CA1182110A - Physiologically active tetrapeptides - Google Patents
Physiologically active tetrapeptidesInfo
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- CA1182110A CA1182110A CA000355887A CA355887A CA1182110A CA 1182110 A CA1182110 A CA 1182110A CA 000355887 A CA000355887 A CA 000355887A CA 355887 A CA355887 A CA 355887A CA 1182110 A CA1182110 A CA 1182110A
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Abstract
PHYSIOLOGICALLY ACTIVE TETRAPEPTIDES
ABSTRACT
This invention provides tetrapeptide derivatives of the formula (I):
(I) wherein R1 and R2 are methyl, 1-methylethyl, 2-methylpropyl, 1-hydroxyethyl, 2-carboxyethyl or 4-aminobutyl and Y is
ABSTRACT
This invention provides tetrapeptide derivatives of the formula (I):
(I) wherein R1 and R2 are methyl, 1-methylethyl, 2-methylpropyl, 1-hydroxyethyl, 2-carboxyethyl or 4-aminobutyl and Y is
Description
BACKGROU D OF THE I ENTION
1. Field of the Invention This invention rela-tes to synthetic chemical compounds which are tetrapeptides and exhibit inhibitory activity against enzymes such as aminopeptidases.
1. Field of the Invention This invention rela-tes to synthetic chemical compounds which are tetrapeptides and exhibit inhibitory activity against enzymes such as aminopeptidases.
2. Description of the Prior Art __ Several physiologically active pep-tides or N-acylated peptides have been found in the culture broths by some of the present inventors. These substances, e.g.
leupeptin, antipain, chymostatin and pepstatin, inhibi~
trypsin, papain, chymotrypsin and pepsin, respectively, but all these inhibitors have their effects on proteases which act in endo-type reaction. For further ~isclosures of these see Enzyme Inhibitors of Microbial Origin, Hamao Umezawa, University of Tokyo Press (1972) in Chapter IV~
Inhibitors of Proteolytic Enzymes (pages 15-52) as follows:
Page Numbe Leupeptin 15 Antipain 29 Chymostatin ~2 Pepstatin ~4 Bestatin, which has also been found in a microbial culture broth, inhibi-ts an exo-type proteolytic enzyme, i.e.
aminopeptidase B and leucine amînopeptidase, but it does not have any inhibi-tory effect on aminopeptidase A[U.S. Pat. No.
4,029,547~.
~`
Bestatin has the chemical name [(2S,3R)-3-amino-2-hydroxy-4-phenylbutanoyl~-L-]eucine and -the following structure ~C~
Other peptides were disclosed in U. S. 4,189~604 which claimed compounds having the formula R -CH -CH-CO-NH-CH~COOH
I 1 l2 N ~ OH R
wherein R is ( C~2 ) n-wherein R3 is hydrogen, chloro, methyl, nitrog hydroxy or amino and n is O or l and R is (lower)alkyl having l to 6 carbon atoms, hydroxy(lower)alkyl, alkylthioalkyl, carboxamido(lower)alkyl or carboxy(lower)alkyl provided that when Rl is benzyl and R2 is isobutyl the configurat~on of the compound is (2S,3R,2'R), {2S,~S,2'S) or (2S,3S,2'R).
The present authors have discovered that _trept~yces sp. ME98-M3(FERM-P 3722) produces new tetrapeptide compounds of the formula (II) named amastatins which contain at the N-terminal a new ~-amino acid unknown in the literature and having the formula `CH CH3 C ~ CH~ CH3 H2N-CH-C-C-NH~CH-C-NEI-CH-C-N~I-CH-COOH (II) OHO O O
wherein R is a carboxymethyl or 2-carboxyethyl group, and found that these tetrapeptide compounds not only inhibit aminopeptidase A but also stimulate the antibody formation (U. S. Patent 4,167,448; J. Antibiotics ~1(6) 636-638, 1978).
In the present specification, the said new ~-amino acid,
leupeptin, antipain, chymostatin and pepstatin, inhibi~
trypsin, papain, chymotrypsin and pepsin, respectively, but all these inhibitors have their effects on proteases which act in endo-type reaction. For further ~isclosures of these see Enzyme Inhibitors of Microbial Origin, Hamao Umezawa, University of Tokyo Press (1972) in Chapter IV~
Inhibitors of Proteolytic Enzymes (pages 15-52) as follows:
Page Numbe Leupeptin 15 Antipain 29 Chymostatin ~2 Pepstatin ~4 Bestatin, which has also been found in a microbial culture broth, inhibi-ts an exo-type proteolytic enzyme, i.e.
aminopeptidase B and leucine amînopeptidase, but it does not have any inhibi-tory effect on aminopeptidase A[U.S. Pat. No.
4,029,547~.
~`
Bestatin has the chemical name [(2S,3R)-3-amino-2-hydroxy-4-phenylbutanoyl~-L-]eucine and -the following structure ~C~
Other peptides were disclosed in U. S. 4,189~604 which claimed compounds having the formula R -CH -CH-CO-NH-CH~COOH
I 1 l2 N ~ OH R
wherein R is ( C~2 ) n-wherein R3 is hydrogen, chloro, methyl, nitrog hydroxy or amino and n is O or l and R is (lower)alkyl having l to 6 carbon atoms, hydroxy(lower)alkyl, alkylthioalkyl, carboxamido(lower)alkyl or carboxy(lower)alkyl provided that when Rl is benzyl and R2 is isobutyl the configurat~on of the compound is (2S,3R,2'R), {2S,~S,2'S) or (2S,3S,2'R).
The present authors have discovered that _trept~yces sp. ME98-M3(FERM-P 3722) produces new tetrapeptide compounds of the formula (II) named amastatins which contain at the N-terminal a new ~-amino acid unknown in the literature and having the formula `CH CH3 C ~ CH~ CH3 H2N-CH-C-C-NH~CH-C-NEI-CH-C-N~I-CH-COOH (II) OHO O O
wherein R is a carboxymethyl or 2-carboxyethyl group, and found that these tetrapeptide compounds not only inhibit aminopeptidase A but also stimulate the antibody formation (U. S. Patent 4,167,448; J. Antibiotics ~1(6) 636-638, 1978).
In the present specification, the said new ~-amino acid,
3-amino-2-hydroxy-5-methylhexanoic acid, is abbreviated AHMHA and its residue AHMHA-.
Summary of the Invention Using AHMHA as the basic moiety, the present authors have synthesized various peptide derivatives in order to look for new compounds which are more inhib:itory than amastatins ~ld have novel physiological activities. As a result, they have discovered that peptide derivatives of the formula (I) are not only inhibitory on the said types of aminopeptidase but also inhibit triaminopeptidase severely as a new physiological effect. Based on these findings, the present invention has been established.
In particular, khe present invention concern new tetrapeptide derivatives of the forrnula (I) CH~ 5H3 ~IH2 Rl ~2 H N-C~g-C~-C-NH-~-C-NH-CH-C-Y (I) 2 1 1~ 1! 11 OH O O O
wherein R and R are methyl, l-methylethyl~ 2-methylpropyl, l-hydroxyethyl, 2-carboxyethyl or 4-aminobutyl and Y is -N
or -NH-CH~COOH
wherein R3 is methyl~ l-methylethyl, 2-methylpropyl, l-methylpropyl, hydroxymethyl, l-hydroxyethyl, carboxy-methyl, 2-carboxyethyl, 4-aminobutyl, 3-guanidinopropyl~
benzyl or p-hydroxybenzyl excluding -the compounds in which and R2 are each l-methylethyl and also R3 is carboxymethyl or 2-carboxyethyl, the first and leftmost ~-amino acid moiety in said tetrapeptide derivatives having the (2S,3R)-configuration and a primary amino group and the second, third and fourth a-ami~no acid moieties in said tetrapeptide derivatives having the L-configuration.
Preferred embodiments of the present invention are the compounds of Formula I in which R is l-methylethyl and R is not l-methylethyl, and preferably in which R2 is 4-aminobutyl, l-hydroxyethyl, 2-methylpropyl or 2-carboxy-ethyl and especially in which R3 is carboxymethyl.
v Other preferred embodiments of the pre~ent invention are the compounds of Formula I in which R is 1 methylethyl and Rl is not l-methylethyl and preferably in which Rl is
Summary of the Invention Using AHMHA as the basic moiety, the present authors have synthesized various peptide derivatives in order to look for new compounds which are more inhib:itory than amastatins ~ld have novel physiological activities. As a result, they have discovered that peptide derivatives of the formula (I) are not only inhibitory on the said types of aminopeptidase but also inhibit triaminopeptidase severely as a new physiological effect. Based on these findings, the present invention has been established.
In particular, khe present invention concern new tetrapeptide derivatives of the forrnula (I) CH~ 5H3 ~IH2 Rl ~2 H N-C~g-C~-C-NH-~-C-NH-CH-C-Y (I) 2 1 1~ 1! 11 OH O O O
wherein R and R are methyl, l-methylethyl~ 2-methylpropyl, l-hydroxyethyl, 2-carboxyethyl or 4-aminobutyl and Y is -N
or -NH-CH~COOH
wherein R3 is methyl~ l-methylethyl, 2-methylpropyl, l-methylpropyl, hydroxymethyl, l-hydroxyethyl, carboxy-methyl, 2-carboxyethyl, 4-aminobutyl, 3-guanidinopropyl~
benzyl or p-hydroxybenzyl excluding -the compounds in which and R2 are each l-methylethyl and also R3 is carboxymethyl or 2-carboxyethyl, the first and leftmost ~-amino acid moiety in said tetrapeptide derivatives having the (2S,3R)-configuration and a primary amino group and the second, third and fourth a-ami~no acid moieties in said tetrapeptide derivatives having the L-configuration.
Preferred embodiments of the present invention are the compounds of Formula I in which R is l-methylethyl and R is not l-methylethyl, and preferably in which R2 is 4-aminobutyl, l-hydroxyethyl, 2-methylpropyl or 2-carboxy-ethyl and especially in which R3 is carboxymethyl.
v Other preferred embodiments of the pre~ent invention are the compounds of Formula I in which R is 1 methylethyl and Rl is not l-methylethyl and preferably in which Rl is
4-aminobutyl, l-hydroxyethyl, 2-methylpropyl or 2-carboxy-ethyl and especially those in which R3 is carboxymethyl.
~ et other preferred embodiments of the present invention are the compounds of Formula I in which both and R2 are l-methylethyl and preferably in which R~ is l-hydroxyethyl, -N ~ , benzyl, COOH
l-methylethyl, 4-aminobutyl or 3-guanidinopropyl.
In the subsequent explanation of the present invention, the following abbreviations o~ amino acids, peptides and related compounds which accord with the recommendations by the IUPAC-IUB joint cornmittee ~Biochemistry 6~ 362(1967); Biochemistry 11, 1726~1972)) are employed in the present specification. Other relevant compounds will be explained on first appearance and the corresponding abbreviations in parentheses will be used thereafter.
For amino acids, the abbreviations are given in parenthesis as follows: valine(Val)~ leucine(Leu), threonine(Thr), aspartic acid(Asp), glutamic acid(Glu), lysine(Lys), arginine(Arg)~ phenylalanine(Phe) and proline(Pro).
When the linkage position is at the amino terminal, it is shown by drawing the hyphen at the left side of the relevant amino acid. Similarly the linkage at the carboxyl -6~
terminal and those at both terminals are presented with the hyphen(s) at the rlght side and both sides o~ ~he amino acid respectively. With glycine as example, they are shown as follows: -Gly, Gly-, -Gly~.
Thus in a broad aspect the present invention provides a process for the production of ~etrapeptide derivatives of the formula (I~ above, which process comprises first condensing an activated derivative of the carboxylic acid having the formula C~ ~H3 ~H
~ 2 Z-~N-CH-S~ OH
O--Z
and having the (2S~3R)-coufiguration ~herei~ Z is a co~ventional blockin~ group with a blocked amino acid having the formula Rl R2 H ~-CH-C-NH-C~-C-Y
2 1 n O O
in which all three amlno acid moieties have the L-configuration and wherein R 9 R , R3 and Y have the meaning set for the above and any hydroxy, amino or guanidi~o groups present in Rl, R2 and R are blocked to form a blocked derivative of the tesired tetrapeptide and next chemically removing all blocking groups to form the desired tetrapeptide.
In a preferred embodiment the activated derivative may be selected from the group consisting of th~ use of a carbodi-imide, an azi~e, a mixed anhydride, an active ester such as cyanomethyl ester 9 vi~yl ester, a substituted or unsubstituted phenyl ester~ a thioester, N-hydroxy-succinimide ester, the use of an O-acyl hydroxylamine derivative method employing O-acyl-acetoxime or O--acylcyclohexanoneoxime and the use of an ~-acyl derivative employing carbonyldiimidazole.
In other preferred embo~iments the blocking gxoups are selected from the group consistiny o~ an acyl group, a substi-tuted or unsubsti~uted benzyloxycarbonyl group, alkoxycarbonyl, cycloalkanooxycarbonyl, substituted or unsubstituted arylsulfonyl, nitrophenyl and trityl groups.
The details o~ the present invention will be explained with exemplary compounds in th0 :following, as described above, the componds of the formula(II) which are produced by fermentation o~ Streptomyce5 sp~ ME98-M3~ERM-P 3722) ar~ proved to be inhibitory on aminopeptidase In addition, ~hose new pcptide derivatives o~ the formula(I) of the present invention are also aminopeptidase~inhibitory ~nd can be obtained by peptide formation of a new ~-amino acidy AHMHA, contained in the peptide~ of -the formula(II~, wlth major a-amino acids which are common constituents of protein~ such as glycine, valine, leucine, threonine, aspartic acid~ glutamic acid5 lysine, arginine~ phenyl~
alanine, tyrosine and proline.
AHMHA, an essential constituent of the peptide derivatives o~ the present invention, has the particular configuration of ~2S,3Rj in the natural peptide products of the said actinomycete As described later, AHMHA can also be obtained in the other configurations b~ synthetic methods and ma~ be similarly incorporated as a constituent amino acid in the present invention. According to the present invention, : however, the peptide co~pounds of the present invention which are found most suitable are those composed of AHMHA
in the (2S~R)-configuration and a-a~ino acids in the L-form.
- 7a -~'':~`~
AHMHA constitutes the N-te~inal amino acid of these new peptides. As the second and the third a-amino acid consti-tuents, neutral and basic amino acids such as valine, alanine leucine, threonine and lysine are pre~erable. When the fourth and C-terrninal amino acid moiety has a free amino or carbox~1 gro~p~ the corresponding acid or alkali addition salts may be prepared. It ca~ be basic, neutral or acidic and thus common ~-amino acids are employable.
rhe peptide derivatives of the present invention can be produced from the said amino acids by various known methods of peptide synthesis and most profitably by liquid phase peptide synthesis.
In the following, the methods of preparation of the tetrapeptide deri~atives according to the present invention will be explained concretely.
A*ter amastatins are hydrolysed in acidic or alkaline conditions, Al{MHA is separated and purified from the hydrolysate by a suitable combination of various chromato-graphic methods for separation of amino acids. AHMHA thus obtained has the (2S,~R)-configuration.
... .
The above stereoisomer o~ AHMHA can also be obtained by a method described in Agric. Biol. Chem. 4~
591 - 596(1979). First of all~ the amino group of the ~-amino acid isomer which has the stereo-confi~uration corresponding to the final isomer of 3-arnino-2-hydroxycarboxyla-te(for exa~ple, D- or ~-leucine) is protected with a protecting ~roup conventionally known in the peptide chemistry. rhe protected amino acid is treated with a secondary amine such as pyrazole to ~ive amide. The amide is reduced to aldehyde with lithium aluminium hydride, for example, in ether at a low temperature below 0 C. The treatment of the aldehyde with sodium bisulfite gives an addition product, which is further allowed to react with cyanate to provide cyanohydrin.
me cyanohydrin that corresponds to 3(R or ~ }amino-2(R
or S)hydroxynitrile protected in the arnino group is then converted by hydrolysis to the corresponding 3(R or S3-amino-2(R or S)hydroxycarboxylate.
The mixture of the two diastereomers thus obtained can be resolved înto two isomers by chromatography or with an optical resolver. An example of processes for preparation of AHMHA employable in the present invention is illustrated below with experimental results.
The abbreviations o~ the protecting groups used in the present invention are as follows t benzyloxycarbonyl(Z), t-butoxycarbonyl(Boc), benzyl(Bzl), benzyloxy(03zl), tOsyl(ros), methoxy(OMe), trityl(Trt).
Experiment 1 Synthesis of (2S~3R)-A~IA.
1-~1) Synthesis o~ Z-D-Leu.
Benzyloxycarbonyl chloride(abbreviated Z-Cl hereafter) (65 ml) and 114 ml of 4N NaOH were simultaneously added under agitation with ice-cooling to a solution of 50 g of D-Leu in 191 ml of 2N NaOH and stirred for a fur-ther one hour. A~ter the reaction mixture was diluted with one liter of distilled water, it was rinsed three times with 300 ml each of ethyl acetate. The aqueous layer was adjusted to pH 2.0 with 4N HCl under cooling with ice, The oily material formed was extracted with ethyl acetate.
The ethyl acetate extrac~, after being washed with a saturated solut~on of sodium chloride, was dehydrated over anhydrous sodium sulfate and then evaporated under reduced pressure to give 44 g of colorless3 -transparent syrup.
1-(2) Synthesis of Z-D-Leu-(3,5)dimethylpyrazolide.
_ N,N'-Di~-yclohexylcarbodiimide(abbreviated DCCD hereaf-ter) (68.3 g) was put into a solution of 87.8 g of Z-D-Leu in 900 ml of tetrahydrofuran(abbreviated rHF hereafter) with ice-cooling and stirred for 20 minutes. After 31.8 g of 3,5-dimethylpyrazole was added, the reac-tion mixture was agitated for 3 hours. The ~,N'-dicyclohexylurea (abbreviated D^U hereafter) thus precipitated was removed by filtration.
The solvent was evaporated off in vacuo and the residue was taken up in 500 ml of benzene. A~ter insoluble matter was separated by filtration, the filtrate was dehydrated over anhydrous sodium sulfate and then subjected to evaporation under reduced pressure to give pale yellow syrup. Hexane (500 ml~ was added to the syrup-and was allowed to stan~
overnight at 5C. The crystals thus formed were collected by filtration and dried to give 89.6 g of Z-D-Leu-(3,5~-dimethylpyrazolide, 1-(3) Synthesis of Z-D-leucinal.
- A suspension of 25 g of aluminium hydride in 160 ml of THF was slowly added dropwise at -25 C to a solution of 43.3 g o~` Z-D-Leu-(3,5)dimethylpyrazolide in 433 ml of rHF
over a period of one and a half hours and was stirr~d ~or a further 2 hours, A~ter ~eing cooled to -70C~ ~he reaction mixture was acidified to pH 2 with 3N hydrochloric acid, The THF solvent was evaporated off at room temperature in vacuo and the remaining hydrochloric acid solutlon was subjected to extraction twice with 100 ml each of ether and once with 50 ml ether. The ether extracts were combined and then washed once with 20 ml of 1 N hydrochloric acid and three times with 20 ml each of sa~urated soluti~n o~ sodium chlorideO
The dehydration over anhydr~us sodium sulfa~e followed by evaporation under reduced pressure gave 24.7 g of colorless, clear syrup of Z-D-leucinal.
1-(4) Synthesis of ~R~-3-benzyloxycarbonylamino-2-hydr
~ et other preferred embodiments of the present invention are the compounds of Formula I in which both and R2 are l-methylethyl and preferably in which R~ is l-hydroxyethyl, -N ~ , benzyl, COOH
l-methylethyl, 4-aminobutyl or 3-guanidinopropyl.
In the subsequent explanation of the present invention, the following abbreviations o~ amino acids, peptides and related compounds which accord with the recommendations by the IUPAC-IUB joint cornmittee ~Biochemistry 6~ 362(1967); Biochemistry 11, 1726~1972)) are employed in the present specification. Other relevant compounds will be explained on first appearance and the corresponding abbreviations in parentheses will be used thereafter.
For amino acids, the abbreviations are given in parenthesis as follows: valine(Val)~ leucine(Leu), threonine(Thr), aspartic acid(Asp), glutamic acid(Glu), lysine(Lys), arginine(Arg)~ phenylalanine(Phe) and proline(Pro).
When the linkage position is at the amino terminal, it is shown by drawing the hyphen at the left side of the relevant amino acid. Similarly the linkage at the carboxyl -6~
terminal and those at both terminals are presented with the hyphen(s) at the rlght side and both sides o~ ~he amino acid respectively. With glycine as example, they are shown as follows: -Gly, Gly-, -Gly~.
Thus in a broad aspect the present invention provides a process for the production of ~etrapeptide derivatives of the formula (I~ above, which process comprises first condensing an activated derivative of the carboxylic acid having the formula C~ ~H3 ~H
~ 2 Z-~N-CH-S~ OH
O--Z
and having the (2S~3R)-coufiguration ~herei~ Z is a co~ventional blockin~ group with a blocked amino acid having the formula Rl R2 H ~-CH-C-NH-C~-C-Y
2 1 n O O
in which all three amlno acid moieties have the L-configuration and wherein R 9 R , R3 and Y have the meaning set for the above and any hydroxy, amino or guanidi~o groups present in Rl, R2 and R are blocked to form a blocked derivative of the tesired tetrapeptide and next chemically removing all blocking groups to form the desired tetrapeptide.
In a preferred embodiment the activated derivative may be selected from the group consisting of th~ use of a carbodi-imide, an azi~e, a mixed anhydride, an active ester such as cyanomethyl ester 9 vi~yl ester, a substituted or unsubstituted phenyl ester~ a thioester, N-hydroxy-succinimide ester, the use of an O-acyl hydroxylamine derivative method employing O-acyl-acetoxime or O--acylcyclohexanoneoxime and the use of an ~-acyl derivative employing carbonyldiimidazole.
In other preferred embo~iments the blocking gxoups are selected from the group consistiny o~ an acyl group, a substi-tuted or unsubsti~uted benzyloxycarbonyl group, alkoxycarbonyl, cycloalkanooxycarbonyl, substituted or unsubstituted arylsulfonyl, nitrophenyl and trityl groups.
The details o~ the present invention will be explained with exemplary compounds in th0 :following, as described above, the componds of the formula(II) which are produced by fermentation o~ Streptomyce5 sp~ ME98-M3~ERM-P 3722) ar~ proved to be inhibitory on aminopeptidase In addition, ~hose new pcptide derivatives o~ the formula(I) of the present invention are also aminopeptidase~inhibitory ~nd can be obtained by peptide formation of a new ~-amino acidy AHMHA, contained in the peptide~ of -the formula(II~, wlth major a-amino acids which are common constituents of protein~ such as glycine, valine, leucine, threonine, aspartic acid~ glutamic acid5 lysine, arginine~ phenyl~
alanine, tyrosine and proline.
AHMHA, an essential constituent of the peptide derivatives o~ the present invention, has the particular configuration of ~2S,3Rj in the natural peptide products of the said actinomycete As described later, AHMHA can also be obtained in the other configurations b~ synthetic methods and ma~ be similarly incorporated as a constituent amino acid in the present invention. According to the present invention, : however, the peptide co~pounds of the present invention which are found most suitable are those composed of AHMHA
in the (2S~R)-configuration and a-a~ino acids in the L-form.
- 7a -~'':~`~
AHMHA constitutes the N-te~inal amino acid of these new peptides. As the second and the third a-amino acid consti-tuents, neutral and basic amino acids such as valine, alanine leucine, threonine and lysine are pre~erable. When the fourth and C-terrninal amino acid moiety has a free amino or carbox~1 gro~p~ the corresponding acid or alkali addition salts may be prepared. It ca~ be basic, neutral or acidic and thus common ~-amino acids are employable.
rhe peptide derivatives of the present invention can be produced from the said amino acids by various known methods of peptide synthesis and most profitably by liquid phase peptide synthesis.
In the following, the methods of preparation of the tetrapeptide deri~atives according to the present invention will be explained concretely.
A*ter amastatins are hydrolysed in acidic or alkaline conditions, Al{MHA is separated and purified from the hydrolysate by a suitable combination of various chromato-graphic methods for separation of amino acids. AHMHA thus obtained has the (2S,~R)-configuration.
... .
The above stereoisomer o~ AHMHA can also be obtained by a method described in Agric. Biol. Chem. 4~
591 - 596(1979). First of all~ the amino group of the ~-amino acid isomer which has the stereo-confi~uration corresponding to the final isomer of 3-arnino-2-hydroxycarboxyla-te(for exa~ple, D- or ~-leucine) is protected with a protecting ~roup conventionally known in the peptide chemistry. rhe protected amino acid is treated with a secondary amine such as pyrazole to ~ive amide. The amide is reduced to aldehyde with lithium aluminium hydride, for example, in ether at a low temperature below 0 C. The treatment of the aldehyde with sodium bisulfite gives an addition product, which is further allowed to react with cyanate to provide cyanohydrin.
me cyanohydrin that corresponds to 3(R or ~ }amino-2(R
or S)hydroxynitrile protected in the arnino group is then converted by hydrolysis to the corresponding 3(R or S3-amino-2(R or S)hydroxycarboxylate.
The mixture of the two diastereomers thus obtained can be resolved înto two isomers by chromatography or with an optical resolver. An example of processes for preparation of AHMHA employable in the present invention is illustrated below with experimental results.
The abbreviations o~ the protecting groups used in the present invention are as follows t benzyloxycarbonyl(Z), t-butoxycarbonyl(Boc), benzyl(Bzl), benzyloxy(03zl), tOsyl(ros), methoxy(OMe), trityl(Trt).
Experiment 1 Synthesis of (2S~3R)-A~IA.
1-~1) Synthesis o~ Z-D-Leu.
Benzyloxycarbonyl chloride(abbreviated Z-Cl hereafter) (65 ml) and 114 ml of 4N NaOH were simultaneously added under agitation with ice-cooling to a solution of 50 g of D-Leu in 191 ml of 2N NaOH and stirred for a fur-ther one hour. A~ter the reaction mixture was diluted with one liter of distilled water, it was rinsed three times with 300 ml each of ethyl acetate. The aqueous layer was adjusted to pH 2.0 with 4N HCl under cooling with ice, The oily material formed was extracted with ethyl acetate.
The ethyl acetate extrac~, after being washed with a saturated solut~on of sodium chloride, was dehydrated over anhydrous sodium sulfate and then evaporated under reduced pressure to give 44 g of colorless3 -transparent syrup.
1-(2) Synthesis of Z-D-Leu-(3,5)dimethylpyrazolide.
_ N,N'-Di~-yclohexylcarbodiimide(abbreviated DCCD hereaf-ter) (68.3 g) was put into a solution of 87.8 g of Z-D-Leu in 900 ml of tetrahydrofuran(abbreviated rHF hereafter) with ice-cooling and stirred for 20 minutes. After 31.8 g of 3,5-dimethylpyrazole was added, the reac-tion mixture was agitated for 3 hours. The ~,N'-dicyclohexylurea (abbreviated D^U hereafter) thus precipitated was removed by filtration.
The solvent was evaporated off in vacuo and the residue was taken up in 500 ml of benzene. A~ter insoluble matter was separated by filtration, the filtrate was dehydrated over anhydrous sodium sulfate and then subjected to evaporation under reduced pressure to give pale yellow syrup. Hexane (500 ml~ was added to the syrup-and was allowed to stan~
overnight at 5C. The crystals thus formed were collected by filtration and dried to give 89.6 g of Z-D-Leu-(3,5~-dimethylpyrazolide, 1-(3) Synthesis of Z-D-leucinal.
- A suspension of 25 g of aluminium hydride in 160 ml of THF was slowly added dropwise at -25 C to a solution of 43.3 g o~` Z-D-Leu-(3,5)dimethylpyrazolide in 433 ml of rHF
over a period of one and a half hours and was stirr~d ~or a further 2 hours, A~ter ~eing cooled to -70C~ ~he reaction mixture was acidified to pH 2 with 3N hydrochloric acid, The THF solvent was evaporated off at room temperature in vacuo and the remaining hydrochloric acid solutlon was subjected to extraction twice with 100 ml each of ether and once with 50 ml ether. The ether extracts were combined and then washed once with 20 ml of 1 N hydrochloric acid and three times with 20 ml each of sa~urated soluti~n o~ sodium chlorideO
The dehydration over anhydr~us sodium sulfa~e followed by evaporation under reduced pressure gave 24.7 g of colorless, clear syrup of Z-D-leucinal.
1-(4) Synthesis of ~R~-3-benzyloxycarbonylamino-2-hydr
5-methylenantonitrile.
. _ .
Z-D-Leucinal(24.7 g) in 50 ml of ethyl acetate was mixed with 22.7 g of sodium hyposul~ite in 100 ml of distilled water and agi~ated for 16 hours. ~hen 5.35 g of sodium cyanide wa~ added to the solution and it was stirred for 3 hours.
The reaction product was extracted three times with 50 ml each of ethyl acetate and the combined ethyl acetate extracts were washed once with 20 ml of distilled water and thr~ times wi.th 30 ml each o~ saturat~ solution of sodium chloride. After the organic layer was dehydrated over anhydrous sodium sulfate, the solvent was evaporated o.~f under reduced pres~ure to provide 22.5 g of syrup.
The syrup was charged on a silica gel column(Wakogel 200, a product of Wako pure Chemical Industries, Ltdo ~ 125 g) and de~elope~ with a mixture~of benzene and ethyl acetate (10/1) to yield 18.1 g of syrup of (3R)-3-benzyloxycarbonyl-amino-2-hydroxy-5-methylenantonitri~le~
*Trademark.
~:3 a~
~f , 0.29(benzene/ethyl acetate=10/1, a silica gel 60 F254 plate, a product of E. Merck) n. m. r. 3ppm (CDC~3) û.9 (6H, eachd, J 5Hz, 4Hz, --CH(CH3~2 ) 1-3~1.7 (3H, m, H-4.4, ~I-5 ) ~6~4.2 (1H, m, H~3) 4.4~4.6 (lH, m, H-2) ~1 (2H, s, -CH2Ph) 52~~5 ~1Hl ~9 ~nH - ) ~s~5.9 (lH, m, -OH) ?. 3 1 ~ 5 H, 5 , --~H 2 P h ) 1-(5) SJ~nthes_~ ~ A.
A solution of 18.1 g of (3R)-3-benzyloxycarbonylamino-2-hydroxy-5-methylenantonitrile in a solvent mixture of 163 ml of dioxane and 78 ml of ~nisole was mixed with 163 ml of concentrated hydrochloric acid and heated under reflux with agitation for 9 hours at a temperature of 115 120C
in an oil bath. The reaction solution was condensed to 100 ml under reduced pressure and then subjected to extraction twice with 50 ml each of ethyl acetate. After the solvent was removed from the combined extracts by evaporation in vacuo, the evaporation residue was dissolved in 200 ml of distilled water and evaporated ~o dryness under reduced 1~ .
Rressure, ~h~s dissolution and evaporation step was repeated three to four times~ ~he evaporation residue thus obtained was applied on a column of sulfonate-type polystyrene ion exchange resin such as Dowex 50~x10(140 ml, ~00 - 400 mesh, a product o~ Dow Chemical Co.). After washing with ~00 ml of distilled water, the column was eluted with 0.3 N ammonia, Ninhydrin-po~itive eluate fractions were collected and evaporated to drynes~ under reduced pressure.
Recrystallization o~ the solid matter from a solvent mixture of 100 ml o~ hot water and 200 ml of iæopropyl alcohol gave
. _ .
