Process for producing amides using catalytic amounts of an N-hydroxy compound

A process is disclosed for the manufacture of amides, including peptides, in which a carboxylic acid is reacted with a primary or secondary amine in the presence of a carbodiimide and a catalytic amount of N-hydroxy compound.

BACKGROUND OF THE INVENTION 
It is known that amides, including peptides, may be produced by reacting a 
carboxylic acid with a primary or secondary amine in the presence of 
dicyclohexylcarbodiimide (DCC) and a N-hydroxy compound. Zeitschrift fur 
Naturforschung (B), 426 (1966) discloses that peptide formation from 
N-acylpeptides, which contain an optically active amino acid having a 
carboxyl terminal, proceeds practically free from racemization when the 
peptide formation is carried out using DCC with the addition of 2 mol 
equivalents of N-hydroxysuccinimide in tetrahydrofuran or 
dimethylformamide at -20.degree. C. From Chemische Berichte 103 (1970), 
pages 788-798, 2024-2033, 2034-2040, it is known that the effect described 
above can also be observed when 1-2 mol equivalents of various 
1-hydroxybenzotriazoles, 1-hydroxy-2-oxoindolines, 
3-hydroxy-4-oxo-3,4-dihydroquinazolines and 
3-hydroxy-4-oxo-3,4-dihydro-1,2,3-benzotriazines are used in place of said 
2 mol equivalents of N-hydroxysuccinimide. It is also known that certain 
N-hydroxy compounds may suppress the formation of byproducts in DCC 
activation. According to the Journal of Organic Chemistry 37, 288 (1972), 
1 mol equivalent of N-hydroxysuccinimide was successful in suppressing the 
formation of N-acylureas (byproducts in DCC activation) in the reaction of 
one mol equivalent of 
1-(9-adenyl)-2,3-O-isopropylidene-.beta.-D-ribofuranuronic acid with 
benzyl esters of various amino acids and peptides. Chemische Berichte 106, 
3626 (1973) discloses that the aminolysis of phenyl esters of amino acids 
and peptides substituted with electron-withdrawing groups is accelerated 
greatly by those N-hydroxy compounds, which have about the acidity of 
acetic acid, in polar solvents, especially with the use of 1 mol 
equivalent of 1-hydroxybenzotriazole, 1-hydroxy-2(1H)-pyridone and 
3-hydroxy-4-oxo-3,4-dihydroquinazoline. According to Journal of the 
American Chemical Society 94, 3590 (1972), 1.1 mol equivalent of 
N-hydroxysuccinimide is used for the preparation of activated 
N-hydroxysuccinimide esters. 
SUMMARY OF THE INVENTION 
The present invention is a novel process for the production of amides in 
high yields and practically free of racemization. The process in 
accordance with the invention comprises reacting a carboxylic acid with a 
primary or secondary amine in the presence of a carbodiimide dehydrating 
agent and a catalytic amount of an N-hydroxy compound. 
It has been found that when only catalytic amounts of an N-hydroxy compound 
is used in the reaction of a carboxylic acid with a primary or secondary 
amine and a carbodiimide dehydrating agent, amides can be produced with 
considerable reduction in reaction byproducts. Furthermore, such reaction 
yields amides in high yields and practically without racemization.

DETAILED DESCRIPTION OF THE INVENTION 
The present invention is a novel process for the production of amides, 
including peptides, by reacting a carboxylic acid with a primary or 
secondary amine in the presence of a carbodiimide dehydrating agent and a 
catalytic amount of an N-hydroxy compound. 
As used throughout this specification, "alkyl"--alone or in 
combination--refers to any straight or branched-chain alkyl group having 
from 1 to 15 carbon atoms. "Lower alkyl" refers to those straight or 
branched-chain alkyl groups having from 1 to 7 carbon atoms. As used 
throughout this specification, "heteroaryl" or "heteroaromatic" refers to 
any aryl or aromatic ring structure containing at least one heteroatom in 
this ring structure, which heteroatom is selected from the group 
consisting of nitrogen, sulfur and oxygen. As used through this 
specification, "peptide" refers to any amide formed from two or more amino 
acids, such that a --NCO-- functional group is formed by the interaction 
between an amino group of one amino acid and a carboxyl group of another 
amino acid. As used herein, the term "peptide" includes polypeptides. 
This invention provides for the production of amides, including peptides, 
by the interaction of any carboxylic acid and any primary or secondary 
amine in the presence of an organic N-hydroxy compound and a carbodiimide 
dehydrating agent. The novelty of this invention resides in the fact that 
only catalytic amounts of an organic N-hydroxy compound are needed to 
facilitate the interaction between the carboxylic acid and the primary or 
secondary amine, and that in using catalytic amounts of such N-hydroxy 
compound, amides are produced in high yield practically free of 
racemization. 
