CA1256650A - Process of producing 2-azetidinone-4-substituted compounds, and medicaments containing the compounds - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE

A novel 4-substituted-2-azetidillone compound shown by the general formula

Description

4-SUBSTITIJTED-2-~ZETI~INONE COMPOUND, PROCESS OF
PRODUCING THE COMPOUNDS, AND MEDICAMENTS CONTAINING
THE COMPOUNDS

DETAILED EXPLANATION OF THE INVENTION
This invention relates to a 4-substituted-2-a etidinone compound shown by following general formula Rl ~ (CH2)nCONH-C -CON ~ (I) wherein R1, R3, and R4, which may be the same or different, each represents a hydrogen atom or a lower alkyl group; R2 represents an imidazole group shown by ~ N ~ N-R5 R5-N 1~ or ~ (wherein R5 represents a hydrogen atom, a lower alkyl group, an aromatic acyl yroup, or an aryl group); n represents O, 1, 2, or 3; X represents a methylene group, an ethylene group, an oxygen atom, or sulfur atom; and Y represents a hydroxy group, a an aralkoxy ~rou~, lower alkoxy group,/or an unsubsti~uted or substituted amino group shown by the formula -N< 7 (whereln R6 and R7, wh-.ch may be the same or different, each represents a hydrogen atcm, a loweralkyl group, a hydroxy lower alkyl a lower alkoxy lower al yl group, group,/a cycloalkyl group, an aryl group, an amino lower alkyl group, or an acyloxy lower alkyl group;
said R6 and R7 may combine with each other to form a 5- or 6-membered cyclic group which rnay contain an oxygen atom, a sulfur atom, or a nitrogen atom together .~

~:~S6~i5~

with the nitrogen atom bonded thereto) and a salt thereof.
The invention also ~relates to a process of produc-ing a 4-substituted-2-azetidinone compound shown by the foregoing general formula (I) or a salt thereof, which comprises reacting a carboxylic acid epresented by general formula (II) Rl ~ (CH2)nCOOH (II) ~ NH

wherein Rl and n have the same meaning as in general formula (I) or a reactive derivative thereof and an amine represented by general formula (VI) H2N-fH-CON X R3 1 2 ~ (VI) R CO-Y

wherein R2, R3, R4, X, and Y have the same meaning as in general formula (I); when Y in the foregoing expla-nation is a hydroxy group or R6 or R7 r~presents a hydroxy lower alkyl group or an amino lower alkyl group, these groups may have a protective group or a reactive derivative thereof and, when the reaction product has a protective group, removing the group.
Furthermore, the invention relates to a process of producing a ~-~ubstituted-2-~zeti~inone compound shown by the foregoing general formula (I) or a salt thereof, which comprises reacting a carboxylic acid represented by general formula (IV) so Rl ~ (CH2)nCONH-CH-COOH
~ NH CH2 (IV) wherein Rl, n, and R2 have the same meaning as described above or a reactive derivative thereof and an amine repr~-sented by general formula (V) HN X
R3 (V) CO-Y
wherein R3, R4, X and Y have the same meaning as described above and when Y is a hydroxy group or R6 or R7 is a hydroxy lower alkyl group or an amino lower alkyl group in the foregoing definition of Y, these groups may have a protective group or a reactive derivative thereof and, when the reaction product has a protective group, removing the protective group.
The lower alkyl group shown by Rl, R3, R4, R5, R6 and R7 in the foregoing general formulae includes straight chain or branched alkyl groups each having 1 to 5, preferably 1 to 3 carbon atoms, such as a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, a pentyl group, etc. When both R3 and R~ are a lower alkyl group, these lower alkyl groups can bond to a same carbon atom.
The aromatic acyl group shown by R5 is an unsubstituted or substituted benzoyl or benzenesulfonyl ~2566~

group and the substituent is a straight chain or branched alkyl group having 1 to 5, preferably 1 to 3 carbon atoms, such as a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, a pentyl group, etc.
The aryl group shown by R5 is an unsubstituted or substituted phenyl group. The substituent of the phenyl group is, for example, a nitro group and the phenyl group may have 1 to 3 such substituents.
The lower alkoxy group shown by Y includes straight chain or branched lower al]coxy groups having 1 to 5 carbon atoms, such as a methoxy group, an ethoxy group, a propoxy grGup, an isopropoxy group, a butoxy group, a tert-butoxy group, a pentyloxy group, etc.
The aralkoxy group shown by Y includes phenyl lower alkoxy groups such as a benzyloxy group, a phenetyloxy group, a 3-phenylpropyloxy group, an ~-methylphenetyloxy group (-O-CHCH2 ~ ), etc.

The hydroxy lower alkyl group shown by R6 and R7 includes lower alkyl groups having 1 to 5 carbon atoms substituted by a hydroxy group, such as a 2-hydroxy-thyl group, a 2-hydroxypropyl group (-CH2CHCH3 ), OH
a 4-hydroxybutyl group (-CH2CH2CH2CH20H), etc.
The lower alkoxy lower alkyl group shown by R6 and R7 includes the foregoing hydroxy lower alkyl groups the hydrogen atom of the hydroxy group of whlch is ~:~S66~3 substituted by a lower alkyl sroup having 1 to S carbon atoms.
The cycloalkyl group shown by R6 and R7 is cyclo-alkyl groups having 5 to 10 carbon atoms, which may be crosslinked, such as a cyclopentyl group, a cyclohexyl group, an adamantyl group, etc.
The aryi group shown by ~6 and R7 includes aromatic hydrocarbon groups such as phenyl group, a naphthyl group, etc.
The amino lower alkyl group shown by R6 and R7 is straight chain or branched alkyl groups of 1 to 5 carbon atoms having an unsubstituted amino group or a substituted amino group (e. g., a methyl amino group, an ethylamino group, a dimethylamino group, an ethyl-methylamino group, a pyrrolidinyl group, a piperidinyl q group, a 2-ketopiperidino group (-N ~ ), a 2-keto-1-pyrrolidinyl group (-N~ ), etc.
Also, the acyloxy lower alkyl group shown by R6 and R7 is straight chain or branched alkyl groups of 1 to 5 carbon atoms having a lower acyloxy group, such as an ac~yloxy group, a propionyloxy group, an iso-butyryloxy group, a butyryloxy group, etc.

R6 and R7 may combine with each other to form a 5- or 6-membered ring group, which may contain an oxygen atom, a sulfur atom or a nitrogen atom, together with the nitrogen atom to which R6 and R7 are bonded as described above and examples of the 5- or 6-.membered ring group are a l-pyrrolidinyl group (-N ~ ), a piperidino group (-N ~ ), an oxazolidine-3-yl group (-N ), a thiazolidine-3-yl group (-N ~ ), a 2-pyrazolidinyl group (HN ~ ), a morpholino group (-N~_~0), a thiomorpholino group (~N~_~S), a 1-piperazinyl group (-N~_~NH), etcO
The desired compound shown by general formula (I) of this invention has at least 3 asymmeteric carbon atoms and there are sterAeoisomers.
Thus, the desired compound of this invention includes each such separated isomer and a mixture of the isomers.
The desired compound of this invention shown by general formula (I) may form a salt with an acid or a base. The salt of the compound included in this invention includes the salts thereof with nontoxic acids (e. g., an inorganic acid salt s~ch as a hydrochloride, a sulfate, etc., and an organic acid salt such asa citrate, an acetate, a tartarate, etc.), and the salts thereof with non-toxic bases (e. g., the salt with an inorganic base, such as a sodium salt, a potassium salt, etc., and the salt with an organic base, such as an ammonium salt, a trimethylamine salt, etc.).
As a compound having relation to the desired conpound of this invention shown by formula (I), there i5 known L-pyroglutamyl-L-histidyl-L-prolinamide (pGlu-His-Pro-NH2) called as "Thyrotropin Releasing ormone" (TRH).
been The existence of TRH has already/known since the ~:~5~i~5~

1960's but the structure thereof was confirmed in 1970 (Fndocrinology, 86, 1143(1970)). TRH is said to be a hormone controlling the release of thyrotropin (TSH) in the hypophysis of a mammal. However, by the investi-gation made after then, it has been clarified that the biological function of the tripeptide TRH is not limited to the control of the release of TSH but TRH
widely acts to a central nervous system (CNS), and a field o~ ~ew investigations 'nas been developed based on the discovery (Science, 178, 417(1972) and LanCet,

2, 999(1972). Thus, it is known that TRH and the derivatives thereof have actions to CNS, such as the decrease of the continuation time of sleep caused by barbitu~ates or alcohol, the control of hypothermia by the stimulus of various medicaments, the acceleration of motor activity, the prevention of haloperidol-memory enhancing e~ect, induced catalepsy,/the lmprovement of anti-psychtic effect, an anti-depressive effect, etc., in addition to the TRH releasing activity (U. S. Patent Nos.

3,865,934 and 3,932,623). Furthermore, it has been discovered that TRH is useful for improving or treating functional or organic disturbances in the brain, for example, a disturbance of consciousness caused by head injury, brain surgery, cerebro-vascular disorders, brain tumors, etc., in particular, an acute or semiacute disturbance of consciousness (Belgian Patent No. 839,833~.
The development of TRH derivatives showing a weaker TSH releasing activity than TRH or almost no ~:~5~iÇiS~

TSH releasing activity and having actions to CNS same as or higher than the foregoing actions of TRH has been demanded. Thus, various TRH derivatives were synthe-sized for the foregoing purpose and the actions to C~S
have been further enlarged. As the compounds synthe-sized for the purpose, there are known a TRH derivative which has a weaker T~H releasing activity than ~RH, has a narcoticantagonizing action, an action of increasing spontaneous activity, or a dopamine-like action, and is said to be useful for the improvement or the treatment of sonifacients poisoning, disturbance of conscious-ness, hyperactivity child, schizophernia, nervous depression, and Parkinson's di.sease (Japanese Patent Publication (unexamined) No. 116,465/'77) and a TRH
derivative which has action of improving and treating the disturbance of consciousness after an external injury of the head and an action of decreasing the continuation time of sleep by hexobarbital, and is said to be useful for the treatment for a patient having a disturbance of consciousness caused by the organic or functional disturbances in the brain, the treatment for a patient sho~ing senility ormental fatigue, and the treatment for depression state (Japanese Patent Publication (unexamined~ No. 59,714/'81).
The compound of this invention has the structural feature in the point that the pyrc.glutamyl (pGlu) structural moiety of TRH is converted into an azetidinone structuré ~-lactum structure) which has never been employed. As to the me~icinal action, the ~2~66~

compound of thls invention has a more remarlcably strong CN~ actions than TR~ and conventionally known TRH derivatives and hence ls very useful as medicaments.
For example, the compound of this invention is useful for improving a disturbance of consciousness in schizophrenia, nervous depression, the sequels of cerebro-vascular disorders, a head injury, senile dementia, epilepsy, etc., or improving hypobulia, depressive syndrome, memory loss, etc.
The compound of this invention shown by general formula (I) can be orally or parenterally administered as it is or as a mixture with proper phamacologically allowable carrier, excipient, diluent, etc., in the form of powders, granules, tablets, capsules, iniections (intravenous, subcut~neous, or intramuscular injections), or suppositories. The dose of the compound of this invention shown by formula (I) differs accordinq to the kind of the compound of formula (I), the age, weight, and symptom of a patient, the manner of administration, etc., but is about 0.001 to 10 mg, preferably 0~01 to 0.1 mg (one dose) in the case of injection and 0.05 to 500 mg, preferably 0.1 to 10 mg (one dose~ in the case of oral administration.
The following experiments show the action to a low body temperature by pentobarbitol (Experiment 1), the action to the disturbance of consciousness by a head injury (Experiment 2~, and the action to acute toxicity (Experiment 3) about typical compounds in the compounds of this invention shown by formula (I).

~2~66~0 Experiment 1.
Pentobarbital-induced hypothermia:
Nine male mice weighing 18 to ~2 g were used for each dosage of the test compounds. Mice were given i. v. various doses of TRH or tested compounds 10 min.
after pentobarbital (55 mg/kg i.p.). Rectal -tempera-ture was measured before pentobarbital dosing and immediately before and 30 min. after th~ test compounds.
Effects of test compounds were evaluated as ED1 5C~
the dose required to reduce by 1.5C pentobarbital-hypothermia of control group of mice which received only pentobarbital and saline. The results are shown in Table 1.
Experiment ~.
Disturbance of consciousness induced by concussive head injury:
Nine male mice weighing 18 to 2~ g were used for each dosage of the test compounds. An acrylate weight containing lead (20.5 g, 19 mm in both diameter and thickness) was dropped to the head of mice from a 18 cm height. Mice were induced loss of consciousness and they remained motionless for some period. The time ~rom the shock up to the onset of spontaneous movement was recorded as the spontaneous movement time. Test compounds were administered intravenously 10 min.
before adding concussive head injury and effects of test compounds were evaluat~d as ED50%, the dose required to shorten by 50~ the spontaneous movement time of control group. The results are shown in Table 1.

~s~s~

Table 1 Test compound (A)* (B)*

N~-[(S)-2-azetidinone-4-carbonyl]-L-histidyl-L-prolinamide (Example 1) 0.01 0.1 N~-[(S)-2-azetidinone-4-carbonyl]-L-histidyl-L-thiazolidine-4-carboxamide (Example 6) 0.05 N~-[(S~-2-azetidinone-4-carbonyl]-L-N-(2-hydroxyethyl)-L-prolinamide (Example 4) 0.35 N~-[(S)-2-azetidinone-4-carbonyl]-NI-methyl-L-histidyl-L-prolinamide (Example 19) 0.004 TRH 0~1 2.5 ~*): (A) Reversal effect against pentobarbital-hypothermia EDl 5C (mg/kg i.v.) (B) Onset time of spontaneous movement EDso% (mg/kg i.v.) Experiment 3.
Acute toxicity:
An a~ueous physiological saline solution of 1493 mglkg of a test compound, N~-[(S)-2-azetidinone-4-carbonyl]-L-histidyl-L-prolinamide was intravenously administered to one group of nine male mice and they were observed for 24 hours but no example of death was observed. l'hat is, LD50 ~i.v.) of the compound of this invention was higher than 1493 my/kg.
On the other hand, in the case of administering TRH to mice, L~50(i. v.) was 751 mg/kg(i.v.).
The the production process of this invention will be explained in more detail.
The reaction courses for the production processes ~2~

of this invention are shown by the following schemes:

1 (CH2)nCOOH H2NCHCOOH HN X
R~ R/22 \~R4R3 COY
(II) (III) (V) 11 ~ I I . 1 ~X
)nCONHCHCOOH H2NlHcoN ~ R3 (IV) (VI) ~ _ _ , _ ~ ~ ~ ~X

o~ CH2)nCONHCHCON~ ~ ~R3 (Il) The compound of this invention shown by above formula (Il) can be converted into the compound of this invention shown by following formula (I2) by the hydrolysis or the catalytic reduction when Y is an alkoxy group or an aralkoxy group or by the reaction with an unsubstituted or substituted amine when Y is a hydroxy group. Also, when the substituent R5 in the imidazolyl group shown by R2 is an aromatic acyl group or an aryl group, the compound of formula (I2) wherein R5 is hydrogen atom is obtained by removing the substituent by an ordinary manner~

CH2 ~ R

(I2) ~2~6~i5;1:3 In the schemes, Rl, R2, R3, R4, n, X and Y have the same meaning as described above; R2 represents a case that the substituent RS in the imidazolyl group shown by R2 is a hydrogen atom; and Y' represents a hydroxy group or an unsubstituted or substituted amino group.
That is, according to the process of this inven-tion, the desired compound of formula (I) can be produced (a) by reacting the compound of formula (II) and the compound of formula (III) to form the compound of formula (IV) and then reacting the compound of formula (IV) and the compound of formula (V) or (b3 by reacting the compound of formula (III) and compound of formula (V) to form the compound of formula (VI) and then reacting the compound of formula (VI) thus obtained and the compound of formula (II~.
Also, the desired compound of formula (I1) can be induced into the other desired compound of -Eormula (I2) by converting the substituent Y.
The production reaction for the compound of formula (I) employed in the foregoing process (a) or (b) is a peptide synthesis reaction and is performed by a known manner. As such a manner usually used, there are a method of using dicyclohe~yl carbodiimide as a condensirg agent, an azide method, an acid chloride method, an acid anhydride method, an active ester method, etc. These methods are performed as follows:
That is, prior to the performance of the peptide ~s~

forming reaction in each step, the functional groups of the raw material compound, such as an amino group, an imino group, a carboxy group, etc., which do not take part in the reaction, are usually protected and an amino group, an imino group, or a carboxy group of the compound, which takes part in the reaction, is, if necessary, activated. The compound of which the amino group, the imino group or the carboxy group is activated, for example, the active ester may be subjected to the peptide synthesis reaction after once isolated from the reaction mixture or may be subjected to the peptlde synthesis reaction without being isolated.
Examples of the protective group for the amino roup are a benzyloxycarbonyl group, a t-butyloxycarbo-p-methoxybenzyloxycarbonyl group, nyl group,/a phthaloyl group, a trifluoroaceiyl group, etc., and examples of the protective group for the imino group are a tosyl group, a benzyloxycarbonyl group, a p-methoxybenzyloxycarbonyl group, a benzyl group, a 2,4-dinitrophenyl group, etc. ~lso, the protective group for the carboxy group is used as the form of an ester such as a m~thyl ester, an ethyl ester, a benzyl ester, a p-nirtobenzyl ester, a t-butyl ester, etc.
The activation o the group which takes part in the reaction is performed by a phosphazo process using phosphorus trichlorlde, ~n isocyanate process using phosgene, or a phosphorous acid ester process when the group is an amlno group or an imino group or is performed in the form of an active ester (e. g., 2,4-dinitrophenol ester, N-hydroxysuccinimide ester, etc.,), an azide group, or a carboxylic anhydride when the group is a carboxy group.
Among the foregoing methods of performing the eptide synthesis reaction, it is prefered to perform reactions the coupling/ of the compound of formula (IV) and the compound of ~ormula (V) by the azide method or the method of using dicyclohexyl carbodiimide as the condensing agent. Also, a method of dirctly forming peptide using the N-carboxy anhydride of aminoacid without using a protective group may be employed.
Then, the peptide forming reaction is performed in an inert solvent at room tempera-ture or by heating by an ordinary manner. Examples of the suitable solvent used in the reaction are dimethylformamide (DMF), ethyl acetate, dicnloromethane (methylene chloride), tetrahydrofuran, etc.
If it is necessary to remove a protective group from the reaction product, the protective group can be removed by a catalytic reduction when the protective group is benzyl ester; by using anhydrous hydrogen N-hydroxy-1,2,3,-benzotriazole fluoride ~HOBT),or a hydrogen fluoride-pyridine complex when the protective group is p-toluenesulfonyl group; by hydrolysis when the protective group is an alkyl ester; by a catalytic reduction or a hydrobromic acid-acetic acid treatment when the protective group is p-methoxybenzyloxycarbonyl; or an acid decomposition when the protective group is a t-butyloxycarbonyl group.

