BE859026A - ANALGESIC COMPOUNDS - Google Patents

Description

       

  ELI LILLY AND COMPANY

  
 <EMI ID = 1.1>

  
exhibiting analgesic activity on parenteral administration.

  
Recently, endogenous substances with morphine-like properties have been extracted from brain or cerebral fluid- <EMI ID = 2.1> by Hughes et al., Nature, 258, 577 (1975) as pentapeptides of the following formulas:

  
H-Tyr-Gly-Gly-Phe-Met-OH

  
H-Tyr-Gly-Gly-Phe-Leu-OH.

  
These compounds are called methionine-enkephalin and leucine-enkephalin, respectively.

  
Although these compounds have shown analgesic activity in mice when administered to the ventricles of the brain

  
 <EMI ID = 3.1>

  
free of any useful analgesic activity when administered parenterally.

  
A new class of compounds has now been discovered which exhibits significant and demonstrable analgesic activity when administered systemically. It is this class of compounds that this invention relates to.

  
This invention therefore relates to a class of compounds of general formula

  

 <EMI ID = 4.1>


  
and their pharmaceutically non-toxic acid addition salts

  
 <EMI ID = 5.1> <EMI ID = 6.1>

  
a hydrogen atom;

  
R3 is a hydrogen atom or a primary alkyl group

  
 <EMI ID = 7.1>

  
 <EMI ID = 8.1>

  
 <EMI ID = 9.1>

  
R6 is a hydrogen atom or a primary alkyl group

  
 <EMI ID = 10.1>

  
R7 is a hydrogen atom or a primary alkyl group

  
 <EMI ID = 11.1>

  
Y is a hydrogen atom or an acetyl group;

  
0

  
 <EMI ID = 12.1>

  
 <EMI ID = 13.1>

  
 <EMI ID = 14.1>

  
A preferred category of compounds includes those in which W is a -CH2-X-CH3 group and in this category the compounds in which X is sulfur.

  
 <EMI ID = 15.1>

  
acceptable3 included in the field of compounds of formula (I) are the addition salts of organic and inorganic acids, for example those prepared from acids such as hydrochloric, sulfuric, sulphonic, tartaric, fumaric, hydrobromic, glycol-

  
 <EMI ID = 16.1>

  
water, acetic acid or succinic acid. All of the foregoing salts are prepared by conventional methods.

  
As will be seen by referring to the definition of the various

  
 <EMI ID = 17.1> <EMI ID = 18.1>

  
acid of the pentapeptides of this invention are numbered successively from the remainder having the terminal amine function., The asymmetry of the amino acid residues, reading from position 1 to position 5, is thus L, D, L, L and L. When the compounds which are defined by formula (I) constitute a tetrapeptide, the preceding asymmetry sequence applies except that the amino acid residue which initially represents the 5 position is omitted. In addition, it will be noted that the remainder in position 3 is defined so as to include a glycine group. In this case, of course, there is no asymmetry with regard to this remainder. It is simply important to note that, when position 3 defines an amino acid residue having an asymmetry, that asymmetry must be L.

  
 <EMI ID = 19.1>

  
isopropyl.

  
 <EMI ID = 20.1>

  
defined so as to understand the groups "primary alkyl in

  
 <EMI ID = 21.1>

  
an ethyl group. By the term "aralkyl" we mean unsubstituted and substituted aralkyl groups, and preferably

  
 <EMI ID = 22.1> <EMI ID = 23.1>

  
j-

  
when there is one, is placed in the para position. A very clearly preferred substituent is the methoxy group, and the group

  
 <EMI ID = 24.1>

  
With regard to the particular position of the residues of tetrapeptides and pentapeptides of formula (I), the following considerations prevail:
(AT). Position 1.

  
This position represents the terminal amino moiety of the peptide. The rest is the one that comes from L-tyrosine or L- (O-acetyl-) tyrosine. In either case, the rest

  
 <EMI ID = 25.1>

  
two of the hydrogen atoms. It can be substituted by a group-

  
 <EMI ID = 26.1>

  
remainder can be substituted by one or two alkyl groups

  
 <EMI ID = 27.1>

  
is present in position 1 of the peptide of formula (I) is not substituted on nitrogen. Further, it is preferred that the remainder is a tyrosyl residue.

  
(B). Position 2.

  
The amino acid residue which is present in the second position

  
 <EMI ID = 28.1>

  
any of several amino acid residues. They understand the

  
 <EMI ID = 29.1>

  
 <EMI ID = 30.1>

  
 <EMI ID = 31.1>

  
by N-methyl-, N-ethyl- and N-n-propyl-. Preferably, the amino acid in position 2 is not substituted on the nitrogen, that is to say that R- is a hydrogen atom.

  
(VS). Position 3.

  
 <EMI ID = 32.1>

  
from glycine (Gly) or any one of a group of L-amino acids. Amino acids include the following:

  
 <EMI ID = 33.1>

  
L-norleucine, L-leucine and L-isoleucine. Preferably, the remainder at this position of the peptide is that originating from glycine.

  
(D). Position 4.

  
The amino acid residue present in this position is that

  
 <EMI ID = 34.1>

  
N-ethyl- or N-n-propyl: Preferably, the remainder is not substituted

  
 <EMI ID = 35.1>

  
(E). Position 5.

  
(1). Pentapeptide.

  
With regard to the compounds of formula (1) which define

  
0

  
 <EMI ID = 36.1> <EMI ID = 37.1>

  
N-methyl-, N-ethyl-, and N-n-propyl- substituents. Preferably

  
 <EMI ID = 38.1>

  
 <EMI ID = 39.1>

  
represents the amino acid with a terminal carboxyl group, it is present in the form of an amide. Preferably, the amide is not

  
 <EMI ID = 40.1>

  
However, the amide group can be N-monosubstituted, the

  
 <EMI ID = 41.1>

  
terminal amide group is an N-methyl-, N-ethyl-, N-n-propyl- or N-isopropyl- group.

  
(2). Tetrapeptide.

  
It is also possible according to this invention to eliminate the

  
 <EMI ID = 42.1> <EMI ID = 43.1>

  
fluorobenzylthio) ethyl.

  
In this description, the following abbreviations are used, most of which are well known and are commonly used in the art:

  
 <EMI ID = 44.1>

  
Ala - alanine Cys - cysteine Gly - glycine Hse - homoserine Ile - isoleucine Leu - leucine Met - methionine

  
 <EMI ID = 45.1>

  
Nva - norvaline Phe - phenylalanine Ser - serine Tyr - tyrosine Val - valid Ac - acetyl Me - methyl Et - ethyl Ip - isopropyl Pr - n-propyl Bu - n-butyl i-Bu - isobutyl t-Bu - t-butyl

  
 <EMI ID = 46.1>

  
 <EMI ID = 47.1>

  
the following <EMI ID = 48.1>

  
H-L-Tyr-D-Ala-Gly-L-Phe-L-Met-NH2;

  
H-L-Tyr-D-Ala-Gly-L-Phe-L- (N-Me) Met-NH2;

  
 <EMI ID = 49.1>

  
H-L-Tyr-D-Ala-L-Ala-L-Phe-L- (N-Me) Met-NH2;

  
H-L-Tyr-D-Ala-L-Abu-L-Phe-L-Met-NH2;

  
 <EMI ID = 50.1>

  
 <EMI ID = 51.1>

  
H-L-Tyr-D-Ala-Gly-L-Phe-NH (&#65533; -pentyl):

  
H-L-Tyr-D- (N-Me) Ala-Gly-L-Phe-L-Met-NH2;

  
 <EMI ID = 52.1> Met-NH2;

  
(N-Me, N-Et) -L-Tyr (Ac) -D- (N-Pr) Nle-L-Ala-L-Phe-

  
L- (N-Me) Met-NH2;

  
(N, N-Di-Me) -L-Tyr (Ac) -D- (N-Et) Ile-L-Val-L-Phe-

  
L- (N-Pr) Met-NH2;

  
(N-Me) -L-Tyr (Ac) -D- (N-Me) Leu-Gly-L-Phe-L- (N-Et) -

  
Nle-NH2;

  
(N-Me) -L-Tyr (Ac) -D- (N-Me) Nva-L-Nva-L-Phe-L-

  
(N-Me) Hse (Me) -NH2;

  
 <EMI ID = 53.1>

  
methylthiopropyl);

  
(N-Pr) -L-Tyr (Ac) -D- (N-Me) Val-L-Leu-L-Phe-NH (n-

  
pentyl);

  
 <EMI ID = 54.1>

  
H-L-Tyr-D-Ala-Gly-L- (N-Pr) Phe-L- (N-Me) Hse (Me) -

  
NH2;

  
 <EMI ID = 55.1>

  
 <EMI ID = 56.1> <EMI ID = 57.1>

  
H-L-Tyr-D-Abu-Gly-L-Phe-L- (N-Me) Leu-NH2;

  
H-L-Tyr-D-Nva-Gly-L-Phe-L- (N-Me) Leu-NH2;

  
 <EMI ID = 58.1>

  
 <EMI ID = 59.1>

  
chloro-Bzl) -NH 2;

  
H-L-Tyr (Ac) -D-Ala-Gly-L-Phe-L-Ser (m-trifluoro-

  
 <EMI ID = 60.1>

  
Bzl) -NH &#65533;:

  
(N-Allyl) -L-Tyr-D-Ala-Gly-L-Phe-L- (N-Me) Leu-NH2;

  
(N-Et) -L-Tyr-D-Abu-L-Ala-L-Phe-L- (N-Et) Leu-NH2;

  
(N, N-di-Pr) -L-Tyr-D-Val-L-Ala-L-Phe-L- (N-Me) -

  
Leu-NH2;

  
 <EMI ID = 61.1>

  
(N, N-Di-Et) -L-Tyr-D- (N-Pr) Abu-L-Ala-L-Phe-L-

  
Leu-NH2;

  
(N-Me, N-Et) -L-Tyr (Ac) -D- (N-Pr) Nle-L-Ala-L-Phe-

  
 <EMI ID = 62.1>

  
Leu-NH 2;

  
(N-Me) -L-Tyr (Ac) -D- (N-Me) Nva-L-Nva-L-Phe-L-

  
 <EMI ID = 63.1>

  
methylthioethyl);

  
(N-Pr) -L-Tyr (Ac) -D- (N-Me) Val-L-Leu-L-Phe-N (Me) (3-

  
 <EMI ID = 64.1>

  
H-L-Tyr-D-Ala-Gly-L-Phe-L-Ser (Et) -NH (Me);

  
H-L-Tyr-D-Ala-Gly-L-Phe-L- (N-Me) Leu-NH (Me);

  
H-L-Tyr-D-Ala-Gly-L-Phe-L- (N-Et) Leu-NH (Me);

  
 <EMI ID = 65.1>

  
H-L-Tyr-D-Ala-Gly-L-Phe-L- (N-Et) Leu-NH (Et);

  
H-L-Tyr-D-Ala-Gly-L-Phe-L- (N-Me) Leu-NH (Me);

  
 <EMI ID = 66.1>

  
NH (Pr);

  
H-L-Tyr-D-Ala-Gly-L-Phe-L- (N-Pr) Leu-NH (Me);

  
H-L-Tyr-D-Ala-Gly-L-Phe-L-Cys (Ip) -NH (Pr);

  
 <EMI ID = 67.1>

  
L- (N-Me) Leu-NH (Me);

  
(N-Me) -L-Tyr-D-Ala-L-Val-L- {N-Me) Phe-L- (N-Pr) -

  
Ser (Bzl) -NH (Me);

  
 <EMI ID = 68.1> <EMI ID = 69.1>

  
Phe-L- (N-Pr) Leu-NH (Et); and

  
(N-Me) -L-Tyr (Ac) -D- (N-Pr) Leu-L-Abu-L- (N-Me) Phe-

  
 <EMI ID = 70.1>

  
The compounds of formula (I) are prepared by conventional methods for the synthesis of peptides. It is possible during the synthesis of some of the compounds of formula (I) that partial racemization may occur. However, the degree of racemization if it occurs is not sufficient to seriously alter the analgesic activity of the compounds of formula (I).

  
Methods of preparing compounds of formula (I) comprise the coupling of amino acids or peptide fragments by reacting the carboxyl function of one with the amine function of the other to produce an amide bond. In order to effect the coupling efficiently, it is first indicated that all the reactive functions not participating directly in the reaction are inactivated by the use of appropriate blocking groups and then that the carboxyl function which is to be linked is activated from appropriate way to allow copulation to take place. All this requires careful selection of the order of reactions and reaction conditions as well as

  
 <EMI ID = 71.1>

  
 <EMI ID = 72.1>

  
 <EMI ID = 73.1>

  
specially chosen, is prepared using

  
 <EMI ID = 74.1> <EMI ID = 75.1>

  
carbonyl (AdOC) and isobornyloxycarbonyl as blocking groups for the amine group in the synthesis of compounds of formula (I). In addition, the benzyl group (Bzl) is generally used as a protective group for the hydroxy group of the remainder

  
 <EMI ID = 76.1>

  
elements such as p-nitrobenzyl (PNB) and p-methoxybenzyl (PMB) groups.

  
The blocking groups of the carboxyl groups used to prepare the compounds of formula (I) can be any of the typical ester-forming groups, for example

  
 <EMI ID = 77.1>

  
 <EMI ID = 78.1>

  
Coupling of the appropriately protected peptide or N-blocked amine-acid moiety with an appropriately protected amino acid or carboxy moiety peptide moiety in the preparation of the compounds of formula (I) consists of in making the carboxyl function free of the amino acid or of the peptide fragment, active vis-à-vis the coupling reaction. This can be done using any of several well known techniques. Such an activation technique comprises

  
 <EMI ID = 79.1>

  
mixed. The free carboxylic function is activated by reaction with another acid, typically a carbonic acid derivative, such as an acid chloride. Examples of the acid chlorides used to form mixed anhydrides are ethyl chloroformate, phenyl chloroformate, s-butyl chloroformate, isobutyl chloroformate and pivaloyl chloride. Isobutyl chloroformate is preferably used.

  
 <EMI ID = 80.1>

  
for the purpose of carrying out the coupling reaction is its transformation into one of its active ester derivatives. Such active esters

  
 <EMI ID = 81.1>

  
Another method of coupling available is the well known method of coupling with azide.

  
 <EMI ID = 82.1> activation and copulation is implemented using

  
an equimolar amount of DCC relative to the amino acid or to the peptide fragment and it is carried out in the presence of an equimolar amount of 1-hydroxybenzotriazole (HBT). The presence of HBT suppresses unwanted side reactions, including the possibility of racemization.

  
The cleavage of selected blocking groups is necessary at particular times of the synthetic sequence used in the preparation of the compounds of formula (I). A chemist skilled in the synthesis of peptides can easily determine which of the typical protective groups are compatible, in the sense that the selective cleavage of the product can be effected by allowing the removal of one or more.

  
 <EMI ID = 83.1>

  
acid or the peptide fragment. These techniques are well known in the field. A fuller discussion of the techniques available for selective cleavage can be found in

  
 <EMI ID = 84.1>

  
(1965), and in particular in the table on pages 72 and 75 of this work.

  
The cleavage of the protective groups of the carboxyl group can be carried out by alkaline saponification. Relatively strong alkaline conditions are generally used to deesterify the protected carboxyl group, typically using an alkali metal hydroxide, such as sodium hydroxide, potassium hydroxide and lithium hydroxide. The reaction conditions under which saponification takes place are well known in the art. Grouping and blocking

  
of the carboxyl group can also be removed by

  
 <EMI ID = 85.1>

  
the release can be carried out by reduction in the presence of zinc

  
 <EMI ID = 86.1>

  
 <EMI ID = 87.1>

  
corresponding acid addition salt. The cleavage of the blocking group of the amino group can also be carried out by treating the blocked amino acid or peptide with a mixture of

  
 <EMI ID = 88.1>

  
or corresponding hydrochloride. All these deblocking agents are therefore an essentially dry acidic medium. The particular method or reagent that will be used will depend on the chemical or physical characteristics of the substances used in the particular deblocking reaction. We have discovered, in the event that the

  
 <EMI ID = 89.1>

  
a peptide containing at least three amino acid residues, which it is much preferred that the peptide be unblocked by trifluoroacetic acid or formic acid to produce the corresponding acid addition salt. Salt can be made into a forma

  
 <EMI ID = 90.1>

  
Amberlyst A27.

  
 <EMI ID = 91.1>

  
tyrosyl residue can be preserved on the peptide throughout its preparation, being removed during the final synthetic step at the same time as the cleavage of the blocking group of the group

  
 <EMI ID = 92.1>

  
from the blocking group of the carboxyl group, it can be removed earlier in the preparation sequence. When the carboxyl group is cut by alkaline saponification, the protecting group of the hydroxy group is not affected;

  
 <EMI ID = 93.1>

  
 <EMI ID = 94.1>

  
 <EMI ID = 95.1>

  
 <EMI ID = 96.1> <EMI ID = 97.1>

  
illustrating the preparation of a pentapeptide of formula (I), can be described as follows. In the reaction sequence, the symbol AA represents the amino acid residue, and the appended number represents the position of the amino acid in the final peptide sequence.

  

 <EMI ID = 98.1>
 

  
The preceding scheme represents only a sequence for preparing the compounds of formula (I). We have other reaction sequences. Another method that can be used involves the sequential addition of separate amino acids in stages in the construction of the peptide chain, starting with the terminal carboxamide amino acid. Yet another method which can be used comprises solid phase peptide synthesis. The C-terminal residue is attached to an appropriate polymeric support and the peptide is extended by a residue each time until the desired peptide still attached to the polymer has been synthesized. The peptide is removed from the polymer by an appropriate deblocking agent. For example, an N-methylated amino acid

  
 <EMI ID = 99.1>

  
t-Butyloxycarbonyl, can be coupled to a benzhydrylamine polymer by activation with dicyclohexylcarbodiimide. The N-t-butyloxycarbonyl group is removed by reacting the residue attached to the polymer and trifluoroacetic acid in methylene chloride. Neutralization of the polymer salt with an appropriate tertiary base and the addition of a second residue is carried out in the same manner. The blocked peptide is removed from the polymer by processing

  
 <EMI ID = 100.1>

  
particular conditions for this synthesis are known to those skilled in the art. One could use reaction techniques like those described previously in this reaction sequence as well.

