoisomers 23b 128
mg is obtained. Compound 23b (63 mg) was then eluted. The physical properties of compounds 23a and 23b are as follows. 23a; NMR (CD ₃ OD) δppm, 7.66, 7.58,
6.92, 6.90 (imidazole ring C-H), 4.10
(1H, dd, hydrogen at position 4 of azetidinone ring), 2.04 centers, 1.72 centers (21H, piperidine ring methylene hydrogen, adamantyl group hydrogen) IR (KBr) cm ^-1 ,
3250, 1760, 1640 center (broad), 1520,
1440 [α] ²⁶ _D = -74.9° (C = 1.1, methanol) Mass.m/e; 496 (M ⁺ ), 382, 345, 318, 261 23b; NMR (CD ₃ OD + DMSO-d ⁶ ) δppm,
7.64, 6.90 (imidazole ring C-H), 4.10 center (1H, d, d, hydrogen at position 4 of azetidinone ring),
2.02 center, 1.70 center (21H, piperidine ring methylene hydrogen, adamantyl group hydrogen) IR (KBr) cm
^-1 , 3200, 1750, 1640, 1530, 1440 [α] ²⁶ _D = +82.4° (C = 1.3, methanol) Mass.m/e; 496 (M ⁺ ), 345, 318, 261 Reference example 6 N-benzyloxycarbonyl-L-proline
Dissolve 24 (4.99 g) in THF (50 ml) and add triethylamine (2.23 g). After gradually adding ethyl chloroformate (2.39 g) under ice cooling,
React at 5°C for 1 hour. Next, n-butylamine (2.19 g) was gradually added to this under ice cooling, and
React at 0° to 5°C for 1 hour. The solvent is distilled off, and the residue is dissolved in ethyl acetate, which is washed successively with 1N hydrochloric acid, saturated sodium bicarbonate, and saturated brine. The organic layer was dried with Glauber's salt and then concentrated to dryness. This was recrystallized from water to obtain (S)-1-benzyloxycarbonyl-N-butyl-2-pyrrolidinecarboxamide 25a (4.31 g). mp88~90°NMR ( _CDCl3 ), δppm: 7.36 (s, 5H), 5.17
(s, 2H), 4.33 (dd, 1H), 3.51 (t, 2H),
3.21 (dd, 2H), 1.65~2.40 (4H), 1.05~1.65
(4H), 0.70-1.05 (3H) IR (KBr): 3280, 2950, 1715, 1640, 1540cm ^-1 Mass (EI): 304 (M ⁺ ), 232, 204, 91, 70 Reference example 7 Compound 25a (4.41 g) is dissolved in methanol (140 ml) and hydrogenated using 10% palladium on carbon (426 mg) as a catalyst. After removing the catalyst and concentrating the liquid, (S)-N-butyl-2-pyrrolidinecarboxamide 26a (2.41 g) was obtained. NMR ( _CDCl3 ), δppm: 7.3-8.0 (1H), 3.71
(dd, 1H), 2.8~3.4 (4H), 1.1~2.4 (9H),
0.7~1.1 (3H) IR (neat): 3280, 2950, 1645, 1520cm ^-1 Reference example 8 N-benzyloxycarbonyl-L-histidine hydrazide 6 (4.85
Compound 26a (2.23 g) was added to an ethyl acetate solution (75 ml) of N-benzyloxycarbonyl-L-histidine azide 7 synthesized from g) under ice cooling, and the mixture was allowed to react overnight in the refrigerator. The reaction solution is concentrated, and the residue is subjected to silica gel column chromatography.
Chloroform-methanol-ammonia water (95:
5:0.5), N-benzyloxycarbonyl-L-histidyl-N-butyl-L-
Prolinamide 27a (4.13 g) is obtained. NMR ( _CDCl3 ), δppm: 7.51 (1H), 7.34
(s, 5H), 6.85 (s, 1H), 5.76 (d, 1H),
5.10 (s, 2H), 4.3~4.8 (2H), 2.8~3.7
(6H), 1.7~2.3 (4H), 1.1~1.7 (4H), 0.7~
1.1 (3H) IR (KBr): 3250, 2950, 1705, 1635, 1540cm ^-1 Mass (EI): 441 (M ⁺ ), 361, 341, 272, 244,
190, 136, 91, 70 Reference example 9 Add ice-cooled 25% hydrobromic acid-acetic acid solution (20 ml) to Compound 27a (1.77 g), and react at room temperature for 3 hours. The reaction mixture was added to dry ether (200 ml), and the resulting precipitate was quickly collected and dried under reduced pressure in a dessicator containing potassium hydroxide overnight, resulting in L-histidyl-N-butyl-L-prolinamide 2. Hydrobromide 28a (2.14 g) is obtained. Example 11 Compound 28a (938 mg) was dissolved in DMF (10 ml), cooled to -40°C, and triethylamine (415
mg). After reacting at -30°C to -40°C for 1 hour, the resulting precipitate was removed, and L-histidyl-N-butyl-L-prolinamide 29a was dissolved in DMF.