~2~5~

Furthermore, in the reaction of inducing the desired compound of formula (I) into other desired compound by converting the substltuent Y of the compound of formula (I), the reaction conditions maybe suitably selected according to the characters of the compounds taking part in the reaction. The details of these conditions will be explained in the examples.
Furthermore, the invention relates to a novel

4-substituted~-2-azetidinone compound shown by general formula ~VII) o ~ CONH-CIH-COOR8 H CH2 (VII) ~ N
N ll wherein R8 represents a hydrogen atom, a lower alkyl group or an aralkyl group or a salt thereof, and a process of the production thereof.
Examples of the lower alkyl group shown by R8 in the foregoing general formula (VII) are straight chain or branched alkyl groups having i to 5, preferably 1 to 3 carbon atoms, such as a methyl group, an ethyl group, a propyl group, a butyl group, a pentyl group, an isopropyl group, etcq, and examples of the aralkyl group shown by R~ are aryl lower al!~yls such as a benzyl group, a phenetyl group, a naphthylmethyl group, etc.

The novel compound shown by general formula (VII) ~ 2~ ~7~ ~

is useful as an intermediate compound for producing the compound of this invention showr by general formula (I).
The compound shown by general formula (VII) can be produced by reacting a carboxylic acid sho~7n by the formula COOH

O ~ H
or a reactive derivative thereof and an amine shown by the general formula H2N-CH-CooR3 CH2 (IX) ~N
I!
H
wherein R8 has the same meaning as described above or a reactive derivative thèreof. The reaction is a peptide synthesis reaction and can be performed by a known manner as described hereinbefore.
Still further, the invention relates to a novel 4-substituted-2-azetidinone compound represented by the general formula F~
O H

wherein R9 represents ~ or Cl C1 and the production process thereof.
The foregoing compound of this invention is useful as an intermediate compound for producing the compound ~S6~

shown by formula (vII) and further is usful as an inter-mediate compound for producing 3-lactum series antibiotics.
The novel intermediate compound is produced by reacting a carboxylic acid shown by the formula COOH

or a reactive derivative t~ereof and pentachlorophenol or N-hydroxysuccinimide.
The reaction is an ester synthesis reaction and a known esterification method can be suitably selected.
Then, the invention will further explai.ned by the following examples but the examples do not limit the scope of this invention.
In addition, the production processes for the raw materials commonly used in plural examples will first be explained as reference examples.
Also, the abbreviations employed in the examples and the reference examples indicate the following meaning.
TLC Thin layer chromatography NMR Nuclear magnetic resonance spectrum IR Infrared absorption spectrum Mass Mass analaysis spectrum Z Benzyloxycarbonyl Bn Benzyl His Histidine Pro Proline ~5~

DNP 2,4-Dinitrophenyl Ts Tosyl BOC t-Butyloxycarbonyl DMF Dimethylformamide HOBT N-Hydroxy-1,2,3-benzotriazole DCC Dicyclohexylcarbodiimide THF Tetrahydrofuran H~Su N-HydroXysuccinimide Ph Phenyl In addition, the compound containing the mark Nim in the names of the compounds is a mixture of a ~ ~substituent nitrogen atom-substituted (~ ) compound of the imidazole ring of histidine and a~ nitrogen atom-substituted ~ ~ ~ ) compound.
substituent Reference example 1.

~ COOCH2 ~ ~ COOH
O H (1) O H (2) In 350 ml of methanol was dissolved 3.46 g of (S)-4-benzyloxycarbonyl-2-azetidinone (1) and the azetidinone was hydrogenated using 350 mg of 10%
palladium-carbon as a catalyst. The catalyst was removed by filtration and the filtrate was concentrated to dryness to provide 1.94 g of (S)-2-azetidinone-4-carboxylic acid (2).

~2~

NMR (DMSO-d6) Sppm 8.26 (s, lH), 4.02 (dd, lH, J=3.4 Hz, 6.9 Hz), 3.21 (dd, ~H, J=6.9 Hz, 16.0Hz), 2.82 (dd, lH, J-3.4 Hz, 16.0 Hz).
IR (KBr) cm~l: 3320, 1740, 1720 M~ss: 116 (M + 1) Reference example 2.

Pro-OBn~5) æ -His-NHNH2 ~ [Z-His-N3] ~ Z-His-Pro-OBn (3) (4) (6~
In 99 ml of an aqueous solution of lN hydrochloric acid was dissolved 10.01 g of N~-benzyloxycarbonyl-L-histidine hydrazide (3). After adding thereto 132 ml of ethyl acetate, 8.25 ml of an aqueous solution of 2.313 g of sodium nitrite was added to the mixture with stirring vigorously~under ice-cooling. After perform-ing the reaction at 0C for 5 minutes, 39.6 ml of an aqueous 50% potassium carbonate solution was added to the reaction mixture under ice-cooling to alkalify the solution. The reaction mixture was placed in a separation funnel and an or~anic layer thus formed was collected. Furthermore, the aqueous layer was extracted by 20 ml of ethyl acetate and the extract was combined with the foregoing organic layer. The mixture was dried overanhydrous sodium sulfate under ice-cooling for 10 minutes~ By remo~7ing anhydrous sodium sulfate by filtration, 152 ml of an ethyl acetate solution of N~-benzyloxycarbonyl-L-histidine a~ide (4) was obtained.

The product was ice-cooled and 20 ml of an ethyl acetate

5.703 g of solution of~ -proline benzyl ester (5) was added thereto.

~25~ Ej5V

The mixture was reacted overnight at 0C and then the reaction mixture was concentrated to dryness. The residue was dissolved in 22 ml of chloroform~methanol (10 : 1) and subjected to silica gel column chromato-graphy. The eluate by chloroform-methanol (95 : 5) was concentrated to dryness to provide 6~602 g of N~-benzyloxycarbonyl-L-histidyl-L-prol~ine benzyl ester

(6).
NMR (CDC13) ~ppm: 7.45 (lH), 7.14 (s, 5H),

7.10 ~s, 5H), 6.82 (lH), 5.70 (d, lH, J=8.5 Hz), 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=5.7 Hz), 1.6-2.5 (m, 4H) Mass: 476 (M+), 396, 325, 244, 91, 70.
Reference example 3.

(6) ---~His-Pro-OBn.2HBr (7) After ice-cooling 20 ml of an acetic acid solution of 25% hydrobromic acid, 1.91 g of compound (6) was added to the solution followed by reaction for 2 hours at s_loC. The reaction mixture was added to 200 ml of dry ether and after quickly removing the precipitates thus formed by filtration and then the reaction mixture.was dried overnight in a desiccator with potassiwn hydroxide, to provide 1.99 g of L-histidyl-L-proline benzyl ester.2-hydrobromide (7).
NMR (CDC13 * CD30D) ppm: 8.75 !lH), 7.57 (lH), 7,35 (s, 5H), 5.2 (lH).

~2~

Example 1

(8) ---~[His-Pro-NH2] ~ CO-His-Pro-NH2

(9) NH (lQ) In 13 ml of ~ DMF was dissolved 826 mg of L-histidyl-L-prolinamide 2-hydrobrmide (~) and then 2 ml of a DMF solution of 405 mg of triethyl-amine was added to the solution under ice-coolingO
After performing 30 minutes under ice-cooling, the precipitates thus formed were filtered off to provide L-histidyl-L-prolinamide (9). The product was immediately used for the subsequent synthesis reactlon.
In 10 ml of DM~ was dissolved 230 mg of compound (2) and then 351 mg of - HOBT and 453 mg of DCC
were added to the solution under ice-cooling. Then, after stirring the mixture for 15 minutes, the - reaction was performed for 15 minutes at room temperature. The reaction mixture was ice-cooled again and 15 ml of a DMF solution of foregoing compound (9) was added to the reaction mixture followed by reaction overnight at 0C. The precipitates thus formed were filtered off, the flltrate was concentrated to dryness, -the residue was dissolved in 10 ml cf chloroform-methanol (4 : 1) and subjected to silica gel column chromatography. The eluates by chloroform-methanol (7 : 3) were collected and concentrated to dryness to provide 509 mg of crude N~--[(S)-2-azetidinone-4-carbonyl]-L-histidyl-L-prolinamide (10).

When the product was subjected to silica gel column chromatography again and eluted by a mixture of chloroform, rnethanol, and aqueous ammonia (40 : 10 : 1) to provide 394 mg of pure product (10).
NMR (CD30D) ~ppm: 7-59 (s, lH), 6,98 (s, lH), 4.41 (dd, lH), 4.11 (dd, lH, J=3.1 Hz, 5.9 Hz), 3.36-3.96 (m, 2H), 3.05 (dd, lH, J=5.9 Hz, 14.9 Hz), 2.80 ~dd, lH, J=3.1 Hz, 14.9 Hz), 1.72-2.20 (m, 4H) Mass: 348 (M ), 234, 207, 154, 82, 70 [~]D3 = -75.8 (C = 0.6, methanol) [~]D4 = -100.4 (C = 1, water) . When the compound (10) was triturated with a small amount of methanol, the compound crystallized.
M. p. 183-185C
Elemental analysis for Cl5H20N6o4.l ~2 C(%) H~%) N(%) Calculated: 50.41 5.92 23.52 Found: 50~35 6.00 23.64 When the compound (10) was recrystallized from methanol, the product having a different crystal form was obtained according to the crystallization conditions. For eY~ample, products having melting points of 145-149C, 154-157C, 154-163C., 181.5-183.5C, 187-189C, etc., were obtained and they were confirmed to be polymorphous crystals by infrared absorption spectra (KBr tablet), powder X-ray diffraction, differential scanning calorimetry, etc. The difference in mel-ting point was by the miYing ratio of 3~S~65~

different crystal forms. The properties (N~, optical rotation, etc.) of the solution states of these products showed same properties.
Example 2 (2) ~ CO-His-Pro-OCH
(7) --~[His-Pro-OBn] ~ n ~11) O~-NH (12) In 45 ml of dichloromethane was dissolved 1.99 g of L-histidyl-L-proline benzylester.2-hydrobromide (7) and after cooling the solution to -2QC, 5 ml of a dichloromethane solution of 900 mg of triethylamine was added to the solution. After performing the reaction for one hour at -10C to -20C, the precipitates were filtered off to provide a solution containing L-histidyl-L-proline benzyl ester (11). The product was immediately used in the subsequent synthesis reaction.
In 30 ml of dichloromethane was suspended 455 mg of compound (2) and after adding thereto 801 mg of HOBT and 1.059 g of DCC and stirring the mixture for 15 minutes, the reaction was performed for 15 minutes at room temperature. The reaction mixture was ice-coole~ again and then 50 ml of a dichloromethane solution of foregoing compound (11) was added to the reaction mixture. After reacting the mixture for one hour under ice-cooling, the reaction was performed overnight at room temperature. Precipitates thus formed was filtered off and the filtrate was concentrated to dryness. The residue thus obtained was dissolved in 20 ml of water-methanol (4 : 1~ and subjected to HP-20 column chromatography. When the eluate by water-methanol (1 : 4) was concentrated to dryness, 1.135 g of crude N~-[(S)-2-azetidinone-4-carbonyl]-L-histidyl-L-proline benzyl ester (12) was obtained. When the product was subjected to silica gel column chromatography again and eluted with chloroform-methanol-aqueous ammonia (40 : 10 : 1), 827 mg of the pure product (12) was obtained.
NMR (CD30D) ~ppm: 7.58 (lH), 7.34 (s, 5H), 6.88 (lH), 5.15 (s, 2H), 4.50 (dd, lH), 4.08 (dd, lH, J=3~3 Hz, 5.9 Hz), 3,40-3,92 (m, 2H), 3.04 (dd, lH, J=5.9 Hz, 14.9 Hz), 2,76 (dd, lH, J=3.3 Hz, 14.9 Hz), 1.65-2.20 (m, 4H) Mass: 439 (M ), 325 Example 3 H

(12) - - ~ CO-His-Pro-OH
O NH (13) In 150 ml of methanol was dissolved 782 mg of N~-[(S)-2-azetidinone-4-carbonyl]-L-histidyl-L-proline benzyl ester ~12) and the compound (12) was hydrogenated for 2 hours at room temperature using 156 mg of 10~
palladium-carbon as a catalyst. When the catalyst was filtered off and the filtrate was concentrated, 620 mg of N~-[(S)-2-azetidinone-4-carbonyl]-L-histidyl-L-proline (13) was obtained.
NMR (CD30D) ~ppm: 8.44 (lH), 7.28 (lH), 4.94 (lH) - ~2~6Ç~

4.44 (dd, lH), 4.14 (dd, lH, J=3.1 Hz, 5.5 Hz), 3.3-4.0 (m, 2H), 3.25 (dd, lH, 5.9 Hz, 14.9 Hz), 2.86 (dd, lH, J=3.1 Hz, 14.9 Hz), 1.7-2.4 (m, 4H) Mass: (diazomethane treatment, as dimethyl compound): 377 (M ), 263, 221, 96, 70 Example 4 H

(13) ~ ~ ~ CO-His-Pro-NHCH2CH20H
O NH (14) In 8 ml of DMF was dissolved 277 mg of compound (13) and then 168 mg of HOBT and 330 mg of DCC were added to the solution under ice-cooling. After performing a react~on for one our under ice-cooling, the reaction was further performed for 2 hours at room temperature. A~te~ ice-cooling again the reaction mixture, 2 ml of a DMF solution of 80 mg of monoethanol-amine to perform the reaction for one hour and there-a~ter, the reaction was further performed overnight at room temperature. The precipitates were filtered off, the ~iltrate was concentrated to dryness, and the residue thus formed was dissolved in lO ml of chloroform-methanol-aqueous ammonia (40 : lO : l) and subjected to silica gel column chromatography. Then, the product was eluted with the same solvent as above to provide 106 mg of N~-[(S)-2-azetidinone-4-carbonyl]-L-histidyl-N-(2-hydroxyethyl)-L-prolinami.~e (14).
NMR (CD30D) ~ppm 7.74 (lH), 7.00 (lH), 4.41 (lH, d,d), 4.12 (d,d, J=2.9 Hz, 5.5 Hz), 3.08 (2H, t, ~;~56~

J=7.1 Hz), 3.62 (2H, t, J=7.1 Hz), 2.81 (d,d, J=2.9 Hz, 15.7 Hz) Mass: 392 (M ), 279, 207 When the compound (14) was triturated with ether, the compound crystallized. The product was recrystal-lized from ethanol- M. p. 239-241C (dec.) [~]D4 ~ -87.3~(C=0.13, methanol~

IR (KBr) cm~l: 3280l 3180, 2950, 1760, 1650, 1635, 1550 Elemental analysis for C17H2~N6O5:

C~%) H(%) N~%) Calculated: 52.03 6.16 21.42 Found: 51.90 6.16 21.23 Example 5 His~DNP)- ~fi 2HC1 ~[HiS ~DNP) -N ¦ (2) qCO-HiS(DNP)-N~JS
N (16) ~ J FN CO~2 In 30 ml of dichloromethane was dissolved 876 mg of 3_[Nim-(2,4-dinitrophenyl)-L-histidyl]-L-thiazoli-dine-4-carboxamide-2-hydrochloride (15) and after adding thereto 2 ml of a dichloromethane solution of 388 mg of triethylamine under ice-cooling, the reaction was performed for 30 minutes at 0C to provide a so~ution of /3-[Nlm-(2,4 dinitrophenyl)-L-histidyl]-L-thiazolidine-4-carboxamide (16).

