CA1259318A - 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-azetidinone compound shown by the general formula:

Description

3~

4-SUBSTITUT~D-~ E IDINO~E CO~IPOU~D, PROCESS OF
PRODUClN~ IE COMPOU.~DS, AND M,.DICAM~mS CONTAI2~iING
THE COMPOUNDS

DrTAILED EXPLANATIGN or THE IN~TrNTIOI\l This invention relates to a 4-substituted-2-azetidinone compound shown by following general formula (I) R1 ~ (CH2) CONH-fH-CON X (I) ~ H IH2 wherein Rl, R3, and R4, which may be the same or differen,, each represents a hydrogen atom or a lower alkyl group; ~ represents an imidazole group shown by R; ~- ~ or ~ (wherein R5 represents a hydrogen atom, a lower alkyl group, an aroma.ic acyl group, or an aryl g_oup); n _e?resents O, 1, 2, o- 3; X represents a methylene grou?, an ethylene gxou?, an oxygen atom, or sulfur atom; and Y represents a hydroxy yrouD, a an aralkoxy ~rou~, lower 21koxy group,/or an unsubs.l~uted or subs~itu~ed amlno sroup shown by the formula -N< 7 (wherel~ R6 and R7, ~hich may be the same or different, each represents a hydrogen atom, 2 lOweralxyl group, a hydroxy lower alkyl a lower alkoxy lower alKyl grou~, group,/a cycloalkyl group, an aryl g_oup, an amino iower alkvl group, or an acylo~y lowe- alkyl group;
said R6 2n5 ~7 may comblne wi.h eac~ o.he- -o fo~m a ;- or 6-membered cycllc group which mav contain an oxygen ~tom, a sul_ur atom, o- a n~tro~en atom together .

~i~h the nitrogen atom bonded 'hereto) and a salt thereof.
The invention also relates to a process of produc-ing a 4-substituted-2-azetidinone comDound shown by the foregoing general formula (I) or a sal' thereof, which comprises reacting a carboxylic acid represented bv general formula (II) Rl ~ ( 2)n (II) ~ ~iH

wherein Rl and n have the same meaning as in general formula (I) or a reactive derivative 'hereo^ znd an amine represented by general formula (VI) X2N-lH_CO~ , ~3 12 ~ (VI) CO-Y
wherein p~2, R3, ~<~, X, and Y ~.ave he same meaning 25 in general formula (I); when ~ in the foregoing expla-nation is a hydrox~v group o~ R6 or ~7 represenls a hydroxy lower alkyl group or an amino lower aiki~l group, these s-ou?s may have a p,3tec~~e group o~ a reactive derivative thereof and, when the reaction produc, has 2 protective group, removing the gro.lp.
Furthermore, tne inven.ion rela_es to a process o~
proàucing a 4-su~stituted~ zeti~-inone ^ompound shown by the fo-egoing generzl for~ul2 (-~) or a salt thereof, which comprises reacting a ca~boxylic acid reDresented ~y general formula (IV) 3~9 Rl--~ (C~2)nCONH-CH-COO~
o ~ NH CH2 (I'~j l2 wherein Rl, n, and R2 have the same meaning as described above or a reactive derivative thereof and an amine repre-sented by general formula (V) HN X
R3 (v) ¦ R~
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 hydroxv lower alkyl group c- an amino lower alkyl group in the fo_egoing de'ini'ion of v, these sroups may have a protective group or a reactive derivative thereof and, when the reaction product has a ?rotec. ve sroup, removing the protective group.
~ he lower alkyl group shown Dy Rl, R3, R4, R5, R6 a~d R7 in the foregoing gene~al fo_~ulae includes s.-aigh. chain o- 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 bu.yl sroup, a pentyl group, etc. When botn R3 and Rg are 2 lower alkyl gro!p, .hese lower alkyl groups can bond to a same carbon atom.
The arom2tic acyl group shown by R5 is an unsubstituted or substi'uted ber.zoyl o- benzenesulfonyl group and the substituent is z s_raisht chain or branched alkyl group havlng 1 .o 5, preferably 1 to 3 carbon atoms, such as a melhvl group, an ethyl group, a propyl group, an lsopropyl group, a butyl group, a pentyl group, etc.
The aryl group shown by R5 is an unsubstituted or substituted phenyl group. The su~stituent 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 str2ight chain or branched lower alkoxy groups having 1 to 5 ca~bon atoms, such as a methoxy group, an ethoxy group, a propoxy grGu~, 2n isopropoxy group, a butoxy sroup, a tert-butoxy group, a pentyloxy sroup, etc.
The aralkoxy group shown by Y includes phenyl lower alkoxy groups such 2s 2 benzyloxy group, a phenetyloxy group, a 3-phenylpropyloxy group, an c~3 ~-methylphenetyloxy group (-G-C~C~2 ~ ), etc.

The hydroxy lower alXyl grolp shown by R6 ana R7 includes lower alkyl groups having 1 to 5 carbcn atoms substituted by a hydroxy sroup, such as a 2-hydroxy-e.hyl grou?, a 2-hydroxypropyl group (-CH2CHCH3 ), H
a 4-hydroxybutyl group (-CH2CH~CH2C~20H), etc.
The iower alkoxy lower alXyl group shown by R6 and R7 includes the foregoing hyaroxy lower alkyl grou?s the hydrogen atom o. the hydroxy grou? of which is substituted by a lower alkyl group having 1 to 5 carbon at~ms.
The cycloalkyl group shown by R6 and R7 is cyclo-alkyl groups having 5 to 10 carbon atoms, which may be crosslin~ed, such as a cyclopentyl sroup, a cyclohexyl group, an adamantyl group, etc.
The aryl group shown by R6 and R7 includes aromatic hydrocarbon groups such as phenyl group, a naphthyl group, etc.
~ he amino lower alkyl group shown by R6 and R7 is straight chain or branched al~yl 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, 2 py-rolidinyl sroup, a piperi~inyl group, a 2-ketopiperidlno s-oup (-N ~ ), a 2-keto-1-pyrrolidinyl group (-~ ~ ), etc.
Also, the acyloxy lower alkyl sroup shown by R6 and R7 is straight chain o- branched zlkyl groups of 1 to 5 carbon a,oms having a lower acyloxy group, such zs an a~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 ~ing group, which may cont2in an oxygen atom, a sulfur atom or a ni.rogen atom, togethe~
wlth the nitrogen atom to which R6 and R7 are bonàed as described above 2nd examples of the 5- or 6-membe~ed ring sroup are a l-?yrrolidinyl grou? (-N ~ ), a .
: . -pipe-idino sroup (-N ~ ), an oxazolidine-3-yl group (-N D ), a thiazolidine-3-yl group (-N ~ ), a 2-pyrazolidinyl group (~N ~ ), a mo-pholino group (-Nr-~O), a thiomorpholino group (-N~_~S), a 1-pipera-inyl group (-N NH), etc.
The desired compound shown ~y general formula (I) of this in~ention has at least 3 asymmeteric carbon atoms and there are ster~isomers.
Thus ~ . the desired compound o. this invention includes each such separated isomer and a mixture of the isomers.
The desired compound of this invention shown by general for~ula (I) may form a sall with an acid or a base. The salt of the compound included in this i~ver,tion includes the salts the-eof with nontoxic acids (e. g., an inorganic acid salt such as a hyd~ochloride, a sulfate, etc., and an organic acid 521t such as2 citrate, an ace.ate, 2 t2~tar~e, etc.), and the salts thereof with non-toxic bases (e. g., the szl, with an inorganic base, such as 2 sodium salt, a potassium salt, etc., and the salt with an organic base, such 2S an ammonium sal', a t~imethylamine salt, etc.).
As a compound ha~ing r~lation to the desired compound of this in~ention shown Dy formula (I), the~e is known l-pyroglu,amyl-L-histidyl-L-prolinamide (pGlu-~is-P-o-NH2) c211ed as "Thyrot-opin Releasing Hormone" ( TRH ) .
been The existence of TRH has alreaày/known since the . .. ' '. ' :~' ' . - . ' ' .. . .. .
'' ' ' '' ' ' ` ''' "' `''" .' ' `

1960's but the structure thereof was confirmed in 1970 (~ndocrinology, 36, 1143(1970)). ~RH is said to be a hormone controlling the release of thyrotro2in (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 TS~ but TRH
widely acts to a central nervous system ~C~S), and a field of new investigations has 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 2S the decrease ~f the continuation ~ime 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 effect, induced catalepsy,/the lmprovemen. o~ anti-psychtic effect, an anti-depressive e~feot, etc., in addition to the TR~ releasing activity ~U. S. Pate~t Nos.

3,865,934 and 3,932,623). Furth~rmore, it has been discovered that TR-H is l7seful for improving o~
treating functional or organic disturbances in the brain, for example, a distur~ance of consciousness caused by head injury, brain surgery, cerebro-vascular disorders, brain tumors, etc., in pa~ticular, a~ acute or semiacute disturbance o~ consciousness (Belgian Patent No. 839,&33).
The development of TRH derivatives showing a weaker TSH releasing activity than TRH or almost no '''- ' '' ' '''.' '' " ' ' ','', ' ' ~ ,' . ' ',, ' TSH releasing activity and having actions to CNS same as or higher than the foregoing actions of TRH has been demandea. ~hus, various TRH derivatives were synthe-sized for the foregoing purpose and the ac~ions to CNS
have been further enlarged. As the compou~ds synthe-sized for the purpose, there are known a TRH derivative which has a weaker TSH releasing activit~- than TRH, has a narcoticantagonizing action, an a-tion 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 Pa~kinson's disease (Japanese PatPnt Publication (unexamined) NoO 116,465/'7/) and a TRH
derivative which has ac~ion of improving and trea,ing the disturbance of consciousness afte- an e~ternal injury of .he head and an action of decre2sing the continuation tlme Oc sleep by hexoba~bital, anc is said to be use ul for the treatment for 2 ?atient h~ving a disturbance of consciousness caused by the organic or functional distu-bances in the brzin, the treatment for a patient showing s~nility or me~tPl fatigue, and the treatment for depression state (Japanese Patent Publica.ion (unexamined) ~o. 59,71~/'81).
The compound of this invention h2s the structural feature in the point that _he pyrGglutamyl (pGlu) structu al r.oie~y cf TRH is conver.ed into an a2etidinone structure (~-lactum struct~re) which has never been employed. As to the me~icinal action, the ' ' '.'' - . ' - , ' , . . .

compound of this invention has a more remarkably s,ron~ C~S actions .han TRH and conven.ionally known IRH derivatives and hence is very useful as medicaments.
For example, the compound of 'his invention is useful for im2roving 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 o~ this invention shown by general formula (I) can be orally or parenterally administered 2S it is or as a mix~ure with proper phamacologically allowable ca-rier, excipient, diluent, etc., in the fo-m of powders, granules, tablets, capsules, injec_ions (intr~venous, subcutaneous, cr intramuscular injections), or supposito-ies. The dose of the com?ound cf this invention shown by formula (I) differs 2-coId~ng to the k~d of the compound of formula (I), the age, weight, and symptom of a patient, the manner OL administration, etc., but is abou. 0.001 to lC ms, preferably 0.01 to C.l mg (one dose) in the case o, injection and 0.05 to 500 mg, preferably 0.1 to 10 mg (one dose) in the case of oral administratior..
The Iollowins experiments show the action to a low body .emperature by pen~_obarbitol (Exper~ment 1), the action to the disturbance o~ consciousness by a head injury (Experiment 2), and the action to acute toxicity (~xperime~t 3) about typical compounds in the compounds of thfs inven_ion shown by formula (I).

~xperimen~ 1.
Pentoba~bit ~-induced hypothermia:
Nine male mice weighing 18 to 22 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 the test compounds.
~ffects of test compounds were eva,uated as ~Dl 5C~
the dose required to reduce by 1.5C pentobarbital-hypothermia o' control group of mice which received only pentobarbital and saline. The results are shown in Ta~le 1.
Experime~t 2.
Distu-bance of consciousness inauced by concussive head injury:
Nine male mice weishing 18 to 22 g were used for each dosage of the test compounds. P.n ac-ylate weight containing lead ( 0.~ 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 remaineà motionless for some period. The time from the shock up to the onset of spont2neous movement W2S recorded as the spontaneous movement time. Test compounds were administered intravenously 10 min.
~efore adding concussive head inju-y and e fects of test compounds were evalua_ed as D;o%, the dose reauired to shorten by 50% the spontaneous movement time of control group. The ~esults are snown in Table 1.

~, .. . . . . . .. .. . . . . ..

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

N~-[(S)-2-azetidinone~4-carbony'J-T-histidyl-L-prolinamide (Examr~le 1) 0.01 0.1 I~-[(S)-2-azetidinone-4-carbonyl]-L-histidyl-L-thiazolidine-4-carboxamide (Example 6) 0-05 Nd-[(S)-2-azetidinone-4-carbonyl]-L-N-(2-hydroxyethyl)-L-prolinamide (Example 4) 0.35 N~-[(S)-2-azetidinone-4-carbonyl]-N~-methyl-L-histidyl-L-prolinamide (Example 19) 0.004 TRH 0.1 2.~

(*): (A) Reversal effect against pentobarbital-hypothermi 2 ~Dl 5C (mg/kg i.v.) (B) Onset time o~ spontaneous movement ED50% (ms/kg i.v. ) ~xperiment 3.
Acute toxlci~y:
An agueous physiologiczl saline solution of 1493 mg/kg of a test compound, ~K-[(S)-2-azetidinone-4-carbonyl]-L-histidyl-L-prol namide W25 intravenously administered to one group of nine m21e mice and they were observed for 24 hours but no example of death was observed. That is, ~D50 (i.v.) of the compound of this invention was higher than 1~93 mg/kg.
On the other hand, in the case of administering TRH to mice, LD50(i. v.) was 751 mg/~s(i.v.).
The the p~oduction proress o, .his invention will be explained in more detail.
The reaction courses fo- the production processes ', .. :.

~2593~L~

of this inventlon 2re shown by the following schemes:

R ~ (CH2)nCOOH ~2 ~R4 ~OY
tII) ~III) (V) I ~ X
R1 ~ 2)nCONHCHCOOH H2NCH~ON ~ R3 (IV) (VI) .
_ ~
~ ~X
R~ CH2 ) nCONHCE~CON~R3 (Il ) The compound o, this inven.ion shown by above formula (Il) can be conve~ted into the compound of this invention shown by following formula ~I2) by the hydrolysis or ',he ca~alytic reductic~ when Y is an alkoxy group or an a-alkoxy group or by the reaction with an unsubstituted or substituted amine when Y is a hydroxy group. ~lso, when the substituent R~ in the imidazolyl group shown by R2 is an aromatic acyl group or an aryl group, the compound of formula (I2) wherein RS is hydrogen atom is obtained by removing the substituent by zn ordinary manner.

R.l~( CH2 ) nCONHCHCON~

(I2 ) In tne schemes, Rl, R2, R3, R4, n, X and Y have the same meaning as descri3ed abo~Te; R2 represents a case that the substituent R5 in the imidazolyl group shown by R2 is a hydrogen atom; and Y' represents a hydroxy group or an unsubstituted or subs_ituted amino group.
That is, according to the process of thls 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 lIV) and then reacting the compound of formula (IV) and the compound of fo-mula (V) or (b) by reacting the compound of fo_mula (III) and compo~nd o. formula (~r ) 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 deslred compound o~ formula (Il) can De induced into the other desi~ed compound of formula (I2) by converting the subs.ituent v.
The production rea_tlon for t~e compound of fo~mula (I) employed in t~e foregoing prccess (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 dicyclohexyl carbodiimide as a condensing agent, an a~ide methoa, an acid chloride method, an acid annyd-iae method, an active ester method, etc. ~hese methods are per~o-med as follows:
Tha is, prior to the perform2nce of the peptide .
.

.
' ',: .: : ' - .. ' - ' . . - - ....
. .

.. . . .

3~
1~
forming reaction in each step, the functional gro~lps 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 g oup, an imino group, or a carboxy group of the compound, which takes part in the reactio~, is, if necessary, activated. The compound of which the amino group, the imino group or the carbo~y group is activated, for example, the active ester may be subjected to the peptide synthesis reaction after once isolated from the reaction mix~ure or may be subjected to the peptide synthesis reaction without being isolated.
Examples of the protec'ive group for the amino sroup 2re a benzyloxyca-bonyl g oup, a t-butyloxyca-bo-p-methoxybenz~-lc)xyc2Tt)c)nyl ~DUp, nyl group,/a phthaloyl sroup, a trifluoroacetyl group, etc., and examples OI the protective group for the imino group are a tosyl g-ou?, a benzyloxycarbonyl group, a p-methoxy`oenzyloxycarbonyl group, a benzyl group, a 2,4-dinitrophenyl yroup, etc. Also, the protec-.ive group fo- the carboxy group-is used as the form o' an ester such as a methyl 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 trichloride, an isocv~nate p-ocess using phosgene, or a phosphorous acid ester process when the group is an amino sroup o- an imino group or is ,.' , :, ' ~ ' ,' ' '' ' '' '' " ' ' '' .