Z-D-Leucinal(24.7 g) in 50 ml of ethyl acetate was mixed with 22.7 g of sodium hyposul~ite in 100 ml of distilled water and agi~ated for 16 hours. ~hen 5.35 g of sodium cyanide wa~ added to the solution and it was stirred for 3 hours.
The reaction product was extracted three times with 50 ml each of ethyl acetate and the combined ethyl acetate extracts were washed once with 20 ml of distilled water and thr~ times wi.th 30 ml each o~ saturat~ solution of sodium chloride. After the organic layer was dehydrated over anhydrous sodium sulfate, the solvent was evaporated o.~f under reduced pres~ure to provide 22.5 g of syrup.
The syrup was charged on a silica gel column(Wakogel 200, a product of Wako pure Chemical Industries, Ltdo ~ 125 g) and de~elope~ with a mixture~of benzene and ethyl acetate (10/1) to yield 18.1 g of syrup of (3R)-3-benzyloxycarbonyl-amino-2-hydroxy-5-methylenantonitri~le~
*Trademark.
~:3 a~
~f , 0.29(benzene/ethyl acetate=10/1, a silica gel 60 F254 plate, a product of E. Merck) n. m. r. 3ppm (CDC~3) û.9 (6H, eachd, J 5Hz, 4Hz, --CH(CH3~2 ) 1-3~1.7 (3H, m, H-4.4, ~I-5 ) ~6~4.2 (1H, m, H~3) 4.4~4.6 (lH, m, H-2) ~1 (2H, s, -CH2Ph) 52~~5 ~1Hl ~9 ~nH - ) ~s~5.9 (lH, m, -OH) ?. 3 1 ~ 5 H, 5 , --~H 2 P h ) 1-(5) SJ~nthes_~ ~ A.
A solution of 18.1 g of (3R)-3-benzyloxycarbonylamino-2-hydroxy-5-methylenantonitrile in a solvent mixture of 163 ml of dioxane and 78 ml of ~nisole was mixed with 163 ml of concentrated hydrochloric acid and heated under reflux with agitation for 9 hours at a temperature of 115 120C
in an oil bath. The reaction solution was condensed to 100 ml under reduced pressure and then subjected to extraction twice with 50 ml each of ethyl acetate. After the solvent was removed from the combined extracts by evaporation in vacuo, the evaporation residue was dissolved in 200 ml of distilled water and evaporated ~o dryness under reduced 1~ .
Rressure, ~h~s dissolution and evaporation step was repeated three to four times~ ~he evaporation residue thus obtained was applied on a column of sulfonate-type polystyrene ion exchange resin such as Dowex 50~x10(140 ml, ~00 - 400 mesh, a product o~ Dow Chemical Co.). After washing with ~00 ml of distilled water, the column was eluted with 0.3 N ammonia, Ninhydrin-po~itive eluate fractions were collected and evaporated to drynes~ under reduced pressure.
Recrystallization o~ the solid matter from a solvent mixture of 100 ml o~ hot water and 200 ml of iæopropyl alcohol gave
6.9 g of (3R)AHMHA.
Rf s 0.33~butanol/acetic acid/water=4/1/1, a silica gel plate as described above3 1-(6) Isolation of (2S~3R)AH~
Twenty grams of cellulose powder was well mixed with a solution of 2.0 g of (3~)-AHMHA crystals in 109 ml o~
distilled water and then e~aporated to dryness under reduced pressure to give AH~ A-cellulose powder. rhe AH~HA-cellulose powder was placed on top of a column of cellulose powder(l.8 liters) which had been pac~ed with a developing solvent system of ethyl acetate/acetic acid/pyridine/water=5/1/1/1 (upper layer). me column elution with the said solvent system separated 750 mg of (2S,3R)AHMHA ~rom its diastereomer mixture s .
(2S~ 3R)A~IA
* Trademark .
,`1 ;~i Rf ~ 0~31(the above-described developing solvent system, a cellulose thin-layer plate from Funakoshi Pharmaceutical Co., Ltd.) m.p. : 231 - 232C
~323 ~ -28.4 ~c=0.5, CH3COOH) (2R,~R)AHMHA
Rf : 0~27(under the same conditions as described above3 j m.p. s 230 - 231 C
~d~D3 ~ +28.1 (c=0.5, CH3COOH) Synthesis of Z-(2S3R)AHMHA.
(2S,3R)AHMHA(12~.2 mg) and 192.6. mg of sodium bicarbonate were suspended in 3.6 ml of a solvent mixture of THF/distilled water(l/l). Z-Cl(0.16 ml) was added drop by drop to the suspension under cooling with ice and then stirred for 8 hours. After THF was e~aporated off ~Inder reduced pressure, the remaining alkaline aqueous solution was diluted with 10 ml of distilled water and was rinsed twice with 2 ml each of hexane and once wi-th 2 ml of ethyl acetate. The acidification of the aqueous solu-tion to pH 2 with 6 N hydrochloric acid produced oil which was extracted four times with 3 ml each of ethyl acetate. rhe organic , extracts were pooled and washed twice with 2 ml each of saturated solution of sodium chloride. ~he dehydration with anhydrous sodium sulfate and the subsequent remov~l of the solvent under reduced pressure yielded 154.4 m~ of o dry syrup of Z-~2S,3R)~HMHA from which 120 mg of Z-(2S,3R)-AH~i~A crystals were obtained by recrys-tallization from a solvent mixture of ether and hexane.
n. m~ rO 8ppm (CDC~3) ns ( 6H, d 9 J5.65Hz~ -CH(CH~)2 ) .2~1.8 (3H , m , H-4.4 , H~5) ~9~4.5 (2H , m ,H-2 . H-3) 5.93 (2H , s , CH2Ph) s.2~5.8 (2H , m , -OH ,--~nI-~
m. p. : 85 ~ 87~
IR ~ vlCaBx cm 1 ~ 1705(carboxyl C=O), 1640~urethane C=O~, 3310(urethane -NH-), 3520~-OH) 1-(8) Synthesis of Z-(2S,3R)AHMHA(OZ).
Using 300 mg o~ (2S,3R)AHMXA and 783 mg of NaHC~3, the same procedure as detailed in 1-~7) was performed to provide 254 mg of syrup of Z-(2S,3R)AHMHA(OZ).
Rf 1 0.4(benzene/ethyl acetate/isopropyl alcohol=30/20/5, a silica gel plate as described above) n. m. r. ~ppm (CDC~3~ :
(6H , d , ~65Hz , -CH(CH~-)2 ) 1.2~~.8 (3H , m , H-4.4 , H-5) 4.15~4.75 (1H . m . H-3) 4-95 (lH , d ,J 2-5Hz O H-2) 5.05 (2H , ~ 7 -CH2Ph) ~15 ~2H , 8 , C~ Ph)
Rf s 0.33~butanol/acetic acid/water=4/1/1, a silica gel plate as described above3 1-(6) Isolation of (2S~3R)AH~
Twenty grams of cellulose powder was well mixed with a solution of 2.0 g of (3~)-AHMHA crystals in 109 ml o~
distilled water and then e~aporated to dryness under reduced pressure to give AH~ A-cellulose powder. rhe AH~HA-cellulose powder was placed on top of a column of cellulose powder(l.8 liters) which had been pac~ed with a developing solvent system of ethyl acetate/acetic acid/pyridine/water=5/1/1/1 (upper layer). me column elution with the said solvent system separated 750 mg of (2S,3R)AHMHA ~rom its diastereomer mixture s .
(2S~ 3R)A~IA
* Trademark .
,`1 ;~i Rf ~ 0~31(the above-described developing solvent system, a cellulose thin-layer plate from Funakoshi Pharmaceutical Co., Ltd.) m.p. : 231 - 232C
~323 ~ -28.4 ~c=0.5, CH3COOH) (2R,~R)AHMHA
Rf : 0~27(under the same conditions as described above3 j m.p. s 230 - 231 C
~d~D3 ~ +28.1 (c=0.5, CH3COOH) Synthesis of Z-(2S3R)AHMHA.
(2S,3R)AHMHA(12~.2 mg) and 192.6. mg of sodium bicarbonate were suspended in 3.6 ml of a solvent mixture of THF/distilled water(l/l). Z-Cl(0.16 ml) was added drop by drop to the suspension under cooling with ice and then stirred for 8 hours. After THF was e~aporated off ~Inder reduced pressure, the remaining alkaline aqueous solution was diluted with 10 ml of distilled water and was rinsed twice with 2 ml each of hexane and once wi-th 2 ml of ethyl acetate. The acidification of the aqueous solu-tion to pH 2 with 6 N hydrochloric acid produced oil which was extracted four times with 3 ml each of ethyl acetate. rhe organic , extracts were pooled and washed twice with 2 ml each of saturated solution of sodium chloride. ~he dehydration with anhydrous sodium sulfate and the subsequent remov~l of the solvent under reduced pressure yielded 154.4 m~ of o dry syrup of Z-~2S,3R)~HMHA from which 120 mg of Z-(2S,3R)-AH~i~A crystals were obtained by recrys-tallization from a solvent mixture of ether and hexane.
n. m~ rO 8ppm (CDC~3) ns ( 6H, d 9 J5.65Hz~ -CH(CH~)2 ) .2~1.8 (3H , m , H-4.4 , H~5) ~9~4.5 (2H , m ,H-2 . H-3) 5.93 (2H , s , CH2Ph) s.2~5.8 (2H , m , -OH ,--~nI-~
m. p. : 85 ~ 87~
IR ~ vlCaBx cm 1 ~ 1705(carboxyl C=O), 1640~urethane C=O~, 3310(urethane -NH-), 3520~-OH) 1-(8) Synthesis of Z-(2S,3R)AHMHA(OZ).
Using 300 mg o~ (2S,3R)AHMXA and 783 mg of NaHC~3, the same procedure as detailed in 1-~7) was performed to provide 254 mg of syrup of Z-(2S,3R)AHMHA(OZ).
Rf 1 0.4(benzene/ethyl acetate/isopropyl alcohol=30/20/5, a silica gel plate as described above) n. m. r. ~ppm (CDC~3~ :
(6H , d , ~65Hz , -CH(CH~-)2 ) 1.2~~.8 (3H , m , H-4.4 , H-5) 4.15~4.75 (1H . m . H-3) 4-95 (lH , d ,J 2-5Hz O H-2) 5.05 (2H , ~ 7 -CH2Ph) ~15 ~2H , 8 , C~ Ph)
7.2~ (5H , s , - CH2 Ph) 7.31 (5H , s , -CH2Ph) ~0~9.2 (2H t m , -NII- , ~COOH) ~ or preparation of tetrapeptide derivatives of the formula(I) from AH~HA and other constituent amino acids such as L-~al and amino acîd Y in the formula(I), the conventional liquid phase peptide synthesis is most favorable. In brief, for production of peptide derivatives of the present invention, the ~-amino or carboxyl group of any one constituent amino acid in the formula(I) may be linXed sequentially to the neighboring amino acid or peptide by peptide bond formation until the desired length of -the peptide can be obtained. In practice it seems to be advantageous that the dipeptide containing constituent amino acid Y of the formula(I) is ~irst synthesized and then sequentially elongated to tetrapeptides. For this purpose, the amino and carboxyl group~ of amino acid ara usually ! protected as follows ~ after functional groups other than the ~ - amino group of amino acid Y of the formula(I)(for i example, carboxyl, hydroxyl, amino and thiol aroups) are appropriately protected, the amino group of ~-valine is covered with a suitable protecting radical which can be cleaved off without a~fecting ~he protec~ed functional groups of amino acid Y. These two protected amino acids are linXed together to give the dipeptide by means of conventional methods for peptide formation. For example, the carbodiimide method using DCCD, the mixed-acid anhydride method ~lith chloroformic esters, the azide method due to azidation of the carboxyl group, the active ester method by virtue of ester formation wi~h N-hydroxysuccinimide, etc. are profitably employable. ~or connection of the next amino acid similarly protected, only the ~-amino-protectin~ group is cleaved off ~rom the L-valine moiety of the dipeptide by an appropriate method(for example, catalytic reduction, treatment with trifluoroacetic acid(TFA), processin~ with hydrobromide in acetic acid or with metallic sodium in liquid ammonia, saponification with an alkali, treatment with anhydrous hydrofluoric acid, etc,) under preliminarily chosen reaction conditions where the other protecting groups easily survive. (Although any type of the protecting group will do at the final stage of peptide formation, it is favorable to employ such a combination of protecting groups that all of them can be simultaneously cleaved off under the same treatment conditions at the terrnination of synthesis.) After the same procedure of peptide elongation by one amino acid unit and removal of the relevant protecting group is repeated until the desired length of the peptide is obtained, the remaining protecting groups are finally cleaved off to give the peptide derivative of the present invention.
retrapeptide derivatives of the present invention prepared as described above markedly inhibit aminopeptidase A
(abbreviated AP-A hereafter), aminop~ptidase B(abbrevia~ed AP-B hereafter), leucine aminopeptidase(abbreviated ~eu-AP herea~ter) t glycylprolylleucine tripeptidylaminopeptidase (abbreviated Gly-Pro-Leu-AP hereafter) and glycylhistidyllysine tripeptidylaminopeptidase(abbreviated Gly-His-1ys-AP
hereafter). In the following, the assay methods of enzyme activity will be described for the above enzymes:
~l) AP-A(E.C. 3,4,ll,7) ~ he activity of this enzyme was assayed by a modification of the method of Nagatsu Qt al,(I, Nagatsu, ~, Nagatsu, T. Yamamoto and G.G. Glenner: BIOCHIMICA E~ 3IOPHYSICA AGTA
lg8, 255 - 270(1970)). That is, 0,25 ml of 2mM L-~-glutamyl-~-naphthylamide, 0.54 ml of O,l ~ ~ris-HCl buffer, p~ 7.0, O.Ol ml o~ O,l M calcium chloride and O,l ml of a test sample in water were mixed and kept at 37 C for 3 minutes. ~he reaction was initiated by addition of O.l ml of an AP-A solution which wa~ obtained by ammonium sulfate fractionation according to the method of ~lagatsu et al.
After incubation at 37CC for 30 minutes, 1 ml of l,O ~
~19-acetate buffer, pH 4.2, containing 1.0 m~/ml Garnet GBC
(orthoaminoazotoluene diazonlum salt) and 10% Tween 20 (Atlas Chemical Co. 9 U, S . A,) was added to the reaction solution and then allowed to st~nd at room temperature for 15 minutes. ~he absorbance of the test solution(a) was measured at 530 nm. The control solution without the test sample was used as the blank test(absorbance ~b)).
me inhibition percentage on aminopeptidase A was calculated as [(b--a)~b]xloo (2) AP-~(E.C. 3,4,11,6) For activity measurement, the method of Hopsu et al.
(V.K. Hopsu, K.K. Makinen and G.G. Glenner: ARC~l~E~ 0~
BIOCH2MISTRY AND ~IOP~YSICS 114, 557(1966)) was modified.
Namely, a react;ion mixture containing 0.25 ml of 2 mM ~
argin yl-~-naphthylamide~ o.63 ml of 0.1 M ~ris-HCl buffer, pH 7.0, and 0.1 ml of an aqueous solution of a test sample was maintained at 37~C for 3 minutes. ~he enzyme reaction was started by addition o~ 0.02 ml of an AP-~ solution that was purified from the rat liver by the method of ~opsu et al.
using "Sephadex ~-lOd ~ross-l~nked dextran gel, a product o~ pharmacia Fine Chemicals AB, Sweden). After 30 minutes of reaction at 37~C, the inhibition percentage was obtained as described in (1).
(3) Leu-AP(E,~. 3~4~11,1 *~rademark.
. ~ -20-For enzyme assay~ 0.25 ml of 2 mM L~d-leucYl-~-naphthylamide, o~6 ml of 0.1 M Tris~HCl buffer, p~ 7,6, and 0.1 ml of an aqueous solution containing a test compound were mixed and allowed to s-tand at 37C for 3 minutes.
After a 20-fold dilution of Leu-AP(~oehringer-i~lannheim so.) in 0.1 M ~ris-HCl buffer, pH 7.6(o,o5 rnl) ~as added to -the solution, the reaction wa~s performed at 37~C for 30 minutes. The inhibition percentage was measured as detailed in (1), (4) Gly-Pro-Leu~AP
According to the method of Aoyagi et al.(T Aoyagi et al~ ~IOCHIMICA Er BIOPHYSICA ACTA 452, 131 - 143(1976)), a~ter a reaction mixture consisting of 0~25 ml of 2 mM
glycyl-L-~-prolyl-L-~leucyl-~-naphthylamide( 3uchem Fine Chemicals Co.3, o.6 ml of the Hanks solution, pH 7.2, and 0.1 ml of an aqueous test solution wa~ kept at 37 C
for 3 minutes, 0.05 ml of a Gly-Pro-Leu-AP that was purified from the cell membrane fraction of the ra-t liver was added and the rea^tion was carried out at 37C ~or 30 minutes.
The same procedure as described in (1) was used for calculation of the inhibition percentage.
The Hanks solution listed above was prepared by dissolving 8.0 g sodium chloride, 004 g potassium chloride, 0.14 g calcium chlorid~ 0.10 g magnesium chloride, o. o6 g disodium-monohydro~en phosphate, 0. o6 g monosodium-dihydrogen phosphate, 0.10 g magnesium sulfate and l.0 g glucose in l liter of distilled water and adjusting the solution to pH 7.2 with solid sodium bicarbonate(about 350 mg).
(5) Gly-~is-Lys-AP
For en~yme assay, 0.25 ml o~ 0.5 m~ glycyl-L-d-histidyl-L-~lysyl-~-naphthylamide, 0.62 ml of the XanXs solution; pH 7.2, and 0.1 ml of an aqueous solution of a substance to be tested were combined and maintained at 37 G
for 3 minutes. Gly-~is-Lys-AP solution(O.03 ml ) which was puri~ied from the FM3A cells(a line of culture cells derived from mouse mammary tumor) by the method described in the reference of (4) was added to the solution and incubated at 37 C for 30 minutes. The'inhibition percentage was calculated as shown in (l).
Gly-His-Lys-~-naphthylamide used above was prepared as follows~ to a solution containing 1 ~ of Gly His-LY~-acetate(Buchem Fine Chemicals Co.) and 3.0 g of sodium bicarbonate in 60 ml of distilled water, 2.13 ml of Z-Cl was added dropwise under cooling with ice and then stirred for 3 hoursO The same treatment as detailed above,in Experiment 1-(7) was performed to give 605.8 mg of the Z-derivative of the tripeptide. This 7-derivative of the tripeptide and 117 m~ of ~-naphthylamine were condensed as described in Experiment 1-(2) to yield 600 mg of a solid ~aterial. ~he solid material was dissolved in 10 ml ~f a solvent mixture of acetic acid and ~ater(l/l) and then shaken overnight together with 30 mg of palladium-carbon in the presence of hydrcgen(4 atmospheres). ~he removal of the catalyst ~ollowed by evaporation of the solvent under re~uced pressure provided 200 mg of ~ly-His-Iys~-naphthylamide.
n. m. r ~ppm (CD~30D) 1.4~~3 (~H , m , -~nH-CH-~CHz~-) 2.9~~2 (4H , m , -CH2-CH2-MH2 ,~CH-C ~ C=CH) ~~9 (2H . m ~nH2 - CH2 -~- ) ~ 9~3 (9H , m y aromatic~
m. p. : 115 ~ 116~ (3HC~, 3alt) ~D ~ (e - ~1 t ~Z O ) ( 3~C~ ~al~) Table 1 summarizes the inhibitory activity of the new tetrapeptide derivatives o~ the present invention and related peptides on the above-listed enzymes, -2~--- -xl ~ x ~ x x -x x x x j x x ~q ~ x~
P P ! P p~ p~ ~: p~ ,_ ~ ~ ~ ~ 9~ ~ P o- I ,_. . /
,_ l_ ,_ ,_ 7- l ,_ l l ~ ,_ ~ rl _ ~, ~ ~/
<~ ~ <~ ~ ~3 < '< ~ ~ C~ t~ ~3 t' ~ I
P~ P P ~ ~ ~ P .~ ~ P~ 1:: C: ~ ~ P, il ~
~3 ~ ~ ~ :~ 1' ~ ~ ~ ~ :~ P '~ /~r/ ~' ~: ~ :~ ~ :r ~ ~ la co co ~O CO Ul W W Ico /~t~
~q .~ ~ O ~ ~_ (D ~ ~ ~a ~ ~ ~ ~o ~ , /~ ' I . . I ~/
_j -- - - - ~- - - i -~ ~ ' ~ ~ ~ _ o ~ _ m o P~ p _1 - ' ~
_ ~ I ~ ~n ~J ~o 0~ vl o _ o ~ o I o l . a : :~ tD
_ 1~ - - -- 1-- :~ ~-v v v v v vlvlv v v v v v vlv~v-l~
1~ ~ ¦ ~ N 1~ _ ¦ _ ¦ N N ~ N N N I N ~
ul ul I Ul ~11 vi o I o I ~1 ul ~Jl ~Jl ~Jl 01 I ~
__ I O O ~ O I O O O O O I O I O I O I O I ~ I
t I I l ~ r I
~ ~ ~ 1 - I o l ,~ o ~ I P l P l - s~ l o I c I
; , ~I ~ o ~ o ~
~ o_ l' ! ~ P~ P; p o, p~ ~ o ~I oI -I PI PI -I P ~
~ ~ O ~ O ¦ O ~ -- ¦ O ¦ O ¦ N Vl ¦ N ~ N ~ O o I o I l + I rr r~
._ o! _¦ pll -I o ol N Pl l _l -¦ Pl ~ -¦ O j~. !
~ I o 11 ~ I 'o I o I o ~ I ~ I o ' o _ . , ~ I ~, . ,~, I I
I I 1 ~
V ~ o ~ o ~ O ! p~
..~ _~ _ .,~ _ ~ ~_ ~ ~ ~ .c~a I ~ .
v I v v, v v v v v v v v ~ . , .. ~ r.~ ~ ~ ~ ~ ~ ~ ~ ~ ~a ~ l O
p, vO~ vl l o vl o ~n Uol t.n ~n On ~n t~:1 . ;:5 o - j I I _ _ l O ~ O I ~ 0~ V ~ V ~ ~ ~ O
~ _ _. . _ ~ ~ ~
V~ Iv~ c ~On i oll ~~ o ~1 o~ ~1 o¦ ol~
¦ ~ ! ¦
! I l l o ~ o ~o, _, l W ,~ , I o . _ I ~ I o ~ _ I o It is quite obvious froln Table l that the peptide compounds of the present invention have a stron~ inhibitory activity an the tripep-tidyla~inopeptidases(Gly-pro-Leu-Ap and Gly-His-Lys-AP).
Shlesinger et al have recently isolated Gly-His-Lys as a tumor cell growth-promotin~ tripeptide, which seems to be produced in vivo by tripeptidase(EXPERIEN~IA 32, 324-325(1977)).
~herefore it can be expected that suppressive effects on growth of mammalian tumor cells will be obtained by administration of amastatins'and tetrapeptide derivatives of the present invention.
Thus the present invention provides new peptide derivatives -that exhibit inhibitory activity on amino-peptidase and tripeptidylaminopeptidase in vitro and in vivo and that can be expected -to be ef'fective as an antitumor agent.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
Example 1 Synthesis of (2S,3R)-A~ Val-L~s-Asp.
1-(1) Synthesis of Boc-Lys(Z)-Asp(OBæ~.
_ ! DCCD(612 mg) was mixed with a suspension of 1~03 g of Boc-Lys(Z), 1.31 g of Asp~OBz1)2~TosOH and 437 mg of ~OBt in anhydrous tetrahydrofuran(abbreviated ~HF hereafter) and stirred ~or 5 minutes. Then 0.414 ml of anhydrous triethyl-amine was added dropwise to the suspension and ~gitated for
retrapeptide derivatives of the present invention prepared as described above markedly inhibit aminopeptidase A
(abbreviated AP-A hereafter), aminop~ptidase B(abbrevia~ed AP-B hereafter), leucine aminopeptidase(abbreviated ~eu-AP herea~ter) t glycylprolylleucine tripeptidylaminopeptidase (abbreviated Gly-Pro-Leu-AP hereafter) and glycylhistidyllysine tripeptidylaminopeptidase(abbreviated Gly-His-1ys-AP
hereafter). In the following, the assay methods of enzyme activity will be described for the above enzymes:
~l) AP-A(E.C. 3,4,ll,7) ~ he activity of this enzyme was assayed by a modification of the method of Nagatsu Qt al,(I, Nagatsu, ~, Nagatsu, T. Yamamoto and G.G. Glenner: BIOCHIMICA E~ 3IOPHYSICA AGTA
lg8, 255 - 270(1970)). That is, 0,25 ml of 2mM L-~-glutamyl-~-naphthylamide, 0.54 ml of O,l ~ ~ris-HCl buffer, p~ 7.0, O.Ol ml o~ O,l M calcium chloride and O,l ml of a test sample in water were mixed and kept at 37 C for 3 minutes. ~he reaction was initiated by addition of O.l ml of an AP-A solution which wa~ obtained by ammonium sulfate fractionation according to the method of ~lagatsu et al.
After incubation at 37CC for 30 minutes, 1 ml of l,O ~
~19-acetate buffer, pH 4.2, containing 1.0 m~/ml Garnet GBC
(orthoaminoazotoluene diazonlum salt) and 10% Tween 20 (Atlas Chemical Co. 9 U, S . A,) was added to the reaction solution and then allowed to st~nd at room temperature for 15 minutes. ~he absorbance of the test solution(a) was measured at 530 nm. The control solution without the test sample was used as the blank test(absorbance ~b)).
me inhibition percentage on aminopeptidase A was calculated as [(b--a)~b]xloo (2) AP-~(E.C. 3,4,11,6) For activity measurement, the method of Hopsu et al.
(V.K. Hopsu, K.K. Makinen and G.G. Glenner: ARC~l~E~ 0~
BIOCH2MISTRY AND ~IOP~YSICS 114, 557(1966)) was modified.
Namely, a react;ion mixture containing 0.25 ml of 2 mM ~
argin yl-~-naphthylamide~ o.63 ml of 0.1 M ~ris-HCl buffer, pH 7.0, and 0.1 ml of an aqueous solution of a test sample was maintained at 37~C for 3 minutes. ~he enzyme reaction was started by addition o~ 0.02 ml of an AP-~ solution that was purified from the rat liver by the method of ~opsu et al.
using "Sephadex ~-lOd ~ross-l~nked dextran gel, a product o~ pharmacia Fine Chemicals AB, Sweden). After 30 minutes of reaction at 37~C, the inhibition percentage was obtained as described in (1).
(3) Leu-AP(E,~. 3~4~11,1 *~rademark.
. ~ -20-For enzyme assay~ 0.25 ml of 2 mM L~d-leucYl-~-naphthylamide, o~6 ml of 0.1 M Tris~HCl buffer, p~ 7,6, and 0.1 ml of an aqueous solution containing a test compound were mixed and allowed to s-tand at 37C for 3 minutes.
After a 20-fold dilution of Leu-AP(~oehringer-i~lannheim so.) in 0.1 M ~ris-HCl buffer, pH 7.6(o,o5 rnl) ~as added to -the solution, the reaction wa~s performed at 37~C for 30 minutes. The inhibition percentage was measured as detailed in (1), (4) Gly-Pro-Leu~AP
According to the method of Aoyagi et al.(T Aoyagi et al~ ~IOCHIMICA Er BIOPHYSICA ACTA 452, 131 - 143(1976)), a~ter a reaction mixture consisting of 0~25 ml of 2 mM
glycyl-L-~-prolyl-L-~leucyl-~-naphthylamide( 3uchem Fine Chemicals Co.3, o.6 ml of the Hanks solution, pH 7.2, and 0.1 ml of an aqueous test solution wa~ kept at 37 C
for 3 minutes, 0.05 ml of a Gly-Pro-Leu-AP that was purified from the cell membrane fraction of the ra-t liver was added and the rea^tion was carried out at 37C ~or 30 minutes.
The same procedure as described in (1) was used for calculation of the inhibition percentage.
The Hanks solution listed above was prepared by dissolving 8.0 g sodium chloride, 004 g potassium chloride, 0.14 g calcium chlorid~ 0.10 g magnesium chloride, o. o6 g disodium-monohydro~en phosphate, 0. o6 g monosodium-dihydrogen phosphate, 0.10 g magnesium sulfate and l.0 g glucose in l liter of distilled water and adjusting the solution to pH 7.2 with solid sodium bicarbonate(about 350 mg).
(5) Gly-~is-Lys-AP
For en~yme assay, 0.25 ml o~ 0.5 m~ glycyl-L-d-histidyl-L-~lysyl-~-naphthylamide, 0.62 ml of the XanXs solution; pH 7.2, and 0.1 ml of an aqueous solution of a substance to be tested were combined and maintained at 37 G
for 3 minutes. Gly-~is-Lys-AP solution(O.03 ml ) which was puri~ied from the FM3A cells(a line of culture cells derived from mouse mammary tumor) by the method described in the reference of (4) was added to the solution and incubated at 37 C for 30 minutes. The'inhibition percentage was calculated as shown in (l).
Gly-His-Lys-~-naphthylamide used above was prepared as follows~ to a solution containing 1 ~ of Gly His-LY~-acetate(Buchem Fine Chemicals Co.) and 3.0 g of sodium bicarbonate in 60 ml of distilled water, 2.13 ml of Z-Cl was added dropwise under cooling with ice and then stirred for 3 hoursO The same treatment as detailed above,in Experiment 1-(7) was performed to give 605.8 mg of the Z-derivative of the tripeptide. This 7-derivative of the tripeptide and 117 m~ of ~-naphthylamine were condensed as described in Experiment 1-(2) to yield 600 mg of a solid ~aterial. ~he solid material was dissolved in 10 ml ~f a solvent mixture of acetic acid and ~ater(l/l) and then shaken overnight together with 30 mg of palladium-carbon in the presence of hydrcgen(4 atmospheres). ~he removal of the catalyst ~ollowed by evaporation of the solvent under re~uced pressure provided 200 mg of ~ly-His-Iys~-naphthylamide.
n. m. r ~ppm (CD~30D) 1.4~~3 (~H , m , -~nH-CH-~CHz~-) 2.9~~2 (4H , m , -CH2-CH2-MH2 ,~CH-C ~ C=CH) ~~9 (2H . m ~nH2 - CH2 -~- ) ~ 9~3 (9H , m y aromatic~
m. p. : 115 ~ 116~ (3HC~, 3alt) ~D ~ (e - ~1 t ~Z O ) ( 3~C~ ~al~) Table 1 summarizes the inhibitory activity of the new tetrapeptide derivatives o~ the present invention and related peptides on the above-listed enzymes, -2~--- -xl ~ x ~ x x -x x x x j x x ~q ~ x~
P P ! P p~ p~ ~: p~ ,_ ~ ~ ~ ~ 9~ ~ P o- I ,_. . /
,_ l_ ,_ ,_ 7- l ,_ l l ~ ,_ ~ rl _ ~, ~ ~/
<~ ~ <~ ~ ~3 < '< ~ ~ C~ t~ ~3 t' ~ I
P~ P P ~ ~ ~ P .~ ~ P~ 1:: C: ~ ~ P, il ~
~3 ~ ~ ~ :~ 1' ~ ~ ~ ~ :~ P '~ /~r/ ~' ~: ~ :~ ~ :r ~ ~ la co co ~O CO Ul W W Ico /~t~
~q .~ ~ O ~ ~_ (D ~ ~ ~a ~ ~ ~ ~o ~ , /~ ' I . . I ~/
_j -- - - - ~- - - i -~ ~ ' ~ ~ ~ _ o ~ _ m o P~ p _1 - ' ~
_ ~ I ~ ~n ~J ~o 0~ vl o _ o ~ o I o l . a : :~ tD
_ 1~ - - -- 1-- :~ ~-v v v v v vlvlv v v v v v vlv~v-l~
1~ ~ ¦ ~ N 1~ _ ¦ _ ¦ N N ~ N N N I N ~
ul ul I Ul ~11 vi o I o I ~1 ul ~Jl ~Jl ~Jl 01 I ~
__ I O O ~ O I O O O O O I O I O I O I O I ~ I
t I I l ~ r I
~ ~ ~ 1 - I o l ,~ o ~ I P l P l - s~ l o I c I
; , ~I ~ o ~ o ~
~ o_ l' ! ~ P~ P; p o, p~ ~ o ~I oI -I PI PI -I P ~
~ ~ O ~ O ¦ O ~ -- ¦ O ¦ O ¦ N Vl ¦ N ~ N ~ O o I o I l + I rr r~
._ o! _¦ pll -I o ol N Pl l _l -¦ Pl ~ -¦ O j~. !