  
 <EMI ID = 101.1>

  
In some of the compounds of formula (I), one or more of

  
 <EMI ID = 102.1>

  
 <EMI ID = 103.1>

  
 <EMI ID = 104.1>

  
 <EMI ID = 105.1>

  

 <EMI ID = 106.1>


  
As the previous scheme shows, the amino acid is first treated with potassium hydride in the presence of an appropriate crown ether, to form the dianion. The intermediate is then treated with the appropriate alkyl or allyl iodide for

  
 <EMI ID = 107.1>

  
alkaline like those used in the previous alkylation process. The degree of racemization can vary depending on the particular amino acid that is. used. Racemization can be minimized by using an excess of the alkylating agent and maintaining the

  
 <EMI ID = 108.1>

  
even in the case where excessive racemization may occur, the product can be purified by recrystallization as the salt of a suitable asymmetric amine, for example as the salt.

  
 <EMI ID = 109.1>

  
 <EMI ID = 110.1>

  
 <EMI ID = 111.1>

  
disubstituted desired by the following diagram:

  

 <EMI ID = 112.1>


  
 <EMI ID = 113.1> <EMI ID = 114.1>

  
described above.

  
The C-terminal portion of the peptides of formula (I) is converted into its amide. In the pentapeptides of formula (I), the amide is unsubstituted or N-monosubstituted. In the tetrapeptides of formula (I), the amide is N-monosubstituted or N, N-disubstituted. The transformation into an amide is carried out by activating the carboxyl group of the amino acid with N, N'-dicyclohexylcarbodiimide

  
 <EMI ID = 115.1>

  
the ester of HBT. In the production of the pentapeptides of the formula
(I), the ester is then reacted with ammonia or the appropriate primary amine to obtain the unsubstituted amide or the N-monosubstituted amide. Examples of primary amines suitable for the preparation of the pentapeptides of formula (I) include

  
 <EMI ID = 116.1>

  
N- [pound] 3- (methoxy) propylj -N-propylamine, N-n-pentyl-N-propylamine,

  
 <EMI ID = 117.1> <EMI ID = 118.1>

  
2-benzylthioethylamine,

  
 <EMI ID = 119.1>

  
Compounds of formula (I) in which Y is an acetyl group are prepared from the corresponding peptide in which Y is a hydrogen atom and the terminal amino group is suitably blocked. The latter compound is treated with acetic anhydride in the presence of pyridine to form the

  
 <EMI ID = 120.1>

  
mixture of hydrochloric acid and acetic acid, the desired compound is obtained.

  
Compounds of formula (I) are valuable pharmaceutical agents. They exhibit analgesic activity and are particularly useful by parenteral administration in mammals including humans.

  
The compounds of formula (I) can be administered as such, or they can be prepared according to pharmaceutical preparations, in unit dosage forms for parenteral administration. In the preparation, organic or inorganic solids and / or liquids which are pharmaceutically acceptable carriers can be used. Such suitable supports are well known to those skilled in the art. The compositions can take the form of tablets, powder granules, capsules, suspensions, solutions and other suitable forms.

  
The compounds of formula (I), administered in an effective amount,

  
 <EMI ID = 121.1>

  
general from about 0.1 mg to about 100 mg per kilogram of the patient's body weight. The preferred dose interval is generally

  
 <EMI ID = 122.1>

  
 <EMI ID = 123.1>

  
 <EMI ID = 124.1>

  
be considered as limiting the scope of the invention.

  
 <EMI ID = 125.1>

Example 1

  
Preparation of L-tyrosyl-D-alanyl-glycyl-L-phenyl- hydrochloride

  
 <EMI ID = 126.1>

  
monohydrate, 25 g (0.281 mol) of D-alanine are added. We wear

  
the mixture was refluxed and removed therefrom azeotropically in a Dean-Stark apparatus. The mixture was heated for 15 h then cooled to room temperature and diluted with ether. The resulting precipitate was collected and recrystallized from a mixture of methanol and ether to give 55.3 g (56%) of the title compound, m.p. 112-115 [deg.] C.

  
Analysis, calculated for C17H21N05S (351.42):

  
C, 58.10; H, 6.02; N, 3.99

  
Found, C, 58.19; H, 6.06; N, 3.82

  
 <EMI ID = 127.1>

  
 <EMI ID = 128.1>

  
and cooled to 0 ° C. and 11.2 g (0.1 mol) of diazabicyclooctane (DABCO) are added. The mixture is stirred for 10 min at 0 [deg.] C

  
 <EMI ID = 129.1>

  
 <EMI ID = 130.1>

  
room temperature for 24 h. The mixture is then cooled to 0 ° C., the resulting suspension is filtered and the filtrate is concentrated in vacuo. The resulting residue is then dissolved again in

  
 <EMI ID = 131.1>

  
water, cold 0.75 N citric acid and water. Then on <EMI ID = 132.1> <EMI ID = 133.1>

  
of water, 31.2 g (0.06 mol) of the product of part B are added. The resulting solution is cooled to 0 [deg.] C and slowly added.

  
13.2 ml (1.1 equivalent) of 5N sodium hydroxide. The resulting mixture is stirred and allowed to warm slowly until

  
Room temperature. After 5 h, the mixture is distributed between

  
water and ether. The aqueous layer is separated and cooled, the pH is adjusted to 2 by addition of citric acid and the mixture is extracted with ethyl acetate. The ethyl acetate extract is washed with water, dried over magnesium sulfate, filtered and diluted with ether. The resulting precipitate is collected and 17.7 g (67%) of the compound are obtained.

  
 <EMI ID = 134.1>

  
Analysis, calculated for C24H30N2O6 (442.51):

C, 65.14; H, 6.83; N, 6.63

  
Found, C, 64.73; H, 6.70; N, 6.20

  
 <EMI ID = 135.1>

  
benzyl.

  
 <EMI ID = 136.1>

  
p-toluenesulfonic acid of benzyl glycinate. The resulting mixture is cooled to 0 ° C. and 2.24 g (0.020 mol) of DABCO are added. The mixture is stirred for a few minutes then 8.84 g are added.
(0.020 mole) of the product of part C, then 2.7 g (0.020 mole) of HBT and 4.12 g (0.020 mole) of DCC. The reaction mixture is stirred for 2 h at 0 [deg.] C then for 24 h at room temperature. The resulting suspension is cooled to 0 ° C., filtered and the filtrate concentrated in vacuo. The resulting residue is dissolved in ethyl acetate and washed successively with

  
 <EMI ID = 137.1>

  
cold and water. The phase is dried over magnesium sulfate

  
 <EMI ID = 138.1>

  
 <EMI ID = 139.1>

  
of the pure title compound, m.p. 145-147 [deg.] C.

  
Analysis calculated for C33H39N3O7 (589.69):

  
 <EMI ID = 140.1>

  
 <EMI ID = 141.1>

  
nitrogen and hydrogen is introduced through a dispersion tube of <EMI ID = 142.1>

  
 <EMI ID = 143.1>

  
by filtration the catalyst. The filtrate is concentrated in vacuo. Trituration of the residue with ether gives 5.4 g (75%) of the title compound as an amorphous solid.

  
 <EMI ID = 144.1>

  
C, 69.94; H, 5.87; N, 6.27

  
Found, C, 70.08; H, 5.82; N, 6.16

  
 <EMI ID = 145.1>

  
carbonyl-L-methionine (17.2 g, 0.04 mol) between ethyl acetate and cold 0.75 N citric acid. The resulting organic phase is separated, washed with water, dried over magnesium sulfate, filtered and concentrated in vacuo to an oily residue. The residue is dissolved in a mixture of

  
 <EMI ID = 146.1>

  
 <EMI ID = 147.1>

  
to the resulting cooled mixture over 30 min. 2.49 ml are added
(0.04 mole) of methyl iodide, and the mixture is stirred for
24 h at room temperature. Then we cool the mixture

  
 <EMI ID = 148.1>

  
then it is distributed between 1 [deg.] water and ether. We wash the diaper

  
 <EMI ID = 149.1>

  
 <EMI ID = 150.1>

  
acidified to pH 2 and extracted with ethyl acetate. The ethyl acetate extract is dried over magnesium sulfate, filtered and evaporated in vacuo to give 8.4 g of a product.

  
 <EMI ID = 151.1>

  
 <EMI ID = 152.1>

  
 <EMI ID = 153.1>

  
 <EMI ID = 154.1>

  
 <EMI ID = 155.1>

  
fractions from the column and the subsequent combination according to the TLC profile give, after concentration in vacuo, a product which is recrystallized twice from a mixture of ether and petroleum ether to obtain 4.1 g ( 39%) of the title compound, mp 75-78 [deg.] C.