1~
performed in the form of an active ester ~e. g., 2,4-dinitrophenol ester, N-hydroxysucc nimide ester, etc.,), an azide group, or a carboxylic anhydride when the group is a carboxy group.
Among the foregoing methods Or pe-forming the peptide synthesis reaction, it is prefered to perform Teactions the coupling/ of the compound of formula (IV) and the compound of formula (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 temperature o~ by hea~ing by an ordinary manner. Examples o~ the suitable solvent used in the reaction are dimethylfo-mamide (DMF), ethyl acetate, dichloromethane (methvlene chlo_ide), r e~rahvd~ofur2n, etc.
If it is necessary to remove 2 ?rotecti~e grou?
from the reaction pr~duct, the protective group cah be r2moved by a catalytic reduction when the protective group is benzyi ester; by using anhvdrous hvdrogen N-hydroxy- 1, 2, 3, -ber.zotTia z ole fluoride ~.~OBT),or a hydrogen fluo_ide-pyridine complex when the protective group is p-toluenesulfonyl group; by hydrolysis when the protective group is an alkyl este-; by a catalytic -eductio~ o~ a hydrobromic acid-acetic acid treatment wnen the ?rotective group is p-methoxybenzyloxycarbonyl; or an acid decomposition when the protective group is a t-butyloxyczrbonyl group.

, .

Furthermore, in the reaction of inducing the desired compcund of fo-mula ~I) into other desired compound by converting the substituent 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.
~ urthermore, the invention relates to a novel

4-s~bstituted-2-azetidinone compound shown by general formula (VII) o~ CONH-fH-COOR8 N CH2 (VII) ~ N
N

wherein R8 represents a hydrogen atom, a lower alkyl group or an aralkyl group or a salt .hereo , and a prDcess of the produc~ion the re o ,;
~ xamples of the lower alkyl yroup shown by R8 in the foregoing general formula (VII) are straight chain o~ branched alkyl groups having 1 to j, preferably 1 to 3 carbon atoms, such as a methyl group, an ethyl group, a propyl group, 2 butvl group, a pentyl group, an isopropyl group, etc., and examples of the aralkyl group shown by R8 are aryl iower alkyls such zs a ben7yl group, a phenetyl s-ou~, a naphthylme.hyl group, etc.
The no~el compound shown by general ormula (VII) is useful as an intermediate compound 'o. p-oducing the compound of this inven.ion shown by general Lo-mula (I).
The compound shown by general fo~mu'a (VII) can be produced by reacting a carboxvlic acid shown by the formula ~ COOH

O ~ H
or a reactive derivative thereof and an amine shown by the general formula CH2 tIX) ~ N

wherein R8 hzs the same meaning 2S desc-ibea ab~ve or 2 reactive de~ivative thereof. The reaction is a peptide s~nthesis reaction and can be pe formed by a ~own r.anner 2S described hereinbefo~e.
~ Still ,u-'her, the invention ~elates to a novel - 4-substituted-2-a7etidinone compound represented by the general L O~mU12 ~ COO-R9 O H
C1 Cl wherein R9 represents ~ C or -N

and the p~oduction process thereo .
The foregoing compouna of this inven.ion is useful as an intermediate compound ~or producins the compound shown Dy formula (VII) and further is usful as an inter-mediate compound for producing ~-lactum series antibiotics.
~ he novel intermediate compound is produced by reactlng a carboxylic acid shown by the formula ~ COOH

or a reactive derivative thereof and pentachlorophenol or N-hydroxysuccinimide.
The reaction is an ester synthesis reaction and akn~wm esterification method can be suitably selected.
Then, the invention will further explained by the following examples but the examples do not limit the scope of this inven~ion.
In addition, the production processes for the raw materials commonly used in plural examples will ~i-st be explained as reference examples.
Blso, ~he a~revialions em~lo~ed in the examples ~nd the reference examples indicate the f~llowing meaning.
TLC Thin layer chromatogra~hy NMR Nuclear magnetic resonance spectrum IR Infrared aDsorption spectrum Mass Mass analaysis spectrum Z Benzyloxyca~~onyl Bn Benzyl His Histidine Pro Proline . " - . . . .
. ~,. .

DNP 2,4-DinitrOphenyl Ts Tosyl B~C t-Butyloxycarbonyl DMr Dimethylformamide HOBT N-Hydroxy-1,2,3-benzotriazole DCC Dicyclohexylcarbodiimide THF Tetrahydrofuran HOSu N-Hydroxysuccinimide Ph Phenyl In addition, the compound containing ,he mar~
Nim in the names of the compoun~s is a mixture o~ a ~ ~substituent nitrogen atom-substituted (~ ) compound of the imidazole ring of histidine and a~ nitrogen atom-substituted ( ~ ~ ) compound.
substituent Reference example 1.

9 ~ COOC~I2 ~ r ~COOH
~ H (1) H (2) In 350 ml of methanol was dissolved 3.46 g of (S)-4-benzyloxycarbonyl-2-azetidinone (1) and the azetidinone was hydroger,ated using 350 mg of '0%
palladium-carbon as a catalyst. The catalyst was removed by filtration and the filt~ate was concent~ated to dryness to provide 1.9 g of (S)-2-azetidinone-4-carboxylic acid (2).

. ' ' ' ' . . .
' .'~.' ' ' ' ' . .. : .. . .
.

,3~3 N~ (DMSO-d6) Sppm 8.26 (s, lH), 4.02 (dd, lH, J_3.4 Hz, 6.9 Hz), 3.21 (dd, lH, J=6.9 Hz, 16.0Hz), 2.82 (dd, lH, J=3.4 Hz, 16.0 Hz).
IR (KBr) cm~l: 3320, 1740, 1720 Mass: 116 (M ~ 1) Reference example 2.

Pro-OBn(5) Z-His-NHNH2 ~ [Z-His-N33 ~ Z-Hls-Pro-OBn (3) ~4) (6) In 99 ml of an agueous solution of lN hydrochloric acid wzs 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 wzs added to the mixture with s,irring v~gorously under ice-coo'ing. P.fter perform-i~g the reaction at 0C for ~ minutes, 39.6 ml of an aqueous 50% potassium carbon2te solutlon was added to the reaction mixture unâer ice-cooling to alkhlify the sol~.ion. The reaction mixt~re was placed in a separation funnel and an oTganic layer thus forme~ was collected. ~urthermore, the acueous layer was extracted ~y 20 ml of ethyl acetate and the extract was combined with the foregoing organic layer. The mixture was dried ove~anhydrous sodium sulfate under ice-cooling for 10 minutes. By removinq anhvdrous sodium sulfate by filtration, 1;2 ml o' an ethyl acetate solution of Na-benzyloxycarbonyl-~-histidine a_i~ (4) was obta ned.

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

5.70~ g of solution of~ -proline ben7yl es.er ~) was added thereto.

. . ' .' . . ' ,' ..... , ' ~ - ' ,'', " .
.

The mixture was reacted ove-night at 0C and then the reaction mixture W2S 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 Na-benzyloxycarbonyl~L-his~idyl-L-proline benzyl ester

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

7.10 ~s, 5H), 6.82 (lH), 5.70 (d, 1~, 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) ~ass: 476 (M+), 396, 325, 244, 91, 70.
Ref erence ex2mple 3 .

(6) ~ His-Pro-O~n.2~Br (7) Aftel ice-cooling 20 ~1 of an acetic acid solu~ion of 25~ hydrobro~ic acid, 1.91 g of compou~d (6) w2s added to the solution followed ~y reaction for 2 hours at s_loC. The reaction mixture W2S added to 200 ml of dry ether znd after quickly removing .he precipitates thus formed by filtration and then the reaction mixture ~-as dried overnight in ~ desiccatoT
with potassium hydTo~ide, to provide 1.99 g o_ L-his.idyl-L-proline benzyl ester.2-hydrobromide (7).
NMR (CDC13 ~ CD30D) ppm 8-75 (_ ), 7,35 (s, 5~), 5.2 (lH).

.
. ~', . ' ' ' ' ' ' ' ~ ' ..
~' ' ' ' , - ' ' ~ - , Example 1

(8) ~His-pro-~H2~ ~ C~-His-Pro-NH2

(9) NH (10) In 13 ml of ~ DMF wzs dissolYed 826 mg of L-hi,tidy1-L-pTolinamide 2-hydrobrmide (8) and then 2 ml of a DMF solution of 40~ mg of triethyl-amine ~as added to the solution under ice-cooling.
After perfcrming 30 minutes under ice-cooling, the precipitates thus formed were filtered off to provide L-histidyl-~-prolinamide (9). The product was immediately used for the subsequent synthesis reaction.
In 10 ml of DMF was dissolved 230 mg o. compound (2) and then 351 mg of - HOBT and ~53 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 ~t room tem~erature. ~he reaction mixture wzs ice-cooled again and 1~ ml of a DM~ solu.ion of foregoing compoun~ (9) was adaed to the reaction mixture followed by reaction overnight at 0C. The precipitates thus formed were filtered off, the filtrate was concentrated to dryness, the residue was dissolved in 10 ml of chloroform-methanol (4 : 1) znd subjected to silica gel column chromatog-aphy. The eluztes by chloroform-methanol (7 : 3) were collected and concentrated to dryness .o provide 509 mg o crude N~-~(S)-2-azetidinone-4-carbonyl]-L-histidyl-~-prolinami~e (10).

. . . - ,: . . - -.
', .
.- ,. . . . -.

I~Aen the product was subjected to silica gel column chromatography again and eluted by a miYture of chloroform, methanol, and aqueous ammonia (40 : 10 : 1) to provide 394 mg of pure Drodu~t (lo!.
N~IR (CD30D) Gppm 7._9 (s, lH), 6,98 (s, lH), 4.41 (dd, 1~), 4.11 (~d, 1~, J=3.1 Hz, 5.9 Hz), 3.36-3.96 lm, 2H), 3.0~ (dd, 1~, J=5.9 Hz, 1~.9 ~z), 2.80 (dd, lH, J=3.1 Hz, 14.9 Hz), 1.72-2.20 ~m, ~H) Mass: 348 (M ), 234, 207, 154, 82, 70 [~']23 = -75.8 (C = 0.6, methanol) [~24 = -100.4 (C = 1, water) Wnen the compound (10) was triturated with a small amount of methanol, the compound crvstallized.
M. p. 183-185C
Elemen,al analysis for C15H20~6 4 ~ ~2 C(%) H(~) N(~) Calculated: 50.41 5.92 23.52 Found: 50.35 6.00 23.64 When the compound (10) W2S recrystallized from methanol, the product having a different crystal form ~,7as obtained according to the crys-211ization conditions. ~or example, products having melting points of 145-149C, 154-157C, 154-163C, 181.5-183.5C, 187-18gC, etc., were obtained and they were confirmed to be pol~orphous crystzls by infrared absorption s?ectra (XBr tablet), po~der X-ray di_ -action, di_ferential scanning caloTimetry, etc. ~he difference ~ melting poin. was by the miY.ing ra,io of .. , , . . : .
' ' ' ' - .' .: " `".,- ,'-'. .' -: , , ~ ' ': . '., . ,' ' -different crystal forms. The properties (NMR, optical rotation, etc.) of the solution states of these products showed same properties.
Example 2 (7)----~[His-Pro-OBn]____~~ CO-His-Pro-OCH
(11) NH (12) In 45 ml of dichlorometnane was dissolved 1.99 g of L-histidyl-L-proline benzylesler.2-hydrobromide (7) and after cooling the solution to -2DC, 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 o f to provide a solution cont2ining L-histiayl-L-proline ben~yl ester ~11). The product was immediately used in the subseauent synthesis reaction.
In 30 ml of dichloromethane was sus~ended ~55 mg of co~pound (2) and after adding thereto 801 mg of HOBT and 1.0~9 g of DCC and stirring the mixture ~or 1~ mi~u.es, the reaction W2S performed for 15 m~nutes at room temperature. The reaction mixture wzs ice-cooled again ar.d then 50 ml of a dichloromethane solution of foregoing compound (11) was added to the reaction mixture. ~fter reacting the mixture for one hour under ice-cooling, the reaction W25 performed overnight at room temperature. Precipitates thus formed wa_ iltered of I and the ~iltrate wzs concentrated to dryness. The residue thus obtained . ' ' . . , .. ' ' . . ' .,, . .:
- ' - . ~ . . . ..
.. . . .. . ..

.

~2S!93~lB

was dissolved in 20 ml of water-methanol (4 : 1) and subjected to HP-20 column chromatography. When the eluate by water-methanol (1 : ~) was ^oncentrated to dryness, 1.135 g of crude N~-[(S)-2-~zetidinone-4-carbonyl]-L-histidyl-L-proline benzyl ester (12) was obtained. When the prodllct W2S subjected to silica gel column chromatography again and eluted with chloroform-methanol-aqueous ammonia (40 : lC : 1), 827 mg of the pure product (12) was obtained.
NMR (CD30D) ~ppm: 7.58 (lH), 7-34 (s, 5H), 6.88 ~1~), 5.1~ (s, 2H), 4.50 (dd, lH), 4.08 (dd, lH, J=3., Hz, 5.9 Hz), 3,4Q-3,92 (m, 2H), 3.04 (dd, lH, J=5.9 Hz, 14~9 Hz), 2,76 (dd, lH, J=3.3 Y.z, 14.9 ~z), 1.6~-2.20 (m, 4~) -Mass: 439 (M ), 325 ~xample 3 (12) ~ CO-His-~ro-OP

N~ (13) In 150 ml of methanol was dissolved 782 ms of N~-~(S)-2-azetidinone-4-carbonyl]-L-histiayl-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 catalvst. When the catalyst W2S
filtered of 2nd ,he filtrate W2S concentrated, 620 mo o ~-[(S)-2-~ eti~none-~-c2rbQn.yl~-L-histidyl-L-proline (13) was obtained.
NMR (CD30D) ~-pm: 8.44 (lH), 7.28 (1~ .9~ (lP.) ~93~
~7 4.44 (dd, lH), 4.14 (dd, lH, J=3.1 Hz, 5.9 Hz), 3.3-4.0 (mt 2H), 3.25 (dd, lH, 5.9 ~z, 14.9 Hz), 2.86 (dd, lH, J=3.1 Hz, 14.9 Hz), 1.7-2.4 (m, 4H) Mass: (diazomethane treatment, as dimethy compound): 377 (M ), 263, 221, 96, 70 Example 4 H
(13) ~ n~ C~-Hi S -3ro-NHCH2CH20H
o~l NH (14) In 8 ml of DMF was dissolved 277 mg of compound (13) and then 168 mg of HOET and 330 mg of DCC were adàed to the solution under ice-cool_ng. A~ter performing a reaction for one our under ice-cooling, the reaction W2S f~-ther pe-formed for 2 hours at room temperature. After ice-cooling again the reaction mixture, 2 ml of a D~ solution of 80 mg of monoethanol-zmine to ~erform the reaction for one hour and there-after, the-reaction W2S fur~,her performed ove~night at room temperature. The precipitates were filtered ol~-, the filtrate W25 concentrated to dryness, and the residue thus formed was dissolved in lo ml of chloroform-methanol-aqueous ammonia (40 : 10 : 1) and subjected to silica gel column chromatogr2phy. Then, the product W25 eluted with the same solvent 2s above to provide 106 mg of N~-[(S)-2-Gzetidinone-4-carbonyl~-L-histidyl-N-(2-hydroxyethyl)-L-prolin2mide (14).
NMR (CD30D) ~ppm: 7-74 (lH), 7.00 (1~), 4.41 (lH, d,d), .12 (d,d, J=2.9 Hz, ;.; ~.z), 3.08 (2H, t, ' ' .

.

=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.) [~24 = -87.3(C=0.13, methanol) IR (KB.) c~ 3280, 3180, 2950, 1760, 1650, 1635, 1550 Elemental analysis for C17H2~N6O5:

C(%) ~(%) N() Calculated: 52.036.16 21. 2 Found: 51.906.16 21.23 Exzmple 5 ~ ~~ 1 (2) ~CO-His(DNP)-~ S
His(DNP)-N ~ 2HCl _~Hl (DNP)-N ~ J~ F \rJ

In 30 ml of dichloromethane was dissolved 876 mg of 3_[Nim-(2,4-dinitro~henyl)-~-histidyl]-L-thi2zoli-dine-4-carbo~amide 2-hydrochlorïde (15) and after adding thereto 2 ml of a dichloromethane solution of 388 mg of triethylamine under ice-cooling, the -eaction was perf~rmed fcr 30 minutes at 0C to provide a solution o~' .
/3-lN~m-(2,4-dinitrophenyl)-L-histidy~-L-thiazo~idine-4-carboxaioide ( 16 ) .
In 6 ml of DMF W2S aissolved 223 mg of compound (2) and after adding thereto 389 mg of HOBT and 517 mg OT DCC under ice-cooling, the reaction was performed .,- . . . , . - .
. . . . . .
.. . .. . . .

. .