The "carboxylic acids" used in accordance with this invention may be any 
carboxylic acid and include aliphatic, aromatic, aromatic-aliphatic, 
heteroaromatic, heteroaromatic-aliphatic carboxylic acids, which 
carboxylic acids may be substituted or unsubstituted. Other "carboxylic 
acids" which can be utilized include natural N-acylated .alpha.-amino 
acids having the L configuration, homologues of such natural amino acids, 
and epimers of such amino acids with the D-configuration, which amino 
acids, homologues and epimers thereof may be substituted or unsubstituted. 
A "homologue" of a natural N-acylated .alpha.-amino acid refers to such 
amino acids wherein the side-chain of the amino acid is lengthened or 
shortened by one or two methylene groups, or in which a methyl group of 
the side chain is replaced by hydrogen. 
Any natural N-acylated .alpha.-amino acid, or homologue or epimer thereof, 
may be used as the carboxylic acid to react with amines to produce amides. 
Examples of suitable amino acids or homologues or epimers thereof, include 
glycine, alanine, valine, norvaline, leucine, isoleucine, norleucine, 
serine, homoserine, threonine, methionine, cysteine, proline, 
trans-3-hydroxyproline, trans-4-hydroxyproline, phenylalanine, 
4-aminophenylalanine, 4-chlorophenylalanine, 4-nitrophenylalanine, 
tyrosine, tryptophan, cyclohexylalanine, cyclohexylglycine, 
indoline-2-carboxylic acid, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic 
acid, aspartic acid, asparagine, histidine, arginine, lysine, 
.alpha.,.beta.-diaminopropionic acid, .alpha.,.gamma.-diaminobutyric acid, 
ornithine, .delta.-hydroxylysine, glutamic acid mono-t-butyl ester, 
glutamine, N-dimethylglutamine, glutamic acid, aminomalonic acid, 
aminomalonic acid monoamide, N-pivaloylornithine, and 
N-t-butoxycarbonyllysine. 
The N-acylated .alpha.-amino acids also include any N-acylated 
.alpha.-amino acid, or homologue or epimer thereof, wherein the carboxy 
group in the amino acid side-chain is esterified, e.g. as an alkyl ester, 
or amidated as a carbamoyl, alkylcarbamoyl, or dialkylcarbamoyl. Examples 
of such alkyl esters are methoxycarbonyl and t-butoxycarbonyl. When 
t-butoxycarbonyl is the alkyl moiety in the alkyl ester, N-acylated 
glutamic acid mono-t-butyl ester is an example of such carboxylic acid. 
Acylated aminomalonic acid monoamide is an example of a carbamoylated, 
N-acylated .alpha.-amino acid. The preferred alkylcarbamoyl and 
dialkylcarbamoyl groups are methylcarbamoyl and dimethylcarbamoyl, 
respectively. 
Suitable N-acyl amino groups in the N-acylated .alpha.-amino acids or 
homologues or epimers thereof include alkanoylamino, alkoxycarbonylamino, 
or arylalkoxycarbonylamino. When alkanoylamino, acetylamino or 
pivaloylamino are enumerated. When alkoxycarbonylamino, 
t-butoxycarbonylamino is preferred. The preferred arylalkoxycarbonylamino 
is benzyloxycarbonylamino. 
In addition, any N-acylated .alpha.-amino acid, or homologue or epimer 
thereof, wherein the hydroxy group in the side chain is present in 
etherified or esterified form, as an alkoxy group such as methoxy, as an 
arylalkoxy group, such as benzyloxy, or as a lower alkanoyloxy group, such 
as acetoxy, may also be used as a carboxylic acid in accordance with this 
invention. 
The amino acid side-chain of the above-mentioned N-acylated .alpha.-amino 
acids, or homologues or epimers thereof, may be any amino acid side-chain. 
Thus, the amino acid side chain can be arylalkyl, heteroarylalkyl, 
hydrogenated arylalkyl, hydrogenated heteroarylalkyl or alkyl, all of them 
may be substituted or unsubstituted. 
When the side-chain is arylalkyl, the aryl residue may be unsubstituted or 
substituted. 
As an example of an unsubstituted aryl, phenyl is enumerated, and the 
corresponding preferred amino acids are phenylglycine, phenylalanine and 
phenylserine. The aryl residue may be substituted with one or more of the 
following groups: alkyl, halogen, hydroxy, alkoxy, alkanoyloxy, amino, 
alkylamino, dialkylamino, alkanoylamino, alkoxycarbonylamino, 
arylmethoxycarbonylamino and/or nitro. 