  
 <EMI ID = 156.1>

  
 <EMI ID = 157.1>

  
 <EMI ID = 158.1>

  
C, 50.36; H, 8.45; N, 10.68

  
Found, C, 50.59; H, 8.24; N, 10.87

  
G. N * -t-butl, -loxycarbonyl-L-phenylalanyl-Nck-methyl-L-methionylamide.

  
Prepare a mixture of 20 ml of glacial acetic acid,

  
2 ml of anisole, 2 ml of triethylsilane and 3.8 g (0.0144 sole} of the product of part F. Hydrochloric gas is bubbled through

  
 <EMI ID = 159.1>

  
 <EMI ID = 160.1>

  
 <EMI ID = 161.1>

  
the mixture is cooled to 0 [deg.] C and 2.9 ml (0.0146 mol) of

  
 <EMI ID = 162.1>

  
 <EMI ID = 163.1>

  
(0.0146 mol) of DCC. The resulting mixture is stirred for 2 h at 0 [deg.] C and then for 24 h at room temperature. The mixture is cooled to 0 [deg.] C and filtered. Then concentrate BOUS and empty the resulting filtrate. The residue is redissolved in ethyl acetate and

  
 <EMI ID = 164.1>

  
water, 0.75 N citric acid and water. The ethyl acetate solution is then dried over magnesium sulfate and

  
 <EMI ID = 165.1> and methanol. TLC analysis of column fractions and

  
 <EMI ID = 166.1> <EMI ID = 167.1>

  
To a mixture of 20 ml of glacial acetic acid, 3 ml of anisole and 3 ml of triethylsilane, 2.2 g are added.

  
(5.37 mmol) of the product of part G. Dry hydrochloric gas is bubbled through the mixture for 30 min. Ether is added to the mixture and a solid precipitates which is filtered and dried in vacuo. The solid (1.75 g, 5 mmol) was dissolved in 30 ml of dry DMF and the mixture was cooled to 0 [deg.] C. The hydrochloride is then neutralized by adding 0.99 ml (5 mmol) of dicyclohexylamine. After 5 min, 2.05 g (5 mmol) of the product from part E are added, then 0.68 g (5 mmol) of HBT and 1.0 g

  
(5 mmol) of DCC. Then the mixture is stirred for 24 h at 4 [deg.] C.

  
The resulting insoluble substance is filtered off and the filtrate evaporated in vacuo. The resulting residue is redissolved in ethyl acetate, and the ethyl acetate washed successively with aqueous sodium bicarbonate 111, acid.

  
 <EMI ID = 168.1>

  
over magnesium sulfate and applied to a 3 x 50 cm column of silica gel (74-250) and eluted with

  
 <EMI ID = 169.1>

  
TLC analysis of fractions from the column and subsequent combination based on the TLC profile giving

  
 <EMI ID = 170.1>

  
The first batch is further purified by preparative thick layer chromatography on silica gel (chloroform: methanol 9: 1) and 0.62 g of the title compound is obtained in the form of an amorphous solid.

  
 <EMI ID = 171.1> <EMI ID = 172.1> adds 0.72 g (1.03 mmol) of the title compound from part H. Anhydrous hydrochloric gas is then bubbled through the mixture for 20 min. The mixture is lyophilized and 0.74 g of the title compound, Rf 0.3, is obtained.

  
An analytical sample of the product is dried under vacuum at 100 [deg.] C.

  
 <EMI ID = 173.1>

  
C, 54.66; H, 6.49; N, 13.19

  
Found, C, 54.36; H, 6.19: N, 13.00

  
Amino acid analysis, found: Tyr, 1.01; Ala, 0.99;

  
 <EMI ID = 174.1>

Example 2

  
Preparation of L-tyrosyl-D- sesqui-hydrochloride monoacetate

  
 <EMI ID = 175.1>

  
of the D-alinate derivative. Yield 73%, m.p. 155-156 [deg.] C.

  
Analysis, calculated for C20H27N05S (393.50):

  
 <EMI ID = 176.1>

  
 <EMI ID = 177.1>

  
 <EMI ID = 178.1>

  
benzyl

  
 <EMI ID = 179.1>

  
 <EMI ID = 180.1>

  
(0.020 mole) of DABCO. The mixture is stirred for 5 min then

  
 <EMI ID = 181.1>

  
 <EMI ID = 182.1>

  
 <EMI ID = 183.1>

  
 <EMI ID = 184.1> <EMI ID = 185.1>

  
C, 71.06; H, 7.37; N, 4.87

  
Found, C, 71.30; H, 7.15; N, 4.79

  
 <EMI ID = 186.1>

  
 <EMI ID = 187.1>

  
part B. After addition of 20 ml of water, the resulting mixture is cooled to 0 ° C., and 7.25 ml (0.0145 mole) of 2N sodium hydroxide are slowly added. Once the addition is complete, the mixture is stirred at 0 [deg.] C for 30 min and then at room temperature for 4 h. The reaction mixture is then partitioned between water and ether. The aqueous phase is separated, cooled to 0.degree. C., acidified to pH 2 with cold 1N HCl and extracted with ethyl acetate. Then the ethyl acetate extract is washed with water, dried over magnesium sulfate, filtered and

  
it is concentrated in vacuo which gives a syrupy residue. The residue is crystallized from a mixture of ether and petroleum ether, to give 6.4 g (95%) of the title compound, m.p. 90-94 [deg.] C.

  
 <EMI ID = 188.1>

  
C, 66.92; H, 7.49; N, 5.78

  
Found, C, 67.14; H, 7.38; N, 5.76

  
 <EMI ID = 189.1>

  
benzyl.

  
A mixture of 3.37 g (0.010 mole) of benzyl glycinate p-toluenesulfonate and 1.12 g (0.010 mole) of

  
 <EMI ID = 190.1>

  
(0.010 mole) of the compound of part C. Then the mixture is cooled to 0 [deg.] C and 1.35 g (0.010 mole) of HBT and 2.06 g are added.

  
(0.010 mol) of DCC. The resulting mixture is stirred for 2 h at 0 [deg.] C and then for 24 h at room temperature. The mixture is cooled to 0 ° C., filtered and the filtrate is concentrated in vacuo.

  
 <EMI ID = 191.1>

  
wash the ethyl acetate solution successively with IN sodium bicarbonate, water, 0.75 N citric acid

  
 <EMI ID = 192.1> <EMI ID = 193.1>

  
3.9 g (0.006 mol) of the compound of part D are added to 5 ml of anhydrous DMF, followed by 1.5 g of 5% Pd / C. To the mixture was then added 40 ml of ethanol, purged with nitrogen and hydrogen was introduced for 5 h, maintaining the mixture at atmospheric pressure and at room temperature. The catalyst is then filtered from the mixture and the filtrate is evaporated in vacuo. The resulting residue is crystallized from a mixture of ether and ethyl acetate, to give 2.3 g (85%) of the title compound, m.p. 189-190 [deg.] C.

  
 <EMI ID = 194.1>

  
C, 58.52; H, 7.37; N, 9.31

  
Found, C, 58.79; H, 7.48; N, 9.39

  
 <EMI ID = 195.1>

  
as in part H of Example 1) and 0.903 g (0.002 mol) of the product of part E. The resulting mixture is cooled to 0 [deg.] C and

  
 <EMI ID = 196.1>

  
0.27 g (0.002 mole) of HBT and 0.412 g (0.002 mole) of DCC. The mixture is then stirred at 0 [deg.] C for 2 h then at 4 [deg.] C for 24 h. The resulting precipitate is removed by filtration and the filtrate is concentrated in vacuo to a residue which is then dissolved in ethyl acetate. The acetate solution is washed successively

  
 <EMI ID = 197.1>

  
0.75 N cold and water. The organic phase is then dried over magnesium sulfate, filtered and the filtrate is concentrated in vacuo. The residue is applied to two preparative thick layer chromatography plates, and the plates are eluted with a

  
 <EMI ID = 198.1> <EMI ID = 199.1>

  
Gly, 1.00; Phe, 0.99

  
G. L-tyrosyl-D-leucyl- sesquichlorhydrate monoacetate

  
 <EMI ID = 200.1>

  
To 5 ml of glacial acetic acid containing 0.3 ml of anisole, 0.900 g (0.0012 mole) of the compound of part F is added. Dry hydrochloric gas is bubbled through the mixture for 20 minutes. The solvent is then removed by lyophilization of aqueous acetic acid, and the title compound is obtained in the form of an amorphous solid.

  
 <EMI ID = 201.1>

  
C, 53.93; H, 6.79; N, 11.10 Cl, 7.02 Found, C, 54.30; H, 6.64; N, 11.32; Cl, 6.96 Amino acid analysis, found: Tyr, 0.99; Leu, 1.03; Gly, 0.99; Phe, 0.99

Example 3

  
 <EMI ID = 202.1>

  
 <EMI ID = 203.1>

  
methyl.

  
 <EMI ID = 204.1>

  
of methyl L-methionate. The mixture is cooled to 0 [deg.] C and

  
 <EMI ID = 205.1>

  
13.5 g (0.1 mole) of HBT and 20.6 g (0.1 mole) of DCC. The resulting mixture was stirred at 0 [deg.] C for 2 h then at room temperature for 24 h. The mixture was again cooled to 0 ° C. and the resulting precipitate was removed by filtration. The filtrate is concentrated in vacuo. The residue is then dissolved in acetate

  
 <EMI ID = 206.1>

  
 <EMI ID = 207.1>

  
To 60 ml of methanol is added 13.0 g (0.032 mol) of the compound of part A. The resulting suspension is placed in a pressure vessel equipped with a magnetic stir bar. The mixture is cooled to -78 [deg.] C and 60 ml of anhydrous liquid ammonia is added. The reaction vessel is closed and allowed to warm to room temperature. The mixture is stirred for 24 h at room temperature. The jar is slowly cooled again to -78 [deg.] C and opened. The residual ammonia is evaporated off by heating the mixture and recrystallized from

  
 <EMI ID = 208.1>

  
obtains 9.7 g (77%) of the title compound, m.p. 192-195 [deg.] C.