~2593~3 for 30 minutes a. 0C and then for~30 minutes al room temperature. The reaction miY.ture ~7as ice-cooled again and 32 ml of the dichloromethane solution of the compound (16) described above was added to the fore-going reaction mixture. The mixture was reacted 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-methanol-aqueous ammonia (40 : 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-a~etidinone-4-carbonyl~-Nim-(2,4-dinitrophenyl)-L-histidyl~-L-thia70lidine-4-car~o~amide (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.~3 (d, 2~, J=9.; ~z), 4.14 (dd, lH, J=3.1 ~z, i.9 Hz), 2.86 (dd, lH, J=3.1 Hz, 14.7 Hz) Mass: 419 (M -C4HsN2O2), 372, 248, ~1 xample 6 H ~^`S
(17) - - ~ n. Co-His-N ~

In 15 ml of DMF was dissolved 337 mg of compound (17) and after addi~g thereto 2 ml of mercaptoe'~anol, the reaction W2S pe_formed for 30 minu.es at room temp~rature. The reaction mixture was concen.rated to dryness and the residue was dissolved in 20 ml of ~3~

chlorofc-m-methanol-aqueous ammonia (30 : 10 : 1) and subjected to silica gel column chromatography. Then, the product was eluted with the same solvent as described above to provide 214 mg of ~-[NX-[(S)-2-azetidinone-4-carbonyl]-L-histidyl~-L-thia201idine-4-carboxamide (18).
NMR (CD30D) ~ppm: 7-55 (lH), 6.91 (lH), 4.32 (d, 2H, J=9.5 H2), 4.07 (dd, lH, J=3 1 Hz, 5.9 ~z) Mzss: 367 (M + 1), 253, 206, 11 Reference examp~e 4.

Hi (Ts)-~ B3C-His~Ts)- ~ 0 l ?
CCOH COON~ COMHC~2CH20 (19) (20) ~

In 90 ml of dry methylene chlo-ide were dissolved 6.28 g of N~-t-butyioxycarbonyl-Ni~-tosyl-L-histidyl-DL-pi~ecolic acid (19) and 1.39 g of N-hvhToxvsuccinimide (~O~u) and the solution was cooled in an ice bath. Afte, adding thereto 2.74 g of DCC, the resultan. mixtur2 W25 stirred for 3 hours in an ice bath and afte- filtering off insoluble matters, the flltrate W2S csncentrated at reduced pressure. The residue thus formed was aissolved in ethyl acetate and the solution was washed successive~y with an aqueous sodium hydrGgencar~o~ale, water, znd aqueous sodium chloride solution. The orga?.ic layer thus formed W2S
collected and d-ied, and then the solvent W25 removed.
The syrupy product thus obtzined was t_i,urated with a 1 : 1 mixture of ether and pet-oleum ethe~. The . . . -.
, . ':, , -, . .

crystalline compound ~20~ 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 co~.pound (20) and then 5 ml of a THF solu,ion of 251 mg of ethanolamine was added to 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 was dissolved in chloroform and the solution was washed successively with an aqueous sodium hydrogencarbonate solution, water, and an aqueous sodium chloride solu-tion. The organic layer thus formed was collected and a-ter removing therefrom the solvent, the -esidue thus formed was triturated with etheI to provide 1.0~ g OL solid N~-t-butyloxyc2rDonyl-Nim-tosyl-~-histidyl-N-(2-hydroxyethyl)-DL-pipecolamide (21). The mothe-2nd liquor was conc~tra-ed./subjected to silica gel column chromatography. By elu.ing with '% me,hanol-chloroform, ~urth O.66 g of compo~nd (2i) was / obtained.
TR (KBr) cm~l: 3360, 2920, 1680, 1640, 1170 NMR (CD30D) ~ppm: 7.28-8.4 (6H, imidazole ring hydrogen, benzene ring hydrogen), 2.4~ (3H, methyl of tosyl group), 1.0-i.5 (broad, BOC
hydrogen) Example 7 0 H-His(Ts)-~'~ (2).~ A
(21) > ~ ~NH
CONHCH2CH20H ~CONH~2CH20H
~22) (23) .. . , . -.. ""' ' . . .
' ' . ' ' , -.
' ' ' , ' ' ' ' ' ' ' . ''' ~` - ' .

~3~

In 40 ml of methylene chloride w25 dissolved 1.71 g of compound (21) and after aàding thereto 40 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 pressu-e, 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 2S it was. In 8 ml of DM~ was dissolved 1.3 g of the trifluoroacetate and then 274 mg of t-iethylamine was adde~ to the solution unde_ ice-cooling. Then the pH
of the mixture W2S adjusted to 7 to 8 with additional triethylamine while checking the p~ using a pH test paper.
In 2 mixture of 8 ml of methylene chloride and 1.5 ml of D~F were dissolved 28~ ~g c~ compound (2) and ~57 mg of DCC to perform the reaction 2nd to the reaction mixtu~e thus obtained was added the foregoing free amine solution undPr ice-cooling. The reaction mixture thus obtained was stirred overnight in a refriger~tor. Insoluble matters were 'iltered cf~ and the filtr2te was concentrated under reduced pressure.
The residue w2s dissolved in chloroform and the solution was washe~ th-ice each ,ime with water.
The organic layer thus ormed W2S collec.ed, dried, znd then the sol-~en. was removed to provide a syrupy m2terial, which was subjected to column chromatog~aphy . . .
,' ' . , ' ' . ~

of 140 ml of silica gel. By eluting with a mixture of 7~ methanol - chloroform, 537 mg of the des~red ompound ~-[(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), 5.0B center (2H, ~-metnine hdyrogen of histidine, ~-methine hydrogen of pipecolic acid), 4.08 (1~, 4-position hdyrogen of azetidinone ring), 2.44 (3H, methyl cf tosyl group) Mass m/z: 559 (M-l), 472, 4G2, 388 Example 8 O
(23) - ~ Kis-Ct)NHC:H7CH.~O~
(2~) :
In 15 ml of dry me~hylene chloTide were dissolved 250 mg of compound (23) and 73 mg of HOBT
ar.d the reaction was performed for ~ hours at room temperature with stirring, whereby nsoluble matters precipitated. The solvent w2s removed f-om the reaction mixture and the residue thus formed was subjected tocolumn chromatography of 100 ml sil~ ca gel. By eluting with chloroform-methanol-2queous ammonia (~C : 20 : 2), 116 mg of the desired compound, ~-[~5)-2-azetidinone-4-carbonyl~-L-r,istidyl-N-(2-was obtained.
hydroxyethyl)-DL-pipecolamide (24) / The product was a mix.ure of diastereomers andshowed ,wo s?ots on TLC.
IR (The sample was measu_ed by KBr tablet a teT

lyop~.ilization) 5m~1 3250 ~NH, OH), 1750 (4-membered ring lactum), 1630 center(broad, amide) NMR (CD30D) ~ppm: 7.65 (lH, imidazolering hydro-gen), 6.92 (lH, imidazole hydrogen), 4.14 (lH, 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 Refe~ence example 5.

(19) ~ [(20)] - - ) BOC-His(Ts)-CONH
(25) ~ rom 2.8 g of the compound (20) prepared from compound (19) by a simil2r procedure t~ in Reference example 4 and 755 mg of l-aminoadamantane, N~-t-butyl-oxyca~onyl-Nim-tosyl-~-his~idyl-rl-(l-adamantyl)-DB-pipecolar,ide ~25) was obtaineQ at a quantitative yield N~R (CD3~D) ~pp~: 7.1-8~3 (6~, imidazole ring hydrogen, berzene ~ing hydrogen), 2.44 (3H, methyl of tosyl group) Mass- m/z: 653 (Ml), 502, 4~5, 419 Example 9 0 H-His(Ts)-N ~ ~ ~ His(Ts) N

CONH ~ (27) The BOC group was _emove~ from compound (25) by - . . . .
- ~ ' ::, - ., - . - . : . ' . - . . . ... . . . . .
. . .

asrmil~rmanner to in foregoing ~xample 7 and the tri-fluoroacetate of compound (26) thus obtained was converted into the free base using triethylamine as in Example 7. Then, 2.15 m ~ol the free amine and 290 mg of compound (2) were subjected to a coupling reaction by asimilar manner to in ~xample 7. AfteT
the re~ction 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 sol-Jent, the residue was su~jected to column chromatogra~hy of 220 ml of silica gel. By eluting the product with 3~ me.hanol-chlorofosm, 598 mg of the desired product, NK-[(S)-2-azetidinone-4-carbonyl]-Nim-tosyl-L-~ as obtained hi~tiàyl-N-(1-adamantyl)-DL-pipecolamide (27 ~as a powdeT .
NMR (CD30D) ~ pm: 7.2-8.24 (6~, imidazole Ting ~ing hydrogen, benzene/hydrogen), 4.04 cente~
4-position hydrogen of azetidinon~ ring), 2.44 (3~, methyl of tosyl group), 1.2-2.3 (21~, methylene hydrogen of piperidine ring, adaman'yl group hydrogen) IR (KB_) cm~l: 3280, 2900, 1760, 1640 center (broad) Mass m/z: o~0 (Ml), 472, 402, 360 . .
' ' .:

0 Exampie 10 o His~s)-N ~ ~ ~ His-N
- NH
~ ~3 ~OW~

~27) (28) In 10 ml of methylene chloride were dissolved 510 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 col~mn chromatography. By eluting the product with ..
chloroform-methanol-aqueous ammonia (80 : 20 : 2), two kinas of diastere.ome~s were separated. That is, 98 mg of /the compound having a ~llpolarity, N~-[2-azetidinone-4-czrbonyl]-~-histidyl-N-(l-adamzntyl)pipecolamide (28a) was first eluted on the silica gel column and then 128 mg of /a mixture of the compound (28a) and the stereoisome~
(28~) thereof W2S obtaine~. The-eafte~, .he compo~nd (28b) (~3 mg) W2S el~ted. The p-operties o~ the compounds (28a) and (28~) are as follows.
Compound (28a):
NMR (CD30D) ~ppm:7.66, 7.58, 6.92, 6.90 (imidazole ring C-H), 4.10 (lX, d,d, ~-position hydrogen of azetidinone ring), 2.04 cente_, 1.72 center (21H, methylene hydrogen of piperidlne ring, adamantyl group hydrogen), IR (K~r) cm~i: 3250, 1760, 1640 center (broad), 15~0, 1.40 . .: . ' . ' ' .- , .. : : .
- . . . .. . . . .

~L~3~

[K]26 = _74.90 (C = 1.1, methanol) ~ass m/z: 496 (M ), 382, 345, 318, 261 Compound t28b):
NMR (CD30D + ~MSO-d5) ~ppm: 7,64, 6.90 (imida~ole ring C-H), ~,10 center(lH, d,d, ~-position of azetidinone ring), 2.02 center, 1070 center (21H, methylene hydrogen of piperidine rins, adamantyl group hydrogen) IR (KBr) cm~l: 3200, 1750, 1530, 1440, 1640 [~26 = ~82.4 (C = 1.3, meth2nol) Mass m/z: 496 1~'), 345, 318, 261 Reference example 6.

Hz~-(CH2)3CH3 ~-P~o-OH ~ ~_PTO_NH_ ~CH2) ~CH3 (79) (30) In 50 ml of ~F was dissolved 4.99 g o, N-benzyl-oxycarDonyl-~-proline ana the~ 2.23 g OI '~rie'~nylamine was adaed to the solu.ion. Then, afteI slowly addiny thereto 2.39 g oS ethyl chloroformate under ice-cooling, the reac,ion W2S performed fo- one hour at 0C to S~C.
Then, 2.19 g of n-butylamine W25 slowly added to the reaction mixture under ice-cooling, the reaction was further performed or one hour at 0C to 5C. The solvent was removed rom the reaction mixture, the residue thus formed was dissolved in ethyl acetate, and the solution was washed successively with an aqueous solu,ion of 1~ hydrochloric acid, a saLu~ated , .. .. . . .
' .: , . . ., . ,: , . . ~ , . .

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-pyrrolidinecarboxamide (30). M. p. 88-90C.
NMR (CDC13) ~ppm: 7.36 (s, 5H), 5.17 (s, 2H), 4.33 (dd lH), 3.51 (t, 2H), 3,21 (dd, 2~), 1.65-2.40 (4H), 1.05-1.65 (4H), 0.70-1.05 (3H) IR (KBr) cm~l: 3280, 295G, 1715, 1640, 1540 ~ss ~EI)~ 304 (M+), 232, 204, 91, 70 Reference example 7.

2-P~o-NH-~CH2)~,CH3 .. ~ Pro-NTH-(CH2),,CH3 (30) (31) In 140 ml of methanol was àissol~ed 4.~1 g of c~mpound (30) and the compound W25 hydr~gen2ted using 426 my of 10~ palladi~m-carbon as a cat21 yst_ ~he catal~st was flltered off and the filtrate was concentrated .o provide 2.41 g of (S)-N-butyl-2-pyrroli-ainecarboxamide (31).
NM~ (CDC13) ~ppm: 7.3-B.0 (lH), 3.71 (~d, lH), 2.8-3.4 (~H), 1.1-2.4 (9H), 0~7-lol (3H) IR (neat) cm~l: 3280, 2950, 1645, 1520 -Reference example 8.

(4) Pro-l~TH(CH2) ~CH3 ~ Z-His-pr~-NH(cH2)3cH3 (31) (32) To 75 ml of an ethyl acetate solution of N~
benzyloxycarbonyl-L-histidine ~zide ( 4) prepared from 4.85 g of ~-benzyloxycarbonyl-L-histidin~ hydra~ide (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 re_rigerator to perform the reaction. The reaction mixture w2s concentrated and the res~due was subjected to silica gel column chromatography. By eluting the product with chloroform-metha~ol-asueous ammonia (g5 : 5 : 0.~
4.13 g OL N~-be~zyloxycarbonyl-L-histidyl-N-butyl-L-prolinamide was obtained.
NM~ (CDC13~ ~ppm: 7 51 (1~), 7.34 (s, 5~), 6 85 ~s, 1~), 5 76 (d, lH), 5_10 (s, 2~) ! 4_3-~.8 (2H), 2.8-3.7 (6H), 1.7-2.3 (~H), 1.1-1.7 (4~), 0.7-1.1 (3~) IR (KBr) cm~l: 3250, 2950, 1705, 1635, 1540 Mass (FI ): 441 (M+), 361, 341, 272, 244, 190, '36, 91, 70 Reference example 9.

5 p~o-~H(CH2)3CH3 ~ His-p~o-~H~cH2),~Crl (~2) (a~) .
"'- .. ' '- ' :'.. ' ' , .

~l;2S~9 3 r31l~3 To 20 ml o' an acetic acid solu~ion 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 overnigh, in a desiccatorcontaining potassium hydroxide under reduced pressure to provide 2.14 g of L-histidyl-N-but~tl-L-prolinamide 2-hydrobromide (33).
Example 11 (2~ H
~3~) >[His-Pro-NH-(CH2)3CH3] -~ ~ Co-His-pro-NH(cH2)~CH3 (34) (~5) In 10 ml of D~ w2S dissol~ed 938 mg of compound (~3) and a~ter cooling the solution to -40C, 415 mg of triethylamine was added to the solution. After reac.ing for o~e hour at -30C to -40C, precipitates thus formed we-e filtered off to pro~ide a DM~ solution oS ~-histidyl-N-bulyl-~-p~olinamide (34). The product was used for the subsequent synthesis reaction im~ediately after the formation thereof.
'n 5 ml of DMF W25 dissolved 230 mg of (S)-2-azetidinone-4-ca2boxylic acid (2) (prepared in Reference example 1) and after adding thereto 406 mg HOBT and 49~ mgof DCC under ice-cooling, the reaction was perCormed fo- one hou- at 0C to ;C The rs2c.ion mi~ture was cooled to -40C and the DMF solution of the foregoins compound (3~) w2s added to _he reaction , : .: . - . ~ . -,,.. ' . . , - . , ' , ' - ': ' ~ .. ..
. ' ' . , .

:~3:~

mixture. The reaction was perfromed for 30 minutes at -4~C and then was urther performed overnight in a refrigerator. ~recipitates thus formed were filtered off, the filtrate was concentrated, and the residue thus formed was subjected to silica gel colu~n 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-~-prolinamide (35) was ~btained.
NMR (D20, sodium 3-(trimethylsilyl)-1-propane-sulfonate) ~ppm: 7.76 (lH), 7.07 (lH), 4.96 (t, lH), 4.2-4.5 (2H), 2.6-g.0 (8H), 1.7-2.2 (4H), 1.1-1.7 (4H)~ 0.7-1.1 (3H) ~R (KBr) cm~l: 3240, 2950, 1755, 1630, 1540 Mass (EI): ~04 (M+), 305, 290, 235, 207, 165, 110, 70 [~]D = -81.8 (C = 0.50, MeOH) ~eference example 10.

