NO771031L - PRODUCTS AND PROCEDURES FOR THE PREPARATION OF SCHIZOFRENI TREATMENTS. - Google Patents

Abstract

Method and product for the treatment of schizophrenia.

Description

The invention relates to compounds and preparations which

is suitable for the "treatment of schizophrenia. More particularly, the invention relates to the treatment of schizophrenia using polypep-

times, omag-N-acylated and/or hydrocarbon-containing esters of polypeptides. The poppy peptides have from 3 to approx. 8 alpha-amino acid groups and a characteristic structure, particularly N-acylated T-V-L-containing polypeptides and their corresponding amides which

e.g. N-acyl-threonylvalylleucinamides.

Schizophrenia is the most serious of all mental illnesses.

judgments that the psychiatrist has for treatment. The frequency is about

trained 1 per 1000 and prevalence around 1 per 100. The disease is responsible for 25 - 50% of all enrollments in the state's

houses for the mentally ill and psychiatric treatment in regular hospitals (U.S.A.) (Kolb, Modern Clinical Psychiatry, page 311 -

312, 1973).

Most patients who develop the disease experience

a course that is slow and insidious, although a smaller proportion of patients may have an acute development. The disease charac-

seen by disturbance of the emotional reactions together with hazy or inadequate expressions; by disturbance of

the ability to think characterized by. inability to reach a goal, by dividing relationships, by disturbed ability to associate, blocking mechanisms, neologisms, etc., by withdrawal from the environment and preoccupation with inner thoughts, by loss of the ability to experience joy, and most often by perceptual disturbance as gross' set is expressed in hallucinations and imaginings.

Unfortunately, schizophrenia most often begins in it

late adolescence or early adulthood and despite the use of antipsychotic drugs, schizophrenia will most often be a permanent condition and incapacitate the person more or less throughout his or her lifetime.

There is now much evidence that the disease is genetically determined, although environmental factors, particularly early life experiences, are undoubtedly also important. There seems to be no doubt that there is a fundamental metabolic disorder which will be described in more detail below.

Unfortunately, it cannot be proven absolutely that there is an animal model for schizophrenia studies since you cannot talk to the animals to find out if they are schizophrenic. The Macawue monkeys at the Wisconsin Primate Laboratory who are maternally and sensory disturbed (they are anxious, aggressive, and unable to function sexually) closely resemble patients with schizophrenia (Harlow, "The Development of Patterns of Affection", affection monsters), Thomas William Salmon Lectures, New York Academy of Medicine, New York, December 1960). The most severely developmentally disturbed monkeys have been studied and show a change in alpha-2-globulin, described later, in the blood, similar to a disturbance that characterizes such human patients (Beckett, Frohman et al., "Schizophrenic-like Mechanisms in Monkeys" (schizophrenia-like mechanisms in monkeys), The American Journal of Psychiatry, 119:835 - 842, 1943). Furthermore, the monkeys showed electroencephalographic disturbances, particularly in the septum of the brain, similar to what has previously been described as a characteristic feature of schizophrenia patients (Heath: "Electroencephalographic Studies in Isolation-raised Monkeys wit Behavioral Impairments" disturbed monkeys reared in isolation),

Diseases of the Nervous System, 33:157-163, 1972).

The use of animals has so far mainly been to demonstrate the presence of abnormal metabolic substances in the blood of patients with schizophrenia. When abnormal blood protein is given to rats in a food-reward and rope-climbing situation, the rats will significantly reduce their performance and activity (Bergen and colleagues: "Further Experiments with Plasma Proteins from Schizophrenics" (further experiments with plasma proteins from schizophrenia patients) , in: Serological Fractions in Schizophrenia, by R. E. Heath, page 67, Paul B. Hoeber, Inc., Medical Book Department of Harper & Row, New York, 1963).

There are certain indications that extracts from the limbic system in the brains of demented patients, injected into normal volunteers, produced a simulated schizophrenic reaction (Garey: "Focal Electroencephalographic Changes Induced by Anti-septal Antibodies" septal antibodies), Biological Psychiatry, 8:75-88, 1974). The clearest investigations involve the injection of isolated alpha-2-globulin from the blood of schizophrenia patients in microliter quantities into the ventricles of rats that have implanted electrodes in the middle forebrain bundle. Rats with such implantation will engage in pushing down a bar as often as they can at the expense of all other activity. When microscopic amounts of isolated alpha-2-globulin are injected into the ventricle, there is a reduction in rod-pressing activity that does not occur when a similar compound from a control subject is injected. Some of the rats injected with blood protein isolated from schizophrenia patients could swing over the bar as if to press it down, swing around and be unable to continue, resembling a schizophrenic disordered motor state. The interpretation of the reduced ability to push down the bar is that the animal has lost the ability to experience pleasure much like that which characterizes schizophrenia patients (Caldwell et al.: "The Effeet of the S-protein on Intracranial Self-Stimulation in-the Rat", (The effect of S-protein on intracranial self-stimulation in the rat), Biological Psychiatry, 8:235-244, 1974).

For many years, the doctor has been engaged in extensive research to find the cause of schizophrenia on a biochemical basis and to find psychopharmacological treatment methods. During this work, it was found that a protein with a high molecular weight, a molecular weight of approx. 263,000 and with approx. 80% fat content, is more active in schizophrenia patients than normal people, and the faster a schizophrenia patient breaks down, the more active the protein is. This protein is classified as an alpha-2-globulin and has been given the designation S-protein: Frohman, CE. L. K. Latham, P.G.S. Beckett and J.S. Gottlieb: "Biochemical Studies of a Serum Factor in Schizophrenia"

(Biochemical studies on a serum factor in schizophrenia), Molecular Basis of Some Aspects of Mental Activity, Volume 2, 0. Walaas, Publisher, Pages 24-1 - 255, Academic Press , New York

(1967) and CE. Frohman: "Studies on the Plasma Factors in Schizophrenia", Mind as a Tissue, C. Rupp, author, pages 181-195, Hoeber Med. Div., Harper & Row, New York. S-protein will, when given to e.g. rats block the experience of pleasure stimuli in rats, but does not appear to change their avoidance responses.

Attempt to reduce the activity of the S protein

in schizophrenia patients has consisted of taking extracts from brains such as bovine brain, pineal glands from meat, etc., during the search for compounds that regulate the activity of the S protein. Frohman et al. in: "Control of the Plasma Factor in Schizophreniz", Recent advances in Biological Pshychiatry, volume 7, pages 45 - 51, 1964, describe that an isolated protein from animal tissue counteracted the activity of S - protein. This protein is called anti-S protein and is found in both animal and human tissues. In schizophrenic patients, S protein is found in an alpha-helical configuration, while in normal individuals the S protein is predominantly present as a random chain or with a (3-helical (p-helix)) configuration. The production of therapeutic amounts of anti-S protein presents great difficulties and large costs and recovery for general medical

use cannot be carried out in practice. Production of a few

a few milligrams of anti-S protein from cattle will require the slaughter of, for example, 100,000 cattle. Thus, natural supply of anti-S protein is in no way sufficient for the treatment of other than a few individuals and would entail unimaginable costs. Other publications relating to these basic investigations are as follows: CF. Frohman, R.E. Arthur, H.S. Yoon and J.S'. Gottlieb: "Distribution and Mechanism of Action of the Anti-S-Protein in Human Brain", Biological

Psychiatry, Vol. 7, No. 1, pp. 53 - 61, 1973, and CR. Harmison and CE. Frohman: "Conformational Variation in a Human Plasma Lipoprotein", Biochemistry, Volume 11, No. 26, Pages 4485 - 4493, 1972.

It has also been noted in investigations in which the patients have participated that extracted anti-S protein is an antigen. While schizophrenia can be dramatically attenuated by giving anti-S protein to a patient, the anti-S protein will be rapidly inactivated by the formation of antibodies and subsequently administered anti-S protein can be inactivated so quickly that it has no apparent anti-schizophrenic effect . The applicant has been engaged in research to break down the anti-S protein into molecular fragments that can have a useful effect in the treatment of schizophrenia without being exposed to the unwanted antigenic activity.