  
Analysis calculated for C19H29N3O4S (395.52):

  
C, 57.70; H, 7.39; N, 10.62

  
Found, C, 57.41; H, 7.17; N- 10.37

  
C. L-phenylalanyl-L-methionylamide hydrochloride.

  
To 150 ml of glacial acetic acid containing 10 ml of anisole and 10 ml of triethylsilane, 9.6 g (0.024 mol) of the product of part B is added. Then anhydrous hydrochloric gas is introduced through a dispersion tube of gas. After 30 min, the reaction mixture is diluted with ether. The resulting precipitate was collected and recrystallized from a mixture of ethanol and ether to give 7.5 g (94%) of the title compound, m.p., 214-216 [deg.] C.

  
 <EMI ID = 209.1>

  
C, 50.67; H, 6.68; N, 12.66

  
Found, C, 50.75; H, 6.84; N, 12.54

  
 <EMI ID = 210.1>

  
methionylamide.

  
To 40 ml of DMF is added 1.66 g (0.005 mol) of the product from part C. 0.99 ml (0.005 mol) of dicyclohexylamine is added and

  
 <EMI ID = 211.1>

  
0.68 g (0.005 mole) of HBT and 1.03 g (0.005 mole) of DCC. The resulting mixture was stirred for 2 h at 0 [deg.] C then at room temperature for 24 h. After cooling the mixture again

  
 <EMI ID = 212.1>

  
filtrate under vacuum. The residue is dissolved in ethyl acetate.

  
 <EMI ID = 213.1>

  
magnesium, filtered and evaporated in vacuo. Then we <EMI ID = 214.1> crystallization. The gel is filtered and the solid is dried

  
 <EMI ID = 215.1>

  
as monohydrate and the mixture was stirred for 5 h. The resulting precipitate was collected by filtration and dried to give

  
 <EMI ID = 216.1>

  
 <EMI ID = 217.1>

  
0.336 g (0.003 mol) of DABCO then, after 10 min, 0.8 g

  
 <EMI ID = 218.1>

  
 <EMI ID = 219.1>

  
 <EMI ID = 220.1>

  
 <EMI ID = 221.1>

  
 <EMI ID = 222.1>

  
 <EMI ID = 223.1>

  
 <EMI ID = 224.1>

  
water, cold 0.75 N citric acid and water. The organic phase is dried over magnesium sulfate, filtered and concentrated in vacuo. The resulting residue is dissolved in

  
 <EMI ID = 225.1> <EMI ID = 226.1>

  
which precipitates and the filtrate is concentrated in vacuo. Then the resulting residue was dissolved in n-butanol and the solution washed successively with 1N sodium bicarbonate, water, cold 0.75N citric acid and water. The organic phase is then dried over magnesium sulfate and filtered, and the filtrate is concentrated in vacuo. Attempts to recrystallize the residue from ethyl acetate or from ethanol give gels. The residue is then dissolved in hot methanol and the solution is applied to a preparative thick layer chromatography plate and eluted with a 9: 1 mixture of chloroform and

  
 <EMI ID = 227.1>

  
treated with the chloroform-methanol mixture. The solvent is evaporated off in vacuo to give 0.270 g (21%) of the title compound,

  
 <EMI ID = 228.1>

  
Amino acid analysis, found: Tyr, 1.00; Ala, 1.02;

  
 <EMI ID = 229.1>

  
introduced anhydrous hydrochloric gas through a gas dispersion tube for 20 min. Then the resulting mixture is frozen and

  
 <EMI ID = 230.1>

  
 <EMI ID = 231.1>

  
 <EMI ID = 232.1>

  
 <EMI ID = 233.1>

  
Analysis indicates the presence of methionine tallow oxide.

  
Example

  
 <EMI ID = 234.1> <EMI ID = 235.1> of methyl iodide. Stirring is continued at room temperature for 24 h. Then the mixture is poured onto crushed ice and extracted with ether. The aqueous phase is acidified to pH 2 with citric acid and then extracted with ethyl acetate. The organic phase is then washed with water, dried over magnesium sulfate and concentrated in vacuo, which gives a syrup which does not crystallize. The NMR spectrum of the syrup is compatible with the expected derivative:

  
 <EMI ID = 236.1>

  
ether and 4.0 ml of dicyclohexylamine is added. Crystals form on cooling. The precipitate was collected and recrystallized from a mixture of methanol and ether to give 6.8 g (74%) of the title compound, m.p. 171-174 [deg.] C.

  
 <EMI ID = 237.1>

  
C, 70.40; H, 9.63; N, 6.08

  
Found, C, 70.60; H, 9.49; N, 6.19

  
 <EMI ID = 238.1>

  
L-methionylamide.

  
 <EMI ID = 239.1>

  
 <EMI ID = 240.1>

  
 <EMI ID = 241.1>

  
 <EMI ID = 242.1>

  
(0.010 mol) of DCC. The resulting mixture is stirred for 2 h at 0 [deg.] C and then for 24 h at room temperature. The resulting precipitate is removed by filtration and the filtrate is concentrated in vacuo to a syrup which is redissolved in acetate.

  
 <EMI ID = 243.1>

  
 <EMI ID = 244.1>

  
mole) of the product of part-B. Anhydrous hydrochloric gas is introduced through a gas dispersion tube for 30 min, then the reaction mixture is diluted with ether. The resulting precipitate is collected and dried (1.1 g) and then redissolved in 40 ml of DMF. The reaction mixture is then cooled to 0 ° C. and 1.27 g (0.031 mol) of the product of part E of Example 1, 0.420 g (0.0031 mol) of HBT and 0.640 g ( 0.0031 mole)

  
 <EMI ID = 245.1>

  
amine and stirring is continued at 0 [deg.] C for 2 h and then at 4 [deg.] C for 48 h. The resulting precipitate was filtered off and the filtrate was concentrated in vacuo to a syrupy residue which was then dissolved again in ethyl acetate. The ethyl acetate solution is washed successively with bicarbonate

  
 <EMI ID = 246.1>

  
water and then dried over magnesium sulfate, filtered and concentrated in vacuo, which gives 2.0 g of crude product. The product is dissolved in chloroform and applied to a

  
 <EMI ID = 247.1>

  
with chloroform followed by a 9: 1 mixture of chloroform and methanol. TLC analysis of the fractions from the column and subsequent combination of these fractions based on the TLC profile gives, after concentration in vacuo, 1.1 g (47%) of the title compound, uncrystallized.

  
 <EMI ID = 248.1>

  
of anisole, 0.70 g (0.001 mol) of the product of part C.

  
Dry hydrochloric gas is introduced through a dispersion tube

  
 <EMI ID = 249.1>

  
 <EMI ID = 250.1>

  
C, 51.08; H, 7.0; N, 11.91

  
Found, C, 51.13; H, 6.97; N, 11.72

  
Amino acid analysis, found: Tyr, 1.03; Ala, 1.01; Gly, 0.96.

Example 5

  
Preparation of L- hydrochloride mono-acetate (1.25 HCl)

  
 <EMI ID = 251.1>

  
benzyl alinate.

  
To a solution of 3.19 g (0.010 mole) of benzyl alinate p-toluenesulfonate in 30 ml of dry DMF, 4.43 g is added.
(0.010 mole) of the product of part B of 'Example 1.' The resulting mixture is cooled to 0 [deg.] C and 1.12 g (0.010 mole) are added, followed by 1.135 g (0.010 mole) in 10 min. ) HBT and 2.06 g (0.010 mol) this DCC. The resulting mixture is stirred at 0 [deg.] C for 2 h then at

  
 <EMI ID = 252.1>

  
resulting precipitate and the filtrate evaporated in vacuo to a syrup. The syrup is dissolved again in ethyl acetate and the ethyl acetate solution is washed successively with 1N sodium bicarbonate, water, cold 0.75N hydrochloric acid and the water. Then the organic phase is dried over magnesium sulfate and filtered, and the filtrate is concentrated in vacuo, to give a residue which does not crystallize from ethanol or ether. Dilution of the ethereal solution with petroleum ether gives a gel which is collected by filtration and dried in vacuo. The impure amorphous solid is applied
(4.0 g) on a 3 x 50 cm column of silica gel (74-250

  
 <EMI ID = 253.1>

  
 <EMI ID = 254.1>

  
 <EMI ID = 255.1>

  
 <EMI ID = 256.1>

  
solvent in vacuo give a syrupy residue. We dissolve this

  
 <EMI ID = 257.1>

  
 <EMI ID = 258.1>

  
2.9 g (0.0048 mol) of the product from part A are added to 5 ml of dry DMF. To the mixture are then added 1.0 g of 5% Pd / C and then 50 ml of ethanol. Hydrogen is introduced at atmospheric pressure and at room temperature through a gas dispersion tube for 6 h. The reaction vessel is then purged with nitrogen, the catalyst is collected by filtration and the filtrate is concentrated in vacuo. The residue is dissolved in ethyl acetate and the solution is diluted with ether. The resulting precipitate was collected by filtration and dried in vacuo to give 1.5 g (74%) of the title compound as an amorphous solid.