A solution is obtained. This is used for the next synthesis reaction immediately after generation. (S)-2-azetidinone-4-carboxylic acid 2 (obtained in Reference Example 1) (230 mg)
Dissolve in DMF (5 ml) and cool on ice with HOBT (406
mg) and DCC (495 mg), and react at 0° to 5°C for 1 hour. This is cooled to -40°C, the above DMF solution of compound 29a is added, and the mixture is reacted at -40°C for 30 minutes and overnight in a refrigerator. The precipitate is removed, the liquid is concentrated, and the residue is subjected to silica gel column chromatography. When eluted with chloroform-methanol-ammonia water (80:20:2), N〓-
[(S)-2-azetidinone-4-carbonyl]-L
-Histidyl-N-butyl-L-prolinamide
30a (471mg) is obtained. NMR (D ₂ O, Sodium 3-(trimethylsilyl)
-1-propanesulfonate) δppm: 7.76
(1H), 7.07 (1H), 4.96 (t, 1H), 4.2-4.5
(2H), 2.6~4.0 (8H), 1.7~2.2 (4H), 1.1~
1.7 (4H), 0.7~1.1 (3H) IR (KBr): 3240, 2950, 1755, 1630, 1540cm ^-1 Mass (EI): 404 (M ⁺ ), 305, 290, 235, 207,
165, 110, 70 [α] ²⁷ _D = -81.8° (C = 0.50, MeOH) Reference example 10 (S)-N-cyclohexyl-2-pyrrolidinecarboxamide synthesized by a known method was added to an ethyl acetate solution (30 ml) of compound 7 synthesized from compound 6 (1.52 g) by the method of Reference Example 2 under ice cooling.
Add 26b (785 mg) and react overnight in the refrigerator. The reaction solution is concentrated and the residue is subjected to silica gel chromatography. When eluted with chloroform-methanol-ammonia water (90:10:1), N
〓-Benzyloxycarbonyl-L-histidyl-N-cyclohexyl-L-prolinamide 27b
(1.30g) is obtained. NMR (CDCl ₃ ), δppm: 7.54 (s, 1H), 7.32
(s, 5H), 6.88 (s, 1H), 5.77 (d, 1H),
5.09 (s, 2H), 4.61 (dd, 1H), 4.36 (t,
1H), 3.3~4.0 (2H), 2.8~3.3 (3H), 0.9~
2.4 (14H) IR (KBr): 3250, 2920, 1710, 1630, 1525cm ^-1 Mass (EI): 467 (M ⁺ ), 387, 360, 341, 272,
244, 136, 108, 70 Reference example 11 Add ice-cooled 25% hydrobromic acid-acetic acid solution (15 ml) to Compound 27b (1.14 g), and react at room temperature for 3 hours. The reaction mixture was added to dry ether (150 ml), and the resulting precipitate was quickly collected and dried under reduced pressure in a desiccator containing potassium hydroxide overnight, resulting in L-histidyl-N-cyclohexyl-L.
- Prolinamide dihydrobromide 28b (1.53
g) is obtained. Example 12 Compound 28b (991 mg) was dissolved in DMF (10 ml), cooled to -40°C, and triethylamine (415 mg) was added.