According to the present invention, treatment of anti-S protein with e.g. pepsin and typsin, which reduce the protein to smaller peptides with a molecular weight below approx. 1,000, give a tripeptide-containing fraction which has been shown to be active at

to reduce the unwanted activity of S-protein. The tripeptide exhibits an activity by causing the conversion of alpha-helix-S protein to the random chain configuration. The tripeptide is characterized as threonyl-valyl-leucine. Work has been done to synthesize the tripeptide and a substantially pure, i.e. crystalline, form of the tripeptide has been produced. The tripeptide has an effect against S-protein in schizophrenia patients, i.e. the alpha-helix configuration of the S-protein is transferred to the random configuration. When the compound at forsbk is administered to counteract the action of S protein in rats,

however, the effective duration of action for the pripeptide is small and

therefore, frequent periodic doses of the tripeptide will be nbd-friendly in the effective treatment of schizophrenia.

According to the present invention, it has also been found that T-V-L peptides and their derivatives can be used to attenuate or suppress schizophrenia symptoms in mammals having such symptoms. These polypeptides contain residues of at least three alpha-amino acids and have the indicated T-V-L structure. The polypeptide can be an amino acid, i.e. have one terminally placed amino acid group in acid form and the other terminally placed amino acid group in alpha-amino form (-NF^). However, it is preferred that the polypeptides used to suppress symptoms of schizophrenia contain at least one of the terminal amino acid groups in derivative form, in particular that the terminal alpha-amino group is acyl-substituted or the terminal carboxylic acid group is in amide or ester form. Preferably, both end groups are substituted in this way and acylated amides are particularly preferred. These polypeptides can also be further substituted and their pharmaceutically non-toxic derivatives such as acid or base salts or derivative forms can be used to alleviate symptoms of schizophrenia according to the present invention.

T-V-L-containing polypeptides according to the invention prove effective in the treatment of schizophrenia symptoms by restructuring the alpha-helix configuration of S protein found in the nervous system, e.g. the brain, in schizophrenia patients, to the randomly oriented form. Polypeptides or acylated polypeptides, esters or amides have struc-

where Ra denotes a saturated aliphatic hydrocarbon group such as alkylene with 1-20 C atoms, preferably lower alkylene with 1-4 C atoms; R13 denotes -0(R<e>) where Re is hydrogen or

a saturated aliphatic hydrocarbon such as alkyl of 1-20 C atoms,

or preferably lower alkyl with 1-4 C atoms, or aryl or aralkyl with 1-4 rings, preferably 6-24 C atoms, or R "denotes -NR R where R and R may be the same or different and may be hydrogen or lower alkyl with eg 1-6

c d

C atoms, or R and R can be lower alkylene and together form a cyclic structure including the nitrogen atom to which they are attached; A and B are both a monoiminoacyl group, preferably an oc-monoiminoacyl group containing 2-10 C atoms and B is hydrolyzable from the (L) component of the essential T-V-L-• structure so that the carboxyl function in the (L) component becomes

available at the host; p is equal to 0 or 1; t is 0 to 5;

x is 0 to approx. 5; and t plus x is from 0 to 5. When t is 2 or more then all A-groups can be the same or different, e.g. can (A)^. be phenylalanylprolyl. Similarly, when x is 2 or more, all B groups can be the same or different. In a preferred group of substances - R° is equal to -NR°R^ or at least one of p, t or x is different from zero. The (T) component shown in the above structural formula can exist either in diastereoisomer form, e.g. such as threonyl or allotreonyl whose stereochemistry at the cc carbon. is reversed. Preferably, the (T) component is threonyl. (L) component i

•the structural formulas can be

(usually designated leucyl), which is preferred, or take another form such as

The compounds according to the invention include substances where any stub stituent, e.g. a monoiminoacyl radical, denoted here by B, on the carboxyl group in the (L) residue in the essential T-V-L-fblge is hydrolyzable. In order to achieve the desired activity, the substituent must be hydrolyzable under the conditions that prevail in the host environment in question. Suitable hydrolysable substituents are those which are hydrolysed in the presence of red blood cells at an incubation temperature of around the host's body temperature, i.e. approx. 35 - 40°C. A suitable method for determining whether a substituent is hydrolyzable or not consists of incubating a compound containing the essential T-V-L structure and a substituent on the (lu residue) in a liver medium at about 37°C

The peptide residues in compounds according to the present invention comprise peptide groups which have optical

Activity. Different peptide groups can have configurations labeled L- or B-. Monoiminoacyl groups with meaning A in formula II and the essential T-V-L part of the compounds can be in L or D form or mixtures thereof

as racemic mixtures. Because of frequent difficulties in hydrolyzing D-peptides which are substituents on carboxyl functions in peptides while maintaining the carboxyl function, monoiminoacyl radicals containing group B in formula II will advantageously not have optical activity or, when optically active, will be present in L configuration. Among preferred compounds are those where the (L) group in the essential T-V-L sequence has D configuration.

The tripeptide threonyl-valyl-leucine or its derivatives as described, e.g. the corresponding amides can be produced in a relatively high degree of purity and can be crystalline, e.g. be approx. 90 - 95% pure, and preferably around 100% pure.

Various compounds of formula II can be intermediates for the production of substances that are active in the treatment of schizophrenia. When e.g. p is equal to zero and R*3 is hydrogen, the compound may be an intermediate for the preparation of a compound where p is 1 and/or R is different from hydrogen. Compounds that are particularly beneficial for the treatment of schizophrenia are polypeptides where p is equal to 0 or 1 and polypeptides containing at least three peptide or amino acid residues and t is equal to 0 or more, in amino acid or derivative form. Polypeptides where at least one of the parameters p,. t or x is different from zero may exhibit a longer period of activity than when the tripeptide T-V-L is in amino acid form.

Preferred acylated polypeptides for use as active agents or their intermediates have the structure:

where the group 4NH-CH-C-) is a monoiminoacyl radical and R e.g.

in

R<1>

hydrogen or a lower alkyl group such as e.g. has 1 - 4 C-

atoms or a

2 ^5

group where R and R-^ denote hydrogen

or lower alkyl with e.g. 1-4 C atoms, or denotes aryl or aralkyl with 1-4 rings and preferably with 6-24 C atoms. Such groups can be substituted as e.g. in

a b

tyrosyl. The symbols R , R , p, t and x have the same meaning as in formula II.

In compounds of formula III, R^ forms part of an alpha-amino acid or peptide residue such as e.g. in threonyl or allotreonyl where R^" denotes an alpha-hydroxyethyl group, leucyl where R"*" is a 2-methylpropyl group, valyl where R denotes an isopropyl group, seryl where R"'" denotes a hydroxymethyl group, isoleucyl where R"* " denotes 1-methylpropyl, glycyl where

R"<*>" denotes hydrogen, or alpha-alanyl where R"*" denotes methyl,

arginyl where R"*" denotes

tyrosyl where R"*"

and. the like. When the group containing R<1>

has an alpha-hydroxy group, it can be transferred to a lower alkoxy or aryloxy group, preferably benzyloxy or t-butyloxy. This can be done during synthesis to protect

the hydroxyl group. Peptide residues such as monoiminoacyl radicals which are bound to the essential T-V-L structure can also be prolyl, arginyl and the like. Furthermore, these groups can be a dicarboxylic acid residue such as e.g. a glutamic or aspartic acid residue.

The R group in the present compounds can have up to 20 C atoms, although the alkylene group preferably has up to 4 C atoms.

0

IN!

Thus, the N-acyl group H-(Ra<->C) can e.g. be acetyl propanoyl, butynoyl, pentanoyl, hexanoyl, heptanoyl, octanoyl, nona-noyl, decanoyl, undecanoyl, dodecanoyl, tridecanoyl, tetradecanoyl, pentadecanoyl, palmitoyl, heptadecanoyl, stearyl, etc. The ester group R can be methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, heptadecyl, octadecyl, phenyl, benzyl, tertiary butoxycarbonyl, t-butyl , dihydro-epi-hydroxy-sy-androsteronyl and others. Preferably, the N-acyl group and the ester group are normal groups and thus have straight C-C chains. The compounds of the formulas in question or their intermediates may have substituents which do not affect their chemical or pharmacological activity.