When the substitution group is alkyl, methyl is preferred. When the 
substitution group is halogen, any halogen may be used, such as fluorine, 
chlorine, bromine or iodine, and the corresponding amino acid is e.g. 
4-cholorophenylalanine. Methoxy is the preferred alkoxy group, and acetoxy 
is the preferred alkanoyloxy group. When the substitution group is 
alkylamino, methylamino may be enumerated. When amino is the substitution 
group, 4-aminophenylalanine is the preferred amino acid. When the 
substitution group is dialkylamino, dimethylamino is enumerated. The 
preferred alkanoylamino groups are acetylamino and pivaloylamino. The 
preferred alkoxycarbonylamino group is t-butoxycarbonylamino. When the 
substitution group is arylmethoxycarbonylamino, benzyloxy-carbonylamino 
and 9-fluorenylmethoxycarbonylamino are enumerated. 4-Nitrophenylalanine 
is the preferred amino acid when nitro is the substitution group. 
N-acylated .alpha.-amino acids also include N-acylated benz-fused 
phenylalanine or phenylglycine, such as a-naphthylalanine, or a 5- or 
6-membered cyclic benz-fused N-acylated .alpha.-amino acid, e.g. 
indoline-2-carboxylic acid or 1,2,3,4-tetrahydroisoquinoline-3-carboxylic 
acid. 
When the amino acid side-chain is a hydrogenated arylalkyl, cyclohexylalkyl 
is the preferred group, such as in cyclohexylalanine or cyclohexylglycine. 
The N-acyl groups of the N-acyl .alpha.-amino acids, or homologues or 
epimers thereof, may be alkanoyl, alkoxycarbonyl, arylalkoxycarbonyl, an 
acyl residue of an aromatic-aliphatic or heteroaromatic carboxylic acid 
referred to above, an amino acid referred to above, including homologues 
or epimers thereof; or a dipeptide consisting of two of the aforementioned 
amino acids, including homologues and epimers thereof. 
The carboxylic acids used in this invention to react with an amine to 
produce amides may be aliphatic, which aliphatic group may be substituted 
or unsubstituted. Examples of unsubstituted aliphatic acids are propionic 
acid and isobutyric acid. Examples of a suitable substituted aliphatic 
carboxylic acid include 2-phthalimidoisobutyric acid, (R)-lactic acid and 
(S)-lactic acid. 
When the N-acyl group is alkanoyl, acetyl or pivaloyl, e.g. 
N-pivaloyl-ornithine, is preferred. When alkoxycarbonyl, t-butoxycarbonyl 
may be enumerated. The preferred arylmethoxy-carbonyl is 
benzyloxy-carbonyl, and N-(benzyloxycarbonyl)-L-asparagine and 
N-(benzyloxycarbonyl)-L-tyrosine are examples of corresponding N-acylated 
amino acids. As examples of acyl residues of aromatic-aliphatic carboxylic 
acids, (S)-.alpha.-(t-butylsulphonyl)-1-methyl!-.beta.-phenylpropionyl 
and (S)-.alpha.-1-(morpholinocarbonyl)-1-methylethyl!sulphonyl!methyl!- 
.beta.-phenylpropionyl are enumerated. An example of an acyl residue of a 
heteroaromatic carboxylic acid includes 2-quinolylcarbonyl. When the acyl 
residue is 2-quinolylcarbonyl, then N-(2-quinolylcarbonyl)-L-asparagine is 
the preferred N-acylated .alpha.-amino acid. 
The carboxylic acid may also be substituted or unsubstituted aromatic. A 
suitable unsubstituted aromatic carboxylic acid is benzoic acid. Examples 
of a suitable substituted aromatic carboxylic acid includes 
2-phthalimidoxyisobutyric acid, 3,4-dihydroxybenzoic acid, salicylic acid, 
1-naphthoic acid, and 2-naphthoic acid. 
The carboxylic acid used in accordance with this invention may also be 
aromatic-aliphatic, which aromatic-aliphatic group may be substituted or 
unsubstituted. The preferred unsubstituted aromatic-aliphatic carboxylic 
acid is phenylacetic acid. The preferred substituted aromatic-aliphatic 
carboxylic acid includes p-hydroxyphenylacetic acid, 
(S)-.alpha.-(t-butylsulphonyl)-methyl!hydrocinnamic acid, and 
(S)-.alpha.-1-(morpholinocarbonyl)-1-methylethyl!sulphonyl!-methyl!hydr 
ocinnamic acid. 