Add. After reacting for 1 hour at -30°C to -40°C, the resulting precipitate was removed and L-histidyl-
N-cyclohexyl-L-prolinamide 29b
A DMF solution is obtained. This is used for the next synthetic reaction immediately after generation. Compound 2 (230mg)
Dissolve in DMF (5 ml) and cool on ice with HOBT (406
mg) and DCC (495 mg), and react at 0° to 5°C for 1 hour. This is cooled to -40°C, the DMF solution of the above compound 29b is added, and the mixture is reacted at -40°C for 30 minutes and overnight in the refrigerator. The precipitate is removed, the liquid is concentrated, and the residue is subjected to silica gel column chromatography. When eluted with chloroform-methanol aqueous ammonia (80:20:2), N〓-
[(S)-2-azetidinone-4-carbonyl]-L
-Histidyl-N-cyclohexyl-L-prolinamide 30b (305 mg) is obtained. NMR ( _CD3OD ), δppm: 7.64 (1H), 6.98
(1H), 4.2-4.5 (1H), 4.12 (dd, 1H), 2.81
(dd, 1H), 0.9~1.2 (14H) IR (KBr): 3220, 2920, 1750, 1620, 1540cm ^-1 Mass (EI): 430 (M ⁺ ), 316, 235, 180, 152,
99, 70 [α] ²⁷ _D = -69.2℃ (C = 1.90, MeOH) Reference example 12 D-Aspartic acid dibenzyl ester paratoluene sulfonate 31 71g (146.6mmol) was suspended in 1150ml of ether, and the mixture was cooled with ice (0° to 5°C).
°C) 22.5 ml of triethylamine while stirring
(146.6×1.1 mmol) was added dropwise. 2 hours 0°~5
After stirring at ℃, add 450ml of water at that temperature.
Stir for 30 minutes. The ether layer was separated and the aqueous layer was extracted with 200 ml of ether. The ether layer and the extract were combined, washed with 400 ml of saturated sodium sulfate solution, and then dried over anhydrous magnesium sulfate. The ether was distilled off under reduced pressure to obtain 45 g of D-aspartic acid dibenzyl ester 32 as a colorless oil. Reference example 13 D-Aspartic acid dibenzyl ester 32 45
g (143.8 mmol) was dissolved in 485 ml of dry ether and cooled to 0°C under an argon atmosphere to dissolve triethylamine.
20 ml (143.8 mmol) was added dropwise. Next, at the same temperature, add 15.6 g of trimethylsilyl chloride (143.8 m
mol) was added dropwise and stirred for 1 hour. The resulting precipitate was removed under an argon atmosphere, the liquid was cooled to 0 to -5°C, and an ether solution of tert-butylmagnesium chloride (143.8 x 1.01 mmol) was added with stirring.
was dripped. The mixture was stirred at 0° C. for 2 hours and at room temperature for 3 hours, then cooled to 0° C., 100 ml of 2N-HCl (saturated with NH ₄ Cl) was added, stirred for 30 minutes, and then 100 ml of saturated ammonium chloride solution was added. After separating the ether layer, the aqueous layer was diluted with 200 ml of ethyl acetate.
Extracted twice. The ether layer and the acetic acid ethyl ester extract were combined, washed with 300 ml of saturated ammonium chloride solution, dried over anhydrous magnesium sulfate, and washed with ether,
Ethyl acetate was distilled off under reduced pressure. Add 10 ml of ethyl acetate to the residue, crystallize it, and obtain the desired product (R)-4-benzyloxycarbonyl-
13.7 g of 2-azetidinone 33 was obtained. After concentrating the mother liquor, silica gel chromatography (eluent: acetic acid ethyl ester-n-hexane (2:
1)) to obtain 5.1 g of the target product. m.
p.136゜～138℃ [α] _D +33.7゜ (C=1, MeOH) NMR (DMSO-d ⁶ ) δppm: 8.40 (1H, NH),
7.40 (5H, s, phenyl group), 5.20 (2H, s,
methylene of benzyl group), 4.22 (1H, d, d,
4th position hydrogen), 3.27 (1H, d, d, d, 3rd position hydrogen), 2.89 (1H, d, d, d, 3rd position hydrogen) IR (KBr) cm ^-1 : 3200, 1760, 1725, 1280 Reference Example 14 Dissolve 5g of compound 33 in 250ml of methanol and make 10%
Catalytic reduction was carried out at room temperature and pressure in the presence of 500 mg of Pd-C in a hydrogen atmosphere. After the catalyst was removed, methanol was distilled off under reduced pressure. The residue was crystallized from ether and collected to obtain 2.5 g of the target product (R)-2-azetidinone-4-carboxylic acid 34 as colorless crystals. mp97
~101℃ NMR (DMSO- ^d6 , _CD3OD ) δppm: 4.60
(1H, d, d, hydrogen at position 4), 3.23 (1H, d,
d, 3rd position hydrogen), 2.85 (1H, d, d, 3rd position hydrogen) IR (KBr) cm ^-1 : 3310, 1735 (broad), 860 Example 13 826 mg (2 mmol) of L-histidyl-L-prolinamide dihydrobromide 10a was dissolved in 13 ml of anhydrous dimethylformamide and cooled to -10°C. Add 404 mg (2 x 2 mmol) of triethylamine to this solution.
