AU592117B2 - Dipeptide derivative of fatty acid - Google Patents

Description

Patent and Trade Mark Attorneys 367 Collins Street Melbourne, Australia Stuart Taylor Fine Printers 592117

AUSTRALIA

Patents Act COMPLETE SPECIFICATION

(ORIGINAL)

Class Int. Class Application Number: I Lodged: Complete Specification Lodged: Accepted: Published: Priority Related Art: Cc ate c [C Ca C I 4 4 ftS

C

APPLICANT'S REF.: FP/S-38-120 Name(s) of Applicant(s): Address(es) of Applicant(s): SUNTORY LIMITED 1-40, Dojimahama 2-chome, Kita-ku, Osaka-shi, Osaka, Japan Actual Inventor(s): TAKAHARU TANAKA NAOKI HIGUCHI MASAYUKI SAITOH MASAKI HASHIMOTO Address for Service is: PHILLIPS, ORMONDE AND FITZPATRICK Patent and Trade Mark Attorneys 367 Collins Street Melbourne, Australia, 3000 Complete Specification for the invention entitled: DIPEPTIDE DERIVATIVE OF FATTY ACID The following statement is a full description of this invention, including the best method of performing it known to applicant(s): P19/3/84

.WWW

D1P8PTIDE DERIVATIVE.OF PATVTY ACID Background of the invention: The present invention relates to a novel compound that exhibits enzyme inhibiting activit against prolyl 3 endopeptidase (EC# 3.4.21.26). The invention also relates to a method for chemical synthesis of such novel compounds, as well as its use as a prolyl endopeptidase activity inhibitor and a drug, especially an anti-amnesic agent, that contains it as the active ingredient.

Prolyl endopeptidase is known to inactivate neurotransmnitters such as Substance thyrotropin-.releasing hormone (TPDH) and neurotensin, or vasopressin speculatively associated with memory. Trsuru and Yoshimoto of the Department of Pharmaceutical Sciences, Nagasaki University, 13 found that compounds capable of inhibiting the prolyl endopeptidase activity were effective for preventing experimental amnesia caused in tats by coopolamine. Based on this discovery, they suggested the potential use of prolyl endolpeptidase activity inhibitors as anti-amnesic agents.

a'umxnary of t he Invention: Miotivated by the report of ST'uru and Yoshimotoo the present inventors made various efforts to find novel ~"compounds that exhibitLed Atrong inhibiting activity against prolyl endopeptidase as well as anti-amnesic activity and which yet had sat'4afactorily low toxicity levels. As a result, the inventors have synthesized compounds which are j close to natural substances by a combination of fatty acids, which enjoy a high safety level as natural compounds, and amino acids or peptide compounds, and found that novel compounds with anti-prolyl endopeptidase activity and having the general formula shown below exhibited excellent effects against amnesia. The present invention has been accomplished on the basis of this finding.

netailed Description of the Inventi:~ The Okpez~ttbi dephva-iyo of foit 1q'diu2Xof the present invention is represent-ed \py the general formula Mt) 2-

R

12

(CH

2 )n O C N C C (I) H m

CHO

(wherein m is an integer of 0, 1 or 2; n is an integer of 0 to 12; R 1 is a saturated or unsaturated unbranched hydrocarbon group having 5 to 25, preferably 10 to 20, most preferably 15 to 17 carbon atoms (the unsaturated carbon chain may include a plurality of double bonds); R 2 represents, when n is 0, an unbranched or branched alkyl group having 1 to 5 carbon atoms, whereas, when n is an integer of 1 to 12, R 2 is methyl group, phenyl group, hydroxyphenyl group, formyl group, amino group, hydroxy group, methylthio group, hydroxyphenyl group whose hydroxy S group is protected, protected amino group, protected hydroxy S group or a hydroxyalkyl group; R 5 is hydrogen atom, or when n is 3, R 2 and R 5 together may represent a single bond between carbon atom and nitrogen atom).

In formula when n represents an integer of 1 or more, it is preferably 1 to 12, more preferably 1 to 6.

The compounds of formula in which R and R together form a single bond between carbon atom ,nd nitrogen atom have the formula (Ib): o O C (Ib) 0 N?

CHO

36" The compounds of formula differ greatly from the known piracetam derivative based anti-amnesic agents in that the former contains a proline residue and an unbranched fatty acid chain. Because they are amino acid or peptide derivatives, the compounds of formula present extremely low toxicity levels in organisms.

ii -3- The following compounds of formula are particularly preferred because of their high anti-prolyl endopeptidaze activities (the following compounds may be sometimes referred to by the numbers in parentheses, hereinafter): 0

II

(SUAM 1155)

CHO

H 0 IN N (SUAM 1140)

H

CHO

~AMA/c?~,b N (SUAM 1154)

IN

i *r Ii 'a *o a a a a.

a a.I a..

0

H

(SUAM 1157) (SUAM 1139)

AAV/\\ANC

0

CHO

(SUAM 1134) -I I -4- H 0 Ii

CHO

(SUAM 1156) 44 41 4 4 4 4 4- 4 4 4: 4 $4 4 4- t 4 4 4t I 4$ ~'rN

N"'

I H 1 0

C

0

CHO

(SUAM 1158) (SUAM 1092) 44** 4. a 44 4 4 4 4 4* 4 i

S

tiLt 0

NCH

H L 0 0o,, CHO (SUAv 1166) (SUAM 1212) N L (SUAM 1214) 0

"I

HN>iA 00

Y

*1F *I t a.I U

S.

S

a.

tttftzr

/NAAA±~'

SUAM 1216) (SUAM 1217) "N(SUAM 1215) 0 The compounds of the present invention may be synthesized by any of the convention &I methods of peptide synthesis, but they may be convenien'tly synthesized by the following procedures in accordance with the present invention, in which each of the abbreviations represents the following meanings: Z benzyloxycarbonyl group Soc t-butyJloxycarbonyl group Pro proline residue Ala alanine residue -Pro-OMe -Pro-CM CM; -14 2 CH OH r -Pro-CHO :-N a. norLeu: norleusin residue Phe :phenylalanine residue Met :methionine residue Lys lysine residue Val :valine residue Leu :leusin residue Lys(Z): Nc-benzyloxycarbonyl lysine residue Glu(Bzl): glutamic acid-Y-benzylester residue a I 0 H i Glu(CH OM): -HN-C-C- CH 2

OH

-7- Glu(CHO)t 0 H II

-NH-C-C-

CHO

CHO

OMe methylester residue WSCD N-ethyl-N'-N'-dimethylaminopropyl carbodiimide TEA triethylamine The compounds of formula of the present invention may be synthesized by the following procedures: If the compounds have a lower alkyl ester group as R 3 in formula and are expressed by the formula (Id): or- 6

R

7 i f ?I Im1 R, -C CH CO 1 m N? 00P, 7 (Id) *4 4

S

.4 4 4..

*9 4.

4

S

.4.

a 9 4 4441 4 644~4 111

I

(wherein m is an integer of 0, 1 or 2; n is an integer of 0 to 12; R 1 is a saturated or unsaturated unbranched organic group having 5 to 25 carbon atoms (the unsaturated carbon sO chain may include a plurality of double bonds); R- is a lower alkyl group; R 6 represents, when n is 0, an unbranched *4 or branched alkyl group having 1 to 5 carbon atoms, whereas, S when n is an integer of 1 to 12, R 6 is methyl group, phenyl S group, hydroxyphenyl group whose hydroxyl group is 25 protected, protected carboxyl group, protected amino group, protected hydroxyl group or methylthio group; R 5 is hydrogen atom, or when n is 3, R 6 and R 5 together may represent a single bond between carbon atom and nitrogen atom), they may readily be synthesized by any of the conventional methods 10 from carboxylic acid, acid anhydride or carbonyl chloride of Sthe formula: 0 0 o Ii If II RICOOH, RI-C-O-C-R l or RI-C-Cl (wherein R 1 is a iaturated or unsaturated unbranched organic group having 5 to 25 carbon atoms (the unsaturated carbon chain may include a plurality of double bonds)), and peptide or amino acid having a proline residue, whose carboxyl group -6has been converted to a lower alkylester, of the formula (IIIn (wherein R R6 R, n and m are as defined above).

The protected groups of the compounds of formula (1d) are removed by any of the known methods in relation to amino acid or peptide, so as to obtain dipeptide derivative of fatty acid of the present invention represented by the formula (ie) RJ 5f CH-Co N (1e)

COOR

7 (wherein R 1 I2 r R 5 r RV n and m are as defined above).