~ I o 11 ~ I 'o I o I o ~ I ~ I o ' o _ . , ~ I ~, . ,~, I I
I I 1 ~
V ~ o ~ o ~ O ! p~
..~ _~ _ .,~ _ ~ ~_ ~ ~ ~ .c~a I ~ .
v I v v, v v v v v v v v ~ . , .. ~ r.~ ~ ~ ~ ~ ~ ~ ~ ~ ~a ~ l O
p, vO~ vl l o vl o ~n Uol t.n ~n On ~n t~:1 . ;:5 o - j I I _ _ l O ~ O I ~ 0~ V ~ V ~ ~ ~ O
~ _ _. . _ ~ ~ ~
V~ Iv~ c ~On i oll ~~ o ~1 o~ ~1 o¦ ol~
¦ ~ ! ¦
! I l l o ~ o ~o, _, l W ,~ , I o . _ I ~ I o ~ _ I o It is quite obvious froln Table l that the peptide compounds of the present invention have a stron~ inhibitory activity an the tripep-tidyla~inopeptidases(Gly-pro-Leu-Ap and Gly-His-Lys-AP).
Shlesinger et al have recently isolated Gly-His-Lys as a tumor cell growth-promotin~ tripeptide, which seems to be produced in vivo by tripeptidase(EXPERIEN~IA 32, 324-325(1977)).
~herefore it can be expected that suppressive effects on growth of mammalian tumor cells will be obtained by administration of amastatins'and tetrapeptide derivatives of the present invention.
Thus the present invention provides new peptide derivatives -that exhibit inhibitory activity on amino-peptidase and tripeptidylaminopeptidase in vitro and in vivo and that can be expected -to be ef'fective as an antitumor agent.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
Example 1 Synthesis of (2S,3R)-A~ Val-L~s-Asp.
1-(1) Synthesis of Boc-Lys(Z)-Asp(OBæ~.
_ ! DCCD(612 mg) was mixed with a suspension of 1~03 g of Boc-Lys(Z), 1.31 g of Asp~OBz1)2~TosOH and 437 mg of ~OBt in anhydrous tetrahydrofuran(abbreviated ~HF hereafter) and stirred ~or 5 minutes. Then 0.414 ml of anhydrous triethyl-amine was added dropwise to the suspension and ~gitated for
8 hours, After DCU formed was removed by filtration, the evaporation of the solvent from the filtrate under reduced pressure yielded 4.8 g of syrup. This syrup was puri~ied by silica gel column chromatography(~akogel C-200, 50 g~
~Yako Pure Chemical Industries, Ltd.) using a solvent system of benzene and ethyl acetate(5/1) to give 1.74 g of cryskals of the title compound.
Rf t 0.12(benzene/ethyl acetate - 5/1; a Silica gel 60 F254 plate, E, Merck~
p~ : 106 ~ 107.5~
~ a ~ - 1 2.4 ~c-2-0 1 CH3OH) n. m. r 8ppm (CDC43) t.3~1.9 ~6H , m ~ - (C~2 )3 - ) 1.44 ~9H . ~ , -C(CH3)~) æ9~~4 (4H , m , -CH2CO- , -CH2~nH-) 4.7~5.2 (2H 9 m , -C~NH-x2) ~05 (2H , ~ 9 -CH2Ph) ~11 (4H ~ g , -CH2Ph X 2) ~8~7.2 (2H , m , -NH~ x 2) ~34 ~15H , g , ~CH2Ph x 3) (Ph means the phenyl group) IR tv KBr cm 1 s 3325(=NHCo )~ 1735(ester C=O), 1690(urethane C=0), 1650(amide C=0) . . .
1-(2) Synthesis of ~oc-Val I~(Z)-Asp(03Zl)~.
rrifluoroacetic acidtO.6 ml) was added dropwise to a solution of 1.14 g of Boc-LyS(Z)-ASp(OBZ) in 11 ml of metnylene chloride under cooling with ice and then war~ed to room temperature, Three hours later, the solvent was evaporated off. The evaporation residue wa~ dissolved in 20 ml of ethyl acetate and washed three times with 5 ~1 each of cold saturated solution of sodium bicarbonate and then twice with 5 ml each o~ saturated solution of sodium chloride.
A~ter dehydration over anhydrous sodium sulfate, the removal of the solvent by evaporation in vacuo provided 873 mg of free amine. This free amine~R73 mg), 314 mg of Boc-Val and 234 mg of l-hydroxybenzotriazole(abbreviated HOBt hereafter) were dissolved in 17 ml of anhydrous ~HF. A~ter 328 m~ of DCCD was added under ica-cooling, the mixture was stirred for 4 hours at room temperature. DCU formed was filtered off and the filtrate was evaporated to dryness under reduced pressure. 'rhe evaporation residue was chromatographed on a silica gel column(40 g, same supporting ma~r1al as described above) using a developing solvent mixture of benzene, ethyl acetate and chloroform(2/1/1~
to yield 906 mg o~ crystals of Boc Val-Lys(Z)-Asp~OBzl)2.
Rf : 0~54(benzene/ethyl acetate/chloroform=l/l/l, a silica gel plate as described above) n. m. r ~ppm ~CDC~3 ) 0.8andD.91 (6H, each d, J-4Hp,~CH~C~I ) .39 (9H, ~ ~--C(CH3 )3 ) 1. 3~2.2 ( 7H , m 9--( CH 2 ~ CH~C~ ) 2.~4 ( 4H, m, -CH2NH- ~ CH2~-~
~&~4.1 ( 1H 9 m, ~
4.2~4.7 ( 1H[, n" --NH--CH--(,'--) 4.8--~ ( 1H , m , -NH~CH~--) 0 2 and 5. 0 8 ~ 6H, each ~ H 2 Ph X 3 ~0~~5 ( 2H 9 m, -NH-x2 ) ~8--7.0 ( 2H, m, -NH-X2) 7.26and7.30 (15H 9 each3,--CH2Phx3) m. p : 13B ~ 139~
25.1 ~c-1.52 , C~30H) IR ~ KBrx cm 1 : 3325(-NH-Co-), 1740, 1730~ester C-O), 1690(urethane C=O), 1645(amide C=O~
1-(3) Synthesis of Z-(2S 3~)-A~MHA-Val-Lys(Z)-Asp(OBzl)2.
-One milliliter of trif'luoroacetic acid was addeddropwise to a soluti.on of 503 mg of 30c-Val-Lys-Asp(03zl)2 in 4 ml of ethylene chloride under cooling with ice and allowed to stand for 2 hours at room temperaturec After the solvent was evaporated o~f in vacuo, the evaporation residue was dissolved in 20 ml of ethyl acetate. ~he ethyl acetate solution was washed three time~ with 2 ml each of cold saturated solution of sodium bicarbonate and then twice ~ith 2 ml each o~ saturated solution of sodium chloride. After dehydration over anhydrous sodium sulfate, the solvent was removed by evaporation under reduced pressure to provide 350 mg of fr~e amine, Thi~ free amine, 177 mO of Z-(2S,~R)-AH,~A(Z) and 107 mg of HOBt were dissolved in 13 ml of anhy~ THF~ Under cooling with ice, 150 mg of DCCD was added to the. mixture and stirred for ~ hours. DCU formed was removed by filtration and the solvent was evaporated off in vacuo to give 502 mg of solid matter. Silica gel column chromatography(20 g silica gel; same supporting material as described above~ using a solvent mixture of benzene~ chloroform and acetonetl/l/l) yielded 387 mg of Z-(2S,~R)-AHMHA(Z)-Val~Lys(Z)-Asp(OBzl)2 in the crystalli~e form.
Rf s 0.57(benzene/chloroform/acetone-1/1/1, a silica gel plate as described above~
m. pO : 162 ~ 163~
~a)D : -18.0 (c-0.88 , CH3 OH) n. m. r ~ppm(CDC~3) 0.8~Q95 (12H , m , -CH(CH3)2 x2) 1.2 ~1.8 (loH ,m ,-~CH2)-x3 ,_CH(~H3)2X2 ~H2--CH ( CH3 ) 2 ) -~ -2.8~~2 (4H , m , -CH2NH- , -CH2CO-) ~8~4.25 (2H ,m , -OH , -NH-~H-~-~
4.25~4.~ (2H , m , N-cH~ox2) 4.8~~1 (1H , m , N-CH-CO) ~03, ~06, ~ 12 (8H , each 9, CH2Phx4) 7.35 (2 oH, ~ , CH2 Phx4 ) IR sv KaBr cm 1 ~ 3325(-NH-Co-), 1750, 1735(ester C=0), 1695(urethane C=0), 1645(amide C=0) 1-(4) Synthesis of (2s,~R~-AHMHA-Val-Lys-Asp.
_ _ _ Z (2S93R)-AHMHA-~al-L~s(Z)-Asp(OBzl)2(372 mg) was dissolved in 13 ml of a solvent mixture of acetic acid~
dioxane and distilled water(mixing ratio 11/2/1) and then shaken for 3 hours in the pre,sence of 38 mg of 10 %
palladium-carbon catalyst in the atmosphere of h~drogen at 3.5 kg/cm2. After the palladium-carbon catalyst was removed by filtration, the solvent was evaporated off under reduced pressure. The evaporation residue was dissolved in 10 ml of distilled water and the evaporation followed by dissolution in water ~as repeated 3-4 times.
Recrystallization in a solvent mixture of 5 ml of distilled water and 10 ml of isopropyl alcohol provided 152 mg of (2S,~R~-AHMHA-Val-~ys-Asp.
Rf ~ 0.22~butanol/a~etic acid/water=4/1/2, a silica gel plate as described abo~e) mlp. ~ 240 ~ 260 C under decomposition Ld~22 1 -29,9(c=2.0, CH3COO~) Elemental analysis(for C22H41N508~
Found C:52.40 ~, H~8.$6 ~0, N:13~58 ~D
Calculated C:52.47 %, Ht8.21 %, N:13.91 %
Example 2 Synthesis of (2S,~R~-AHMHA-Val-Thr-Asp.
2-(1) Synthesis of Boc~Thr(Bzl)-Asp(OBzl)~.
The procedure described in Example 1-(1) was followed with 1.00 g of Boc-Thr(Bzl) and 1.57 g of Asp(O~z1)2 to give 1.69 g of crystals of Boc-Thr(Bzl)-Asp(OBzl)2.
Rf ~ 0.55(benzerle/ethyl acetate-5/1, a silica gel plate as described above) m. p. : ~55 ~ 765C
~22: ~225 (c=10 CH OH~
nr m. r, dppm tCDC~3) 1.15 (3H , d , J6Hz , ~CH-CH3) 1.45 (9H , ~ , -CtCH3)3) 2.92 (2~ , dd t ~sHz- 5Hz , ~H-CH2 CO-) ~-4.4 (2H . m , ~CH-OCH2Ph , fCH-NH-~-O~
4.52~ 4.98and~1~ (6H , each 8 ,-C~-Ph.X~) 4.85 (lH ,--HN_~ ~ CH2 ~
L
s.4S ~1H , m , -NH-) 73 (15H , s , CH2 Ph 7.4~7.6 ~lH , m , -NH-) IR ~ KBr cm 1 s 3340(NH-Co-), l74~, 1735(ester C--O), 1700(urethane C~O), 1650(amide C--O) 2-(2) Synthesis of Boc-Val-Thr(Bzl)~Asp(OBzl)2.
As detailed in Example l-(2)~ 9 g of Boc-Thr(Bzl)-Asp(QBzl)2 was treated with trifluoroacetic acid and then condensed with 569 mg of Boc~Val to yield le64 g of crystals of Boc~Val-Thr(Bzl)-Asp(OBzl)2.
R~ t 0038(benzene/ethyl acetate=5/l, a silica gel plate as described above) m. p. : 124.5 ~ 125.5~
.75 ~=2.0 ~ ~H30~I) n. mv r. ~ppm(CDC~) ~zl ~9, ~96, 1.11 (9H ~ each d , J6Hz , CH(CH3)2~CH-CE~) .9~2.3 (1H , m , -CH(CH~2 ) .9 (2H , dd , -CH2-CO) ~~4-4 (3H . m , -H~N-cH-~-x2 , -~H~CH 3 ) 4.ss, 4.96, 5.09 (6H , each ~ , -CH2 Phx3) .. . . .... .. .
48~~1 (1H , m , N-~H~
7.28 (15H , s , -CH2Phx3) ~8~7.8 (3H ,m , NH-x3) IR Iv K3rx cm 1 ~ 3300tNH-Co-), 1735(ester C=0), 1690(urethane C=0), 1650~amide C=0) 2-(3) Synthesis of Z-(2S,~R)-AH~IA-Val-Thr)Bzl)-Asp(OBzl)2.
_ .
As detailed in Example 1-(3), 676.5 mg of Boc-Val-Thr(Bzl~-Asp(03zl)2 was treated with trifluoroacetic acid and then co~densed with 283 ~g of (2S,~R)-A~HA t~ yield 555 mg of the title compound.
R~ ~ 0.58(benzene/ethyl acetate=l/l, a silica gel plate a~ described above) m. p. : 126 ~ 127.5~
~22 -~91 (c=~ , CH~ OH) n. m. r. ~ppm ~CDC~
0.88,0.91, 1.1Q (15H , each d , J6H~, -cH(cH3)2x2 .
~Bzl -~H-CHs) 1.2~2.1 (4H I m , -CH(CH3)2 x2 , -CH2-CH(CH3~) 2.9 (2H , dd , CH2-CO) ~8~4.5 (5H $ m , N-CH-CH2, -~H~ CH-~X3) 4.55, 4.98, ~05, 5.08 (2H , ~ , -C~2PhX4) 4.8~~2 (1H , m , -NH-~H-~) 4.5~4.7 (1H , m , -OH) ~4~5.8 (2~ nHX2) A8~%1 (1H ~ m , -~nH) 7.~-t.8 (1H , m , -~nH) 7.~ (20H I ~ , CH2 PhX4) -~4-IR Iv K9r cm 1 , 3300(NH CO-), 1740(ester C=O), 1690(urethane C--O), 1650(amide C=O) 2-(4) Synthesis of (2S _R)-AHM~IA-Val-r~hr-Asp.
Z-(2s~3R)-AH~IA-val-Thr(Bzl)-Asp(oB2l)2(468 mg) was dissolved in 3.0 ml of a solvent mixture o~ acetic acid, dioxane and distilled water(mixing ratio 11/2/1) and then shaken with 46 mg of 10 ~0 palladium-carbon catalyst in the atmosphere of hydrogen at 3. 6 kg/cm2 for 26 hours. After the c&talyst was filtered off, the filtrate was e~aporated to dryness. The evaporation residue was dissolved in 10 ml of distilled water and a~ain evaporated to dryness. Complete removal of the organic solvents by repeating the above evaporation p~ocedure 3-4 times provided 250 mg of the title compound.
Rf ~ 0.37(bu1;anol/acetic acid/water=4/1/1, a silica gel plate as descl~ibed above) m.p. s 196 - 198C(transformation into needle crystals around 166 - 168 C) ~3 22 s -19.9~(c=2.0, CH3COOH) Elemental analy~is(for C20H3~N40 Found C~50.18 ~0, Hs7086 ~o, Ntll.43 ~;~
Calculated Cs50.41 5;, Ht7.62 i~, N111.76 ~0 Example 3 Synthe~i~ of (2s~3R)-AH~A-val-Leu-A
3-~l) Synthesi~ of Boc-Leu-Asp(0Bæl)2.
~y the same procedure as presented in Example l-(l~, 1.04 g of Bo -Leu and 2.19 g of Asp(0Bzl)2~os0H were conden~ed and then subjected to silica gel column(~0 g~
same supporting material a~ described above) chromatogr~phy using a developin~ ~olvent of benzene and ethyl acetate (mixing ratio l0/l) to yield 2.39 g o~ syrup of the title compound, Rf 8 0.49(benzene/ethyl acetate=5/l, a silica gel plate as described above) D -30~7 (C=2-0, CE~3 OH~
n. m. r~ ~ppm (CDC~3) ~88 .(6H , d , J5Hz, -CH(C~3 )2 ) 1.40 (9H , s , -C~.CH3 ~ ) 1.2~1.7 (3H , m , -CH2 -, -CH(CH3)2) 2.9 b (ZH , dd , -CH2-C-) ~8~4;4 (1H , m , ~-CH-NH-) 4.7~~1 (2H , m , -~-CH-NH-) 5D6, ~13 (4H , each s , C~ Phx2?
6B~7.2 (1H , m , NH) 73~ (10H . s 1 CH2PhX2) _36-~ 7 IR ~v ~a~rx Gm 1 1 3350(-NI~-Co-), 1740(ester C-0), 1690(urethane C=0), 1670(amide C=0) 3-(2) Synthesis of ~oc-Val Leu-Asp(03zl)2.
By the same procedure as presented in Ex~mple 1-(2), 1.80 g of Boc-Leu Asp(03z1)2 was treated ~ith trifluoro-acetic acid; condensed with 651 mg of Boc-Val and then submitted to silica gel column(50 g, same supporting material as described above) chromatography employing a solvent mixture of benzene and ethyl acetate(mixing ratio 5/1) to ~ive 1.53 g of crystals of the title compound, Rf ~ 0.22~benzene/ethyl acetate=5/1, a silica gel plate as descrlbed above) m. p. : 112 ~ 113~
~22 ~ -39.8 (c=1.57.~ CH30H) n-mO r- ~ppm ~CDC~3) Q8~Q~5 (12H , m , -CH(CH3)2 x2 1.41 (9H , 9, -C(CHs)3 ) 1.3~2.1 (4H ,m ,~CH(CHs)2 X2 ,~C_2~CH(CHs~2 ) ~94 (2H , t , -CHz ~C0-) ~89 (lH , dd , -N~I-CH~
4.2~~3 (3H , m , -~nH-CH ~-X2 , ~ ) ~05. 510 (4H , each 9 , -CHz~PhX2) ~37-7.~0 (10H , ~ , -cH2phx2) 66 (1H , m , ~nH~) 7.08 ~lH , m g ~NH-) IR ~v m~Brx om 1 ~ 3340(-NH-Co-), 1750(e~ter C=O), 1695 (urethane C=O), 1650(amide C=O) 3 (3) Synthesis of Z-(~S,~R)-AH~IA-Val-Leu-Asp(OBzl)2.
By the same procedure as presented in Example 1-(3)0 700 mg o~ BoC Val-~eu-Asp(OBzl)2 was treated with trifluoro_ acetic acid~ condensed with 281 mg o~ Z-~2S, 3R)-AHMHA and recrystallized from a mixture of ethyl acetate and hexane t~ provide 417 mg of crystals of the title compound~
Rf s 0,60(benzene/ethyl acetate=l/l, a silica gel plate as described above) m. p~ : 156 ~ 157 [a~ _34l (c=20 , CH30H) n.m. r. ~ppm (CDC~3) ~8~1.0 (18H , m , -CH~CM3)2 x3) 1.3~æ3 (7H ,m ,-CH2~CH(,C~I3~ x2 ,-CH -(CH3~ ) 2.9 (2~ ~ m , -~H2-CO-) ~8~4.7 (3H , m , -NH~H-CO-x3) 4.7~s3 (lH ,m t -~nl-CH-CO-) ~02 (4H , ~ , -C~ -p~x2) -38~
~06 (2H 5 S ~ -C~I2-Ph) 5.5~~9 (1H ,m , -NH) 7.0~~.6 (3H , m , ~NHX3) 73 (15H , ~ ~ -CHz Phx~) IR ~v KEr cm 1 ~ 3325( NH-~-), 1745(ester C-O), 1680(urethane C=O), 1640(amide ~~0~
3 (4) Synthesis of (2S,3R)-A~HA-~al-Leu-Asp.
Under the same conditions as described in Example 2-(4), 416 mg o~ z-(2s~3R)AHMHA-~al-Leu-Asp(~Bzl~2 was hydroge~oly~ed to give 223 mg of (2S,3R~-AHMHA-Val-Leu-Asp.
R* ~ 0.38(butanol/acetic acid/water=4/1/1, a silica gel as described above) mOp, ~ 264 - 266C
~22 ~ 41~7(c=2.0, C~3COOH) Elemental analysis(for C22H40N408) Found C~53,79.%, ~:8.63 %1 N~ 19 ~a Calculated c 54.o8 yfO, Hl8.25 jS, N~11.47 ~0 Example 4 Synthesis of (2S,3R)-AHr~HA-Val-Glu-As~.
4-(1) Synthesis o~ Boc-Glu(OBzl)-Asp~03zl ?~
Boc-Glu(OBzl) (1 7 04 g) was subjected to condensation with Asp~OBzl)~ under the same reaction conditions as prese~ted in Example 1-~1) and then chromatographed on a silica _ ~; 9 _ gel column~50 g, same supporting material a~ described above) using a solvent mixture of benzene and ethyl acetate(mixing ratio 10~1). The recrystallization from a solvent mixture of ether and hexane yielded 1.73 g of crystals of the title compound~
Rf ~ 0.73(benzene/ethyl acetate=2/l, a silica gel plate as described above ) m. p. : b9.s ~ 70.5~ !
~ ~2 -10.7 (c=20 , CH30H) n.m.r. : ~ppm (CDC~3) 1.41 (9H , s , -C(C~ )3 ~0 (2H 9 m , CH2-CH2-CH-~ ~
~4 (2H , m , -~CH2 CH2-CH-) 2.94. (2H ,dd , -~-CH-C,H
~9~4.4 (1]H , m , -~nH-CH~
; 4.7~~1 (1H , m , -~nH-CH-~-) 505 (2H-, 8 ~-CH2 Ph) 5.10 (4H , 8 , -cH2PhX2) 7.0~7.5 (2H ,~ , -~n~- x2) .. !
7.27,7.30, 733 ~15H , each 3 ,-cH2phx3) IR ~v ~Br cm 1 ~ 3330(-~H-~-), 1750, 1735(es-ter C=0~, 1690~urethane G--0~, 1655(amide C~0) --~0 -4-(2) Synthesis of ~oc~Val-Glu(OBzl)-Asp(OBzl)~.
As described in Example 1-(2), 1.63 g of Boc-Glu(OBzl)-Asp(OBzl)2 was treated with trifluoroacetic acid and then condensed with 559 mg o* ~oc-Val 3y silica ~el column (20 g, same supporting material as described above) chromato-graphy usin~ a developing solvent system of b~nzene-ethyl acetate(3/l), 1.38 g of crystals of the title compound was obtained, Rf s 0.16(benzene/ethyl acetate-5/l, a silica gel plate as described above3 m. p. : 113 ~ 114 ~a322 : -25.2 (c=2-0 , CH3 OH~
n.m. r. 8ppm tCDC~ 2) 0.88~ 0.91 (6H , each d , J7H~ , -CH(CH3 )2 ) 1.41 (9H , s , ~C~CH~ )3 ) 1.8~2.2 (5H , m ~cH-c~2-cH~2 ~ CH(CH3 )2 2.94 (2H , dd , ~CH-CHz~
~91 (1~I , dd , J6Hz, 8H7,-N~I-CH~
...... ~
4.3~5.0 (2H ~ m , -NH-CH~
s.os (2H . 9 ~ - CH2 - Ph) ~10 ~4H , ~ , -C ~ -PhX2 ~0~~3 (1H , m , -NH-) 68~-~.1 (2H , m , -NHX2) 7.2~ 7.~, 7.34 (15H ~ each 3, - CH2-PhX3) IR ~v KBr cm 1 s 3330(-NH-~-), 1730(ester ~=o)~ 1685 (urethane C=O), l650(arnide c=oj 4-(3) Synthe~îs of Z-(2S,3R) AH~HA-Val-Glu(OBzl)~Asp(O~zl) ~ mploying the same procedure as detailed in ~xample l-(3), 707 mg of 30c-Val-Glu(OBzl)-Asp(OBzl)2 was treated with trifluoroacetic acid and then submitted to condensation with 280 mg of Z-(2S,3R)-AHI~HA. Crystals of the title compound(343 mg) was recovered by recrystallization from a chloroform-ether mixture.
Rf s 0.53(benzene/ethyl acetate=l/l, a silica ~el plate as described above) ml p. : 157 ~ 15~
~a~22 : -22.1 (c=2.0 , CH30~) . m. r. ~ppm (CDC~3 ) :
~75~Q95 (12H , m , -CH(CH~)2 x2) 1.2~1.7 (2H , m , -CH~CH3 )2X2) 1.7~ æ2 (4H ,m ,-N~- OEI-C~ -CH(CH3)2, -CHz-C7I2CO-) 2.2~2.5 ~2H , m , ~CH2-CH2-~-) 2.7~~0 (2H , m , >CH-CH2-~-) ~8~ ~0 (4H , m , -~nH-CH-~X4) ~0, S05 (8H , ~ , -CH2 PhX4) . .. . .. . . .. .
5.4~~8 (lH ,m , ~NH) 7.1~7.8 ~3H , m 9 - NHX3) 7.25, 7.2B ~20H ~ -CH2PhX4) IR sv~aBr cm 1 ~ 3425(-NH-~-), 1735(ester C=O), 1680 (urethane C=O), 1640(amide C=O) 4-(4) Synthesis of (2S,3R)-AHMHA-Val-Glu-Asp.
Under the same reaction conditions as detailed in ~xample 1-(4), 341 mg o~ z-(2S~R)-AHMHA-Val-Glu(OBzl)-Asp~OBz1)2 was hydrogenolysed to provide 247 mg of crystals of the title compound.
Rf s 0.39(butanol/acetic acid/water=4/1/1, a silica gelplate as described aboye)m.p. : 159 - 161C
2 ~ -29.4 (c=l.O, 80 % CH3COOH in water) Elemental a~alysis(for C22~40N408) Found C:54.35 %, H:8.21 %, N:11.24 o~
Calculated C:54~08 %, H18. 25 ~, N:11.47 %
2xample 5 Synthesis of (2S,3R)-AHMHA-Thr-~Jal-Asp, _ . .
5~ Synthesis of 30c-Val-Asp(03zl ~.
~ ~ _ . . _ Under the same reaction conditions as specified in Example 1~ , the condensation produc~ of 4.0 g of ~oc-Val with 8.94 g o~ Asp(OBz1)2 was chro~atographed on a silica gel column(100 g~ same supp~rting ~aterial as -~3-described above~ using a developing solvent system of benzene anq ethyl aceta~te~5/1) and then recrystallized from a solvent mixture of chloroform and hexane to give 8,?7 g o~ crystals of the title compound, R~ ~ 0.39(benzene/ethyl acetate=5/1, a silica gel plate as described above) m~ p. : 80 ~ 81~
~3 -24.6~ (c--~O i CH30~) n~m- rO ~ppm (CDC~3) Q8, ~92 (6H geach d ,J5H~, ~CH(CH3 )2 ) 1.42 (9H , ~ , -C(CH3)3 ) 1.9~2.3 ~1H , m , ~CH(CH3)2 ) 2.99 (2H , dd , -CHz~
~92 (1H , dd ,35H~. 9H~ CH~
4.~~~1 (1H ,m , ~NnI~~H~
5.05, ~12 (4H , each si -CH2-PhX2) ~7~7.0 (1H ~ m , -~nH) 7.2~7.4 (1H , m ,--N~I~
7.30 (10H , 8 , -CH2-PhX2) IR ~ KBrx cm 1 ~ 3325(-NH-~-), 1730(ester C=O), 1690 (urethane C=O), 1650(amide C=O) .. . .
5-(2) Synthesis of Boc-~hr(Bzl)-Va.l-Asp(OBzl)z.
~y the same procedure as detailed in Example 1-(2), 1.0 g of Boc-~al-Asp(O~zl)2 was treated with tri~luoro-acetic acid and then subjected ~o ~ondensation with 535 mg of Boc-Thr(gzl), Silica gel column(25 g, same supporting material as described above~ chromatography with a developing solvent mixture of benzene and ethyl acetate(l0~1) followed by recrystallization in an ether hex~ne m~ ~re yielded 611 mg o~ the title compou.~d.
Rf ~ 0.86(benzene/ethyl acetate=2/1, a silica gel plate as described above) m.p. : 136 - 137C
[~D ~ -17.5 (c-2,0, C~ 0~) I~ sv Ka~r cm 1 ~ 3300(-NH~ 1730(ester C=0), 1685 (urethane ~-0), 1640(amide C=0 n~ m. r ~ppm (CDC~3) Q75and ns 7 (6H , each d. J55Hz, -CH(CH3)2) ~Bzl 1.1~1.2 (3H .m , -~H-CH3) 1-43 (~H , 9 9 -O-C(CH~)3 ) 1.7~2.3 (4H ,m , CH2-CL(CH3)2 . ~ H-OBzl) 2.89 ~2H ,dd ~--C~-I2-C-) 41~4-~ (3H , m ~ H-NH X3) ~os (2H , ABq , -CH2Ph) , ~o~, 6.95 (~H .m , -NH-X3) 7.29 (sH , ~ , -CH~ Ph) 5- ( 3 ~ Synthesis of Z- ( 2S, 3R) -AHMHA-Thr ~Bzl ) -Val-Asp (OBzl ) 2 .
3y the same procedure as described in Example 1-(2)~
520 mg of Boc-~hr(~æl)~Val-Asp(OBzl)2 was treated with trifluoroacetic acid and then subjected to condensation ~ith 187 mg of ~-(2~3R)-AHMHA. ~he condensation product was charged on a silica gel column(30 ~; same supporting material as described above) and eluted with a developing 501vent mixture of benzene ,and ethyl acetate(3/1). 3y recrystallization from an ether-hexane mixture, 386 mg of the title compound in the crystalline fQrm was obtained.
Rf ~ 0,37(benzene/ethyl acetate=l/l, a silica gel plate as described above) m.p. t 93.0 - 96.05C
~D ~ ~18,5 ( c=l . o, CH OH) IR ~ KEr cm 1 ~ 3325(-NH-~-), 1730(ester C=O), 1630(amide C=O) n, m. r ~ppm (CDC43) : I
~72, ~82, ~09 ~91,1.08 (15H ,each d,-CH-CH3X5) 1.2~2.0 (5H , m , -~H-CH3X3 , -CH2-CH-) 2.Bg (2H , dd , -CH2~
~9~A5 (3H , m , -~nH-CH-x3) 45~4-9 (2H 9 m ~ ~nH-C~-X2) 4.55 (2H p ABq , -CH2Ph~
~J~
., ' . , . . . ~ . .. . . . . . . . . .