was gradually added and stirred at the same temperature for 30 minutes. The precipitated triethylamine hydrobromide was removed in an argon stream. This solution was mixed with 230 mg (2 mmol) of (R)-2-azetidinone-4-carboxylic acid 34 (obtained in Reference Examples 12 to 14) and 351 mg of HOBT (2×
1.3 mmol), DCC453 mg (2 x 1.1 mmol) and
−20% in an active ester solution prepared from 10ml of DMF.
Add dropwise at ℃. After stirring at the same temperature for 1.5 hours, it was left in the refrigerator overnight. DMF under reduced pressure from the reaction solution
was distilled off and methylene chloride-methanol was added to the residue.
10 ml of concentrated ammonia water (80:20:2) was added to remove the precipitated crystals. The solution was subjected to silica gel column chromatography, and the developing solution was methylene chloride-methanol-concentrated ammonia water (80:20:
2) to produce N-[(R)-2-azetidinone-4-carbonyl]-L-histidyl-L
- 370 mg of prolinamide 35 was obtained as an amorphous powder. [α] _D −21.5° (C=1, methanol) NMR (D ₂ O) δppm 7.03 (1H, imidazole ring), 6.72 (1H, imidazole ring), 4.95
(1H, m), 4.42 (1H, m), 4.27 (1H, d,
d, hydrogen at position 4 of azetidinone ring), 3.40-4.00
(2H, m), 3.32 (1H, d, d, hydrogen at 3-position of azetidinone acid), 2.74 (1H, d, d, hydrogen at 3-position of azetidinone ring), 2.00 (4H, m) IR (KBr) cm ^-1 : 3350, 3150, 1745, 1660,
1625, 1440 Mass: 348 (M ⁺ ), 304, 278, 234, 207, 190 Example 14 N-[(S)-2-azetidinone-4-carbonyl]-L-histidyl-L-proline 3c (obtained in Example 3) 300 mg, HOBT (116 mg) and
Dissolve DCC (177 mg) in 2 ml DMF and
After stirring for an hour, cool in an ice bath. Add 0.6 ml of 30% methylamine methanol solution and react at 2-6°C with stirring overnight. remove insoluble matter,
The liquid was concentrated to dryness under reduced pressure. The residue is purified by column chromatography using Ricroprep Si-60B size. N-[(S)-2-azetidinone-4-carbonyl]-L-histidyl-N-methyl-L-prolinamide 48 was obtained using chloroform-methanol-ammonia water (40:10:1) as the eluent. can get. This is dissolved in water and then freeze-dried. Yield 149 mg NMR (CD ₃ DO) δppm: 7.62 (1H, imidazole ring), 6.94 (1H, imidazole ring), 4.8
(1H, methine group), 4.10 (1H, hydrogen at 4-position of azetidinone ring), 2.76 (3H, N-methyl group), 1.6
~2.3 (4H, proline ring hydrogen) IR (KBr) cm ^-1 : 3250, 1750, 1630, 1540, 1440 Mass m/e: 362 (M ⁺ ), 305, 292, 248,
235, 207 Reference example 15 N-t-butyloxycarbonyl-N ^im -tosyl-L-histidine 37 (10 g) and L-proline benzyl ester hydrochloride (6.50 g) are mixed with 100 ml of dry methylene chloride and cooled in an ice bath. After adding triethylamine (2.72g), DCC
(6.05g). The mixture is stirred in an ice bath for 30 minutes and then at room temperature overnight. After removing the insoluble materials, the liquid is concentrated. The residue is purified by silica gel column chromatography. N〓-t- from a mixture of ethyl acetate and benzene (1:1)
Butyloxycarbonyl-N ^im -tosyl-L-histidyl-L-proline benzyl ester 39 is eluted. Yield 13.5g NMR (CD ₃ OD) δppm: 8.14, 7.92, 7.84,
7.40, 7.30 (total 11H, imidazole ring, benzene ring), 5.12 (2H, q, benzyl group), 4.5 center (2H, two types of methine groups), 2.36 (3H, methyl of tosyl group), 1.30 (9H, t-butyl group) IR (KBr) cm ^-1 : 3400, 3280, 2970, 1740,
1700, 1640, 1590 Mass m/e: 596 (M ⁺ ), 523, 480, 364,
290, 155, 91 Reference example 16 Compound 39 (13.5 g) was dissolved in 150 ml of methanol and subjected to catalytic reduction in the presence of 10% palladium-carbon for 5 hours. The catalyst was removed and the liquid was concentrated under reduced pressure. The residue is dissolved in ethyl acetate and extracted three times with aqueous sodium bicarbonate solution. The combined extracts were washed once with ethyl acetate. After acidifying the aqueous layer with 1N hydrochloric acid, the target compound is extracted with ethyl acetate. N〓-t-butyloxycarbonyl-N ^im −
Tosyl-L-histidyl-L-proline 40 (2.0
g) is obtained as a foam. Starting material 39 is extracted from the organic layer after extraction with aqueous sodium bicarbonate solution.