Methanol is added dropwise to a mixture of the compound of formula (1d) and sodiumborohydride suspended in t-butyl alcohol or tetrahydrofurane, so as to reduce the 4 1. a compound, thereby obtaining a compound (If) which has hydroxymethyl group converted from the carboxyl group:

R

8 R C N CH -CO (f aS. CH 2 0OH (wherein R, R 5 n and m are as defined above; when n is 0, R is an unbranched or branched alkyl group having 1 to carbon atoms whereas, when n is an integer of 1 to 12, Rais methyl group, phenyl group, hydroxyphenyl group whose hydroxyl group is protected, protected amino group, protected hydroxyl group, hydroxyalkyl group whose hydroxyl group is protected, methylthio group or hydroxyinethyl group, or when n is 3f R and R together may represent a single bond between carbon atom and nitrogen atom).

-9- The compound of formula (if) is subjected to a deprotect raction commonly used in the field of amino acid and peptide, so as to obtain dipeptide derivative of fatty acid of the present invention having the formula (Ig): CR01 (1g) (wherein R 1 n arid m are as defined above, when n is 0, R 9is an unbranched or branched alkyl group having 1 to carbon atoms, whereas, when n is an integer of 1 to 12, R 9 is methyl group, phenyl group, hydroxyphenyl group, amino group, hydroxyl group, hydroxymethyl group or methylthio group, or when n is 3, R 9 and R 5 together may represent a single bond between carbon atom and nitrogen atom).

The compound of formula (If) is oxidized so as to obtain dipeptide derivative of fatty acid represented by the formula (1h)t *4 4 p 4 4p 4 t 444 #4 4 4 4 4 #4 0 4 44.4

R

101 R1SCH2I R 1- N n (Ib) (wherein Kl, RS1 n and m are as defined above; Rl 0 represents,, when n is 0, an unbranched or branched alkyl group having 1 to 5 carbon atoms, whereas, when n is an integer of 1 to 12, R 10 is methyl group, phenyl group, hydroxyphenyl group whose hydroxyl group is protected, protected amino group, protected hydroxyl group, hydroxyalkyl group whose hydroxyl group is protected, methylthio group or formyl group, or when n is 3f R 1 andR together may represent a single bond between carbon atom and nitrogen atom).

in this oxidation, it is most preferable to employ sulfur trioxide-pyridine complex as an oxydizing agent. A suitable reaction solvent is dimethy. sulfoxide and the

H--

j reaction may be carried out at room temperature. A period of about 1 hour will be sufficient.

The compound (Ih) is subjected to the same de-protect reaction as that mentioned above so as to obtain dipeptide derivative of fatty acid represented by the fomula (Ii)s

R

I I I1 2 )n C N CH

CO

CHO

(Ii) (wherein R 1

R

5 n and m are as defined above, RIl represents, when n is 0, an unbranched or branched alkyl group having 1 to 5 carbon atoms, whereas, when n is an integer of 1 to 12, R1 is methyl group, phenyl group, hydroxyphenyl group, amino group, hydroxyl group, hydroxyalkyl group, methylthio group or formyl group, or when n is 3, R11 and R 5 together may represent a single bond between carbon and nitrogen atom).

The present invention is hereinunder described in 420 greater detail by way of an Example for Reference and Practical Examples.

o 4.

es at .30 44*4 St i t i t C L1,S t'-11- SyntheLLAJLYA stiartn compoun1 r-Presonted by foQrmula t U)-L wzAla-Pro-OMa Z-Alla-OH (1 equivalent), Pro-OMe hydrochloride (1 equivalent) and TEA (1 equivalent) were dissolved in dry methylene chloride, and WSCD (1 equivalent) was added under Cooling With ice. Thereaftor, the mixture was stirred at room temperature for 20 hours, and the reaction mixture Was washed successively with IN HClj water, saturated aqueous sodium bicarbonate, water and saturated brine. After drying over anhydrous magneuium sulfate, the solvent was distilled off under vacuum. The resulting crude product was purified by medium-pressure liquid column chromatography on silica gel. The obtainAd Z-Aa-Pro-OMe (1 equivalent) was dissolved in ethanol, and boron trifluoride-ether complex (1 equivalent) and palladium on carbon (a smal. amount) were added. The mixture was subjected to catalytic reduction to remove the z group, And the solvent was distilled off under vacuum to obtain the end compound.

Instead of Z-Ala-OH; a) Z-Val-OH, b) Z-Leu-OHj C) Z- ?he-OH, d) Z-Met-OH and e) Z-norLeu-OH were used as starting compounds to obtain H-Val-Pro-OMe, ll-Leu-Pro'-OH# c') I-Phe--Pro-OH, H-Met-Pro-OMe and el) I-norLeu-Pro-OMe as oils, respectively, H-Lys(Z)-pro-OMe trifluoroacetate Boc-'Lys(Z)-OH (1 equivalenE), Pro-OMe hydrochloride equivalent) and TEA (I equivalent) were dissolved in dry methylene chloride, and WSC~I (I equivalent) was added under cooling with ice. Thereafter, the mixture was stirred at room temperature for 20 hours# and the reaction mixture was washed auccesaively with XN 1HClp water, saturated aqueous sodium bicarbonate, water and saturated b~rine. After drying over anhydrous magnesium sulfate, the solvent was distilled off under vaCUUm, The resulting crude product was purified by medium-presaure liquid column chromatography on silica !'nl to) obtain the end compound. Boc-Ly$(Z)-Pto-OMO (1 equivalent) thus obtained was dissolved in dry methylene chloride# and an excess of trifluoroacetic acid was added.

The mixture was was distilled o -12stirred (for about 6 hours), and the solvent ff under vacuum to obtain the end compound

I

II

I

t

I

a

I

I

4..

Instead of Soc-Lys(Z)-OH in Boc-GlU(Bzl)-OH was used as a starting compound to obtain H-Glu(Bzl)-Pro-OMe trifluoto.Acetate (oil).

H-Leu-Leu-Pro-OMe Z-'Leu-OH equivalent), H-Leu-Pro-OMe (I equivalent) synthesize4 in and TEA (1 equivalent) were dissolved in dry methylene chloride# and WSCD (1 equivalent) was added under cooling with ice. Thereafter, the mixture was stirred at room temperature for 20 hours, and the reaction mixture was washed successively with IN HCl, water, saturated aqueous sodium bicarbon 'ate, water arnd saturated brine.

After dryingj over anhydrous magnesium sulfatel the solvent was distilled off under vacuum. The resulting crude product was purified by medium-pressure liquid column chromatography to obtain the end compound. Z-Leu-Leu-Pro-OMe (I equivalent) thus obtained was dissolved in ethanol, and boron trifluoride-ether complex (1 equivalent) and palladium on carbon (a small amount) were added. The mixture was subjected to catalytic reduction in a hydrogen atmosphere to remove the Z group, and the solvent was distilled off under 4 vacuum to obtain the end compound as an oil.

f-Pro-Pro-OMe Z-Pro-OH (l equivalent), H-Pro-OMe hydrochloride (1 ;1 equivalent) and TEA (I equivalent) were dissolved in dry methylene chloride, and WSCD (1 equivalent) was added under ,cooling with ice. Thereafter, the mixture was stirred at '30 room temperature for 20 hours, and the reaction mixture was washed successively with IN HCl, water, saturated aqueous sodium bicarbonate, water and saturated brine. After drying over anhydrous magnesium sulfate, the solvent was distilled off under vacuum. The resulting crude product was purified by medium-pressure liquid column chromatography on silica gel to ob)tain the end compound. Z-Pro-Pro-OMe (1 equivalent) thus obtained was dissolved in ethanol, and boron trifluoride-ether complex (I equivalent) and palladium a

I

~a.

*6 'a 4~) *41 £111,

_LY_

-13on carbon (a small amount) were added. The mixture was subjected to catalytic reduction in a hydrogen atmosphere to remove the Z grup, and the solvent was distilled off under vacuum to obtain the end compound as an oil.

Example 1A N-oleoyl-Pro-OMe (SUAM 1131) H-Pro-OMe hydrochloride. (1 equivalent) and TEA (2 equivalents) were dissolved in dry tetrahydrofurane, and oleoyl chloride (1 equivalent) was added dropwise under cooling with ice. Thereafter, the mixture was stirred at room temperature for 6 hours, and the hydrochloride of TEA separted out was filtered off. The solvent was distilled off under vacuum, and the resulting crude product was dissolved in a small amount of ether. Thereafter, the mixture was washed successively with IN HC1, saturated brine, saturated aqueous sodium bicarbonate and saturated brine and dried over anhydrous magnesium sulfate. After concentrating under vacuum, an excess of diazomethane in ether was added to the reaction mixture, and unreacted oleic '0 acid was converted to methylester. The solvent was distilled off under vacuum, and the resulting crude product was purified by medium-pressure liquid column chromatography on silica gel to obtain the end compound (oil).