Intermediate products for the production of active substances according to the invention are compounds with formula

where r, x, A and B have the previously stated meaning, D denotes H or an α-amino protecting group, E is equal to -OH, a terminally placed carboxylic acid group, a metal complex of e.g. zinc, copper, nickel, cobalt, iron, magnesium or aluminium, and preferably a metal hydroxide complex, or an acid or base addition salt, R denotes H, aliphatic hydrocarbon, preferably saturated, for example alkyl with 1-20 C atoms as lower alkyl with 1-4 C atoms or an araliphatic

or aryl group with 1-4 rings and preferably 6-24 C atoms, acyl incl. alkanoyl or aralkanoyl with 1-20 C atoms, preferably lower acyclic acyl with 1-4 C atoms, tetrahydropyranyl, a monovalent metal such as an alkali metal of the type sodium etc. A metal such as zinc,. copper, nickel, cobalt, iron, magnesium or aluminum can form a complex with functions of the lead peptide, such as e.g. a carbonyl group.

The oc-amino protecting group can be (1) of the acyl or thioacyl type, with preferably 2-21 C atoms, e.g. lower aliphatic acyl with 2-7 C atoms, for example acetyl, etc; lower araliphatic acyl with 8-15 C atoms such as phthalyl, naphthoyl, benzoyl etc; trifluoroacetyl; chloroacetyl; Y-chloro-butyryl; toluenesulfonyl; benzenesulfonyl; nitrophenylsulfenyl; tritylsulfenyl; o-nitrophenoxyacetyl etc., (2) an aromatic urethane group illustrated by benzyloxycarbonyl and substituted benzyloxycarbonyl such as p-chlorobenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl, (3) aliphatic urethane as a protecting group illustrated by tert-butyloxycarbonyl, diisopropylmethoxycarbonyl, isopropyloxycarbonyl,. ethoxycarbonyl, allyloxycarbonyl, (4) cycloalkyl urethane protecting groups as illustrated by cyclopentyloxycarbonyl, cyclohexyloxycarbonyl, (5) thiourethane protecting groups such as phenylthiocarbonyl,

(6) alkyl and aralkyl protecting groups as illustrated by triphenylmethyl (trityl) and benzyl, (7) trialkylsilane groups such as trimethylsilane. The end-placed carboxylic acid groups can be (.1) -OG where G e.g. denotes an aliphatic group with preferably 1-20 C atoms or an araliphatic group with 1-4 rings and preferably 6-24 C atoms, and containing an ester terminated with a carbon-containing radical or acyl such as lower acyclic acyl preferably with 1-6 C atoms, (2) an amide-containing group such as -NH^, aminoaliphatic or amino-araliphatic group (for example monocyclic araliphatic) with 1 - 10 C atoms, thus the amine can be primary, secondary or tertiary, for example -NR cR d where R c and R d have the meaning as above, or (3) a means of anchoring which

resin as carrier material; - 0 - CHp - CgH^- resin support material, chloromethylated resin, hydroxymethyl resin, Merrifield resin, etc. Preferably, said aliphatic or araliphatic groups are hydrocarbyl.

Polypeptides according to the invention, e.g. substances with formula II can be used to eliminate symptoms of schizophrenia in humans and mammals and it is assumed that this occurs by reducing the activity of S-protein, i.e. by producing a reduction in the degree of alpha-helix configuration in The S-protein in the warm-blooded animal. These active substances can be effective in counteracting symptoms of schizophrenia and their forms of expression such as nervous tension states, manic-depressive psychosis, etc., when given pharmacologically internally to a living animal. When providing an active compound according to the invention, e.g. mixed with a pharmaceutical carrier, the preparation is given internally, e.g. in the digestive tract, and parenteral administration is preferred. The active compound can be in solid or liquid form and. can be combined with a pharmaceutical carrier such as e.g. is in solid form, solution, suspension, emulsion or other form. Parenteral administration can take place e.g. subcutaneous, intraperitoneal, intravenous, intraarterial, intramuscular or the like. For oral administration, the active compound and the pharmaceutical carrier can e.g. be a pill, drops, tablet, capsule or liquid suspension. Examples of carriers are solid types such as lactose, magnesium stearate, calcium stearate, starch, terra alba, dicalcium phosphate, sucrose, talc, stearic acid, gelatin, agar, pectin or acacia gum and liquids such as sesame oil, olive oil, water etc. An enteric coating such as cellulose acetate phthalate can be used. The compounds according to the invention may also contain preservatives, stabilizers, wetting agents, emulsifiers and buffers or salts etc. The active substances used according to the invention or preparations containing them can either be given together with or contain other physiologically active substances or drugs such as e.g. buffers, acid scavengers, sedatives, analgesics, hormones etc.

Varying doses of the active substances can be used depending on the chosen compound, the condition, the route of administration, the desired effect on the host, etc., the species of the mammal, etc. Thus, the amount of active substance can vary, but should be sufficient to cancel schizophrenia symptoms in the mammal to a significant degree. The doses will generally vary between at least approx. 0.01 mg per kg per day (milligrams per kg body weight per day) e.g. of up to 2 or more mg per kg per day and preferably between approx.; 0.1 and 1 mg per kg per day.- The treatment can consist of a single daily dose or the above doses can be given divided over a period, e.g. 2-4 doses to approx. 0.05 - 0.2 mg per kg can be given per day. The active compound can be incorporated into long-acting preparations so that these amounts are made available to the body over a longer period of time. The activity time can be extended by incorporating the polypeptides into organic and usually polymeric compounds such as gelatin, polychloroethene phosphate or polyglutamic acid.

The compounds according to the invention, including such as

are active in the treatment of schizophrenia symptoms or their precursors or intermediates can be synthetically produced from individual amino acids or from polypeptides, forming the desired T-V-L peptide sequence. According to a synthesis route, amino acid components are reacted in the desired order to produce the peptide structure, e.g. the amine group in a first amino acid can be condensed with the carboxylic acid group in a second amino acid to form a dipeptide structure that can be further converted to the desired structure with 3 - approx. 8 amino acid groups or units. In order to arrive at the desired combination of amino acid groups, it is usually necessary to block or protect the places on the amino acid molecule that could give rise to unwanted side reactions, e.g. the carboxylic acid group in a first amino acid that could react with the amine group in a first amino acid or a second amino acid. Such blocking can take place in a known manner. Particularly favorable protecting groups for the amino group on amino acids are α-amino protecting groups of the type phthalyl, carbobenzyloxy and t-butyloxycarbonyl, because they can be easily attached to the amino group without causing racemization of the amino acid, are relatively inert to the reaction conditions and can easily be removed from the amino group without adverse effect on the amino acid. The protecting groups are substituted as acid halides of the chloride, ester or acid anhydride type, e.g. a mixed acid anhydride with alkyl-

formate, for example lower alkyl formate. The reaction can be carried out at room temperature in an inert organic medium such as benzene. Higher or lower temperatures of between 0 and 50°C can possibly be used. The carboxylic acid group on the amino acid can be blocked by reaction with the amino group in the preceding amino acid in the peptide series or with another suitable blocking group when the acid is the first acid in the series.

For easy extraction of the amino acid synthesis product after each reaction step, it may be advantageous to use the first or final amino acid group as a resin ester or resin amide. Particularly favorable resins are e.g. benzhydrylamine resins, chloromethylated resins, Merrifield resin and hydroxymethyl resin. Preparation of benzhydrylamine resin is described by Rivallile et al., Heiv. 54, 2772 (1971), and the preparation of hydroxymethyl resin is described by Bodanszky et al., Chem. Ind. (London) 38, 1597-98 (1968).

The protective groups must be stable in the reaction medium under the reaction conditions used to produce the peptide. Protecting groups for terminal carboxylic acid groups should be stable under the conditions used to remove the alpha-amino protecting group at each synthetic step. The blocking group must retain the protective properties, i.e. not be cleaved off under the synthesis conditions and when they are removed after completion of synthesis, the reaction conditions used must not change the peptide chain in an undesired way. The blocking groups can be removed in various known ways depending on the type of group used, e.g. with trifluoroacetic acid, by hydrogenolysis with, e.g. hydrogen and a catalyst such as platinum, palladium or the like or with hydrogen bromide in glacial acetic acid or trifluoroacetic acid or with anhydrous hydrofluoric acid. Hydrogenolysis medium is preferably water-free.

The reaction between a present peptide unit and a subsequent amino acid can take place in the solid phase. When producing resin esters or amides, the resin support material and the amino acid can be carefully mixed in an inert organic medium such as e.g. tetrahydrofuran, dioxane, dimethylformamide, benzene, ethanol etc. The reaction proceeds at room temperature even if higher temperatures of e.g. between 0 and 80°C can possibly be used. The reaction is usually continued to completion to maximize the yield of peptide product. Use of substantial excess of amino acid reactant, e.g. excesses of at least 1.5 - 200 or more times the required stoichiometric amount and long reaction times of at least 1 hour and preferably 5,500 hours or more give increased yields. Whether the reaction is complete can be determined by the Kaiser color reaction. A positive Kaiser color reaction indicates unsubstituted groups on the amino acid. Dorman titration can also be used to determine if the reaction is complete.