(9.1 g) is recovered. The physical property values of the target compound produced are shown. NMR ( _CD3OD ) δppm: 8.16, 7.96, 7.86,
7.46, 7.40 (total 6H, imidazole ring hydrogen, benzene ring hydrogen), 4.48 center (2H, two types of methine hydrogen), 2.42 (3H, s, methyl of tosyl group), 1.32 (9H, t-butyl group) IR (KBr)cm ^-1 : 3300, 3100, 2970, 2500~
2600, 1710, 1640 Mass m/e: 388 (M-118), 308, 234 Reference example 17 Compound 40 (3.25g), monoethanolamine (0.79g) and HOBT (2.61g) in methylene chloride
Mix with 70 ml and cool in an ice bath. DMF (20ml)
Add to make a homogeneous solution. After adding DCC (1.99 g), stir in an ice bath for 2 hours, then stir at room temperature overnight. After removing the insoluble matter, the liquid is concentrated under reduced pressure. Dissolve the residue in 70ml of methylene chloride,
Add HOBT (1.3 g) and stir again at room temperature for 20 hours. After the solvent is distilled off under reduced pressure, the residue is purified by column chromatography on silica gel.
Chloroform-methanol-ammonia water (40:
By developing with a mixture of 10:1), N〓-t
-butyloxycarbonyl-L-histidyl-N
1.35 g of -(2-hydroxyethyl)-L-prolinamide 41 is obtained. NMR (CD ₃ OD) δppm: 7.64 (1H, imidazole ring), 6.96 (1H, imidazole ring), 4.48 centers (2H, two types of methine groups), 2.0 centers (4H, proline ring), 1.40 (9H, t -butyl group) IR (KBr) cm ^-1 : 3250, 2960, 1700, 1630 Mass m/e: 395 (M ⁺ ), 365, 322, 307, 278 Example 15 Compound 41 (790 mg) is dissolved in 25 ml of methylene chloride, and 20 ml of trifluoroacetic acid is added dropwise at 0 to 5°C. After stirring in an ice bath for 2.5 hours, concentrate to dryness under reduced pressure. Furthermore, it is azeotropically dried several times using toluene. Trituration of the residue with dry ether gives powdered L-histidyl-N-(2-hydroxyethyl)-L-prolinamide 2-trifluoroacetate 42 in quantitative yield. Compound 2
(253 mg) and HOBT (350 mg) were dissolved in a mixture of DMF (7 ml) and methylene chloride (8 ml) and cooled on ice.
Add DCC (530 mg) and stir for 1.5 hours. A reaction solution obtained by neutralizing the above compound 42 with triethylamine (445 mg) in a mixture of DMF (4 ml) and methylene chloride (4 ml) is added to this reaction solution. The mixture is stirred and reacted in the refrigerator overnight. After removing the insoluble matter, the liquid is concentrated and the residue is purified by column chromatography on silica gel. Compound 3d (313 mg) identical to that obtained in Example 4 is obtained by developing with a mixture of chloroform-methanol-aqueous ammonia (40:40:1). Reference example 18 N-benzyloxycarbonyl-L- synthesized from N-benzyloxycarbonyl-L-histidine hydrazide (6.07 g) by a known method.