Among the carboxylic acids used to react with an amine to produce amides 
include heteroaromatic carboxylic acids, such as 2-pyridinecarboxylic 
acid, 3-pyridine carboxylic acid, 4-pyridinecarboxylic acid, 
2-pyrimidinecarboxylic acid, 4-pyrimidinecarboxylic acid, 
5-chloro-2-pyridine carboxylic acid, 2-quinoline carboxylic acid, 
3-quinoline carboxylic acid and isoquinoline-1-carboxylic acid. 
When the carboxylic acid is heteroaromatic-aliphatic, 
(S)-1-(t-butyoxycarbonyl)-.alpha.-(S)-.alpha.-(t-butyl-sulphonyl)methyl! 
hydrocinnamamido!imidazole-4-propionic acid, and 
(S)-(t-butoxycarbonyl)-.alpha.-1-(morpholinocarbonyl)-1-methyl-ethyl!su 
lphonyl!-methyl!hydrocinnamamido!imidazole-4-propionic acid, 
2-pyridylacetic acid, 3-indolylacetic acid, 3-(3-indolyl)propionic acid, 
and (4-imidazolyl)acetic acid are preferred. 
Any of the carboxylic acids enumerated above can be reacted with any 
primary or secondary amine in the presence of an N-hydroxy compound and a 
carbodiimide dehydrating agent to produce amides, in accordance with this 
invention. Any primary or secondary amine may be used in this invention. 
The primary or secondary amine to be reacted with a carboxylic acid to 
produce an amide may include any amino acid enumerated above, including 
any homologue or epimer thereof, wherein there is at least one 
non-acylated amino group, and the acid group is protected. Any means of 
protecting the acid group may be used, such as by esterification. 
Among the primary or secondary amines to be used in this invention include 
methyl esters of the amino acids enumerated above, such as histidine 
methyl ester, as well as t-butyl (2-aminoethyl)-carbamate, 
(1S,2R,3S)-3-amino-2(R)-hydroxy-4-phenylbutyl)-N-t-butyl-decahydro-(4aS,8a 
S)-isoquinoline-3(S)-carboxamide, 
(1S,2R,3S)-3-amino-4-cyclohexyl-1-cyclopropyl-butene-1,2 diol and 
2(3(S)-amine-2(R)-hydroxy-4-phenylbutyl)-N-t-butyl-decahydro-(4aS,8aS)-iso 
quinoline-3(S)-carboxamide, and t-butyl(4-piperidinyloxy)acetate. 
In addition, primary or secondary amines to be utilized in the production 
of amides include alkylamines, dialkylamines, aralkylamines or C.sub.3-6 
-alkylene-disubstituted amines, or hydrazine, which hydrazine may be 
substituted or unsubstituted. The hydrazine may be substituted with alkyl, 
such as methylhydrazine, or acyl, such as 3,4-dihydroxybenzoic acid 
hydrazide. 
Any primary or secondary amine which can be used to react with a carboxylic 
acid to produce amides may contain any other groups which are inert under 
the reaction conditions. These primary or secondary amines may be 
optionally interrupted by an oxygen, sulphur, or a substituted nitrogen 
atom. The nitrogen atom may be substituted with an alkyl, phenylalkyl, 
alkanoyl or alkanoyloxy. 
As set forth above, any carboxylic acid can be reacted with any amine in 
the presence of a carbodiimide dehydrating agent and an organic N-hydroxy 
compound to produce amides. Carbodiimides are known in the art to function 
as dehydrating agents in organic reactions. Any conventional carbodiimide 
dehydrating agent may be used. 
Examples of preferable carbodiimides dehydrating agents are 
dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), 
N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), 
N-cyclohexyl-N'-(.beta.-N-methylmorpholino!ethyl)carbodiimide 
p-toluenesulphonate and the like. 
In carrying out this reaction of a carboxylic acid with an amine to produce 
an amide, the amount of carbodiimide dehydrating agent is not critical as 
long as said carbodiimide dehydrating agent is present in sufficient 
amounts to facilitate such reaction by dehydration to yield an amide. It 
is generally preferred when said carbodiimide dehydrating agent is present 
in molar amounts equivalent to the molar amount of carboxylic acid present 
in the reaction mixture. 
The novelty of this invention directed to a process for producing amides 
from the reaction of a carboxylic acid with primary or secondary amines, 
in the presence of a carbodiimide dehydrating agent and an N-hydroxy 
compound, resides in the fact that the reaction proceeds in the presence 
of only catalytic amounts of an N-hydroxy compound to produce amides in 
high yield practically free of racemization. 