  
 <EMI ID = 259.1>

  
Analysis, calculated for C20H29N3O7 (423.47):

C, 56.73; H, 6.90; N, 9.92

  
 <EMI ID = 260.1>

  
 <EMI ID = 261.1>

  
known in part H of Example 1). The mixture is cooled to 0 ° C. and 0.28 ml (0.002 mol) of triethylamine is added. The reaction mixture is stirred for 10 min, and 0.846 g (0.002 mole) of the product of part B is added, followed by 0.270 g (0.002 mole) of HBT and 0.412 g (0.002 mole) of DCC. The resulting mixture was stirred at 0 [deg.] C for 2 h and then at room temperature for 48 h. After cooling the mixture again to 0 [deg.] C, it is filtered and the filtrate is concentrated in vacuo. The residue is redissolved in ethyl acetate and the ethyl acetate solution washed.

  
 <EMI ID = 262.1>

  
cold 0.75N citric acid and water. Then we skip the phase

  
 <EMI ID = 263.1>

  
 <EMI ID = 264.1>

  
the product in chloroform and applied to two plates

  
 <EMI ID = 265.1>

  
 <EMI ID = 266.1>

  
 <EMI ID = 267.1> <EMI ID = 268.1>

  
 <EMI ID = 269.1>

  
MeOH)

  
 <EMI ID = 270.1>

  
C, 58.80; H, 7.05; N, 11.76

  
Found, C, 58.60; H, 6.87; N, 11.53

  
D. L-tyrosyl-D-alanyl-L- hydrochloride mono-acetate

  
 <EMI ID = 271.1>

  
To 5 ml of glacial acetic acid containing 0.5 ml of anisole, 0.880 g (0.0011 mol) of the product of part C. is added. Anhydrous hydrochloric gas is introduced by one. gas dispersion tube for 20 min. The reaction mixture is then frozen and lyophilized to give 0.704 g of the title compound;

  
 <EMI ID = 272.1>

  
 <EMI ID = 273.1>

  
C, 53.43; H, 6.45; N, 11.68; Cl, 6.16 Found, C, 53.48; H, 6.47; N, 11.62; Cl, 6.50 Amino acid analysis, found: Tyr, 1.00; Ala, 1.99; Phe, 1.01.

Example 6

  
Preparation of L-tyrosyl-D-alanyl-glycyl-L- acetate

  
 <EMI ID = 274.1>

  
nel 44.8 ml (0.336 mol) of tetramethylguanidine and 66.8 ml
(0.336 mol) of dicyclohexylamine. Then we add drop by drop

  
 <EMI ID = 275.1>

  
 <EMI ID = 276.1>

  
 <EMI ID = 277.1>

  
aqueous layer. The organic layer is separated and discarded. Then

  
 <EMI ID = 278.1> <EMI ID = 279.1>

  
 <EMI ID = 280.1>

  
159 [deg.] C.

  
 <EMI ID = 281.1>

  
Analysis, calculated for C22H42N2O4S (430.6):

C, 61.36; H, 9.83; N, 6.51

  
Found, C, 61.37; H, 9.98; N, 6.26

  
 <EMI ID = 282.1>

  
by neutralization of the product of part A and extraction in ethyl acetate). The resulting mixture is added dropwise over 30 min to a mechanically stirred suspension of 42.45 g of a suspension of potassium hydride (22.1% KH in mineral oil; 0.234 mol of KH) in 375. ml of THF at 0 [deg.] C and containing 0.5 g of ether (6) in a crown (18). We add drop by drop in
15-20 minutes 9.25 ml (0.149 mole) of methyl iodide in 20 ml

  
 <EMI ID = 283.1>

  
 <EMI ID = 284.1>

  
5 ml of ethanol. The resulting reaction mixture is then poured onto ice and the pH of the mixture is adjusted to about 9 by addition of 2N sodium hydroxide. The resulting aqueous solution was extracted with ether. The pH of the aqueous layer is then adjusted to 3 by adding solid citric acid and extracted again with three 300 ml portions of ether. The ethereal extracts are combined, extracted back with water, dried over magnesium sulphate and concentrated in vacuo. The resulting residue is dissolved in 200 ml of ether, and added.

  
 <EMI ID = 285.1>

  
mixture and 500 ml of petroleum ether are added. It does not happen

  
 <EMI ID = 286.1>

  
 <EMI ID = 287.1>

  
The product (6.5 g, 0.025 mol) is dissolved in 80 ml of DMF and <EMI ID = 288.1>

  
the mixture is cooled to -15 [deg.] C. 3.6 ml (0.027 mol) of

  
 <EMI ID = 289.1>

  
morpholine. The resulting mixture was stirred for 10 min at -15 [deg.] C and then anhydrous ammonia was bubbled through the reaction mixture for 1 h. The mixture is stirred for an additional 4 h

  
at -15 [deg.] C and then poured onto a mixture of ice and IN sodium bicarbonate. The cold aqueous layer is extracted with ether. The ethereal extract was then extracted with cold 0.75N citric acid and water, dried over magnesium sulfate and concentrated in vacuo to give a residue which crystallized from a mixture of ether and petroleum ether giving 1.7 g (26%) of the title compound, mp 56-59 [deg.] C.

  
 <EMI ID = 290.1>

  
 <EMI ID = 291.1>

  
 <EMI ID = 292.1>

  
 <EMI ID = 293.1>

  
 <EMI ID = 294.1>

  
 <EMI ID = 295.1>

  
cysteinylamide.

  
 <EMI ID = 296.1>

  
 <EMI ID = 297.1>

  
 <EMI ID = 298.1>

  
bubbling dry hydrochloric gas in the mixture for 30 minutes

  
 <EMI ID = 299.1>

  
(1.8 g). The precipitate is dissolved in 25 ml of DMF. We cool

  
 <EMI ID = 300.1> <EMI ID = 301.1>

  
 <EMI ID = 302.1>

  
combination of the appropriate fractions and evaporation of the solvent in vacuo, 3.0 g of the title compound.

  
 <EMI ID = 303.1>

  
 <EMI ID = 304.1>

  
C, 58.65; H, 7.63; N, 10.26

  
 <EMI ID = 305.1>

  
 <EMI ID = 306.1>

  
The product of the hydrogenolysis is dissolved in 150 ml of isopropyl alcohol (according to the process of part E. of Example 1)

  
 <EMI ID = 307.1>

  
benzyl glycinate, and 16 ml (0.081 mol) of dicyclohexylamine are added. Ether is added to bring the volume to about 1.5 1. The semi-solid mass is triturated until it is

  
 <EMI ID = 308.1>

  
 <EMI ID = 309.1>

  
 <EMI ID = 310.1>

  
isopropyl alcohol. The volume of the solution is reduced, under a stream of nitrogen, to approximately 150 ml. On cooling, crystallization begins. The mixture is left to stand overnight and the precipitate is collected and dried, which gives
41.44 g (88%) of the title compound, m.p. 198-200.5 [deg.] C.

  
 <EMI ID = 311.1>

  
 <EMI ID = 312.1>

  
 <EMI ID = 313.1>

  
 <EMI ID = 314.1> the filtrate under vacuum. The residue was dissolved in ethyl acetate and the ethyl acetate solution was extracted with 1N sodium bicarbonate, water, 0.75 N citric acid and water. The organic phase is dried over magnesium sulfate and evaporated in vacuo, to give 3.5 g of the crude title compound. The product is dissolved in chloroform and applied to a 3 x 45 cm column of quality silica gel.
62 from Grace and Davidson and we elect with a continuous gradient

  
 <EMI ID = 315.1>

  
fractions based on their CCM profile and evaporated in vacuo, to give 2.4 g (62%) of the compound mentioned in

  
 <EMI ID = 316.1>

  
 <EMI ID = 317.1>

  
C, 58.27; H, 6.90; N, 11.99

  
 <EMI ID = 318.1>

  
 <EMI ID = 319.1>

  
To 20 ml of glacial acetic acid containing 2 ml of anisole and 2 ml of triethylsilane, 2.2 g (3 mmol) of the product of part E is added. Dry hydrochloric gas is bubbled through the reaction mixture for 25 mn. Then ether is added to the mixture and cooled. Filter and dry the resulting precipitate (2.0 g). A portion of the precipitate (1.2 g) was dissolved in sufficient buffer (1% pyridine and 0.05% acetic acid in water) to make a total of 10 ml. The solution is deposited on a 2.5 x 99 cm column of DEAE-Sephadex A 25

  
 <EMI ID = 320.1> <EMI ID = 321.1>

Example 7

  
Preparation of L-tyrosyl-D-alanyl-glycyl-L-phenyl- acetate

  
 <EMI ID = 322.1>

  
hydrated oxycarbonyl-L-leucine. The mixture is dried over magnesium sulfate and concentrated in vacuo. The residue is dissolved in
75 ml of THF and the resulting ionized solution is added dropwise to a cooled (0 [deg.] C) mechanically stirred suspension of

  
 <EMI ID = 323.1>

  
 <EMI ID = 324.1>

  
containing 0.25 g of ether (6) in a crown (18). We add drop to

  
 <EMI ID = 325.1>

  
maintains the mixture at 0 [deg.] C for 3 h then added dropwise

  
 <EMI ID = 326.1>

  
 <EMI ID = 327.1>

  
 <EMI ID = 328.1>

  
The aqueous solution is extracted with ether and then acidified.

  
at pH 3 by addition of solid citric acid. The acidified aqueous suspension is then extracted with ether. The combined ethereal extracts are washed with water, dried over magnesium sulfate.