Add (S)-N-(2-hydroxyethyl)-2-pyrrolidinecarboxamide 43 (2.31 g) to a DMF solution (10 g) in ethyl acetate solution of histidine azide 7 under ice cooling.
ml) and react overnight in the refrigerator. The reaction solution is concentrated, and the residue is subjected to silica gel column chromatography. When eluted with chloroform-methanol-aqueous ammonia (90:10:1), N-benzyloxycarbonyl-L-histidyl-N-
(2-hydroxyethyl)-L-prolinamide 44
(3.43g) is obtained. NMR ( _CDCl3 ), δppm: 8.3-8.7 (1H), 7.55
(s, 1H), 7.34 (s, 5H), 6.87 (s, 1H),
5.93 (d, 2H), 5.10 (s, 2H), 4.3-4.8
(2H), 2.8~3.8 (8H), 1.6~2.3 (4H) Mass (EI): 429 (M ⁺ ), 341, 272, 244, 136,
108, 79 Reference example 19 An ice-cooled 25% hydrobromic acid-acetic acid solution (37.5 ml) was added to Compound 44 (3.22 g), and the mixture was allowed to react at room temperature for 3 hours. The reaction mixture was added to dry ether (375 ml) and the resulting precipitate was quickly removed and dried overnight in a dessicator containing potassium hydroxide.
L-Histidyl-N-(2-acetoxyethyl)-
L-prolinamide dihydrobromide 45 (4.43
g) is obtained. Example 16 Compound 45 (4.43 g) was dissolved in DMF (35 ml), cooled to -40°C, and triethylamine (1.82 g) was dissolved in DMF (35 ml).
g) was added and reacted for 1 hour at -30° to -40°C, and the resulting precipitate was filtered off, resulting in a DMF solution of L-histidyl-N-(2-acetoxyethyl)-L-prolinamide 46. is obtained. This is used for the next reaction immediately after its formation. Compound 2 (863mg)
Dissolved in DMF (17.5ml), HOBT (1.52g),
Add DCC (1.86 g) and react for 30 minutes under water cooling. This was cooled to -40℃ to form the above compound 46.
Add DMF solution and react at -40°C for 30 minutes and overnight in the refrigerator. The precipitate is removed, the liquid is concentrated, and the residue is subjected to silica gel column chromatography. When eluted with chloroform-methanol-aqueous ammonia (80:20:2), N〓-[(S)-2-
Azetidinone-4-carbonyl]-L-histidyl-N-(2-acetoxyethyl)-L-prolinamide 47 (1.60 g) is obtained. NMR (D ₂ O), δppm: 7.74 (s, 1H), 7.05
(1H), 4.93 (t, 1H), 4.1~4.5 (4H), 2.9~
3.9 (7H), 2.76 (dd, 1H), 2.7~3.2 (7H) IR (KBr): 3230, 2950, 2860, 1755, 1730,
1630, 1540cm ^-1 Mass (EI): 434 (M ⁺ ), 364, 320, 262, 235,
154, 70, 43 [α] ²⁷ _D = -86.2° (C = 0.45, MeOH) Potassium carbonate (56 mg) was dissolved in methanol (12 ml), compound 47 (348 mg) was added under ice cooling, and the mixture was reacted for 2 hours under ice cooling. The reaction solution was subjected to silica gel column chromatography and eluted with chloroform-methanol-aqueous ammonia (80:20:2), resulting in N-[(S)-2-azetidinone-4
-Carbonyl]-L-histidyl-N-(2-hydroxyethyl)-L-prolinamide 48 (294 mg)
is obtained. This compound had the same physical and chemical properties as Compound 3d obtained in Example 4. Reference example 20 (S)-2-azetidinone-4-carboxylic acid 210
g (8.68 mmol) and pentachlorophenol
24.4 g (8.68 mmol) was dissolved in 200 ml of DMF, and 17.93 g (8.70 mmol) of DCC was added under cooling (0 to 5°C). The mixture was stirred at room temperature for 5 hours to remove precipitated dicyclohexylurea, and the liquid was concentrated under reduced pressure. The obtained residue was dissolved in 200 ml of ethyl acetate under heating and then cooled to collect precipitated crystals. 25.9 g of yellowish crystals (S)-4-pentachlorophenoxycarbonyl-2-azetidinone 49 having a melting point of 177-179°C were obtained. NMR (90MHz, ^d6 -DMSO- _CD3OD ) δppm 3.23 (1H, q, azetidinone ring 3rd position), 3.57 (1H, q, azetidinone ring 3rd position) 4.70 (1H, d, d, azetidinone ring 4th position) IR (KBr) cm ^-1 3200, 1775, 1755, 1720 Mass 363 (M ⁺ ), 335, 266, 237 Reference example 21 L-Histidine methyl ester dihydrochloride 50