An N-hydroxy compound is any conventional organic compound having a 
&gt;N--OH-- functional group. Any conventional N-hydroxy compound may be used 
in this invention, such as N-hydroxysuccinimide, 1-hydroxybenzotriazole, 
3-hydroxy-4-oxo-3,4-dihydro-1,2,3-benzotriazine, 1-hydroxy-2-oxoindoline, 
3-hydroxy-4-oxo-3,4-dihydroquinazoline, 1-hydroxy-2(1H)-pyridone and the 
like. 
The organic N-hydroxy compound must be present in sufficient amounts to 
catalyze the reaction. Preferably, the N-hydroxy compound is present in a 
molar equivalent amount of not less than 1% and not more than 60% of the 
molar equivalent of carboxylic acid present in the reaction mixture. Best 
results are obtained when the N-hydroxy compound is present in the amount 
of 3-25% of the molar amount of carboxylic acid. 
The reaction of a carboxylic acid with a primary or secondary amine is 
carried out in a manner known per se, conveniently in an organic solvent 
or solvent mixture which is inert under the reaction conditions. Any 
conventional organic solvent may be used. Among the preferred solvents 
include such as ethyl acetate, acetone, acetonitrile, dimethylformamide, 
methylene chloride, tetrahydrofuran and the like. 
The temperature is not critical; however a temperature between about 
0.degree. C. and 50.degree. C. is preferred. Best results are obtained 
when the temperature is room temperature. 
Solid phase synthesis methods can also be used to react a carboxylic acid 
with an amine to produce amides in accordance with this invention, when 
the primary of secondary amine contains a carboxyl group. Any solid phase 
synthesis method may be used. These solid phase synthesis methods are 
especially suitable for the manufacture of peptides, in which case the 
amide coupling is carried out several times in succession. When solid 
phase synthesis methods are used, it is preferable to use as the 
N-acylated .alpha.-amino acids described above, which are protected at the 
amino group by t-butoxycarbonyl or 9-fluorenylmethoxycarbonyl. Suitable 
carriers are polystyrene or polyamide resins, such as p-benzyloxy-benzyl 
alcohol-polystyrene resin, p-hydroxymethyl-phenoxy-polystyrene resin, 
4-(2',4'-dimethoxyphenyl-hydroxymethyl)-phenoxy-polystyrene resin, 
dimethylacrylamide-polyamide resin, glycylacrylamide-polyamide resin, 
siliceous earth/polyamide resin and the like. The desired peptide can be 
cleaved off readily from the carrier resin, for example using 
trifluoroacetic acid and the like. 
In the following Examples, which illustrate the present invention but which 
are not intended to limit its scope in any manner, all temperatures are 
given in degrees Celsius. 
EXAMPLE 1 
17.9 g (74 mmol) of histidine methyl ester dihydrochloride, 70 ml of 
acetonitrile and 20.6 ml (148 mmol) of triethylamine were stirred at 
20.degree. for 2 hours. 20 g (70 mmol) of 
(S)-.alpha.-(t-butylsulphonyl)methyl!hydrocinnamic acid, 0.78 g (7 mmol) 
of 1-hydroxy-2(1H)-pyridone and 140 ml of ethyl acetate were subsequently 
added. After 15 minutes a solution of 15.2 g (74 mmol) of 
dicyclohexylcarbodiimide in 90 ml of ethyl acetate was added within 30 
minutes. The reaction had finished after stirring at 20.degree. for 18 
hours. The solid (dicyclohexylurea) was filtered off under suction and the 
filtrate was washed with aqueous sodium bicarbonate solution and water. 
32.3 g of methyl 
(S)-.alpha.(t-butyl-sulphonyl)methyl!hydrocinnamamido!imidazolepropionate 
were obtained as a white foam (HPLC content 94%; content-corrected yield: 
98%). 
EXAMPLE 2 
2.5 g (10.5 mmol) of histidine methyl ester dihydrochloride, 10 ml of 
acetonitrile and 2.9 ml (148 mmol) of triethylamine were stirred at 200 
for 20 hours. 3.8 g (10 mmol) of 
(S)-.alpha.1-(morpholinocarbonyl)-1-methylethyl!sulphonyl!methyl!hydroc 
innamic acid, 0.11 g (1 mmol) of 1-hydroxy-2(1H)-pyridone and 20 ml of 
ethyl acetate were subsequently added. After 15 minutes a solution of 2.2 
g (10.5 mmol) of dicyclohexylcarbodiimide in 15 ml of ethyl acetate was 
added within 30 minutes. The reaction had finished after stirring at 
20.degree. for 18 hours. The solid (dicyclohexylurea) was filtered off 
under suction and the filtrate was washed with aqueous sodium bicarbonate 
solution and water. 5.4 g of 
N-(S)-.alpha.-1-methyl-1-(morpholinocarbonyl)ethyl!sulphonyl!methyl!hy 
drocinnamoyl!-L-histidine methyl ester were obtained. 