Instead of oleoyl chloride in a) palmitoyl chloride was used to obtain palmitoyl-Pro-OMe (SUAM 1135).

N-oleoyl-Leu-Pro-OMe (SUAM 1136) H-Leu-Pro-OMe (1 equivalent) and TEA (1 equivalent) were dissolved in dry tetrahydrofurane, and oleoyl chloride ,30 (1 equivalent) was added dropwise under cooling with ice.

The mixture was stirred at room temperature for 6 hours, and

S

t the hydrochloride of TEA which precipitated was filtered off. The solvent was distilled off under vacuum, and the resulting crude product was dissolved in a small amount of ether. The mixture was washed successively with IN HC1, saturated brine, saturated aqueous sodium bicarbonate and saturated brine and dried over anhydrous magnesium sulfate.

Aftet concentrating under vacuum, an excess of diazomethane -14in ether was added 9 and unreacted oleic acid was converted to methylester. The solvent was distilled off under vacuum, pressure liquid column chromatography on silica gel to obtain the end compound (oil).

Instead of oleoyl chloride in a) palmitoyl chloride, b) stearoy. chloride and c) linoleolyl chloride were used to obtain palmitoyl-Leu-Pro-QMe (SUAM 1141)t stearoy3.-Leu-Pro--OMe (SUAM 1142) and linoleoyl-Leu- Pro-OMe (SIUhM 1143), respectively. Further, instead of H- Leu-Pro-OMe in (b)f d) H-Val-Pro-OMe, e) H-Ala-Pro-OMe, f) H-Leu-Leu-Pro-QMe, g) H-Phe-Pro-OMe, h) H-Met-Pro-OMe and i) H-norLeu-Pro-OMe were used to obtain oleoyl-Val-Pro-OMe (SUAM 1147), oleoyl-Ala-Pro-OMe (SrJAM 1076), fl) oleoyl- Leu-Leu-Pro-OMe (StJAM 1159), oleoyl-Phe-Pro-OMe (SUAM 1194), oleoyl-Met-Pro-OMe (SUAM 1198) and il) oleoylnorLeu-Pro-ome (SUAM 1164), as oils, respectively.

Instead of H-Phe-Pro-Ome equivalent) and TEA (1 equivalent) in C) H-Lys(Z)-Pro-OMe trifluoroacetate (1 .t*s.0 equivalent) and TEA (2 equivalents) were used to obtain c') oleoyl-Lys(Z)-Pro-oMe (StJAM 1197) as an oil, and d) H- Glu(Bzl)-Pro-QMe triflujoroacetate (1 equivalent) and TEA (2equivalents) were used to obtain oleoyl-Glu(Bzl)-Pro-oi~e (SUAM 1196) as an oil.

N-oleoyl-Pro-Pro-OMe (SUAM 1195) H-Pro-Pro-OMe (1 equivalent) and TEA (1 equivalent) were dissolved in dry tetrahydrofurane, and oleoyl chloride (I equivalent) was added dropwise under cooling with iize.

The mixture was stirred at room temperature for 6 hours, and 3Othe hydrochloride of TEA precipitated was filtered off. The solvent was distilled off under vacuum, and the resulting crude product was dissolved in a small amount of ether, The mixture was washed successively with IN IHCl, saturated brine, saturated aqueous sodium bicarbonate and saturated brine and dried over anhydrous magnesium sulfate. After concentrating under vacuum# an excess of diazomethane in ether wa2 added, and unreacted oleic acid was converted to methylester. The solvent was distilled off under vacuum, and the resulting crude product was purified by mediumpressure liquid columan chromatography on silica gel to obtain the end compound (oil).

Oleoyl-Leu-Pro-CH 2 ON (SUAM 1138) Oleoyl-Leu-Pro-OMe (SUAM 1136) (2 g) obtained in Example 1 and sodiumborohydride (600 mg) were dissolved in t-butyl alcohol (60 nil), and the mixture was stirred under heating (800C). Then, absolute methanol (50 ml) was added dropwise under reflux. Thereafter, the mixture was refluxed under heating and stirring for two hours. After allowing the mixture to warm to room temperature, water (several ml) was added under cooling with ice, and unreacted sodiumborohydride was inactivated. Methanol and t-butyl alcohol were distilled off under vacuum and the residue was subjected to extraction with ethyl acetate. The extract was washed with saturated brine and dried over anhydrous magnesium smlphate. The solvent was distilled off under vacuum and the resulting crude product was purified by ~omedium-pressure liquid column chromatography on silica gel to obtain the end compound as an oil (1 g).

instead of oleoy3.-Leu-Pro-OMe in a) oleoyl-Pro- Oe (SUAM 1131), b) palmitoyl-Pro-OMe (SUAM 1135), c) palmitoyl-Leu-Pro-oMO (SUAM 1141), d) stearoyl-Leu-Pro-OMe (SUAM 1142), e) linoleoyl-Leu-Pro-OMe (SUAM 1143)l f) oleoyl-Ala-Pro-OMe (SUAM 1076), g) oleoyl-Val-Pro-OMe (SUAM 1147)r h) oleoyl-Leu-LeU-Pro-OMe (SUAM 1159)p i) oleoyl-Phe- 0 Pro-OMe (SUAM 1194), J) oleoyl-Met-Pro-OMe (811AM 1198), k) V oleoyl-norLeu-Pro-oMe (SUAM 1164), 1) oleoyl-Lys(Z)-Pro-ome C~3 0 (SUAM 1197) and m) oIeoyI-Glu(3zl)-Pro-o0Me were useO as starting compounds to obtain oleoyl-Pro-CH 2 0H (SUAM 1133)t palmitoyl-Pro-C1 2 0 (SUAM 1137)t palmitoyl- Leu-Pro-CH 2 0H (SUAM 1144), stearoyl-Leu-Pro-CH 2 0H (SUAM 1145), el) linoleoyl-Leu-Pro-CH 2 OH (SUAM 1153)r fl) oleoyl- Ala-Pro-cH 2 0H (SUAM 1077), oleoyl-Val-Pro-CH 2 0H (SUAM 1151), oleoyl-Leu-Leu-Pro-CH 2 0H (SUAM 1152) oleoyl- Phe-Pro-CH 2 0H (SUAM 1205), oleoyl-Met-Pro-CH 2 OH (SOAK -16- 1206), cleoyl-nor-Leu-Pro-C 2 OR (SUAM 1165), oleoyl- Lys(Z)-Pro-CH 2 OR (SUAM 1207) and in') oleoyl-Glu(CH 2 OH)-Pro- CH 2 OR (SUAM 1209) as oils, respectively.

oleoyl-Pro-Pro-CH 2 OR (SUAM 1204) Qleoyl-Pro-Pro-OMe (SUAM 1195) (2 g) obtained in Example 1 and sodiumborohydride (600 mg) were dissolved in t-'butyl alcohol (60 ml), and the mixture was stirred under heating (80 0 Then., absolute methanol (50 ml) was added dropwise under reflux. Thereafter, the mixture was reflUxed under heating and stirring for two hours. After allowing the reaction solution to wArm to room temperature, water (sevez-.' was added under cooling with ice, and unreacted sodiumborohydride was inactivated. methanol and tbutylalcohol were distilled off under vacuum and the residue was subjected to extraction with ethyl acetate. The extract was washed with saturated brine and dried over magnesium sulphate anhydride. The solvent was distilled off under vacuum and the resulting Crude product was purified by medium-pressure liquid column chromatography on silica gel to obtain the end compound as an oil (1 g).

EX-amle2I3-.

Oleoyl-Leu-Pro-CHO (SUAM 1140) UOleoyl-Leu-Pro-CH 2 OH (SUAM 1138) (1 g) obtained in Example 2 and TEA (800 mg) were dissolved in dimethyl sulfoxide anhydride (8 ml), and sulfur tioxide-pyridine complex (700 mng) in dimethylsulfoxide (S ml; was added under '...'stirring. The mixture was stirred at room temperature for about 1 hour, and was then poured into ice water (100 ml).

The reaction mixture was then subjected to extraction with ethyl acetate. The extract was washed successively with l0t citric acid solution, saturated brine, saturated aqueous sodium bicarbonate and saturated brine an-d dried over anhydrous magnesium sulfate. The solvent was distilled off under vacuum, and the resulting crude product was purified by medium-pressure liquid column chromatography on silica gel to obtain the end compound as an oil (700 mng).