In 6 ml of DMF was dissolved 223 mg of compound (2) and after adding thereto 389 mg of HOBT and 517 mg of DCC under ice-cooling, the reaction was performed ~:~5~

for 30 ~inutes at 0C and then for 30 minutes at room temperature. The reaction mixture was ice-cooled again and 32 ~1 of the dichloromethane solution of the compound (16) described above was added to the fore-going reaction mixture. The mixture was react2d overnight at 0C. Precipitates thus formed were filtered off, the filtrate was concentrated to dryness, and the residue was dissolved in 20 ml of chloroform-met~anol-aqueous ammonia (40 0 10 . 1) and subjected to silica gel column chromatography. The product was eluted with the same solvent as above to provide 347 mg of 3-~N~-[(S)-2-azetidinone-4-carbonyl]-Nim-(2,4-dinitrophenyl)-L-histidyl]-L-thiazolidine-4-carboxamide (17).
NMR (CD30D) ~ppm: 8.93 (d, lH, J=3.1 Hz), 8.63 (dd, lH, J=3.1 Hz, 9.0 Hz), 7.92 (d, lH, J=
9.0 Hz), 7.87 (s, lH), 7.26 (s, lH), 4.43 (d, 2H, J=9.5 Hz), 4.14 (dd, lH, J=3.1 Hz, 5.9 Hz), 2.86 (dd, lH, J=3.1 Hz, 14.7 Hz) Mass: 419 (M+_C4HsN202), 372, 248, 81 Example 6 H ~~`S
(17) ~ C0-Hls-N ~
ol NH (18) CONH2 In 15 ml of DMF was dissolved 337 mg of compound (17) and after addinq thereto 2 ml of mercaptoethanol, the reaction was performed for 30 minutes at room temperature. The reaction mixture was concentrated to dryness and the residue was dissolved in 20 ml of ~:256~

chloroform-methanol-aqueous ammonia (30 : 10 : 1) and subjected to silica gel column chroma-tography. Then, the product was eluted with the same solvent as described above to provide 214 mg of 3-[N~-[(S)-2-azetidinone-4-carbonyl]-L-histidyl]-L-thiazolidine-4-carboxamide ~18).
NMR (CD30D) ~ppm: 7-55 (lH), 6.91 (lH), 4.32 (d, 2H, J=9.S Hz), 4.07 (dd, lH, J=3.1 Hz, 5.9 Hz) Mass: 367 (M ~ 1), 253, 206, 115 Re erence example 4.

BOC-His(Ts)-N ~ BOC-His(ls)- ~ 0 1 ~ BOC-His(Ts)-~ ~
CCOH COON~ ¦ CONHCH2CH20H

(19) (20) J (21) In 90 ml of dry methylene chloride were dissolved 6.28 g of N~-t-butylox~rcarbonyl-Nim-tosyl-L-histidyl-DL-pipecolic acid (19) and 1.39 g of N-hydroxysuccinimide (HOSu) and the solution was cooled in an ice bath. After adding thereto 2~74 g of DCC, the resultant mixture was stirred for 3 hours in an ice bath and after filtering off insoluble matters, the filtrate was concentrated at reduced pressure. The residue thus formed was dissolved in ethyl acetate and the solution was washed successively with an aqueous sodium hydrogencarbonate, water, and aqueous sodium chloride solution. The organic layer thus formed was collected and dried, and then the solvent was removed.
The syrupy product thus obtained was triturated with a 1 : 1 mixture of ether and petroleum ether. The ~ 25665 crystalline cornpound (20t thus~obtained was dried and was used in the subsequent reaction as it was. In 40 ml of tetrahydrofuran (THF) was dissolved 2.3 g of the compound (20) and then 5 ml of a ~HF solution of 251 mg of ethanolamine was added ~o the solution under ice-cooling. After performing the reaction for one hour with stirring under ice-cooling, the solvent was removed under reduced p~essure. The residue Wa5 dissolved in chloroform and the solution was washed successively with an aqueous sodium hydrogencarbonate solution, water, and an aqueous sodium chloride solu-tion. The org~nic layer thus formed was collected and after removing therefrom the solvent, the residue thus foxmed was triturated with ether to provide 1.05 g of solid N~-t-butyloxycarbonyl-Nim-tosyl-L-histidyl-N-2-hydroxyethyl)-DL-pipecolamide (21). The mother and liquor was concelltra~ed/subjected to silica gel column hromatography. By eluting with 1% methanol-chloroform, furthe~, 0.66 g of compound ~ 21) was ~ btained.
IR (KBr) cm~l: 3360, 2920, 1680, 1640, 1170 NMR (CD30D) ~ppm: 7.28-8.4 (6H, imidazole ring hydrogen, benzene ring hydrogen), 2.44 (3H, methyl of tosyl group), 1.0-1.5 (broad, BOC
hydroyen) Example 7 0 H-His(Ts) -N~ ~ ~ His (Ts)-N~) (22) (23) ~ ~5 In 40 ml of methylene chloride was dissolved 1.71 g of compound (21) and after adding thereto ~0 ml of trifluoroacetic acid under ice-cooling, the reaction was performed for 2 hours with stirring. The reaction mixture thus obtained was dried at reduced pressure, the residue thus formed was azeotropically dehydrated several times using toluene and then dried. By triturating the residue with ether, the solid tri-fluoroacetate of compound (22) was obtained. The product was dried and used for the subsequent reaction as it was. ln 8 ml of DMF was dissolved 1.3 g of the trifluoroacetate and then 274 mg of trieth~ylamine was added to the solution under ice-cooling. Then the pH
of the mixture was adjusted to 7 to 8 with additional triethylamine while checking the pH using a pH test paper.
In a mixture of 8 ml of methylene chloride and 1.5 ml of DMF were dissolved 286 mg of compound (2) and 557 mg of DCC to perform the reaction and to the reaction mixture thus obtained was added the foregoing free amine solution under ice-cooling. The reaction mixture thus obtained was stirred overnight in a refrigerator. Insoluble matters were filtered off and the filtrate was concentrated under reduced pressure.
The residue was dissolved in chloroform and the solution was washed thrice each time with water.
The organic layer thus formed was collected, dried, and then the solvent was removed to provide a syrupy material, which was subjected to column chromatography :.' s~

of 140 ml of silica gel. By eluting wlth a mixture of 7~ methanol - chloroform, 537 mg of the deslred ompound N~-[(S)-azetidinone-4-carbonyl]-Nim-tosyl-L-obtained.
histidyl-N-(2-hydroxyethyl)-DL-pipecolamide (23) was/
NMR (CDC13) ~ppm: 7.0-8.3 (9H, imidazole hydrogen, benzene hydrogen, NH), 5.08 center (2H, ~-methine hdyrogen of histidine, ~-methine hydrogen of pipecolic acid), 4.08 ~lH, 4-position hdyrogen of azetidinone ring), 2.44 (3H, methyl of tosyl group) Mass m/z: 559 (M-l), 472, 402, 388 Example 8 (23) - - - ~ His- ~

~24) In 15 ml of dry methylene chloride were dissolved 250 mg of compound (23) and 73 mg of HOBT
and the reaction was performed forr 5 hours at room temperature with stirring, whereby insoluble matters precipitated. The solvent was removed from the reaction mixture and the residue thus formed was subjected tocolumn chromatography of 100 ml silica gel. By eluting with chloroform-methanol-aqueous ammonia ~30 : 20 : 2), 116 mg of the desired compound, -[(S~-2-azetidinone-4-carbonyl]-L-histidyl-N-(2-was obtained.
hydroxyethyl)-DL-pipecolamide (24) / The product was a mixture of diastereomers andshowed two spots on TLC.
IR (The sample was measured by KBr tablet after S6~5~
; 33 lyophilization) cm~l: 3250 (NH, OH), 1750 (4-mer.lbered ring lactum), 1630 center(broad, amlde) NMR (CD30D) ~ppm: 7.65 (lH, imidazolering hydro-gen), 6.92 (lH, imidazole hydrogen), 4.14 ~
d,d, 4-position hydrogen of azetidinone ring), 1.2-1..8 (6H, methylene hydrogen of piperidine ring) Mass m/z: 406 (M ), 388, 345, 318 Reference example S.

(19) > [~20)] ~ BOC-His(Ts)-CoNH ~3 (25) From 2. 8 g of the compound (20) prepared from compound (19) by a similar procedure to in Referance example 4 and 755 mg of l-aminoadamantane, NX-t-butyl-oxycarbonyl-Nim-tosyl-L-histidyl-N-(l-adamantyl)-DL-pipecola~ide ( 25) was obtained at a quantitative yield.
NMR (CD30D) ~ppm: 7.1-8.3 ?6H, imidazole ring hydrogen, benzene ring hydrogen), 2.44 13H, methyl of tosyl group) Mass: m/z: 653 (M~), 502, 475, 419 Example 9 O

H-His(rs)-N ~ ~ ~ His(Ts)-N

CONH ~ CONH

The BOC group was removed from compound (25) by 'i 31 25~5~

a similar manner to in foregoing Example 7 and the tri-fluoroacetate of compound (26) thus obtained was converted lnto the free base using triethylamine as in Example 7. Then, 2.15 m . mol the free amine and 290 mg of compound (2) were subjected -to a coupling reaction by a simllar manner ~o in Example 7. After the reaction was over, insoluble matters were filtered off and the filtrate was concentrated under reduced pressure. The residue thus formed was dissolved in ethyl acetate and the solution was washed thrice each time with water. The organic layer thus formed was collected and dried and after removing the solvent, the residue was subjected to column chromatography of 220 ml of silica gel. By eluting the product with 3% methanol-chloroform, 598 mg of the desired product, NK-[(S)-2-azetidinone-4-carbonyl]-Nim-tosyl-L-was obtained histidyl-N-(l-adamantyl)-DL-pipecolamide (27 ~as a powder.
NMR (CD30D) ~Sppm 7.2-8.24 (6H, imidazole ring ring hydrogen, benzene/hydrogen), 4.04 center (lH, 4-position hydrogen of azetidinone ring), 2.44 (3H, methyl of tosyl group), 1.2-2.3 (21H, methylene hydrogen of piperidine ring, adamantyl group hydrogen) IR (KBr) cm~l: 3280, 2900, 1760, 1640 center (broad~
Mass m/z: 650 (M+~, 472, 402, 360 ~25~i~i5~

O Example 10 o His(Ts)-N ~ -- ~ ~ His-N
NH ~ , , ~ ~
CONH ~ ~ONH

(27) (28) In 10 ml of methylene chloride were dissolved ~10 mg of compound (27) and 130 mg of HOBT and the solution was stirred for 7 hours at room temperature.
After removing the solvent from the reaction mixture, the residue thus obtained was subjected to silica gel column chromatograpny. By eluting the product with chloroform-methanol-aqueous ammonia (80 : 20 : 2), two kinds of diastereomers wereseparated. That is, 98 mg of /the compound having a smallpolarity, N~-[2-azetidinone-4-carbonyl]-L-histidyl-N-(l-adamantyl)pipecolamide (28a) was first eluted on the silica gel column and then 128 mg of /a mixture of the compound (28a) and the stereoisomer (28b) thereof was obtained. Thereafter, the compound (28b) (63 mg) was eluted. The properties of the compounds (28a) and (28b) are as follows.
Compound (28a):
NMR (CD30D) ~ppm 7~66~ 7~58, 6.92, 6.90 (imidazole ring C-H), 4.10 ~lH, d,d, 4-position hydrogen of azetidinone ring), 2.04 center, 1.72 center (21H, methylene hydrogen of piperidine ring, adamantyl group hydrogen), IR (KBr) cm~l: 3250, 1760, 1640 center (broad), 1S20, 1440 ~256~S~

[~]26 = _74.9O (C = 1.1, methanol) Mass m/z: 496 (M ), 382, 345, 318, 261 Compound (28b):
NMR (CD30D + DMSO-d6) ~ppm: 7,64, 6.90 (imidazole ring C-H), 4,10 center(lH, d,d, 4-position of azetidinone ring), 2.02 center, 1.70 center (21H, methylene hydrogen of piperidine ring, adamantyl group hydrogen) IR (KBr) cm~l: 3200, 1750, 1530~ 1440, 1640 [~]26 = ~32.4 (C = 1.3, methanol) Mass m/z: 496 (M~), 345, 318, 261 Reference example 6.

H2N-(CH2)3CH3 Z-Pro-OH - ~ Z-Pro-NH-(cH2)3cH3 (29) (30) In 50 ml of THF was dissolved 4.99 g of N ben2yl-oxycarbonyl-L-proline and then 2.23 g of triethylamine was added to the solution. Then, after slowly adding thereto 2.39 g of ethyl chloroformate under ice-cooling, the reaction was performed for one hour at 0C to 5C.
Then, 2.19 g Gf n-butylamine was slowly added to the reaction mixture under ice-cooling, the reaction was further performed for one hour at 0C to 5C~ The solvent was removed from the reaction mixture, the residue thus formed was dissolved in ethyl acetate, and the solution was washed successively with an aqueous solution of lN hydrochloric acid, a saturated aqueous sodium hydrogencarbonate solution, and a saturated aqueous sodium chloride solution~ The organic layer this formed was collected, dried by Glauber's salt, and then concentrated to dryness. The product was recrystallized from water to provide 4.31 g of (S~-l-benzyloxycarbonyl-N-butyl-2-pyrrolidinecarbsxamide (30). M. p. 88-9oQC.
NMR (CDCl~) ~ppm: 7.36 (s, 5H), 5.17 (s, 2H), 4.33 (dd lH), 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) cm~l: 3280, 2950, 1715, 1640, 1540 Mass (EI): 304 (M+), 232, 204, 91, 70 Reference example 7~

~--Pro-NH-(CH2)3CH3 ~ Pro-NH-~cH2)3cH3 (30) (31) In 140 ml of methanol was dissolved 4~41 g of compound (30) and the compound was hydroyenated using 426 mg of 10% palladium-carbon as a catalySt. The catalyst was filtered off and the filtrate was concentrated to provide 2.41 g of IS)-N-butyl-2-pyrroli-dinecarboxamide (31).
NMR (CDC13) ~ppm: 7.3-8.0 (lH), 3.71 (dd, lH), 2.8-3.4 (4H), 1.1-2.4 (9H), 0.7-1.1 (3H) IR (neat) cm~l: 3280, 2950, 1645, 1520 ~l~25~i~iS~3 Reference example 8.

(4) Pro-NH(cH2)3cH3 - ~ Z-His-pro-NH(cH2)3cH3 (31) ~32) To 75 ml of an ethyl acetate solution of N~-benzyloxycarbonyl-L-histidine azide ( 4) prepared from 4.85 g of N~-benzyloxycarbonyl-L-histidine hydrazide ~ 3) by the method of Reference example 2 was added 2.23 g of compound ( 31) under ice-cooling and the mixture was placed overnight in a refrigerator to perform the reaction. The reaction mixture was concentrated and the residue was subjected to silica gel column chromatography. By eluting the product with chloroform-rnethanol-aqueous ammonia (95 : 5 : 0. 5), 4.13 g of N~-benzyloxycarbonyl-L-histidyl-N-butyl-L-prolinamide was obtained.

NMR (CDC13) ~ppm: 7.51 (lH), 7.34 (s, 5H), 6.85 (s, lH), 5.76 (d, lH), 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) cm~l: 3250, 2950, 1705, 1635, 1540 Mass (EI): 441 (M+~, 361, 341, 272, 244, 190, 136, 91, 70 Reference example 9.

Z-His-Pro-NH(CH2) 3GH3 ~ His-Pro-NH(CH2) 3CH3 - 2HBr (32) (33) To 20 ml of an acetic acid solution of 25~ hydro-bromic acid was added 1.77 g of compound (32) under ice-cooling and the reaction was performed for 3 hours at room temperature. The reaction mixture was added to 200 ml of dry ether, the precipitates thus formed were quickly collected by filtration and dried overnight in a desiccatorcontaining potassium hydroxide under reduced pressure co provide 2.14 g of T-histidyl-butyl-L-prolinamide 2 hydrobromide (33).
Example 11 ~2) H
(33)--~[His-Pro-NH-~CH2)3~3]~~ C0 His-pro-~l(cH2)3cH3 ,~
~4) (35) Tn 10 ml of DMF was dissolved 938 mg of compound (33) and after cooling the solution to -40C, 415 mg of triethylamine was added to-the solution. After reacting for one hour at -30C to -40C, precipitates thus formed were filtered off to provide a DMF solution of L-histidyl-N-butyl-L-prolinamlde (34). The product was used for the subsequent synthesis reaction immediately after the formation thereof.
In 5 ml of DMF was dissolved 230 mg of (S)-2-azetidinone-4-carboxylic acid (2) (prepared in Reference exampie 1) and after adding thereto 406 mg HOBT and 495 mgof DCC under ice-cooling, the reaction was performed for one hour at 0C to 5C. The reaction mixture was cooled to -40C and the DMF solution of the foregoing compound (34) was added to the reaction ~;25~

mixture. The reaction was perfromed for 30 minutes at -40C and then was further performed overnight in a refrigerator. Precipitates thus formed were filtered off, the filtrate was concentrated, and the residue thus form~d was subjected to silica gel column chromatography. By eluting the product with chloroform-methanol-aqueous ammonia (80 : 20 : 2),471 mg of Nd-[(S)-2-azetidinone-4-carbonyl]-L-histidyl-N-butyl-L-prolinamide (35) was obtained NMR (D2O, sodium 3-(trimethylsilyl)-1-propane-sulfonate) ~ppm: 7.76 (lH), 7 07 (lH), 4~96 (t, lH), 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) cm~l: 3240, 2950, 1755, 1630, 1540 Mass (EI). 404 (M+), 305, 290, 235, 207, 165, 110, 70 [~]D = -81.8 (C - 0.50, MeOH~
Reference example 10.

Pro-NH- O ~4) -~ Z-His-Pro-NH

(35) (37) To 30 ml of an ethyl acetate solution of compound (4) prepared from 1.52 g of compound (3) by the method shown in Reference example 2 was added 785 mg of (S)-N-cyclohexyl-2-pyrrolidinecarboxamide (36) prepared by a known method and then the reaction was performed overnight in a refrigerator. The reaction mixture was ~:~S6~

concentrated and the residue thus formed was subjected to silica gel column chromatography. By eluting the product with chloroform-methanol-aqueous ammonia (90 : 10 : 1), 1.30 g of N~-benzyloxycarbonyl-L-histidyl-N-cyclohexyl-L-prolinamîde (37) was ob~ained NMR (CDC13) ~ pm: 7.54 ts, lH), 7.32 (s, 5H), 6O88 (s, lH), 5.77 (d, lH), 5,09 (s, 2H), 4.61 (dd, lH), 4.36 (t, lH), 3.3-4.0 (2H), 2.8-3~3 (3H), 0.9-2.4 (14H) IR (KBr) cm~l: 3250~ 2920, 1710, 1630, 1525 Mass (EI): 467 ~M ), 387, 360, 341, 272, 244, i3~, 108, 70 ~eference example 11.