EXAMPLE 3 
89 g (170 mmol) of 
(S)-1-(t-butoxycarbonyl)-.alpha.-(S)-.alpha.-(t-butylsulphonyl)methyl!hy 
drocinnamamido!imidazole-4-propionic acid, 1 g (8.5 mmol) of 
N-hydroxysuccinimide, 35.5 g (155 mmol) of 
(1S,2R,3S)-3-amino-4-cyclohexyl-1-cyclopropyl-butane-1,2-diol and 800 ml 
of ethyl acetate were stirred at 20.degree.. A solution of 37 g (178 mmol) 
of dicyclohexylcarbodiimide in 110 ml of ethyl acetate was added within 10 
minutes. The reaction had finished after stirring at 20.degree. for 17 
hours. The solid (dicyclohexylurea) was filtered off and the filtrate was 
treated while stirring firstly with 68 ml of deionized water and then with 
1100 ml of hexane. After one hour at 0.degree. the crystallizate was 
filtered off under suction and triturated in methanol at 20.degree.. The 
suspension was cooled to -15.degree. and the product was filtered off. 
98.8 g (86%) of t-butyl 
4-(S)-2-(S)-2-t-butanesulphonylmethyl-3-phenylpropionylamino!-2-(1S,2R, 
3S)-1-cyclohexylmethyl-3-cyclopropyl-2,3-dihydroxy-propylcarbamoyl!ethyl!-1 
H-imidazole-1-carboxylate were obtained. 
EXAMPLE 4 
11.7 g (44 mmol) of N-(benzyloxycarbonyl)-L-asparagine, 16.0 g (40 mmol) of 
2-3(S)-amino-2(R)-hydroxy-4-phenylbutyl!-N-t-butyl-decahydro-(4aS,8aS)-is 
oquinoline-3(S)-carboxamide and 0.46 g (4 mmol) of N-hydroxysuccinimide 
were suspended in 160 ml of tetrahydrofuran and 80 ml of ethyl acetate at 
20.degree.. A solution of 9.1 g (44 mmol) of dicyclohexylcarbodiimide and 
80 ml of ethyl acetate was added dropwise within 15 minutes and the 
reaction mixture was stirred at 20.degree.. The reaction had finished 
after 18 hours. The solid (dicyclohexylurea) was filtered off under 
suction. The filtrate was freed from tetrahydrofuran and the product was 
crystallized from ethyl acetate/hexane. The yield of 
cis-2-3(S)-N-(benzyloxycarbonyl)-L-asparaginyl!aminol-2(R)-hydroxy-4-ph 
enylbutyl!-N-t-butyldecahydro-(4aS,8aS)-isoquinoline-3(S)-carboxamide was 
23.0 g (88%). 
EXAMPLE 5 
10.2 g (30 mmol) of N-(benzyloxycarbonyl)-L-tyrosine dihydrate, 6.48 g (30 
mmol) of t-butyl (4-piperidinyloxy)acetate and 0.17 g (1.5 mmol) of 
N-hydroxysuccinimide were dissolved in 200 ml of ethyl acetate while 
stirring under argon. 6.5 g (31.5 mmol) of dicyclohexylcarbodiimide 
dissolved in 33 ml of ethyl acetate were added dropwise to the yellowish 
solution within 19 minutes. A suspension formed slowly and this was 
stirred at 22.degree. for 22 hours. The precipitated dicyclohexylurea was 
filtered off and the filtrate was extracted with 1N hydrochloric acid, 
water and sodium chloride solution. The organic phase was dried over 
sodium sulphate and, after removing the drying agent, evaporated. 13.1 g 
of t-butyl 
1-N-(benzyloxy)carbonyl!-L-tyrosyl!-4-piperidinyl!oxy!acetate were 
obtained as a white, hard foam, the microanalysis of which corresponded to 
that of authentic material. 
EXAMPLE 6 
11.14 g (30 mmol) of N-(benzyloxycarbonyl)-O-(1,1-dimethylethyl)-L-tyrosine 
were dissolved in 65 ml of ethyl acetate while warming to 40.degree.. The 
solution was treated at 20.degree. with 0.1 g (0.9 mmol) of 
N-hydroxysuccinimide. Subsequently, a solution of 7.5 g (36 mmol) of 
dicyclohexylcarbodiimide in 40 ml of ethyl acetate was added dropwise at 
22.degree. within 25 minutes. A white suspension formed during the 
addition and this was stirred for 30 minutes. A solution of 7.8 g (36 
mmol) of t-butyl (4-piperidinyloxy)acetate in 100 ml of ethyl acetate was 
then added dropwise to the suspension within 30 minutes. A suspension 
formed and this was stirred at 22.degree. for 3 hours. The precipitated 
dicyclohexylurea was filtered off and the filtrate was washed with 2N 
hydrochloric acid, water and semi-saturated sodium bicarbonate solution. 