Prc~-~ (~) ` Z~~~~ ~0 (~) (~7) To 30 ml of an ethyl acetate solution of compound (4) prepared from 1.52 q of compound (3) by the method shown in Reference example 2 was added 78~ mg of (S)-~-cyclohexyl-2-pyrrolidinecarboxamide (36) prep2red by a known method and .hen the reaction w~s perormed overn'ght ln a refriger2to-. The reaction mixture was .

concent~ated 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 o~ N~-benzyloxycar~onyl-L-histidyl-N-cyclohexyl-L-prolinamide t37) was obtained.
N~R (CDC13) ~ppm: 7 54 ~5, lR), 7.32 ~s, 5~), 6.88 (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): 457 (M ), 387, 360, 341, 272, 244, 136, 108, 70 Re erence example 11.

(37) (38) To 1~ ~.1 of ice-cooled acetic acid solution of 25% hydrobromic acid was added 1.14 g o~ compound (37) and the reaction was per~romed fo- 3 hou-s a~ room temperature. The reaction mixture thus o~tained was added to 150 ml of desicated ether, the precipitates thus formed we_e quickly collected by filtration and dried overnight in a desicator at reduced pressure to provide 1.53 g of L-histidyl-N-cyclohexyl-L-prolin-amide~2-hydrob-omide.

$~3~

~xample 12 (~) H
(38)-~[His-Pro-N~ O ~ ~ Co-His-pro-NH O

0~
(39) (40) In 10 ml of DMF was dissolved 991 mg of compound (38) 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 -4CC, precipitates thus formed were filtered off to provide a DMF solution of L-histidyl-N-cyclohexyl-L-prolin-amide (39).
Tn; ml of DMF was dissolved Z30 mg of compound (2) and after adding theret~ 406 mg of HOB~ and g95 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 foregoing D~ sol~'ion o~ ,he compo~nd (39) W25 added to the re2ction mixt~re, and then ~he reaction was pe-~ormed for 30 minutes at -40C a~d then ove-night in a refrigerato-. Precipitates thus formed were filtered off, the filtrate was concentrated, and the residue was subjected tD silica gel column chromatography.
By eluting the product with chlorofrom-methanol-aaueous ammonia (80 : 20 : 2), 30~ mg of NX-[(S)-2-azetidinone-4-c2rbonvl3-~-his.i~yl-N-cyclohexyl-L-prolinamide (40) W25 obtained.
( 30D) ~ppm: 7.64 (i~), 6.98 (lH), 4 2-4 5 .
.
-- . .

(lH~, 4.12 (dd, lH), 2.81 (dd, lH), 0.9-1.2 (14~) IR (KBr) cm~l: 3220, 2920, 1750, i620, 1540 Mass ~EI): 430 (M ), 316, 235, 180, 152, 99, 70 [~]D = -69.2C (C = 1.90, MeOH) Reference example 12 ~ .Ts0H NH2 ~02~COz~2~ > ~22cr~2~2~
(41) (47) In 1150 ml of ether was suspended 71 g (146.6 m mol) of D-aspartic acid dibenzyl ester p-toluene-sulfoante (41) and while stirring the suspension under ice-cooling (0C to 5C), 22.5 ~1 (146.6 x 1.1 m mol) of t-iethylamine was added dropwise tc the suspension. After stirring the mixture for 2 hours at 0C to 5C, 4~0 ml of wate- was added theretc at the same tempera'~re ana the m xture was ~ur'~her stirred for 30 minutes. The ether layer tnus formea W2S separated, the ayueous layer was ext~acted with 2~0 ml of eth~r, and the foregoing ether layer was combined with the ethe- extract. The mixture was washed with 400 ml of a saturated aqueous sodium sulfate solution and dried by anhydrous ~.agr.esium sulfate. Ether was distilled off under reduced pressure to provide 4; g of D-aspartic acid dibenzyl ester (42) 2S a colo-less oily product.

.', ' ', ' ' .. . .
' . ' :' ~ ' ' . . ' . . : - .
.. -: .- . . . . .. . . .

3~L~

Reference example 13.

2 2 ~ H C2CH2 CH202C ~H2 ) ~NH

(42) (43) In 4~5 ml of dry ether was dissolved 45 g (143.~ m mol) of D-asparti~ acid di~enzyl ester (42) and after cooling the solution to 0C under an argon atmosphere, 20 ml (143.8 m mol) of trie.hylamine was added dropwise tG the SO1ULiOn. Then, 15.6 g (1~3.9 m mo~) of trimethy~si~yl ch~oride was furthe~ added dropwise ,o the mixture at the same temperature as above and the resultant mixture was stirred for one hour. Precipitates thus formed were iltered of' unde~
an argon atmosphere and the riltrate was cooled to 0C ~o -5C and then 134.8 x 1 01 m mol of an ether solution of t-butyl magnesium chloride was added a-cpwise to the m~xi~re wi~h st~rring_ ~t~r ~urther s~riDg the mixture Sor 2 hou~s a~ ~C and ~hen ~or 3 hours at room temperature, the mixture was cooled to OC, 100 ml of 2N HCl (saturated with N~gCl) was added to the mixture and after sti ring the mixture Lor 30 minutes, 100 ml of a saturated aqueous ammonium chloride solution was added to the mixture. The ether layer thus formed was separated and the aqueous layer was extracted twice each time with 200 ml of ethyl acetate. The ether layer was combined with the ethyl acetzte extract, and after washing the mix.ure with 300 ml o_a saturated aqueous ammonium chlo_ide solution and .: . . . . . .
. . .
.. . . ..
- ~ ' . ' ' ' ' '' ' ' :' ' ~L~9~
drying with anhvd_ous magnesium sulfate, e_he~ and ethyl acetate were dis,illed off under reduced pressure. To the resiàue was added 10 ml of ethyl acetate to form crystals, which were collected by filtration to provide 13.7 q of (~)-4-benzyloxycarbonyl-2-azetidinone (43).
In addition, the mother li~uor was concent~ated and purified by silica gel column ^hromatosraphy (eluen': ethyl acetate-n-hexane (2 : 1)) to provide 5.1 g of the desired product. M. p. 136-138C
[~ D = '33 7 (C = 1, MeOH) NMR (DM5~-d6) ~ppm: 8.40 (lH, N~), 7.40 (5~, s, phenyl group), 5.2D (2H, s, methylene of benzyl group), 4.22 (lH, d,d, 4-position hydrogen), 3.27 (lH, d,d,d, 3-position hyàrogen), 2.89 (1~, d,d,d, 3-posi,ion hydrogen) IR (RBr) cm~l:3200, 1760, 1'25, 1280 Reference example 14.

E, COOC~ ~ E, COOH

O ~
(43) (44) In 250 ml of methanol was dissolved ~ g of compound (43) 2nd the compound was cataly.~cally reduced in the presence of 5G0 mg of pa'ladium-carbon zt normal temperature and normal pressure in a hdyrogen atmosphere. After filtering o~ the catalyst, methanol was distilled orf under reduced p~essure.

The resiàue thus formed was c-ys,allized from ether and the crystals thus formed were collected by 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 hyd-ogen),3.23 (lH, d,d, 3-position hydrogen), 2.85 (lH, d,d, 3-position hydrogen) IR (KBr) cm~l: 3310, 1735 (b-oad), 860 ~xample 13.
~,, Coo~
2~ ~ C
(44 ) ~ 1is-PrD-N~.2 (8)~ [}~s-ProNE~2] o (9) (45) In 13 ml of anhydrous D~IF was dissolved 826 mg (2 m mol) of ~-hist_dyl-L-prolin-amide 2-hydrobromide (8) followed by cooling to -lCC.
~o the solutior. W2S slowly added 4~4 mg (2 x ~ m mol~
of triethylamine an~ ~he mixt~re was stirred for 30 minutes at the same temperature. ~hen, triethyla~ine hy~ro~rcmide thus preci2itated w25 ~ iltered off unde~ an aryon atmospnere. The solution was added dropwise to an active ester solution prepared from 230 mg (2 m mol) of (R)-2-azetidinone-4-carbox-~lic acid (44) (obtained in Reference examples 12 to i4), 351 mg (2 x 1.3 m mol) G' ~03T, 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 '' . , " ' ' ' . - ' ' ' .
., , : . -. - . . . . . .

~5~13~

overnight in a re.-igerator Then, DMF was dis'illed off from the reaction mixture under reàuced ~ressure, to the residue thus formed was added 10 ml of methylene chloride-methanol-concentrated aqueous ammonia (80 :
20 : 2), and cryst~ls thus precipitated were filtered off. ~he filtrate was subjected to silica gel column chromatography and purified using ~ethylene chloride-methanol-concentrated aqueous ammonia (80 :
501~ent to 20 : 2) as the de~-eloping / provide 370 mgof N~-[(R)-2-azetid_none-4-ca-bonyl]-L-histidyl-L-prolinamide (45) as an amor~hous powder.
[~]D =-21.5 (C = 1, MeOH) NMR (D2O) ~ppm: 7 03 (lH, imidazole ring), 6.72 ~lH, imidazole ring), 4,c~ (lH~ m), 4.42 (lH, m)4,27 (1~, d,d, 4-position hydrogen o aze,idinone -ing), 3. 40-4.00 (2~, m), 3,32~1~, d,d, 3-position hydrogen o aze~idinone ring), 2,74 (lH, ~,a, 3-position hycrogen OI aZetldiIlOne ring), 2.00 (4X, m) IR (~B~) cm~l: 33~0, 31~0, 174~, 1660, 162~, Mass: 348 (M ), 304, 278, 234, 207, 190 ~xampl e 14 C-31s-Pr~03 ' -3is-~r~-NACH3 ~13~ (46~

.
- . - - ' ' ., : ~ . . - -..

In 2 ml of DMF were dissolved 300 ms of ~-[~S)-2-azetidinone-4-carbonylj-L-~is.idyl-1-proline (13) (obtained in Example 3), 116 mg of HOBT, and 177 mg of DC~ and after stirring for 7 hours a, 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 aryness under reduced pressure. The residue thus formed was purified by column chromatoyraphy using LiCh~opTep Si 60(size ~). By using chloroform-methanol-aqueous a~monia (40 : 10 : 1) as the eluent, ~-[(S)-2-azetidinone-4-car~onyl]-L-histidyl-~-methyl-was obtained.
~-prolinamiQe (46) / The p-oauct ~2S dissolved ln water and then lyophilized. The amount oI the pIoduct thus obtained was 149 mg.
~M~ (CD30D) Sppm 7.62 (lU, imidzzole ring), 6 94 (lH, imidazole ring), 4.8 (1~, methine group), 4.10 (lH, ~-position hydrogen of zzetidinore ring), 2.76 (3~, N-me~-hyl sroup), ~ 1,6-2.3 (4~, prollne ring hydrogen) IR (XBr) cm~l: 3250, 175C, 1630, 1540, 1440, ~ass m/z: 362 (M+), 304, 252, 248, 235, 207 Reference example 15.

BOC-Eis(Ts)-O~ ~ ~ro-Osn ~C1 (47) (48) ~OC-~is(Ts)-Pr~-OCH

(49) æ~

To 100 ml of desi~ated me~hylene chloride were added 10 g of N~-t-butyloxycarbonyl-Nim-tosyl-~-histidine (47) and 6.50 g of L-proline benzyl ester hydrochloride and the mixture W25 cooled in an ice bath. After addins thereto 2.72 g of triethylamine, 6.G5 g of DCC was furthe- added to the mixture and the resultant mixture was stirred for 30 minutes in an ice-bath and stirred overnight at room temperature.
Insoluble mazters 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 NX-t-~utvloxycarbonyl-Nim-tosyl-L~histidyl-~-proline benzyl ester (~9) was obtained.
NMR (CD30D) ~ppm: 8.14, 7.92, 7.84, 7.40, 7.30 (total llH, imidazol ring, benzene ring), 5.i2 (2H, q, benzyl group), 4.5 center (2X, 2 kin~s of methine groups), 2 36 (3H, methyl of tosyl group) t 1.30 ~9~/ t-~ut~l group) IR (K~r) cm~l~ 3400, 3280, 2970, 1740, 1700, 1640, sso ~ass m/z: ~96 (M+), 523, 480, 364, 290, 155, 91 Reference example 16.

BOC-His(Ts)-Pro-OC~2 ~ , BOC-His(Ts)-Pr~-OH
(49) (50) .
... . . . . .
.
.: . - .- . . . ; .
- . .. . :
.

33~

In i50 ml o~ methanol was dissolved 13.5 g of compound (99) and the compound was catalytically reduced for ~ hours in the presence of 10% palladium-carbon. The catalyst was filtered off and the filtrate was concentrated under reduced pressure. The residue thus for~ed was dissolved in ethyl acetate and extracted thrice each time with an aqueous sodium hydrogen carbonate solution. The extracts were combined with each ot~er and washed once with ethyl acetate. After acidifying the aqueous layer with 1 N hydrochloric acid, the desired compcl~nd was extracted with ethyl acetate. Thus, 2.0 g of N~-t-butyloxycarbonyl-Nim-tosyl-L-histicyl-L-proline (50) was obta'ned as a oamy product. Also, from the org~anic layer formed after ex.-2cting ,he produc, with an aqueous sodium nydrogencarbonate solution, 9.1 g of the sta-'ing material (49) was -ecovered.
~0) The properties of the desire~ compound/thus obtained a~e shown below.
N~ (CD30D) ~PPm 8.16, 7.96, 7.86, 7.46, 7.40, (total ~E, imi~a~ole ring hydrogen, benzene ring hydrogen), 4,4B center (2~,tw~ kinds of methine groups), 2,42 (3~, s, methyl of tosyl group), 1.32 (9H, t-butvl sroup) IR !KB-) cm~l: 3300, 3100, 2970, 2500-2600, 1710, Mass m/z: 388 (M-118), 3G8, 234 . . - , :
- . . . . . . . . .
, . ~ . . ... . - . . .. . .

Reference example 17.

BOC -Hi s ( Ts ) -Pr ~-OH ` BOC-Hi s -Pr o-N~CH2 C~12 OH

(50 ) (51 ) To 70 ml of methylene chloride were added 3.25 g of compound ~50), 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 si~red fo~ 2 hours in an ice-bath and then stirred overnight at room temperature.
Insoluble matters were filtered off and the filtrate w2S concentrated under reduced pressure. The residue was dissolved in 70 ml of methylene chloride and after further adding thereto 1.3 5 ol H03T, the mixture was stirred again for 20 hours at room te~.perature ~he solvent w2s distille~ OII îrom tne reaction mixture under reduced pressure and the resiàue thus formed was su~jected to silica gel co~ n chromatography. By eluting th~ product with chloroform-methanol-aqueous ammonia (40 : 10 : 1), 1.35 g of N~-t-butyloxyc2rbonyl-L-histidyl-N-(2-hydroxyethyl)-L-prolinamide (Sl)was obtained.
NMR (CD30~) ~ppm: 7.64 (1~, imidazole ring), 6.96 (lH, imidazole -ing), 4.4~ center (2H, L ~;10/
inds of methine groups), 2.0 cente~ (4H, porline ringj,l.40 (9H, t-butyl group) I~ (KBr) cm~l: 32;0, 2960, 1700, 1630 . .
., , , . - . .

Mass m/~: 395 (M+), 365, 322, 307, 278 Example 15 (2) BOC-His-Prc~-~HCX,,CX20H- ~His-Pr~-NHCH2C~.20H = ~
(51) 2CF COOH
(52) 3 ~ CO-Xis-Pro-NHCH2CP~2OH

0~- (14) In 25 ml of methylene chloride was dissolved 790 mg of compound (51) a~d then 20 ml of 'ri'l~oroacet'c acid was added àropwise to the solution at 0C to 5C.
After stirring the mixture for 2.5 hours in an ice-bath, the reaction mixture was concentrated to à-yness under reduced pressu-e. ~urthermore, the product was azeotropically Qlied several 'imes using toluene and he residue W25 trltu-2ted with d~y ethe- to of provide powde~ /~-his-tidvl~ 2-hyaroxyethyl)-L-prolinamide 2-trifluoroacetate with 2 quantitctive yield.
In a mixture of 7 ml o DMF an~ 8 ml of methylene chloride were dissol~ed 2_3 mg of compound ~2) and ~ DCC
350 mg of HOBT and after adding thereto ~30 mg/unaer ice-coolins, the mixture was stirred for 1.5 hours.
To the reaction mixture was added a reaction mix~ure obtained by neutralizing the foregoing compound (52) in a mixture of 4 ml of DM~ and 4 ml of methylene chlor~de with .4~ mg of t~iethyl~mine, and the resultant mixture was reactQd overnight with s,i-ring in a refrigerator. Insolu~le matters were fil,ered of~, the Cilt-ate wcs concent_~,ed, and the residue 3~
;3 was subjected to silica gel column ch-omatography.
By eluti~g with chloroform-methanol -aqueous ammonia (4Q : 40 : 1), 313 mg of the compcun~ (14) which was identical with hat obta~ned in Example 4 was obtained.
~eference example 18.