Z-His-Pro-NH O ~ His-Pro-NH- O 2HBr ~37) ~38) To 15 ml of ice-cooled acetic acid solution of 25% hydrobromic acid was added 1.14 g of compound (37) and the reaction was perfromed for 3 hours at room temperature. The reaction mixture thus obtained was added to 150 ml of desicated ether, the precipitates thus formed were quickly collected by filtration and dried overnight in a desicator at reduced pressure to providel.53 g of L-histidyl-N-cyclohexyl-L-prolin-amide-?-hydrobromidP.

~665i~

Example 12 (38)--3[His-Pro-NH ~ ]____~ CO-His-Pro-NH O
0~

; (39) ~40) : In 10 ml of DMF was dissolved 991 mg of compound t38~ and after cooling the solution to -40C, 415 mg o~ triethylamine was added to the solution. After performing the reaction for one hour at -30C to -40C, precipitates thus formed were filtered off to provide a DMF solution of L-hist.idyl-N-cyclohexyl-L-prolin-amide (39).
In 5 ml of DMF was dlssolved 230 mg of compound (2) and after adding thereto 406 mg of HOBT and 495 mg of DCC under ice-cooling, the reaction was performed for one hour at 0C to 5C. The reaction mixture thus obtained was cooled to -40C, the .oregoing DMF solution of the compound (39) was added to the reaction mixture, and then the reaction was performed for 30 minutes at -40C and then overnight in a refrigerator~ Precipitates tnus formed were filtered off, the filtrate was concentrated, and the residue was subjected to silica gel column chromatography.
By eluting the product with chlorofrom-methanol-aqueous ammonia (80 : 20 : 2), 305 mg of ~X-[~S)-2-azetidinone-4-carbonyl]-L-histidyl-rl-cyclohexyl-L-prolinamide (40) was obtained.
NMR (CD30D) ~ppm: 7.64 (lH), 6.98 (lH), 4.2-4.5 ~2S Ei~5~

(lH), 4~12 (dd, lH), 2.81 (dd, lH), 0.9-1.2 tl4H) IR (KBr) cm~l: 3220, 2920, 1750, 1620, 1540 Mass (EI): 430 (M ), 316, 235, 180, 152, 99, 70 [~]D ~ -69.2C (C = 1.90, MeOH) ; Reference example 12 ~12.TsOH ~ NH2 2 C2CH2- ~ > ~ CH202C C02CH
(41) ~42) In 1150 ml of ether was suspended 71 g (146.6 rn mol) of D-aspartic acid dibenzyl ester p-toluene-sulfoante t41) and while stirring the suspension under ice-cooling (0C to 5C), 22.5 ml (146.6 x 1.1 m mol) of triethylamine was added dropwise to the sllspension. After stirring the mixture for 2 hours at 0C to 5C, 450 ml of water was added thereto at the same temperature and the mixture was further stirred for 30 minutes. The ether layer thus formed was separated, the aqueous layer was extracted with 200 ml of ether, and the foregoing ether layer was combined with the ether extract. The mixture was washed with 400 rnl of a satuxated aqueous sodium sulfate solution and dried by anhydrous magnesium. sulfate. Ether was distilled off under reduced pressure to provlde 45 g of D-aspartic acid dibenzyl ester (42) as a colorless oily product.

~25~

Rererence example 13.

r~ 2 2 ~ H C02CH2 22C NH2 ~ O rNH
(42) ~43) In 485 ml of dry ether was dissolved 45 g (143.8 m mol~ of D-aspartic acid dibenzyl ester (42) and after cooling the solution to 0C under an argon atmosphere, 20 ml (143.8 m mol) of triethylamine was added dropwise to the solution. Then, 15.6 g (143.9 m mol) of trimethylsilyl chloride was further added dropwise to the mixture at the same temperature as above and the resultant mixture was stirred for one hour. Precipitates thus formed were filtered off under an argon atmosphere and the filtrate was cooled to 0C to -5C and then 134.8 x 1 01 m mol of an ether solution of t-butyl magnesium chloride was added dropwise to the mixture with stirring. After further stirring the mixture for 2 hours at 0C and then for 3 hours at room temperature, the mixture was cooled to OC, 100 ml of 2N HCl (saturated with NH4Cl) was added to the mixture and after stirring the mixture for 30 minutes, 100 ml of a saturated aqueous ammonium chloride solution was added to the mixture. The ether layer thus formed was se2arated and the aqueous layer was extracted twice each time with 200 ml of ethyl acetate. The ether layer was combined with the ethyl acetate extract, and after washing the mixture with 300 ml ofasaturated aqueous ammonium chloride solution and ~2~J5 drying with anhydrous magnesium sulfate, ether and ethyl acetate were distilled off under reduced pressure. To the residue was added 10 ml of ethyl acetate to form crystals, which were collected by filtration to provide 13.7 g of (R)-4-benzyloxycarbonyl-2-azetidinone (43~.
In addition, the mother liquor was concentrated and purified by silica gel column chromatography (eluent: ethyl acetate-n-hexane (2 : 1)) to provide 5.1 g of the desired product. M. p. 136-138~C
[oiD = +33 7 (C = 1, MeOH) NMR (DMSO-d6) ~ppm: 8.40 (lH, NH), 7.40 (5H, s, phenyl group), 5.20 (2H, s, methylene of benzyl grGup), 4.22 (lH, d,d, 4-position hydrogen), 3.27 (lH~ d,d,d, 3-position hydrogen), 2.89 (lH, d,d,d, 3-position hydrogen) IR (KBr) cm~l:3200, 1760, 1725, 1280 Reference example 14.

~ ,COOCH~ ~ H ,CooH
N~ o NH
(43) (44) In 250 ml of methanol was dissolved 5 g of compound (43) and the compound was catalytically reduced in the presence of 500 mg of palladium-carbon at normal temperature and normal pressure in a hdyrogen atmosphere. After filtering off the catalyst, methanol was distilled off under reduced pressure.

rj~J

The residue thus formed was crystallized from ether and the crystals thus formed wexe collected bv filtration to provide 2.5 g of the desired product, (R)-2-azetidinone-4-carboxylic acid (44) as the colorless crystals. M. p. 97-101C.
NMR (DMSO-d6, CD30D) ~ppm: 4.60 (lH, d,d, 4-position hydrogen),3.23 (lH, d,d, 3-position hydrogen), 2.85 ~lH, d,d, 3-position hydrogen) IR (K~r) cm~l: 3310, 1735 (broad), 860 Example 13.
H COOH
n~- ~
o~ NH H

(44) ~ His-Pro-NH2 (8)--~[His-P~oNH2] ~ ~ NH

(~) (45) In 13 ml of anhydrous DMF was dissolved 826 mg (2 m mol) of L-histidyl-L-prolin-amide 2-hydrobromide (8) followed by cooling to -10C.
To the solution was s]owly added 404 mg ~2 x 2 m mol) of triethylamine and the mixture was stirred for 30 minutes at the same temperature. Then, triethylamine hydrobromide thus precipitated was filtered of~ under an argon atmosphere. The solution was added dropwise to an active ester solutlon prepared from 230 mg (2 m mol) of (R)-2-azetidinone-4-carboxylic acid (44) (obtained in Reference examples 12 to 14), 351 mg (2 x 1.3 m mol) of HOBT, 453 mg (2 x 1.1 m mol) of DCC, and 10 ml of DMF at -20 C. After stirring the mixture for 1.5 hours at the same temperature, the mixture was stirred ~2566~

overnight in a refrigerator. Then, DMF was distilled off from the reaction mixture under reduced pressure, to the residue thusformed was added 10 ml of methylene chloride-methanol-concentrated aqueous ammonia (80 :
20 : 2), and crystals thus precipitated were filtered off. The filtrate was subjected to silica gel columnchromatography and purified using methylene chloride-methanol-concentrated aqueous ammonia (80 :
solvent to 20 : 2) as the developing / provide 370 mgof N~-[(R)-2-azetidinone-4-carbonyl]-L-histidyl-L-prolinamide (45) as an amorphous powder.
[~]D =-21.5 (C = 1, MeOH) NMR (D2O) ~ppm: 7-03 (lH, imidazole ring), 6.72 (lH, imidazole ring)~ 4,95 (lH, m), 4.42 (lH, m)4,27 (lH, d,d, 4-position hydrogen of azetidinone ring), 3.40-4.00 (2H, m), 3,32tlH, d,d, 3-position hydrogen of azetidinone rin~), 2,74 (lH, d,d, 3-position hydrogen of azetidinone ring), 2.00 (4H, m) IR (KBr) cm~l: 3350, 3150, 1745, 1660, 1625, Mass: 348 (M ), 304, 278, 234, 207, 190 Example 14 , ,. H, ,, ,~C- Hls-ProOH ~ -His-Pro-NHCH3 O NH 0~ NH
(13) (46) s~

In 2 ml of DMF were dissolved 300 mg of N~-[(S)-2-azetidinone-4-carbonyl]-L-histidyl-L-proline (13) (obtained in Example 3), 116 mg of HOBT, and 177 mg of DCC and after stirring for 7 hours at room temperature, the solution was cooled in an ice bath. Then 0.6 ml of a methanol solution of 30% methylamine was added to the solution and the mixture was reacted onvernight with stirring at 2 to 6C. Insolub~e matters were filtered off and the filtrate was concentrated to dryness under reduced pressure. The residue thus formed was purified by col~lmn chromatography using LiChroprep Si 60~size B). By usinq chloroform-methanol-aqueous ammonia (40 : 10 : 1) as the eluent, ~-[(S)-2-azetidinone-4-carbonyl]-L-histidyl-N-methyl-was obtained.
L-prolinamide (46~ / The product was dissolved in water and then lyophilized. The amount of the product thus obtained was 149 mg.
NMR (CD30D) dppm 7.62 (lH, imidazole ring), 6.94 (lH, imidazole ring), 4~8 (lH, methine group), 4.10 (lH, 4-position hydrogen of azetidinone ring), 2.76 (3H, N-methyl group), 1,6-2.3 (4H, proline ring hydrogen) IR (KBr) cm~l: 3250, 1750, 1630, 1540, 1440, Mass m/z: 362 (M+), 304, 292, 248, 235, 207 Reference example 15.

BOC-His(Ts)-OH ~ Pro-OBn HCl -(47) (~8) BOC-His(Ts)-Pro-OCH
(49) 5r~

To 100 ml of desicated methylene chloride were added 10 g of N~-t-butyloxycarbonyl-Nim-tosyl-L-histidine ~47) and 6.50 g of L-proline benzyl ester hydrochloride and the mixture was cooled in an ice bath. After adding thereto 2.72 g of triethylamine, 6.05 g of DCC was further added to the mixture and the resultant mixture was stirred for 30 minutes in an ice~bath and stirred overnight at room temperature.
Insoluble matters were filtered off and the filtrate was concentrated. The residue thus formed was purified by silica gel column chromatography. By eluting the product with ethyl acetate-benzene (1 : 1), 13.5 g of N~-t-butyloxycarbonyl-Nim-tosyl-L-histidyl-L-proline benzyl ester (49) was obtained.
NMR (CD30D~ ~ppm: 8.14, 7.92, 7.84, 7.40, 7~30 (total llH, im~dazol ring, benzene ring), 5.12 (2H, q, benzyl group), 4.5 center (2H, 2 kinds of methine groups), 2.36 (3H, methyl of tosyl group), 1.30 (9H, t-butyl group) IR (KBr) cm~l: 3400, 3280, 2970, 1740, 1700, 1640, i590 Mass m/z: 596 (Ml), 523, 480, 364, 290, 155, 91 Reference example 16.

BOC-His (Ts)-Pro-OCH24~ 3, BOC-E~is (Ts) -Pro-OH
(49) (50) ~56~S~

In 150 ml of methanol was dissolved 13.5 g of compound (49) and the compound was catalytically reduced for 5 hours in the presence of 10% palladium carbon. The catalyst was filtered off and the filtrate was concentrated under reduced pressure. The residue thus formed was dissolved in ethyl acetate and extracted thrice each time with an aqueous sodium hydrogen carbonate solution. The extracts were combined with each other and washed once with ethyl acetate. After acidifying the aqueous layer with 1 N hydrochloric acid, the desired compound was extracted with ethyl acetate. Thus, 2.0 g of N~-t-butyloxycarbonyl-Nim-tosyl-L-histidyl-L-proline (50) was obtained as a foamy product. Also, from the organic layer formed after extracting the product with an aqueous sodium hydrogencarbonate solution, 9.1 g of the starting material (49) was recovered.
(50 ) The properties of the desired compound/thus obtained are shown below.
NMR (CD30D) ~PPm: 8.16, 7.96, 7.86, 7.46, 7.40, (total 6H, imidazole ring hydrogen, benzene ring hydrogen), 4,48 center (2H, two kinds of methine groups), 2,42 (3H, s, methyl of tosyl group), 1.'2 (9H, t-butyl group) IR (KBr) cm~l: 3300, 3100, 2970, 2500-2600, 1710, Mass m/z: 388 (M-118), 308, 234 Reference example 17.

BOC-His (Ts)-Pro OH ~ BOC-His-Pro-NHCH2CH20H

(50) (51) To 70 ml of methylene chloride were added 3.25 g of compoun~ 150), 0.79 g of monoethanolamine, and 2.61 g of HOBT and the mixture was ice-cooled in an ice-bath. Then, 20 ml of DMF was added to the mixture to form a uniform solution. After further adding 1.99 g of DCC, the mixture was sirred for 2 hours in an ice-bath and then stirred overniyht at room temperature.
Insoluble matters were filtered off and the filtrate was concentrated under reduced pressure. The residue was dissolved in 70 ml of methylene chloride and after further adding thereto 1.3 g of HOBT, the mixture was stirred again for 20 hours at room temperature~ The solvent was distilled off from the reaction mixture under reduced pressure and the residue thus formed was sub,ected to silica gel column chromatography. By eluting the product with chloroform-methanol-aqueous ammonia (40 : 10 : 1), 1.35 g of N~-t-butyloxycarbonyl-L-histidyl-N-(2-hydroxyethyl)-L-prolinamide (51) was obtained.
NMR ~CD3OD) ~ppm: 7.64 (lH, imidazole ring), 6.96 IlH, imida~ole ring), 4.48 center (2H, two/
inds o~ methine groups), 2.0 center (~H, porline ring),1.40 (9H, t-butyl group) IR (KBr) cm~1: 3250, 2960, 1700, 1630 ~25~ S~

Mass m/z: 3~5 (M+), 365, 322, 307, 278 Example 15 (2) BOC-His-Pro-NHCH2CH20H--~His-Pro-~CH2CH20H --- 3 (51) 2CF C00H
(52) 3 ~ CO-His-Pro-NHCH2CH20H
o~NH (14) In ?5 ml of methylene chloride was dissolved 790 mg of compound ~51) and then 20 ml of trifluoroacetic acid was added dropwise to the solution at 0C to 5c.
~fter stirring the mixture for 2.5 hours in an ice-bath, the reaction mixture was concentrated to dryness under reduced pressure. Furthermore, the product was azeotropically dried several times using toluene and the residue was triturated with dry ether to provide powder /L-histidyl-N-(2-hydroxyethyl)-L-prolinamide 2-trifluoroacetate with a quantitative yield.
In a mixture of 7 ml of DMF and 8 ml of methylene chloride were dissolved 253 mg of compound (2) and oE DCC
350 mg of HOBT and after adding thereto 530 mg/under ice-cooling, the mixture was stirred for 1.5 hours.
To the reaction mixture was added a reaction mixture obtained by neutralizing the foregoing compound (52) in a mixture of 4 ml of DMF and 4 ml of methylene chloride wil,h 445 mg of triethylamine, and the resultant mixture was reacted overnight with stirring in a refrigerator. Insoluble matters were filtered off, the filtrate was concentrated, and the residue ~5~

was subjected to silica gel column chromatography.
By eluting with chloroform-methanol -aqueous ammonia (40 O 40 : 1), 313 mg of the compound (14) which was identical with hat obtained in Example 4 was obtained.
Reference example 18.

(4) Fro-NH-CH2CH20H ~ Z-His-Pro-NH-CH2CH20H
(53) (54) To an ethyl acetate solution o N-benzyloxy-carbonyl-L-histidinazide (4~ prepared from N -benzyl-(3), oxycarbonyl-L-histidine hydrazid~/(6.07 g) by a known method was added 10 ml of a DMF solution or 2.31 g of (S)-N-(2-hydroxyethyl)-2-pyrrolidinecarboxamide (53) under ice-cooling and they were reacted overnight in a refrigerator. The reaction mixture was concentrated and the residue thus formed was subjected to silica gel column chromatography. By eluting the product with chloroform-methanol-aqueous ammonia (90 :

10 : 1), 3.43 g of N~-benzyloxycarbonyl-L-histidyl-N-(2-hydroxyethyl)-L-prolinamide (54) was obteined.
NMR (CDC13) ~pp~: 8.3-8.7 (lH), 7.55 (s, lH), 7.34 (s, 5H), 6.87 (s, lH), 5.93 (d, 2H), 5.10 (s, 2H), 4.3-4.8 (2H), 2.8 3.8 (8 L~ ), 1.6-2.3 (4H) Mass (EI): 429 (M ), 341, 272, 244, 136, 108, 79 ~;~S~i~i5~

Reference example 19.