50~ (4~ CH2PhX2) ~09 (2H ~ s t ~CH2Ph) 5~7 (2H , m , ~NH) ~~77 (3H , m ~ nIX3) 7.29 (20H , ~ , -CH~PhX4) 5-(4) Synthesis of (2S,~R~-AHMHA-Thr-Val-A~p, Under the same conditions of hydrog3nolysis as detailed in Example 1-(4), 200 mg of the title compound was recovered from 386 mg of Z (2s~R)-A~MHA-Thr(~zl)-val-Asp(o~l)2 by using 10 ~0 palladium-carbon.
R~ ~ 0.41(butanol/acetic acid/water=4/1/1, a s;lica gel plate as described aboYe ) m.p. ~ o~er 180 C under decomposition Elemental analysis(for C2oH36N409) Found C~50.27 %, ~7.95 ~o, N~11.49 %
Calculated C~50.41 %~ Ht7.62 ,~0, Nt11.76 ~
Example 6 Synthesis of (2s,3R)-AH~IA-Leu-Val-Asp.
. _ 6-(1) Synthesis of Boc-Leu-Val-Asp(OBzl~.
Boc-Val~Asp~OBzl)2(708 mg) prepared by the same method as presented Ln Example 5-(1) was treated with trifluoro-acetic acid un~er the conditions speci~ied in Example 1-(2) and then subjected to condensation with 397 mg of Boc-Leu.
Silica gel column(30 g~ same supportin~ material as described F~
,....... . .. ..
above ) chromato~raphy with a developing solvent mixture of benzene and ethyl acetate(3/2~ gave 673 mg of crystals of the title compound~ , Rf ~ 0.56~benzene/ethyl acetate=3/2, a silica gel plate as described above ) m.p. ~ 135 - 136 C
D t -36.7 (c=2.0, CH30H) IR ~v K~r cm 1 ~ 33lO(-NH-~-), l730(ester C=0), 169Q
~urethane C-0), l640~amide C~0) n~ m. r. 8ppm fCDCL3) Q~~1.1 (~2H , m , -CH(C~2 x2 1.42 (9H , 8 , C-(CH~)3 ~
1.5~~.3 ~4H , m , -CH2 -CH ~ , -CH( CH3 )2 ~ 2 ) ~95 (2H , ~d , -CH2~-) Ao~4.6 (2H , m , -~nH-CH-~-X2) 4.6s~~4 (2H ,m , -NH-CH~
~06, 5.11 (4H , each ~ , -cH2phx2) 67~7.~ (ZH ~m , -NH-X2) 7.~2 (1oH , s , -cH2phx2) 6-(2) Synthesis of z-(2s,3R)-AHMHA(Z~-Leu-Val-Asp(OBzl)2.
By the same method as ~etailed in Example l-(2), 509 m~ of Boc-Leu-Val-Asp(OBzl)2 was treated with trifluoro_ acetic acid and then conden~ed with z-(2s~R)-AHMHA~z) (285 mg). Af'ter c:olumn chromatography on silica gel (30 g, ~ame supporting ma-t~rial as ~escribed above ) with a developing solven-t system of benæene and ethyl acetate (3/1 ) 490 mg of the title compound was ~btained.
Rf s 0.45(benzene/ethyl acetate~2/1, a silica gel plate as described above ) m.p. s 140 ~ 143 G
~d~ 22, -17 . 6 ( c=l . 0 , CH3 01{/CHC1 3=5/1 3 XR ~ v ICBr cm 1 ~ 3320(-NH-~), 17.50(ester C=0), 1695 (urethane C-0) j 1645(amide C=0) n. m. r. ~ppm (CDC,C3 ) o 7~0 ~5 ( 1 8H,--CH(CH3 )2 x3) 2.3 (7H ,m .~~2~ x 2 ,-OEI(CH3)2x3) ~9 2 ( 2EI, d d ~ CH2--~--0- ~
4.1~4.6 ( 4H , m , NH--CH-~--x 4 ) 48~5.0 (1H ,m ,NH-CH- ~) ~0~~2 ( 9H ~--CH2 -Phx4 , --CH--0-~) ~ 2~ 6 ( 1 H , -NH ~ ) 6. 5~7. 0 ( 3 lH ~ --NH--X 3 ) r Z3~7.4 ( 2 oE~, CH2 -PhX4 ) 6_(3) SynthesiS o~ (2s~3R)-AHMEA-Leu-val-Asp.
Under the same operational condîtions as presented in Example 1-(4), 200 mg of Z~(2S,3R)-~H~(Z)-Leu-V~l--49~
... . . , ., . . . . ~
Asp~OBz1)2 wa~ hydro~enolysed to give 72 mg o~ the title compound.
Rf t 0~42(butanol/acetic acid/water=4/1/1, a silica gel plate as described above~
m.p. ~ over 249C under decomposition ~D s -30~2 (c=l.O, CH3COOH~
Elemental analysis(for C22H40N408) Found C:53.75 %, H~8.18 ~0, N,11.28 ~0 Calculated Cs54.08 ~0, H,8.25 ~, N,11.47 -~0 Example 7 Synthesis of (2s,3R)-AHMHA~Lys-Val~Asp.
7-(1) Synthesis o~ Boc-l~s(Z)-Val-Asp(OBzl)2.
Under the same reaction conditions as specified in Example 1-~2), 1.0 g of ~oc-Val-Asp(OBzl)2 prepared by the method detailed in Example 5-(1) was treated with trifluoro_ acetic acid and then subjected to condensation with Boc-Lys(Z).
Silica gel column(30 g, same suppor-ting material as described above) chromatography with a developing solvent mi.xture of benzen~ and ethyl acetate(3/2) yielded 866 mg o~ crystals of the title compound.
Rf ~ 0.26(benzene/ethyl acetate=2/1, a silica Oel plate as described above) m.p. 1 122 - 124C
2 1 -21.2 ~c-3.0, CH30H) IR Iv mKaBx cm l ~ 332S(~NH-~-), 1740(ester C=0), 1685 . (urethane C=~), 1640(amide C~0) n.m.r. 8ppm ~CDC~3) 0.86ando.90 (6H 7 each d , J7Hz ~ -CH(C~)2 1.41 (9H , 3 ~-C(CH3)3 1.2~2.1 (7H 3 m ~--(CH2~ CH~CH3) 2.~-~3 ~4H ,m , -CH2-NH- , -CH2~C0-~
~9~4-5 (2H , m , -~nH--CH-~-x2) 4-7~5~ (lH , m , -~nH-CH~
~05~~ (6H ~, -CH2-PHx3) ~3~~5 (1H 9 ~ ~ - ~nH ~
~7~7.2 ~2H ~-m , -~H~x2) 733 (15~ , -c~2phx3~
7~(2~ Synthesi~ o~ 2-(2S,3R)-AHMHA(Z)-Lys(Z)-Val-Asp(OBzl)2.
Boc-Lys(Z)-Val-Asp(OBzl)2(534.4 mg) was treated with tri~luoroacetic acid and th~n subjected to condensation with 258 mg of Z-(2S,3R)-AHMHA(Z) by the same prodedure as specified in Example 1-(3). m e condensation product was puri~ied by silica gel column(30 g, same supporting material as dsscribed above) chroma-tography with a developing solvent system of benzene and ethyl acetate~2/l) to yield 510 mg of the title compound.
Rf t 0~53~benzene/ethyl acetate=l/l, a silica ge-~.plate as described above~
. .. . . . . . . .. . .. ... . .. ...
v2 , _g ~ 6 ( c- O . 85, CHcl 3/c}{3 oH=l/6 ) IR, " KBr cm 1, 3320(~NH~ ), 1730(ester C=O). 1690 ( urethane C=O), 1640 (amide C=O) n.m~ r . ~ppm ~CI)C~3 ) Q75~Q95 ( 1 2H .,--CH(CH3 )2 x2) -1~22 ( 1 ~H .--(CH2 )3-- ,--CH(CH3 )2 x2, --CH2--CH 1 CH 3 )2 2.8~~3 7 4H, -CH2 ~ C~2-NH~) 4.0~4.6 ( 4H,--NH--CH--~--x ~ ) 4.~~~2 ~ 1 2H ~--CH2--Phx5 ,/CH~O--6s~7. o ( 4H ~--NH-x4 ~
7. 2~7. 4 ( 2 5H, CH2--Phx 5 ) 7-(3~ Synthesis of (2S,~R)-AHMHA-Lys-Val-Asp.
. . _ .
Hydrogenolysis of 230 mg of Z-(2S,~ HMHA(Z)-Lys(Z)-Val-Asp(OBz:L)2 under the operational conditions specified in Example 1-(4) gave 84 mg of the above-listed compound.
Rf t O.l~bu-tanol/acetic acid/water=4/1/1~ a silica gel plate as described above) m.p. s over 170~C under decomposition C~D2 S -28.6 (C=1.0, CH3COOH) Elemental analysis(for C22H40N508) Found Ct52.72 %, H~8.54 9~, Nsl3.57 5~
Calculated C~52.47 ~0~ Hs8.21 ~D~ N,13.91 YD
. . .
D
Example 8 Synthe~i~ of (2S,~R)-~HM~IA-Glu-Val~Asp.
8-(1) Synthesis of Boc-Glu(OBzl)-Val-Asp(OBzl)2.
By the same procedure as presented in Example 1-(2~, loO g of Boc-Val-Asp(oBzl)2 was treated with trifluoro-acetic aGid and then submitted to condensation with Boc-Glu(OBzl). After column chromatography on silica gel (30 g, same supporting ma~erial as described above) with a developing solvent system of benzene and ~thyl acetate(3/1)3 780 mg of the title compound was obtained.
Rf a 0,51(benzene5ethyl acetate-2/1, a silica gel plate as described above) m.p. ~ 124 - 126C
~d~22 ~ -27.7(c=1.5, CH30H) IR 5~ KBr cm 1 ~ 3300(-NH-~-), 1730(~ster C=0),1690 (urethane C=O), 1640(amide C=O) n.m.r. 8ppm (CDC~3) 0~85~ndo.38 (6H ., each d , J6.5Hz , CH(CH3)2) 1.4 (9H , 8 , -CHtCH3)3) 1.8~2.3 (3H , m , -CHtCH3 )2 , -cH 2 -cH 2 ~ ) 2.3~2.7 (2H , m , -CH2~CH2-CO-) 2.9s (2H ~ ~CH-CH2CO-) .. .. . .. . .
4.0~50 (3H ~ -rnH-CH-CO-x3) ~05~~1 (6H ~ -CH2Phx3 (lH , -~nH~) , . , .. , , ..... , .. . ., ., . ... . , ., .,,, .. , . ~ .. , .. , . . ., ~ , . . . ... .
~8~7.1 (2H , -NH-x2) 7.3 (15H , -CH2Ph~33 8-(2) Synthesis of Z-(2S,3R)-AHMHA(Z~-Glu(OBzl)-Val-~ . ~ . . . i . __ Asp(OBz1)2.
Under the same operational conditions as detailed in Example 1-(2); 510 mg of Boc' Glu(OBzl)-Val-Asp(O~zl)2 was treated with trifluoroacetic acid and then condensed with 270 mg of Z-(2~,3R)~ HA(Z). The condensation product was purified by column chromatography on silica gel (30 g, same supporting material as described above) using an elution solvent system of benzene and ethyl acetate(Z/1) to pr~vide 500 mg of the title compound.
R~ ~ 0.75(benzene/ethyl acetate=l/l~ a silica gel plate as described abo~e) ~d~22 ~ -10,,8 (c=l.O, CH30H) IR ~v K~r cm 1 ~ 3300(-NH-~-), 1740(ester C-O), 1690 (urethane C~O), 1630(amide C=O) n.m,r. ~ppm ~CDC~
(12H , -CH~CH~)2 x2) 1.0~1.8 (3H , -CH(CH 3)2 x2 ~ -CH2-CH(CH~)2 ) æ~ (2H , -CH2-CH2 -~-) æ2~æ6 ( 2H,--CH2--CH2~
2.8~1 (2H , ~CH-CH2- ) -~4~
, ., , , , , . , . _. _ _.. . .. . .. . . ..
q~
~9~4.6 ( 3H ~,--NH--CH--~X3) __ 4.7~5.1 ( 2H,--NH--C --~ ,>CH~O~
~o~s2 (l~H,--CH2--Phx5~) 5 ~~5.4 ~ NH--68~7.1 (3H ~-NH-7.2~7.4 (25H ~-CH2Phx5~
8-~3) Synthesis of (2S~3R)-AH~A-Glu-Val-Asp.
.. .. ,. ,,, .~._ The same conditions o* hydrogenolysis as detailed in Example 1-(4) yielded 65 mg of the title compound from 190 mg of Z-(2s~3R)-AHMHA(Z)-Glu(OBzl~-Val-Asp(O~zl)2~
Rf 1 0. 22(butanol/acetic acid/water-4,~ a silica gel plate as descri~ed above ) m.p. J over 165 C under decomposition 27 . 2 ( c-0 . 5, CH3COOH ) Elemental analysis(for C~8H42N4 ~0) Found C:50.36 ~0, H~7.12 ,~ N:10.86 Calculated C:49.99 i~, H~7.19 ,~, Ntll.ll ~
Example 9 Synthesis o~ (2S,~R)-AHMHA-Val-Thr-Phc.
~Yako Pure Chemical Industries, Ltd.) using a solvent system of benzene and ethyl acetate(5/1) to give 1.74 g of cryskals of the title compound.
Rf t 0.12(benzene/ethyl acetate - 5/1; a Silica gel 60 F254 plate, E, Merck~
p~ : 106 ~ 107.5~
~ a ~ - 1 2.4 ~c-2-0 1 CH3OH) n. m. r 8ppm (CDC43) t.3~1.9 ~6H , m ~ - (C~2 )3 - ) 1.44 ~9H . ~ , -C(CH3)~) æ9~~4 (4H , m , -CH2CO- , -CH2~nH-) 4.7~5.2 (2H 9 m , -C~NH-x2) ~05 (2H , ~ 9 -CH2Ph) ~11 (4H ~ g , -CH2Ph X 2) ~8~7.2 (2H , m , -NH~ x 2) ~34 ~15H , g , ~CH2Ph x 3) (Ph means the phenyl group) IR tv KBr cm 1 s 3325(=NHCo )~ 1735(ester C=O), 1690(urethane C=0), 1650(amide C=0) . . .
1-(2) Synthesis of ~oc-Val I~(Z)-Asp(03Zl)~.
rrifluoroacetic acidtO.6 ml) was added dropwise to a solution of 1.14 g of Boc-LyS(Z)-ASp(OBZ) in 11 ml of metnylene chloride under cooling with ice and then war~ed to room temperature, Three hours later, the solvent was evaporated off. The evaporation residue wa~ dissolved in 20 ml of ethyl acetate and washed three times with 5 ~1 each of cold saturated solution of sodium bicarbonate and then twice with 5 ml each o~ saturated solution of sodium chloride.
A~ter dehydration over anhydrous sodium sulfate, the removal of the solvent by evaporation in vacuo provided 873 mg of free amine. This free amine~R73 mg), 314 mg of Boc-Val and 234 mg of l-hydroxybenzotriazole(abbreviated HOBt hereafter) were dissolved in 17 ml of anhydrous ~HF. A~ter 328 m~ of DCCD was added under ica-cooling, the mixture was stirred for 4 hours at room temperature. DCU formed was filtered off and the filtrate was evaporated to dryness under reduced pressure. 'rhe evaporation residue was chromatographed on a silica gel column(40 g, same supporting ma~r1al as described above) using a developing solvent mixture of benzene, ethyl acetate and chloroform(2/1/1~
to yield 906 mg o~ crystals of Boc Val-Lys(Z)-Asp~OBzl)2.
Rf : 0~54(benzene/ethyl acetate/chloroform=l/l/l, a silica gel plate as described above) n. m. r ~ppm ~CDC~3 ) 0.8andD.91 (6H, each d, J-4Hp,~CH~C~I ) .39 (9H, ~ ~--C(CH3 )3 ) 1. 3~2.2 ( 7H , m 9--( CH 2 ~ CH~C~ ) 2.~4 ( 4H, m, -CH2NH- ~ CH2~-~
~&~4.1 ( 1H 9 m, ~
4.2~4.7 ( 1H[, n" --NH--CH--(,'--) 4.8--~ ( 1H , m , -NH~CH~--) 0 2 and 5. 0 8 ~ 6H, each ~ H 2 Ph X 3 ~0~~5 ( 2H 9 m, -NH-x2 ) ~8--7.0 ( 2H, m, -NH-X2) 7.26and7.30 (15H 9 each3,--CH2Phx3) m. p : 13B ~ 139~
25.1 ~c-1.52 , C~30H) IR ~ KBrx cm 1 : 3325(-NH-Co-), 1740, 1730~ester C-O), 1690(urethane C=O), 1645(amide C=O~
1-(3) Synthesis of Z-(2S 3~)-A~MHA-Val-Lys(Z)-Asp(OBzl)2.
-One milliliter of trif'luoroacetic acid was addeddropwise to a soluti.on of 503 mg of 30c-Val-Lys-Asp(03zl)2 in 4 ml of ethylene chloride under cooling with ice and allowed to stand for 2 hours at room temperaturec After the solvent was evaporated o~f in vacuo, the evaporation residue was dissolved in 20 ml of ethyl acetate. ~he ethyl acetate solution was washed three time~ with 2 ml each of cold saturated solution of sodium bicarbonate and then twice ~ith 2 ml each o~ saturated solution of sodium chloride. After dehydration over anhydrous sodium sulfate, the solvent was removed by evaporation under reduced pressure to provide 350 mg of fr~e amine, Thi~ free amine, 177 mO of Z-(2S,~R)-AH,~A(Z) and 107 mg of HOBt were dissolved in 13 ml of anhy~ THF~ Under cooling with ice, 150 mg of DCCD was added to the. mixture and stirred for ~ hours. DCU formed was removed by filtration and the solvent was evaporated off in vacuo to give 502 mg of solid matter. Silica gel column chromatography(20 g silica gel; same supporting material as described above~ using a solvent mixture of benzene~ chloroform and acetonetl/l/l) yielded 387 mg of Z-(2S,~R)-AHMHA(Z)-Val~Lys(Z)-Asp(OBzl)2 in the crystalli~e form.
Rf s 0.57(benzene/chloroform/acetone-1/1/1, a silica gel plate as described above~
m. pO : 162 ~ 163~
~a)D : -18.0 (c-0.88 , CH3 OH) n. m. r ~ppm(CDC~3) 0.8~Q95 (12H , m , -CH(CH3)2 x2) 1.2 ~1.8 (loH ,m ,-~CH2)-x3 ,_CH(~H3)2X2 ~H2--CH ( CH3 ) 2 ) -~ -2.8~~2 (4H , m , -CH2NH- , -CH2CO-) ~8~4.25 (2H ,m , -OH , -NH-~H-~-~
4.25~4.~ (2H , m , N-cH~ox2) 4.8~~1 (1H , m , N-CH-CO) ~03, ~06, ~ 12 (8H , each 9, CH2Phx4) 7.35 (2 oH, ~ , CH2 Phx4 ) IR sv KaBr cm 1 ~ 3325(-NH-Co-), 1750, 1735(ester C=0), 1695(urethane C=0), 1645(amide C=0) 1-(4) Synthesis of (2s,~R~-AHMHA-Val-Lys-Asp.
_ _ _ Z (2S93R)-AHMHA-~al-L~s(Z)-Asp(OBzl)2(372 mg) was dissolved in 13 ml of a solvent mixture of acetic acid~
dioxane and distilled water(mixing ratio 11/2/1) and then shaken for 3 hours in the pre,sence of 38 mg of 10 %
palladium-carbon catalyst in the atmosphere of h~drogen at 3.5 kg/cm2. After the palladium-carbon catalyst was removed by filtration, the solvent was evaporated off under reduced pressure. The evaporation residue was dissolved in 10 ml of distilled water and the evaporation followed by dissolution in water ~as repeated 3-4 times.
Recrystallization in a solvent mixture of 5 ml of distilled water and 10 ml of isopropyl alcohol provided 152 mg of (2S,~R~-AHMHA-Val-~ys-Asp.
Rf ~ 0.22~butanol/a~etic acid/water=4/1/2, a silica gel plate as described abo~e) mlp. ~ 240 ~ 260 C under decomposition Ld~22 1 -29,9(c=2.0, CH3COO~) Elemental analysis(for C22H41N508~
Found C:52.40 ~, H~8.$6 ~0, N:13~58 ~D
Calculated C:52.47 %, Ht8.21 %, N:13.91 %
Example 2 Synthesis of (2S,~R~-AHMHA-Val-Thr-Asp.
2-(1) Synthesis of Boc~Thr(Bzl)-Asp(OBzl)~.
The procedure described in Example 1-(1) was followed with 1.00 g of Boc-Thr(Bzl) and 1.57 g of Asp(O~z1)2 to give 1.69 g of crystals of Boc-Thr(Bzl)-Asp(OBzl)2.
Rf ~ 0.55(benzerle/ethyl acetate-5/1, a silica gel plate as described above) m. p. : ~55 ~ 765C
~22: ~225 (c=10 CH OH~
nr m. r, dppm tCDC~3) 1.15 (3H , d , J6Hz , ~CH-CH3) 1.45 (9H , ~ , -CtCH3)3) 2.92 (2~ , dd t ~sHz- 5Hz , ~H-CH2 CO-) ~-4.4 (2H . m , ~CH-OCH2Ph , fCH-NH-~-O~
4.52~ 4.98and~1~ (6H , each 8 ,-C~-Ph.X~) 4.85 (lH ,--HN_~ ~ CH2 ~
L
s.4S ~1H , m , -NH-) 73 (15H , s , CH2 Ph 7.4~7.6 ~lH , m , -NH-) IR ~ KBr cm 1 s 3340(NH-Co-), l74~, 1735(ester C--O), 1700(urethane C~O), 1650(amide C--O) 2-(2) Synthesis of Boc-Val-Thr(Bzl)~Asp(OBzl)2.
As detailed in Example l-(2)~ 9 g of Boc-Thr(Bzl)-Asp(QBzl)2 was treated with trifluoroacetic acid and then condensed with 569 mg of Boc~Val to yield le64 g of crystals of Boc~Val-Thr(Bzl)-Asp(OBzl)2.
R~ t 0038(benzene/ethyl acetate=5/l, a silica gel plate as described above) m. p. : 124.5 ~ 125.5~
.75 ~=2.0 ~ ~H30~I) n. mv r. ~ppm(CDC~) ~zl ~9, ~96, 1.11 (9H ~ each d , J6Hz , CH(CH3)2~CH-CE~) .9~2.3 (1H , m , -CH(CH~2 ) .9 (2H , dd , -CH2-CO) ~~4-4 (3H . m , -H~N-cH-~-x2 , -~H~CH 3 ) 4.ss, 4.96, 5.09 (6H , each ~ , -CH2 Phx3) .. . . .... .. .
48~~1 (1H , m , N-~H~
7.28 (15H , s , -CH2Phx3) ~8~7.8 (3H ,m , NH-x3) IR Iv K3rx cm 1 ~ 3300tNH-Co-), 1735(ester C=0), 1690(urethane C=0), 1650~amide C=0) 2-(3) Synthesis of Z-(2S,~R)-AH~IA-Val-Thr)Bzl)-Asp(OBzl)2.
_ .
As detailed in Example 1-(3), 676.5 mg of Boc-Val-Thr(Bzl~-Asp(03zl)2 was treated with trifluoroacetic acid and then co~densed with 283 ~g of (2S,~R)-A~HA t~ yield 555 mg of the title compound.
R~ ~ 0.58(benzene/ethyl acetate=l/l, a silica gel plate a~ described above) m. p. : 126 ~ 127.5~
~22 -~91 (c=~ , CH~ OH) n. m. r. ~ppm ~CDC~
0.88,0.91, 1.1Q (15H , each d , J6H~, -cH(cH3)2x2 .
~Bzl -~H-CHs) 1.2~2.1 (4H I m , -CH(CH3)2 x2 , -CH2-CH(CH3~) 2.9 (2H , dd , CH2-CO) ~8~4.5 (5H $ m , N-CH-CH2, -~H~ CH-~X3) 4.55, 4.98, ~05, 5.08 (2H , ~ , -C~2PhX4) 4.8~~2 (1H , m , -NH-~H-~) 4.5~4.7 (1H , m , -OH) ~4~5.8 (2~ nHX2) A8~%1 (1H ~ m , -~nH) 7.~-t.8 (1H , m , -~nH) 7.~ (20H I ~ , CH2 PhX4) -~4-IR Iv K9r cm 1 , 3300(NH CO-), 1740(ester C=O), 1690(urethane C--O), 1650(amide C=O) 2-(4) Synthesis of (2S _R)-AHM~IA-Val-r~hr-Asp.
Z-(2s~3R)-AH~IA-val-Thr(Bzl)-Asp(oB2l)2(468 mg) was dissolved in 3.0 ml of a solvent mixture o~ acetic acid, dioxane and distilled water(mixing ratio 11/2/1) and then shaken with 46 mg of 10 ~0 palladium-carbon catalyst in the atmosphere of hydrogen at 3. 6 kg/cm2 for 26 hours. After the c&talyst was filtered off, the filtrate was e~aporated to dryness. The evaporation residue was dissolved in 10 ml of distilled water and a~ain evaporated to dryness. Complete removal of the organic solvents by repeating the above evaporation p~ocedure 3-4 times provided 250 mg of the title compound.
Rf ~ 0.37(bu1;anol/acetic acid/water=4/1/1, a silica gel plate as descl~ibed above) m.p. s 196 - 198C(transformation into needle crystals around 166 - 168 C) ~3 22 s -19.9~(c=2.0, CH3COOH) Elemental analy~is(for C20H3~N40 Found C~50.18 ~0, Hs7086 ~o, Ntll.43 ~;~
Calculated Cs50.41 5;, Ht7.62 i~, N111.76 ~0 Example 3 Synthe~i~ of (2s~3R)-AH~A-val-Leu-A
3-~l) Synthesi~ of Boc-Leu-Asp(0Bæl)2.
~y the same procedure as presented in Example l-(l~, 1.04 g of Bo -Leu and 2.19 g of Asp(0Bzl)2~os0H were conden~ed and then subjected to silica gel column(~0 g~
same supporting material a~ described above) chromatogr~phy using a developin~ ~olvent of benzene and ethyl acetate (mixing ratio l0/l) to yield 2.39 g o~ syrup of the title compound, Rf 8 0.49(benzene/ethyl acetate=5/l, a silica gel plate as described above) D -30~7 (C=2-0, CE~3 OH~
n. m. r~ ~ppm (CDC~3) ~88 .(6H , d , J5Hz, -CH(C~3 )2 ) 1.40 (9H , s , -C~.CH3 ~ ) 1.2~1.7 (3H , m , -CH2 -, -CH(CH3)2) 2.9 b (ZH , dd , -CH2-C-) ~8~4;4 (1H , m , ~-CH-NH-) 4.7~~1 (2H , m , -~-CH-NH-) 5D6, ~13 (4H , each s , C~ Phx2?
6B~7.2 (1H , m , NH) 73~ (10H . s 1 CH2PhX2) _36-~ 7 IR ~v ~a~rx Gm 1 1 3350(-NI~-Co-), 1740(ester C-0), 1690(urethane C=0), 1670(amide C=0) 3-(2) Synthesis of ~oc-Val Leu-Asp(03zl)2.
By the same procedure as presented in Ex~mple 1-(2), 1.80 g of Boc-Leu Asp(03z1)2 was treated ~ith trifluoro-acetic acid; condensed with 651 mg of Boc-Val and then submitted to silica gel column(50 g, same supporting material as described above) chromatography employing a solvent mixture of benzene and ethyl acetate(mixing ratio 5/1) to ~ive 1.53 g of crystals of the title compound, Rf ~ 0.22~benzene/ethyl acetate=5/1, a silica gel plate as descrlbed above) m. p. : 112 ~ 113~
~22 ~ -39.8 (c=1.57.~ CH30H) n-mO r- ~ppm ~CDC~3) Q8~Q~5 (12H , m , -CH(CH3)2 x2 1.41 (9H , 9, -C(CHs)3 ) 1.3~2.1 (4H ,m ,~CH(CHs)2 X2 ,~C_2~CH(CHs~2 ) ~94 (2H , t , -CHz ~C0-) ~89 (lH , dd , -N~I-CH~
4.2~~3 (3H , m , -~nH-CH ~-X2 , ~ ) ~05. 510 (4H , each 9 , -CHz~PhX2) ~37-7.~0 (10H , ~ , -cH2phx2) 66 (1H , m , ~nH~) 7.08 ~lH , m g ~NH-) IR ~v m~Brx om 1 ~ 3340(-NH-Co-), 1750(e~ter C=O), 1695 (urethane C=O), 1650(amide C=O) 3 (3) Synthesis of Z-(~S,~R)-AH~IA-Val-Leu-Asp(OBzl)2.
By the same procedure as presented in Example 1-(3)0 700 mg o~ BoC Val-~eu-Asp(OBzl)2 was treated with trifluoro_ acetic acid~ condensed with 281 mg o~ Z-~2S, 3R)-AHMHA and recrystallized from a mixture of ethyl acetate and hexane t~ provide 417 mg of crystals of the title compound~
Rf s 0,60(benzene/ethyl acetate=l/l, a silica gel plate as described above) m. p~ : 156 ~ 157 [a~ _34l (c=20 , CH30H) n.m. r. ~ppm (CDC~3) ~8~1.0 (18H , m , -CH~CM3)2 x3) 1.3~æ3 (7H ,m ,-CH2~CH(,C~I3~ x2 ,-CH -(CH3~ ) 2.9 (2~ ~ m , -~H2-CO-) ~8~4.7 (3H , m , -NH~H-CO-x3) 4.7~s3 (lH ,m t -~nl-CH-CO-) ~02 (4H , ~ , -C~ -p~x2) -38~
~06 (2H 5 S ~ -C~I2-Ph) 5.5~~9 (1H ,m , -NH) 7.0~~.6 (3H , m , ~NHX3) 73 (15H , ~ ~ -CHz Phx~) IR ~v KEr cm 1 ~ 3325( NH-~-), 1745(ester C-O), 1680(urethane C=O), 1640(amide ~~0~
3 (4) Synthesis of (2S,3R)-A~HA-~al-Leu-Asp.
Under the same conditions as described in Example 2-(4), 416 mg o~ z-(2s~3R)AHMHA-~al-Leu-Asp(~Bzl~2 was hydroge~oly~ed to give 223 mg of (2S,3R~-AHMHA-Val-Leu-Asp.
R* ~ 0.38(butanol/acetic acid/water=4/1/1, a silica gel as described above) mOp, ~ 264 - 266C
~22 ~ 41~7(c=2.0, C~3COOH) Elemental analysis(for C22H40N408) Found C~53,79.%, ~:8.63 %1 N~ 19 ~a Calculated c 54.o8 yfO, Hl8.25 jS, N~11.47 ~0 Example 4 Synthesis of (2S,3R)-AHr~HA-Val-Glu-As~.
4-(1) Synthesis o~ Boc-Glu(OBzl)-Asp~03zl ?~
Boc-Glu(OBzl) (1 7 04 g) was subjected to condensation with Asp~OBzl)~ under the same reaction conditions as prese~ted in Example 1-~1) and then chromatographed on a silica _ ~; 9 _ gel column~50 g, same supporting material a~ described above) using a solvent mixture of benzene and ethyl acetate(mixing ratio 10~1). The recrystallization from a solvent mixture of ether and hexane yielded 1.73 g of crystals of the title compound~
Rf ~ 0.73(benzene/ethyl acetate=2/l, a silica gel plate as described above ) m. p. : b9.s ~ 70.5~ !