Instead of oleoyl-Leu-Pro-CH 2 OH (SUAM 1138) in a) oleoyl-Pro-CH 2 OH (SUAM 1133), b) palinitoyl-Pro-CH 2

OR

-1 7- (SAN 1137)t c) palm:Ltoyl-Leu-Pro-CH OH (811AM 1144), d) stearoyl-Leu-Pro-CH 2 OH (SUAM 1145), e) linoleoyl-Leu-Pro- CH 2 0H (eVJAM 1153), f) Oleoy1l-Ala-Pro-CH 2 OH (SUAM 1077), g) Oleoyl-Val-Pro-C 2 OH (SUAM 1151)l h) oleoyl-Leu-Leu-Pro-

CHI

2 OH (811AM 1152), 1) Oleoyl-Phe-Pro-CH OH (SUAM 1205), j) oleoyl-Met-Pro-CH 2 OH (SUAM 1206), k) oleoyl-norLeu-Pro-CH 2

OH

(SUAM 1207), 1) oleoyl-Lys(Z)-Pro-CH.

2 oH (511AM 1207) and mn) oleoyl-Glu(,H 2 OH)-pro-CH 2 OH (811AM 1209) were used as qtartinq compounds to obtain aV) oleoyl-PL-o-CHO (SUAM 1134), PalMitojrl-pro-CH) (SUAM 1139), palmitoyl-Leu-Pro-CHO (SEJAM 1155), stearoyl-Leu-Pro-CHO (811AM 1154), el) linoleoyl-Leu-Pro-CH) (811AM 1157), fl) oleoyl-Ala-Pro-CHO (SUAM 1092), oleo~yl-Val-Pro-CHO (811AM 1156)t h') oleoyl-Leu-L~eu-Pro-Cno (811AM 1158), il) oleoyl-Phe-Pro-CHiO (SUAM 1212)p JI) oleoyl-Met-Pro-CHO (SUAM 1214), oleoylflOrLeu-Pro-CHQ (SUAM 1166), oJleoyl-Ly(Z)-Pro-CHQ (St.AM 1216) and inl) oleoy1--Glu(CHO)-Pro-CHO (811AM 1217), respectively.

oleoyl-Pro-Pro-CZIO (SUAM 1215) Oleoyl-Pro-pro-CH OH (511AM 1204) (1 g) obtained in *2 xample 2 and TEA (800 mg) were dissolved in anhydrous dimethylsulfoxide (8 ml), and sulfur trioxide-pyridine complex (700 mg) in dimethylsulfoxide (8 ml) was added under stirring. T1he mixture was stirred at room temperature for about 1 hour, and poured into ice water (100 ml). The 'Po reaction Solution was subjected to extraction with ethyl acetate, and the extract was washed successively with citric acid solution, saturated brine, saturated aqueous sodium bicarbonaite and saturated brine and dried over ,,30 anhydrous magneu-ium sulfate. The solvent was distilled off under vacuum, and the resulting crude product was purified by medium-pressure liquid column chromatography on silica gel to obtain the end compound as an oil (700 mg).

The analytical data for the compounds obtained are listed in Table 1. All the conipouds irn Table 1 are oils which are soluble in CCIV ether, CHC1 3

CH

2 C1 2 ACO~t and MeOH. SUAM 1194 is also soluble in benzene.

I

j The present inventors examined the capability of the compounds of the present invention to inhibit the decomposition of 2-glycyl-prolyl-B-naphthylamlde by proly.

endopeptidase and found that they exhibit strong anti-prolyl endopeptidase activity and yet show no inhibiting activity against proteinase such as papain, bronielain, trypsin, chymotrypsii, therrnolysin and pepsin, as shown in the Experimental Examplegs described later.

The compounds thus obtained are novel and exhibit excellent effects against amnesia.

li .et 2-S a.

a a *0 *4 4 a a a a S S a a a Table 1 7 7 Ex.-

NO.

Compound (SIJAM number) Molecular formula (molecular weight) IR spectrum film (cm- 1 Proton NMR spectrum 6 (ppm) from T14S in CDC1 3 (mAz)

D

I oleoyl-L- C 2 4

H

4 3 NO D~ ()Pro-OMe 24433),260H~m) 32(21{,rti) (SUAZ4-1131) (393-6) 42U~) .22~, l plmiC224114030.8-l.0(3H,m), 1.1-2..4(32H, Pro-OMe(376m) .02m) 7(Hs, (SUAK-11135) (6.)4.24U(H,rn) oleoyl-L- C0.8-1-1(9H,m), 1.2-2.3(3511, 1 Leu-L-Pro- C 3 0

H

54

N

2 0 4 in), 3.-7O(3H,s), 3.70(211,m), 014e (506.8) 4.50(1H,m), 4..84(lE,m), (SUAK-1136) 5.32(2H,m), 6.26(IH,d,J=8) palraitoyl-L- 0.8-l.l(9H,xn), 1.1-2.4(35H, 1 Leu-L-P~ro- C 28 11 52

N

2 0 4 3_70(211,m), 3.72(311,s), OMe (480.73) 4.5O(1l[,m), 4.84(lH,m), (SUAM-1141) G.25(1H,d,J=8.2) stearoyl-L- 1.1l-2-3(37H, 1 Leu-L-Pro- C 30

H

56

N

2 0 4 m)d, 3.60(4H,m), 3.66(3H,s); OMe (508.8) 4.42(lH,m), 4.78 (IH,m), (SUAR-1142) 16. 26 (1H, d, J=8 2) a e f* tfl~ a.

a S S a a S a ma F4~ 9.01% 0 Table 1 (cont'd) I -Molecular E-x. Compound formula IR spectrum Proton NMR spectrumE-S No. (SUAJ4 number). (molecular film (crW-) 6 (ppm) from TMS in CDC1 3

[D

lincleoyl-L- wih)0.8-1..l(9H,i), 1-1-2..3(29H, 014e (504.8) 3..66(311,s), 4-44(lH,m) ,I 4.BD(lH,m), 5..28(4H.,m), (SUAP4-114 3) 6. 1Hrdyj'=8) oleoyl-L-0.86(3H,t,J=G), 0..98(6H,,dd, 1 Val-L-o C 2

R

2 2 4 j-9, l.1-2.3(331,m), I Va-L-Po- C9H522043.70(3H,s), 3-76(2B,m), O[4e (492.7) 4.4-4-7(2H,m), 5-32(2H,ni', (SUAN-1147) 6.20 (lR,d,J=9) oleoyl-L- 3300, 2920, 0.88(3H,m), l..30-2-18(35Hmi). 49 1 Ala-L-Pro- C 2 7

H

4 8

N

2 0 4 2840, 1740, 3.70(3H~s), 3-58-3.8l(2H,m) -54.96 i 64 Wb Ome (464.7) 1630,, 1530, 4.51(1H,i), 4-76{lIH,m)ffc-.6i (SUAM-1076) 1170, 1190, 5..30(21,n), 6-72(1B~dJ=9) CHC1 3 0-B-1.0(15H,m), l 1-2.4(38H, oleoyl-L C65Nm), 3.70C2H~m), 3.72(3H,s), 1 Leu-L-Leu- C 3

H

5 3 0 5 4.50(2H,m), 4-74 (lH~rn), Pro-OI-e (619.9) 5.32(2H,m), 5-88(lH,d~J-=8), (SUAM-1159) 6.54 (Il,d,J=8) Jt-H n 4, 4, A a ft Table 1 (cont'ci) Ex. Compound foleular I spectrum Proton NMR spectrujm El-MS No. (SUM number) (molecular film (cm- 1 6 (ppm) from TMS in CDC1 3 Im ID (m/z) weight) 3400, 3330, 3250, 2920, oleoyl-L- 2850, 1740, 0-9(311,m), 1.0-2.4(34H,m), 1 Phe-L-I1ro- C 33 11 52

N

2 0 4 1630, 1600, 3.70(2H,m), 3.76(3His) (b)O~e(50.8 L150, 1420 ,4.50(lli,m), O Ne(540.8 1370, 1200, 5.35(2H,m), 6.40(lH,d,J=9.0), (SAM114)1090, 980, 7.24(5f,s) 3300. 2920, 0..90(31,m), oleoyl-L- 2850, 1740, 1.1-2.4(32H,m), 1 Met-L-Pro- C 2 9 11 5 2

N

2 0 4 S 1630, 1530, 3.12(31t,s), 2..58(2H,m), Wb OMe (524.8) 1440, 1360, 3,73(3H1s), 3-75(.2Hm), (SUAI-1198) 1270, 1190, 4.54(1ll,m), 4.94(lH,m), 1170, 950, 5..34(2H,m), 6-32(111d,J=8-0) 720, 680 Oleoyl-L- 0.8-1.0(611,m), 1.0-2.4(38H, 1 norLeu-L- C 30

H

54

N

2 0 4 in), 3..70(211,m), 3-.72(3f1,s), Pro-OMe (506.8) 14.50 (111,m) 4.76 (111,i) j(SUAM-1164) 6.28(lIH,d,J=8.0)

CO

a 0 S 0* *05 44. 0 *0s *050e4 S C

I.