Z-His-Pro-NH-CH2CH20H----~ 2 2 3 (54) (5~) To 37.5 ml of ice-cooled acetic acid solution of 25~ hydrobromic acid was added 3.2~ g of compound (54) and the reaction was performed for 3 hours at room temperature. The reaction mixture was added to 375 ml of dry ether and the precipitates thus formed were quickly collected by filtration and dried overnight in a desiccatorcontaining potassium hydroxide to provide 4.43 g of L-histidyl-N-(2-acetoxyethyl)-L-prolinamide 2-hydrobromide.
Example 16 a) E~i.s-Pro-NH-CH2CH20COCH32HBr--3[His-Pro-NH-CH2CH20COCH3]
(55) (56) H

(2) ~ Co-His-pro-NH-cH2cH2ococH3 ~ n ~ NH (57) In 35 ml of DMF was dissolved 4~43 g of compound 155) and after cooling the solution to -40C, 1.ô2 g of triethylamine was added to the solution followed by performing the reaction for one hour at -30 to -40C.
Then, precipitates thus formed were removed to provide a DMF solution of L-histidyl-N-(2-actoxyethyl)-L-prolin amide (56). The product was immediately used for ~.256~5~

the subsequent reaction.
In 17~5 ml of DMF was dissolved 863 mg of compound (2) and after adding thereto 1.52 g of HOBT and 1.86 g of DCC, the reaction was performed for 30 minutes under ice-cooling. The reaction mixture was cooled to -40C and after adding thereto the DMF
solution of the foregoing compound (56), the reaction was per-formed for 30 minutes at -40 C and then performed overnight in a refrigerator. Precipitates thus formed were filtered off, the filtrate was concentrated, and the residue thus obtained was subjected to silica gel column chromatography. By eluting the product with chloroform-methanol-aqueous ammonia (80 : 20 : 2), 1.60 g o~ NX-[(S)-2-azetidinone-4-carbonyl]-L-histidyl-N-(2-acetoxyethyl)-L-prolin-amide.
NMR (D2O) ~ppm: 7.74 (s, lH), 7.05 (lH), 4.93 (t, lH), 4.1-4.5 (4H), 2.9-3.9 (7H), 2.76 (dd, lH), 2.7-3.2 (7H) IR (KBr) cm~l: 3230, 2950, 2860, 1755, 1730, 1630, 1540 Mass (EI): 434 (M~), 364, 320, 262, 235, 154, 70, 43 [a]D7 = -86.2 (C= 0.45, MeOH) b~ H
~ CO-His-Pro-NHCH2CH20COCH3 --- , ~ H (57) H
~ ~ CO-His-Pro-NHCH2CH20H
o (14) ~ ~6~5~

In 12 ml of methanol was dissolved 56 mg of potassium carbonate and after adding thereto 348 mg of compound (57) under ice-coolins, the reaction was performed for 2 hours under ice-cooling. The reaction mixture thus obtained was subjected to silica gel column chromatography and by eluting the product with chloroform-methanol-aqueous ammonia (80 : 20 : 2), 294 mg o~N~-[(S)-2-azetidinone-4-carbonyl]-L-histidyl-N-(2-hydroxyethyl)-L-prolinamide was obtained. The physicochemical properties of the product were same as those of the compound (14) obtained in Example 4.

(Reference example 20) Example 17-a Cl C1 COOR C00 ~ Cl Cl Cl O (58 ~) In 200 ml of DMF were dissolved 10 g (8.68 m mol) of (S)-2-azetidinone-4-carboxylic acid (2) and 24.4 g ~ 8.70 m mol) (8.68 m mol) of pentachlorophenol and then 17.93 g/of DCC was added to the solution under cooling (0 to 5C).
After stirring the mixture for 5 hours at room temperature, dicyclohexylurea thus precipitated was filtered off and the filtrate was concentrated under reduced pressure. The residue thus formed was dissolved in 200 ml of ethyl acetate by heating and then cooled. The crystals thus precipiated were collected by filtration to provide 25.6 g of the yellowish crystals of (S)-4-pentachlorophenoxy-6~

57carbonyl-2-azetidinone (58) having a melting point of 177 to 179C.
NMR (90 MHz, d6-DMSO-D20) ~ppm: 3.23 (lH, q, azetidinone rlng 3-position), 3.57 (lH, q, azetidinone ring 3-position), 4.70 (lH, q, azetidinone ring 4-position) IR (KBr) cm~l: 3200, 1775, 1755, 1720 Mass: 363 (M ), 335, 266, 237 Example 17-b oF~ co2~
In 20 ~1 of DM~ was dissolved 690 mg of (S)-2-azetidinone-4-carboxylic acid (2) and after adding thereto 690 mg of HOSu and 1.236 g of DCC under ice~
cooling, the re~ction was performed for 30 minutes under ice-cooling and then for 4 hours at room temperature. Insoluble matters were filtered off from the reaction mixture and then the solvent was distilled offto pxovide a light-hrown solid product. When the product ~as recrystallized from dioxane petroleum ether (5 : 1), 750 mg of (S)-4-(2,5-dioxopyrrolidine-l-yl)-oxycarbonyl-2-azetidinone was obtained~
NMR (DMSO-d6, TMS) ~ppm: 8.70 (bro2d, lM), 4.62 (dd, lH), 3.84 ls, 4H) I~ (KBr) cm~l: 3320, 2920, 2840, ]810, 1780, 1750, 1730, 17~0, 1650, 1620, 1570 Mass ~CI in beam): 213 (M +]),185, 171, 116 s~

(Reference example 21) Example 17-c O

His-OCH3 2HCl ~ C ~

(59~ (60) In 75 ml of DMF was suspended 6.05 g (25 m mol) of L-histidine methyl ester 2-hydrochlor1de (59) and after cooling the suspension to 0 to 5C, 5.05 g (50 m mol) of triethylamine was slowly added dropwise to the suspension~ Thereafter, the mixture was stirred for 15 minutes at the same temperature.
Then, 9.50 g (25 m mol) of compound (58) was added to the mixture as a powder and after stirring the mixture for one hour at the same temperature, the mixture was allowed to stand overnight at room temperature. Triethylamine hydrochloride thus precipitated was filtered off, the filtrate was concentrated under reduced pressure. The residue thus formed was mixed with 40 ml of ethyl acetate and 30 ml of water followedby shaking and then the aqueous layer thus formed was collected. The ethyl acetate layer was extracted twice each time with 20 ml of water. The aqueous layers were combined with each other and water was distilled off under reduced pressure. The residue was mixed with acetonitrile and benzene and the mixture was concentrated under reduced pressure. The rasidue was crystallized from 30 ml of methanol and the crystals thus formed were collected to s~
5~
provide 4.1 g of the colorless crystals of N~-[(S)-2-azetidinone-4-carbonyl]-L-histidine methyl ester (60) haviny a melting point of 142 to 147C.
NMR (90 MHz, d6-DMSO) ~ppm: 2.94 (2H, d, ~-position methylene oE histidine group), 3.60 (3H, s, methyl group), 4.02 (lH, q, azetidi-none ring 4-position), 4.54 ~lH, m, a-position methine of hlstidine group), 6.72 (lH, s, imidazole ring), 7.56 (lH, s, imidazole ring), 8.20 (lH, s, NH), 8.56 (lH, d, azetidinone ring NH) IR (KBr) cm~l: 3250, 3100, 2g50, 1770, 1750, 1740, 1720, 1650, 1550 Example 17-d (58) His-OCH
His-OCH2 ~ ~ H 2 (60-1) In 20 ml of chloroform was dissolved 1.178 y (2 m mol) of hi~tidine benzyl ester-2-p-toluenesulfonate and then 404 mg (2 m mol) of triethylamine was slowly added to the solution under cooling to 0C. To the solution was added 766 mg (2 m mol) of (S)-4-penta-chlorophenox~carbonyl-2-azetidinone/as a powder and the mixture was stirred overnight at O to 5C. To the reaction mixture was added 30 ml of chloroform and the desired product was extracted twice each time with 30 ml of water. Then, water was distilled off under ~256~J~i~

reduced ~ressure and the xesidue thus formed was azeotropically dehydrated with benzene-acetonitrile to provide a colorless sticky product. The product was subjected to silica gel column chromato-graphy using 50 ml ~ako gel C-200 and elution with ethyl acetate-methanol-concentrated aqueous ammonia (60 : 30 : 3) ~ 70 mg of N -~IS)-2-azetidinone-(60-1), ~-carbonyl]-L-histidine benzyl ester/having a melting point of 196 to 199C as colorless crystals.
NMR (90 MHz, d5-DMSO) ~ppm: 2.58 (lH, m, azetidinone rlng 3-position), 2.96 (2H, d, histidine group ~-position methylene), 3.12 (lH, m, azetidinone ring 3-position), 4.00 (lH, m, azetidinone ring ~-position), 4,62 (lH, m, histidine group ~-position methine), 5.10 (2H, s, benzyl position), 6.80 (lH, s, imidazole ring), 7.36 (SH, s, benzene ring), 7.56 (lH, s, imidazole ring), 8.20 (lH, s, NH), - 8.58 (lH, d, NH) IR (KBr) cm~l: 3260, 2980, 2760, 1750, 1650, 1540 Example 17-e His-OCH2-~ ~His-OH
NH

(60-l) (61) In 20 ml of methanol was suspended 342 mg of compound (60-l)and the compound was catalytically 20 mg of reduced with the addition of/10~ palladium-carbon at ~2~

ambierlt temperature andordinary pressure. After the had absorption of hydrogen/stopped, the catalyst was filtered off and methanol was distilled oif under reduced pressure to provide 230 mg of N~[(S)-2-(61) azetidinone-4-carbonyl]-L-histidine/having a melting point of 213 to 215C (decO) as colorless crystals.
NMR (9O MHz, D2O) ~ppm: 2.80 (lH, q, azetidinone ring 3-position), 3.~0 (2H, m, histidine group ~position), 3.38 (lH, q, azetidinone ring 3-position), 4.28 (lH, q, azetidinone ring 4-position), 4.58 (lH, m, histidine X-position methine), 7.30 (lH, s, imidazole ring), 8.60 (lH, s, imidazole ring) IR (KBr) cm~l: 3400, 3260, 2560, 1750, 1630, 1570, 1390 Example 17-A
O O

~ His-OCH3 ~ ~ ~His-OH ~ Pro-NH2 (62) l TsOH ¦
(60) (61-1) C

His-Pro -NH2 NH
(10) After cooling 20 ml of an aqueous solution of 0.1 N sodium hydroxide to O to 5C, 532 mg (2 m mol) of compound (60) was added thereto and the mixture was stirred for 1.5 hours at the same temperature. Then, 760 mg (4 m mol) of p-toluenesulfonic acid monohydrate was added to the mixture at the same temperature and water was distilled off under reduced pressure. The ~5~5~

residue thus obtained was azeotropically dehydrated with acetonitrile and benzene and then dried under reduced pressure. The powder obtained was dissolved in 20 ml of DMF and after adding thereto 228 mg (2 m mol) of L-prolinamide (62) and 412 mg (2 m mol~ of ~CC, the mixture was stirred overnight at room temperature.
Dicyclohexylurea thus precipitated was filtered off, the filtrate was concentrated under reduced pressure, and the residue thus formed was dissolved in 20 ml of water~
After filtering o-ff insoluble matters, water was distilled off unde~ reduced pressure. After drying the residue thus formed under reduced perssure, the residue was dissolved in 7 ml of methanol by heating and after stirring the solution under cooling, crystals thus precipitated were collected by filtration to provide 500 mg of the colorless crystals of N~-[(S)-2-azetidinone-4-carbonyl]-L-histidyl-L-prolin~mide having a melting point of 179 to 184C. The physico-chemical properties of the product were same as those of the compound (10) obtained in Example 1 Example 17-B

His-OH ~ ~His-Pro-NH
L NH + Pro NH ~ L 2 (61) (10) In 10 ml of DMF was suspencled 252 mg (1 m mol) of compound (61) and after adding thereto ll5my (1 m mol) ~:25~;65V

of N-hydroxysuccinimide and then 114 mg (1 m mol) of prolinamide and 206 mg (1 m mole) of DCC under cooling to 0C, the resultant mixture was allowed to stand overnight at 0 to 5C and then stirred for 2 days at room temperature. After filtering off the crystals thus precipitated, DMF was distilled off under reduced pressure. The residue formed was mixed with 5 ml of water and after filtering off insoluble matters, water was distilled off under reduced pressure. After azeotropically dehydrating the residue with the addition of benzene-acetonitrile, 3 ml of methanol was added to the residue and the mixture was stirred to provide 82 mg of the crystals of N~-~(S)-2-azetidinone-4-carbonyl]-L-histidyl-L-prolinamide (10). The physicochemical properties of the product were same as those of the product obtained in Example 17-A.
Example 18 + His-N ~ C~3 ~ ~ ~ CH
~ NH CH ~, NH 3 o ~ 3 o CONH2 (2) t62) (63) In a mixture of 2 ml of DMF and 10 ml of methylene chloride were dissolved 211 mg of (S)-2-azetidinone-4-carboxylic acid (2) and 248 mg of HOBT and the solution was ice-cooled. After adding thereto 472 mg of DCC, the mixture was stirred for one hour at room temperature~
Then, a 10 ml of a DMF solution of 426 mg of L-histidyl-DL-(3,3-dimethyl)prolinamide (62) was added ~:~566~ 3 to the mixture and the resultant mixture was stirred for 2 days at 0 to 4C. Insoluble mat-ters were collected by filtration and washed with DMF. The filtrate was combined with the washings and the solvent was distilled off ~rom the mixture under reduced pressure. The residue was subjected to column chromatography of 150 ml of silica gel. By eluting with chloroform-methanol-aqueous ammonia (80 : 20 : 2), N~-[(S)-2-azetidinone-4-sarbonyl]-L-histidyl-DL-(3,3-dimethyl)prolinamide (63) was obtained. The aqueous solution was lyophilized to provide 63 mg oE a white powder. 1'he product was a mixture of diastereomers showing two spots on TLC.
IR (KBr) C~ 1 3250 (broad), 1750, 1670, 1630, 1540, 1440 NMR (CD30D) ~ppm: 7.64, 6.96, 6.88 (2Hr imidazole ring), 4.14 (lH, d,d, azetidinone ring 4-position), 1.80 center (2H, proline ring), 1.06, 1.12, 1.06, 0.92 (6H, proline ring 3-position dimethyl) Mass m/z: 376 (M ), 343, 316, 306, 262, 98 Reference example 22 (raw materials for Examples 19 and 22) a) (NT-CH3)~-lis-OH 2HC1 , Z-(NT-CH3)His-OH

(64) (65) ~S6~

In 130 ml of water was dissolved 9.9 g of L-NT-methyl-histidine 2-hydrochloride (64) and the solution was cooled in an ice-sodium chloride cooling bath. The pH of the solution was adjusted to 11 by the addition of an aqueous solution of 2N sodium hydroxide and while maintaining the inner temperature at 0 to 5C, 10.5 g of carbobenzoxy chloride was added dropwise to the mixture~ During the operation, the pH
of the system w~s controlled to 11 to 12 by the addition of an aqueous solution of 2N sodium hydroxide.
Thereafter, the mixture was stirred for one hour at 0 to 5C while keeping the pH thereof at 12 1 0.5 by adding occasionally an ayueous solution of 2N sodium had hydroxide. After the change in pH/stopped, the solution was further stirred for 1.5 hours at 5 to 10C. The reaction mixture was washed twice each time with ethyl acetate, the aqueous solution thus formed was collected, and the pH of ~he solution was adjusted to 3.9 with 4N hydrochloric acid. Then, the pH thereof was adjusted to 2.2 by the addition of 2N p-toluenesulfonic acid.
The reaction mixture was saturated with sodium chloride and extracted four times with acetonitrile-isobutanol-ethyl acetate (1 : 1 : 2~. The organic layer thus obtained was concentrated under reduced pressure and the residue was triturated with aceto-nitrile. After filtering off insoluble matters, the filtra-te was concentrated to provide a yellow syrupy product. The product was subjected to column chromato-graphy using 600 ml of silica gel and by eluting the product with chloroform-methanol-aqueous ammonia (60 :
40 : 3), 8.8 g of N~ benzyloxycarbonyl-N~-methyl-L
histidine (65) was obtained as a foamy material.
IR (KBr) cm : 3100 center (broad3, 1700, 1590, NMR (CD30D) ~ppm: 7.86 (lH, imidazole ring), 7.32 ~5H, benzene ring), 6.96 (lH, imidazole ring), 5.04 (2H, benzyl 4.30 center (lH, d,d, methine group), 3.68 (3H, N-methyl) ~]D = +~2.2 (C = 1, methanol) b) Z-(N -CH3)His-OH + Pro-NH2 -~ Z-(NT-CH3)His-Pro-NH2 (65) (66) In 25 ml of dry DMF were dissolved 1.65 g of compound (65) and 621 mg of L-prolinamide and the solution was cooled to S to 10C. Then, 1.03 g of p-toluenesulfonic acid and 1.35 g of DCC were added to the solution and the mixture was allowed to stand overnight in a refrigerator. Insoluble matters were filtered off, the filtrate was concentrated under and reduced pressure,/the the residue was triturated with a mixture of ethyl aceate and ether. Then, 3.2 g of the insoluble matters was sub~ected to column chromatography of 500 ml of silica gel and by eluting using chloroform-methanol-aqueous ammonla (80 : 20 : 2), 2.0 g of NC~-benzyloxycarbonyl-N~-methyl-L-histidyl--L-5~ S~
67prolinamide (66) was obtained.
IR (KBr) cm~l: 3300 center(broad), 1620-1720 (broad), 1510, 1440 NMR (CD3aD) ~ppm: 7.48 (lH, imldazole ring), 7032 (5H, benzene ring), 6.90 (lH, imidazole ring), 5,04 (2H, benzyl 3.64 (3H, N-methyl), 2.0 center(4H, proline ring~