~ ~2 -10.7 (c=20 , CH30H) n.m.r. : ~ppm (CDC~3) 1.41 (9H , s , -C(C~ )3 ~0 (2H 9 m , CH2-CH2-CH-~ ~
~4 (2H , m , -~CH2 CH2-CH-) 2.94. (2H ,dd , -~-CH-C,H
~9~4.4 (1]H , m , -~nH-CH~
; 4.7~~1 (1H , m , -~nH-CH-~-) 505 (2H-, 8 ~-CH2 Ph) 5.10 (4H , 8 , -cH2PhX2) 7.0~7.5 (2H ,~ , -~n~- x2) .. !
7.27,7.30, 733 ~15H , each 3 ,-cH2phx3) IR ~v ~Br cm 1 ~ 3330(-~H-~-), 1750, 1735(es-ter C=0~, 1690~urethane G--0~, 1655(amide C~0) --~0 -4-(2) Synthesis of ~oc~Val-Glu(OBzl)-Asp(OBzl)~.
As described in Example 1-(2), 1.63 g of Boc-Glu(OBzl)-Asp(OBzl)2 was treated with trifluoroacetic acid and then condensed with 559 mg o* ~oc-Val 3y silica ~el column (20 g, same supporting material as described above) chromato-graphy usin~ a developing solvent system of b~nzene-ethyl acetate(3/l), 1.38 g of crystals of the title compound was obtained, Rf s 0.16(benzene/ethyl acetate-5/l, a silica gel plate as described above3 m. p. : 113 ~ 114 ~a322 : -25.2 (c=2-0 , CH3 OH~
n.m. r. 8ppm tCDC~ 2) 0.88~ 0.91 (6H , each d , J7H~ , -CH(CH3 )2 ) 1.41 (9H , s , ~C~CH~ )3 ) 1.8~2.2 (5H , m ~cH-c~2-cH~2 ~ CH(CH3 )2 2.94 (2H , dd , ~CH-CHz~
~91 (1~I , dd , J6Hz, 8H7,-N~I-CH~
...... ~
4.3~5.0 (2H ~ m , -NH-CH~
s.os (2H . 9 ~ - CH2 - Ph) ~10 ~4H , ~ , -C ~ -PhX2 ~0~~3 (1H , m , -NH-) 68~-~.1 (2H , m , -NHX2) 7.2~ 7.~, 7.34 (15H ~ each 3, - CH2-PhX3) IR ~v KBr cm 1 s 3330(-NH-~-), 1730(ester ~=o)~ 1685 (urethane C=O), l650(arnide c=oj 4-(3) Synthe~îs of Z-(2S,3R) AH~HA-Val-Glu(OBzl)~Asp(O~zl) ~ mploying the same procedure as detailed in ~xample l-(3), 707 mg of 30c-Val-Glu(OBzl)-Asp(OBzl)2 was treated with trifluoroacetic acid and then submitted to condensation with 280 mg of Z-(2S,3R)-AHI~HA. Crystals of the title compound(343 mg) was recovered by recrystallization from a chloroform-ether mixture.
Rf s 0.53(benzene/ethyl acetate=l/l, a silica ~el plate as described above) ml p. : 157 ~ 15~
~a~22 : -22.1 (c=2.0 , CH30~) . m. r. ~ppm (CDC~3 ) :
~75~Q95 (12H , m , -CH(CH~)2 x2) 1.2~1.7 (2H , m , -CH~CH3 )2X2) 1.7~ æ2 (4H ,m ,-N~- OEI-C~ -CH(CH3)2, -CHz-C7I2CO-) 2.2~2.5 ~2H , m , ~CH2-CH2-~-) 2.7~~0 (2H , m , >CH-CH2-~-) ~8~ ~0 (4H , m , -~nH-CH-~X4) ~0, S05 (8H , ~ , -CH2 PhX4) . .. . .. . . .. .
5.4~~8 (lH ,m , ~NH) 7.1~7.8 ~3H , m 9 - NHX3) 7.25, 7.2B ~20H ~ -CH2PhX4) IR sv~aBr cm 1 ~ 3425(-NH-~-), 1735(ester C=O), 1680 (urethane C=O), 1640(amide C=O) 4-(4) Synthesis of (2S,3R)-AHMHA-Val-Glu-Asp.
Under the same reaction conditions as detailed in ~xample 1-(4), 341 mg o~ z-(2S~R)-AHMHA-Val-Glu(OBzl)-Asp~OBz1)2 was hydrogenolysed to provide 247 mg of crystals of the title compound.
Rf s 0.39(butanol/acetic acid/water=4/1/1, a silica gelplate as described aboye)m.p. : 159 - 161C
2 ~ -29.4 (c=l.O, 80 % CH3COOH in water) Elemental a~alysis(for C22~40N408) Found C:54.35 %, H:8.21 %, N:11.24 o~
Calculated C:54~08 %, H18. 25 ~, N:11.47 %
2xample 5 Synthesis of (2S,3R)-AHMHA-Thr-~Jal-Asp, _ . .
5~ Synthesis of 30c-Val-Asp(03zl ~.
~ ~ _ . . _ Under the same reaction conditions as specified in Example 1~ , the condensation produc~ of 4.0 g of ~oc-Val with 8.94 g o~ Asp(OBz1)2 was chro~atographed on a silica gel column(100 g~ same supp~rting ~aterial as -~3-described above~ using a developing solvent system of benzene anq ethyl aceta~te~5/1) and then recrystallized from a solvent mixture of chloroform and hexane to give 8,?7 g o~ crystals of the title compound, R~ ~ 0.39(benzene/ethyl acetate=5/1, a silica gel plate as described above) m~ p. : 80 ~ 81~
~3 -24.6~ (c--~O i CH30~) n~m- rO ~ppm (CDC~3) Q8, ~92 (6H geach d ,J5H~, ~CH(CH3 )2 ) 1.42 (9H , ~ , -C(CH3)3 ) 1.9~2.3 ~1H , m , ~CH(CH3)2 ) 2.99 (2H , dd , -CHz~
~92 (1H , dd ,35H~. 9H~ CH~
4.~~~1 (1H ,m , ~NnI~~H~
5.05, ~12 (4H , each si -CH2-PhX2) ~7~7.0 (1H ~ m , -~nH) 7.2~7.4 (1H , m ,--N~I~
7.30 (10H , 8 , -CH2-PhX2) IR ~ KBrx cm 1 ~ 3325(-NH-~-), 1730(ester C=O), 1690 (urethane C=O), 1650(amide C=O) .. . .
5-(2) Synthesis of Boc-~hr(Bzl)-Va.l-Asp(OBzl)z.
~y the same procedure as detailed in Example 1-(2), 1.0 g of Boc-~al-Asp(O~zl)2 was treated with tri~luoro-acetic acid and then subjected ~o ~ondensation with 535 mg of Boc-Thr(gzl), Silica gel column(25 g, same supporting material as described above~ chromatography with a developing solvent mixture of benzene and ethyl acetate(l0~1) followed by recrystallization in an ether hex~ne m~ ~re yielded 611 mg o~ the title compou.~d.
Rf ~ 0.86(benzene/ethyl acetate=2/1, a silica gel plate as described above) m.p. : 136 - 137C
[~D ~ -17.5 (c-2,0, C~ 0~) I~ sv Ka~r cm 1 ~ 3300(-NH~ 1730(ester C=0), 1685 (urethane ~-0), 1640(amide C=0 n~ m. r ~ppm (CDC~3) Q75and ns 7 (6H , each d. J55Hz, -CH(CH3)2) ~Bzl 1.1~1.2 (3H .m , -~H-CH3) 1-43 (~H , 9 9 -O-C(CH~)3 ) 1.7~2.3 (4H ,m , CH2-CL(CH3)2 . ~ H-OBzl) 2.89 ~2H ,dd ~--C~-I2-C-) 41~4-~ (3H , m ~ H-NH X3) ~os (2H , ABq , -CH2Ph) , ~o~, 6.95 (~H .m , -NH-X3) 7.29 (sH , ~ , -CH~ Ph) 5- ( 3 ~ Synthesis of Z- ( 2S, 3R) -AHMHA-Thr ~Bzl ) -Val-Asp (OBzl ) 2 .
3y the same procedure as described in Example 1-(2)~
520 mg of Boc-~hr(~æl)~Val-Asp(OBzl)2 was treated with trifluoroacetic acid and then subjected to condensation ~ith 187 mg of ~-(2~3R)-AHMHA. ~he condensation product was charged on a silica gel column(30 ~; same supporting material as described above) and eluted with a developing 501vent mixture of benzene ,and ethyl acetate(3/1). 3y recrystallization from an ether-hexane mixture, 386 mg of the title compound in the crystalline fQrm was obtained.
Rf ~ 0,37(benzene/ethyl acetate=l/l, a silica gel plate as described above) m.p. t 93.0 - 96.05C
~D ~ ~18,5 ( c=l . o, CH OH) IR ~ KEr cm 1 ~ 3325(-NH-~-), 1730(ester C=O), 1630(amide C=O) n, m. r ~ppm (CDC43) : I
~72, ~82, ~09 ~91,1.08 (15H ,each d,-CH-CH3X5) 1.2~2.0 (5H , m , -~H-CH3X3 , -CH2-CH-) 2.Bg (2H , dd , -CH2~
~9~A5 (3H , m , -~nH-CH-x3) 45~4-9 (2H 9 m ~ ~nH-C~-X2) 4.55 (2H p ABq , -CH2Ph~
~J~
., ' . , . . . ~ . .. . . . . . . . . .
50~ (4~ CH2PhX2) ~09 (2H ~ s t ~CH2Ph) 5~7 (2H , m , ~NH) ~~77 (3H , m ~ nIX3) 7.29 (20H , ~ , -CH~PhX4) 5-(4) Synthesis of (2S,~R~-AHMHA-Thr-Val-A~p, Under the same conditions of hydrog3nolysis as detailed in Example 1-(4), 200 mg of the title compound was recovered from 386 mg of Z (2s~R)-A~MHA-Thr(~zl)-val-Asp(o~l)2 by using 10 ~0 palladium-carbon.
R~ ~ 0.41(butanol/acetic acid/water=4/1/1, a s;lica gel plate as described aboYe ) m.p. ~ o~er 180 C under decomposition Elemental analysis(for C2oH36N409) Found C~50.27 %, ~7.95 ~o, N~11.49 %
Calculated C~50.41 %~ Ht7.62 ,~0, Nt11.76 ~
Example 6 Synthesis of (2s,3R)-AH~IA-Leu-Val-Asp.
. _ 6-(1) Synthesis of Boc-Leu-Val-Asp(OBzl~.
Boc-Val~Asp~OBzl)2(708 mg) prepared by the same method as presented Ln Example 5-(1) was treated with trifluoro-acetic acid un~er the conditions speci~ied in Example 1-(2) and then subjected to condensation with 397 mg of Boc-Leu.
Silica gel column(30 g~ same supportin~ material as described F~
,....... . .. ..
above ) chromato~raphy with a developing solvent mixture of benzene and ethyl acetate(3/2~ gave 673 mg of crystals of the title compound~ , Rf ~ 0.56~benzene/ethyl acetate=3/2, a silica gel plate as described above ) m.p. ~ 135 - 136 C
D t -36.7 (c=2.0, CH30H) IR ~v K~r cm 1 ~ 33lO(-NH-~-), l730(ester C=0), 169Q
~urethane C-0), l640~amide C~0) n~ m. r. 8ppm fCDCL3) Q~~1.1 (~2H , m , -CH(C~2 x2 1.42 (9H , 8 , C-(CH~)3 ~
1.5~~.3 ~4H , m , -CH2 -CH ~ , -CH( CH3 )2 ~ 2 ) ~95 (2H , ~d , -CH2~-) Ao~4.6 (2H , m , -~nH-CH-~-X2) 4.6s~~4 (2H ,m , -NH-CH~
~06, 5.11 (4H , each ~ , -cH2phx2) 67~7.~ (ZH ~m , -NH-X2) 7.~2 (1oH , s , -cH2phx2) 6-(2) Synthesis of z-(2s,3R)-AHMHA(Z~-Leu-Val-Asp(OBzl)2.
By the same method as ~etailed in Example l-(2), 509 m~ of Boc-Leu-Val-Asp(OBzl)2 was treated with trifluoro_ acetic acid and then conden~ed with z-(2s~R)-AHMHA~z) (285 mg). Af'ter c:olumn chromatography on silica gel (30 g, ~ame supporting ma-t~rial as ~escribed above ) with a developing solven-t system of benæene and ethyl acetate (3/1 ) 490 mg of the title compound was ~btained.
Rf s 0.45(benzene/ethyl acetate~2/1, a silica gel plate as described above ) m.p. s 140 ~ 143 G
~d~ 22, -17 . 6 ( c=l . 0 , CH3 01{/CHC1 3=5/1 3 XR ~ v ICBr cm 1 ~ 3320(-NH-~), 17.50(ester C=0), 1695 (urethane C-0) j 1645(amide C=0) n. m. r. ~ppm (CDC,C3 ) o 7~0 ~5 ( 1 8H,--CH(CH3 )2 x3) 2.3 (7H ,m .~~2~ x 2 ,-OEI(CH3)2x3) ~9 2 ( 2EI, d d ~ CH2--~--0- ~
4.1~4.6 ( 4H , m , NH--CH-~--x 4 ) 48~5.0 (1H ,m ,NH-CH- ~) ~0~~2 ( 9H ~--CH2 -Phx4 , --CH--0-~) ~ 2~ 6 ( 1 H , -NH ~ ) 6. 5~7. 0 ( 3 lH ~ --NH--X 3 ) r Z3~7.4 ( 2 oE~, CH2 -PhX4 ) 6_(3) SynthesiS o~ (2s~3R)-AHMEA-Leu-val-Asp.
Under the same operational condîtions as presented in Example 1-(4), 200 mg of Z~(2S,3R)-~H~(Z)-Leu-V~l--49~
... . . , ., . . . . ~
Asp~OBz1)2 wa~ hydro~enolysed to give 72 mg o~ the title compound.
Rf t 0~42(butanol/acetic acid/water=4/1/1, a silica gel plate as described above~
m.p. ~ over 249C under decomposition ~D s -30~2 (c=l.O, CH3COOH~
Elemental analysis(for C22H40N408) Found C:53.75 %, H~8.18 ~0, N,11.28 ~0 Calculated Cs54.08 ~0, H,8.25 ~, N,11.47 -~0 Example 7 Synthesis of (2s,3R)-AHMHA~Lys-Val~Asp.
7-(1) Synthesis o~ Boc-l~s(Z)-Val-Asp(OBzl)2.
Under the same reaction conditions as specified in Example 1-~2), 1.0 g of ~oc-Val-Asp(OBzl)2 prepared by the method detailed in Example 5-(1) was treated with trifluoro_ acetic acid and then subjected to condensation with Boc-Lys(Z).
Silica gel column(30 g, same suppor-ting material as described above) chromatography with a developing solvent mi.xture of benzen~ and ethyl acetate(3/2) yielded 866 mg o~ crystals of the title compound.
Rf ~ 0.26(benzene/ethyl acetate=2/1, a silica Oel plate as described above) m.p. 1 122 - 124C
2 1 -21.2 ~c-3.0, CH30H) IR Iv mKaBx cm l ~ 332S(~NH-~-), 1740(ester C=0), 1685 . (urethane C=~), 1640(amide C~0) n.m.r. 8ppm ~CDC~3) 0.86ando.90 (6H 7 each d , J7Hz ~ -CH(C~)2 1.41 (9H , 3 ~-C(CH3)3 1.2~2.1 (7H 3 m ~--(CH2~ CH~CH3) 2.~-~3 ~4H ,m , -CH2-NH- , -CH2~C0-~
~9~4-5 (2H , m , -~nH--CH-~-x2) 4-7~5~ (lH , m , -~nH-CH~
~05~~ (6H ~, -CH2-PHx3) ~3~~5 (1H 9 ~ ~ - ~nH ~
~7~7.2 ~2H ~-m , -~H~x2) 733 (15~ , -c~2phx3~
7~(2~ Synthesi~ o~ 2-(2S,3R)-AHMHA(Z)-Lys(Z)-Val-Asp(OBzl)2.
Boc-Lys(Z)-Val-Asp(OBzl)2(534.4 mg) was treated with tri~luoroacetic acid and th~n subjected to condensation with 258 mg of Z-(2S,3R)-AHMHA(Z) by the same prodedure as specified in Example 1-(3). m e condensation product was puri~ied by silica gel column(30 g, same supporting material as dsscribed above) chroma-tography with a developing solvent system of benzene and ethyl acetate~2/l) to yield 510 mg of the title compound.
Rf t 0~53~benzene/ethyl acetate=l/l, a silica ge-~.plate as described above~
. .. . . . . . . .. . .. ... . .. ...
v2 , _g ~ 6 ( c- O . 85, CHcl 3/c}{3 oH=l/6 ) IR, " KBr cm 1, 3320(~NH~ ), 1730(ester C=O). 1690 ( urethane C=O), 1640 (amide C=O) n.m~ r . ~ppm ~CI)C~3 ) Q75~Q95 ( 1 2H .,--CH(CH3 )2 x2) -1~22 ( 1 ~H .--(CH2 )3-- ,--CH(CH3 )2 x2, --CH2--CH 1 CH 3 )2 2.8~~3 7 4H, -CH2 ~ C~2-NH~) 4.0~4.6 ( 4H,--NH--CH--~--x ~ ) 4.~~~2 ~ 1 2H ~--CH2--Phx5 ,/CH~O--6s~7. o ( 4H ~--NH-x4 ~
7. 2~7. 4 ( 2 5H, CH2--Phx 5 ) 7-(3~ Synthesis of (2S,~R)-AHMHA-Lys-Val-Asp.
. . _ .
Hydrogenolysis of 230 mg of Z-(2S,~ HMHA(Z)-Lys(Z)-Val-Asp(OBz:L)2 under the operational conditions specified in Example 1-(4) gave 84 mg of the above-listed compound.
Rf t O.l~bu-tanol/acetic acid/water=4/1/1~ a silica gel plate as described above) m.p. s over 170~C under decomposition C~D2 S -28.6 (C=1.0, CH3COOH) Elemental analysis(for C22H40N508) Found Ct52.72 %, H~8.54 9~, Nsl3.57 5~
Calculated C~52.47 ~0~ Hs8.21 ~D~ N,13.91 YD
. . .
D
Example 8 Synthe~i~ of (2S,~R)-~HM~IA-Glu-Val~Asp.
8-(1) Synthesis of Boc-Glu(OBzl)-Val-Asp(OBzl)2.
By the same procedure as presented in Example 1-(2~, loO g of Boc-Val-Asp(oBzl)2 was treated with trifluoro-acetic aGid and then submitted to condensation with Boc-Glu(OBzl). After column chromatography on silica gel (30 g, same supporting ma~erial as described above) with a developing solvent system of benzene and ~thyl acetate(3/1)3 780 mg of the title compound was obtained.
Rf a 0,51(benzene5ethyl acetate-2/1, a silica gel plate as described above) m.p. ~ 124 - 126C
~d~22 ~ -27.7(c=1.5, CH30H) IR 5~ KBr cm 1 ~ 3300(-NH-~-), 1730(~ster C=0),1690 (urethane C=O), 1640(amide C=O) n.m.r. 8ppm (CDC~3) 0~85~ndo.38 (6H ., each d , J6.5Hz , CH(CH3)2) 1.4 (9H , 8 , -CHtCH3)3) 1.8~2.3 (3H , m , -CHtCH3 )2 , -cH 2 -cH 2 ~ ) 2.3~2.7 (2H , m , -CH2~CH2-CO-) 2.9s (2H ~ ~CH-CH2CO-) .. .. . .. . .
4.0~50 (3H ~ -rnH-CH-CO-x3) ~05~~1 (6H ~ -CH2Phx3 (lH , -~nH~) , . , .. , , ..... , .. . ., ., . ... . , ., .,,, .. , . ~ .. , .. , . . ., ~ , . . . ... .
~8~7.1 (2H , -NH-x2) 7.3 (15H , -CH2Ph~33 8-(2) Synthesis of Z-(2S,3R)-AHMHA(Z~-Glu(OBzl)-Val-~ . ~ . . . i . __ Asp(OBz1)2.
Under the same operational conditions as detailed in Example 1-(2); 510 mg of Boc' Glu(OBzl)-Val-Asp(O~zl)2 was treated with trifluoroacetic acid and then condensed with 270 mg of Z-(2~,3R)~ HA(Z). The condensation product was purified by column chromatography on silica gel (30 g, same supporting material as described above) using an elution solvent system of benzene and ethyl acetate(Z/1) to pr~vide 500 mg of the title compound.
R~ ~ 0.75(benzene/ethyl acetate=l/l~ a silica gel plate as described abo~e) ~d~22 ~ -10,,8 (c=l.O, CH30H) IR ~v K~r cm 1 ~ 3300(-NH-~-), 1740(ester C-O), 1690 (urethane C~O), 1630(amide C=O) n.m,r. ~ppm ~CDC~
(12H , -CH~CH~)2 x2) 1.0~1.8 (3H , -CH(CH 3)2 x2 ~ -CH2-CH(CH~)2 ) æ~ (2H , -CH2-CH2 -~-) æ2~æ6 ( 2H,--CH2--CH2~
2.8~1 (2H , ~CH-CH2- ) -~4~
, ., , , , , . , . _. _ _.. . .. . .. . . ..
q~
~9~4.6 ( 3H ~,--NH--CH--~X3) __ 4.7~5.1 ( 2H,--NH--C --~ ,>CH~O~
~o~s2 (l~H,--CH2--Phx5~) 5 ~~5.4 ~ NH--68~7.1 (3H ~-NH-7.2~7.4 (25H ~-CH2Phx5~
8-~3) Synthesis of (2S~3R)-AH~A-Glu-Val-Asp.
.. .. ,. ,,, .~._ The same conditions o* hydrogenolysis as detailed in Example 1-(4) yielded 65 mg of the title compound from 190 mg of Z-(2s~3R)-AHMHA(Z)-Glu(OBzl~-Val-Asp(O~zl)2~
Rf 1 0. 22(butanol/acetic acid/water-4,~ a silica gel plate as descri~ed above ) m.p. J over 165 C under decomposition 27 . 2 ( c-0 . 5, CH3COOH ) Elemental analysis(for C~8H42N4 ~0) Found C:50.36 ~0, H~7.12 ,~ N:10.86 Calculated C:49.99 i~, H~7.19 ,~, Ntll.ll ~
Example 9 Synthesis o~ (2S,~R)-AHMHA-Val-Thr-Phc.
9- (lj Synthes s of Boc-Thr (OBzl) -Phe (OBzl) .
By the same method as detailed in Example 1-(1), 2.9 g of Boc-Thr(OB~) and 4.0 g of Phe(OBzl)~osOX were subjected to oondensation and then purified by column chromatography E
.. . . .. ~ . . . .. .. . .. .. . . . . . . ... .. . .. ....
on silica gel(50 g, Kiesel gel 60, E~ Merck3 usin~ a developing solvent mixture of benzene and ethyl acetate tlO/l ) to pro~ide 4. 8 g of the title compound.
m.p. s 138 - 139C
21 s _4,0(c-l.0, CH30H) IR sv KaRr cm 1 ~ 332o(-NH-co ), 1730(ester C=O), 1680 (urethane C=O), 1660(amide C=O) nrm.r. ~ppm ~CDC~
1.14 (3H ~ d , J6H ,~CH-CH3) 1.4 ~9H , 3 D -Ct~H3)3 ) ~02 (2H 9 d , J6Hx ~ -CH2Ph) ~9~~0 (3H , -NH-cH-~-x2 9 ~CH-OCH2Ph) 4.53 ~2H ~ ~OCH~Ph) 5.09 (2H ~-oCH2Ph) 5.3~55 (1H , -~nH-) 6~-7.3 (16H ~ -~nH- , -CH2Phx3~
9-(2) Synthesis of Boc-Val-Thr(OBzl)-Phe(OBzl).
Under the same reaction conditions as specified in Example 1-(2)t 4.8 g of Boc-Thr(OBzl)-Phe(03zl) was treated with trifluoroacetic acid and then ~ubjected to condensation with 1.9 g of Boc-Val. ~y column chromatography on silica : gel(Kieselgel 60, 60 g) with an eluant of benzene and ethyl acetate(5/1), 4.8 g o~ the title compound was obtained.
*Tr~demark.
Rf s 0.28(benzene/ethyl acetate=5/1, a silica gel plate as de~cribed above) )D ~ -13,7 (c=l,O, CH OH~
m.p. , 13f - 138 C
IR v KBrx cm 1 , 3280(-NH-Co-), 3740(ester C=O), 1690 (urethane C--~), 1640(amide C=o) n.m~r~ ~ppm (CDC~3) 0.8~1.0 (9H ,-CH(CH3)2 I~CH-CH3) .4 (~H , -C~CH3)3 ) ts~2.2 ~ CH~CH~) ~o (2H ,-CH2Ph) ~8~4.9 (4H , -~nH-CH-CO~x3 , ~CH-O-CH2Ph) 4s2 (2H , -O-C 2Ph3 5.08 (2H , -O-CH2Ph) . 4.9~5.2 (1H , ~n~
: ~8~7.3 (17H , -CH2Phx3 , ~nH-X23 9-(3) Synthesis of Z-(2St~R)-AHM~IA-Val-Thr(OBzl)-Phe(OBzl).
~ ... . _._ .., .. _ . __ By the same procedure as presented in Example 1-(3)t 600 mg o~ Boc-Val-~hr(OBzl) Phe(OBzl) was treated with trifluoroacetic acid and then condensed with 259 mg of z-(2S, 3R)-AX~A. Recrystallization of the condensation product from a solvent mixtur~ of chloroform and hexane provided 650 mg of the ti~lc compound.
Rf ~ 0.37(benzene/ethyl acetate=l/l, a silica gel plate as described above ) m . p . 2 168 - 169 C
[d~Dl ~ -11.9 (c-O.6s C~cl3/cH3oH=3/l) IR ~ ~! KBrx cm 1 1 3290(-NH-CO-)t 1730(ester C=O), 1680 ~urethane C=O), 1630 (amide C=O~
n.m. rc ~ppm t CDC~
Q8~1.0 ( 1 5~ ,--CH ( CH3 )2 x 2 , ,~CH--CH3 ) 1.1~1.7 ( 4H,--CH(CH3 )2 x2, CH2--CH~CH~)2 ) o ( 2H "--CH 2Ph ) 3.~4.9 ( 6H,--NH-CH-CO--x3 ~--NH-~H--~H--OHt 4-5 ( 2H , -O-CH2Ph ) S.Oand ~0 5 ( 4H,--O-CH~Ph ) 5.2~s.7 ( 2~ s -NH--xZ~
68~7. 3 ( 2 2H 7--NH-x 2, CH2PhX 4 ) 9-(4) Synthesis of (2s,3R)-A~IA-Val-Thr-Phe.
The same hydrogenoly~is conditions as descri.bed in Example 2-(4) produced 100 mg o~ (2s,3R)-AHMHA-Val-Thr~Phe ~rom 300 mg of Z-(2S,3R)-AHMHA-ValiThr(OBzl)-Phe(OBzl~
Rf ~ 0.55(butanol/acetic acid~water-4/1/1~ a sllica gel plate as described abov~) m.p. t 159 ~ 161C
~21 1 -14.2(c=0.5i CX3COOH) Elemental analysis ( for C25H40N4û7 ) Found Ct58.76 ~, H~8.o6 ~0, N~10.95 ~0 Calculated C~59.04 ~0, H~.93 ~, N~11.02 5~0' Example lO Synthesis o~ ( 2S ,3R) ~ qHA-Lys-Val-Val,
By the same method as detailed in Example 1-(1), 2.9 g of Boc-Thr(OB~) and 4.0 g of Phe(OBzl)~osOX were subjected to oondensation and then purified by column chromatography E
.. . . .. ~ . . . .. .. . .. .. . . . . . . ... .. . .. ....
on silica gel(50 g, Kiesel gel 60, E~ Merck3 usin~ a developing solvent mixture of benzene and ethyl acetate tlO/l ) to pro~ide 4. 8 g of the title compound.
m.p. s 138 - 139C
21 s _4,0(c-l.0, CH30H) IR sv KaRr cm 1 ~ 332o(-NH-co ), 1730(ester C=O), 1680 (urethane C=O), 1660(amide C=O) nrm.r. ~ppm ~CDC~
1.14 (3H ~ d , J6H ,~CH-CH3) 1.4 ~9H , 3 D -Ct~H3)3 ) ~02 (2H 9 d , J6Hx ~ -CH2Ph) ~9~~0 (3H , -NH-cH-~-x2 9 ~CH-OCH2Ph) 4.53 ~2H ~ ~OCH~Ph) 5.09 (2H ~-oCH2Ph) 5.3~55 (1H , -~nH-) 6~-7.3 (16H ~ -~nH- , -CH2Phx3~
9-(2) Synthesis of Boc-Val-Thr(OBzl)-Phe(OBzl).
Under the same reaction conditions as specified in Example 1-(2)t 4.8 g of Boc-Thr(OBzl)-Phe(03zl) was treated with trifluoroacetic acid and then ~ubjected to condensation with 1.9 g of Boc-Val. ~y column chromatography on silica : gel(Kieselgel 60, 60 g) with an eluant of benzene and ethyl acetate(5/1), 4.8 g o~ the title compound was obtained.
*Tr~demark.
Rf s 0.28(benzene/ethyl acetate=5/1, a silica gel plate as de~cribed above) )D ~ -13,7 (c=l,O, CH OH~
m.p. , 13f - 138 C
IR v KBrx cm 1 , 3280(-NH-Co-), 3740(ester C=O), 1690 (urethane C--~), 1640(amide C=o) n.m~r~ ~ppm (CDC~3) 0.8~1.0 (9H ,-CH(CH3)2 I~CH-CH3) .4 (~H , -C~CH3)3 ) ts~2.2 ~ CH~CH~) ~o (2H ,-CH2Ph) ~8~4.9 (4H , -~nH-CH-CO~x3 , ~CH-O-CH2Ph) 4s2 (2H , -O-C 2Ph3 5.08 (2H , -O-CH2Ph) . 4.9~5.2 (1H , ~n~
: ~8~7.3 (17H , -CH2Phx3 , ~nH-X23 9-(3) Synthesis of Z-(2St~R)-AHM~IA-Val-Thr(OBzl)-Phe(OBzl).
~ ... . _._ .., .. _ . __ By the same procedure as presented in Example 1-(3)t 600 mg o~ Boc-Val-~hr(OBzl) Phe(OBzl) was treated with trifluoroacetic acid and then condensed with 259 mg of z-(2S, 3R)-AX~A. Recrystallization of the condensation product from a solvent mixtur~ of chloroform and hexane provided 650 mg of the ti~lc compound.