S 0 Table 1 (cont'd) Ex.

NO.

Compound (SUAM number) Molecular f ormula (molecular weight) IR spectrum film (cm'1) Proton NMR spectrum 6 (ppm) from TMS in CDC1 3 EI-14S (m/z) -4 I P o leoyl-L- Lys L-Pro-1e (SUAM-1197)

C

3 8

H

6 lN 3 0 6 (655.9) 3300, 2850, 1710, 1530, 1240, 1170, 690 2920, 1740, 1630, 1450, 1190, 730, 0.90 (31,m), 1.1-2.4 (33H, 3.22(2Hrnij, 3. 7 2(211,rm) 4. ao (H,nd, 5.35 (2H,n) 7.35 (511,s) 3.660(1,s), 4.50 (lH,m), 5.10 (2H,m}, 6-34 (lH,d,J=a.G) oleoyl-L- 13300, 2920, '0.90[3H,m), l.0-2.8(36f1,m), 1 Blu(Bzl)- C 36 11 55

N

2 0 6 2850, 1730, 3-70(2H,m), 3.72(3H,s) L-Pro--OMe (611.8) 1630, 1450, 4.50(1R,m), 4.90(1H,m), (SU14116)1170, 730, 5.14 (2H,dd,j=15 0), (SU~4-196)690 5-35(2fi,rn), 6.38(H,d,J=9.0) oleoyl-L- 3470, 2920, 1 Pro-L-Pzo- C 29

H

50

N

2 0) 4 2850, 1740, 0.90(311,m), 1-2-2.4(3GH,m), C)ONe (490.7) 1640, 1430, 3.4-4.0(4H,m), 3.72(3H.,s), SAM115 1320, 119.0, 4.5-4.8(21,m), 5.35(2B-Im) (SUM-195)1170, 720 oleoyl-L- 0.8-1.1(911,mn), 1.2-2.3(35H1, 2 Leu-L-,Pro- C 2 9

H

5 4

N

2 0) 3 Mn), 3.6 (311,m) 3.9 (1H,m) C20H (78.7)4.2(flH,m), 4-54(IH,dd,j=7, (S(a UCH 2 H (48.7 4.9(1B,m), 5..34(2H,m), 4SU~t41138)6.08 (1l,d,J-=S) 9 0 9 5. 4 *4* 9 4e frB5* rAe 4, 9 5. N 5~ 44 4* C 44 4444 N ITable 1 (corit'd) MolecularI Ex. Compound formula IIR spectrum Proton NMR spectrum [a jEI-lI-S No. (SUAM number) (molecular film (cm- 1 6~ (ppm) from TH4S in CDC1 3 D oleoyl-L- f -1.1-2-4(32H1, 2 Pro-CH 2 0H C 23 iI 4 3

NO

2 mn), 3.48(211,m) 3. 62 (2H,n) (UR13) (365.6) I4-20(lH,m), 5.1811H,dd,J=7, I jJ=4) 5. 332P,2) 2 palMitoyl-L- C 21

H

4 1 N0 2 0 1-1-2-40(211, PrO-C11 2 0H I 3.4-3-8(41,i), (UM13)r339.)4-20(lIH,m), 5.18(1H,n) palmitoyl-L- O.8-l.l(9Hi,m),11-.35, 2 Lu--Po- C2B5N23 2a CH20-Pro (4 7 5 2 .7) 3 4.16U(H,m), 4 .50f1H,dd,J=7,r

C

2 OH 4.82lF,mi), [SUM-1-1144) 6.06 (lH,d,J=8) stearoyl-L- O.8-l.O(9H,m), 1.1-2-3(37H1, 2 Leu-L-Pro- C 29

H

56

NI

2 0 3 xi), 3..3-4.0(61,mn), CH20H (480.81 4-20(1H,mu), 4.50(1H,iu), (SUAM-1145) 4.85(lH,m), 6-12(lH,d,J--8) 0.8-1.0(91-,ni), 1l-2.4{29H1, linoleoyl-L- CHN 0, 2.72fH,x), 3.3-3-7(3]1, 2 Leu-L--Pro- C 29 5 2 2 3 In), 4.16(1H,un),

CH

2 0H (476-7) 4.56(lH,dd,J=-7, J=4), (SUN415- .8 0 UH,io) 5 -28 (4H (SUAM-153) .53(lH,d,J=8) .4 Table 1 (cont'd) IMolecular EX. Compound formula IR spectrum Proton WNIR spectrum E-4 No. (SUAM number) I(molecular f Ilm (CM- 1 6 (ppm) from TMS in CDC1 3 D (mfz) weight) oleoyl-L- 3400, 3300, 1 O.88(311,m), 1.29-2-19(35H, 2 Ala-L-Pro- C 26

H

4 8M 2

Q

3 2920, 2840, mn), 3.59(4[i,in), 4.20(lH,m), 294436

CH

2 0H (436.7) 1620, 1530, 4-51L(lH,m), 4.74(1E1,m), c=0..89 In W+ (SUAI4-1077) 1450, 1040 j5-31(2H,m) 6..68(1H1,d,,T=9) CRC1 3 (CDC1 3 int T14S) 2 Val-L-Pro- C 28

H

52

N

2 0 3 mn), 3.90(111,I

CH

2 0H (464.7) mn), 4-22(1H,in), 4.58{11,i), (CDC1 3 nt, TNS) o'Leoyl-L- 0.3-1.0(15H,m), 1.1-2.3(38H.

2 Leu-L-Leu-L- C 35

H

65

M

3 0 4 mn), 3.4-3.9(41,i), 4.26(lH, Pro-CH 2 CII (591.9) 4.50(2H,mn), 4.80(l.In), ISUAM-1152) 5.34 (2H,mt) ,5.84(1H,d,J=8), 6.84 (lHf,d,J=8) oleoyl-L- 3400, 2920, 0.88(311,m), 432,l 2 Phe-L-Pro- C 3 2 8 5 2

N

2 0- 3 2850, 1610, 3.00(211,m), 3.2-3.,8(4H,m), CH20HI (512.8) I1540, 1440, 4.20(21,in), 4.96(1Hf,m), 1040, 690 5-34(2H,in), 6-28(IIH,d,J=8iJ), (SUAM-KL205) 17-26 (511,s) I 1 111-1 "1 1.-11-1 -11.11, I' ll, 1-1-11 ft a 4. 0 .4 4. Table 1 (cont'd) r Molecularj EX. Cunipound formula IR spec trumi Proton NNIR spectrum El-MS No- (SUAM number) (moeclar, film 6 (ppm) from TMS in COC1 3 JcI (M/z) 1lolL 30 90 0-90(31,m), 1.0-2.4(34,n),} 2 Met-L-Pro- C 2

H

52

!N

2

O

3 S 2850, 1620, 3.160(H,), 2.4.2ff,m~ CAeo l-L 330 2 -7 5 0, 2935, 2. .03 H, r, 2.544 2H,, t~i CI 2 H(42.7) 150, 1445 4.94{1Rin) 5..34(2Hi,m), (SUAK-l206) OSr70 6.2B(1H,d,J=8-0) oleoyl-L- 0.,84(6H,xn), l.0-2..3(38n,ni), 2 norLeu-L- C 29 11 54 1N 2 0 3 3..56(3Hi,m), 80 (li,m) PrO-CE{20H (478.7) 4-12(111,m), 4..70(2H,m), (SUAN-1165) 5.38(2H,m), 6..46(2H,d,J=-8.0) 3300, 2925, 0..88(3H,m), 1..0-2.3{38H,zi), oleoyl-L- 376125 2850, 1700, 3.18(2ff,m), 3-4-3.9(3H,nd, 2 Lys(Z)-L- 1 3 1 6 N0 620, 1530, 4..30(2H,md, 4-.6-5-2(3H,m), (SUAM-1207) 1050, 730, G.38(1H,d,J=8.0), 7.34(5H,s) 700 2 I(a) oleoyl-L- Glu (CH20H) I5-Pro-CH,2OH (SUAK-12 09)

C

2 8

H

5 2

N

2 0 4 (480-7) 3300, 2850, 1610, 1450, 2920, 1720, 1530, 1180, 0.82(3H,m), 1.0-2.2(36H,m), 4.76(2H,M), 5-28(211,m), 7. 04 lH, d, J=8 -0) 720, 590 *0 a Table I (cont'd) M~olecularI EX. Comipound formula IR spectrum Proton NMIR spectrum []EI-?4S No. (STJAM num~ber) (molecular film (crn-i) J (ppm) from TMS in CDC1 3 [aD (m/z) weight) oleoyl-L- 3400, 2920, 0.86(3n,m), 1.0-2.4(36H,m), 2 Pxo-L-Pro- C 2 0H 50 1 2