Suitable binding methods for producing the polypeptide chain of compounds according to the invention are described in the literature. In general, the amino acid and/or peptide fragments are bound to each other so that e.g. an amino acid or peptide containing a protected alpha-amino group and a terminal carboxyl group is reacted with an amino acid or peptide containing a free alpha-amino group and a protected terminal carboxyl group, or an amino acid or peptide containing an active alpha-amino group and a protected terminal carboxylic acid group is reacted with an amino acid or peptide containing a free terminal carboxylic acid and a protected alpha-amino group. The carboxylic acid group can be activated, e.g. by conversion to an acid azide, -anhydride or -imidazolide or to an activated ester such as cyanoethyl ester, thiophenyl ester, p-nitrothiophenyl ester, thiocresyl-, p-methanesulfonylphenyl-, p-nitrogenyl-, 2,4-dinitrophenyl-, 2,4 ,5- or 2,4,6-trichlorophenyl-, pentachlorophenyl-, N-hydroxysuccinimide-, N-hydroxyphthalimide-, 8-hydroxyquinoline-, N-hydroxypiperidine ester, or by reaction with a carbodiimide (possibly with the addition of N- hydroxysuccinimide) or N,N'-carbonyldiimidazole or an isoxazolinium salt, e.g. "Woodward's" reagent, and the amino group is activated, e.g. by reaction with a phosphite. Frequently used methods include the carbodiimide method, the Weygand-Wuensch method (carbodiimide in the presence of an N-hydroxysuccinimide), the azide method, the method with activated esters and the anhydride method, and further the Merrifield method and the method with N-carboxycyananhydrides or N-thiocarboxylic anhydrides .

The carbodiimide method can be used with advantage and the carbodiimide coupling agent can e.g. be N-ethyl-N'-(~)f-dimethylamino-propyl-carbodiimide) or a dihydrocarbyl-carbodiimide such as e.g. a dialkyl with 1-20 C atoms, for example dicyclohexylcarbodiimide. The carbodiimide can be placed in an inert solvent such as e.g. methylene chloride to facilitate turnover. Often the molar ratio between carbodiimide and the number of moles of the least abundant amino acid is approx. 0,):1 to 10:1. The binding reaction can be carried out in an inert medium such as dichloromethane, in quantities which provide sufficient solvent, e.g. 1 - 100 ml or more per grams of amino acid. The reaction proceeds at room temperature, although one can use higher or lower temperatures of e.g. between -30 and +50°C as desired.

Release or cleavage of protecting groups from an amino acid or a peptide fragment for further reaction can be carried out in any known manner. Advantageously, blocking agents on amino groups, e.g. acyl protecting groups are removed in two steps with a hydrogen halide such as hydrogen chloride, in each step. The product is the acid addition salt such as the hydrogen chloride, which can be neutralized with a base such as triethylamine, pyridine, sodium or potassium hydroxide, sodium or potassium carbonate, etc. The hydrogen halide can be prepared by bubbling hydrogen halide through a liquid medium such as e.g. dioxane or methylene chloride. A resin ester can be converted to an unprotected peptide by bubbling through hydrogen halide such as hydromide gas in a medium containing peptide resin ester<p>g trifluoroacetic acid.

As mentioned above, the aim is to modify the polypeptide chain containing the T-V-L sequence into a compound that shows a longer effective lifetime in the treatment of schizophrenic symptoms than the amino acid tripeptide and thus the latter polypeptide can be converted into an omega-N-acylated polypeptide. Known acylation methods can be used, although it would be most advantageous to use a process which does not interfere with the optical activity of the amino acid groups. The alpha-amino group on the polypeptide can be acylated by reaction with the corresponding acid halide, or acid anhydride, according to known methods. End-placed carboxylic acid or ester groups can be esterified or transesterified according to known methods for producing a peptide that will show a higher effective dose. Esterification can take place by reacting the polypeptide which has a free terminal carboxylic acid group with the corresponding alcohol. Amides with a characteristic T-V-L sequence according to the invention can be prepared in known ways. E.g. can the corresponding acid or acid halide, e.g. -chloride of the peptide is reacted with ammonia or a primary or secondary amine. Ammonia or amide is often used in maximum stdchiometric amount to achieve complete reaction with the peptide. The reaction can be carried out in an inert solvent such as tetrahydrofuran and at a temperature of about 10 to 50°C or more. The higher the molecular weight of the acyl and ester groups on the peptide, the slower and of longer duration the effect will usually be in the treatment of schizophrenic symptoms.

The functional derivatives of compounds and intermediates according to the invention comprise pharmaceutical salts including acid and base addition salts of the polypeptides. The acid addition salts can be prepared by reacting the polypeptide with an organic or inorganic acid such as hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, acetic acid, maleic acid, tartaric acid, citric acid, malic acid, ascorbic acid, benzoic acid and the like. Compounds with the T-V-L sequence can also be found in the form of metal complexes which are produced by reacting the polypeptides with a sparingly soluble salt, hydroxide or oxide of the metal. Metals that can be used include cobalt, copper, calcium, iron, zinc, magnesium, sodium, potassium and ammonium. Other metals that can be used are nickel and aluminium. E.g. a metal complex can be obtained by adding the polypeptide and a sparingly soluble metal salt, metal hydroxide or metal oxide to an aqueous medium or by adding an alkaline medium to an aqueous solution of the polypeptide and a substantially insoluble metal salt to form an insoluble polypeptide-metal hydroxide complex . An insoluble polypeptide metal salt complex can also be prepared in situ by adding the polypeptide and a metal salt to an aqueous alkaline medium.

The invention shall be described below by means of examples. In the examples, there are peptide groups with optical activity in L-configuration where nothing else is indicated.

Example 1

Production of leucine-benzyl ester-p-toluenesulfonate

In a 200 ml three-necked flask fitted with a Dean and Stark trap, reflux condenser (with tube) and mechanical stirrer, 15 g (0.0789 mol) p-toluenesulfonic acid^ monohydrate is boiled until one equivalent of water is given off. At this point, 10 g (0.07634 mol) of leucine and 17.0 g (0.15 mol) of benzyl alcohol are added. The mixture is refluxed for 4 hours and allowed to cool to room temperature. The solvents are removed under vacuum and a white, solid residue is obtained. The white substance is washed with anhydrous ether and recrystallized from ethanol/ether and after three crystallization steps yields 28.2 g of leucine benzyl ester p-toluenesulfonate (94% yield) with m.p. 157 - 158°C. (decomp.).

Example 2

Preparation of benzyl-N-q-t-butyloxycarbonylvalylleucinate

About. 13.2 g (0.0336 mol) benzyl-leucinate-p-toluenesul-. phonate is treated with sodium bicarbonate to produce benzyl leucinate, which is recovered by methylene chloride extraction.

In a 200 ml three-necked flask supplied with nitrogen inlnlbpsrbr and

-exhaust and magnetic stirrer dissolve 6.25 g (0.34 mol) of dicyclohexylcarbodiimide in 30 ml of freshly distilled dry CH2CI2 and the worked-up benzyl leucinate is added. The mixture is kept under a nitrogen atmosphere and cooled to approx. -30°C with ice and carbon tetrachloride. 7.45 g (0.0336 mol) of N-a-t-butyloxycarbonylvaline dissolved in 50 ml of dry Ct 2 C 2 are added dropwise to the mixture. The resulting mixture is stored at -10°C for 2 days. After removal of the white precipitate formed by filtration, the methylene chloride layer is washed successively with 25% aqueous acetic acid, aqueous sodium bicarbonate solution, water, and saturated aqueous brine. The organic layer is dried over anhydrous MgSO^ and the solvent is removed in vacuo. The evaporation residue is taken up in ethyl acetate, filtered and concentrated again in vacuo. The solid formed is recrystallized from ethanol/hexane and, after two crystallisation steps, yields 9.17 g of benzyl-N-oc-t-butyloxycarbonylvalyl-leucinate (65% yield), m.p. 90 - 91°C Example 5

Preparation of benzyl-N-cc-t-butyloxycarbonyl-0-benzylthreonyl-valylleucinate

4.08 g (0.00972 mol) of benzyl-N-a-t-butyloxycarbonylvalylleucinate dissolved in 50 ml of glacial acetic acid are filled into a 100 ml three-necked flask equipped with a gas inlet and outlet and a magnetic stirrer. The mixture is cooled to 5°C and hydrogen chloride gas is bubbled through the mixture for 30 minutes. It is then evaporated to dryness in vacuo. The white powder formed, which is benzyl-valyl-leucine hydrogen chloride, is washed several times with anhydrous ether. The powder is suspended in 30 ml of dry Ct 2 C 2 , cooled to -30°C and treated with 1.38 ml (0.010 mol) triethylamine and stirred for 30 minutes until a solution is formed.