Rf ~ 0.37(benzene/ethyl acetate=l/l, a silica gel plate as described above ) m . p . 2 168 - 169 C
[d~Dl ~ -11.9 (c-O.6s C~cl3/cH3oH=3/l) IR ~ ~! KBrx cm 1 1 3290(-NH-CO-)t 1730(ester C=O), 1680 ~urethane C=O), 1630 (amide C=O~
n.m. rc ~ppm t CDC~
Q8~1.0 ( 1 5~ ,--CH ( CH3 )2 x 2 , ,~CH--CH3 ) 1.1~1.7 ( 4H,--CH(CH3 )2 x2, CH2--CH~CH~)2 ) o ( 2H "--CH 2Ph ) 3.~4.9 ( 6H,--NH-CH-CO--x3 ~--NH-~H--~H--OHt 4-5 ( 2H , -O-CH2Ph ) S.Oand ~0 5 ( 4H,--O-CH~Ph ) 5.2~s.7 ( 2~ s -NH--xZ~
68~7. 3 ( 2 2H 7--NH-x 2, CH2PhX 4 ) 9-(4) Synthesis of (2s,3R)-A~IA-Val-Thr-Phe.
The same hydrogenoly~is conditions as descri.bed in Example 2-(4) produced 100 mg o~ (2s,3R)-AHMHA-Val-Thr~Phe ~rom 300 mg of Z-(2S,3R)-AHMHA-ValiThr(OBzl)-Phe(OBzl~
Rf ~ 0.55(butanol/acetic acid~water-4/1/1~ a sllica gel plate as described abov~) m.p. t 159 ~ 161C
~21 1 -14.2(c=0.5i CX3COOH) Elemental analysis ( for C25H40N4û7 ) Found Ct58.76 ~, H~8.o6 ~0, N~10.95 ~0 Calculated C~59.04 ~0, H~.93 ~, N~11.02 5~0' Example lO Synthesis o~ ( 2S ,3R) ~ qHA-Lys-Val-Val,
10- (1 ) Preparation of Boc-Val-Val ( OBzl ) .
U8ing l.l g o~ Boc-Val and 2.0 g of Val-OB~l~ TosOH, the condensation reaction as specified in ~xample l- (1 ) was repeated. Column chromatography on silica gel~Kiesel~el 60, 50 g) with a developing solvent oP benzene and ethyl acetate(mixing ratio 10 ~ 1~ provided 2 . O ~ ~f the title compound.
Rf s 0.41(benzene/ ethyl acetate-5/l, a silica gel plate as describ~d abo~e) (~ 321 4900 (c=2-0, CH30X) IR ~v m~Brx cm 1 ~ 3310(-NX-Co-), 1740(ester C-0), 1685 ( urethane C=O), 1655 (amide C=O) nOm.r. ~ppm (CDC~3 ):
Q8~1.0 ( 1 2H,--CH(CH3 )2 x2) 1.4~ (9H, 9 It--C(CH3 )3 ?
17~2-5 ( 2H, m, -CH ( CH ~, )2 x.2 3 ~93 ( 1H, dd, J9Hz, 7Hz . ~NH--CH~CO--) 4-57 . ( ~H 7 dd 9 J9Hz ~ 5Hz,--NH--CH--CO--5 0~53 ( lH,--NH-) ( 2H, ~, -O-C 2Ph ) 64~66 ( 1H, -NH ) 7.33 ( sH ~ 9 ~ -OC~I2P~
10-~2) Preparation of Boo-hYs ~ ~ ~ -Val-Val~o~zl), . _ .
Using the same method as detailed in Example 1-(2), 1.4 g of Boc-Val-Val(OBzl) was treated ~ith trifluoroacetic acid and then submitted to condensation with ~oc-Lys(Z)(1,3 g), After silica gel column(60 ~, Kieselgel 60) chromatography with a developing solvent system of benzene and ethyl acetate(mixLng ratio 4 s 1), 2.0 g of the title compound was obtained.
R~ s 0.48(benzene/ethyl acetate=l/l, a silica gel plate as descr;.bed above) .p. s 14S - 146 C
321 5 -47,5 (c=l.O, CH30H) IR sv K~r cm 1 s 3320(-NH-Co-), 1730(ester C=~), 1685 (urethane C=O) 9 1640~amide C=O) n ~m. r ~ ~ppm (CDC~3) 5~1.0 ~12H , -CH(CH3)2 X2) 1.4 (9H ~ -C(C~ )~ ) 1.3~2.3 (8H , -~CH2)~ CHtCH~ )2 x23 ~0~3 (~H -CH2N~
~9~4.8 (3H ,-N~-CH~CO-x3) 5.0~5.15 (4H ,CH2Phx2) 5.0~~6 (2H ,~NH-x2) ~8~7.1 (2H ,-NH~x2) 7.3 (10~ ,-cH2phx2) lO-(3) Preparation of Z-(2SI~R)~AHMHA-Lys~Z)-V~l-Val(OBzl).
~y the same procedure a~ specified in Example 1-(3) 600 mg of Boc-Lys~Z)-Val-Val~OBzl) was treated with tri-*luoroacetic ac.id and then submitted to condensation with 222 m~ of Z-(2S,~R)-A~A. The above-listed compound (500 mg) was produced by recrystallization in a solvent - mixture of chloroform and hexane.
Rf ~ 0.l5(benzene/ethyl acetate=l/l, a silica gel plate as described above) m.p. ~ 176 - 178C
Ld~21 ~ -35.1 (c=l.0, CH30M) IR ~ " Ka~x cm 1 ~ 3290(-NH-C0-)~ 1730(ester C=0), 1685 (urethane C=O), 1635(amide C=O) n.m.r. ~ppm (CDC~3) Q7~1.0 (18H 1~CH(CH3)~ x3) 1.1~2.3 (11H ~-cH(cH3)2x3 ,-CH2~I~CH3~.-(CH~
- 2.8~2 (2H ~-CH2NH~~
4-0~4.9 ~6H ,-NH ~ -CO-x3 ,-NH-C_ ~ H~QH) 5.0~5.2 (6H , ~CH2PhX3) 5~~5.~ (4H , ~NH-x4) 7.2~7.4 ( 1 k~l t --NH[-- ~ --CH 2Ph X 3) 10-t4) Pre~___tion of (2St~R~-A~IA-Lys-Val-Val.
Three hundred milligrams of Z-(2S,~R)-AHMHA-Lys(Z)-Val~Va~(OBzl) was hydrogenolysed under the same operational conditions as detailed in Example 1-(4) to yield 109 mg of (2S~R)-AMMHA-Lys-V~l-Val.
Rf : 0.25(butanol/acetic acid/water=4/1/1, a silica gel plate as described above) m.p. , over 170~C under decomposition ~21 ~ _39.6~(c=0,7, CH3COOH) Elemental analysis(for C23H45N506~
Found C~56.51 %, H~9.17 ~o, N~13098 ~0 Calculated C~56.65 %, H59.30 ~0, N:14.36 ~0 Example 11 Synthesis o~ (2 _3R)-A~IA-Val-~al-Thr,
U8ing l.l g o~ Boc-Val and 2.0 g of Val-OB~l~ TosOH, the condensation reaction as specified in ~xample l- (1 ) was repeated. Column chromatography on silica gel~Kiesel~el 60, 50 g) with a developing solvent oP benzene and ethyl acetate(mixing ratio 10 ~ 1~ provided 2 . O ~ ~f the title compound.
Rf s 0.41(benzene/ ethyl acetate-5/l, a silica gel plate as describ~d abo~e) (~ 321 4900 (c=2-0, CH30X) IR ~v m~Brx cm 1 ~ 3310(-NX-Co-), 1740(ester C-0), 1685 ( urethane C=O), 1655 (amide C=O) nOm.r. ~ppm (CDC~3 ):
Q8~1.0 ( 1 2H,--CH(CH3 )2 x2) 1.4~ (9H, 9 It--C(CH3 )3 ?
17~2-5 ( 2H, m, -CH ( CH ~, )2 x.2 3 ~93 ( 1H, dd, J9Hz, 7Hz . ~NH--CH~CO--) 4-57 . ( ~H 7 dd 9 J9Hz ~ 5Hz,--NH--CH--CO--5 0~53 ( lH,--NH-) ( 2H, ~, -O-C 2Ph ) 64~66 ( 1H, -NH ) 7.33 ( sH ~ 9 ~ -OC~I2P~
10-~2) Preparation of Boo-hYs ~ ~ ~ -Val-Val~o~zl), . _ .
Using the same method as detailed in Example 1-(2), 1.4 g of Boc-Val-Val(OBzl) was treated ~ith trifluoroacetic acid and then submitted to condensation with ~oc-Lys(Z)(1,3 g), After silica gel column(60 ~, Kieselgel 60) chromatography with a developing solvent system of benzene and ethyl acetate(mixLng ratio 4 s 1), 2.0 g of the title compound was obtained.
R~ s 0.48(benzene/ethyl acetate=l/l, a silica gel plate as descr;.bed above) .p. s 14S - 146 C
321 5 -47,5 (c=l.O, CH30H) IR sv K~r cm 1 s 3320(-NH-Co-), 1730(ester C=~), 1685 (urethane C=O) 9 1640~amide C=O) n ~m. r ~ ~ppm (CDC~3) 5~1.0 ~12H , -CH(CH3)2 X2) 1.4 (9H ~ -C(C~ )~ ) 1.3~2.3 (8H , -~CH2)~ CHtCH~ )2 x23 ~0~3 (~H -CH2N~
~9~4.8 (3H ,-N~-CH~CO-x3) 5.0~5.15 (4H ,CH2Phx2) 5.0~~6 (2H ,~NH-x2) ~8~7.1 (2H ,-NH~x2) 7.3 (10~ ,-cH2phx2) lO-(3) Preparation of Z-(2SI~R)~AHMHA-Lys~Z)-V~l-Val(OBzl).
~y the same procedure a~ specified in Example 1-(3) 600 mg of Boc-Lys~Z)-Val-Val~OBzl) was treated with tri-*luoroacetic ac.id and then submitted to condensation with 222 m~ of Z-(2S,~R)-A~A. The above-listed compound (500 mg) was produced by recrystallization in a solvent - mixture of chloroform and hexane.
Rf ~ 0.l5(benzene/ethyl acetate=l/l, a silica gel plate as described above) m.p. ~ 176 - 178C
Ld~21 ~ -35.1 (c=l.0, CH30M) IR ~ " Ka~x cm 1 ~ 3290(-NH-C0-)~ 1730(ester C=0), 1685 (urethane C=O), 1635(amide C=O) n.m.r. ~ppm (CDC~3) Q7~1.0 (18H 1~CH(CH3)~ x3) 1.1~2.3 (11H ~-cH(cH3)2x3 ,-CH2~I~CH3~.-(CH~
- 2.8~2 (2H ~-CH2NH~~
4-0~4.9 ~6H ,-NH ~ -CO-x3 ,-NH-C_ ~ H~QH) 5.0~5.2 (6H , ~CH2PhX3) 5~~5.~ (4H , ~NH-x4) 7.2~7.4 ( 1 k~l t --NH[-- ~ --CH 2Ph X 3) 10-t4) Pre~___tion of (2St~R~-A~IA-Lys-Val-Val.
Three hundred milligrams of Z-(2S,~R)-AHMHA-Lys(Z)-Val~Va~(OBzl) was hydrogenolysed under the same operational conditions as detailed in Example 1-(4) to yield 109 mg of (2S~R)-AMMHA-Lys-V~l-Val.
Rf : 0.25(butanol/acetic acid/water=4/1/1, a silica gel plate as described above) m.p. , over 170~C under decomposition ~21 ~ _39.6~(c=0,7, CH3COOH) Elemental analysis(for C23H45N506~
Found C~56.51 %, H~9.17 ~o, N~13098 ~0 Calculated C~56.65 %, H59.30 ~0, N:14.36 ~0 Example 11 Synthesis o~ (2 _3R)-A~IA-Val-~al-Thr,
11-(1) S~nthesis of Boc-Val-Thr(Bzl)OBzl.
__ _ Thr(Bzl)oBzl~ oxalate(406.4 mg) was dissolved in 50 ml of ethyl acetate and washed three times with 20 ml each of 4 % sodium bicarbonate and twice with 20 ml each of saturated solution of sodium chloride. After the organic solution ~as dehydrated over anhydrous sodium sulfate, the solvent was removed by evaporation under reduced pressure ~62-.L~l~
to give m r(Bzl)OBzl. The total amount of Thr(Bzl)OBzl thus prepared~ 217.3 mg of Boc-Val and 148.6 mg of HOBt were dissolved in 10 ml of THF and cooled with ice. After 227 mg o~ DCCD was added, the reaction mixture Nas allowed to react for 2 hours under cooling with ice and for one hour at room temperature. The precipitates o~ DCU were filtered off and the solvent was removed b~ evaporation _n vacuo. The evaporation residue was dissolved in 5Q ml of ethyl acetate and extracted three times with 20 ml each of 10 ~ citric acid~
three time~ wlth 20 ml each o~ 4 ~0 sodium bicarbonate and twice with 20 ml each of saturated solution of sodium chloride. The organic solution was dehydrated over a suitable amount o~ anhydrous sodium sulfate and the ef,hyl acetate was evaporated off under reduced pressure. The evaporation residue was ~ubjeo-ted to silica gel column(10 g, Kiesel gel 60, 70 - 230 me~h, E. Merck) chromatography using a developing solvcnt ~ystem of chloroform and methanol (40~13 to give 30~--mg of the title compound.
n.m.r. ~ppm tCDC~3 ):
Q91andQ95 (6H, each d 9 J=7Hz, --CH(CH~)2 ) 1.18 ( 3H, d,--O--~H--CH3 ) 1. 4 0 ( 9H . ~ ,--C ~ C~
9~~ ( 1H , m , ~CH ( CH3 )2 ~8~A3 (2H ,m,--CH~CH~,)~OBzl ,~NH-CHCO-) ~ . ~ . . . . ~
~ 3 4.1~4.6 (2H ,ABq~J=jem12Hz .-OCH2Ph) 4.70 (1H I dd ~ J=5Hz., 9Hz , -Nn~CHCO-~
5.06 (2H , 3 , -COOCH2Ph) 51~~3 ~1H . ~ ~ ~~
s~~7 (1H , ~nH-3 7.22 ~ loH t S t -CH2PhX2 ) 11-(2) Synthesi5 of Bv_-Val-Val-Thr(Bzl)OBzl.
~ y treatment with trifluoroacetic acid under the same reaction conditions as described in Example 1-(2), 305 m~
of Boc-Val-Thr(B2l)0Bzl was converted to its free amine.
This amine was condensed wi~h 168 mg of Boc-Val to yield 438 mg of crystals of the title compound.
Rf ~ 0.94(benzene/acetone=4/l), a silica gel pl~te as described above .m.r. ~ppm (CDC~3) :
~8~1.n5 (12H , m , -CH(CH3 )2 x2) 1.18 (3H , d , J6H2 , ~CH-CH3) 1.42 .(9H t 9 ~ - C(CH3)3 ) 1.8~2.3 (2H , m , -CH(CH3 )2x2) ~7~45 (3H ,m ,-CH(CH3)-OBzl , -NH CH-CO-x2) 4.36 (2H , ABq , -O-CH2Ph) 4.b~4.8 (1H ,m , -~nH-CH-CO-) 5.06 (2H , 8 , -C02-C~2Ph) S.1~5.25 (1H , -NH ) ~5~~9 (2H ~ -NH~X2) 7.25 (1oH , ~ , -CH2PhX2) 11-(3) Synthesis o~ Z-(2S,3R)-AHMHA-Val~Val-Thr(Bzl)OBzl.
- Boc-Val-Val-Thr(Bzl)OBzl(418 mg~ was treated with trifluoroacetic acid under the same reaction conditions as speci~ied in Example 1-~3) to provide its free amine, from which 246.1 mg of the ~itle compound wa~ produced by condensation with 161.8 mg of Z-(2S,~R) ~HMHA.
n.m.r. ~ppm (CD3COCD3) Q75~10 (18H , m , -CH(CH3)2 x3) 1.20 (3H t d , J~oH~, /CH-CH3) 1.3~1.7 (5H , m , -CH(CH3)2 x3 ,-C~ -CH(CH3~2 ) 4.D~4.8 (7H ,m ,-NH-CH-X4 ,-NH-C_-Ç~
CH(CH3~-OBzl~
S.Oand~12 (4H , each 9 , -CH2PhX2) 5.2~~4 (1H , m , -NH-) 7,22 .... ~5H , ~ , -CH2Ph) 7.29 (10H , 3 , -c~I~PhX2) 7.3~7.6 (3H / m , -~nH-x3) 11-(4~ Synthesis o~ (2s,3R)-AHMHA-Val-Val-Thr.
z-(2s,~R)-AHMHA-Val-V~l-Thr(Bzl 3 OBz1(246.1 mg) and -~5-50 mg of 5 y. palladium~charcoal catalyst was ~uspended in a solvent mixture of methanol, acetic acid and water(4/2/1) and shaken for catalytic reduction a-t room temperature in a hydrogen atmosphere~ After the catalyst w~s separated by filtration, the filtrate was diluted with water and e~aporated to dryness under reduced pressure to yield 137,4 mg of the title compound, ~f ~ 0,62(butanol/acetic acid/water=4/1/2, a silica gel . plate as described above~
m.p. , 229,0C
cd~ 53,2(c-0.5, CH3COOH3 Elemental analysis~for C21H40N4o7j Found C~54.62 %, Hs8.83 %, N:12,05 ~0 Calculated C~54~76 %, H:8.75 ~0, N:12.17 ~0 Example 12 Synthesis of (2S 3R)-AHMHA- al-Val-Pro.
__ _ Thr(Bzl)oBzl~ oxalate(406.4 mg) was dissolved in 50 ml of ethyl acetate and washed three times with 20 ml each of 4 % sodium bicarbonate and twice with 20 ml each of saturated solution of sodium chloride. After the organic solution ~as dehydrated over anhydrous sodium sulfate, the solvent was removed by evaporation under reduced pressure ~62-.L~l~
to give m r(Bzl)OBzl. The total amount of Thr(Bzl)OBzl thus prepared~ 217.3 mg of Boc-Val and 148.6 mg of HOBt were dissolved in 10 ml of THF and cooled with ice. After 227 mg o~ DCCD was added, the reaction mixture Nas allowed to react for 2 hours under cooling with ice and for one hour at room temperature. The precipitates o~ DCU were filtered off and the solvent was removed b~ evaporation _n vacuo. The evaporation residue was dissolved in 5Q ml of ethyl acetate and extracted three times with 20 ml each of 10 ~ citric acid~
three time~ wlth 20 ml each o~ 4 ~0 sodium bicarbonate and twice with 20 ml each of saturated solution of sodium chloride. The organic solution was dehydrated over a suitable amount o~ anhydrous sodium sulfate and the ef,hyl acetate was evaporated off under reduced pressure. The evaporation residue was ~ubjeo-ted to silica gel column(10 g, Kiesel gel 60, 70 - 230 me~h, E. Merck) chromatography using a developing solvcnt ~ystem of chloroform and methanol (40~13 to give 30~--mg of the title compound.
n.m.r. ~ppm tCDC~3 ):
Q91andQ95 (6H, each d 9 J=7Hz, --CH(CH~)2 ) 1.18 ( 3H, d,--O--~H--CH3 ) 1. 4 0 ( 9H . ~ ,--C ~ C~
9~~ ( 1H , m , ~CH ( CH3 )2 ~8~A3 (2H ,m,--CH~CH~,)~OBzl ,~NH-CHCO-) ~ . ~ . . . . ~
~ 3 4.1~4.6 (2H ,ABq~J=jem12Hz .-OCH2Ph) 4.70 (1H I dd ~ J=5Hz., 9Hz , -Nn~CHCO-~
5.06 (2H , 3 , -COOCH2Ph) 51~~3 ~1H . ~ ~ ~~
s~~7 (1H , ~nH-3 7.22 ~ loH t S t -CH2PhX2 ) 11-(2) Synthesi5 of Bv_-Val-Val-Thr(Bzl)OBzl.
~ y treatment with trifluoroacetic acid under the same reaction conditions as described in Example 1-(2), 305 m~
of Boc-Val-Thr(B2l)0Bzl was converted to its free amine.
This amine was condensed wi~h 168 mg of Boc-Val to yield 438 mg of crystals of the title compound.
Rf ~ 0.94(benzene/acetone=4/l), a silica gel pl~te as described above .m.r. ~ppm (CDC~3) :
~8~1.n5 (12H , m , -CH(CH3 )2 x2) 1.18 (3H , d , J6H2 , ~CH-CH3) 1.42 .(9H t 9 ~ - C(CH3)3 ) 1.8~2.3 (2H , m , -CH(CH3 )2x2) ~7~45 (3H ,m ,-CH(CH3)-OBzl , -NH CH-CO-x2) 4.36 (2H , ABq , -O-CH2Ph) 4.b~4.8 (1H ,m , -~nH-CH-CO-) 5.06 (2H , 8 , -C02-C~2Ph) S.1~5.25 (1H , -NH ) ~5~~9 (2H ~ -NH~X2) 7.25 (1oH , ~ , -CH2PhX2) 11-(3) Synthesis o~ Z-(2S,3R)-AHMHA-Val~Val-Thr(Bzl)OBzl.
- Boc-Val-Val-Thr(Bzl)OBzl(418 mg~ was treated with trifluoroacetic acid under the same reaction conditions as speci~ied in Example 1-~3) to provide its free amine, from which 246.1 mg of the ~itle compound wa~ produced by condensation with 161.8 mg of Z-(2S,~R) ~HMHA.
n.m.r. ~ppm (CD3COCD3) Q75~10 (18H , m , -CH(CH3)2 x3) 1.20 (3H t d , J~oH~, /CH-CH3) 1.3~1.7 (5H , m , -CH(CH3)2 x3 ,-C~ -CH(CH3~2 ) 4.D~4.8 (7H ,m ,-NH-CH-X4 ,-NH-C_-Ç~
CH(CH3~-OBzl~
S.Oand~12 (4H , each 9 , -CH2PhX2) 5.2~~4 (1H , m , -NH-) 7,22 .... ~5H , ~ , -CH2Ph) 7.29 (10H , 3 , -c~I~PhX2) 7.3~7.6 (3H / m , -~nH-x3) 11-(4~ Synthesis o~ (2s,3R)-AHMHA-Val-Val-Thr.
z-(2s,~R)-AHMHA-Val-V~l-Thr(Bzl 3 OBz1(246.1 mg) and -~5-50 mg of 5 y. palladium~charcoal catalyst was ~uspended in a solvent mixture of methanol, acetic acid and water(4/2/1) and shaken for catalytic reduction a-t room temperature in a hydrogen atmosphere~ After the catalyst w~s separated by filtration, the filtrate was diluted with water and e~aporated to dryness under reduced pressure to yield 137,4 mg of the title compound, ~f ~ 0,62(butanol/acetic acid/water=4/1/2, a silica gel . plate as described above~
m.p. , 229,0C
cd~ 53,2(c-0.5, CH3COOH3 Elemental analysis~for C21H40N4o7j Found C~54.62 %, Hs8.83 %, N:12,05 ~0 Calculated C~54~76 %, H:8.75 ~0, N:12.17 ~0 Example 12 Synthesis of (2S 3R)-AHMHA- al-Val-Pro.
12-(1) Pre~aration of Boc-Val-Val.
Two grams of Boc-Val-Val(OBzl) which was synthesized by the same method as described in Example 10-(1) was dissolved in 50 ml of methanol and then subjected to catalytic reduction overnight in the presence of 300 mg of 10 ~o palladium-charcoal catalyst. After the catalys~ was removed by filtration, the total volume of the filtrate was evaporated to dryness under reduced pressur8 to provide l.53 g of the aimed product.
n.m~r O ~ppm (CDC~3) 0.95 (12H , d ,J7Hz , ~GH(CH3)2X2) 1.42 (9H , 5, -C~CH~
t8~2.4 (2H , m , -CH(CH3)2 x~) ~8~4.1 (1H , m , -NH-~H-CO-) 4.4~4.7 (1H ~ m . -NH-~H-CO-~64~7.2 (2H , m g ~nHx2) 12-(2) Preparation of Boc-Val-Val-ProtoBzl~.
~ o a mixture of l.17 g of Pro(OBzl)~HCl and 540 ~l of NMM in 40 ml of dimethylformamide(abbreviated DMF hereafter) and 20 ml o~ THF, l~53 g of ~oc-Val-Val and then O.72 g of HOBt were adcled under cooling with ice~ DCCD(l~lO g) was further added to the solution under cooling with ice and then reacted for 2 hours at 0C and for one hour a-t room temperature. After precipitates of DCU thus formed were filtered off, the filtrate was evaporated to dryness under reduced pressure. The evaporation residue was dissolved in 370 ml of ethyl acetate and was washed three times with lOO ml each of lO ~0 citric acid, three times with lOO ml each of 4 % sodium bicarbonate and twice with 80 ml each o~ saturated solution of sodium chloride. me organic ~olution was . -67 dehydrate~ over anhydrous sodium sulfate. After the sodium sulfate was removed by ~iltration, the filtrate was evaporated to dryness to provide 1.16 g of the aimed compound.
n.m.r. ~ppm (CDC~3) Q85~~.05 (12H , m , -CH(CH~)2 X2) 1.42 (9~ ~ 8 ~ -C(CH3)3 ) .7~2.3 (6H , m , -C (CH3)2 X2 , -(CH ~)2-~H-C'O ) ~4~-4.1 (2H , m , -~-CH2-) 44~4~ (2H , m , -CH-CO-x2) 5.11 (2H , ~ ~ -CH2Ph) 5.0~5.3 (1H , -NH-) 5.6~s.8 (1H ~ -NH-~
7.29 (5H , s , ~CH2Ph) 12-(3) Preparation of z-(2s,3R)-AE~EU~ Val-Pro(oBzl~.
3y the same procedure as specified in Example 1-(3), 1.16 g of Boc-Val-Val-Pro(OBzl) was treated ~ith trifluoro-acetic acid and 717 mg of Vai-Val-Pro(O~zl) thus formed was subjected to condensat.ion with 525 mg of Z-(2S,3R)-AHM~A to give 568 mg of the title compound.
Rf 1 0,64(benzene/aceto.ne-4/1, a silica gel plate as described above) m,p. : 6~0~
~21 -7~.4 (C=1.0 , CH3COOH) n.m.rO ~ppm (CDC~3) ~8~1.1 (18H , m , -CH(~H3)~ x3) 1.3~2.4 (9H ,m ,-CH(CH3~ x~ 9 - CH2-CH(~-I3 )2 -(CH2)2. ) ~4.8 (8H ,m ,> N~ x4 ,~N-CH2- ,~CH-~_-O~I) ~02and~10 (4H ~ each 5 ~-C~2-PhX2) ~3~61 (2~ nH-X2) 7.25 (10H , s , -CH2Phx2) 7.3~7.6 (1H , -NH~) 12-(4) Preparation o~ (2s~3R)-AHMHA-val-val-pro z-(2S,3R)-AHMHA-Val-Val-Pro(OBzl)(204 mg) was dissolved in 30 ml of a solvent mixture o~ methanol, acetic acid and water(4/2/1) and, under the same operational c~nditions as presented in Example 1-(4), submitted to catalytic reduction with 50 mg of 10 ~0 palladium-'charcoal catalyst to give 128 mg of the desired compound.
R~ , Or61(butanol/acetic acid/water=4/1/2, a silica gel plate) m.p. a 176.0C
~21 ~ -88.6 (c=0.5, CH3CO~H) .. . ,, . . , . . . _ , . . .. .. .
Elemental analysis(for C~2H40N406) Found Gl58tO4 ~0, H,8.~5 ~0, N~12.05 ~0 Calculated C~57.87 %, H,~.83 ~0, NJ12~27 ~0 Example 13 Synthesis of (2S,~R~-AHMHA-Val-Val-Phe.
Two grams of Boc-Val-Val(OBzl) which was synthesized by the same method as described in Example 10-(1) was dissolved in 50 ml of methanol and then subjected to catalytic reduction overnight in the presence of 300 mg of 10 ~o palladium-charcoal catalyst. After the catalys~ was removed by filtration, the total volume of the filtrate was evaporated to dryness under reduced pressur8 to provide l.53 g of the aimed product.
n.m~r O ~ppm (CDC~3) 0.95 (12H , d ,J7Hz , ~GH(CH3)2X2) 1.42 (9H , 5, -C~CH~
t8~2.4 (2H , m , -CH(CH3)2 x~) ~8~4.1 (1H , m , -NH-~H-CO-) 4.4~4.7 (1H ~ m . -NH-~H-CO-~64~7.2 (2H , m g ~nHx2) 12-(2) Preparation of Boc-Val-Val-ProtoBzl~.
~ o a mixture of l.17 g of Pro(OBzl)~HCl and 540 ~l of NMM in 40 ml of dimethylformamide(abbreviated DMF hereafter) and 20 ml o~ THF, l~53 g of ~oc-Val-Val and then O.72 g of HOBt were adcled under cooling with ice~ DCCD(l~lO g) was further added to the solution under cooling with ice and then reacted for 2 hours at 0C and for one hour a-t room temperature. After precipitates of DCU thus formed were filtered off, the filtrate was evaporated to dryness under reduced pressure. The evaporation residue was dissolved in 370 ml of ethyl acetate and was washed three times with lOO ml each of lO ~0 citric acid, three times with lOO ml each of 4 % sodium bicarbonate and twice with 80 ml each o~ saturated solution of sodium chloride. me organic ~olution was . -67 dehydrate~ over anhydrous sodium sulfate. After the sodium sulfate was removed by ~iltration, the filtrate was evaporated to dryness to provide 1.16 g of the aimed compound.
n.m.r. ~ppm (CDC~3) Q85~~.05 (12H , m , -CH(CH~)2 X2) 1.42 (9~ ~ 8 ~ -C(CH3)3 ) .7~2.3 (6H , m , -C (CH3)2 X2 , -(CH ~)2-~H-C'O ) ~4~-4.1 (2H , m , -~-CH2-) 44~4~ (2H , m , -CH-CO-x2) 5.11 (2H , ~ ~ -CH2Ph) 5.0~5.3 (1H , -NH-) 5.6~s.8 (1H ~ -NH-~
7.29 (5H , s , ~CH2Ph) 12-(3) Preparation of z-(2s,3R)-AE~EU~ Val-Pro(oBzl~.
3y the same procedure as specified in Example 1-(3), 1.16 g of Boc-Val-Val-Pro(OBzl) was treated ~ith trifluoro-acetic acid and 717 mg of Vai-Val-Pro(O~zl) thus formed was subjected to condensat.ion with 525 mg of Z-(2S,3R)-AHM~A to give 568 mg of the title compound.
Rf 1 0,64(benzene/aceto.ne-4/1, a silica gel plate as described above) m,p. : 6~0~
~21 -7~.4 (C=1.0 , CH3COOH) n.m.rO ~ppm (CDC~3) ~8~1.1 (18H , m , -CH(~H3)~ x3) 1.3~2.4 (9H ,m ,-CH(CH3~ x~ 9 - CH2-CH(~-I3 )2 -(CH2)2. ) ~4.8 (8H ,m ,> N~ x4 ,~N-CH2- ,~CH-~_-O~I) ~02and~10 (4H ~ each 5 ~-C~2-PhX2) ~3~61 (2~ nH-X2) 7.25 (10H , s , -CH2Phx2) 7.3~7.6 (1H , -NH~) 12-(4) Preparation o~ (2s~3R)-AHMHA-val-val-pro z-(2S,3R)-AHMHA-Val-Val-Pro(OBzl)(204 mg) was dissolved in 30 ml of a solvent mixture o~ methanol, acetic acid and water(4/2/1) and, under the same operational c~nditions as presented in Example 1-(4), submitted to catalytic reduction with 50 mg of 10 ~0 palladium-'charcoal catalyst to give 128 mg of the desired compound.