Mass (FD) m/z: 399 (M ) c ) Z-(N -CH3~His-Pro-NH2--~(N -CH3)His-Pro-WH22HBr (66) (67) To 24 ml of an acetic acid solution of 25%
hydrobromic acid was added 2.04 g of compound (66) and the mixture was stirred for 1.5 hours at room tempera-ture. The reaction mixture was poured in 2~0 ml of dry ether and white precipitates thus formed were collected by filtration. The precipitates were dried o~er potassium hydroxide under reduced pressure to provide 2.2 g of a hygroscopic solid of NT-methyl-L-histidyl-L-prolin-amide-2-hydrobromide (67).
Example 19 COO~
~ + (N~-CH3)His-Pro-NH2~2HBr >
" NH
0 (67) o (2) C
r~ \His(NT-cH3)-pro-NH2 ~'-NH

(68) ~25~i~5~

In a mixture of 7 ml of DMF and 7 ml of methylene hloride were dissolved 323 mg of compound (2) and the 380 mg of HO~T and after ice-cooling/solution, 579 mg of DCC was added to the solution, whereby crystals precipitated soon. After stirring for about 20 minutes, a solution of the free amine compound prepared from 1.0 g of compound (67) and 521 mg of triethylamine in DMF
under ice~cooling was added to the reaction mixture.
The reaction mixtur~ thus obtained was stirred for 20 hours in a refrigerator, insoluble matters were filtered off, and the filtrate was concentrated to provide a syrupy residue. The residue was subjected to silica gel column chromatography and by eluting with chloroform-methanol-aqueous ammonia (80 : 20 : 2), 543 mg of N~-[(s)-2-azetidinone-4-carbonyl]-NT -methyl-L-histidyl-L-prolinamide (68) was obtained.
IR (XBr) cm~l: 3250, 1750, 1670, 1630 NMR (CD30D) '5ppm 7.52 (lH, imidazole ring), 6.96 (lH, imidazole ring), 4.44 center (lH, m, methine hydrogen), 4.12 center (lH, d,d, azetidinone ring 4-position hydrogen), 3.70 (3H, s, N-methyl), 2.0 center (4H, proline ring) Mass m/z: 362 (M ), 31g, 292, 249, 221 [~]D5 = -68.6 (C=l, methanol) ,, ~Z5~Çi 5~

Reference example 23 IRaw material for Example 20) a) ~ CH3 3 ~N ~ CONH2 (65) Z-(NT-CH3)His-N~ x H3 3 (70) 2 By reacting 2.24 g of compound (65) and 1.05 g of 3,3-dimethyl-DL-prolinamide (69) in a simil~r method to that in Refernce /example 22 b), 3.2 g of N~ -benzyloxycarbonyl-N~ -methyl-L-histidyl-3,3-dimethyl-DL-prolinamide (70) was obtained, which was a mixture of diastereomers.
NMR (CD30D) ~ppm: 7.52, 7.48 (lH, imidazole ring), 7.32 (5H, benzene ring), 6.88, 6.84 (lH, imidazole ring), 5.04 (2H, benzyl), 3.64, 3.60 (3H, N-methyl) IR (KBr) cm~l: 3300, 2920, 1620-1720(broad) Mass m/z: 427 (M ), 383, 319, 286, 277, 258 b) Z-(N -CH3)His- ~ H3 (N -CH3)His-N ~ X cH3 2Hsr (71) CONH2 By following the procedure described in Reference example 22 c), N~T-methyl-L-histidyl-3,3-dimethyl-DL-prolinamide 2-hydrobromide (71) was obtained with a quantitative yield from 3.2 g of compound (70). The product was used for the subsequent reaction as it was.

~2566~

COOH Example 20 C~ + (N -CH3)His ~CH3 2HBr (71) ( 2 ) ,O, r~ His (NT-CH3) ~ X 3 (72a), (72b) By following a similar manner to that in E~ample 19, 329 mg of compound (2) was reacted with 1.3 g of compound (71) and the reaction product thus obtained was subjected to sllica gel column chromatography.
The product was eluted with chloroform-methanol-aqueous arnmonia (80 : 20 : 2). The desired reaction product, N d _ [ ( S ) -2-azetidinone-4-carbonyl]-NT -methyl-L-histidyl-3,3-dimethyl-DL-prolinamide was a mixture of a diastereomer ( 72al having a weak polarity on chromatograph and a diastereomer (72b) having a strong polarity. The product first eluted was 244 mg of a mixture of ( 72a) and (72b) in a ratio of 8 : 2, 254 mg of the product eluted in the next was/a mixture of ( 72~
and (72b) of 1 : 1, and the product finally eluted was 182 mg of a mixture of ( 72a) and (72b) of 2 : 8.
Properties of the diastereomer having weak polarity [ratio . Of (72a) and ( 72b) = 8 : 2 ]
IR (KBr) cm~l: 3250 center (broad), 1750, 1670, 1630, 1540, 1510, 1440 NMR (CD30D) ~ppm: 7 50 (lH, irnidazole ring), 6,92 (lH, imidazole ring), 4.8 (lH, methine), 4.10 (lH, d,d, azetidinone 4-positlon hydrogen), 4.0 (lH, methine~, 3,66 (3H, N-methyl ), 1.12, ~2S6~

1.08 (6H, two kinds of m~thyl) Mass m/z: 390 (M ), 373, 347, 320, 278, 249, 221 [~]D = -27.3 (C=l, methanol) Properties of the diastereomer having strong polarity ~ratio of (72~ and (72b) = 2 : 8]
IR (KBr) cm~1: 3250 (broad), 1750, 1670, 1630, 1~40 NMR (CD30D) ~ppm: 7;56 (lH, imidazole ring), 6~90 (lH, imidazole ring), 4.14 ~lH, d,d, azetidinone ring 4-position hydrogen), 3.68 (3H, N-methyl~, 1.08, 0.92 (6H,two kinds of methyl Mass m/z: 390 (M ), 347, 320, 277, 249, 221 ~]D = 7-9 (C = 1, methanol) Reference example 24 ~Raw material for Example 21) a) S
Z-(NT-CH3)HiS-OH ~ >

(65) ~
(73) Z-(NT-CH3)His-N ~ S

(74) CONH2 By reacting 2.25 g of compound (65) and 0.89 g of L-thiazolidine-4-carboxamide (73)in a similar manner to that in Reference example 2 ~ 1.72 g of 3-~N~ -benzyloxy-carbonyl-N~-methyl-L-histidyl]-L-thiazolidine-4-carboxamide (74) was obtained as a foamy material.

IR (KBr) cm~l: 3270, 1640-1720 (broad), 1510, 1410, 1250 NMR (CD30D) ~ppm: 7.48 (lH, imdiazole ring), :~5~i&95~.~

7.32 (5H, benzene ring), 6.90 (lH, imidazole ring), 5.06 (2H, benzyl 3.64 (3H, N-methyl) Mass m/z: 417 (M ), 373, 346, 286, 258 b) T ~
Z-(N -CH3)His-N S ~ _ _ ~ 9 ~ONH2 (74) (N -CH3)His-N S 2HBr (75) CONH2 descrlbed By following the procedure / ln Reference example 22 c), 1.9 g of 3-[NT-methyl-L-histidyl]-L-thiazolidine-4-carboxamide-2-hydrobromide (75) was obtained from 1.70 g of compound (74~. The product was used for the subse~uent reaction as it was.
Example 21 COO~
~ + (NT-CH3)His-N S ~ 2HBr (75) (2) ~ ~His(NT-CH3)-N ~ S

0 ~ NH (76) CONH2 By following t~.e procedure described in Ex2mple 19, 416 mg of 3-[N~ -[(S)-2-azetidinone-4-carbonyl]-NT -methyl-L-histi.dyl]-L thiazolidine-4-carboxamide (76) was obtained as a foamy material from 230 mg of compound (2) and 900 mg of compound (75).
IR (KBr) cm~l: 3250, 1750, 1630-1680 (broad), NMR (CD30D) dppm 7.46 (lH, imidazole rlng), 6,90 (lH, imidazole ring), 4.9 center (3H, ~ 2 methine, methylene), 4.4 (lH, methine), 4.10 (lH, d,d, azetidinone ring 4-position hydrogen), 3.64 (3H, N-methyl) Mass m/z: 381 (M+), 326, 309, 281, 267, 249 Example 22 CH~CH2-COOH
FH + tNI-CH3)HiS-PrO-N~I2 2HBr (77) (N -CH3)-PrO-NH2 N~ (78) By performing a similar reaction/in Example 19 using 413 mg of DL-4-~2-carboxyethyl)-2-azetidinone (77) and 1.12 g of compound (67), 540 mg of N~-[(RS)-3-(2-oxo-4-azetidinyl)propionyl]_~T -methyl-L-histidyl-L-prolinamide (78) was obtained. The product was a mixture of diastereomers.
IR (KBr) cm~l: 3250, 1730, 1660, 1630, 1540, 1510, NMR (CD30D) ~ppm: 7.52 (lH, imidazole ring), 6.96 (lH, imidazole ring), 4.80 (lH, methine), 4.44 (lH, methine), 3,68 (3H, N-methyl), 3~3-3.96 (3H, azetidinone ring 4-position hydrogen), proline ring), 1.94 center (4H, proline ring) Mass m/z: 390 (M ), 348, 320, 307, 277, 249 Reference Example 25 (Raw matexial for Example 23) a) Z-Pro-OH ~ Z-Pro-NHCH2CH20H
(29) ~79) ~256~

In 40 ml of THF were dissolved 9.96 g of N-benzyl-oxycarbonyl-L~proline (29) and 4O45 g of triethylamine and the solution was ice-cooled. Then, 6.10 g of ethyl chloroformate was slowly added to the solution under ice-cooling and then a soiution o-f 5.13 g of monoethanolamine~was added to the mixture.
The reaction mixture thus obtained was stirred for 15 minutes under ice-cooling and then stirred for 1.5 hours at room temperature. After distillir.g off THF
under xeduced pressure, 150 ml of ethyl acetate and 50 ml of water were added to the residue and the organic layer thus formed was separated from the aqueous layer.
The organic layer was washed in succession with an aqueous solution of lN hydrochloric acid, an aqueous solution of O.lN sodium hydroxide, water, and then an aqueous sodium chloride solution. After drying the organic layer, the solvent was removed, whereby precipitating crystals, which were collected by filtration and recrystallized from ethyl acetate to provide 5.23 g of (S)-l-benzyloxycarbonyl-N-2-hydroxy-ethyl-2-pyrrolidinecarboxamide (79).
M. p. 104-106C
IR (KBr) cm 1 3420, 3270, 16i30, 1640, 1540 NMR (CDC13) ~ppm: 7.36 (5H, s, benzene ring), 5.16 (2H, q, benzyl 4,30 (lH, t, methine 2,04 center (5H, proline ring, OH) ~25~6~i;0 b) Z-pro-NHcH2cH2oH ~ Pro-NHCH2CH20H
(79) (53) In 70 ml of methanol was dissolved 5.92 g of compound (79) and the compound was catalytically reduced by an ordinary method using 10% palladium-carbon as a catalyst. After the reaction~ the catalyst and the solvent were removed to provide 3.2 g of syrupy (S)-N-2-hydroxyethyl-2-pyrrolidinecarboxamide ~53). The product was solidified when it was refrige-rated.
NMR (CD30D) ~ppm: 2.96-3.84 (7H, proline ring, hydroxyethyl~, 1.64-2.36 (4H, proline ring) IR (neat) cm~l: 3250, 1640 (broad), 1530 (broad) Mass m/z: 159 (M + 1), 127, 70 c ) Z-(N -CH3)His-OH + Pro-NHCH2CH2OH
(65) (53) Z-(N -CH3)His-Pro-NHCH2CH2oH
(80) In 25 ml of dTy - DMF were dissolved 1.54 g of N~-benzyloxycarbonyl-NT -methyl-L-histidine (65) and 0.80 g of compound (53) and the solution was ice-cooled.
After adding 1.01 g of p-toluenesulfonic acid monohydrate to the solution, 1.36 g of DCC was also added to the mixture~ The mixture wa5 stirred over-night in a refrigerator to perform the reaction.
Thereafter, the reaction mixture was stirred for 2.5 76 ~2~6~Sf~
hours at room temperature. After filtering off insoluble matters, the filtrate was concentrated under reduced pressure, the residue thus formed was subjected to column chramatography of 300 ml of silica gel, and the product was eluted with chloroform-methanol-aqueous ammonia (80 : 20 : 2) to provide 1.7 g of N~ -benzyl-oxycarbonyl-NI -methyl-L-histidyl-N-2-hydroxyethyl-L-prolinamide (80) as a foamy material.
NMR (CD30D) ~ppm: 7 54 (lH, imidazole ring), 7.32 (5H, benzene ring), 6.92 (lH, imidazole ring), 5.04 (2H, benzyl 3.64 (3~i, N-methyl), 2.0 center (4H, proline ring) Mass m/z: 443 (M+), 413, 355, 286, 258 IR (neat) cm~l: 3250 (broad), 1620-1720 (broad) d) Z-(N -cH3)His-pro-NHcH2cH2oH ~
(80) 0

11 (N -CH3)His-Pro-NHCH2CH20CCH3 2HBr (81) To 22 ml of an acetic acid solution of 25% hydro-bromic acid was added 1.7 g of compound (80) and the reaction was performed for 1.5 hours at room temperature. The reaction mixture was added to 250 ml of dry ether to form white precipitates. The precipitates were collected by filtration and dried under reduced pressure to provide N T -methyl-L-histidyl-N-2-acetoxyethyl-L-pxolinamide 2-hydrobromide~81) with a quantitative yield. The product was used for the subsequent reaction as it was~

~5665~1 ~ COOH Example 23 l!

o ( 3) 2CH2OCCH3 HBr (81) (2) o, C O
His(NT~CH3)-Pro-NHCH2Cl~20CCH3 ~_N~
~ (82) In a mixture of 7 ml of methylene chloride and 7 ml of DMF were dissolved 317 mg of compound (2) and 448 mg of HOBT and the solution was ice-cooled. Then, after adding thereto 683 mg of DCC, the mixture was stirred for 20 minutes under ice-cooling. To the reaction mixture was added a free amine solution prepared by reacting 1.37 g of compound (81) and 670 mg of triethylamine in 12 ml of DMF under ice-cooling followed by filtration. The mixture was stirred for 38 hours in a refrigerator to perform the reaction. Insoluble matters were filtered off, the filtrate was dried under reduced pressure, and the residue was subjected to column chromatography using 300 ml of silica gel. By eluting the product with chloroform-methanol-aqueous ammonia (85 : 15 : 2), 593 mg of N~-[(S)-azetidinone-4-carbonyl]-N~-methyl-L-histidyl-N-2-acetoxyethyl-L-prolinamide (8~) was as foamy material.
obtaine ~ The product was dissolved in water and then lyophilized.
IR (KBr) cm~1: 3250, 1750, 1730, 1640, 1540, NMR (CD30D) ~ppm: 7.56 (lH, imidazole ring), 6~

6.96 (lH, imidazole ring), ~.80 (lH, methine hydrogen), 4.40 (lH, methine hydrogen), 4 16 center (3H, azetidinone ring ~-position hydrogen, -CH2O-~CH3~, 2.0 center (7H, proline ring, acetyl) Mass (FD) m/z: 449 (M ~ 1) Reference example 26 (Raw material for Example 24) a) r~~
Z-Pro-OH ~ Z-Pro-N~_~O
(29) (83) In 80 ml of dry tetrahydrofuran were dissol-ved 4.9 g (20 m mol) of Z-proline (29) and 3.5 g (26 m mol) of HOBT and then 4.53 g (22 m mol) of DCC was slowly added to the solution at 0C. After stirring the mixture for 30 minutes at the same temperature, a solution of 1.74 g (20 m mol) o~ morpholine dissolved in 20 ml of dry ~ was gradually addecl dropwise to the mixture. The resultant mixture was allowed to stand for 18 hours at room tamperature and then the solvent was distilled off under reduced pressure. The residue thus formed was dissolved in 200 ml of ethyl acetate and the solution was washed, in succession, with 75 ml of an aqueous solution of 0.5N
hydrochloric acid, 75 ml of a saturated aqueous solution of sodium hydrogen carbonate, and then 50 ml of water. After drying the solution with anhydrous magnesium sulfate, ethyl acetate was distilled off and the residue thus formed was purified by silica gel ~ 4g0 ml of column chromatography/ Wako Gel C-200;