O

3 2850, 1620, I8--02~) CH20H (462.7) 1430, 1320r 3.70(5H,m), 40(2H~m,m (SUAfK-1204) 72110, 330, 220, 0.8l~l9H~r~) :L2-2.](35nl, oleoyl-L- 3300,I 2 0 17301 .3-7(2H,mn), 4_52(11m), 47 3 Leu-L-Pro- C95123 2825, 1730,Hm) 477(2~) 79 CHO (476-7) 1620, 1540, 4.85(lH,d,=8, 3(2H04) in.; (M-I (SUA-110) 4409.50(IH,d,J=21z) CHC1 3 oleoyl-L- CH O 30,22, 0,8-.0(3Hm),

D

3 Pro-CHO C2341N2 2850, 1252, 1.1-2.4(32H,m), 3.54(2HEm), D40 364 (363.6) 2650, 1420 4.24(1H,m), 5.33(2H,m), -46.00 i M1 (SUAN--1134) 160 40 9.51 (fl,d,J=2) cC103I C3 pa.lmitoyl-L- 330090 .8KL.0(3H,m), [a]I3 3 Pro-CHIO 281192 350, 2120, .1-24(32,m), D3.2 338 (337.5) 2630, 1430 3.56(211,m). 4_24(11T,mi), -39.53 i (KM++1) (SUAL4-1139) 160 40 9.52 (1H,d,J=2) c1 3 i at a a a a a a ?i Table 1 (cont'd) Ex.i compound formula Mlcar IR spectrum Pro~ton NMR spectrum IIS No.j (SUAM4 number) (molecular film (cxn 1 6 (ppm) from TMS in CDC1 3 D mz weight) palwitoyl-L- 3300, 2920 0.8-1.1(911,m), 1.2-2-.4 1C 3 euL-ro C 7

H

0 N0 3 280,170 (35H,m), 3.4-4-0(2H,m),D 3 e u L r o 2 7 5 0 2 0 8 5 7 3 4 5 2 1 H m 4 8 8 1 H m 4 2 5 0 4 1 CHO (4507) 1620. 1540, 604lIHda-8) c=1.70 in (SUAK-1155) 1440 9.52(iF,dJ=i) CHC1 3 starylL-320,290 1.1-2.3 1 L 3 searo-L-- 3290. 292 28 0, 37H,nj), D47 -LC140 2 4 8 2 8) 1250, 1755 AU,) 4-80(lH,m), -41.9 0 CAi (478.8 1620, 55 16 .77(IH,d,J=8), c=1.16 in (SUA-114) 4409.40(1H,d.J=2) CEIC1 3 08-.l(H~r),1.1-2.3(2911, linoleoyl-L- 3280, 2920, mn), 2.77(21,in), 3-5Q(1H,n), 4a] 3 Lu-Po C 2

H

0 0 3 2850, 1725, 3.83(lfI,rwd, 4.56(1H.m),-13947 CHO (474.7) 1620, 1535, 4.86(Ifi,m), 5.35(4Hbm), ci0in (SUAR-1157) 1440 6.02(1H,d,J CHC13 9.50 (lII,d,J=2) 0.80(3H,in), 1.18-2.19(35H,m), 28 oleoyl-L- 3300, 2920, 3.50(211,m), 4-43(1ll,m), [olD 3 Ala-L-Pro- C 26 11 46

N

2 0 3 2850, 2720, 4.66(111,in), S.25(21,rn), -35.10 434 CHO (434.7) 1730, 1620, 6.64(1HdJ9), c=0.73 in SUJMA-1092) :1530, 1450 .(Hd,=)CC1 *0 V C. C *C C C 5 0e C S S 4$ Table I. (cont'd) Molecular EX. Compound formula jIR spectrum' Proton 14MR spectrum El -KS No. (SUAI number) (mo1e"'la if ilm (crirC) 6 (ppm) from TMS in CDC1 3 (GID (m/z) weight)I oleoyl-L- CH O 3300, 2920, 10.8-1.1(911,m), 1.1--2.3(33H, 3 Val-L-Pro- C2850N23 2850, 1725, 3.72(2H,m), 4-54(111,m), D 463 CHO (462.7) 1620, 1525, 4.66(1Hf,dd,J -35.8 (4+1 (SAK116)1405.34(2H,m), 6.08(IH,d,J=8), c=1.4; in +1 (SUM-156 1409.53 (111d,32) CRCl 3 0.8-l-l(15Hm), 1.1-2-3 oleoyl-l- C 3280, 2920, (3811,n), 3.3-4.0(2i,m), [a]D 9 3 Leu-L--Leu-L- C31 5

H

63

N

3 0 4 2850, 1730, 4.50(211,m), 4.80(lf,ni),D59 Pro-CHO (589.9) 1635, 1540, 5.34(2H~m), 5.80(1H,d,J=8), -92.8o(+1 (SUA-1158) 1440 6.50(1H,d,JT=8), C1i 9.50 (1H,d,J=21 H1 oleoyl-L- 3300, 2920, 0.88(3H,m), 1-0-2.4(32H,m}, 3 Phe-L-Pro- C 32

H

50 tJ 2

O

3 2850, 1730, 3.08(2fl,m), 3.60(2H,m), ()CHO (510.8) 1620, 1530, 4.38(lli,m), S-0O(lH,nt), (SAM122)740, 700 7.26(51[,s), 9.32(lH,d~J=-8.0) 3300,2920, 0-88(3Hl,m), 1.0-2.3(34{,m), oleoyl-L- 30 90 2.12(3H,s), 2-58(211,m), 3 Met-L-Pro- C 2 8

H

50

N

2 0 3 S 2850, 1730, 3.74(2fl1m), 4..54(lHf,m), CHO (494-8) 1620, 1530, 4.96(1H,m), 5..33(2f],m), {SUAM-1214) 1440, 1340, 6..26(IH,d,J=S-0), 1110, 750 9.50(1H,d,J=2-01 ft S S ft.

S 5 0 5 S ft ft rft ft Table 1 (cont'd) Molecular Ex. Compound formula IR spectrum Proton NMR spectrum El-14S No. (SUAM number) (molecular film (cu-1) 6 (ppui) from TMS in CDC1 3 D (M/z) weight) oleoyl-L- 3300, 2920, 0-84(6H,rn), 1.0-2.3(38H,m), 3 norLeu-L- C 29

H

52

N

2 0 3 2850, 1730, 3.70(2H,m), 4-50(lni,m), (a)ProCHO(47.7) 1620, 1540, 4.77(lH,m), 5.30(2H,m), Pr-UO(47.7 1450, 1110, 6_4O(1H,d,J (SUJ4116)990. 72-0 9.47(lB,d,j 3310, 2930, 0.88(31,n) 1_0-2.4(381.rn), oleoyl-L- 2850, 1710, 3.20(2F1,m), 4.54(lJ1m), 3 Lys(Z)-L- C 3 7 H1 5 9

N

3 0 5 1620, 1530, 4.82(211,m), 5.08(211,sJ 1 Pro-CHO (625-9) 1450, 1340, 5.32(2H,xn), (SUM-116)1250, 1130, 6.26(1ll,d,J=8.0), (SUZ4126)1020, 740, 7.33(5ll,s), 700 9.48(1H,d,J=I.5) O.86(3HEm), 1..0-2.6(3611,mn), oleoyl-L- 3300, 2920, 3.70(211,M), 4.60(lH.m), 3 Glu(CHO)- C 28

H

4 8

N

2 0 4 2850, 1720, 4.84(IH,m), 5.32(2Hni), L-Pro-CHO (476.7) 1620, 1530, 6.34(1H,d,J=8.01, (SUAM-1217) 1440, 1080 9_50(IH,dd,J=3.0, 9. 78 (1U, d,JT2 0) oleoyl-L- 33,50, 2920, 0.86(311,m), 1.0-2.4(361-1,m), 3 Pro-L-Pro- C 2 8 11 4 8

N'

2 0 3 2850, 1720, 3.60(411m), 4..60(21I,m), CHO -(460-7) 1640, 1430, 5.32(2H,m), (SUM-115)1320, 1200, 9.50(l1_,d,J=2.0) ISUN1-15 7501 The method of Yoshimoto and TsuUu (To Yoshimoto and D. Tsuru, Aqr. Biol. Chem. All, 2417, 1978) was used to measure the anti-prolyl endopeptidase activities of several compounds of the present invention. A mixture of 0.0025M zglycyl-proline-0-naphthylamide (0.25 mil), 0.1M phosphate buffer (PH, 7.01 0.99 ml) and a solutioi of a particular anti-prolyl endopeptidase compound (0.01 ml) was incubated in a test tube at 3700 for 3 minutes. Thereafter, 0.1 mil of a solution of prolyl endopeptidase (0.2 U/mi) was added and the mixture was heated at 3500 for 10 minutes. After the reaction, 2.0 ml of rriton x-100 in 1M4 acetatze buffer (pHi, was added to the reaction mixture until the final concentration of the surfactant was 10%. The mixture was left at room temperature for 15 minutes and the absorbance at 410 nm was mpeasured.