R~ , Or61(butanol/acetic acid/water=4/1/2, a silica gel plate) m.p. a 176.0C
~21 ~ -88.6 (c=0.5, CH3CO~H) .. . ,, . . , . . . _ , . . .. .. .
Elemental analysis(for C~2H40N406) Found Gl58tO4 ~0, H,8.~5 ~0, N~12.05 ~0 Calculated C~57.87 %, H,~.83 ~0, NJ12~27 ~0 Example 13 Synthesis of (2S,~R~-AHMHA-Val-Val-Phe.
13-(1) Synthesls of_Boc-Val-Phe(OBzl).
Phe(OB21) TosOHt427 mg) and llO yl of N-methylmorpholine (abbreviated NMM hereafter) were dissolved in 20 ml of THF
and then, under the same reaction conditions as detailed in Example 1-~1), treated with 217 mg of Boc-Val, 148 mg of HbBt and 227 mg of DCCD to give 430 mg of the title compound.
Rf ~ 0.93(benzene/acetone=4~1, a silica gel plate as described above ) n.m.r . ~ppm ~CDC~3 ~ :
0.86, Q89( 6H, each d,--CH~CH3 )2 ) 1 4 3 ( 9H 9 3, O--C--( CH3 )3 ) 1.80~2.40 ( 1H, m,--CH(CH3 )2 ) ~1 0 ( 2H, d ,>CH--CH2~Ph ) 7 0~4.1 0 ( 2H , m , -NH-(~ . CO-x 2 ) ~1 2 ( 2H, ~ , ~O--CHz VPh) ~0~7.50 (5H ,m ,~CH2Ph) 7. 3 2 ,~ 5H, ~,--OCH 2 Ph ) 13-(2) Synthesis of Boc-Val-Val-Phe(03zll.
3y the same method as explained in Example 1-(2), 530 mg of Boc-Val-~Phe~OBzl) was treated wi.th -trifluoroacetic acid to provide 484 mg of Val-Phe(OBzl)~ wi~h which 296 mg of Boc-Val was condensed to yield 469 mg of the title compound.
Rf ~ 0,62(benzene/ace-tone=4/1, a silica gel plate as described above) m.pO : 114.0~
n,m. r C ~ppm ~CDC~3) o ~12H , broad d , -CH(CH3 )2 x2) 1.42 (9H , ~ , -C(CH3)~ ) ~17 (2H , d , -C-CH2Ph~
5.09 ~2~ , ~ , ~O-C~2P~
7.0~7.6 ~10H 7m+s , Phx2) 13-(3) Syn-thesis of Z-t2S,~R~-AHMHA-Val-Val-Phe(OBzl¦.
According to the same method as described in Example 1 (3), the treatmen~ o.~ 342 mg o~ Boc--Val-Val-Phe(OBzl) with trifluoroacetic acid resulted in 270 mg o~ Val-Val-Phe(OBzl), ~rom which 343 mg o~ the title compound was obtained by condensation with 163 mg of Z-(2S,3R~-AHMHA.
R~ t 0.76(benzene/acetone=4/1, a silica gel plate as described above) .. . . _ .. . _ _ -- ... .. , ..... , .. . _ .. .. _, _ . . ..
m.P~: 6 1.5 21, -80.6 ~--0.5, CH3C~
n.m. r . ~PPm (CDC~
0.8~1.0 (18H tm l-CH(CH~)2 X3) ~05 t2H, d, J=7HZ, ~CH--C~I2-Ph) s.o3 (2H , 9 , O - CH2-Ph) 5.08 (2H , ~ , -O-CH2-Ph~
~8~7.4 ( ~ 5H,--CH2PhX3) 13-(~) Synthesis of (2S,3R) -A~HA-Val-Val-Phe .
~ he same treatment as specified in Example 1-(4) was repeated to yield 213 mg of the desired derivative, excep-t that 320 mg of Z-(2s~3R)-AHMHA-val-val-phe(oBzl) and 80 mg of 10 % palladium-charcoal catalyst was suspended in 16 ml of a 501vent miXtllre of methanol, acetic acid and water ( L~/2/1 ~ .
Rf : 0.77(butanol/acetic acid/water=~/1/2, a silica gel plate as described above) m.p. , 164.7~C
~D : 18.8 (c=0.5, CH3COOH) Elemental analysis(for G26H42N406) Found Cs61.38 ~o, H~B.61 ~o, Nslo~B4 /~to Calculated C~61.63 ~, H~8r36 ~o~ N,11.06 ~0 -72~
Example 14 Synthesi~ of (2s~R)-AHMHA~val-~al-val.
Phe(OB21) TosOHt427 mg) and llO yl of N-methylmorpholine (abbreviated NMM hereafter) were dissolved in 20 ml of THF
and then, under the same reaction conditions as detailed in Example 1-~1), treated with 217 mg of Boc-Val, 148 mg of HbBt and 227 mg of DCCD to give 430 mg of the title compound.
Rf ~ 0.93(benzene/acetone=4~1, a silica gel plate as described above ) n.m.r . ~ppm ~CDC~3 ~ :
0.86, Q89( 6H, each d,--CH~CH3 )2 ) 1 4 3 ( 9H 9 3, O--C--( CH3 )3 ) 1.80~2.40 ( 1H, m,--CH(CH3 )2 ) ~1 0 ( 2H, d ,>CH--CH2~Ph ) 7 0~4.1 0 ( 2H , m , -NH-(~ . CO-x 2 ) ~1 2 ( 2H, ~ , ~O--CHz VPh) ~0~7.50 (5H ,m ,~CH2Ph) 7. 3 2 ,~ 5H, ~,--OCH 2 Ph ) 13-(2) Synthesis of Boc-Val-Val-Phe(03zll.
3y the same method as explained in Example 1-(2), 530 mg of Boc-Val-~Phe~OBzl) was treated wi.th -trifluoroacetic acid to provide 484 mg of Val-Phe(OBzl)~ wi~h which 296 mg of Boc-Val was condensed to yield 469 mg of the title compound.
Rf ~ 0,62(benzene/ace-tone=4/1, a silica gel plate as described above) m.pO : 114.0~
n,m. r C ~ppm ~CDC~3) o ~12H , broad d , -CH(CH3 )2 x2) 1.42 (9H , ~ , -C(CH3)~ ) ~17 (2H , d , -C-CH2Ph~
5.09 ~2~ , ~ , ~O-C~2P~
7.0~7.6 ~10H 7m+s , Phx2) 13-(3) Syn-thesis of Z-t2S,~R~-AHMHA-Val-Val-Phe(OBzl¦.
According to the same method as described in Example 1 (3), the treatmen~ o.~ 342 mg o~ Boc--Val-Val-Phe(OBzl) with trifluoroacetic acid resulted in 270 mg o~ Val-Val-Phe(OBzl), ~rom which 343 mg o~ the title compound was obtained by condensation with 163 mg of Z-(2S,3R~-AHMHA.
R~ t 0.76(benzene/acetone=4/1, a silica gel plate as described above) .. . . _ .. . _ _ -- ... .. , ..... , .. . _ .. .. _, _ . . ..
m.P~: 6 1.5 21, -80.6 ~--0.5, CH3C~
n.m. r . ~PPm (CDC~
0.8~1.0 (18H tm l-CH(CH~)2 X3) ~05 t2H, d, J=7HZ, ~CH--C~I2-Ph) s.o3 (2H , 9 , O - CH2-Ph) 5.08 (2H , ~ , -O-CH2-Ph~
~8~7.4 ( ~ 5H,--CH2PhX3) 13-(~) Synthesis of (2S,3R) -A~HA-Val-Val-Phe .
~ he same treatment as specified in Example 1-(4) was repeated to yield 213 mg of the desired derivative, excep-t that 320 mg of Z-(2s~3R)-AHMHA-val-val-phe(oBzl) and 80 mg of 10 % palladium-charcoal catalyst was suspended in 16 ml of a 501vent miXtllre of methanol, acetic acid and water ( L~/2/1 ~ .
Rf : 0.77(butanol/acetic acid/water=~/1/2, a silica gel plate as described above) m.p. , 164.7~C
~D : 18.8 (c=0.5, CH3COOH) Elemental analysis(for G26H42N406) Found Cs61.38 ~o, H~B.61 ~o, Nslo~B4 /~to Calculated C~61.63 ~, H~8r36 ~o~ N,11.06 ~0 -72~
Example 14 Synthesi~ of (2s~R)-AHMHA~val-~al-val.
14-(1) Preparation of Boc-Val-Val-Val(OBZl).
Boc-Val-Val(395 mg ) obtained ~y the same method as specified in Example 12-(1) and 474 mg of Val~OBzl~ rosOH
were subjected to condensation and processed by the same procedure as explained in Example 1-(2) to provide 6G5 mg of the desired derivative.
n.m.r, ~ppm (CDC~3) n (18H 9 ~CH(C 3)2x3) 1-4 (9H , s , C~CH3)~ ) 1.8~~3 (3H , -CH(CH3)2 x3 ~8~4.1 (1H , -~nH-CH-CO-) 4.2~4.7 (2H , -~nH-CH-CO-X2) ~05~~15 (2~ , -OCH2P~l) 5.4~5.7 1~1H ~ -~nH-) ~7~7..2 2H , -~nH-X2) 7.28 (5H . ~ , -O-CH2Ph) 14-(2) Preparation o~ Z-(~S!~R)-AH~TA-Val-Val-Val(OBzl).
.,1 The treatment of 278 mg of Boc-Val-Val-Val ( OBzl ) with trifluoroacetic acid under the same operatlonal conditions a~ described in Example 1 (3) produced a free amine which, on condensatlon with 129 mg of Z-t2S, ~R)-AH~A, resulted in 250 mg of crystals of the title compound.
n.m,rO ~ppm (CDC~31 ~7~1.05 ~24H , -CH(CH~)2 x4 50~~18 (4H , -OCH2Phx2) 7.28 (10H ~ s , -OCH2Phx2) (Qnly characteristic signals are listed) 14-(3) Preparation_of (2s,3R~-AHMHA-Val-Val-Val.
Z-(2S,~R~-AHMHA-Val-Val-Val(OBzl) (246 mg) was dissolved in a mixed solvent o~ methanol, acetic acid and water(8/2/1) (15 ml) and then subjected to catalytic reduction in the presence of 10 ~O-palladium-charcoal catalyst (50 mg) under the same operational conditions as detailed in Example 1-(4) to yield 114.8 mg of the title compound.
R~ s 0077(butanol/acetic acid/water=4,/1/2, a silica gel plate as described above) m.p. ~ 79.~C
1 ~ -62.6(c=0.5, CH3COOH) Elemental analysis(for C22H42NL~06) Found Cs57.37 ~0, ~:9.28 ~0, N:11.94 ~'0 Calculated Cl57.62 ~0, H~9.23 %, ~:12.22 ~0 ~xample 15 ~ynthesis of (2~R)-AHMHA-val-val-Lys-
Boc-Val-Val(395 mg ) obtained ~y the same method as specified in Example 12-(1) and 474 mg of Val~OBzl~ rosOH
were subjected to condensation and processed by the same procedure as explained in Example 1-(2) to provide 6G5 mg of the desired derivative.
n.m.r, ~ppm (CDC~3) n (18H 9 ~CH(C 3)2x3) 1-4 (9H , s , C~CH3)~ ) 1.8~~3 (3H , -CH(CH3)2 x3 ~8~4.1 (1H , -~nH-CH-CO-) 4.2~4.7 (2H , -~nH-CH-CO-X2) ~05~~15 (2~ , -OCH2P~l) 5.4~5.7 1~1H ~ -~nH-) ~7~7..2 2H , -~nH-X2) 7.28 (5H . ~ , -O-CH2Ph) 14-(2) Preparation o~ Z-(~S!~R)-AH~TA-Val-Val-Val(OBzl).
.,1 The treatment of 278 mg of Boc-Val-Val-Val ( OBzl ) with trifluoroacetic acid under the same operatlonal conditions a~ described in Example 1 (3) produced a free amine which, on condensatlon with 129 mg of Z-t2S, ~R)-AH~A, resulted in 250 mg of crystals of the title compound.
n.m,rO ~ppm (CDC~31 ~7~1.05 ~24H , -CH(CH~)2 x4 50~~18 (4H , -OCH2Phx2) 7.28 (10H ~ s , -OCH2Phx2) (Qnly characteristic signals are listed) 14-(3) Preparation_of (2s,3R~-AHMHA-Val-Val-Val.
Z-(2S,~R~-AHMHA-Val-Val-Val(OBzl) (246 mg) was dissolved in a mixed solvent o~ methanol, acetic acid and water(8/2/1) (15 ml) and then subjected to catalytic reduction in the presence of 10 ~O-palladium-charcoal catalyst (50 mg) under the same operational conditions as detailed in Example 1-(4) to yield 114.8 mg of the title compound.
R~ s 0077(butanol/acetic acid/water=4,/1/2, a silica gel plate as described above) m.p. ~ 79.~C
1 ~ -62.6(c=0.5, CH3COOH) Elemental analysis(for C22H42NL~06) Found Cs57.37 ~0, ~:9.28 ~0, N:11.94 ~'0 Calculated Cl57.62 ~0, H~9.23 %, ~:12.22 ~0 ~xample 15 ~ynthesis of (2~R)-AHMHA-val-val-Lys-
15-(1) Synthesis of Boc-Val I,ys~Z)-OBzl.
Us.-ng 217.3 mg of 30c-val and 407 mg of Lys(Z)-O~zl ~Cl~
the same reaction and treatment as explained in ~xample 1-(1) was performed to give 613 mg of the title derivative.
n.m.r. ~ppm (CDC~3) 0.82, n.95 (6H ,each d , J=4Hz ~ -CHtC~ ) 1.40 (9H , s , -C(CH3)~ ) 1~1~æ1 (~H , m , -(CH2)3 - , -CH(CH3)2) .9~~2 ~2H ,m , -NH-CH2 ~) ~7~45 (2H ,m , -NH-CH-C0-) 5.08, 5.15 (4H 9 each ~, -cH2phx2 ~5~7.0 (2H , m , -NH-X2) 735 (lO~ , s , CH2Phx2) 15-(2~ Synthesis of ~oc Val-Val-~s(Z~-O~zl.
~ y the same procedure as detailed in Example 1-(2), 362 mg of Boc-Val-Val-Lys(Z)-OBzl~rosOH was treated with tri~luoroacetic acid to give a free amine which was condensed with 122 mg of Boc-Val for production of 286 mg of the aimed compound.
n.m~r. 3ppm (CDC~3-CD30D) Q9 (12H , d , J=6Hz , -CH(CH ~ x2) ~-45 (9H , s , -C~CH~33 ) 1.0~2.4 (8H , -CH(CH ~ X2 ~~(CH2 ~3 - ) 2.9~~2 (2H . -CH 2 -NHZ~
~8~4.9 (3H , -NH~CH CO-x3 ~1and5.15 (4H , each ~ , -CH2Ph 732 - (10H , s , -CH2Phx2) 15-~3) Synthesis of Z-( 2S, 3R) -AHMHA-Val-Val-Lys (Z ) -OBzl .
~ ree amine which was derived ~rom 286 mg of 30c-Val-Val-lys(Z)-03zl by treatment with trifluoroacetic acid under the same reaction conditions as detailed in '~xample 1-(3) was submitted to condensation with 124 mg of Z-(2S,3R)- ~MHA
to yield 177 mg of the desire'd compound~
n.m.r. ~ppm (CDC~3) 0.87 (18H , d-like , -CH(CH3)2 x3) 5.0, 5.05and~12 (6H , each ~ ~ -CH2Phx3) 7.30 (15H , 9, -CH2PhX3) 15-(4~ Synthesis of (2S,~R~-A~IA-Val-Val-l.ys.
Solution of 175 mg of Z~(2S,~R)-AHMHA-Val-Val-Lys(Z)-OBzl in 30 ml of a mixed solvent of methanol, acetic acid and water(4/2/1) was hydrogenolysed under the same reaction conditions as defined in Example 2-(4) to yield 77.1 mg of the title compound.
Rf s 0.27(butanol/acetic acid/water=4/l/2, a silica gel plate as described above) m.p. , 224.2C
[~21 ~ -51,2 (c=0.5, CH3COOH) ~lemental analysis(for C23H45N506) Found C:56.41 %, H:9.56 ~0, N~14.11 ~0 Calculated C:56.65 ~, H~9.30 ~0, N:14.36 ~0 Example 16 Synthesis of (2S,~R)-AHMXA-Val-Val-Arg.
.
Us.-ng 217.3 mg of 30c-val and 407 mg of Lys(Z)-O~zl ~Cl~
the same reaction and treatment as explained in ~xample 1-(1) was performed to give 613 mg of the title derivative.
n.m.r. ~ppm (CDC~3) 0.82, n.95 (6H ,each d , J=4Hz ~ -CHtC~ ) 1.40 (9H , s , -C(CH3)~ ) 1~1~æ1 (~H , m , -(CH2)3 - , -CH(CH3)2) .9~~2 ~2H ,m , -NH-CH2 ~) ~7~45 (2H ,m , -NH-CH-C0-) 5.08, 5.15 (4H 9 each ~, -cH2phx2 ~5~7.0 (2H , m , -NH-X2) 735 (lO~ , s , CH2Phx2) 15-(2~ Synthesis of ~oc Val-Val-~s(Z~-O~zl.
~ y the same procedure as detailed in Example 1-(2), 362 mg of Boc-Val-Val-Lys(Z)-OBzl~rosOH was treated with tri~luoroacetic acid to give a free amine which was condensed with 122 mg of Boc-Val for production of 286 mg of the aimed compound.
n.m~r. 3ppm (CDC~3-CD30D) Q9 (12H , d , J=6Hz , -CH(CH ~ x2) ~-45 (9H , s , -C~CH~33 ) 1.0~2.4 (8H , -CH(CH ~ X2 ~~(CH2 ~3 - ) 2.9~~2 (2H . -CH 2 -NHZ~
~8~4.9 (3H , -NH~CH CO-x3 ~1and5.15 (4H , each ~ , -CH2Ph 732 - (10H , s , -CH2Phx2) 15-~3) Synthesis of Z-( 2S, 3R) -AHMHA-Val-Val-Lys (Z ) -OBzl .
~ ree amine which was derived ~rom 286 mg of 30c-Val-Val-lys(Z)-03zl by treatment with trifluoroacetic acid under the same reaction conditions as detailed in '~xample 1-(3) was submitted to condensation with 124 mg of Z-(2S,3R)- ~MHA
to yield 177 mg of the desire'd compound~
n.m.r. ~ppm (CDC~3) 0.87 (18H , d-like , -CH(CH3)2 x3) 5.0, 5.05and~12 (6H , each ~ ~ -CH2Phx3) 7.30 (15H , 9, -CH2PhX3) 15-(4~ Synthesis of (2S,~R~-A~IA-Val-Val-l.ys.
Solution of 175 mg of Z~(2S,~R)-AHMHA-Val-Val-Lys(Z)-OBzl in 30 ml of a mixed solvent of methanol, acetic acid and water(4/2/1) was hydrogenolysed under the same reaction conditions as defined in Example 2-(4) to yield 77.1 mg of the title compound.
Rf s 0.27(butanol/acetic acid/water=4/l/2, a silica gel plate as described above) m.p. , 224.2C
[~21 ~ -51,2 (c=0.5, CH3COOH) ~lemental analysis(for C23H45N506) Found C:56.41 %, H:9.56 ~0, N~14.11 ~0 Calculated C:56.65 ~, H~9.30 ~0, N:14.36 ~0 Example 16 Synthesis of (2S,~R)-AHMXA-Val-Val-Arg.
.
16-(l) Preparation of BocVal-Ar~NO~J-03zl.
utilizing 0.439 g of Boc-Val and 103Q7 ~ of Arg~N02I-03zl~2TosOX, the s~me reaction and treatment as described in Example l-(l) was repeated to g;ve 0.968 J of the desired product.
n.m.r. ~ppm (CDC~3) 0.95 (6H / d , J-6Hz , -CH(CH3)2) 1-4 (9H , s ~ -C(CH3 )3 ) 1.3~2.2 (5H , -COCH(CH 2)2 - , -CH-(CH3)2) ~0~~7 (2H , -CH2-NH-C=N-) ~7~4.2 (1H , -NH-~H-CO-) 4.4~4.8 ~lH , -NH-CH-CO-) 517- (2H 3 S ~ - ~C~2 51~54 (1H ,~N~
B
7.0~.2 (1~ NII-) 7.34 (5H , s , -OCH2Ph) 73~79 (3H , -NH~x3~
16-(2) Preparation of 3Oc-Val-Val-Arg(N02~-BOzl.
~ he same trifluoroacetic acid treatment of 0.960 g of 3Oc-~al-Arg~N02~-03zl as specif.ied in ~xample 1-(2) led to the formation of a free amine Ylhich was subjected to conden-sation with 0.304 g of 3Oc-Val to provide 0.605 g of the desired compound.
n~rn.r. ~ppm (CDC~3~ :
~9 (-2H ,d-like , -CH (CH3)2 x2) 1-4 (9H , s, -C(CH3)3 ) ~15 (2H , ~ 9 -0-CH2Ph) .32 (5H ,~s , -0-C~2Ph) (Qnly characteristic signals are listed) 16-(3~ Preparation of Z-(2S,3R)-A ~A-Val-Val-Arg[N02]-OBzl.
Free amine that ~s obtained from 143 mg of 3Oc-Val-Val-ArgLN021-03zl by the same trifluoroacetic acid trea-tment as explained in Example 1-(3) was subjected to condensation with 81.6 mg of Z-(2S, 3R)-AH~A to give 152 rng of the title compound.
n.m~r. ~ppm ~CDC~3-CD~COCD~CDjOD) Q8~1.05 ~12H ~ broad d , -CH(CH~)2 x2 ~02 (2H , g , -OCH~Ph) 516 (2H , 5 , -OCH2Ph) 735 (~oH 9 ~ I - CH2PhX2) ~Only characteristic signals are listed~
16-~43 Preparation o~ (2S,~R)-AHMHA-Val-Val~Arg.
.. . . ..
Solution of 114 mg of Z-(2S,3R)-AHMHA~Val-Val~Arg~N02]-OBzl in 30 ml o~ a solvent mixture o~ methanol, acetic acid and water(4/2/1) was hydro~enolysed under the same reaction conditions as detailed in Example 1~(4~ to provide 48.7 mg of the title compound.
R~ i 0.41(butanol/acetic acid/water=4/1/2, a silica gel pl~te aæ described above~
m.p. 1 97.5C
~d~21 ~ ~38.6 (c--Oo5~ CH3COOH) Elemental analysis(for C23H45N706) Fou.nd Cl53.44 %, H~9.08 j~, N~18.75 %
Calculated Ct53.57 ~0, H.8.80 ,.~" N~19.02 %
utilizing 0.439 g of Boc-Val and 103Q7 ~ of Arg~N02I-03zl~2TosOX, the s~me reaction and treatment as described in Example l-(l) was repeated to g;ve 0.968 J of the desired product.
n.m.r. ~ppm (CDC~3) 0.95 (6H / d , J-6Hz , -CH(CH3)2) 1-4 (9H , s ~ -C(CH3 )3 ) 1.3~2.2 (5H , -COCH(CH 2)2 - , -CH-(CH3)2) ~0~~7 (2H , -CH2-NH-C=N-) ~7~4.2 (1H , -NH-~H-CO-) 4.4~4.8 ~lH , -NH-CH-CO-) 517- (2H 3 S ~ - ~C~2 51~54 (1H ,~N~
B
7.0~.2 (1~ NII-) 7.34 (5H , s , -OCH2Ph) 73~79 (3H , -NH~x3~
16-(2) Preparation of 3Oc-Val-Val-Arg(N02~-BOzl.
~ he same trifluoroacetic acid treatment of 0.960 g of 3Oc-~al-Arg~N02~-03zl as specif.ied in ~xample 1-(2) led to the formation of a free amine Ylhich was subjected to conden-sation with 0.304 g of 3Oc-Val to provide 0.605 g of the desired compound.
n~rn.r. ~ppm (CDC~3~ :
~9 (-2H ,d-like , -CH (CH3)2 x2) 1-4 (9H , s, -C(CH3)3 ) ~15 (2H , ~ 9 -0-CH2Ph) .32 (5H ,~s , -0-C~2Ph) (Qnly characteristic signals are listed) 16-(3~ Preparation of Z-(2S,3R)-A ~A-Val-Val-Arg[N02]-OBzl.
Free amine that ~s obtained from 143 mg of 3Oc-Val-Val-ArgLN021-03zl by the same trifluoroacetic acid trea-tment as explained in Example 1-(3) was subjected to condensation with 81.6 mg of Z-(2S, 3R)-AH~A to give 152 rng of the title compound.
n.m~r. ~ppm ~CDC~3-CD~COCD~CDjOD) Q8~1.05 ~12H ~ broad d , -CH(CH~)2 x2 ~02 (2H , g , -OCH~Ph) 516 (2H , 5 , -OCH2Ph) 735 (~oH 9 ~ I - CH2PhX2) ~Only characteristic signals are listed~
16-~43 Preparation o~ (2S,~R)-AHMHA-Val-Val~Arg.
.. . . ..
Solution of 114 mg of Z-(2S,3R)-AHMHA~Val-Val~Arg~N02]-OBzl in 30 ml o~ a solvent mixture o~ methanol, acetic acid and water(4/2/1) was hydro~enolysed under the same reaction conditions as detailed in Example 1~(4~ to provide 48.7 mg of the title compound.
R~ i 0.41(butanol/acetic acid/water=4/1/2, a silica gel pl~te aæ described above~
m.p. 1 97.5C
~d~21 ~ ~38.6 (c--Oo5~ CH3COOH) Elemental analysis(for C23H45N706) Fou.nd Cl53.44 %, H~9.08 j~, N~18.75 %
Calculated Ct53.57 ~0, H.8.80 ,.~" N~19.02 %
Claims (59)
1. The process for the production of tetrapeptide deri-vatives of the formula (I):
(I) wherein R1 and R2 are methyl, 1-methylethyl, 2-methylpropyl, 1-hydroxyethyl, 2-carboxyethyl or 4-aminobutyl and Y is wherein R3 is methyl, 1-methylethyl, 2-methylpropyl, 1-methyl-propyl, hydroxymethyl, 1-hydroxyethyl, carboxymethyl, 2-carboxy-ethyl, 4-aminobutyl, 3-guanidinopropyl, benzyl or p-hydroxybenzyl excluding the compounds in which R1 and R2 are each 1-methyl-ethyl and also R3 is carboxymethyl or 2-carboxyethyl, the first and leftmost .beta.-amino acid moiety in said tetrapeptide deratives having the (2S,3R)-configuration and a primary amino group and the second, third and fourth .alpha.-amino acid moieties in said tetrapeptide derivatives having the L-configuration which comprises first condensing an activated derivative of the carboxylic acid having the formula and having the (2S,3R)-configuration wherein Z is a conventional blocking group with a blocked amino acid having the formula in which all three amino acid moieties have the L-configuration and wherein R1, R2, R3 and Y have the meaning set for the above and any hydroxy, amino or guanidino groups present in R1, R2 and R3 are blocked to form a blocked derivative of the desired tetrapeptide and next chemically removing all blocking groups to form the desired tetrapeptide.
(I) wherein R1 and R2 are methyl, 1-methylethyl, 2-methylpropyl, 1-hydroxyethyl, 2-carboxyethyl or 4-aminobutyl and Y is wherein R3 is methyl, 1-methylethyl, 2-methylpropyl, 1-methyl-propyl, hydroxymethyl, 1-hydroxyethyl, carboxymethyl, 2-carboxy-ethyl, 4-aminobutyl, 3-guanidinopropyl, benzyl or p-hydroxybenzyl excluding the compounds in which R1 and R2 are each 1-methyl-ethyl and also R3 is carboxymethyl or 2-carboxyethyl, the first and leftmost .beta.-amino acid moiety in said tetrapeptide deratives having the (2S,3R)-configuration and a primary amino group and the second, third and fourth .alpha.-amino acid moieties in said tetrapeptide derivatives having the L-configuration which comprises first condensing an activated derivative of the carboxylic acid having the formula and having the (2S,3R)-configuration wherein Z is a conventional blocking group with a blocked amino acid having the formula in which all three amino acid moieties have the L-configuration and wherein R1, R2, R3 and Y have the meaning set for the above and any hydroxy, amino or guanidino groups present in R1, R2 and R3 are blocked to form a blocked derivative of the desired tetrapeptide and next chemically removing all blocking groups to form the desired tetrapeptide.
2. The process of claim 1 in which the activated derivative is selected from the group consisting of the use of a carbodi-imide, an azide, a mixed anhydride, an active ester such as cyanomethyl ester, vinyl ester, a substituted or unsubstituted phenyl ester, a thioester, N-hydroxy-succinimide ester, the use of an O-acyl hydroxylamine derivative method employing O-acyl-acetoxime or O-acylcyclohexanoneoxime and the use of an N-acyl derivative employing carbonyldiimidazole.
3. The process of claim 1 in which the blocking groups are selected from the group consisting of an acyl group, a substituted or unsubstituted benzyloxycarbonyl group, alkoxy-carbonyl, cycloalkanooxycarbonyl, substituted or unsubstituted arylsulfonyl, nitrophenyl and trityl groups.
4. The process of claim 1 in which the chemical deblock-ing is performed by the use of catalytic hydrogenation or saponification with an alkali or acidolysis with hydrogen bromide in acetic acid or with trifluoroacetic acid or with hydrogen chloride in dioxane, tetrahydrofuran or ethyl acetate or with liquid hydrogen fluoride or to obtain hydra-zinolysis with hydrazine or treatment with sodium in liquid ammonia.
5. The process for the production of tetrapeptide derivatives of the formula (I):
(I) wherein R1 and R2 are methyl, 1-methylethyl, 2-methylpropyl, 1-hydroxyethyl, 2-carboxyethyl or 4-aminobutyl and Y is wherein R3 is methyl, 1-methylethyl, 2-methylpropyl, 1-methyl-propyl, hydroxymethyl, 1-hydroxyethyl, carboxymethyl, 2-carboxyethyl, 4-aminobutyl, 3-guanidinopropyl, benzyl or p-hydroxybenzyl excluding the compounds in which R1 and R2 are each 1-methylethyl and also R3 is carboxymethyl or 2-carboxyethyl, the first and leftmost .beta.-amino acid moiety in said tetrapeptide derivatives having the (2S,3R) configuration and a primary amino group and the second, third and fourth .alpha.-amino acid moieties in said tetrapeptide derivatives having the L-configuration which comprises first condensing an activated derivative of the carboxylic acid having the formula and having the (2S, 3R)-configuration wherein Z is a con-ventional blocking group with a blocked amino acid having the formula in which all three amino acid moieties have the L-configuration and wherein R1, R2, R3 and Y have the meaning set for the above and any hydroxy, amino or guanidino groups present in R1, R2 and R are blocked to form a blocked derivative of the desired tetrapeptide and next chemically removing all blocking groups to form the desired tetrapeptide.