Suspend 6.05 g (25 mmol) in 75 ml of DMF and
The mixture was cooled to 5° C. and 5.05 g (50 mmol) of triethylamine was gradually added dropwise. After the dropwise addition was completed, the mixture was stirred at the same temperature for 15 minutes. 9.50 g (25 mmol) of Compound 49 was added as a powder, stirred at the same temperature for 1 hour, and then left at room temperature overnight. After removing the precipitated triethylamine hydrochloride,
The liquid was concentrated under reduced pressure. After adding 40 ml of ethyl acetate and 30 ml of water to the obtained residue and shaking, the aqueous layer was taken, and the ethyl acetate layer was extracted twice with 20 ml each of water. The aqueous layers were combined and water was distilled off under reduced pressure. Acetonitrile and benzene were added to the obtained residue, and the mixture was concentrated under reduced pressure. The residue was crystallized from 30 ml of methanol. Colorless crystal N〓-[(S) with a melting point of 142-147℃
4.1 g of -2-azetidinone-4-carbonyl]-L-histidine methyl ester 51 was obtained. This compound 51 has crystalline polymorphism, with a melting point of 209.5~
Crystals showing a temperature of 212.5°C are also obtained. NMR (90MHz, ^d6 -DMSO) δppm 2.94 (2H, d, histidine group β-position methylene), 3.60 (3H, s, methyl group), 4.02 (1H, d, d, azetidinone ring 4-position), 4.54 (1H , m, histidine group α-position methine), 6.72 (1H, s, imidazole ring), 7.56 (1H, s, imidazole ring), 8.20 (1H, s, NH), 8.56 (1H, d, azetidinone ring NH) IR (KBr) cm ^-1 3250, 3100, 2950, 1770, 1750, 1740, 1720,
1650, 1550 Example 17 20 ml of 0.1N sodium hydroxide was cooled to 0-5°C, 532 mg (2 mmol) of Compound 51 was added, and the mixture was stirred at the same temperature for 1.5 hours. Next, 760 mg (4 mmol) of para-toluenesulfonic acid monohydrate was added at the same temperature, and water was distilled off under reduced pressure. The obtained residue was azeotropically dehydrated with acetonitrile and benzene and dried under reduced pressure. Dissolve the obtained powder in 20 ml of DMF and L-prolinamide 53 228
mg (2 mmol) and 412 mg (2 mmol) of DCC were added, and the mixture was stirred at room temperature overnight. After removing the precipitated dicyclohexylurea, the liquid was concentrated under reduced pressure, the resulting residue was dissolved in 20 ml, insoluble matter was removed, and water was distilled off under reduced pressure. The residue was dried under reduced pressure, then dissolved under heating in 7 ml of methanol, stirred while cooling, and the precipitated crystals were collected. Melting point 179-184
500 mg of colorless crystal N-[(S)-2-azetidinone-4-carbonyl]-L-histidyl-L-prolinamide 3a having a temperature of 0.degree. C. was obtained. The NMR, IR, and Mass spectra of this product matched those of Compound 3a obtained in Example 1 above. Prescription example Injection 1-In an ampoule, along with 10 mg of mannitol, N
-((S)-2-azetidinone-4-carbonyl)-L-histidyl-L-prolinamide
Freeze-dried preparations containing 0.025 mg or 0.05 mg are prepared and each is dissolved in 1 ml of sterile physiological saline to prepare an injection. Tablet N-((S)-2-azetidinone-4-carbonyl)-L-histidyl-L-prolinamide
0.25 parts by weight and 7.5 parts by weight of lactose are mixed and ground, and this mixture contains 44.4 parts by weight of lactose and 2.25 parts by weight of crystalline cellulose.