A sample for blind test was prepared by using the ;buffer instead of the anti-prolyl endopeptidase compound and absorbance was also measured. The percent t inhibition of prolyl endopeptidase was calculated by the formula: -a/)x30,ndte amount of a speoific compound to achieve 50% inhibition (Ic 5 o) was determined.

The results are shown in Table 2.

4 6 6 t tL -31is CompoundIcso (rig/test tube) BtJAM 1155 0.7 SUAM 1154 SUAM 1158 80.0 STJAM 1092 12.0 SUAM 1156 0.7 SUAM 1157 0.6 SUAM 1140 0.8 SUAM 1166 0.8 SUAM 12:12 0.4 SUA.1 1214 0.6 StJAM 1216 0.6 SUAM 1217 0.3 SUAM 1215 0.3

V

V. t~ r .t o

I

0* Of

S*

V

*000 *0 *600

I#

ft 20 Ex am'p1e Measu remeii of ireventive erff-ect againast experimnta1 amn esia& ca used -n r-a-tsty sc~noa ine int r aeRiji.Le a 1 0 administratin) Several of the anti-prolyl endopeptidase comnpounds of the present invention were checked for their ability to prevent the inhibition of long-term memory fixation by 25 scopolamine. Solutions of physiological saline (0.3 ml) t that contained selected compounds of the present inventions t in varying amounts (11 mg, 0.25 mg, 0.1 mg, 0.025 mg and 0.010 mg/kg) were administered intraperitoneally once a day to Wister male rats (100 120 one hour after the administration, electric shocks (1.7 m.A) were applied to the rats so that they would acquire passive avoidance learning.

Immediately thereafter, scopolamine was administered intraperitoneally to each rat in an amount of 3 mg per kg of body weight.

The result of the test war, assessed both 24 hours and 48 hours after the administration of scopolamine. The number of amnesic rats and of sound rats was counted for each of the control group (rats to which the test compounds -32had not been administered but to which only scopolamine and physiological saline had been adminisee rrprtnal and the treated group (rats to which both the test compound and scopolamine had been administered). The resp~lts are shown in Table 3.

t It.4 6* 4 *1000 ~ii~iiils an~i i- si~i -r S 5 4 a 4. n i Table 3 Amnesia test with rats (intraperitoneal administration) Learning Avoidance time Pharmacological Drug(sec.)i effects amice No. of Initial No. of Leaffc Saleafte rats avoid- avoid- No of Percenttesedane nc ing 24 hrs.'1 48 hrs. ammesic ag learningafte dring tim later later rats/Mo. of amiesia learning (sec.) rats tested 1 PYSiological physioloqicaiil

J

saline saline 9 62 2-3 100 230-0 209-3 3/9 3 2) physiological scopolamine 10 2.7 2.9 100 90.6 110.4 8/10 saline (3 g/k-i 3) SUAM 1156 scopolamine 184-0 119-8 5/10 (1 mg/kg-i-p.) (3 ng/kg-i. p.) 4) SUN.! 1156 scopolamine (250 pg/g-ip.) (3 ma/kg-i.p.) 10 2.1 2.8 100 240-6 226.0 2/10 SUAM 1156 scopolamine (100 pg/kg-i.p-) (3 uq/kg-i. p.) 6) SUAM 1140 scopolamine 5 95 i6 100 73.0 1030 3/5 (1 mg/kg-i-p.) (3 mg/kgi.p.) 7) SUAM 1140 scopolamine (20 3 gicgi~4 5 1.7 2.4 100 263 284 1/5 (250,p/kq-iip.) (3 mg/kg-i~p.) 8) SUAM .140 scopolaminne 5 3.1 1.8 100 300 300 0/5 0 (100 pg/kg'i-p.) (3 mw/kgi.p.) r' f. 4.

a.

'-a S a a at L 4 4 a C. ear 4** aF t15 4 a a 5 4* 5* a 49 a Table 3 (cont'd) Learning Avoidance tiwe Pharacological Drug NCo. of I nta 1 No. of (sec.) effectsj admninisee I _ar-r Sait~le afte rats avoid- avoid- inj No. of Percentlerig -ested ance ance tie 24 hrs. 48 hirs. agesii Ulmmin during tie later later rats/,No. of anirisia learning rats tested M% jag/kg- i. (3 nu/kg 0 0 0 SUIAM 1140 scopolanine 5 47 20 10 150 190254 pg/kg-Lp.) (3 img/kg-i.p.) 5 47 20 10 15. 8. 11) SWAM 1158 scopolamin 10 28 30 10 30 3001 (100 ig/kg-i.p.) (3 rim/gi.ip.) 0 2. 00 30 3001 12) SUAM 1158 scopolamine 10 17 2300 0000/0 pg/kgi.p.) (3 Kq/kg-i-p. 10 17 2300 0000/00 13) SUiAM 1155 scopolamine 0 22 30 10 0000/00 (100 pt/kgi.p.) (3 urjkg i. p. 10 22 3000 30 30/0 14) SUAiM 1154 scopolamine 147 2- 10 30 30 /00 (100 p~q/kg-i.p.) (3 mq/kg i. p. 10.7 2000 300/0 SU7IP 1214 s~mpolamne 19- 1.10730 0.06/10 (I irqAg-i-P.) (3 mg/kg-i-p. 10 95.600 70 130 16) SUAM 1214 scopolamine, 12. 2- 10220 0 (250 pg/kg-i.p-) (3 nnkg-i.p.) 1 0 4. 2. /02 4,t, a.' t f t ft a. ft ft Table 3 (cont'd) Iarning Avoidance tirne Phannacological DrugI (sc. effects No. of Initial No. of a I e satleem rats avoid- avoid- No- of Percentafter tested ance arbe iig 24 hrs. 48 hrs. anresic age learning I time during tine later later rats/No. of. aesia (sec.) (learni rats tested (100%Jr p Ip (3214 n'Oj/Wi~p. 10 1.8 1.6 100 300 2764 1/10 (100 Pq~kg-i--) (3 ing/kg-i-.) 18) ST.4 1214 scopolnnlfe, 10 2-2 2.0 100 300 300 0/10 0 pg/kg-ii~p~p. (3 nxj/q-.ip.

19) SUAM 1214 scopolamine 10 4.6 1.9 100 300 300 0/10 0 pgckg-i-p.) (3 mgkg-i.p.) SUeM 1166 scopolamine p/kgi.Lp.) (3 zq/kg-i.p.)100 300 300 0 21) SUAM 1212 scopolamine 2.7 2.4 r 100 1300 (3285 n0/0 0 22) SUAR 1215 scopolamine 10 3-2 1.8 100 300 300 0/10 0 pg/kg-i-p.) (3 mng/kgi.p.) 23) SUArM 1215 scxnpoiland me1ne pg/kg-i.p.) (3 mj/g-i-p.) 10 2.2 2.4 100 300 00 0/10 0 i.p. intraperitoneal administration 1 r -36- SUAM 1156 exhibited no remarkable ef L-ect when administered orally in an amount of 1 mg/kg, but exhibited a considerably excellent anti-amnesic action when administered orally in an amount of 0.1 mg/kg. Although SUAV 1140 and SUAM 1214 exhibited no remarkable effect when administered orally in art amount of 1 mg/kq. they oxhibited a considerably excellent anti-amnesic action when administered orally in amouhts of 0.1 mg/kg and 0.025 mg/kg, respectively. SUAM 1214 exhibited a rema~rkable antil0 amnesic action even when the dose was lowered to 0.01 mg/kq.

~SUAM 1158, J.155 and 1154 exhibited a strong anti-amnesic action when administered orally in an amount of 0.1 mg/kg.

SUAM 1158 exhbited excellent effect even at a close of 0.025 mg/kg. In addition, SUAM 1166, 1212 and 1215 also exhibited a strong anti-amnesic: action when administered orally in an amount of 0.025 mg/kg. SUAM 1215 exhibited a strong antiamnesic action. even at a dose of 0.01 mg/kg. The action of all the compounds depends upon the dose, Ant1i-hvnnviA ant-inn~ tL t~ I t tT~4 t Lit IL I.

t

L,

t It I I 9 0 Se '23 0* 9 9 p *e 9*9.~ S 9

I

C I The compounds of the present invention were checked for their anti-hypoxia action in ddY strain male mice (body weight: 25 34 Each of the treated groups consisted of 7 to 10 Mice. Test samples were prepared by adding 2 to 3 drops Of Tween 8&to physiological saline and suspending the respective compounds in the saline. The so conditioned test samples were administered (0.1 m/10 9) to each of the mice used.