The organic phase was dried over sodium sulphate and, after removing the 
drying agent, evaporated. The residue was a viscous oil and weighed 18.5 
g. The oil was diluted with a mixture of 10 ml of ethyl acetate and 60 ml 
of hexane and warmed to 50.degree.. The insoluble solid was filtered off 
under suction and the filtrate was treated with 20 ml of hexane and seeded 
with a small amount of crystalline product at 30.degree.. The mixture was 
cooled to 0.degree. within one hour and stirred at this temperature for 30 
minutes. The crystals were then filtered off under suction and washed on 
the filter with 20 ml of hexane. 14.5 g (85%) of benzyl 
(S)-p-t-butoxy-.alpha.-4-(t-butoxycarbonyl)methoxy!-piperidino!carbony 
l!phenethyl!carbamate were obtained in this manner. Its content was 96% 
according to HPLC. 
EXAMPLE 7 
125.9 g (312 mmol) of 
2-(3(S)-amino-2(R)-hydroxy-4-phenylbutyl)-N-t-butyl-decahydro-(4aS,8aS)-is 
oquinoline-3(S)-carboxamide, 94.6 g (328 mmol) of 
N-(2-quinolylcarbonyl)-L-asparagine and 3.5 g (31 mmol) of 
1-hydroxy-2(1H)-pyridone were treated with a mixture of 75 ml of 
tetrahydrofuran and 1925 ml of ethyl acetate. A solution of 70.9 g (344 
mmol) of dicyclohexylcarbodiimide in 500 ml of ethyl acetate was 
subsequently added dropwise at 25.degree. within 30 minutes while 
stirring. The reaction mixture was stirred for 10 hours. Then, 50 ml of 
water of low ion contents were added and the suspension was cooled to 
2.degree.-3.degree. and stirred at this temperature for 1 hour. The 
dicyclohexylurea was then filtered off and washed on the filter with 2 500 
ml portions of ethyl acetate. The filtrates were combined and treated 
slowly at 50.degree. with a solution of 32.5 g (338 mmol) of 
methanesulphonic acid and 250 ml of ethyl acetate. The suspension was 
subsequently stirred at 20.degree. for 14 hours. The crystals were then 
filtered off under suction, washed with a total of 600 ml of ethyl acetate 
and subsequently dried at 45.degree./2000 Pa for 24 hours. 229.5 g (95%) 
of N-t-butyl-decahydro-2-2(R)-hydroxy-4-phenyl-3(S)-N-(2-quinolylcarbon 
yl)-L-asparaginyl!amino!butyl!-(4aS,8aS)-isoquinoline-3(S)-carboxamide 
methanesulphonate having a HPLC content of 97.1% were obtained. 
EXAMPLE 8 
H-Val-Gln-Ala-Ala-Ile-Asp-Tyr-Ile-Asn-Gly-OH (SEQ ID NO:1) was prepared by 
solid phase synthesis using base-labile N-fluorenylmethoxy-carbonyl-amino 
acids (Fmoc-amino acids), t-butyl side-chain protecting functions and 
p-benzyloxybenzyl alcohol-polystyrene resin as described by Atherton and 
Sheppard in "The Peptides; Analysis, Synthesis, Biology", vol. 9 (S. 
Udenfriend and J. Meienhofer, Eds.; Academic Press, New York 1987!). The 
synthesis was started using 4 g of Fmoc-Gly-OCH.sub.2 C.sub.6 H.sub.4 
O-CH.sub.2 C.sub.6 H.sub.4 -resin having a loading of 0.5 mmol of Fmoc-Gly 
per gram of resin, i.e. using 2 mmol of substrate. A semi-automatic 
"Peptide Synthesizer" SP 640 from Labortec AG, CH-4416 Bubendorf was used. 
The following groups were used as side-chain protecting functions: The 
t-butyl ether for tyrosine and the t-butyl ester for aspartic acid. 2.5 
mol equivalents of the subsequent Fmoc-amino acids based on the amino 
components coupled to the resin were used for each coupling step. 1 mol 
equivalent of DCC in the presence of 0.1 mol equivalent of 
1-hydroxy-2(1H)-pyridone, based on the Fmoc-amino acid, was used as the 
coupling reagent. In difficult coupling steps, such as the coupling of 
valine to glutamine, a 1:1 solvent mixture of dimethylformamide (DMF) and 
1,3-dimethyl-2-imidazolidinone was used. 