Parts by weight and 0.4 parts by weight of magnesium stearate are added, mixed uniformly, and pressed using a tablet machine to form tablets of 75 mg/tablet. Capsule N-((S)-2-azetidinone-4-carbonyl)-L-histidyl-L-prolinamide 0.5
137.5 parts by weight of lactose and 60 parts by weight of corn starch are mixed and ground into a mixture of 10 parts by weight of lactose and 10 parts by weight of lactose.
parts by weight and 2.0 parts by weight of magnesium stearate are added and mixed uniformly. This is filled into No. 3 hard gelatin capsules at a rate of 210 mg per capsule to prepare a capsule preparation. Example 18 (i) N-[(S)-2-azetidinone-4-carbonyl]-L-histidine methyl ester 51
Add 13.4 g to 504 ml of 0.1N sodium hydroxide cooled to 0-5°C. This mixture was heated at 0-5℃.
Stir for 1 hour. Then, 50.4 ml of 1.0N hydrochloric acid was gradually added to neutralize. Water is distilled off from the reaction mixture under reduced pressure, and the residue is dried by azeotropic dehydration using toluene. When the precipitated crystals are washed with methanol and collected, crude N-[(S)
-2-azetidinone-4-carbonyl]-L-
Histidine 54 is obtained in quantitative yield. This product is used in the next synthesis reaction without purification. (ii) 1.553 g of compound 54 obtained as above, 2.
1.012 g of 4-dinitrophenol and 570 mg of L-prolinamide 53 are suspended in 20 ml of dry DMF and cooled on ice. Next, add 1.13g of DCC to 0-5
Stir at ℃ for 1 hour. Bring this mixture to room temperature for 20 minutes.
After stirring for an hour, insoluble matter is removed. Remove DMF from the solution under reduced pressure and add 50 ml of water to the residue. Remove the precipitated insoluble matter, combine the liquid and washing liquid, and then add 5.5 ml of 1N hydrochloric acid. This aqueous solution was washed three times with 30 ml each of methylene chloride. aqueous solution
After neutralizing with 5.5 ml of 1N sodium hydroxide, water is distilled off under reduced pressure. The residue is azeotropically dehydrated using toluene-ethanol and dried. Add 40 ml of methanol to this to remove insoluble matter.
After concentrating the liquid to a volume of approximately 10 ml, cool it on ice.
When the precipitated crystals are taken, N〓-[(S)-2-
1.3 g of azetidinone-4-carbonyl]-L-histidyl-L-prolinamide 3a are obtained. This product had the same physical and chemical properties as 3 obtained in Example 1. Reference example 22 N was prepared by the same method using D-histidine methyl ester dihydrochloride 55 instead of L-histidine methyl ester dihydrochloride 50 in Reference Example 21.
-[(S)-2-azetidinone-4-carbonyl]-D-histidine methyl ester 56 is obtained. NMR ( ^d6 -DMSO) δppm: 2.6-3.24 (4H,
m, histidine group β-position methylene, azetidinone ring 3-position), 3.60 (3H, s, methyl), 4.0 (1H,
d, d, azetidinone ring 4th position), 4.56 (1H, histidine group α-position methine), 6.82 (1H, imidazole ring), 7.56 (1H, imidazole ring), 8.22
(1H, NH), 8.56 (1H, NH) IR (KBr) cm ^-1 : 3230, 3000, 2950, 1780,
1730, 1710, 1660, 1580, 1440, 1230 Example 19 Compound 56 instead of compound 51 in Example 17
Amorphous N-[(S)-2-azetidinone-4-carbonyl]-D-histidyl-L-prolinamide 57 is obtained by carrying out the same reaction using . NMR (D ₂ O) δppm: 2.0 center (4H, m, pyrrolidine ring), 2.72, 2.90 (1H, d, d, azetidinone ring 3rd position), 3.0-3.48 (3H, histidine β position methylene, azetidinone ring 3rd position) ), 4.30 (2H,
azetidinone ring position 4, and methine), 7.0
(1H, imidazole ring), 7.72 (1H, imidazole ring) IR (KBr) cm ^-1 : 3250 (broad), 1750,
1670, 1630, 1540, 1440 [α] _D = -76.2° (C = 1, water)

Claims (1)

[Claims] 1. General formula (In the formula, R ¹ is [Formula] or [Formula] (In the formula, R ² means a hydrogen atom, an aromatic acyl group or an aryl group (Aryl).), X is a methylene group, an ethylene group, Oxygen atom or sulfur atom Y means a hydroxyl group, a lower alkoxy group, an aralkoxy group, or an amino group substituted with an unsubstituted or lower alkyl group, a hydroxy lower alkyl group, a lower alkoxy lower alkyl group, or a cycloalkyl group.) Derivatives of 2-azetidinone-4-carboxylic acid and salts thereof as shown.