(4) ~H CH2CH20H ) L-His-Pro-NH-CH2CH20H
(53) (54) To an ethyl acetate solution o~ ~C-benzyloxy-carbonyl-L-histidir.azide (4) pre?ared from N -benzyl-(3)~
oxycarbonyl-L-histidine hydrazid~(6.07 g) by a known method was added 10 ml of a DMF solution of 2.31 g of (S)-N-(2-hydroxyethyl)-2-pyrroliQinec2_boxamide (53) unde~ i~e-cooling and they were rea~ed overnight in a refrlgerator. The reaction mixture was concentrated and the residue thus formed wzs subjected to silica gel column chromatography. By eluting the p_od-~ct with chloroform-methanol-aqueous ammonia ~gO :

10 : 1), 3.43 g of N~-benzyloxycarbonyl-L-his.idyl-N-(2-hydroxyethyl)-L-prolinamide (a`4) was ~btai~ed.
NMR (CDC13) ~ppm: 8.3-8.7 (lX), 7.;a (s, lH), 7.34 (s, aH), 6.87 (s, lH), 5.93 (d, 2H), 5.10 (s, 2H), 4.3-~.8 (2X), 2.8-3.8 (~H), 1.6-2.3 (4H) Mass (EI): 429 (M ), 341, 272, 244, 136, 108, 79 ' ' ,'' ','' ' ~ ' ' '. ,, ' .
., . . . , . -... . . . .. . .
.

Reference exam~le 1~.

Z-His-Pro-NH-CH2CH20H ~ ~s-Pro-NH-C~2cH2ococH3 2HBr (54) (55) To 37.5 ml of ice-cooled acetic acid solution of 25% hydrobromic acid was added 3.22 g of compound (54) and the reaction was perfo-med for 3 hours at room tempe-ature. The reaction mi~,ure was added to 37; ml Gf dry ether and the precipitates thus fo_med ~ere quickly collecte~ by fil.ra'ion and dried overnight in a desiccatoTcontaining potassium hydroxide to provide 4.43 g o' L-histidyl-N-(2-acetoxyethyl)-L-prolinamiae ~2-hydrobromide~

~ xam?le 16 a) ~.s-Pr~ -CE12C~20COC~3 2~ r ~ [H:Ls-Pr~-NH-CX2CX20COCX3]
(55) (i6) ~2) ~ C0- ;s-~ -C~2 ~ oCoC~3 0~ (57) In 35 ml of DMF was dissolved 4.43 g of compound ~55) and after cooling the solution to -40C, 1.82 g of triethyl2mine was added to the solution followed by pe-forming the reaction -o- one hou- at -30 to -40C.
Then, precipi=ates thus formed were -emoved to provide a DMF solution of L-histiayl-N-(2-actoxyethyl)-L-prolin amide (56). ~he ~roauc. w~s i.,l~edia.ely used for .',., ' , ' ' ' - ' ', ' ' '. ' . '"" -'-'. "- ' : . - . . ~ - . .

~al~

the subsequent reaction.
In 17.5 ml of DM~ was dissclved 863 mg of compound (~) and after adding thereto 1.52 g of HOBT and 1.86 g of DCC, the reaction was performed for 30 mlnutes under ice-cooling. The reaction mixture was cooled to -40C and after adding the~eto the DMF
solution of the foregoing compound (56), the reaction was performed for 30 minutes at -40 C and then perfor~ed overnlght in a refrigerator. Precipitates thus formed were filtered olf, the filtrate was concentrated, and the residue thus obtained was subjected t2 s_lica gel column chromatography. By eluting the product with chloroform-me.hanol-aqueous ammonia (~0 : 20 : 2), 1.60 g of N~-[(S)-2-azetidinone-~-carbonyl~-L-histidyl-3~-,-(2-acetoxyethyl)-~-prolin-amide.
NMR (D20) ~ppm: 7 74 (5, lH), 7.05 (lH), 4.93 (t, lH), 4 1~ (4H~, 2.5-3.9 (7E), 2.76 (dd, 1~), 2.7-3 2 (7E) IR (RBr) c~-l: 3230, 2950, 2860, 1755, 1730, 1630, 1510 Mass (~ 434 (Ml), 364, 320, 262, 23~, 154, 70, 43 [~]27 = -86.2 (C= 0.45, MeOH) b) R
~CO-E~is-Pro-NHCFi2C~20COCEl~

O (~7) R
~ CD-~is-Pro-N~Crl2CH~Dr3 0~
(1~) --: . . .
:. . : - ' . -In 12 ml of methanol was dissolved 56 mg of potassium carbonate and afte~ adding thereto 3~8 mg of compound (57) under ice-cooling, the reaction was pe-formed for 2 hours under ice-cooling. The reaction mixture thus obtained was s~bjected to silica gel column chromatography and by eluting the product with chlorofor~-methanol-aqueous ammonia (80 : 20 : 2), 294 mg ofNa-[(S)-2-azetidinone-4-car~onyl]-L-histidyl-N-(2-hydraxyethyl)-L-prolinamide was obtained. The physicochemical properties of the product were same as those of the compound (i4) obtained in Example 4.

~Xefere~ce example 20) ~xa~.ple 17-a CDOE ~ CO~ ~ C1 (2) (58) In 200 ml of DM~ were dissolved 10 g (8~58 m mol) of (S)-2-azetidinone- -carboxylic acid (2) and 29.4 g (8~70 m mol) (8.68 m mol) of pentachloropher.ol ~d then 17.93 g/of 3CC wzs added to the solution under oooli~g (~ to 5C).
After sti~-ins ,he mixture for ~ hours at room temperature, dicyclohexylurea thus precipitated was filtered off and the filtrate was concentrated under reauced pressure. The residue thus fc~med was dissolved in 200 ml of ethyl acetate by heating and then cocled. The c ystals thus precipiated were collected by filtration to provide 2~.6 ~ Of .he yellowish c_ys~als o (S) 4-pentachlo ophenoxy-.. . . . .
. . , .. . . . .

gL;2~3~
57carbonyl-2-azetldinone (58) h~ving a melt~ng point o 177 to 179C.
NMR ~9C MHz, d6-DMSO-D~O) dppm 3.23 (lH, q, azetidinone ring 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 Exam~le 17-b F~ C02H ~ C2-~

o (2) In 20 ml o DMF was dissolved 690 mg of (S)-2-azetidinone-4-carboxylic acid (~) and after adding therets 690 ~g Gf '~OS~ and 1 236 g o~ DCC under ice-cooling, the reaction was performed fo- 30 minutes under ice-cooling and then for 4 hours at room tempe_ature. Insoluble matters were filtered o~~ ~rom .he reaction mixtu~e and ~hen the solvent was distilled ofto pro~ide a li~ht-brown solid product. ~hen the product was recrystallized from dioxane-petroleum ether (5 : 1), 750 mg of (S)-4-(2,5-dioxopyrrolidine-l-yl)-oxyc~rbonyl-2-~7etldinone was obtained.
NMR (DMSO-d6, TMS) ~ppm: 8.70 (broa~, lH), 4.62 (dd, lH), 3.84 (s, 4H) IR (XBr) cm~l: 3320, 2920, 2840, 1810, 1780, 1750, '730, 1l20, '6;0, i620, 1570 Mass (CI in beam): 213 (M +1),185, 171, 116 .
.. . . . . .
. . . . .

~Z593~8 (Re,erence example 21) E~ample 17-c His-OCH3 2HCl ~NH

(5g) (60) Ir. 75 ml of DM~ was suspended 6.05 g (25 m mol) of L-histidine ~ethyl ester 2-hydro~ori~ (59) and after cooling the suspension to 0 to 5C, 5.05 ~ (50 m mol) of triethylamine was slowly added dropwise to the suspension. Thereafter, the mixture was stirred for 15 ~inutes 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 houl at the same tempe_ature, the mixture wzs allowed to sta~d ove night at room temperature. Triethylamine hydrochlo~ide thus precipitated was filtered off, the Siltrate wzs concentrated ~de~ reduced pressure. ~'qe residue thus ~ormea was mixed with ~0 ml of ethyl acetate and 30 ml of water followedby shakihg 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 com~ined with each other and water was distilled off under reduced ~ressure. The residue w2s mixed w~th acetonitrile and benzene and tne mixture w2s concen~ra.ed under reduced pressure. The residue wzs crystallized rom 30 ml o_ methanol and the crystals thus fo_med -~ere collected to ,: . . . :

~;93~8 ss provide 4.1 g of the colorless c-ystals of N~-[(S)-2-azetidinone-4-carbonyl]-L-histidine me~hyl este- (60) having a melting point of 142 to 147C.
NMR (90 MHz, d6-DMSO) dppm 2.94 ~2H, d, B-position methylene of his,idine group), 3.60 (3H, s, methyl grou?), 4.02 (lH, q, azetidi-none ring 4-position), 4.54 (lH, m, ~-posi,ion methine of histidine group), 6.72 (lH, s, imidazole ring), 7.56 (lH, s, imidazole ring), 8.20 (lH, s, NH), 8.55 (1~, d, azetidinone ring NH) IR (KBr) cm~l: 3250, 3100, 29S0, 1770, 1750, 1740, 1/20, 1650, 1550 Example 17-d (;8) ~ ~His-OCH
His-OCH2 ~ ` 2 - NH

t60-1) In 20 ml o~ chlorolorm W25 dissolved 1.178 y (2 m mol) of histiaine benzyl este- 2-p-toluenesul,onate and then 404 mg (2 m mol) of triethylamine was slowly added to the solution unde- cooling to 0C. ~o the solution was added 766 mg (2 m mol) of (S)-4-penta-chlorophenoxycarbonyl-2-2zetidinone/zs a powder and the mixture was stirred overnight at 0 to ;C. To the reaction mix~ure was added 30 ml OL chloroform and he desired product was extracted twice each time with 30 ml Oc water. Then, wa,e- W25 Gistilled O__ unde-: , : , . . .

reduced pressu,e and the res- due thus formed was azeotropically dehyd-ated with benzene-acetonitrile to provide a colorless sticky product. The product was subjected to silica gel column chromato-graphy using 50 ml Wako gel C-200 and eluti~n with ethyl acetate-methanol-concentrated aqueous mmonia (60 : 30 : 3) ~ 70 mg of N -[(S)-2-azetidinone-(60~
4-carbonyl]-L-histidine benzyl ester/having a melting point OI 196 to 199C as colorless crystals.
NMR (90 MHz, d6-DMSo) ppm: 2.58 (1~, m, azetidinone r~'ng 3-positio~), 2.96 (2~, d;
his.idine group ~-posi'ion methylene), 3.12 (1~, m, azetidinone ring 3-position), 4.00 (lH, m, azetidinone ring ~-position), 4,62 (1~, m, histiaine sroup ~-position methine), ~.10 (_~, s, benzyl posi_ion), 6.8C (lH, s, imid2zole ring), 7.36 (3~, S, benzene ring~, 7.~6 (1~, s, imidazole ring), 8.20 (lR, s, N~), ~.~8 (1~, d, N~) IR ~B-) cm~l: 3260, 2930, 2760, 17~0, 1650, 1540 ~xample 17-e His-OCH2- ~ ~His-OH
NH

(60-l) (61) In 20 ml o_ methanol was suspenaed 342 ms o compound (60-l)and the compound was catalytically 20 mg of reauced with .he adci'ion o /10% palladium-ca_bon a.

ambient temperature andordinzr~; p-essure. Afte- the had absorption of hydrogen/stopped, the catalyst was filtered off ~nd methanol was distilled off under educed pressure to provide 230 mg of N~-[(S)-2-(61) azetidinone-4-carbonyl]-L-histidine/hz~ing a melting polnt o, 213 to 215C (dec.) as colorless crystals.
NMR (90 ~Z, D2O) ~ppm: 2.80 (1~, q, azetidinone ring 3-position), 3.20 ~2H, m, histidine group ~-posltion), 3.38 (1~, q, azetidinone ring 3-position), 4.28 (1~, ~, azeti~none r1ng 4-position), 4.58 (1~, m, histidine ~-position methine), 7.30 (lH, s, imidazole ring), 8.60 (lH, s, i~idazole -ing) IR (KB-) cm~l: 3400, 3260, 2560, 1750, 1630, 570, 1390 ~xample 17-A
O O

~is-OC~3 ~ is-O~ ~ Pr~ (62) (60) (61-1) C
~ ` \Eis-Pr~ -~H
d (10) After cooling 20 ml of an aqueous solution of 0.1 N sodium hydro~ide to 0 to 5C, 532 m~ (2 m mol) of compound (60) was adaed thereto and the mixture was s.irred for ~.S hours a' the same temperature. Then, 760 mg ( m mol) of p-Loluenesulronic acid monohydrate was added to the mixture at the same tempe_2ture and water was distilled of^ un~e~ reauced pressure. The residue thus ob'ained was azeo~ropically dehydrated with acetonitrile and benzene and ~he~ dried unde~ reduced pressuTe. The powder obtained was dissolved in 20 ml of DMF and after addin~ ,hereto 228 mg (2 m mol~ of T-prolinamide (62) and 412 mg (2 m mol) of DCC, the mixture was stlrred overnight at room te~perature.
Dicycl~he~ylurea thus precipitated was filtered off, the filtrate ~as concentrated under reduced pressure, and the residue thus formed was dissolved i~ 20 ml o, water.
After filtering o_f insoluble matters, water was distilled off under reduced pressure. After d-ying the residue thus formed under reduced perssure, the residue W25 dissol-~ed in 7 ml of methanol by heating and af~er stirring the solution unde~ cooling, crystals thus ?recipitated were collected by S~ltration ~o p ovide 500 mg of the colorless crystals of N~-[(S)-2-azetidinone-~-carbonyl~-L-histidyl-L-prolina~ide ha~ing a melting point Or 179 ~0 l~gC. The physico-chemical prope~ties of the product were same zs those of the compound ~10) obtained in Example 1 Example 17-B

~ \His-O~ ;s-Pro NH
L + Pro ~ 2 (61) . (10) In 10 ml OL- DMB was suspended 252 mg (1 m mol) of compound (61) and after adding thereto ll~mg (1 m mol) .'-. -' , , - ' , :-., :

~2593~18 of N-hyGroxysuc_inimide and Ihen 114 mg (1 m mol) of prolinamide and 206 mg (1 m mole) of DCC under coo'ing to 0C, the resultant mixture was allowed to stand overnight at 0 to 5C and then stirred ~or 2 days at room temperature. After filte~ing off the crystals thus precipltated, DM~ was distilled off unde~ reduced pressure. The residue formed was mixed with 5 ml of water and after filtering off insoluble ma.ters, water was distilled off under reduced pressure. After azeotropically dehydrating the residue with the addition of benzene-acetonitrile, 3 ml o_ methanol was added to the residue and the mixture was stirred to provide 82 mg o' the c-ystals of ~-[(S)-2-azetidinone-4-carbonyl~-L-histidyl-~-prolinamide (1~). The physicochemiczl p_ope-_ies o_ _he product were same 2S
those of the product obtained in Example 17-~.
Example 18 ~c~or~ ~3 > r~ s ~ C~3 F ~ C~3 0 C~2 3 C01~2 (2) (62) (63) In a mixture of 2 ml of DM~ and 10 ml of methylene chloriae were dissolved 211 mg of (S)-2-azetidinone-4-carboxylic acid (2) 2nd 248 mg of HO~T and the solution w2s ice-cooled. After adding .hereto 472 mg of DCC, the mixture w~s stirred ~or one hour 2_ -oom tem?e-ature.
Then, a 10 ml of a DMF solution of 426 ms of L-h' stidyl-DL-(3,~-dlme.hyl)prolinaJIlide (62) was adGed ~ . . . . -~ 2593~
6~
to the mixtu-e and the .esul~an' m~xture was stirrec for 2 da~s at 0 to 4C. Insoluble matters were collected by filtration and washed with DMF. The filtrate was combined with the washings and the solvent was distilled oLf fro~ the mixtu~e under reduced pressure. The residue was subjected to column chromatography of 150 m7 of silica gel. 3y elu'ing with chloroform-methanol-aqueous ammonia (80 : 20 : 2), N~-[(S)-2-azetidinone-4-carbonyl]-L-histidyl-DL-(3,3-dimethyl)prolinamide (63) was obtained. The aqueous solution was lyophilized to provide 63 mg of a white powder. The product w25 a mixture of diastereome-s showing two spots on TLC.
IR (KBr) C~ l: 3250 (broad), 1750, 1670, 1630, 1540, 1440 ~MR (CD30D) ~ppm: 7.64, 6.96, 6.88 (2H, lmidazole r ng), 4.14 (lH, d,d, azetidinone ring ~-pcs'ti~n), 1.80 cente- (2~, p_oline ~ing), 1 06, 1 12, 1.06, 0.92 (5H, prol~n~ _ing 3-position dime.hyl) ~ass m/z: 376 (M ), 343, 316, 306, 262, 98 Reference exam~le 22 (raw materials for Examples 19 a~d 22) a) (NT-CH3)His-OH 2HCl , "- (NT-CH3)His-OH

(6~) (6;) In 130 ml of wate- W2S dissolved 9 9 g of ~-N'-methyl-hlstidine 2-hycrochlo~ide (D~) and tAe solution was cooled in an ice-sodium chloride cooling bath The pH of the sol~tion 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 mix~ure During the operation, the pH
of the system was con.rolled to 11 to 12 by the addition o, 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 ~ 0 5 by adding occasionally an aqueous solution of 2N sodium had hydroxide ~fter the chanye in pH/stopped, the solution wzs fur.her sti-red fo- 1 5 r,ours a, ~ to 10C TAe reaction mixture w~s washed twice each time with ethyl acetate, the aqueous solution thus formed was collected, and the p~ of the solution was aajusted to 3 9 with 4N hydroc~lo-ic acid~ Then, the p~ thereGf was adjusted to 2 2 by the addition of 2N p-tolu~es~l~onic acid ~he ~eaction mixture was saturated with sodium chloride and extracted four times with acetonitrile-isobutanol-ethyl acetate (1 1 2) The or~anic layer thus obtained was concentrated under reduced pressure and the residue was tritura.ed wi'h aceto-nit_ile After _iltering off insoluble matte~s, .he ~ l._ate W2S concent~ated .o p-ovide a yeliow syrupy product The product W25 subjected to column chromato-g-aphv usins 600 ml of silica gel and by eluting 'he " :' ' ' ' ' .