The formed triethylamine solution is added to 1.94 g (0.010 mol) of dicyclohexylcarbodiimide dissolved in 30 ml of freshly distilled dry C₂C₂. The solution is kept under a nitrogen atmosphere and 3.0 g (0.00972 mol) of N-a-t-butyloxycarbonyl-O-benzylthreonine dissolved in 30 ml of dry ChL-jC^ is added dropwise. The reaction mixture is kept at -10°C for 2 days. The white precipitate formed is filtered off and the methylene chloride layer is washed successively with water, 25% aqueous acetic acid, aqueous sodium bicarbonate solution, water and saturated aqueous saline. The organic layer is applied; anhydrous MgSO^ and the solvent is removed in vacuo. The residue is taken up in ethyl acetate, filtered and concentrated in vacuo to give 3.56 g of white powder which is

■benzyl-N-a-t-butyloxycarbonylrO-benzylthreonyl-valylleucinate (60% yield, m.p. 115 - 119°C).

Example 4

Preparation of benzyl-O-benzylthreonylvalylleucinate hydrochloride

Hydrogen chloride gas is bubbled slowly for 50 minutes through a cold (5°C) and stirred solution of 4.1 g (6.71 mmol) of benzyl-N-oc-t-butyloxycarbonyl-O-benzyl-threonylvalylleucinate in 50 ml of glacial acetic acid. The solvent is removed under vacuum. The residue is washed with ether and recrystallized from ethanol/ether to give 3.14 g of benzyl-O-benzylthreonylvalylleucinate hydrochloride (86% yield with m.p. 200 - 202.5°C).

Example 5

Production of threonyl-valyl-leucine

1 g of benzyl-O-benzylthreonyl-valylleucinate hydrochloride is neutralized to benzyl-O-benzylthreonyl-valylleucinate. A hydrogenolysis mixture is prepared containing benzyl-O-benzylthreonylvalylleucinate, 0.5 g of 10% palladium on charcoal per

0.01 mole of the compound to be hydrogenated, and absolute ethanol. 1.1 molar equivalents of acetic acid based on the benzyl ester are added to the mixture. The mixture is hydrogenated for 4 hours. The catalyst is filtered off and the solvents are removed in vacuo. The evaporation residue is washed with anhydrous ether and recrystallized from ethanol/ether to give 544 mg of the free peptide, threonylvalyl leucine (85%' yield).

Example 6

Preparation of benzyl-N-q-acetyl-O-benzylthreonylvalylleucinate

Benzyl-O-benzylthreonylvalylleucinate is prepared from

1 g of benzyl-O-benzylthreonylvalylleucinate hydrochloride as in example 5 and dissolved in acetic acid, after which 2 equivalents of acetic anhydride, based on the benzyl ester, are added. The mixture is stirred overnight. The solvents are removed in vacuo and the evaporation residue is recrystallized from methanol/ether to give 806 mg of benzyl-N-α-acetyl-O-benzylthreonylvalylleucinate (85% yield).

Example 7

Preparation of N-cc-acetylthreonylvalyl leucinamide

1 g of benzyl-N-oc-acetyl-O-benzylthreonylvalyl leucinate is dissolved in 95% ethanol. Ammonia is bubbled through the solution

for 8 hours. After stirring at room temperature for 48 hours, the solvent is removed in vacuo. The evaporation residue is washed with ether to give 795 mg (99%) of N-α-acetyl-O-benzylthreonylvalylleucinamide. 1 g of this amide undergoes hydrogenolysis in absolute ethanol to give 705 mg (90% yield) of N-cc-acetylthreonylvalyl leucinamide.

Example 8

Merrifield synthesis of threonyl-valyl-leucine

A solution of 5.3 g of t-butyloxycarbonyl-L-leucine (21.2 mmol), 2.5 ml of triethylamine (18 mmol) in 2-methyltetrahydrofuran as solvent is prepared and 20 g of Merrifield resin, which is polystyrene tyre-bonded with 2% divinylbenzene,

and which gives 21.2 mmol chlorine, is added. The Merrifield resin is analyzed and shows a content of 1.06 mmol chlorine per grams of resin, and before use the resin is washed with methanol, distilled water, with ethanol and methylene chloride and then dried in a vacuum at 100°C. For use, 2-methyltetrahydrofuran is distilled over sodium dispersion and the triethylamine is distilled over phenyl isocyanate and redistilled over sodium dispersion. The mixture is boiled by adding

bakelbp for about 70 hours to carry out esterification of the blocked amino acids to the resin. After the esterification reaction, the resin is washed with tetrahydrofuran, ethanol, glacial acetic acid, ethanol, distilled water, ethanol and methylene chloride and dried in a vacuum at 100°C for 3 hours. About. 21.5 - 22 g of t-butyloxycarbonyl-L-leucine resin ester are prepared.

The t-butyloxycarbonyl-L-leucine resin ester is suspended

in 200 ml of methylene chloride and stirred by bubbling through pure nitrogen, the t-butyloxycarbonyl protecting group is removed by adding 400 ml of a mixture of equal parts of trifluoroacetic acid and methylene chloride. The resin is washed and neutralized with 400 ml of 10% triethylamine in chloroform. The resin is waxed with chloroform and methylene chloride. About. 4.82 g of t-butyloxycarbonyl-L-valine is added to the resin followed by an equivalent amount, i.e. approx.

4.57 g of dicyclohexylcarbodiimide (24 mmol) and the coupling reaction is continued for at least 16 hours. The t-butyloxycarbonyl-L-valyl-L-leucyl resin ester formed is washed with methylene chloride, especially anhydrous ethanol, glacial acetic acid, ethanol and methylene chloride to remove the dicyclohexylurea by-product. Completion of the reaction is checked by the Kaisér color reaction.

The same method as above is used to remove the t-butyloxycarbonyl (boc) protecting group. 5.00 g of t-boc-O-benzyl-L-threo-

nin and an equivalent amount (3.6 g) of dicyclohexylcarbodiimide (18.4 mmol) and the coupling reaction proceeds for 4 hours. The resin ester formed is washed in the same way as before and dried overnight at room temperature in a vacuum over phosphorus pentoxide. About 22 - 23 g of t-butyloxycarbonyl-O-benzyl-L-threonyl-L-valyl-L-leucyl resin ester are prepared.

Cleavage of the tripeptide from the resin takes place by

to suspend the t-butyloxycarbonyl-O-benzyltrebnyl-valylleucine resin ester in 100 ml of 100% trifluoroacetic acid through which anhydrous hydrogen bromide is bubbled. During this process the resin ester bond and at the same time the O-benzyl protecting group on the threonine is cleaved and gives the soluble hydrobromide and trifluoroacetate salts of threonyl valylleucine in trifluoroacetic acid.

The trifluoroacetic acid solution is evaporated to dryness, the resin is dissolved in 100 ml of an equal volume of methanol-distilled water. The mixture is evaporated to dryness under reduced pressure and the evaporation residue is dissolved in 100 ml of ethanol which is again evaporated under reduced pressure. The dry material is dissolved in 10 - 20 ml in as little as possible 30% glacial acetic acid and filtered. Solid sodium carbonate or sodium bicarbonate is added slowly until precipitation appears. The finished tripeptide product is recrystallized from ethanol/water by dissolving the product in a large excess of distilled water, concentrating the solution by distilling off part of the water and carrying out crystal formation in a dresser at approx. 5°C. You get approx. 657 mg L-threonyl-L-valyl-L-

leucine (75% yield) with m.p. range 240 - 242°C (decomp.).