~2S66~5~

as a eluent) ethyl acetate /to provide 5.0 g of N-[N-benzyloxy-carbonyl-L-prolyl]morpholine (83) having a melting point of 139-140C.
NMR: 90 MH2 (CDC13) ~ppm: 1.70 2.40 (m, 4H, proline ring), 3.20-4.00 (m, 10H, proline ring, morpholine ring), 4.40-4.90 (m, lH, proline ring methine), 5.10, 5~14 (q, q, 2H, benzyl ), 7.32, 7.34 (s, s, SH, benzene ring) IR (KBr) cm~l: 2960, 2910, 2860, 2830, 1680, b) Z-Pro-N~_~O -~ Pro-N~_~O
(83) (84) In 100 ml of ethanol was suspended 4.9 g of compound (83) and after adding thereto 250 mg of 10%
palladium carbon, the mixture was stirred for 4 hours in a hydrogen stream. After filtering off 10% palladium-carbon, ethanol was distilled off under reduced pressure from the filtrate to provide 2.8 g of crude N-(L-prolyl)morpholine (84).
NMR: 90 MHz (CDC13) ~ppm: 1.40-2.30 (m, 4H, proline ring), 2.60-3.40 (m, 2H, proline ring), 2.97 (s, 1~), 3,40-4.10 (m, 9H, morphiline ring) IR (neat) cm~1: 3280, 2960, 2840, 1635 Mass: 185 (M ~ 1), 142, 114, 98, 70, 43 ~2~6~i5~

c ) Z-His-NHNH2-- 3 [Z-His (3) (4) Z-His-Pro-N~_Jo (85) In 54 ml of an a~ueous solution of lN hydrochloric acid was dissolved 5.46 g (18 m mol) of L-Z-histidine (3) hydrazide/and after adding thereto 72 ml of ethyl acetate, the mixture was cooled to 0C. Then, 5.4 ml of an aqueous solution of 4N-NaNO2 was added to the mixture at the same temperature followed by stirring for 5 minutes and after adding thereto 21.6 ml of an aqueous solution of 5G% potassium carbonate followed by stirring vigorously, the ethyl acetate layerthus formed was separated. The aqueous layer was extracted with 1~ ml of cooled ethyl acetate, the ethyl acetate extract was combined with the foregoing ethyl acetate layer, and the mixture was dried with anhydrous sodium sulfate for 5 minutes with stirring under ice-cooling.
After filtering off sodium sulfate, the filtrate was cooled to -20C and after slowly adding dropwise a solution of 2.76 g (15 m mol) of compound (8~) dissolved in 10 ml of ethyl acetate to the filtrate, the mixture was allowed to stand overnight in a refrigerator at 4C. After allowing to raise the temperature to room temperature, ethyl acetate was distilled off under reduced pressure /and the residue thus formed was purified by silica gel (600 ml of column chromatography /~ako gel ~ 200, chloroform-methanol-aqueous ammonia (10 : 1 : 0.1)) to ~2S6&~J

provide 6.46 g of N-[N~-benzyloxy-carbonyl-L-histidyl-L-prolyl]morpholine (85) as a colorIess/
NMR: 90 MHz (CDC13) ~ppm: 1.60-2.40 (m, 4H, proline ring), 3.80 (d, 2H, methylene of His moiety), 3.20-4.00 (m, lOH, morpholine ring, proline ring), 4.40-5.00 (m, 2H, methine) 5.08 (s, 2H, benzyl ), 6.09 (m, lH, amide), 6.86 (s, lH, imidazole ring), 7.12 (s, 5H, benzene ring), 7.52 (s, lH, imidazole ring) IR (KBr) cm 1 3250, 2950, 2840, 1710, 1640, 1630 Mass: 455 (M~), 374, 341, 305, 272, 244 d) Z-His-Pro-N O ~ His-Pro-N~_~02HBr (85) (86) In 21.4 ml of acetic acid was dissolved 6.46 g (14.2 m mol) of compound (85) and after adding thereto 42.8 g of an acetic acid solution of 25% hydrobromic acid under ice-cooling, the mixture was stirred for After 1.5 hours. /the reaction mixture was added 600 ml of desicated ether, precipitates were filtered to provide 6.50 g of the crude crystals of N-[L-histidyl-L-prolyl]morpholine.2-hydrobromide (86) ~25 Ei~5C~

Example 24 o His-Pro-N O 2HBr (2) ~ His-Pro-N O

(86) (87) Solution A]
dry In 10 ml of/DMF was dissolved 230 mg (2 m mol) of compound (2) and after adding thereto, in succesion, 351 mg of HOBT and 453 mg of DCC with stirring under cooling to 0, the mixture was stirred for 40 minutes at the same temperature.
~Solution B]
In 13 ml of dry DMF was dissolved 966 mg (2 m mol) of compound (86) and the solution was cooled to -15C. After adding thereto 404 mg of triethylamine with stirring, the mixture was stirred for 30 minutes at the same temperature and then triethylamine hydro-bromide was filtered off udner cooling.
To solution A cooled to 0C was added solution B
cooled to -15C and after stirring the .nixture for 2 hours at -10C, the mixture was allowed to stand overnight in a refrigerator at 4C
After rasing the temperature of the reaction mixture to room temperature, insoluble matters were filtered off, DMF was distilled off under reduced pressure, and the residue thus obtained was purified by (400 ml of silica gel column chromatography ~'ako gel C-200 chloroform-methanol-aqueous ammonia (40 : 10 : l)) to -` ~Z5~S~

provide 480 ms of the colorless crystals of the desired product, 4-[N~-[(S)-2-azetidinone-4-carbonyl]-L-histidyl}L-prolyl]morpholine ~87) having a melting point of 148-150C.
NMR: 90 MHz ~CD30D) ~ppm: 1.60-2.40 (m, 4H, proline ring), 2.72 (d,d, lH, azetidinone ring 3-position), 4.11 (d,d, l~t azetidinone ring 4-position), 6.96 (s, lH, imidazole ring), 7.63 (s, lH, imidazole ring) IR (KBr) cm~l: 3200, 3040, 2850, 1755, 1650, 1625, 1555 Mass: 418 (M ), 348, 304, 235, 207 [~]~ -69.1 (C = 1, methanol) Reference example 27 (Raw material for Example 25) a) CH3 Z-Pro-OH ) Z-Pro-N <

(29) (88) In 80 ml of dry THF were dissolved 4.9 g (20 m mol) of N-benzyloxycarbonyl-L-proline (29) and 3.5 g (26 m mole) of HOBT and then 4.53 g (22 m mol) of DCC was slowly added to the solution at 0C. After for 30 minutes stirring the mixture/at the same temperature, 10 ml of was added.
solution of 2M dimethylamine tetrahydrofuran/
resulting The /mixture was allowed to stand for 12 hours at room temperature. THF was distllled off from the reaction mixture under reduced pressure, the residue thus formed was dissolved in 200 ml of ethyl acetate, and the solution was washed, in succesion, with 75 ml of an aqueous solution of 0.5N hydrochloric acid, 75 ml 8~ ~2~6~.5~
of a saturated aqueous sodium hydrogencarbonate solu-tion, and ~0 ml of water. After drving the organic layer with anhydrous magnesium sulfate, ethyl acetate was distilled off and the residue thus formed was (400 ml of purified by silica gel column chroma,ography /Wako gel C-200, . ethyl acetate) to provide 4.8 g of (S)-l-benzyloxycarbonyl-N,N-dimethyl-2-pyrrolidinecarbo-xamide (88) having a melting point of 66-67C~
NMR: 100 MHz (DMSO-d5) ~ppm: i.50-2.40 (m, 4H, proline ring), 2.74, 2.78, 2.86, 2.98 (s,s,s,s, 6H, N-dimethyl), 3.38 (m, 2H, proline ring), 4.70 (m, lH, methine), 4,g6 and 5.02 (q and s, 2H, benzyl IR (KBr) cm : 3020, 2960, 2.940, 2860, 1700, 1640 b) Z-Pro-N ~ 3 . ~ Pro-N ~ CH3 (88) (89) , In 86 ml of ethanol was dlssolved 4.3 g of com?ound (88) znd after adding thereto 210 mg of 10%
pz ladiu~-carbon, the mixture was slirred vigorously ~o- 90 minutesin2 hydrogen stre~m,. After filte-~ng o~f the ca~alyst, ethanol was distilled off under reduced ~-essu-e to provide 2.19 g of crude (S)-N,N-dimethyl-2-py-rolidlnecarboxamide (89).
NMR: 90 MH~ (CDC13) ~ppm: '.40-2.40 (m, 4H, proline -ing), 2.84 (s, lF;, NH), 3.00 (s, 3n, N-methyl), 3.04 (m, 3H, N-methyl), 3.80-4.00 (m, lH, methine) * Trade Mark .. .. .

IR (neat) cm~l: 3280, 2940, 2850, 1635 c ) Z-His-NHNH2 --~[Z-His-N3] - (89) _~

(3) Z-His-Pro-N~CH3 (90) In 53.3 ml of an aqueous solution of lN hydro-chloric acid was dissolved 5.38 g (14.8 x 1.2 m mol) of L-z- histidine hydrazide/and after adding thereto 71 ml of ethyl acetate, the mixture was cooled to 0C.
Then, 5.33 ml of an aqueous solution of 4N-NaNO2 was added to the mixture at the same temperature followed by stirring for 5 minutes and after adding thereto 21.3 ml of an aqueous solution of 50% potassium carbonate followed by stirring for 3 minutes, the ethvl acetate layer thus formed was collected. The aqueous layer was extracted with 18 ml of cooled ethyl acetate, the extract was combined with the foregoing ethyl acetate layer and the mixture was dried by anhydrous sodium sulfate for 5 minutes with stirring under ice-cooling.
After filtering off sodium sulfate, the filtrate was cooled to -20C and a solution of 2.10 g (14.8 m mol) of compound (89) dissolved in 10 ml of ethyl acetate was added slowly dropwise to the fore-going solution. The mixture was allowed to stand overnight in a refrigerator at4 C. Ethyl acetate was distilled off under reduced pressure and the residue was purified by silica gel column chromatography ~:2S6~5~

(600 ml of /Wako gel C-200, chloroform-methanol-conc.
aqueous ammonia (10 : 1 : O.l))to provide 5.86 g of oily N~-benzyloxycarbonyl-L-histidyl-N,N-dimethyl-L-- prolinamide (gO).
NMR: 9G MHz (CDC13) dppm 1.60-2.40 (m, 4H, proline ring), 3.05 (s, 3H, N-methyl), 3.16 (s, 3H, N-methyl), 4.40-5.00 (m, 2H, methine), 5.12 (s, 2H, benzyl ), 5.64 (m, lH, amide), 6.90 (s, lH, imidazole ring), 7.38 (s, 5H, b~nzene ring), 7.56 ~s, lH, imidazole ring), 11.70 (m, lH, imidazole -NH) IR (neat) cm~l: 3250, 2950, 2840, 1710, 1635 Mass: 413 (M ), 341, 332, 272, 262, 244 d) Z-His-Pro-N < 3 > His-Pro-N ~ 3 2Br ~so) '9l?

dissolved - In 15 ml of acetic acid was/4.13 g (10 m mol) of compound (90) and after adding thereto 30.12 g of an acetic acid solution of 25~ hydrobromic acid under ice-cooling, the mixture was stirred for 1.5 hours at room temperature. To the reaction mixture was added 450 ml of dry ether and the precipitates thus deposited were collected by filtration to provide 3.75 g of L-histidyl-N,N-dimethyl-L-prolinamide 2-hydrobromide (91).

~25~

Example 25 His-Pro-N< CH3 2~r ( ) (91) ~ His-Pro-N <

[Solution A] (92) dry In 10 ml of/DMF was dissolved 230 mg (2 m mol) of compound (2) followed by cooling to 0C and after adding thereto, in succesion, 351 mg of HOBT and 453 mg of DCC with stirring, the mixture was stirred for 40 minutes at the same temperature.
[Solution B]
In 13 ml of dry DMF was dissolved 966 mg (2 m mol) of compound (91) followed by cooling to -15C, and after slowly adding 404 mg of ~riethylamine to the solution with stirring, the mixture was stirred for 30 minutes. Thereafter, triethylamine hdyrobromide was filtered off under cooling.
To solution A cooled to 0C was added solution B
cooled to -15C and after stirring the mixture for 2 hours at -10C, the m~xture was allowed to stand over-night in a refrigera~or at 4C.
The reaction mixture was allowed to raise to room temperature and insoluble matters deposited were filtered off. DMF was distilled off from the filtrate under reduced pressure and the residue thus formed was purified by silica gel column chromatography ~Wako gel C-200, 400 ml, chloroform-methanol-aqueous ammonia ~256~

(100 : 10 : 1)) to provide 520 mg of the colorless crystals of N~--[(S)-2-azetidinone-4-carbonyl]-L-histidyl-N,N-dimethyl-L-prolinamide/having a melting point of 133-1~0C.
N~R: 90 Mhz (D2O~ ~ppm: 1.60-2.50 (m, 4H, proline ring), 2.71 (d,d, lH, azetidinone ring, 3-position), 2.92 (s, 3H, N-methyl), 3.17 (s, 3H, N-methyl), 4.15 (d~d, lH, azetidinone ring, 4-position), 7.40 (s, lH, imidazole ring), 7.74 (s, lH, imidazole ring) IR (KBr) cm~l: 3180, 1755, 1630, 1560 Mass: 376 (M+), 306, 262, 235, 207 [~]D0 -73.1 (C = 1, methanol) Reference example 28 ~Raw material for Example 26) a) Z-Pro-OH ) Z-Pro NHPh (29) (93) In 50 ml of THF was dissolved 4.99 g of N-benzyloxycarbonyl-L-proline (29) and after adding thereto 2.23 g of triethylamine and 2.39 g of ethyl chloroformate, the reaction was performed for 20 minutes under ice-cooling. To the reaction mixture was added 2.79 g of aniline and the reaction was performed for one hour under ice-cooling. The solvent was distilled off, the residue thus formed was dissolved in ethyl acetate, and the solution was washed, in succession, with an aqueous solution of lN hydro-chloric acid, a saturated aqueous sodium hydrogen-carbonate solution, and then a saturated aqueous sodium ;66S~

chloride solution. The organic layer thus formed was sodium sulfate dried by anhydrous /and then concentrated to dryness. The residue was recrystallized from chloroform-et~yl acetate-hexane to pro~ide 5.20 g of (S)-1 benzyloxycarbonyl-N-phenyl-2-pyrroiidine-carboxamide (93) having a melting point of 143-144C.
NMR (CDC13) ~ppm: 6.9-7.7 (lOH), 5.17 (s, 2H), 4.43 (t, lH), 3.4-3.8 (2H), 1.7-2.5 (4H) IR (KBr) cm~l: 3260, 1690, 1660, 1595, 1545 Mass (EI): 324 (M+), 204, 160, 91, 70 b) Z-Pro-NHPh ~ > Pro-NHPh (93) (94) In 150 ml of methanol was dissolved 4.87 g of compound (93) and the compound was hydrogenated using 487 mg of 10% palladium-carbon as a catalyst. The catalyst was filtered off and the filtrate was concentrated to provide 2.79 g of (S)-N-phenyl-2-pyrrolidinecarboxamide ( 94) .
NMR (CDC13) ~ppm: 9.5-10.0 (lH), 6.95-7.75 (lOH), > 3.86 (dd, lH), 2.75-3.25 (2H), 1.5-2.5 (5H) Mass (EI): 190 (M ), 93, 70 c ) Z_His-NHNH2 --~[Z-His-N3]-- ~94! _, ~3) (4) Z-His-Pro-NHPh (95) To 45 ml of an ethyl acetate solution of N ~ -benzyloxycarbonyl-L-histidine azide (4) prepared from 3.03 g of N~ -benzyloxycarbonyl-L-histidine hydrazide ¦ IR (KBr) cm : 3340, 3220, 2950, 2850, 1660, 1595, 1515 J

~s~

9o ( 3~ by a known method was added 1.52 g of compound (94) under ice-cooling and the reaction was performed overnight in a refrigerator. The reaction mixture was concentrated ~nd the residue thus formed was subjected to silica gel column chromatography. By eluting the product with chloroform-methanol (95 : 5), 2.49 g of N~ -benzyloxycarbonyl-L-histidyl-N-phenyl-L-prolinamide (95) was obtained.
NMR (CDC13) ~ppm: 6.9-7.7 (llH), 6.78 (s, lH), 5.86 (d, lH), 5.08 (s, 2H), 4.5-4.8 (2Hj, 2.8-3.9 (4H), 1.5-2.5 (4H) IR (KBr) cm~l: '250, 2950, 1700, 1630, 1590, 1535 Mass (EI): 461 (M+), 342, 310, 272, 245, 191, 136, 107, 91, 70 d) Z-His-Pro-NHPh ~ His-Pro-NHPh.2HBr (9S) (96) To 1.79 g of compound (95) was added 19 ml of 25 % hydrobromic acid - acetic acid cooled in an i~e bath followed by stirring one hour at ~oom temperature, The reaction mixture was added to 190 ml of desicated ether and precipitates thus formed were quickly collected by filtration ,and dried overnight in a desicator containing potassium hydroxide to provide 2.05 g of L-histidyl-N-phenyl-L-prolinamide.2-hydrobromide (96)-~56~5`~

Example 6 His -Pro -l~rPh - 2HBr ~ [His -Pro -~lPh]
(96~ (97) ~CO-His -Pro-~Ph ~1~ (98) In 10 ml of DMF was dissolved 979 mg of compound ( 96) and after cooling the solution to -40C, 415 mg of triethylamine was added to the solution. After performing the reaction for one hour at -30C to -40C, precipitates thus formed were filtered off to provide a DMF solution of L-histidyl-N-phenyl-L-prolinamide (97). The product was used for the subsequent reaction immediately after the foramtion thereof.
In 5 ml o~ DMF wzs dissolved 230 mg o (S)-2 azetidinone-4-carboxylic acid (2) and after adding hereto 406 mg of HOBT and 495 mg of DCC under ice-hour cooling, the reaction was performed for one/at o~C.
The reaction mixture was cooled to -40C
and after adding thereto a DMF solution of the fore-going compound (97), the reaction was performed for 30 minutes at -40C and then overnight in a refrigerator. Precipitates were filtered off, the filtrate was concentrated to dryness, and the residue was subjected to silica gel column chromatography.
By eluting the product with chloroform-methanol-aqueous ammonia (~0 : 20 : 2), 652 mg of M~-[(S)-~-azetidinone-4-carbonyl]-L-histidyl-N-phenyl-L-prolinarnide (98) was obtained.