At 30 minutes after the administration, each of the mice was placed in a desiccator having a volume of 1 Lj and the desiccator was evacuated to 180 mmHg using a vacuum pump. The period of time from the start of the evacuation until the arrest of breathing was defined as Survival Time (mirvute).

When a mouse survived for 15 minutes or more, the sur .:ival time was recorded as being 15 minutes. Student's t-test was employed for significance analysis.

The results are shown in Table 4 below.

.4.4.4 a a *a a a a AS t444fl, .4 flu .4 4* r .4 Table 4 Compound (m/kr uhsed f(~l F 'ai structure Control Drug Control Drug 1al 1 0/10 0/7 2-32 0.11 2.94 0.27* 1.27 0 SUAM 1212I N N 1N H 0 10 0/10 0/7 2.25 ±0-15 2.89 ±0-29# 1.28 0.05 Sp 0-1 -38- The prescint invention aloo relates to an anti-iimr-,ic agent useful for the treatment of diseases originating from organic disorders in the brain, The anti-amnesic agent comprises at least one compound of the formula ti) together with a pharmaceutically acceptable carrier.

The formulation of the agent of the invention includes either solid formulations such as capsules, tablets and powders, a liquid formulations such as elixirs, syrups and suspensions for oral administration. Alternatively, the active compounds may be formulated as injections or suppositories.

The carrier included in the agent of the invention may be selected from pulverulent solid carriers such as lactose, saccharose, dextrose, mannitoll sorbitol, cellulose, and glycine etc.

The agent of the invention may further contain a lubricant, a binder or a disintegrater. Examples of lubricant are silicon dioxide, talc, magnesium stearate and polyethylene glycol. Examples of binder are starch, :0120 gelatin, tragacantht methyl cellulose and polyvinyl pyrrolidone. Examples of disintegrator are starch and agar etc.

The active ingredient of the agent of the invention is orally administered to an adult patient in a dose of 10 to 4,000 mg, preferably 100 to 1,000 mg/day, or administered parenterally in a dose of I to 2,000 mig, preferably 50 to 500 mg/day. The dose may be varied *:..,~depending on the disease, age, weight, or condition of the patient or the formulation of the drugs, Formulation

I

Ingredient Part Compound of the formula Lactose Magnesium oxide (MgO >96t) is The ingredients are mixed thoroughly, and tablests or capsules are formulated from the mixture.

Formulation 2 Ingredient Part -39- Compound of the formula Starch Lactose The above ingredients are mixed thoroughly, and s powders or fine granules are formulated from the mixture.

Formulation 3 Ingredient Part: Compound of the formula 1.

Surface active agent Physiological saline 94 The above ingredients are mixed under heating, and 14 dispensed uider sterile conditions into amipoules to obtain injections.

It

SO

*of

Claims (8)

1. A compound of formula (I) R O S (CH 2 n 0 R C N C C (I) H m CHO (wherein m is a number of 0, 1 or 2; n is a number of 0 to 12; R 1 is a saturated or unsaturated unbranched hydrocarbon group having 5 to 25 carbon atoms (the unsaturated carbon chain may include a plurality of double bonds); R 2 represents, when n is 0, an unbranched or branched alkyl group having 1 to 5 carbon atoms, whereas, when n is an integer of 1 to 12, R 2 is methyl group, phenyl group, hydroxyphenyl group, formyl group, amino a group, hydroxyl group, methylthio group, hydroxyphenyl group whose hydroxy group is protected, protected amino group, protected hydroxy group or a hydroxyalkyl group; R 5 is hydrogen atom, or when n is 3, R 2 and R 5 together may represent a single bond between carbon atom and nitrogen atom).

2. A compound according to Claim 1, represented by the formula (Ia): 9 0 O (CH2)n O II I 11 S R1---C-N-C C N (la) H H (wherein n is an integer of 1 to 12; R 1 represents the C30 meaning given in Claim 1; R 2 is methyl group, phenyl group, hydroxyphenyl gioup, formyl group, amino group, hydroxyl group, methylthio group, hydroxyphenyl group whose hydroxy group is protected, protected amino group, protected hydroxy group or a hydroxyalkyl group,

3. A compound according to Claim 1, represented by the i ~~p 41 formula (Ib); R C -N 1 (Ib) O N CHO (wherein R 1 represents the meaning given in Claim 1).

4. A compound according to Claim 1, represented by the formula (Ic): 0 H R 0 II2 II (Ic) R1--C CH -C (Ic) CHO t ,t (wherein m and R 1 represent the meaning given in Claim 1; R 2 is: CH -CH -CH -CH-CH -CH 3 2 '-CH 2 3 3 CH 3 CH 3 S' -CH or -CH 3 CH3 3 A compound according to Claim 1 or Claim 2, wherein n represents a number of 1 to 6.

6. A compound according to Claim 1 substantially as hereinbefore described with reference to any one of the examples.

7. A process for producing a compound as defined in Claim 1 said process comprising reacting a carboxylic acid, carboxylic acid anhydride or carbonyl chloride represented by the formula: 0 0 0 II II ii R -COOH, R -C-O-C-R 1 or R -C-C1 (wherein R 1 is a saturated or unsaturated unbranched hydrocarbon group having 5 to 25 carbon atoms (the unsaturated carbon chain may include a plurality of double bonds)), and peptide or amino acid, having a proline residue whose carboxyl group has been converted to a lower alkylester, of 42 the formula (II): R CH n CH (II) COOR 7 (wherein m is a number of 0, 1 or 2; R7 is a lower alkyl group; R 6 represents, when n is 0, an unbranched or branched alkyl group having 1 to 5 carbon atoms, whereas, when n is an integer of 1 to 12, R 6 is methyl group, phenyl group, hydroxyphenyl group whose hydroxyl group is protected, protected group or methylthio group; R 5 is hydrogen atom, or when n is 3, R 6 and R 5 together may represent a single bond between carbon atom and nitrogen atom); to form a compound of the formula (Id): R R- C--N--CH CO N (Id) m COOR 7 (wherein R 1 R 2 R 5 R 6 R 7 n and m are as defined above), (ii) reducing said compound of the formula (Id) to form a °o compound of formula (If): R 18 R 5 (CH 2 CH CO N (If) m 30 CH 2 OH (wherein R 1 R 5 n and m are as defined above; R represents, when n is 0, an unbranched or branched alkyl group having 1 to 5 carbon atoms, whereas, when n is an integer of 1 to 12, R 8 is methyl group, phenyl group, hydroxyphenyl whose hydroxyl is protected, protected amino group, protected hydroxyl group, methylthio group or hydroxymethyl group, or when n is 3, R 8 and R 5 together may represent a single bond between carbon atom and nitrogen atom), and I 43 (iii) oxidizing said compound of the formula (If) to form a compound of formula (Ih): 0 R 5 (CH2) n R 1 CH- CO N (Ih) CHO (wherein R 1 R 5 n and m are as defined above; represents, when n is 0, an unbranched or branched alkyl group having 1 to 5 carbon atoms, whereas, when n is an integer of 1 to 12, R10 is methyl group, phenyl group, hydroxyphenyl group whose hydroxyl group is protected, protected amino group, protected hydroxyl group, methylthio group or formyl group, or when n is 3, R10 and R together may represent a single bond between carbon atom and nitrogen atom), S* and if a compound of the formula (Ih) has a protected group, said group is removed to form a compound of formula (Ii): -R fll illm r i 11 i 85 (CH2 n CH CO--(i) CHO S (wherein R 1 R 5 n and m are as defined above; R1 represents, when n is 0, an unbranched or branched alkyl group having 1 to 5 carbon atoms, whereas, when n is an integer of 1 to 12, R 1 1 is methyl group, phenyl group, hydroxyphenyl group, amino group, hydroxyl group, hydroxyalkyl group, methylthio group or formyl group, or when n is 3, R11 and R 5 together may represent a single bond between carbon atom and nitrogen atom).

8. A process according to Claim 7 substantially as lhereinbefore described with reference to any one of the examples.

9. A pharmaceutical composition comprising a compound as defined by any one of Claims 1 to 7 and a pharmaceutically 44 acceptable carrier. DATED: 16 October 1989 PHILLIPS ORMONDE FITZPATRICK Attorneys For: SUNTORY LIMITED 5773h V 9* 94 4 9 S *9 4e 4. 9* 4 *1 #9 *9 4 4 9, 9. 9 *4 9 4 9 4 1 I 4