~25~

~roauct with chloroform-me~hano;-aqueous am~.onia (60 :
40 : 3), 8.8 g of ~ - ben~yloxyca-bonyl-N`-methyl-L-histidine (65) was obtained as a foamy material.
IR (KBr) cm : 3100 cente_ (broad), 1700, 1590, N.~ (CD3OD) ~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 grou?), 3.68 (3H, N-methyl) [~]D = ~22 2 (C = 1, methanol) b) Z-(N -CH3)His-OH ~ Prc-NH2 ~ (N~-CH~)His-Pr~-~H2 (65) (66) In 25 ml of d~y D~ we~e dissolved 1.65 g of compound (65) and 621 mg of L-prolinamide and the solu,ion -w~s cooled to ~ to 10C. Then, 1.03 y o p-.o1u~esul~onic acid 2nd 1_'~ g of 3CC were added to ,he solution and _he mixtu_e ~-as allowed to stand o~ernight in a re~rigerator. lnsoluble matters were iltered off, the filtrate was concentrated under and reduced pressure,/the the residue was triturated with a mixture of ethyl aceate and e.her. Then, 3.2 g o' the insoluble ma_ters l~as subjec~ed to column chromatography of 500 ml of silica gel and by eluting using chloroform-methanol-aqueous ammonia (80 20 : 2), 2.0 g of N~-~enzylox~c2r~0nyl-N~-me.hyl-J-histidyl-L-.

~L25g~

prollnamide (66) was obtalned.
IR tK3r) cm~l: 3300 center(broad), 1620-1720 ~broad), 1510, 1440 NMR (CD3aD) ~ppm: 7.43 (lH, imidazole -ing), 7.32 (5H, benzene ring), 6.90 (lH, imidazcle ring), 5.04 (2~, benzyl 3.64 (3H, N-methvl), 2.0 center(4H, proline ring) Mass (FD) m/z: 399 (M ) c~
Z-(N -CH3)His-Pro-NH2 -~ (N -CH3)His-Pro-NP~22~Br (66) (67) To 24 ml of an acetic acid solution of 25 hydrobromic acid was added 2.0~ g of compound (66) an~
the mixture was s irred for 1 ~ hours a. room tempe~a- .
ture. The reaction mixture W25 poured in 260 ml of dry ether and white precip.itates thus formed were collected ~y ,i7tra-,io~. ~he DreCipitateS were dried ove~ potassium hydroxide under reduced pressure ,o provide 2.2 g of a hvgroscoplc solid o_ N~-meth~ L-his,idyl-L-prolin-amide-2-hydrobromide (67).
Example 19 ~COOH
~ ~ (N~-CH3)His-Pro-NH2 2HBr " N~
O (67) (2) C
\His(Nl-c~3)-pro-~2 (68) 6~
In a mixture of 7 ml of D.~5F and 7 ml Oc methylene hloride were dissolved 323 mg of compouna (2) and the 380 mg of HOBT and after ice-cooling/solution, 579 mg of DCC was adde~ to the solution, whereby crystals ~recipitated soon. After sti--ing for about 20 minutes, a solution of the free amine compound prepared from 1.0 g of compound (6,) and ~21 mg o triethylamine in DM~
under ice-cooling was added to the reaction ~ixture.
~he reaction mixture thus obtained was stirred for 20 hours in a refrigerator, insolu~le matters were filtered off, and the riltrate was concentrated to provide a syrupy residue. The residue was subjected to silica gel colwmn chromatography and by eluting with chloroform-methanol-aqueous ammonia (80 : 20 : 2), ~43 mg of N~-~(sj-~-azetidinone-4-carbonyl3-N' -methyl-L-histidyl-~-prolinamide (68) was obtained.
IR (KBr) cm~l: 3250, 17~0, 1670, 1630 ~R (CD30D) ~pm: 7..2 (1;~, ~midazole ring), 6.96 (1~, i~id2zole _ing), 4.44 center (1~, m, methine hydrogen); 4.12 cen.e- (lH, d,d, azetidinone ring 4-posiLion hydrogen), 3.70 (3H, s, N-methyl), 2.0 center (4~, proline ring) Mass m/z: 362 (M ), 319, 292, 2ag~ 221 -[~]2~ = -68.6 (C=l, metha"ol) Refexence example 23 (Raw mate~ial for Example 20) C~3 a) ~ CH3 (NT-cH3)His-oH ~ ~ NA CoNH2 (65) Z- ~ ~ ' -CE~3 ) Hi S -~Ç~3 3 ( 70 ) 2 By reac,ing 2.24 g of compound (65) and 1.0~ g of 3,3-dimethyl-DL-prolinamide (69) i~ a similar method to that in Refernce /example 22 b), 3.2 g o~ N~ -benz~loxycarbonyl-N~ -methvl-L-histidyl-3,3-dime'hyl-DL-prolinamide (70) w2S
obtain~d, which was a mi~ture of diastereome~5.
NMR (CD30D) ~ppm: 7.;2, 7.~8 (lH, imidazole -ing), 7.32 (~H, benzene ring), 6.88, 6.34 (lH, imidazole rlng), ~.04 (2H, benz~l), 3.64, 3.60 (3H, ~-methyl) IR (KBr) cm~l: 3300, 2920, 1620-1720(broad) Mass m/z: 427 ~M ), 383, 319, 286, 277, 258 b) Z-(N -CH3)~is- ~ 3 _____~
CO~-2 ~3 ) ~S-~<c~3 (71) CONH2 By following the ~r~ceduTe desc~ibedin Re,erence example 22 c), N`-methyl-L-histidyl-3,3-dimethyl-DL-prolinamide 2-hydrobromide (/1) was obtained with 2 au2ntitative yield from 3.2 g of com~ound (7D). The product was used for the subsequent reaction as it was.

- . . , . -3L~

Example 20 COO~.
CH3)His ~ ~3 2-~r (71) (2) o ~ His(~T-CH3)-~ ~ c33 NH CONP.2 (72a),(72b) By following a si~ilar 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 .hus obtained was subjecteo to silica gel column chromatographv.
The product wzs eluted wit~ chlorofo-m-me~hanol-aqueous ammonia (80 : 20 : ). The desired reaction product, N d-~S)-2-azetidinone-4-c2rbonyl]-N' -methyl-L-histidyl-3,3-dimethvl-DL-prolir~amide was a mlxture of a diastereomer (72a) having a -~eak pola_itv on chromatograph znd a diastereomer ~2b) having 2 strong polarity. The product irst eluted W2S 244 mg of 2 mixt~re of ~7~ and (7~ a la~ f 8 : ~, 2;4 mg of the product eluted in ~he next was/2 mixture of 172~
and ~72b) of 1 : 1, and ~he product Linally elu-ed was 182 mg of a mixture of (72a) and ~72b) of 2 : 8.
Properties o~ the dizstereomer having weak polarity [ratio . of ~72a) and ~72b) = 8 : 2]
; IR ~KB-) cm~l: 3250 center ~broad), 1750, 1670, 1630, 1540, 1510, 1440 NMR ~CD30D) cppm: 7-50 (1~, imiàazole ring), 6,92 (lP., imiaazoie ~ing), 4.8 (lH, me,:~ine), 4.10 (lH, d,d, azetidinone 4-position hyd ogen), 4.0 (lH, methine), 3,66 ~3H, N-meth~l ), 1.12, 7 l 1.08 (6H, two kinds of methyl) Mass m/z: 390 (M ), 373, 347, 320, 278, 249, 221 [~]27 = -27.3 (C=l, methanol) Properties of the diastereomer having strong polarity [ratio of (72~ and (/2b) = 2 : 8~
IR (KBr) cm~l: 32~0 (broad), 1750, 1670, 1630, 14~0 NMR (CD30D) ~ppm: 7.56 (lH, imiàazole rlng), 6.90 ~lH, imldazole -ing), 4.14 (lH, d,d, aæetidinone ring 4-position hvd~ogen), 3.68 (3H, N-methyl), 1.08, 0.92 (6H, tw~ kinds of methyl Mass m/z: 390 ~M ), 347, 320, 2/7, 749, 221 ~]27 = _7,9o (C = 1, melhznol) Refe_ence example 24 (Raw material for Example 21) a) S
Z--(NT-C~13)~is-0~

(6~) (~3) Z-(N~-C~3)~is-N S

(74) CO~E2 By reacting 2.25 g of compound (65) and 0.89 g o~ L-thiazolidine-4-carboxamide (73)in a similar ~a~ner tD Shat in Reference example 2 ~ 1.72 g of 3-[N~ -benzyloxy-ca~bonyl-N~-methyl-L-histidyl~-L-thiazolidine-4-carbox2mide (74) was obtained as a foamy material.
IR (KBr) cm~l: 3270, 16AO-1720 (broad), 1510, 1410, 1250 NMR (CD30D) ~ppm: 7.48 (1~, imdiazole ring), . ~ ', . ,' ' ' '. . .

7.32 (~;~, benzene -ing), 6.90 (lH, imidazole ring), ~.06 (2H, benzyl 3.6~ (3~, N-methyi) Mass m/z: 417 (M ), 373, 346, 286, 258 b) ~ ,^~
Z-(N -CH3)~is-~ ~ s (74) (N -CH3)~is-~ S ~ 2~Br ( 75 ) CON~2 describe~
By followin~ the prccedure / in Re_erence example 22 c), 1.9 g of 3-[NT-methyl-L-his,idyl]-L-thiazolidine-4-carDoxamide 2-hydrobromide (7i) was ob'ained from i.70 g of compound (74). ~he product w2s used for the subsequent reaction as it W25.
Example 21 COO~
C~3)~is-N~,5 2~r 3 CON~2 (7~) is (NT--(~i3)--~Y' S
N~ (7S~ CO ~
By following the proceàure aesc~ibed i~ F~ple 19, 416 mg of 3-~N -~(S)-2-azetidinone- A -car~onyli-N T _ methyl-L-histidyli-~-thiazolidine-~-Carboxamide (76) was obLained as a foamy ma~erial from 230 mg of compound (2) and 900 mg of compo~nd (75).
: IR (KBr) cm~-: 3250, 17~0, 1630-1680 (b-ozd), 1.20 NMR (CD30D~ '~ppm 7.46 (1~, ~midazole ring), 6,90 (lH, imidz-ole ring), 4.9 cente- (3~, .. . . . . .
... . . . . .

methine, methylene), 4.4 (lH, met~.lne), 4.10 ~lH, d,d, azetldlnone i~s 4-posi-ion hydrogen), 3.64 (3H, N-methyl) Mass m/z: 381 (M+), 326, 309, 281, 267, 249 Example 22 + (N~-CH3)'~is-Pro-NH2 2XBr G"~ NH ( 6 7 ) t77) o ^2 2 \HiS(~T-C~ r~-NH (78) By performlng 2 similzr re2ction/in ~xample 19 usins 413 mg of DL-4-(2-carboxyethyl)-2-azetid~none (77) and 1.12 g of c~mpound (67), 540 mg o~ Na-!(RS)-3-(2-cxo-9-azetidinyl)propionyl]-N' -methyl-L-histi~yl-L-prolinamide (78) was obtainec.. ~;ne ~roauct ~-as a.
mixture of diastereomers.
IR (~Br) cm~l: 3250, 1730, 1660, 1630, 1540, 1510, ~ MR (CD30D) ~ppm: 7.52 (1 , ~mia2zole ,ins), 6.96 (lH, imidazole rins), 4.80 (lH, methine), 4.44 (lH, methine), 3,68 (3~, N-methyl), 3,3-3.96 (3H, azetidlnone _ing 4-position hydrogen), proline rina), 1.94 center (4H, proline ring) Mass m/z: 390 (M ), 348, 320, 307, 277, 299 Reference Example 25 (Raw material fo- Example 23) 2 ) .
Z-g-o-O~ Z-?-o-N~CH2CY:20H
(~9) (79) In 40 ml o_ TH~ were dissolved 9.95 g o~ ~-benzyl-oxycarbonyl-L-p-oline (29) and ~.45 g of t~iethylamine and the solution was ice-cooled. Then, 6.10 g of ethyl chloro~ormate was slowly added to the solutio~
under ice-cooling and then a solution of 5.13 g of monoethanolamlne~was added to the mixture.
The reaction mixture thus obtained ~Jas stirred for 15 minutes under ice-cooling and then stirred for 1.5 hours at room temperature. After distilling off THF
unde~ reduced pressure, 150 ml of eth~l acetate and 50 ml OL water were added to the residue and the organic layer thus formed was separated from the aqueous laver.
The organic laver was washed in succession with an acueous solution of lN hyd-ochlo-ic acid, an aqueous solution of O.lN sodium hydroxide, water, and then an aqueous sodium chloride sol1~tion. After drying the or~anic layer, the solvent was ,emoved, whereby precipitating crystals, -~hich were collected by filtration and rec-ystallized ~rom ethyl acetate to provide 5.23 g of (S)-l-benzyloxycarbonyl-N-2-hydroxy-ethyl-2-pyrrolidinecarboxamide (79).
. p. 104-106~C
IR (KBr) cm~l: 3420, 3270, 1680, 1640, 1~40 NMR (CDC13) Gppm 7.36 (5H, s, benzene _ing), 5.16 (2X, c, benzy' - 4,30 (1~, t, methine 2,0. center (5H, proline ~ing, 3H) ~5~313~

b) ~_prO-NHCH2CH20H ~ P-~-NHcH2cH2oH
(79) (53) In 70 m1 of methanol was dissolved 5.92 g Of compound (79) and the compound was catalytically reduced by an or-inary method using 10% palladium-carbon as a catalyst. After the reactionJ the catalyst and the solvent were removed to pro~ide 3.2 g of SYrUPY (S)-N-2-hYdrOXYethY1-2-PYrrO1idineCarbOXamide (5~). The product was solidifie~ when it was refrige-rated.
NMR (CD30D) ~ppm: 2.96-3.84 (7H, proline ring~
hydroxyQthyl), 1.64-2.36 (4H, proline ring) IR (neat) cm~l: 3250, 1640 (broad), 1530 (~road) Mass m/z: 159 (M + 1), 127, 70 c ) Z~ C~3)~is-0~ + ~r~ C~C 2 (~5) r~

Z-(N~-c~3)~is-pr~
(80) In 23 m1 OL dry--- DMP were dissolved 1.54 g of N -benzyloxycarbonyl-NT -methyl-L-histidine (6~) and 0.80 g of compound (~3) and the solution was ice-cooled.
After adding 1.01 g of ~-toluenesulfonic acid monohydrate to the solution, 1.36 g of DCC was also added to the mixture. The mixture was stirred over-night in a refriger2tor to ~er_o-m the reaction.

Therea .er, the reaction mixtl~re w2s stirred for 2.j ~ .'. ,." ' ,,' ,'', ,,,' . .
' .,: .. . . . .. .

hours at oom temDerz_~-e. A~te~ filterins Or~
insoluble matlers, the 'iltrate was concentrated under reduced pressure, the Tesidue thus formed was subjected to column chramatography of 300 ml of silica ~el, and the product W25 eluted wi~h chloroform-methanol-acueous ammonia (80 : 20 : 2) to provide 1.7 g of N~ -benzyl-oxycarbonyl-Nr -methyl-L-histidyl-~-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, imiàa~ole ring), 5.04 (2n, benzyl 3.64 (3H, N-methyl), 2.0 center (4H, proline ring) Mass m/-: 443 (M~), 413, 355, 286, 2i8 IR (neat) cm~l: 3250 (broad), 1620-1~20 (broad) d) Z-(N'-ce3)~is-pr~-N~c~2C~2 (80) 0 (~T C~ is P~o ~ 2occ~
( ~1) To 22 m~ of an acetic acid solution of 25% hydro-bromic acid was added 1.7 g of compound (~0) 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 unaer reduced pressure to provide N~ -methvl-L-histidvl-~-2-acetoxyethyl-L-prolinamide 2-hyarobromide(81) with a quantitative vield. The p~oduct was used for the subsequent reac~ior. as it W2S.