  
 <EMI ID = 329.1>

  
 <EMI ID = 330.1>

  
indicates the presence of a certain amount of unreacted starting material. The product is dissolved in ether and 5.25 ml (0.05 mole) of t-butylamine is added. The ethereal solution is diluted with petroleum ether and cooled.

  
 <EMI ID = 331.1>

  
 <EMI ID = 332.1>

  
 <EMI ID = 333.1>

  
 <EMI ID = 334.1>

  
 <EMI ID = 335.1>

  
product of part A with citric acid and extraction in ether). The mixture is cooled to -15 [deg.] C then 6.7 ml are added.
(0.052 mole) of isobutyl chloroformate and 5.7 ml (0.052 mole) of N-methylmorpholine. The mixture is stirred for 10 min at -15 [deg.] C and anhydrous ammonia is bubbled through the reaction mixture for 1 hour. Stirring is continued for 4 h

  
at -15 [deg.] C. The reaction mixture is poured onto a mixture of

  
 <EMI ID = 336.1>

  
with ether. Then the ethereal layer is extracted with 0.75 N citric acid and water, dried over magnesium sulfate and evaporated in vacuo. The residue is crystallized from a mixture of ether and petroleum ether to give 5.5 g (48 µl of

  
 <EMI ID = 337.1>

  
 <EMI ID = 338.1>

  
C, 58.99; H, 9.90; N, 11.47

  
Found, C, 59.17; H, 9.66; N, 11.21

  
 <EMI ID = 339.1>

  
amide

  
 <EMI ID = 340.1>

  
3 ml of triethylsilane are added 5.0 g (0.02 mol) of the product of part B. Dry hydrochloric gas is bubbled through the reaction mixture for 25 min. Then ether is added and the mixture is cooled. The resulting precipitate was collected and dried (3.6 g). The hydrochloride collected is dissolved in 60 ml of DMF. The resulting solution was cooled to 0 ° C. and 3.99 ml (0.02 mole) of dicyclohexylamine was added. The mixture is stirred at 0 [deg.] C

  
 <EMI ID = 341.1>

  
 <EMI ID = 342.1>

  
0 [deg.] C then at room temperature for 24 h. We cool the

  
 <EMI ID = 343.1>

  
 <EMI ID = 344.1>

  
 <EMI ID = 345.1>

  
 <EMI ID = 346.1>

  
Apply it to a 3 x 45 cm column of silica gel quality 62 <EMI ID = 347.1>

  
 <EMI ID = 348.1>

  
combines the fractions based on their TLC profile and after evaporation of the solvent, 5.7 g (73%) of the compound are obtained

  
 <EMI ID = 349.1>

  
 <EMI ID = 350.1>

  
alanyl-N &#65533; -methyl-L-leucylamide.

  
 <EMI ID = 351.1>

  
ice-cold containing 1 ml of anisole, 2.0 g of the product from part C are added. The mixture is kept at room temperature for
30 minutes then ether is added. The mixture was cooled, and the resulting precipitate was collected and dried (1.63 g). We

  
 <EMI ID = 352.1>

  
 <EMI ID = 353.1>

  
carbonyl-L-tyrosyl-D-alanyl-glycine. The mixture is stirred for

  
15 min at 0 [deg.] C and 0.675 g (0.005 mol) of HBT and 1.3 g are added
(0.005 mol) of DCC. The reaction mixture is then stirred for
24 h at 4 [deg.] C. The resulting precipitate is collected and the filtrate is concentrated in vacuo. The residue was dissolved in ethyl acetate and the ethyl acetate solution was extracted with 1N sodium bicarbonate, water, 0.75 N citric acid and water. Then the ethyl acetate solution is dried over magnesium sulfate and concentrated in vacuo. The resulting residue was dissolved in chloroform and the chloroform solution was deposited on a 3 x 45 cm column of Woelm grade III silica gel. We elected

  
 <EMI ID = 354.1>

  
on their CCM profile. After evaporation of the solvent, 2.3 g (67%) of the title compound are obtained.

  
 <EMI ID = 355.1>

  
Analysis, calculated for C35H50N608 (682.8):

C, 61.57; H, 7.38; N, 12.31

  
 <EMI ID = 356.1>

  
 <EMI ID = 357.1>

  
of triethylsilane, 1.8 g (0.003 mol) of the product of <EMI ID = 358.1> are added

  
3 hrs. Then the reaction mixture is diluted with ether and allowed to stand for 1 hour. The ether is decanted from the resulting oil and the oil is dissolved in ethanol. Addition of ether gives a precipitate which is filtered and dried to give 0.9 g of the crude title compound. The product is dissolved in enough buffer solution (1% pyridine and 0.05% formic acid in water) to make a total of

  
5.0 ml. The solution was applied to a 2.5 x 100 cm column of DEAE-Sephadex A-25 (formate) and eluted with the same buffer. We gather the appropriate fractions based on their profile

  
 <EMI ID = 359.1>

  
Sations in 10% acetic acid and a 75:25 mixture of water and acetonitrile give 0.852 g of the title compound.

  
 <EMI ID = 360.1>

  
Amino acid analysis, found: Tyr, 1.02; Ala, 1.00 Gly, 1.01; Phe, 0.96; NH3, 1.03.

Example 8

  
Preparation of L-tyrosyl-D-alanyl-glycyl-L-phenyl- hydrochloride

  
 <EMI ID = 361.1>

  
When cooled, 2.88 ml (0.022 mole) of isobutyl chloroformate and 2.42 ml (0.022 mole) of N-methylmorpholine are added. After 15 min, anhydrous ammonia is bubbled through the reaction mixture for 1.5 h. Stirring is continued at -15 [deg.] C for a further 2 hours. The reaction mixture is poured into a mixture of ice and 1N sodium bicarbonate. We extract the

  
 <EMI ID = 362.1>

  
citric 0.75 N and water. The organic layer is then dried over magnesium sulfate and concentrated in vacuo. We recrystallize

  
 <EMI ID = 363.1>

  
 <EMI ID = 364.1>

  
Analysis, calculated for C16H24N2O4S (340.4):

  
 <EMI ID = 365.1> <EMI ID = 366.1>

  
cysteinylamide.

  
Anhydrous hydrochloric gas is bubbled through a solution of 4.1 g (0.012 mole) of the product of part A in

  
45 ml of glacial acetic acid, 5 ml of anisole and 5 ml of triethylsilane. After 20 min, ether is added and the resulting precipitate is collected and dried (3.3 g). The hydrochloride collected is dissolved in 50 ml of DMF and 2.92 g (0.012 mole) of dicyclohexylamine, 3.19 g (0.012 mole) of

  
 <EMI ID = 367.1>

  
HBT. The mixture is stirred for 10 min at 0 [deg.] C and 2.47 g are added.
(0.012 mol) of DCC. After 2 h at 0 [deg.] C, the reaction mixture is stirred at room temperature for 24 h and then cooled to 0 [deg.] C. The resulting precipitate is filtered off. The filtrate is concentrated in vacuo and the resulting residue dissolved in n-butyl alcohol. The solution is extracted with IN sodium bicarbonate and water, then dried over magnesium sulfate and evaporated in vacuo. The resulting residue is recrystallized from ethanol to give 4.95 g (85%) of the compound mentioned in

  
 <EMI ID = 368.1>

  
 <EMI ID = 369.1>

  
C, 61.58: H, 6.82; N, 8.62

  
Found, C, 61.78; H, 6.78; N, 8.28

  
 <EMI ID = 370.1>

  
Anhydrous hydrochloric gas is bubbled through a solution of 1.3 g (0.027 mol) of the product of part B in 40 ml of glacial acetic acid, 4 ml of anisole and 4 ml of triethylsilane. After 20 min, ether is added to the mixture and the mixture is collected.

  
 <EMI ID = 371.1>

  
 <EMI ID = 372.1>

  
0.75 N citric acid and water. Then the extract is dried over magnesium sulphate and concentrated in vacuo. The resulting residue is dissolved in ethyl acetate and purified by dry column chromatography on quality silica gel.
62 from Grâce and Davidson. The fractions were pooled based on their TLC profile and concentrated to give 1.1 g

  
 <EMI ID = 373.1>

  
 <EMI ID = 374.1>

  
 <EMI ID = 375.1>

  
C, 60.14; H, 6.14; N, 10.79

  
Found, C, 59.95; H, 6.24; N, 10.53

  
Amino acid analysis, found: Tyr, 0.98; Ala, 1.03;

  
 <EMI ID = 376.1>

  
D. L-tyrosyl-D-alanyl-glycyl-L-phenylalanyl-S- hydrochloride

  
 <EMI ID = 377.1>

  
 <EMI ID = 378.1>

  
 <EMI ID = 379.1>

  
 <EMI ID = 380.1>

  
Found, C, 56.85; H, 6.06; N, 11.48; Cl, 5.21

  
Amino acid analysis, found; Tyr, 0.99; Ala, 1.01;

  
 <EMI ID = 381.1> <EMI ID = 382.1>

  
then 40 ml of ethanol. The mixture is stirred for 30 minutes and then poured onto 2 l of ice. The pH of the aqueous mixture is adjusted to 7 with 2N potassium hydroxide. The aqueous mixture was entered three times with 400 ml of ether and then acidified to pH 3 with solid citric acid. The mixture is extracted three times with 500 ml of ether. The ethereal extracts are combined, extracted, dried over magnesium sulfate and evaporated in vacuo to a syrup (44.76 g, 84% of the theoretical yield). The syrup is dissolved in 450 ml of ethyl acetate and 25.78 ml is added.