Example 9

Production of L-threonyl-L-valy-D-leucine

The procedure from example 8 is repeated, except that D-leucine is used instead of L-leucine and L-threonyl-L-valyl-D-leucine is produced. The product is a white crystalline mixture which has a decomposition point

about. 240°C under charring.

Example 10

Preparation of N-q-acetyl-threonylvalyl leucine

t-butyloxycarbonyl-O-benzylthreonylvalylleucine resin ester is prepared in the same way as described in example 8. The t-butyloxycarbonyl protecting group is removed on

the same way as the groups on the leucyl resin ester and the valyl leucyl resin ester are removed in Example 8 to form O-benzylthreonyl valyl leucine resin ester. About. 5.0 g of used 0-benzylthreonylvalyl leucine resin ester is acylated by first washing the resin ester with dimethylformamide and then reacting the resin with acetylation reagent consisting of 100 ml 44 parts by volume dimethylformamide, 5 parts by volume acetic anhydride and 1 part by volume triethylamine for 20 - 50 minutes and the resin is washed with dimethylformamide, and with methylene chloride, after which it is dried in vacuo at room temperature overnight.

The N-acetyl peptide is cleaved from the resin as described in example 8. The trifluoroacetic acid solution is evaporated to dryness under reduced pressure. The residue is washed with equal parts methanol and water, evaporated to dryness under reduced pressure, washed with ethanol and worked up by evaporation again. to dryness under reduced pressure. The final product is crystallized from ethanol/ether and gives 400 mg of N-α-acetyl-threonylvalylleucine (55% yield) with melting point 210 - 214° C (decomp.).

Example 11

Merrifield synthesis of the hydrochloride salt of glycylthreonylvalylleucine •From 10 g of O-benzylthreonylvalylleucine resin ester prepared as described in Example 10, the coupling of t-butyloxycarbonylglycine was carried out by suspending the resin ester in 100 ml of methylene chloride, adding 1.94 g of t- butyloxycarbonylglycine (10 mmol), followed by an equimolar amount, i.e. 2.06 g, of dicyclohexylcarbodiimide (10 mmol) and allowed the condensation to proceed for 4 hours. The t-butyloxycarbonyl-glycyl-O-benzyl-threonylvalylleucine resin ester is cleaved from the resin as described in Example 8 above. After suitable washing in methanol, distilled water, ethanol and the addition of sodium carbonate to keep the pH at 4.5, the product is evaporated to dryness under reduced pressure.

The glycylthreonylvalyl leucine is transferred to the hydrochloride salt by dissolving in 100 ml of a mixture of 3 parts by volume ethanol and 1 part by volume ether and bubbling anhydrous hydrogen chloride gas through the solution for approximately 10 minutes. The product is filtered and concentrated overnight at room temperature in a vacuum flask and gives 600 mg of the hydrochloride salt of glycylthreonyl valylleucine (60% yield) with a melting point of 225 - 226°C (decomp.).

Example 12

Merrifield synthesis of N-ct-acetyl-tyrosyl-threonylvalylleucine

O-benzyl-threonylvalylleucine resin ester (prepared as in Example 10) in an amount of 7.4 g is added to a reaction vessel and suspended in 75 ml of methylene chloride. 3.735 g of t-butyloxycarbonyl-O-benzyltyrosine (10 mmol) are added to the solution followed by an equivalent amount of dicyclohexylcarbodiimide (2.06 g = 10 mmol) and the coupling reaction proceeds for 4 hours. Mix by bubbling pure nitrogen through the suspension* The resin is washed with methylene chloride, ethanol, acetic acid, ethanol and methylene chloride again to remove the dicyclohexylurea by-product, and dried overnight at room temperature in a vacuum over phosphorus pentoxide. The dry product O-benzyltyrosyl-O-benzylthreonyl valylleucine resin ester typically increases in weight to 7.50-7.55 g.

The N-acetylation is carried out in essentially the same way as

in Example 10 for the synthesis of N-oc-acetyl-threonylvalylleucine except that 7.5 ml of acetic anhydride, 2.5 ml of triethylamine and 50 ml of dimethylformamide are used. After the N-acetylation, the resin is washed with dimethylformamide, then methylene chloride and dried in a vacuum at room temperature overnight.

Cleavage of the peptide from the Merrifield resin takes place in the same way as described in example 8, except that to avoid possible electrophilic aromatic substitution on the tyrosine ring with bromine, hydrogen bromide is first bubbled through a resorcinol-trifluoroacetic acid mixture in a ratio of 2 g of resorcinol per 100 ml of trifluoroacetic acid to remove traces of Br As a further precaution against electrophilic substitution, 20 ml of anisole is added directly to the cleavage vessel. The final product, N-α-acetyl-tyrosylthreonylvalylleucine is recrystallized from ethanol-ether to give 900 mg of tetrapeptide (5% yield)

with melting point range.234.5 - 235°C (decomp.). Amino acid analysis of the product gives 0.93 tyrosine, 1.00 threonine, 1.00 valih and 1.00 leucine.

Example 13

Merrifield synthesis of threonylvalylisoleucine

A solution of 5.3 g of t-butyloxycarbonyl-L-isoleucine (21.2 mmol), 2.5 ml of triethylamine (18 mmol) in 2-methyltetrahydrofuran as solvent is prepared and 20 g of Merrifield resin is added. Before use, the resin is washed with methanol, distilled water, ethanol and methylene chloride and dried in a vacuum at 100°C. For use, 2-methyltetrahydrofuran is distilled over a sodium dispersion and the triethylamine is distilled over phenylisocyanate and redistilled over a sodium dispersion. The mixture is refluxed for about 72 hours to remove the esterification of the blocked amino acids to the resin. After the esterification reaction, the resin is washed with tetrahydrofuran, ethanol, glacial acetic acid, ethanol, distilled water, ethanol and methylene chloride and dried in a vacuum at 100°C for 3 hours. About. 21.5

- 22 g of t-butyloxycarbonyl-L-isoleucine resin ester are yielded. This coupling of t-butyloxycarbonylvaline to t-butyloxycarbonylisoleucine resin ester takes place in the same way as the coupling of t-butyloxycarbonyl-L-valine to the t-butyloxycarbonyl-L-leucine resin ester as described in Example 8. Due

of the still greater steric hindrance allows, however,

The session will last for 24 hours. The resin is then washed as usual to remove by-products and one examines whether the reaction is complete with the Kaiser color reaction. The addition reaction to t-butyloxycarbonyl-threonine for the preparation of t-butyloxycarbonyl-threonyl valyl isoleucine resin ester, cleavage of the peptide from the resin to form t-butyloxycarbonyl-threonyl valyl isoleucine and the following isolation and purification takes place as described for the preparation of threonyl valyl leucine in Example 8. Man receives the tripeptide, threonylvalylisoleucine in an amount of approx. 600 mg (68% yield).

Example 14

Merrifield synthesis of threonylvalyleucinylarginine

The same procedure as in Example 8 is followed, except that 6.78 g of t-butyloxycarbonyl-nitro-(guanidinyl)-L-arginine (21.2 mmol) are used as amino acid esterified to 20 g of Merrifield resin. The esterification reaction is carried out by refluxing the mixture for about 72 hours and the resin ester washed and dried as in Example 8 to about 22 or 23 g of t-butyloxycarbonyl-nitroarginine resin ester About 5 g of ,t-butyloxycarbonyl-L-leucine (24 mmol) is coupled to the nitroarginine resin ester by adding an equivalent amount of dicyclohexylcarbodiimide (4.6 g = 24 mmol), and the reaction is allowed to proceed for 16 hours. After washing the resin ester, the coupling of t-butyloxycarbonyl-L-valine and t-butyloxycarbonyl- O-benzyl-L-threonine to the resin ester in the same way as described in example 8. Approximately 24 g of t-butyloxycarbonyl-O-benzoyl-L-threonyl-L-valyl-L-leucyl-arginine resin ester are obtained.

Due to the stability of the nitro group towards hydrogen bromide, the protected peptide resin ester is cleaved with anhydrous hydrogen fluoride to also remove the nitro group. After passing through a suitable ion exchange resin, the eluent is concentrated to an oil and isoelectrically precipitated by the addition of solid sodium carbonate. The product L-threonyl-L-valyl-L-leucyl-L-arginine is recrystallized from ethanol-water and gives approx. 900 mg of the tetrapeptide.