C00~ Example ~3 _c~3)HiS-pr~-NHc~2cy~oc~3 2H~r -(2) ( (81) C O
His(N~CH~Pro-NHCH2CH20CCU.3 L NH
~ (82) In a mlxture of 7 ml of methylene chloride and 7 ml of DMF were dissolved 317 mg of compound (2) and 44& mg of HOBT and the solution was ice-cooled. Then, afte~ adding thereto 683 mg of DCC, 'he ~ixture was stirred for 20 minutes under ice-coolins. To the reac~ion mixture was added a free amine solution prepared by reacting 1.37 g of compo~1nd (~1) and 670 mg of triethylamine in 12 ml of DM~ under ice-cooling followed by ~iltration. The mixture was stirrea for 38 hours in a refrigerator to perform the reaction. Insoluble matters weIe Ciltered ofl, the fi''~-ate w~s c-ied ~nde_ _ed-,~cer' ~-ess~-e, and Lhe resldue w2s su~jec.ed ~o col~ ch-om~log_2phy using 300 ml o~ silica gel. By eluting the pro~uct with chloroform-~ethanol-aqueous ammoni2 (85 : 15 : 2), 593 mg oi N~-~(S)-azetidinone-4-car~onyl~-N~-methyl-L-histidyl-N-2-acetoxyethyl-L-prolinamide (82) was as f~a~y material.
obtaine ~ The product was dissolved in water and then lyophilized.
IR ~K~r) cm~l: 3250, 17;0, 1730, 1640, 1~40, NMR (CD30D) ~pp~: 7.56 (1~, imidazole ring~, ' .., . ' ' ' ' ' . - , ' . .. . .
.

~3~

6.96 (lH, imidazoie _lng), 4.80 (lH, melhine hydrogen), 4.40 (1'., me,hine hydrogen), 4.16 center (3~, azetidinone ring 4-position hydrogen, -CH2O-~C~3), 2.0 center (7H, proline ring, acetyl) Mass (FD) m/z: 449 (M ~ 1) Reference example 26 (~aw mate_ial for Example 24) a) ~_~
Z-~ro-Oi ) Z-Pro-N~_~O
(29) (83) In 80 ml of dry te_rahydrofuran were dissol-ved 4.9 g (20 m mol) of Z-proline (~9) 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 2' the same temperature, a solution of 1.74 g (20 m mol) of morpholine dissol~ed in 20 ml cf d~y D~ was gradually added dropwise to the mixture ~he resultant mixture was allowed to stand for 18 ho~-s ~_ room temperature ana ~en _he s~l~ent w2s ~is.illed O r~ unde_ reduced pressure. The residue thus formed was dissolved in 200 ml of ethyl acetate and the solution was washed, in succession, with 7~ ml of an aqueous solution of 0.SN
hydrochloric acid, 75 ml of a saturated aqueous solution of sodium hydrogen c2rbonate, and then 50 ml o wate~. Afte~ dryins ~he solu,ion wi.h anhydrous magnesium sulfate, ethyl acel2.e was distilled off and the residue thus formed w2s pU-i ~ ed by silica gel (490 ml of column chromatogr2phy/ Wako G_l C-200;

' . ' , , ' . . . ' . ' ',, ' '' ' as a eluen.~ ! 79 ethyl acetate /to ~~ov~de 5.0 g of ~-[~-ber.-yloxy-carbonyl-L-prolyl]mo_pholine (8;) havlng a melting point of 139-140C.
NMR: 90 MHz (CDC13) ~ppm: 1.70-2.40 (m, 4H, proline ring), 3.20~4.00 (m, lOH, proline rins, mo~pholine ring), 4.40-4.90 (m, lH, proline ring methine), 5.10, 5.14 (q, ~, 2H, benzyl ), 7.32, 7.34 (s, s, SH, benzene ~ing) IR (KBr) cm 1 2960, 2910, 2860, 2S30, 1680, b) Z-Pro-N~_JO ~ Pro-N O
(8~) (84) In 100 ml of ethanol W2S suspended 4.9 g of compound ~8~) ar.d acter adding thereto 250 mg of iO~
palladium carbon, the mixture W2S s,irred fo_ 4 hours in a hydrogen s_ream. A~te~ ~;ltering ofC 10~ palladium-carbon, ethanol was distilled Oc under reduced pressure from the filtrate to provide 2.B g of crude ~-(L-prolyl)morpholine ~84~. .
N~R: 90 ~z (CDC13) ~ppm: 1.40-2.30 (m, 4~, proline ring), 2.60-3.40 (m, 2H, proline ring), 2097 (s, lX), 3,40-4.10 (m, 9H, morphiline ring) R (neat) cm~l: 3,80, 2960, 2840, 1635 Mass: 185 (M i 1), 142, 114, 98, 70, 43 : . .

Z-His-NHNH2 > [z-His 3] (84 (3) (4) Z -Hi s -P r o -1~0 (85) In 5~ ml of an aqueous solution of lN hydrochloric acid w2s dissolved 5.46 g (18 m mol) of L-Z-histidine ( ~ ) hydrazide/and after adding thereto ~2 ml of ethyl acetate, the mixture was cooled to 0C. Ihen, 5.4 ml of an aqueous solution of 4~-NaNO2 was added to the mixture at the same temperature followed by sti~rins for 5 minutes and after addin5 thereto 21.6 ml of an aqueous solution of 50% potassium carbonate followed by stirring vigorously, the ethyl acetate laveTthus formed was separated. The aqueous 12ver was extracted with 18 ml of cooled eth~l acetate, the e,hyl acetate extract was combined with the foregoing ethyl acetate 12yC~, 2~ e ~.i~ture w2s a~ eA with 2~hyd~0us sod~m sulfate fo- ~ m7 nutes with stlr-ing under ice-cooling.
ATter filte~ing off sodium sulfate, the filtrate w2s cooled .o -20C and acte, slowly adding dropwise a solution Ol ~.76 g ~'~ m mol) of compound (84) diss~lved in 10 ml of ethyl acetate to the filtrate, the mixture was allowed tD stand overnight in a refrigerator at 4C. After allowing to raise the temperature to room tempe~2ture, ethyl 2cet2te w2s distilled o unde~ reduced ~ressure /and the residue thus ~ormed w2S ?ur-fied by silica gel (600 ml of column chrom2tos_aphy /Wako gel C ~00, chioro~orm-methanol-aqueous ammonia (10 : 1 : 0.1)) to .

provide 6.46 g of N-[N -benzyloxv-carbonyl-L-histidyl-T-prolyl~mo-pholine (85) as a colo.iess/
NMR: 90 MHz (CDC13) ~ppm: 1.60-2.40 (m, 4H, proline ring), 3.80 (d, 2~, methylene of His moiety), 3.2~-4.00 (m, 10~, morpholine rin~, proline ring), 4.40-5.00 (m, 2H, me~hine) 5.08 (s, 2H, benzyl ), 6.09 (m, 'H, amide), 6.86 (s, 1~, imid2zole ring~, ~.12 (s, 5H, benzene ring), 7.52 (s, lH, imiàazole ring) TR ~KB-) cm 1 3250, 2950, 2340, 1710, 1640, 1630 M2ss: 455 (M+), 374, 341, 305, 272, 244 d) Z-Eis-Pro-N o ~ His-Pro N~_~0 2~Br (85) (~6) ~ n 21~g ml o, acetic acid w-s ~csolved 6.46 g (14.2 m mol) OL compound (85) and afte~ aadi~g .hereto ~2.B g of an acetic acid solution of 25% hydrobromic acià under ice-cooling, the mixture ~-as stirred IOr hours. /the reaction mixture W2S added 600 ml of desicated ether, precipitates . were filtered to provide 6.50 g of the crude cryst~ls of N-[L-histidyl-L-p-olyl]morpholine.2-hydrobromide (8S) , -- .

3~

Example 24 o r~ (2) C` r-~
~is-Pro-N O 2xsr - - 3 ~ His-Pro-N O

(86) (87) [Solution A]
d~y In 10 ml of/DMF was dlssolved 230 mg (2 m mol) of compound (2) and after adding ,hereto; in succesion, 351 mg of HOBT and 453 mg of DCC with s.i-ring under coolirg to 0, the mixture was stirred for 40 minutes at the same temperature.
[Solution 3]
In i3 ml of dry DMP 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 s,irred for 30 minutes at the same temperature and then t~ieth~-l,amlne hydr~-~r~mide was ~iltered of~ udner coolins.
To solution A cooled to 0C was 2~ded solu,ion B
cooled to -15C an~ after sLirrins Lhe mixture for 2 hours at -10C, the mixture W25 allowed to stand overnight i~ a refrigerator at 4C.
After rasing the temperature OT the reaction mixture to room temperature, insoluble ma~ters were filte-ed off, D~F was distilled ofS under reduced pressure, and the residue thus obtained W25 pu-i_ied by (400 ml of silica gel column chromatography ~ ko gel C-200 chloroform-meth2nol-aqueous ammonia (40 : 10 : 1)) to .. . . .
.. . . . ~ .. .

~25~3~
~ 3 provide 480 mg of the colo-less crystals of ~;~e desired p-oduct, 4-[N~~~(S)-2-azeticinone-4-carbonyl]-L-histidyl-L prolyl]morphollne (87) having a meltin~
point of 148-150C.
NMR: 90 M~z (CD30D) C?p~ 1.60-2.40 (m, 4H, proline ring), 2.72 (d,d, lH, azetidinone ring 3-position), 4.11 (d,d, lHI a2etidinone ring 4-position), 6.96 (s, lH, imidazole .ing), 7.63 (s, lH, imldazole ring) IR (KBr) cm 1 3200, 30~0, 2850, 1755, 1650, 1625, 1555 Mass: 18 (M ), 348, 30~, 235, 207 [~ -69.1 (C = 1, methanol) Reference example 27 (Raw material for ~xample 25) 2 ) Z-Pro-OH ) Z-~ro-N < 3 c~3 (29) (g8) 1~ 80 ml of dl~r T~ were dissolved ~.9 ~
(20 m mol) of N-ben7yloxyczr30ny1-L-prol;ne (29) and 3.5 q (26 m mole) of ~03T and .hen 4.~3 g (22 m mol) of DCC w2s slowly added .o the solution ~t 0C. After IO~ ~0 minutes s~irring the mixture/at the same temperature, 10 ml of uas adde~.
solu~ion of 2M dimethylamine tetrahydrofuran/
resulting The /mixture was allowed to stand for 12 hours at -oom temperature. TX~ W2S distilled o~~f from the reaction mixture under reàucea p-essure, the residue thus formed was dissolved in 200 ml of ethyl acetate, an~ the solution was washed, in succesion, with 75 ml o~ 2n aqueous solu_ior, o- 0..~' h-~drochloric acid, 7; ml ": -. ' ' '. . ' ' ' ' . ' 8~ ~
of a saturated aqueous sodium hydrogencarbonate solu-tion, and 50 ml of water After drying the organic layer with anhydrous magnesium sulfate, ethyl a~etate was distilled off and the residue thus formed was (400 ml of *
purified by silica gel column chromatography /Wako gel C-200, ethyl acetate) to provide 4 8 g of (S)-1-benzyloxycarbonyl-N,N-dimethyl-2-pyrrolidinecarbo-xamide (88) having a melting point of 66-67~C
NMR 100 MHz (DMSO-d ) ~ppm: 1 50-2 40 (m, 4H, proline ring), 2 74, 2 78, 2 86, 2 98 (s,sts,s, 6H, N-dimethyl), 3 38 (m, 2H, proline ring), 4.70 (m, lH, methine), 4,96 and 5 02 (q and s, 2H, benzyl IR (KBr) cm 1 3020, 2960, ~940, 2860, 1700, 1640 b) Z-Pro-N ~ 3 ~- ~ Pro-N < 3 (88) (89) , In 86 ml of eth~nol was dissolved 4 3 g of compound (88) 2nd after adding thereto 210 mg of 10%
pz ladium-carbon, the mixture was s_irred vigorously for 90 minutesi~a hydrogen stream, After filte.ing off the ca.alyst, ethanol was distilled off under reduced pressure to provide 2 19 g of crude (S)-N,M-dimethyl-2-pyrrolidinecarboxamide (89) NMR: 90 MHz (CDC13) ~ppm: 40-2 40 (m, 4H, proline ring), 2 84 (s, lH, NH), 3 00 (s, 3H, N-methyl), 3 04 (m, 3H, N-methyl), 3 80-4 00 (m, lH, methine) Trade-Mark *
.

, ~2S~

IR (neat) cm~l: 3280, 2940, 28~0, 1635 c ) Z-His~ P.2 ~[Z His 3 (89) ~, ) ( 4 ) ~ -~ s -Pro-N < CH3 (90) In 53.3 ml of an aqueous solution of lN hydro-chlo-ic 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 W2S cooled to O~C.
~hen, 5.33 ml of an aqueous solu,ion of 4~-NaNO2 was added to the mixture at the szme temperature followed by stir~ing for 5 minutes and a~te~ adding thereto 21.3 ml of an aqueo~s solution OL 50~ potassium cz~bonate followed by stirring for 3 minutes, the ethyl ace,ate layer thus formed was collected. The aqueous layer was extracted with 18 ml of cooled etnyl acetate, the extra~t was combined with the foregoing et;q~l ace~ate layer and tne mixture was cried ~y annvdrous soaium s~al~ate _or ~ minutes ~-ith stirring under ice-cooling.
~ After filtering off sodium sulfate, the ; fil,rate was cooied 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 reIrig~ra~or 2t4 C. Ethyl acetate w2s disLilled cff under reduced pressu-e and the residue wzs purified by silica gel column chromatography (600 ml of /Wako gel C-200, chlo~oform-methanol-conc.
aqueous ammonia (10 : 1 : O.l))to provide 5.86 g Oc oily N~-benzyloxycarbonyl-L-histidyl-N,N-dimethyl-L-p.olinamide (gO).
NMR: 90 MHz (CDC13) ~ppm: 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, 1~, amide), 6.90 (s, lH,imidazole ring), 7.38 (s, 5~, benzene -ing), 7.56 (s, 1~, imidazole ring), 11.70 (m, lH, imidazole -N~) IR (nea.) cm~l: 3250, 2950, 2840, 1710, 1635 Mass: 413 (M ), 341, 332, 272, 262, 244 d) Z- ~ -Pro-N< 3 > Eis-Pro-N~ 3 2~3I
~ C~3 (90) (91) dissolved In 15 ml of acetic acid was/4.13 g (10 m mol) o_ compound ~90) and afte- adding thereto 30.12 g of an acetic acid solution of 25% hydrobromic acid under ice-cooling, the mixture was sti_red for 1.5 hours at room temperature. ~o the reaction mixture was added A50 ml of d~y etner and the p~ecipitates thus deposited were collected bv filtration to provide 3.75 g o~ ~-histidyl-~',N-dimethyl-L-prolinamide 2-hydrobromide (91).

.. . .