  
 <EMI ID = 383.1>

  
friction, crystallization takes place. The title compound, 51.05 g (66%), m.p. 131-134 [deg.] C.

  
 <EMI ID = 384.1>

  
 <EMI ID = 385.1>

  
C, 59.35; H, 8.39; 'he, 7.29

  
 <EMI ID = 386.1>

  
 <EMI ID = 387.1>

  
ether). The solution is cooled to -15 [deg.] C and 18.3 ml are added.
(0.14 mole) of isobutyl chloroformate and 15.4 ml (0.14 mole)

  
 <EMI ID = 388.1>

  
and anhydrous ammonia is bubbled through the mixture through a gas dispersion tube for 1 hour. We stir the mixture

  
 <EMI ID = 389.1>

  
 <EMI ID = 390.1>

  
aqueous with ether. The ethereal extract is washed with water, cold 0.75N citric acid and water, dried over

  
 <EMI ID = 391.1> <EMI ID = 392.1>

  
of the product of Part B. Dry hydrochloric gas is bubbled through the resulting mixture for 27 min. The mixture was then poured into ether, and the resulting precipitate was collected and dried (9.9 g). The hydrochloride is dissolved in 200 ml of DMF. The mixture is cooled to 0 ° C. and 9.9 ml (0.05 mol) of dicyclohexylamine are added. After 10 min of stirring, 6.8 g are added

  
 <EMI ID = 393.1>

  
phenylalanine and 10.3 g (0.05 mol) of DCC. We stir the mixture

  
 <EMI ID = 394.1>

  
ambient. The mixture is cooled to 0 [deg.] C and filtered. The resulting filtrate is then concentrated in vacuo to an oil. The oil is dissolved in ethyl acetate and the mixture is washed.

  
 <EMI ID = 395.1>

  
 <EMI ID = 396.1>

  
 <EMI ID = 397.1>

  
empty, giving a residue which crystallizes from ether, 16.4 g

  
 <EMI ID = 398.1>

  
 <EMI ID = 399.1>

  
 <EMI ID = 400.1>

  
 <EMI ID = 401.1>

  
Found, C, 58.76; H, 7.42; N, 10.30

  
 <EMI ID = 402.1>

  
To a mixture of 20 ml of glacial acetic acid, 2 ml of anisole and 2 ml of triethylsilane is added 3.5 g (8.56 mmol) of the product of part C. Hydrochloric gas is bubbled through. anhydrous in the mixture for 25 min. Ether is added to the mixture and the hydrochloride is precipitated which is filtered.

  
 <EMI ID = 403.1>

  
 <EMI ID = 404.1> <EMI ID = 405.1>

  
magnesium sulfate and evaporated 1 [deg.] under vacuum. The residue is chromatographed on Grace and Davidson silica gel of grade 62, this

  
 <EMI ID = 406.1>

  
 <EMI ID = 407.1>

  
Analysis, calculated for C34H48N608S (700.86):

  
 <EMI ID = 408.1>

  
Found, C, 58.05; H, 6.62; N, 11.73

  
Amino acid analysis, found: Tyr, 1.00; Ala, 1.01; Gly, 0.99; Phe, 1.00; NH3, 1.01.

  
 <EMI ID = 409.1>

  
8.3 g (0.012 mol) of the product from part D are added to 15 ml of thioanisole. The mixture is cooled to 0 [deg.] C and added.

  
 <EMI ID = 410.1>

  
 <EMI ID = 411.1>

  
 <EMI ID = 412.1>

  
 <EMI ID = 413.1>

  
 <EMI ID = 414.1>

  
 <EMI ID = 415.1>

  
of DEAE Sephadex A-25 (acetate form) previously balanced

  
 <EMI ID = 416.1>

  
 <EMI ID = 417.1>

  
buffer. The residue is dissolved in about 200 ml of acetic acid

  
 <EMI ID = 418.1>

  
a 3: 1 mixture of water and acetonitrile gives 6.64 g (83%) of

  
 <EMI ID = 419.1>

  
 <EMI ID = 420.1>

  
 <EMI ID = 421.1>

  
 <EMI ID = 422.1>

  
 <EMI ID = 423.1>

  
 <EMI ID = 424.1>

  
The analgesic activity of compounds of formula I is demonstrated by the hot plate test on the sciais. In this try a

  
 <EMI ID = 425.1> <EMI ID = 426.1>

  
test and then place the mouse on the surface of the hot plate. The latency time is then noted, in seconds until the appearance of each of two different phenomena. The latency time is measured first until the mouse lifts its hind legs and then the latency time until the mouse jumps off the surface of the hot plate. An agent exhibiting analgesic activity produces an increase in these two latency times compared with those of control mice which are injected only with the carrier. This should occur for a dose interval which does not produce incoordination or motor disability. The following tables give the results obtained in this test,

  
by comparing them with a control, with natural enkephalin and with natural enkephalin transformed into amide. Table I gives the latency time for the lifting of the hind leg; Table II gives the latency time until the jump marking the

  
 <EMI ID = 427.1>

  
percentage of animals in each test group that exhibit analgesic effect. The criterion for asserting that there is an analgesic effect is as follows: the latency time for raising the hind legs or jumping in a treated animal must be equal to or greater than the mean control latency time plus two standard deviations of the average. Each result provided in Tables I and II represents the mean value plus or minus the standard error and Table III gives the percentage obtained for a number of mice giving at least 9 up to 40.

  

 <EMI ID = 428.1>


  

 <EMI ID = 429.1>
 

  

 <EMI ID = 430.1>


  

 <EMI ID = 431.1>
 

  

 <EMI ID = 432.1>


  

 <EMI ID = 433.1>
 

  

 <EMI ID = 434.1>


  

 <EMI ID = 435.1>
 

  

 <EMI ID = 436.1>


  

 <EMI ID = 437.1>
 

  

 <EMI ID = 438.1>


  

 <EMI ID = 439.1>
 

  
Notes

  
at. Unless otherwise indicated, the tests are carried out using a control which is the saline solution. The numbers "1" and "2" appearing in upper indices indicate that the

  
 <EMI ID = 440.1>

  
 <EMI ID = 441.1>

  
present as a suspension.

  
vs. The designations refer to the following compounds:
A. L-tyrosyl-D-alanyl-glycyl-L-phenylalanyl- hydrochloride

  
 <EMI ID = 442.1>

  
B. L-tyrosyl-D-alanyl-glycyl-L-phenylalanylL-methionylamide hydrochloride (Example 3)

  
C. L-tyrosyl-D- hydrochloride (1.25 HCl) monoacetate

  
 <EMI ID = 443.1>

  
5).

  
 <EMI ID = 444.1>

  
 <EMI ID = 445.1>

  
EJ = escape jump

  
e. When tested at 0.3 mg / kg, the lag time for hind leg lift is 33.9 + 2.3 seconds, the time to

  
 <EMI ID = 446.1>

  
Analgesic response is 10% for HPL and 60% for EJ.


    

Claims (1)

<EMI ID = 447.1> 1. Compound of general formula <EMI ID = 448.1> <EMI ID = 449.1> acceptable, where L and D, when they apply, define <EMI ID = 450.1> R. is an atose of hydrogen or a primary alkyl group <EMI ID = 451.1> R2 is a hydrogen atom or a primary alkyl group <EMI ID = 452.1> a hydrogen atom; <EMI ID = 453.1> <EMI ID = 454.1> <EMI ID = 455.1> <EMI ID = 456.1> <EMI ID = 457.1> <EMI ID = 458.1> <EMI ID = 459.1> R7 is a hydrogen atom or a primary alkyl group <EMI ID = 460.1> <EMI ID = 461.1> 0 n <EMI ID = 462.1> 2. A compound according to claim 1, where Y is an atom <EMI ID = 463.1> of hydrogen. <EMI ID = 464.1> hydrogen atoms. <EMI ID = 465.1> of hydrogen. 5. A compound according to claim 1, wherein R4 is a methyl group. 6. A compound according to claim 1, wherein R. is a hydrogen atom. <EMI ID = 466.1> of hydrogen. 8. A compound according to claim 1, wherein Z is a group O -C-NHR8. <EMI ID = 467.1> of hydrogen. 10. A compound according to claim 1, wherein W is a group -CH2-X-CH3. 11. A compound according to claim 10, wherein X is sulfur. <EMI ID = 468.1> <EMI ID = 469.1> 13. A compound according to claim 12, wherein R7 is a methyl group. <EMI ID = 470.1> methionyâamide, or its monoacetate - sesquichlorhydrate. 16. L-Tyrosyl-D-alanyl-glycyl-L-phenylalanyl-L-methionylamide, or its hydrochloride. <EMI ID = 471.1> <EMI ID = 472.1> 1 which consists in cleaving the blocking groups of a corresponding protected compound of formula I, with an essentially dry acidic medium. 23. A pharmaceutical composition comprising an excipient and, as an active ingredient, a compound of formula I according to claim 1. 24. A compound according to claim 1, substantially as described above with particular reference to any one of the examples. 25. The method of claim 22, substantially as described above with particular reference to any one of the examples. 26. A pharmaceutical composition according to claim 23, in substance as described above with particular reference to any one of the examples.