~s~

NMR (CD30D) ~ppm: 6.9-7.7 (7H), 4.56 (dd, lH) 4.12 ~dd, lH), 3.7-3.9 (lH), 2.81 (dd, lH), 1.7-2~3 (4H) IR (KBr) cm~l: 3250, 2910, 1750, 1620, 1540 Mass (EI)o 425 (M~ + 1), 305, 262, 250, 208, 191, 154, 93, 70 [~]D = -103.5 (C = 1.35, methanol) Reference example 29 (Raw material for Example 27) a) Z-Pro-OH ~ Z-Pro-N~

(29) (99) In 100 ml of THF was dissolved 9.97 g of slowly compound (Z9) and after adding/thereto 4.45 g of triethylamine and then 6.01 g of isobutyl chloroformate under ice-cooling, the reaction was performed under ice-cooling. To the reaction mixture was slowly added 11.37 g of 3-(2-oxo-1-pyrrolidinyl)-propylamine and then the reaction was performed for one hour under ice-cooling. Precipitates were filtered off, the filtrate was concentrated, and the residue thus formed was subjected to silica gel column chromato-graphy. By eluting the product with ethyl acetate-methanol (4 ~ .43 g of (S)-l-benzyloxycarbonyl-N-[3-(2-oxo-pyrrolidinyl)propyl]-2-pyrrolidine carboxamide (99) was obtained.
NMR (CDC13) ~ppm: 7.36 (s, 5H), 5.16 ~2H), 4.33 (t, lH), 2.8-3.8 (8H), 1.7-2.5 (lOH) IR (neat) cm~1: 3280, 2930, 2860, 1700, 1660, 1530 ~S6~;S~

Mass (EI): 373 (M ), 238, 204, 160, 91, 70 b) Z-Pro-NH N~ o ~ Pro-NH ~ N ~ >

(99) (100) In 150 ml of methanol was dissolved 4.32 g of compound (99) and the compound was hydrogenated using 432 mg of 10~ palladium-carbon as a catalyst. Then, the catalyst was filtered off from the reaction mixture and the filtrate was concentrated to provide 1.99 g of (S)-N-[3-(2-oxo-1-pyrrolidinyl)propyl]-2-pyrrolidine-carboxiamide (100) was obtained.
NMR (CDC13) ~ppm: 7.6-8.2 (lH), 3.72 (d,d, lH), 2.8-3.5 (8H), 1.5-2.5 (lOH) IR (neat) cm~l: 3280, 2920, 2850, 1650, 1530 Mass (EI): 239 (M ), 197, 141, 99, 70 c ) ~~\~ '' ' Pro-N~iv^~N ~ ~ Z-His-Pro-NH ~ N`~

tlOO) (101) To 30 ml of a ethyl aceta~e solu~ion of compound ( 4 ) prepared from 2.12 g of compound (3) by a known method was added 5 ml of a DMF solution of ~.17 g of compound (100) under ice-cooling and the reaction was per~ormed overnight in a refrigerator. The reaction mixture was concentrated and the residue thus formed was subjeoted to silica gel column chromatography. By eluting the product with chloroform-methanol-aqueous ammonia (90 : 10 : 1), ~L~S6~

9~
2.00 g of N~-benzyloxycarbonyl-L-histidyl-N-[3-(2-oxo-(101) l-pyrrolidinyl)-propyl]-L-prolinamide~was obtained.
NMR (CDC13) ~ppm: 8.0-8.4 (lH), 7.56 (lH), 7.35 ts, 5~), 6.97 (s, lH), 5.87 (d, 2H), 5.10 (s, 2H), 4~3-4.8 !2H)/ 2.9-3.7 (8H), 1.5-2.6 (lOH) IR (KBr) cm~l: 3220, 2930, 2850, 1700, 1640, 1530 Mass (EI): 510 (M~), 430, 402, 359, 267, 239, 136, 1~8, 79 d) ~

~0 (101) ~\
His-Pro-NH~f~N ~ 2HBr (102) To 1.02 g of compound (101) was added 10 ml of ice-cooled an/acetic acid solution of 25% hydrobromic acid and the reaction was performed for 2 hours at room temperature.
The reaction mixture was added to 100 ml of dry ether and t-hen the precipitates thus formed were quickly collected by filtration and dried overnight in a desicator containing potassium hydroxide, 1.28 g of L-histidyl-N-[3-(2-oxo-1-pyrrolidinyl)propyl]-L-prolinamide.2-hydrobromide (102) was obtained.
Example 27 ~
His-Pro-NH~N ~ 2HBr ~-~ [His-Pro-NH~vN ~ ]
(102) (103) ,H C0-His-Pro-NH~N
(2) o ~ NH (104) i6~5~
In 10 ml of DMF was dissolved 1.28 g of compound (102) and after cooling to -40C, 415 mg of triethylamine was added to the solution. After performing the reaction for one hour at -30 to -40C, precipitates thus formed were filtered off to provide a DMF solution of L-histidyl-N-[3-(2-oxo-1-pyrrolidinyl)-propyl]-L-prolinamide (103). The product was used for the subsequent reaction immediately after the formation thereof.
In 5 ml of DMF was dissolved 230 mg of compound

12) and after adding thereto 406 mg of HOBT and 495 mg of DCC, the reaction was performed for one hour under ice-cooling. The reaction mixture was cooled to -40C
and after adding thereto the foregoing DMF solution of compound (103~,the reaction was performed for 30 minutes at -40C and then overnight in a refrigerator. Precipi-tates thus formed were filtered off, the filtrate was concentrated to dryness, and the residue was subjected to silica gel column chromatography. By eluting the product with chloroform-methanol-aqueous amonnia (80 :
20 ~ 2), 461 mg of N~-[(S)-2-azetidinone-4-carbonyl]-L-histidyl-N [3~(2-oxo-1-pyrrolidinyl)propyl]-L-prolinamide (104) was obtained.
NMR (D20) ~ppm: 7-74 (lH), 7.05 (lH~, 5.96 (dd, lH), 5.2-5.5 (2H), 2.9-3.9 (llH), 2.72 (dd, lH), 1.6-2.6 (lOH) ---~ ~"-IR (KBr) cm~l: 3240, 2950, 2850, 1755, 1630 Mass (EI): 473 (M+), 304, 262, 235, 154, 70 ~2S6~iS;~3 [~]27: -75.6 (C = 0~55, methanol) Reference example 30 a) CH3 ~ COOH

Si(CH3)2 Bu Si(CH3)2 Bu (105) (106) In 6 ml of dry THF was dissolved 836.3 mg (8.28 m mol) of dilsopropylamine and the solution was cooled to 0C under,\nitrogen atmosphere. To the solution was added 5.2 ml of a n-hexane solution containing 530 mg (8.28 m mol) of n-butyl lithium at 0C and the mixture was stirred for 10 minutes at the same temperature. To the solution was added a solution of 92û mg (4 m mol) of (S~ t-butyldimethylsilyl-2-azetidinone-4-carboxylic acid (105) dissolved in 8 ml of dry THF at 0C and then the mixture was stirred for 30 minutes at room temperature. The solution was cooled to 0C and after adding thereto 682 mg (~.8 m mol) of methyl iodide, the mixture was stirred for 30 minutes ~t room temperature. The reaction mixture thus obtained was cooled again to 0C, acidified with the addition of an aqueous l0%
citric acid solution, and after addition of ether and water, the organic layer was separated. The ether layer was separated from the aqueous layer, dried, and the solvent was distilled off to provide 860 mg of l-t-butyldimethyl-silyl-3(R)-methyl-2-azetidinone-4(S)-carboxylic acid (106) as colorless crystals.

.~ ~2~ S¢~

~23 = 36.1 (C = 0.5, methanol) NMR (90 MHz, CDC13) ~ppm: 0.16 (3H, s, Si-methyl), 0.34 (3H, s, Si-methyl), 0.98 (9H, s, t-butyl), 1.42 (3H, d, azetidinone ring 3-position methyl), 3.37 (lH, q,d, azetidinone ring 3-position), 3.74 (lH, d, J=3.5 Hz, azetidinone ring 4-position), 9.60 (lH, s, carboxy group) IR (KBr) cm~1: 2940, 2920, 2840, 1740, 1680 Mass m/z: 244 (M + 1), 200, 186, 143 b) CH~ COOH CH3 COOH

~Si(CH3)2tBu NH

(106) ~107) In 20 ml of a mixture of water, methanol, and concentrated hydrochlorlc acid (10: 90 : 1~ 7) was dissolved 641 mg ~2.63 m mol) of compound (106) and the solution was stirred for 1.5 hours at room temperature. The reaction mixture was cooled to 0C, neutralized with 4 ml of an aqueous solution of lN sodium hydroxide, and the solvent was distilled off under reduced pressure to provide 3(R)-methyl-2-azetidinone-4(S~-carboxylic acid (107), which was used in the subsequent reaction without being purified.
NMR (60 MHz, D2O) ~ppm: 1.25 (3H, d, methyl group), 3~20 (lH, q,d, azetidlnone ring 3-position), 3.88 (lH, d, azetidinone ring 4-position) ~Z~66S~

Example 28 His-Pro-~l2 2HBr > ~ His-Pro-NH2 (107) (8) ~108) In 13 ml of dry DMF was dissolved compound (107) obtained in the foregoing step followed by cooling to 0C and after adding thereto 461.6 mg (3.42 m mol) of HOBT and 596 mg (2.89 m mol) of DCC, the mixture was stirred for 15 minutes at the same tempera-ture (solution A).
In 30 ml of dry DMF was dissolved 1.086 g (2.63 m mol) of L-histidyl-L-proLinamide.2-hydrobromide (8) and after cooling the solution to -10C, 0.733 ml (2.63 m mol) of triethylamine was added to the solution.
After stirring the mixture for 30 minutes at the same temperature, triethylamine hydrobromide thus precipita-ted was filtered off in~nitrogen atmosphere to provide a clear filtrate (solution B).
To solution A was added solution B and the mixture was stirred overnight at 0 to 5C and then for 3 hours at room temperature. Crystals thus precipitated were filtered off, the filtrate was concentrated under reduced pressure, and the residue thus formed was subjected to silica gel column chromatography using 200 ml of silica gel (~lako gel C-200). By eluting the product with chloroform-methanol-aqueous ammonia (80 :
20 : 2), 200 mg of N~-~3(R)-methyl-2-azetidinone-4(S~-~;25~

-carbonyl]-L-histidyl-L-prolinamide (108) was obtained.

~]~3 = -33.8 (C = 0.5, methanol) NMR (100 MHz, D2O) ~ppm~ 1.60-2.40 (4H, m, prolïne ring) 2.80-3.20 (3H, m, histidine ring, ~-methylene, proline ring), 3.40-4.00 (2H, m, azetidinone ring 3-position, proline ring), 3.90 (lH, d, J = 3.0 Hz, azetidinone ring 4-position), 4.40 (lH, m, met~ine), 4.88 (lH, m, metine~, 7.Q0 (lH, s, imidazole ring), 7.68 (lH, s, imidazole ring), 1.32 (3H, d, methyl) IR (KBr) cm~l: 3450, 2960, 2860, 1750, 1670, 1630 Mass m/z- 362 (M~), 318, 278, 249, 234, 221 Preparation examples:
Injection A lyophilized formulation containing 0.025 mg or 0.05 mg of N~-[(S)-2-azetidione-4-carbonyl]-L-histidyl-L-prolinamide toyether with 10 ml of mannitol in one ampule was prepared and each of the formulations was dissolved in 1 ml of a sterilized physiological saline solution to provide an injection.

Tablets .
A mixture of 0.25 part by weight of N~-[(S)--azetidinone-4-carbonyl~-L-histidyl-L-prolinamide and uniformly 7.5 parts by weight of lactose was puvlerized, and mixed/
with 44.4 parts by weight of lactose , 22.5 parts by weight of crystalline cellulose, and 0.4 part by weight of magnesium stearate The resultant mixture was compacted to form tablets of 75 mg/tablet.

~s~

- ~apsules A mixture of 0.5 part by weight of N~-[~S)-2-azetidinone-4-carbonyl]-L-histidyl-L-prolinamide and 10 parts by weigllt of lactose was pulverized, and mixed uniformly with 137.5 parts by weight of lactose, 6G parts by weight of corn starch, and 2.0 parts by weight of magnesium stearate. The mixture was filled into gelatin hard capsules, to provide a capsulated preparations of 210 mg/capsule.

Claims (13)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A 4-substituted-Z-azetidinone compound represented by general formula (I) (I) wherein R1, R3 and R4, which may be the same or different, each represents a hydrogen atom or a lower alkyl group; R2 represents an imidazolyl group shown by (wherein R5 represents a hydrogen atom, a lower alkyl group, an aromatic acyl group, or an aryl group); n represents 0, 1, 2 or 3;
X represents a methylene group, an ethylene group, an oxygen atom, or a sulfur atom; and Y represents a hydroxy group, a lower alkoxy group, an aralkoxy group or an unsubstituted or substituted amino group shown (wherein R6 and R7, which may be the same or different, each represents a hydrogen atom, a lower alkyl group, a hydroxy lower alkyl group, a lower alkoxy lower alkyl group, a cycloalkyl group, an aryl group, an amino lower alkyl group or an acyloxy lower alkyl group; said R6 and R7 may combine with each other to form, together with the nitrogen atom to which these groups are bonded, a 5-membered or 6-membered ring, which may contain an oxygen atom, a sulfur atom or a nitrogen atom) or a salt thereof.

2. A 4-substituted-2-azetidinone compound as claimed in claim 1, wherein in the formula (I), R2 is a group shown by (wherein R5 has the same meaning as defined in general formula (I)); X
is a methylene group, an ethylene group, or a sulfur atom; and R1, R3, R4, n, and Y are same as described above, or a salt thereof.

3. A 4-substituted-2-azetidinone compound as claimed in claim 1, wherein in the formula (I), R1, R3, and R4 are a hydrogen atom; R2 is a group shown by ( wherein R5 is a hydrogen atom, a lower alkyl group, a tosyl group, or a mono-or dinitro-aryl group); X is a methylene group or a sulfur atom; Y is a group shown by ( wherein R6 and R7 has the same meaning as described above); and n has the same meaning as described above, or a salt thereof.

4. A 4-substituted-2-azetidinone compound as claimed in claim 1, wherein in the formula (I), R1, R3 and R4 are a hydrogen atom; R2 is a group shown by (wherein R5 is a hydrogen or a lower alkyl group); n is 0; X is a methylene group, and Y is a group shown by ( wherein R6 and R7, which may be the same or different, each is a hydrogen atom, a lower alkyl group, a hydroxy lower alkyl group, or a cylo-alkyl group, or a salt thereof.

5 . N.alpha.- [(S) - 2-Azetidinone-4-carbonyl]-L-histidyl-L,-prolinamide.

6. N.alpha.-[(S)-2-Azetidinone-4-carbonyl]-L-histidyl-L-thiazolidine-4-carboxamide.

7 . N.alpha.[(S)-2-Azetidinone-4-carbonyl]-L-histidyl-N-(2-hydroxyethyl)-L-prolinamide.

8. N.alpha.-[(S)-2-Azetidinone-4-carbonyl]-NT-methyl-L-histidyl-L-prolinamide.

9. A process of producing a 4-substituted-2-azetidinone compound represented by general formula (I) ( I ) wherein R1, R3 and R4, which may be the same or different, each represents a hydrogen atom or a lower alkyl group; R2 represents an imidazolyl group shown by (wherein R5 represents a hydrogen atom, a lower alkyl group, an aromatic acyl group, or an aryl group); n represents 0, 1, 2 or 3;
X represents a methylene group, an ethylene group, an oxygen atom, or a sulfur atom; and Y represents a hydroxy group, a lower alkoxy group, an aralkoxy group or an unsubstituted or substituted amino group shown (wherein R6 and R7, which may be the same or different, each represents a hydrogen atom, a lower alkyl group, a hydroxy lower alkyl group, a lower alkoxy lower alkyl group, a cycloalkyl group, an aryl group, an amino lower alkyl group, or an acyloxy lower alkyl group; said R6 and R7 may be comine with each other to form, together with the nitrogen atom to which these groups are bonded, a 5-membered or 6-membered ring, which may contain an oxygen atom, a sulfur atom or a nitrogen atom) or a salt thereof, which comprises reacting a carbo-xylic acid represented by general formula (II) (II) wherein R1 and n have the same meaning as defined in foregoing general formula (I) or a reactive derivative thereof and an amine represented by general formula (VI) (VI) wherein R2, R3, R4, X, and Y have the same meaning as defined in general formula (I) ; when Y is a hydroxy group or?
or a reactive derivative thereof and, when the reaction product has a protective group, removing the protective group.

10. A process of producing a 4-substituted-2-azetidinone compound represented by general formula (I) R6 or R7 represents a hydroxy lower alkyl group or an amino lower alkyl group, these groups may have a protective group (I) wherein R1, R3 and R4, which may be the same or different, each represents a hydrogen atom or a lower alkyl group; R2 represents an imidazolyl group shown by (wherein R5 represents a hydrogen atom, a lower alkyl group, an aromatic acyl group, or an aryl group); n represents 0, 1, 2 or 3;
X represents a methylene group, an ethylene group, an oxygen atom, or a sulfur atom; and Y represents a hydroxy group, a lower alkoxy group, an aralkoxy group or an unsubstituted or substituted amino group shown by (wherein R6 and R7, which may be the same or different, each represents a hydrogen atom, a lower alkyl group, a hydroxy lower alkyl group, a lower alkoxy lower alkyl group, a cycloalkyl group, an aryl group, an amino lower alkyl group, or an acyloxy lower alkyl group; said R6 and R7 may combine with each other to form, together with the nitrogen atom to which these groups are bonded, a 5-membered or 6-membered ring, which may contain an oxygen atom, a sulfur atom or a nitrogen atom) or a salt thereof, which comprises reacting a carboxylic acid represented by general formula (IV) ( IV ) wherein R1, R2 and n have the same meaning as defined in general formula (I) or a reactive derivative thereof and a cyclic amine represented by general formula (V) (V) wherein R3, R4, X and Y have the same meaning as defined in general formula (I); when Y is a hydroxy group ?
or a reactive derivative thereof and, when the reaction product has a protective group, removing the protective group.

11. A medicament formulation containing a compound as claimed in any of claims 1, 2 or 3 or a salt thereof and a pharmaceutically acceptable carrier or excipient.

12. A medicament formulation containing a compound as claimed in any of claims 4, 5 or 6 or a salt thereof and a pharmaceutically acceptable carrier or excipient.

13. A medicament formulation containing a compound as claimed in any of claims 7, 8 or 9 or a salt thereof and a pharmaceutically acceptable carrier or excipient.