~;~3~8 ~xample 25 ~is-Vro-N<C~3 2Br ~

(91) ~His-Pr~-N<

[Solution A] (92) dr~
In 10 ml of/DMF was dissolved 230 mg (2 m mol) of compound (2) followed by cooling to 0CC and after adding thereto, in succesion, 351 mg o- HOBT and 453 mg of DCC with stirringj the mixture was sti_red for 40 minutes at the same temperatLre.
[Solution ~]
In 13 ml of dTy DMF was dissolved 966 mg (2 m mol) of compound (91) followed bv cooling to -15C, and after slowly adding 404 mg of triethyla~ ~e to the solution with stirring, the mixture was stirred for 30 minutes; Thereafter, triethylamine hdyrobromlde was ~iltered o~~ ~de- cooli~.
~ o sol~ ion A cDoled to 0C w~s zdded solution 3 cooled to -1~C and after stirring the mixture fo- 2 hours at -10C, the mixture was allowed to stand over-night in a refrigerat~r at 4 C.
The reaction mixture was allowed to raise to room temperature and insoluble matters deposited were filtered off. DMF was distilled Or_ _rom .he filt-ate under reduced pTessure and the residue .hus formed wzs (600 ~1 of/
purified by silica gel column chromatog-aphy ~ ako gel C-200, 400 ml, chloroform-methanol-aqueous a~monia ' . ., . ' . - . ' . -~L2S93~8 (100 : 10 : 1)) to provide 520 mg of the colorless rystals of N~-[(S)-2-azetidinone-4-carbonyl]-L-(92) histidyl-N,N-dimethyl-L-prolinamide/h2ving a melting point of 1~3-140C.
NMR: 90 Mhz (~2) ~ppm: 1.60-2.50 (m, 4H, proline ring), 2.71 (d,d, lH, azetidinone rinS, 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), ~.74 (s, lH, imidazole ring) IR (KBr) cm~l: 3180, 17~5, 1630, 1560 Mass: 376 (M~), 306, 262, 235, 207 [~]D -73.1 (C = 1, methanol) Referen_e example 28 (Raw material for ~xample 26) a) Z-Pro-OH -) Z-Pro-NHPh ~2g) (93) In ~0 ml OI TH~ was dissolved 4.9 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 reac,ion was performed for one hour under ice-cooling. The solvent was distilled off, the residue thus formed was dissolved in ethyl acetate, and the soiution was washed, in succession, wi.h an aqueous solution c~ lN hyd-o-chloric acid, a saturated aqueous sodium hydrogen-czrbonate solution, and then a saturated aqueous sodium " ', -.' ., '. - ' . - ' , chloride solu_ior.. The organic layer thus formed was sodium sul f a te dried by anhydTous /and then co~cen,rated ~o dryness. The residue was recrystallized f-om chloroform-ethyl acetate-hexane to provide 5.20 g of (S)-l-benzyloxyc2-3:)onyl-N-phenyl-2-pyrrolidlne-carboxa~ide (93) having a melting point of 143-144C.
NMR (CDC13) ~ppm: 6.9-7.7 (lOH), 5.17 (s, 2H), 4.43 (_, lH), 3.4 3.8 ~2H), 1.7-2.5 (4H) IR (KB-) c~,-l: 3260, 1690, 1660, 1595, 1545 Mass (EI): 324 (M+), 204, 160, 91, 70 b) Z-Pro-NHPh > Pr~-NHPh (93) (94) In 150 ml of methanol was dissolved 4.87 g of compound (9~) and the compound was hydrogenated using 487 mg of 10% palladium-carbor. 2s a catzlys~. The catalyst was filtered o_~ and the ~iltrate W2S
~oncentrated to provide 2.79 g of (S)-~-phenyl-2-pyrroliainec rboxa~niQe (94).
NMR tCDC13) ~p,~: 9.~-10.0 (lH), 6.9~-7.7~ (lC~'), ~ 3.86 (dd, l'H.), 2.75-3.2~ (2~), 1.5-2.5 (5H) Mass (~I): 190 (M ), 93, 70 c ) Z-His-NHNH2 --~[Z-His-N3] ~ 3 t3) (4) 2-His-Pro-~HPh (9~) ~ o 9~ ml c_ an ethyl aceta,e sol~ti~r. c~ N'~-benzyloxvca-bonyl-L-h~stidlne azide (4) prepare~ ~rom 3.03 g of ~'~ -benzyloxycar~onyl-L-histidine hy~razide ~(K3~) C2 1 : ;3!0, ,220, ~SO, ~SO, 16SO, 5~5, '515 ~

9o ( ;) by a known method was added 1.52 g of compound (94) under ice-cooling and the reaction was pe-formed overnight ln a refrigerator. The reaction mixture was concentrated and the residue thus formed was subjected tc silica gel column chromatog-a?hy. By eluting the product with chloroform-methanol (95 : 5), 2.49 g of N~ -benzyloxycarbonyl-L~his'idyl-N-phenyl-L-prolinamide (g5) 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 (2H), 2.8-3.9 (4H), 1.5-2.5 (4H) IR (KBr) cm~l: 3250, 2950, 1700, 1630, 1590, 1535 Mass (~I): 461 (M~ 42, 310, 272, 245, 191, 136, 107, 91, 7 d) Z-His-Pro-N~h ~ ~is-~ro-NHPh.2~Br (95) (96) To 1.79 g of co~pound (95) uas a~ded 19 ~1 of 25 % hydrobromic .~ acid - ~ce ic acid cooled i~ an ioe bz~h followæd by stirring one nour a, room temperature.

~ he 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 o' L-his.idyl-N-phenyl-L-prolinamide.2-hydrobromide (96)~

~xample 26 His-Pro-NHPh 7H3r ~> [His-Pro-NHPh]
(96) (97) . CO-His-Pro-NHPh (2) ~ (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. ~fter performing the reaction for one hour at -30C to -40C, precipitates thus formed were filtered o.f to provide a DMF solution of L-histidyl-N-phenyi-L-prolinamide (97). The product was used for the subsequen~
reaction immediately after the foramtion thereof.
In 5 ml OI D~L~ wzs dissolved 230 mg of (S)-2-azetidinone-4-carboxylic acid (2) and after adaing thereto 406 mg of HOB~ a~d 495 mg of DCC under ice-hour cooling, tne reaction wzs per~ormed for one/at o~C.
~ he Ie2c+.~0n m Ytll~e W25 cooled to -~0CC
2nd after zdding thereto a D~ solution of th~ fo_e-going compound (97), the reaction W2S performed for 30 minutes at -40C and then overnight in a refrigerator. Precipitates were filtered off, the filtrate was concentrated to dryness, an~ the residue was subjected to silica gel column chromatography.
By eluting the product with chloroform-methanol-aqueous ammonia (80 : 20 : 2), 652 mg of NO~-[(S)-2-zzetidinone-4-ca-bonyl]-L-histidyl-N-phenyl-L-prolinamide (98) W2S obtained.

. ~ ~ , . . , ~ . ' - - . .. .

~MR (CD30D) ~ppm: 6.9-7.7 (7~), 4.56 (dd, lH) ~.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): 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) L-Pro-OH ~ L-P.~-NH~

(29) (99) In 100 ml of ~P was dissol~ed 9.97 g o, slowly compound (29) and after adding/thereto 4.45 g of triethylamine and then 6.01 g o isobu-yl chloroformate under ice-cooling, the rea~tion W25 pe-~~ormed under ice-cooling. To the reaction mixture was slowly added 11.37 g of 3-(2-oxo-1-pyrrolidlnyl)-propylamine and then tne ~eac.ion was pe-iormea ,or one hour ~nde- ic~-coolins. Precipitates ~e_e _ilt~red o~ , the filtrate was concentrated, and the residue hus formed was subjected to silica gel column chromato-graphy. By eluting the product with ethyl acetate-methanol (~ : 1), 4.43 g of (S)-l-benz~loxycarbonyl-3-(2-oxo-pyrrolidinyl)pro?yl]-2-py_rolidine ca~boxamide (99) was obtained.
NMP~ (CDC13) ~ppm: 7.36 (s, 5H), 5.16 (2H), 4.33 (t, lHi, 2.8-3.8 (8H), 1.7-2.5 (lOH) IR (neat) cm~l: 3280, 2930, 2860, 1700, 1660, 153C

.- ' . . :

. ~ , '.'. ~:' ' , 1~3`~
o~
MaSS (EI): 373 (A; ), 238, 204, 160, 91, 70 b) Z-PT~-N~ ~h~O ~ P~ T~
(99) (100) In 150 ml of methanol was dissolved 4.32 g of compound (99) and the compound was hydrogenated using 432 mg of 1~% palladium-ca=bon as a catalyst. Ther., the catalyst was filtered off from the reaction mixture and the filtrate was concentrated to provide l.99 g of (S)-N-[3-(2-oxo-l-py-rolidinyl)propyl]-2-pyrrolidine-carboxiamide (100) was obtained.
NMR (CD~13) ~ppm: 7.6-8.2 (lH), 3.72 (~,d, lH), 2.8-3.5 (8H), 1.5-2.~ H) IR (neat) cm~l: 3280, 2920, 2850, 16~0, 1~0 Mass (EI): 239 (M ), '97, l4l, 99, 70 c ) r~
PTO NH~N~ - ~ Z-H~ s-Pr~-NH ~ ~y~

(100) (101) To 30 ~.1 of ,~ ethyl ace~a_e solution of compound ( 4 ) prepared from 2.12 g of compound (3) by a known method was added 5 ml of 2 DM~ solution of 1.17 g of compound (lO0) under ice-cooling and the reaction was performed overniyht in a refrigerator. The reaction mixture was concentrated and the residue thus formed was subjected to silica gel column cn-oma~ography. ~y elu~ins .he product wi.h chloroform-methanol-a~ueous ammonia (90 : lO : l), ~IL2S~13~8 2.00 g of N~-benzylGxycarbonyl-L-his'idyl-~-[3-(2-oxo-(101) l-pyrrolidiny')-p~opyl]-L-prolinamide/was obtained.
NMR (CDC13) ~ppm: 8.0-8.4 (lH), 7.56 (lH), 7.35 (s, 5~), 6.97 (s, lH), 5.87 (d, 2~), 5.10 (s, 2H), 4.3-~.8 (2H), 2.9-3.7 (8H), 1.5-2.6 (lOH) IR (XBr) cm-l: 3220, 2930, 2850, 1700, 1690, 1530 Mass ~EI): 510 (~+), 430, 402, 359, 267, 239, 136, 108, 79 d)~
Z-His-Pro-NH ~ ~ >

(101) ~
His PT~ NH~N~ 2HBT

(10~ ) To 1.02 g of compound (101) ~as added 10 ml oS
ice-cooled an/acetic acid solution of 25~ hydrobromic acid and the reaction was performed for 2 hours at r~om temper ture.
~he reaction mixture was added to 100 ml of dry ether 2nd the~ ~e precipi ~tes ~hl~s fo~e~ we~e yuickly colle~ted by filtration and dried o~er~ight in a desicato- containing potassium hydroxide, 1.28 g of L-histidyl-N-[3-(2-oxo-1-pyrrolidinyl)propyl]-L-prolinamide.2-hydrobromide (102) was obtained.
E~ample 27 ~
~is-Pro-N~ 2EBr~ [R;s-Pro-N~v~? ]
(102) (iO3) CO ~iis -Pro-~

.

1' ,~U , In 10 mi Oc DMr was dissolved 1.28 g o' co~pound ~102) and a'ter cooling to -40C, 415 mg of triethylamine was added to the solution. After per'orming the reaction for one hour at -30 to -40C, precipltates thus formed were '-ltered o.f to provlde a D~F solution of L-histidyl-N-[3-(2-oxo-1-pyrroliàinyl)-propyl]-L-prollnamide (103). The product was used Lor the subsequent reaction i~mediately after the formation thereof In 5 ml of DMF was dissolved 230 mg of _ompound ( ) and after adding thereto 406 mg of HOBT and 495 mg of DCC, the reaction was perfo~med for one hour under ice-cooling. The reaction mix,~-e w25 cooled LO -40C
and after adding thereto the fo-egoing DMF solution of compound (lO~),the reaction was performed for 30 minutes at _40r and then overnigh, in z re,rigerator. P-ecipi-tates thus fDrmed were filtered o'f, the filtrate was concen~ratea to àryness, and _he residue w2s subjecte~
to silica gel column chromatog_aph~. By eluting the product with chloroform-methanol-aqueous amonnia (80 :
20 : 2), 461 mg of NQ-~(S)-2-zzetidinone-4-carbonyl]-~-histidyl-N-[3-(2-oxo-1-pyrrolidinyl)propyl]-~-prolinamide (104) was obtained.
N~ (D20) ~pp~: 7.74 (1~), 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) I~ (X~r) cm~l 3240, 2g50, 2850, 1755, 1630 Mass (EI): 473 (M+), 304, 262, 235, 154, 70 [~]27: -75.6 (C = 0.55, methanol) Reference exa~p e 30 a) COOH CH3 ~C00H

0 ~ ~ t 0~ \
si(c~3)2 ~u Si(C~.3)2 Bu (10~) (106) In 6 ml of dTy TH~ was dissolved B36.3 mg (8.28 m mol) of diisopropylamine and the solution was cooled to 0~C unde-~nitrcgen atmosphere. To the solution was added ~.2 ml of a n-hexane solution containing 530 mg (8.28 m mol) of n-butyl lithium at 0C and the mixture was sti~ed for 10 minutes at the same temperature. To the solution was added a solution of 920 mg (4 m mol)of(S) -l-t -butyldimethylsilyl-2-azetidinone-4-ca-boxylic acid (105) dissolved in 8 ml of dry T~ at 0C and then the mixture was stirred fox 30 minutes at room temperature. The solution was coole~ to 0C and 2fte~ adding thereto 682 mg (4.8 m mol~ Df me.hyl iodide, the mixture was stirred for 30 minutes t room temperature. ~he reaction mixture thus obtained was cooled again to 0C, acidified with the addition of an aqueous 10%
citric acid solution, and a~ter addition of ether and w2ter, the organic layer was sep~rated. The ether layer was separated from the aaueous layer, dried, and the solvent was distilled off to p-ovide 860 mg OL l-t-butyldimethyl-silyl-3~R)-methyl-2-a2etidinone-'lS)-c2rboxylic acid (106) as colorless crystals.

3t~

[~]D3 = -36.1 (C -, 0.~, met;~anol) NMR (90 MHz, CDC13) ~ppm: 0.16 ~3H, s, Si-methyl), 0.3 4 ( 3H, s, Si -methyl), 0.98 (9H, s, t-butyl), 1.42 (3H, d, azetidinone -ing 3-posi'ion methyl), 3.37 (lR, q,d, aze~idlnone ring 3-position), 3.74 (lH, d, J=3.5 Hz, azetidinone ring 4-position), 9. 60 (1~, s, carboxy group) IR ~KBr) ~m~l: 2940, 2920, 28~0, 1740, 1680 Mass m/z: 244 ~M + 1), 200, 186, 1-43 b) `r~ `n~
dr-N~ t ~ ~H
Si(CH3)2 Bu (106) (107) 7n 20 ml of a mixtu~e Oc wate-, me,'h2nol, and concen.rate~ hydrochloric a~d (10: 90 : 1.7) was dissolved 6 1 mg (2.63 m mol) o~ compound ~106) ~nd the solution w,~s stirred ior 1.~ ho~rs 2t room tempe-a~ure. The reaction mixture was cooleQ to 0C, neutrali2ed with 4 ml o~ an agueous solution cf lN sodium hyaroxide, ar,d the s41vent was distilled of r under reduced pressure.to pro~ide 3(R)-methyl-2-azetidinone-4(S)-carboxylic acid (107), which w~s used in the subsequent reaction without being purified.
~z~ D2O) ~ppm: 1.25 (3H, d, methyl group), 3.20 (lH, q,d, azetidinone -ing ~~position), 3.88 ~1~, d, zzetidinone ~ing 4-position) - . . ..

~ '. .. . . -: . , " .. . . . . .

98 ~3~
Example 28 CH3~ COOH CH C0 ~ ~ His-Pro-NH2 2HBr > ~ His-Pro-NH2 o"L~ ~NH

(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-a 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 precipitatPd were filtered off, the filtrate was concentrated under reduced pressure, and the residue thus formed was subjected LO silica gel column chromatography using 200 ml of silica gel (Wako gel C-200). By eluting the product with chloroform-methanol-aqueous ammonia (80 : -20 : 2), 200 mg of NQ-[3~R)-methyl-2-azetidinone-4(S)-. :-99 ~
-carbonyl]-L-histidyl-L-prolinamide (108) ~as obtained.

~]23 = -33.8 (C = 0.5, methanol) NMR (100 MHz, D20) ~ppm: 1.60-2.40 (4H, m, proline 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, methine), 4.88 (lH, m, metine), 7.00 (lH, s, imidazole ring), 7.68 llH, s, imidazole ring), 1.32 (3H, d, methyl) IR (KBr~ cm~l: 3450, 2960, 2860, 1750, 1670, 1630 Mass m/~: 362 (Mt), 318, 278, ~49, 234, 221 Preparation examples:
Iniection A lyophilized formulation containing o.o25 mg or 0.05 mg of N~-[(S)-2-azetidione-4-carbonyl]-L-histidyl-L-prolinamide together with 10 ml of mannitol in one ampule was p~epared 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)-2-azetidinone-4-carbonyl]-L-histidyl-L-prolinamide and uniformly 7.5 parts by weight of laetose was puvlerized, and ~ix~d/
with 44.4 parts by weight of lac~ose , 22.5 parts by weight of crystalline cellulose, and 0.4 part by weight of magnesium stearate The resultant mixture was compacted to form tabletsof 75 mgltablet.
.

.. ' -~ ' ' . .

~S93~6'i Ca~sules A mixture of 0.5 paAt by weigh. of ~-[(S)-2-azetidinone-4-carbonyl] _T -histidyl-L-prolinamide and ln parts by weight of lactose WâS pulverized, and mixed uniformly with 1~7.~ paTts by wei~ht of lactose, 60 paTts by weight of corn starch, and 2.0 parts by ~-eight of magnesium stearate. The mixture ~as filled into gelatin ha.d capsules, to provide a capsulated preparations oS 210 mg/capsule.

.
.
.. .: ., ., . . , - .......................... .
.

Claims (2)

C L A I M S

1. A 4-substituted-2-azetidinone compound represented by general formula (VII) (VII) wherein R8 represents a hydrogen atom, a lower alkyl group or an aralkyl group or salt thereof.

2. A process of producing a 4-substituted-2-azetidinone compound represented by general formula (VII) (VII) wherein R8 represents a hydrogen atom, a lower alkyl group or an aralkyl group or a salt thereof, which comprises reacting a carboxylic acid represented by formula (VIII) (VIII) or a reactive derivative thereof and an amine represented by general formula (IX) (IX) wherein R8 has the same meaning as defined above or a reactive derivative thereof.