Peptides with a characteristic T-V-L sequence can also be prepared in solid phase by reaction with chloromethylated resin which is a polystyrene cross-linked with divinylbenzene resin and sold by Bio Rad Laboratories, Richmond, California, U.S.A., under the brand "Bio Beads SX-1". For the addition of peptide residues, e.g. a t-butyloxycarbonylleucine resin ester suspended in dioxane and the protecting groups released with 4N hydrochloric acid. The hydrochloride formed is neutralized with triethylamine and washed. The coupling of further amino acid residues can be carried out by using a coupling agent such as dicyclohexylcarbodiimide. 'This method has not been shown to be as cheap as other synthesis methods such as e.g. The Merrifield Synthesis. Example 15 Threonylvalyleucine methyl ester

,Crude threonylvalyleucine (0.066 g = 0.2 mmol) is added

a 5 ml reaction ampoule and dried at 100°C in vacuum overnight over phosphoric anhydride. The dry tripeptide is dissolved in 0.54 ml of anhydrous methanol. A previously prepared mixture of dimethoxypropane and pure hydrogen chloride in a weight ratio of 542:83 is added in an amount of 0.63 ml to the reaction mixture. The reaction vial is closed. The mixture is shaken thoroughly and set aside

night for the formation of the threonyl valyl leucine methyl ester. The solvent is evaporated under nitrogen and dried in a vacuum at 100°C over phosphoric anhydride overnight.

Example 16

N- O- diacetyl- threonyl valyl leucine methyl ester

The product from example 15 is dissolved in 0.38 ml of pyridine. Acetic anhydride in an amount of 0.1 ml (2 mmol) is added as an acylating agent and the reaction mixture is kept at 37°C for 20 minutes in a closed ampoule. The solvent is evaporated under nitrogen and the acylated product is dried in a vacuum at 100°C

over phosphoric anhydride overnight.

The acylated tripeptide ester N-O-diacetyl-threonylvalylleucine methyl ester can be purified by dissolving the crude product in methanol and passing the sample through one high-pressure liquid chromato-

graph. A modular liquid chromatograph of the Waters type equipped with a preparation column 3 m high and 9 mm in diameter filled with "Phenylporasil-B" is used with methanol as eluent.

Refractive index and UV absorption at 225 nm are measured for the eluate and the methanol's flow rate is 1 ml per minute.

An absorbent fraction that makes up about 100 ml of eluate volume is collected. The fraction's solvent is evaporated under a stream of dry nitrogen and dried in a vacuum at 100°C over phosphorus

acid anhydride, overnight.

The following example illustrates the isolation and preparation of the tripeptide threonylvalylleucine from animals.

Example 17

Extraction and isolation of threonylvalylleucine-containing polypeptide from cattle. iron

The hypothalamus from several bovine brains is dissected out and homogenized in a buffer solution, the following termed "tris-citrate buffer", which is prepared as follows:

Tris-citrate buffer

A 0.498 molar aqueous solution of tris-citrate buffer is prepared as follows: 9.58 g of citric acid and 49.9 g of tris(hydroxymethyl)aminomethane are added to 1 liter of distilled water. The pH of the solution formed is adjusted to 8.65 by adding 0.1 molar aqueous sodium hydroxide. Homogenization of the hypothalamus is carried out in four volumes (ml) of tris-citrate buffer per weight part (g) hypothalamus. The homogenized mixture is centrifuged at 15,000 rpm. minute for 20 minutes at 4°C and the supernatant containing extracted anti-S protein factor and a variety of contaminants is collected. 4 kg of starch hydrolyzate is washed once with distilled water and twice with the tris-citrate buffer. The moist starch is shaped into a block measuring 123 x 60 cm x height 5 cm.

The two ends of the block are held in place with filter paper

that excess buffers should be able to flow out. After such drainage, the block has a thickness of only 1.5 cm.

Along the width of the starch block, at a distance

43.5 cm from one end, dig a 2.5 cm wide channel in the block. The block's temperature is set to 4°C. The liquid from the above-mentioned centrifugation (approx. 35 - 40 ml) containing the anti-S protein factor is mixed with 10 drops of bromine-phenol blue. The solution is mixed with the excavated starch and this mixture is poured into the 2.5 cm wide channel in the block.

The block is then subjected to an electrophoretic current of 750 volts and 50 milliamperes, the cathode being connected to the end of the block which is 43.5 cm from the chute, and the anode to the other end. The stream is fed continuously through the block to the blue line above. the width of the block (which is due to bromo-phenol blue) moves towards the anode a distance equal to 42 cm. This takes approx. 18 hours. During the first half of this time, a light-rbd line (which comes from the hemoglobin in the extract from the hypothalamus) moves towards the cathode at a distance of 5 cm from the trough.

The area between the blue and red lines after the electrophoresis is divided into 10 strips, where each strip runs across the entire width of the block and is itself approx. 5 cm wide. Each strip is separately and completely excavated from the block and resuspended in 12 ml of Tris-citrate buffer for approx. 10 minutes. The slurry is vacuum-filtered through a Buchner funnel. The 10 fractions undergo a tropt.ofan uptake test described in Biological Psychiatry, Vol. 7, No. 1, page 53 (1973), as an indication of the amount of anti-S protein factor. Two or three of the active fractions are retained. By numbering the bands on the starch block from 1 - 10 and starting with the blue line, the fractions containing the greatest anti-S-protein activity are usually found in bands or strips No. 2, 9 and 10.

The above procedure is repeated a sufficient number of times to obtain 20 fractions with relatively high anti-S protein factor activity. These 20 fractions are combined and evaporated through dialysis tubes to reduce the volume from the initial 100 - 160 ml to approx. 40 - 60 ml.

A packed column is then prepared for chromatographing the concentrated solution of crude anti-S protein factor as follows: DEAE cellulose column

800 g of "Selectacel" which is a DEAS type of cellulose (diethylaminoethyl cellulose sold by the Brown Company of Berlin, New Hampshire, U.S.A.) is waxed twice with 20 L portions of 0.19N aqueous sodium hydroxide. The filling material is then washed with 20 1 portions of 0.19N hydrochloric acid. The material is then washed with 0.005 molar phosphate buffer until the rinse buffer is chloride-free (usually 25 times). The filling material is then filled on a 110

cm hby glass column with inner diameter 2.5 cm.

The concentrated crude solution of anti-S protein factor is loaded onto the DEAE column and it is then eluted with a 2-flask gradient system where flask A contains 1,000 ml of 0.005 molar aqueous cation phosphate buffer solution (pH 7.4) and flask B contains 1,000 ml of 0 .04 M aqueous cation phosphate buffer solution (pH 4.3) to which sodium chloride has been added in a ratio of 405.5 g sodium chloride per 50 liters of buffer solution. The eluate is collected in 15 ml fractions and you get approx. 120 fractions in total that are numbered. Each fraction undergoes a test to determine the activity of the anti-S-protein factor by the tryptophan absorption method, and the 10 fractions with the greatest anti-S-factor activity are combined and concentrated by freeze-drying to approx. 10% of the original volume.

This procedure is repeated a sufficient number of times until there is enough concentrate containing the anti-S-protein factor to give a total protein content of approx. 200 mg according to the Lowry method. This requires approx. 300 - 400 ml concentrate which again requires approx. 400 - 1,000 g of bovine hypothalamus, which again contains RNA, is dissected from around 160 cattle.

The concentrate, which contains around 200 mg of protein, is dialysed against distilled water for 24 hours. For every 17 ml of concentrate, 4.25 ml of 0.1 molar trypsin and 2.1 ml of 1 molar CaC3 are added. The pH of the entire mixture is adjusted to 7.8 with 1 molar NaOH and incubated for 2 hours on a water bath shaker at 37°C. After incubation, the solution is filtered through a "Diaflo<u>membrane with a molecular weight of 10,000 ("Amicon UM-10"). After filtration, 4.25 ml of the 0.1 molar pepsin solution per 17 ml of filtrate mixture. The pH is adjusted to 1.5 with 1 molar HCl and the mixture is again incubated for 2 hours at 37°C. After incubation, the pH is raised to 7.8 with 1 molar NaOH and the solution are filtered through a Diaflo membrane ("Amicon UM-2"), with a molecular weight of 1,000.