(I) wherein R1 and R2 are methyl, 1-methylethyl, 2-methylpropyl, 1-hydroxyethyl, 2-carboxyethyl or 4-aminobutyl and Y is wherein R3 is methyl, 1-methylethyl, 2-methylpropyl, 1-methyl-propyl, hydroxymethyl, 1-hydroxyethyl, carboxymethyl, 2-carboxyethyl, 4-aminobutyl, 3-guanidinopropyl, benzyl or p-hydroxybenzyl excluding the compounds in which R1 and R2 are each 1-methylethyl and also R3 is carboxymethyl or 2-carboxyethyl, the first and leftmost .beta.-amino acid moiety in said tetrapeptide derivatives having the (2S,3R) configuration and a primary amino group and the second, third and fourth .alpha.-amino acid moieties in said tetrapeptide derivatives having the L-configuration which comprises first condensing an activated derivative of the carboxylic acid having the formula and having the (2S, 3R)-configuration wherein Z is a con-ventional blocking group with a blocked amino acid having the formula in which all three amino acid moieties have the L-configuration and wherein R1, R2, R3 and Y have the meaning set for the above and any hydroxy, amino or guanidino groups present in R1, R2 and R are blocked to form a blocked derivative of the desired tetrapeptide and next chemically removing all blocking groups to form the desired tetrapeptide.
6. The process of Claim 5 in which the activated derivative is selected from the group consisting of the use of a carbodiimide, an azide, a mixed anhydride, an active ester such as cyanomethyl ester, vinyl ester, a substituted or unsubstituted phenyl ester, a thioester, N-hydroxy-succinimide ester, the use of an O-acyl hydroxylamine derivative method employing O-acyl-acetoxime or O-acylcyclohexanoneoxime and the use of an N-acyl derivative employing carbonyldiimidazole.
7. The process of Claim 5 in which the blocking groups are selected from the group consisting of an acyl group, a substituted or unsubstituted benzyloxycarbonyl group, alkoxycarbonyl, cycloalkanooxycarbonyl, substituted or unsubstituted arylsulfonyl, nitrophenyl and trityl groups.
8. The process of Claim 5 in which the chemical deblocking is performed by the use of catalytic hydrogenation or saponi-fication with an alkali or acidolysis with hydrogen bromide in acetic acid or with trifluoroacetic acid or with hydrogen chloride in dioxane, tetrahydrofuran or ethyl acetate or with liquid hydrogen fluoride or to obtain hydrazinolysis with hydrazine or treatment with sodium in liquid ammonia.
9. A process according to Claim 5 in which R1 is 1-methylethyl and R2 is not 1-methylethyl, in the starting material and in the final product.
10. A process according to Claim 1 in which R2 is 4-aminobutyl, 1-hydroxyethyl, 2-methylpropyl or 2-carboxyethyl, in the starting material and in the final product.
11. A process according to Claim 5 in which R3 is carboxymethyl, in the starting material and in the final product.
12. A process according to Claim 5 in which R2 is 1-methylethyl and R1 is not 1-methylethyl, in the starting material and in the final product.
13. A process according to Claim 5 in which R1 is 4-aminobutyl, 1-hydroxyethyl, 2-methylpropyl or 2-carboxyethyl, in the starting material and in the final product.
14. A process according to Claim 5 in which R3 is carboxymethyl, in the starting material and in the final product.
15. A process according to Claim 5 in which both R1 and R2 are 1-methylethyl, in the starting material and in the final product.
16. A process according to Claim 5 in which both R1 and R2 are 1-methylethyl, and in which R3 is 1-hydroxyethyl, benzyl, 1-methyl-ethyl, 4-aminobutyl or 3-guanidinopropyl, in the starting material and in the final product.
17. A process according to Claim 5 in which both R1 and R2 are 1-methylethyl, and in which R3 is 1-hydroxyethyl, in the starting material and in the final product.
18. A process according to Claim 5 in which both R1 and R2 are 1-methylethyl, and in which R3 is benzyl, in the starting material and in the final product.
19. A process according to Claim 5 in which both R1 and R2 are 1-methylethyl, and in which R3 is 1-methylethyl, in the starting material and in the final product.
20. A process according to Claim 5 in which both R1 and R2 are 1-methylethyl, and in which R3 is 4-aminobutyl, in the starting material and in the final product.
21. A process according to Claim 5 in which both R1 and R2 are 1-methylethyl, and in which R3 is 3-guanidinopropyl, in the starting material and in the final product.
22. A process according to Claim 6, 7 or 8 in which R1 is 1-methylethyl and R2 is not 1-methylethyl, in the starting material and in the final product.
23. A process according to Claim 6, 7 or 8 in which R2 is 4-aminobutyl, 1-hydroxyethyl, 2-methylpropyl or 2-carboxyethyl, in the starting material and in the final product.
24. A process according to Claim 6, 7 or 8 in which R3 is carboxymethyl, in the starting material and in the final product.
25. A process according to Claim 6, 7 or 8 in which R2 is 1-methylethyl and R1 is not 1-methylethyl, in the starting material and in the final product.
26. A process according to Claim 6, 7 or 8 in which R1 is 4-aminobutyl, 1-hydroxyethyl, 2-methylpropyl or 2-carboxyethyl, in the starting material and in the final product.
27. A process according to Claim 6, 7 or 8 in which R3 is carboxymethyl, in the starting material and in the final product.
28. A process according to Claim 6, 7 or 8 in which both R1 and R2 are 1-methylethyl, in the starting material and in the final product.
29. A process according to Claim 6, 7 or 8 in which both R1 and R2 are 1-methylethyl, and in which R3 is 1-hydroxyethyl, benzyl, 1-methylethyl, 4-aminobutyl or 3-guanidinopropyl, in the starting material and in the final product.
30. A process according to Claim 6, 7 or 8 in which both R1 and R2 are 1-methylethyl, and in which R3 is 1-hydroxyethyl, in the starting material and in the final product.
31. A process according to Claim 6, 7 or 8 in which both R1 and R2 are 1-methylethyl, and in which R3 is benzyl, in the starting material and in the final product.
32. A process according to Claim 6, 7 or 8 in which both R1 and R2 are 1-methylethyl, and in which R3 in 1-methylethyl, in the starting material and in the final product.
33. A process according to Claim 6, 7 or 8 in which both R1 and R2 are 1-methylethyl, and in which R3 is 4-aminobutyl, in the starting material and in the final product.
34. A process according to Claim 6, 7 or 8 in which both R1 and R2 are 1-methylethyl, and in which R3 is 3-guanidinopropyl, in the starting material and in the final product.
35. Tetrapeptide derivatives of the formula (I):
(I) wherein R1 and R2 are methyl, 1-methylethyl, 2-methylpropyl, 1-hydroxyethyl, 2-carboxyethyl or 4-aminobutyl and Y is or wherein R3 is methyl, 1-methylethyl, 2-methylpropyl, 1-methylpropyl, hydroxymethyl, 1-hydroxyethyl, carboxy-methyl, 2-carboxyethyl, 4-aminobutyl, 3-guanidinopropyl, benzyl or p-hydroxybenzyl excluding the compounds in which R1 and R2 are each 1-methylethyl and also R3 is carboxymethyl or 2-carboxyethyl, the first and leftmost .beta.-amino acid moiety in said tetrapeptide derivatives having the (2S,3R)-configuration and a primary amino group and the second, third and fourth .alpha.-amino acid moieties in said tetxapeptide derivatives having the L-configuration, whenever prepared or produced by the process of Claim 1, 2 or 3, or by an obvious chemical equivalent thereof.
(I) wherein R1 and R2 are methyl, 1-methylethyl, 2-methylpropyl, 1-hydroxyethyl, 2-carboxyethyl or 4-aminobutyl and Y is or wherein R3 is methyl, 1-methylethyl, 2-methylpropyl, 1-methylpropyl, hydroxymethyl, 1-hydroxyethyl, carboxy-methyl, 2-carboxyethyl, 4-aminobutyl, 3-guanidinopropyl, benzyl or p-hydroxybenzyl excluding the compounds in which R1 and R2 are each 1-methylethyl and also R3 is carboxymethyl or 2-carboxyethyl, the first and leftmost .beta.-amino acid moiety in said tetrapeptide derivatives having the (2S,3R)-configuration and a primary amino group and the second, third and fourth .alpha.-amino acid moieties in said tetxapeptide derivatives having the L-configuration, whenever prepared or produced by the process of Claim 1, 2 or 3, or by an obvious chemical equivalent thereof.
36. Tetrapeptide derivatives of the formula (I):
(I) wherein R1 and R2 are methyl, 1-methylethyl, 2-methylpropyl, 1-hydroxyethyl, 2-carboxyethyl or 4-aminobutyl and Y is wherein R3 is methyl, 1-methylethyl, 2-methylpropyl, 1-methylpropyl, hydroxymethyl, 1-hydroxyethyl, carboxy-methyl, 2-carboxyethyl, 4-aminobutyl, 3-guanidinopropyl, benzyl or p-hydroxybenzyl excluding the compounds in which R1 and R2 are each 1-methylethyl and also R3 is carboxymethyl or 2-carboxyethyl, the first and leftmost .beta.-amino acid moiety in said tetrapeptide derivatives having the (2S,3R)-configuration and a primary amino group and the second, third and fourth .alpha.-amino acid moieties in said tetrapeptide derivatives having the L-configuration, whenever prepared or produced by the process of Claim 5, or by an obvious chemical equivalent thereof.
(I) wherein R1 and R2 are methyl, 1-methylethyl, 2-methylpropyl, 1-hydroxyethyl, 2-carboxyethyl or 4-aminobutyl and Y is wherein R3 is methyl, 1-methylethyl, 2-methylpropyl, 1-methylpropyl, hydroxymethyl, 1-hydroxyethyl, carboxy-methyl, 2-carboxyethyl, 4-aminobutyl, 3-guanidinopropyl, benzyl or p-hydroxybenzyl excluding the compounds in which R1 and R2 are each 1-methylethyl and also R3 is carboxymethyl or 2-carboxyethyl, the first and leftmost .beta.-amino acid moiety in said tetrapeptide derivatives having the (2S,3R)-configuration and a primary amino group and the second, third and fourth .alpha.-amino acid moieties in said tetrapeptide derivatives having the L-configuration, whenever prepared or produced by the process of Claim 5, or by an obvious chemical equivalent thereof.
37. Tetrapeptide derivatives of the formula (I):
(I) wherein R1 and R2 are methyl, 1-methylethyl, 2-methylpropyl, 1-hydroxyethyl, 2-carboxyethyl or 4-aminobutyl and Y is wherein R3 is methyl, 1-methylethyl, 2-methylpropyl, 1-methylpropyl, hydroxymethyl, 1-hydroxyethyl, carboxy-methyl, 2-carboxyethyl, 4-aminobutyl, 3-guanidinopropyl, benzyl or p-hydroxybenzyl excluding the compounds in which R1 and R2 are each 1-methylethyl and also R3 is carboxymethyl or 2-carboxyethyl, the first and leftmost .beta.-amino acid moiety in said tetrapeptide derivatives having the (2S,3R)-configuration and a primary amino group and the second, third and fourth a-amino acid moieties in said tetrapeptide derivatives having the L-configuration, whenever prepared or produced by the process of Claim 6, 7 or 8, or by an obvious chemical equivalent thereof.
(I) wherein R1 and R2 are methyl, 1-methylethyl, 2-methylpropyl, 1-hydroxyethyl, 2-carboxyethyl or 4-aminobutyl and Y is wherein R3 is methyl, 1-methylethyl, 2-methylpropyl, 1-methylpropyl, hydroxymethyl, 1-hydroxyethyl, carboxy-methyl, 2-carboxyethyl, 4-aminobutyl, 3-guanidinopropyl, benzyl or p-hydroxybenzyl excluding the compounds in which R1 and R2 are each 1-methylethyl and also R3 is carboxymethyl or 2-carboxyethyl, the first and leftmost .beta.-amino acid moiety in said tetrapeptide derivatives having the (2S,3R)-configuration and a primary amino group and the second, third and fourth a-amino acid moieties in said tetrapeptide derivatives having the L-configuration, whenever prepared or produced by the process of Claim 6, 7 or 8, or by an obvious chemical equivalent thereof.
38. A compound of Claim 36 in which R1 is 1-methylethyl and R2 is not 1-methylethyl, whenever prepared or produced by the process of Claim 9, or by an obvious chemical equivalent thereof.
39. A compound of Claim 36 in which R2 is 4-aminobutyl, 1-hydroxyethyl, 2-methylpropyl or 2-carboxyethyl, whenever prepared or produced by the process of Claim 10, or by an obvious chemical equivalent thereof.
40. A compound of Claim 36 in which R3 is carboxymethyl, whenever prepared or produced by the process of Claim 11, or by an obvious chemical equivalent thereof.
41. A compound of Claim 36 in which R2 is 1-methylethyl and R1 is not 1-methylethyl, whenever prepared or produced by the process of Claim 12, or by an obvious chemical equivalent thereof.
42. A compound of Claim 36 in which R1 is 4-aminobutyl, 1-hydroxyethyl, 2-methylpropyl or 2-carboxyethyl, whenever prepared or produced by the process of Claim 13, or by an obvious chemical equivalent thereof.
43. A compound of Claim 36 in which R3 is carboxymethyl, when-ever prepared or produced by the process of Claim 14, or by an obvious chemical equivalent thereof.
44. A compound of Claim 36 in which both R1 and R2 are 1-methyl-ethyl, whenever prepared or produced by the process of Claim 15, or by an obvious chemical equivalent thereof.
45. A compound of Claim 36 in which both R1 and R2 are 1-methyl-ethyl, and R3 is 1-hydroxyethyl, benzyl, 1-methylethyl, 4-amino-butyl or 3-guanidinopropyl, whenever prepared or produced by the process of Claim 16, or by an obvious chemical equivalent thereof.
46. A process for preparing (2S,3R)-3-amino--2-hydroxy-5-methyl-hexanoyl-Val-Leu-Asp, which comprises the steps of:
(a) condensing Boc-Leu and Asp (OBzl)2 Tos OH, wherein Boc is t-butoxycarbonyl, OBzl is benzyloxy and Tos is tosyl, thereby forming Boc-Leu-Asp (OBzl)2;
(b) treating said Boc-Leu-Asp (OBzl)2 with trifluoroacetic acid and then condensing with Boc-Val, thereby forming Boc-Val-Leu-Asp (OBzl)2;
(c) treating said Boc-Val-Leu-Asp (OBzl)2 with trifluoroacetic acid and then condensing with Z-(2S,3R)--3-amino-2-hydroxy-5-methylhexanoic acid, wherein Z is benzyloxycarbonyl, thereby forming Z-(2S,3R)-3-amino-2-hydroxy-5-methylhexanoyl-Val-Leu-Asp-(OBzl)2; and (d) subjecting the Z-(2S,3R)-3-amino-2-hydroxy--5-methylhexanoyl-Val-Leu-Asp(OBzl)2 formed by step (c) to hydrogenolysis to cleave off the protecting groups Z and OBzl and produce (2S,3R)-3-Amino-2-hydroxy-5-methyl-hexanoyl-Val-Leu-Asp.
(a) condensing Boc-Leu and Asp (OBzl)2 Tos OH, wherein Boc is t-butoxycarbonyl, OBzl is benzyloxy and Tos is tosyl, thereby forming Boc-Leu-Asp (OBzl)2;
(b) treating said Boc-Leu-Asp (OBzl)2 with trifluoroacetic acid and then condensing with Boc-Val, thereby forming Boc-Val-Leu-Asp (OBzl)2;
(c) treating said Boc-Val-Leu-Asp (OBzl)2 with trifluoroacetic acid and then condensing with Z-(2S,3R)--3-amino-2-hydroxy-5-methylhexanoic acid, wherein Z is benzyloxycarbonyl, thereby forming Z-(2S,3R)-3-amino-2-hydroxy-5-methylhexanoyl-Val-Leu-Asp-(OBzl)2; and (d) subjecting the Z-(2S,3R)-3-amino-2-hydroxy--5-methylhexanoyl-Val-Leu-Asp(OBzl)2 formed by step (c) to hydrogenolysis to cleave off the protecting groups Z and OBzl and produce (2S,3R)-3-Amino-2-hydroxy-5-methyl-hexanoyl-Val-Leu-Asp.
47. (2S,3R)-3-amino-2-hydroxy-5-methyl-hexanoyl--Val-Leu-Asp, when prepared by the process of claim 46 or by an obvious chemical equivalent thereof.
48. A process for preparing (2S,3R)-3-amino-2-hydroxy-5-methyl-hexanoyl-Val-Glu-Asp, which comprises the steps of:
(a) condensing Boc-Glu(OBzl) with Asp (OBzl)2, wherein Boc is t-butoxycarbonyl and OBzl is benzyloxy, thereby forming Boc-Glu(OBzl)-Asp (OBzl)2;
(b) treating said Boc-Glu(OBzl)-Asp-(OBzl)2 with trifluoroacetic acid and then condensing with Boc-Val, thereby forming Boc-Val-Glu (OBzl)-Asp (OBzl)2;
(c) treating said Boc-Val-Glu (OBzl)-Asp (OBzl)2 with trifluoroacetic acid and then condensing with Z-(2S,3R)-3-amino-2-hydroxy-5-methylhexanoic acid, wherein Z is benzyloxycarbonyl, thereby forming Z-(2S,3R)-3-amino--2-hydroxy-5-methylhexanoyl-Val-Glu(OBzl)-Asp (OBzl)2; and (d) hydrogenolyzing the product of step (c), thereby to cleave off the protecting groups Z and OBzl and produce (2S,3R)-3-amino-2-hydroxy-5-methyl-hexanoyl-Val-Glu-Asp.
(a) condensing Boc-Glu(OBzl) with Asp (OBzl)2, wherein Boc is t-butoxycarbonyl and OBzl is benzyloxy, thereby forming Boc-Glu(OBzl)-Asp (OBzl)2;
(b) treating said Boc-Glu(OBzl)-Asp-(OBzl)2 with trifluoroacetic acid and then condensing with Boc-Val, thereby forming Boc-Val-Glu (OBzl)-Asp (OBzl)2;
(c) treating said Boc-Val-Glu (OBzl)-Asp (OBzl)2 with trifluoroacetic acid and then condensing with Z-(2S,3R)-3-amino-2-hydroxy-5-methylhexanoic acid, wherein Z is benzyloxycarbonyl, thereby forming Z-(2S,3R)-3-amino--2-hydroxy-5-methylhexanoyl-Val-Glu(OBzl)-Asp (OBzl)2; and (d) hydrogenolyzing the product of step (c), thereby to cleave off the protecting groups Z and OBzl and produce (2S,3R)-3-amino-2-hydroxy-5-methyl-hexanoyl-Val-Glu-Asp.
49. (2S, 3R)-3-amino-2-hydroxy-5-methyl-hexanoyl--Val-Glu-Asp, when prepared by the process of claim 48 or by an obvious chemical equivalent thereof.
50. A process for preparing (2S,3R)-3-amino-2--hydroxy-5-methyl-hexanoyl-Thr-Val-Asp, which comprises the steps of:
(a) condensing Boc-Val with Asp (OBzl)2 wherein Boc is t-butoxycarbonyl and OBzl is benzyloxy, thereby forming Boc-Val-Asp(OBzl)2;
(b) treating said Boc-Val-Asp (OBzl)2 with trifluoroacetic acid and then condensing with Boc-Thr (Bzl), thereby forming Boc-Thr (Bzl) - Val-Asp (OBzl)2;
(c) treating said Boc-Thr(Bzl)-Val-Asp (OBzl)2 with trifluoroacetic acid and then condensing with Z-(2S,3R)--3-amino-2-hydroxy-5-methyl hexanoic acid, wherein Z is benzyloxycarbonyl, thereby forming Z-(2S,3R)-3-amino-2-hydroxy-5-methyl-hexanoyl-Thr-(Bzl)-Val-Asp(OBzl)2; and (d) subjecting the product of step (c) to hydrogen-olysis, thereby to cleave off the protecting groups Z,Bzl and OBzl and produce (2s,3R)-3-amino-2-hydxoxy-5-meth--hexanoyl-Thr-Val-Asp.
(a) condensing Boc-Val with Asp (OBzl)2 wherein Boc is t-butoxycarbonyl and OBzl is benzyloxy, thereby forming Boc-Val-Asp(OBzl)2;
(b) treating said Boc-Val-Asp (OBzl)2 with trifluoroacetic acid and then condensing with Boc-Thr (Bzl), thereby forming Boc-Thr (Bzl) - Val-Asp (OBzl)2;
(c) treating said Boc-Thr(Bzl)-Val-Asp (OBzl)2 with trifluoroacetic acid and then condensing with Z-(2S,3R)--3-amino-2-hydroxy-5-methyl hexanoic acid, wherein Z is benzyloxycarbonyl, thereby forming Z-(2S,3R)-3-amino-2-hydroxy-5-methyl-hexanoyl-Thr-(Bzl)-Val-Asp(OBzl)2; and (d) subjecting the product of step (c) to hydrogen-olysis, thereby to cleave off the protecting groups Z,Bzl and OBzl and produce (2s,3R)-3-amino-2-hydxoxy-5-meth--hexanoyl-Thr-Val-Asp.
51. (2S,3R)-3-amino-2-hydroxy-5-methyl-hexanoyl--Thr-Val-Asp, when prepared by the process of claim 50 or by an obvious chemical equivalent thereof.
52. A process for preparing (2S,3R)-3-amino-2-hydroxy-5-methyl-hexanoyl-Leu-Val-Asp, which comprises the steps of:
(a) condensing Boc-Val with Asp (OBzl)2, wherein Boc is t-butoxycarbonyl and OBzl is benzyloxy, thereby forming Boc-Val-Asp (OBzl)2;
(b) treating said Boc-Val-Asp (OBzl)2 with trifluoroacetic acid and then condensing with Boc-Leu, thereby forming Boc-Leu-Val-Asp (OBzl)2;
(c) treating said Boc-Leu-Val-Asp (OBzl)2 with trifluoroacetic acid and then condensing with Z-(2S,3R)--3-amino-2-hydroxy-5-methylhexanoic acid, wherein Z is benzyloxycarbonyl, thereby forming Z-(2S,3R)-3-amino-2--hydroxy-5-methyl-hexanoyl(Z)-Leu-Val-Asp(OBzl)2; and (d) subjecting the product of step (c) to hydrogenolysis, thereby to cleave off the protecting groups Z and OBzl and produce (2S,3R)-3-amino-2-hydroxy-5--methyl-hexanoyl-Leu-Val-Asp.
(a) condensing Boc-Val with Asp (OBzl)2, wherein Boc is t-butoxycarbonyl and OBzl is benzyloxy, thereby forming Boc-Val-Asp (OBzl)2;
(b) treating said Boc-Val-Asp (OBzl)2 with trifluoroacetic acid and then condensing with Boc-Leu, thereby forming Boc-Leu-Val-Asp (OBzl)2;
(c) treating said Boc-Leu-Val-Asp (OBzl)2 with trifluoroacetic acid and then condensing with Z-(2S,3R)--3-amino-2-hydroxy-5-methylhexanoic acid, wherein Z is benzyloxycarbonyl, thereby forming Z-(2S,3R)-3-amino-2--hydroxy-5-methyl-hexanoyl(Z)-Leu-Val-Asp(OBzl)2; and (d) subjecting the product of step (c) to hydrogenolysis, thereby to cleave off the protecting groups Z and OBzl and produce (2S,3R)-3-amino-2-hydroxy-5--methyl-hexanoyl-Leu-Val-Asp.
53. (2S,3R)-3-amino-2-hydroxy-5-methyl-hexanoyl--Leu-Val-Asp, when prepared by the process of claim 52 or by an obvious chemical equivalent thereof.
54. A process for preparing (2S,3R3-3-amino--2-hydroxy-5-methyl-hexanoyl-Val-Thr-Phe, which comprises the steps of:
(a) condensing Boc-Thr (OBzl) with Phe (OBzl), Tos OH, wherein Boc is t-butoxycarbonyl, OBzl is benzyloxy, and Tos is tosyl, thereby forming Boc-Thr (OBzl) - Phe (OBzl);
(b) treating said Boc-Thr (Obzl) - Phe (OBzl) with trifluoroacetic acid and then condensing with Boc-Val, thereby forming Boc-Val-Thr (OBZl) -Phe (OBzl);
(c) treating said Boc-Val-Thr(OBzl)-Phe(OBzl) with trifluoroacetic acid and then condensing with Z-(2S,3R)--3-amino-2-hydroxy-5-methyl-hexanoic acid (wherein Z. is benzyloxycarbonyl), thereby forming Z-(2S,3R)-3-amino-2--hydroxy-5-methyl-hexanoyl-Val-Thr (OBzl)-Phe(OBzl); and (d) subjecting the product of step (c) to hydrogenolysis, thereby to cleave off the protecting groups Z and OBzl and produce (2S,3R)-3-amino-2-hydroxy-5-methyl--hexanoyl-Val-Thr-Phe.
(a) condensing Boc-Thr (OBzl) with Phe (OBzl), Tos OH, wherein Boc is t-butoxycarbonyl, OBzl is benzyloxy, and Tos is tosyl, thereby forming Boc-Thr (OBzl) - Phe (OBzl);
(b) treating said Boc-Thr (Obzl) - Phe (OBzl) with trifluoroacetic acid and then condensing with Boc-Val, thereby forming Boc-Val-Thr (OBZl) -Phe (OBzl);
(c) treating said Boc-Val-Thr(OBzl)-Phe(OBzl) with trifluoroacetic acid and then condensing with Z-(2S,3R)--3-amino-2-hydroxy-5-methyl-hexanoic acid (wherein Z. is benzyloxycarbonyl), thereby forming Z-(2S,3R)-3-amino-2--hydroxy-5-methyl-hexanoyl-Val-Thr (OBzl)-Phe(OBzl); and (d) subjecting the product of step (c) to hydrogenolysis, thereby to cleave off the protecting groups Z and OBzl and produce (2S,3R)-3-amino-2-hydroxy-5-methyl--hexanoyl-Val-Thr-Phe.
55. (2S,3R)-3-amino-2-hydroxy-5-methyl-hexanoyl--Val-Thr-Phe, when prepared by the process of claim 54 or by an obvious chemical equivalent thereof.
56. A process for preparing (2S,3R)-3-amino-2--hydroxy-5-methyl-hexanoyl-Lys-Val-Val, which comprises the steps of:
(a) condensing Boc-Val with Val-OBzl. TosOH
(wherein Boc is t-butoxycarbonyl, OBzl is benzyloxy and Tos is tozyl), thereby forming Boc-Val-Val(OBzl);
(b) treating said Boc-Val-Val(OBzl) with trifluoro-acetic acid and then condensing with Boc-Lys(Z) (wherein Z. is benzyloxycarbonyl), thereby forming Boc-Lys(Z)-Val-Val(OBzl);
(c) treating said Boc-Lys(Z)-Val-Val(OBzl) with trifluoroacetic acid and then condensing with Z-(2S,3R)-3--amino-2-hydroxy-5-methyl-hexanoic acid, thereby forming Z-(2S,3R)-3-amino-2-hydroxy-5-methyl-hexanoyl-Lys(Z)-Val-Val (OBzl); and (d) subjecting the product of step (c) to hydrogenolysis, thereby to cleave off the protecting groups Z and OBzl and produce (2S,3R)-3-amino-2-hydroxy-5--methyl-hexanoyl-Lys-Val-Val.
(a) condensing Boc-Val with Val-OBzl. TosOH
(wherein Boc is t-butoxycarbonyl, OBzl is benzyloxy and Tos is tozyl), thereby forming Boc-Val-Val(OBzl);
(b) treating said Boc-Val-Val(OBzl) with trifluoro-acetic acid and then condensing with Boc-Lys(Z) (wherein Z. is benzyloxycarbonyl), thereby forming Boc-Lys(Z)-Val-Val(OBzl);
(c) treating said Boc-Lys(Z)-Val-Val(OBzl) with trifluoroacetic acid and then condensing with Z-(2S,3R)-3--amino-2-hydroxy-5-methyl-hexanoic acid, thereby forming Z-(2S,3R)-3-amino-2-hydroxy-5-methyl-hexanoyl-Lys(Z)-Val-Val (OBzl); and (d) subjecting the product of step (c) to hydrogenolysis, thereby to cleave off the protecting groups Z and OBzl and produce (2S,3R)-3-amino-2-hydroxy-5--methyl-hexanoyl-Lys-Val-Val.
57. (2S,3R)-3-amino-2-hydroxy-5-methyl-hexannyl-Lys-Val-Val, when prepared by the process of claim 56 or by an obvious chemical equivalent thereof.
58. A process for preparing (2S,3R)-3-amino--2-hydroxy-5-methyl-hexanoyl-Val-Val-Pro, which comprises the steps of:
(a) condensing Boc-Val with Val(OBzl). TosOH
(wherein Boc is t-butoxycarbonyl, OBzl is benzyloxy and Tos is tosyl), thereby forming Boc-Val-Val-(OBzl);
(b) subjecting said Boc-Val-Val-OBzl) to catalytic reduction, thereby forming Boc-Val-Val;
(c) condensing the Boc-Val-Val so formed with Pro(OBzl).HCl, thereby forming Boc-Val-Val-Pro(OBzl);
(d) treating said Boc-Val-Val-Pro(OBzl) with trifluoroacetic acid and then condensing with Z-(2S,3R)-3-amino-2-hydroxy-5-methylhexanoic acid (wherein Z is benzyloxycarbonyl), thereby forming Z-(2S,3R)-3-amino-2--hydroxy-5-methyl-hexanoyl-Val-Val-Pro(OBzl); and (e) subjecting the product of step (d) to hydrogenolysis, thereby cleaving off the protecting groups Z and OBzl and producing (2S,3R)-3-amino-2-hydroxy-5-methyl-hexanoyl-Val-Val-Pro.
(a) condensing Boc-Val with Val(OBzl). TosOH
(wherein Boc is t-butoxycarbonyl, OBzl is benzyloxy and Tos is tosyl), thereby forming Boc-Val-Val-(OBzl);
(b) subjecting said Boc-Val-Val-OBzl) to catalytic reduction, thereby forming Boc-Val-Val;
(c) condensing the Boc-Val-Val so formed with Pro(OBzl).HCl, thereby forming Boc-Val-Val-Pro(OBzl);
(d) treating said Boc-Val-Val-Pro(OBzl) with trifluoroacetic acid and then condensing with Z-(2S,3R)-3-amino-2-hydroxy-5-methylhexanoic acid (wherein Z is benzyloxycarbonyl), thereby forming Z-(2S,3R)-3-amino-2--hydroxy-5-methyl-hexanoyl-Val-Val-Pro(OBzl); and (e) subjecting the product of step (d) to hydrogenolysis, thereby cleaving off the protecting groups Z and OBzl and producing (2S,3R)-3-amino-2-hydroxy-5-methyl-hexanoyl-Val-Val-Pro.
59. (2S,3R)-3-amino-2-hydroxy-5-methyl-hexanoyl--Val-Val-Pro, when prepared by the process of claim 58 or by an obvious chemical equivalent thereof.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA000355887A CA1182110A (en) | 1980-07-10 | 1980-07-10 | Physiologically active tetrapeptides |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA000355887A CA1182110A (en) | 1980-07-10 | 1980-07-10 | Physiologically active tetrapeptides |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1182110A true CA1182110A (en) | 1985-02-05 |
Family
ID=4117389
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000355887A Expired CA1182110A (en) | 1980-07-10 | 1980-07-10 | Physiologically active tetrapeptides |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA1182110A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0498680A1 (en) * | 1991-02-08 | 1992-08-12 | Sankyo Company Limited | New beta-amino-alpha-hydroxycarboxylic acids and their use |
-
1980
- 1980-07-10 CA CA000355887A patent/CA1182110A/en not_active Expired
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0498680A1 (en) * | 1991-02-08 | 1992-08-12 | Sankyo Company Limited | New beta-amino-alpha-hydroxycarboxylic acids and their use |
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