______________________________________ 
Operation 
Solvent/Reagent Repetitions/Duration 
______________________________________ 
Washing DMF 2 .times. 1 min. 
Deprotection 
20% piperidine in DMF 
1 .times. 3 min. 
Deprotection 
20% piperidine in DMF 
1 .times. 10 min. 
Washing DMF 4 .times. 0.5 min. 
washing Isopropanol 2 .times. 0.5 min. 
Stop Ninhydrin test 
(must be positive) 
Washing DMF 2 .times. 1 min. 
Stop Addition of the next Fmoc- 
amino acid and of 0.1 mol 
equivalent of 1-hydroxy-2(1H)- 
pyridone in DMF 
Equilibration 1 min. 
Stop Addition of DCC 
Coupling 30 min. 
Washing DMF 1 .times. 1 min. 
Washing Isopropanol 2 .times. 1 min. 
Stop Ninhydrin test after 
coupling (must be negative) 
______________________________________ 
After completion of the synthesis 0.5 g of decapeptide resin was shaken 
with trifluoroacetic acid/water (4:1) for 2 hours. The resin was filtered 
off and the filtrate was concentrated, the residue was digested with ether 
and the product (insoluble in ether) was lyophilized from water. The 
product weighed 125 mg and had the expected mol mass of 1063 in the mass 
spectrum (FAB). 
EXAMPLE 9 
16 g (0.1 mol) of t-butyl (2-aminoethyl)carbamate, 17.3 g (0.11 mol) of 
5-chloro-2-pyridinecarboxylic acid and 1.1 g (0.01 mol) of 
1-hydroxy-2(1H)-pyridone are stirred in 170 ml of acetonitrile at 
20.degree.. A solution of 22.7 g (0.11 mol) of dicyclohexylcarbodiimide in 
200 ml of acetonitrile is added dropwise to the reaction mixture within 30 
minutes. The reaction mixture is stirred at 20.degree. overnight. 
Subsequently, the solid is filtered off under suction and the filtrate is 
evaporated in a water-jet vacuum. The residue is dissolved in 90 ml of 
methylene chloride and the solution is treated slowly with 120 ml of 
hexane, whereby t-butyl 2-(5-chloro-2-pyridinecarboxamido)ethyl!carbamate 
crystallizes out. The suspension is stirred at -10.degree. for 90 minutes 
and the t-butyl 2-(5-chloro-2-pyridinecarboxamido)ethyl!carbamate is 
subsequently filtered off and dried at 35.degree. in a vacuum drying oven. 
EXAMPLE 10 
16.9 g (0.11 mol) of 3,4-dihydroxybenzoic acid in 170 ml of tetrahydrofuran 
are treated with 5 g (0.1 mol) of hydrazine hydrate and 1.1 g (0.01 mol) 
of 1-hydroxy-2-(1H)-pyridone. A solution of 22.7 g (0.11 mol) of 
dicyclohexylcarbodiimide in 200 ml of tetrahydrofuran is added to the 
reaction mixture at 20.degree. within 15 minutes. The reaction mixture is 
stirred at 20.degree. for 16 hours. The resulting dicyclohexylurea is 
filtered off and the filtrate is evaporated. The residue is stirred with 
60 ml of ethanol at 20.degree. for 2 hours and the crystalline crude 
product is subsequently filtered off under suction and dried firstly in a 
water-jet vacuum and then in a high vacuum at 40.degree., whereby 
3,4-dihydroxybenzoic acid hydrazide is obtained as a beige powder. 
EXAMPLE 11 
16.8 g (0.1 mol) of 3,4-dihydroxybenzoic acid hydrazide, 27.4 g (0.11 mol) 
of 2-phthalimidoxyisobutyric acid and 1.1 g (0.01 mol) of 
N-hydroxysuccinimide are suspended in 400 ml of tetrahydrofuran and the 
reaction mixture is treated at 25.degree. within one hour with a solution 
of 13.9g (0.11 mol) of diisopropylcarbodiimide in 200 ml of 
tetrahydrofuran. The reaction mixture is stirred at 25.degree. for 24 
hours. The solid is subsequently filtered off under suction and stirred 
intensively at 20.degree. for 2 hours with 40 ml of isopropanol and 200 ml 
of t-butyl methyl ether. The crude product is filtered off under suction 
and dried for 15 hours in a water-jet vacuum at 40.degree., whereby 
1-(3,4-dihydroxybenzoyl)-2-2-methyl-2-(phthalimidoxy)propionyl!hydrazine 
is obtained. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 1 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 10 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: protein 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
ValGlnAlaAlaIleAspTyrIleAsnGly 
1510 
__________________________________________________________________________