The filtrate is flash evaporated to reduce the volume to around 50 ml. The mixture is centrifuged and the supernatant liquid is collected. The liquid's pH is set below 2.2 by adding concentrated hydrochloric acid. The acidified liquid is diluted with distilled water to 75 ml. The solution containing anti-S protein factor is column chromatographed on the following column: First sulfonated resin column

The filler material is "Aminex 50-WX2" from Bio Rad Company which is a hydrogen ion form of a "Dowex" cation exchange resin with a particle size of 200 - 325 mesh. The resin is a sulfonated polystyrene with 2% nominal cross-linking with divinylbenzene. The filling material is washed in a Buchner funnel with 0.1N hydrochloric acid until the washing liquid is colourless. The material is then washed with distilled water until the liquid has a pH equal to 5, after which the material is washed with about 3 parts by volume of 2.ON aqueous sodium hydroxide. The filler material is washed again with distilled water until the liquid has a pH equal to 5. It is then transferred to a beaker and suspended in 2 parts by volume IN aqueous sodium hydroxide at 35°C for 2 - 3 hours. The slurry is then passed through a Buchner funnel and the filter cake is washed with distilled water until the liquid has a pH equal to 5. The filler material is slurried in 2 volumes of a 0.2 molar aqueous solution of sodium citrate (pH 3>l) and the slurry is poured into a glass column 150 cm high and 2 cm in inner diameter.

The acidic aqueous solution of crude anti-S protein factor is loaded onto the Aminex column which is then eluted with a 2-flask elution system where flask A contains 2,000 ml of 0.2M aqueous sodium citrate solution (pH 3.1) and flask B contains 2,000 ml acetate citrate buffer [pH 9.1) prepared from 315 g of citric acid and 402 g of sodium acetate in 4 liters of deionized water. The column is kept under a pressure of 0.7 atm nitrogen. The eluate is collected in 10 ml fractions, which gives a total of 120 fractions. The fractions are numbered in the order provided from the column. The fractions are examined by the tryptophan absorption method to determine the activity of the anti-S protein factor. One is set up

curve of the number of fractions along the x-axis and the fractions' activity along the y-axis. You find at least one activity peak on the formed curve, usually in the area between the twentieth and thirtieth fractions and often you see other peaks, e.g. one i

The 40s fractions, one in the 70s fractions and one in the 90s fractions and one peak in the area between the 110s and 120s fractions. The fractions that form each peak, e.g. 2 or 3

fractions in the 20s fractions are merged for further processing. Each such group of fractional sequences is treated separately in the following as indicated below.

Distribution chart

. Method A

The group of fractions with hby anti-S protein factor activity is flash evaporated on a rotary evaporator to approx. 5-10 ml volume. The concentrate undergoes blanket electrophoresis with apparatus Beckman Spinco model CP. 20 liters of electrolyte solution is made from 22.4 ml of 0.5M aqueous KH2PO^ solution with 259.2 ml of 0.5M aqueous solution of Na2HP0^ and diluting the mixture to 20 1 with distilled water and setting the pH of the mixture to 8 .0 with phosphoric acid or sodium hydroxide as required. 50 milliamps 950 volts direct current is used. Only approx. 5 1 electrolyte solution is used for each sample. The electrophoretically fractionated solution of anti-S protein factor plus contaminants is collected from the electrophoresis apparatus in 32 fractions, each of which has a volume of approx. 15 ml. Each of these fractions is tested for anti-S protein factor activity by the tryptophan uptake method. The factions with the greatest activity are held back and merged. This is often the eighth to twelfth fraction taken from the device.

The fraction group that has the most active fractions is flash evaporated in a rotary evaporator to a volume of approx.

1 - 5 ml. The concentrated information that now contains so

as little as approx. 5 mg of polypeptide material is separated in columns of molecular sieves prepared as follows:

Molecular sieve columns

2 glass columns are arranged in series where each column is 200 cm high and 0.81 cm in inner diameter. Both columns are filled with "Sephadex G-15 fine" which is a molecular sieve material supplied by Pharmacia Fine Chemicals, Piscataway, New Jersey,

U.S.A. The filler material is particulate dextran with a particle size melting range of 40 - 120 microns.

The solution containing anti-S protein factor is passed through the two "Sefadex" columns with 0.02M acetic acid as eluent. The eluate is collected in 2 ml fractions which are numbered from 1 - 120 in total. These fractions all undergo the tryptophan absorption method to find anti-S protein factor activity and are examined for total polypeptide content by UV absorption at a wavelength of 220 nanometers. Enough of them

most active fractions (with regard to activity of anti-S protein factor) are combined and give a total polypeptide content in the range of approx. 60 micrograms to 1.5 milligrams. The solution is flash evaporated on a rotary evaporator to reduce the volume to 1 - 3 ml.

The concentrated solution of anti-S protein factor plus impurities is purified by ion exchange chromatography on the following column:

Sulfonated Resin Column No. 2

The glass column is 200 cm high and has an inner diameter of 0.81 cm. The filler material is "Aminex 50-WX2" which is a cation exchange resin described earlier. The filling material is filled onto the column and brought into equilibrium with 0.2N sodium citrate buffer (pH 3.0) and the system is heated to 35°C.

The solution to be chromatographed is filled onto the column which is eluted with a 2-flask elution system where flask A contains 1 liter of 0.2N sodium citrate buffer solution (pH 3.0) and flask B contains 250 ml buffer solution prepared as follows: 14.6 g citric acid, 20 .6 g of sodium acetate, 3.1 ml of glacial acetic acid and 800 ml of distilled water are dissolved, the pH of the solution is set to 4.0 by adding concentrated hydrochloric acid and the volume of the solution is set to 1 liter by adding distilled water.

The eluate from the column is collected in 5 ml fractions i

a speed of approx. 4-5 fractions per hour. Around 130 - 150 such fractions are collected in total. Each fraction is tested for anti-S protein factor activity by the tryptophan uptake method. The fraction(s) that have the highest activity form solutions of relatively pure anti-S protein factor, i.e. material that is free of most other peptides, cell debris, etc. that were associated with the factor when it was extracted from bovine hypothalamus in tris - the citrate buffer. The fractions with the highest concentration of anti-S protein factor are often found in fraction numbers 80 - 86. Amino analysis of these fractions shows that the factor is a polypeptide that contains a combination of some of the following amino acids: glutamic acid, threonine, valine, leucine, phenylalanine , tyrosine, aspartic acid, serine and glycine. Statistical comparison of the figures from the amino acid analysis combined with enzyme studies suggests that the anti-S protein factor contains the tripeptide L-threonyl-L-valyl-L-leucine.

Method B

Alternatively, each group of fractions from the above fractionation and tryptophan uptake sample is processed separately and purified by reverse phase partition chromatography. Reverse phase partition chromatography is carried out in a Waters Associates model 660 Solvent Programmer and a Waters Associates model 6000 Solvent Delivery System in connection with a column of length 1.8 or 3 m x 9 mm diameter filled with "Phenyl Porasil B" from Waters Associates. The column is placed in a constant temperature housing made of Styrofoam blocks surrounding a constant temperature bath of the "Haake FE" type. The samples are loaded onto the column through a Waters Associates model "U6K Universal Injector" (1 microliter to 10 ml) and the eluate from the column is collected in a "Fraction Collector 1205" from Scientific Manufacturing Industries. The eluate from the column is monitored with a Beckman model 25 ultra-violet spectrophotometer equipped with a Waters Associates "LC-25" micro-cell unit and a Waters Associates "R-401" differential refractometer.

For each fraction group from the sulphonated resin column, the fraction is dried and dissolved in 5 - 10 ml of solvent and injected onto the column. The elution is followed with regard to refractive index and absorption at 225 nanometers. As activity peaks elute, the flow from the column is stopped and the sample is examined for UV absorption from 350 - 210 nanometers. Fractions of 5 ml each are collected and homogeneous peaks are pooled. The fraction or several fractions are evaporated to dryness and undergo amino acid analysis to find trypto-pan uptake and the peaks that have an inhibitory effect.

Fractions containing L-threonyl-L-valyl-L-leucine can be N-acylated and/or esterified as described in examples 10, 15 or 16.

As indicated above, research regarding schizophrenia has led to the isolation of α-5-globulin (alpha-helix S-protein) from the plasma of schizophrenia patients, and which has different activities than similar α-2-globulin (randomly oriented S-protein) from plasma in normal subjects. See Frohman et al. Recent Advances in Biological Psychiatry mentioned earlier. Alpha-helix-S protein isolated from schizophrenia patients produces observable psychological reactions when they e.g. given to rats. Caldwell and co-workers in the above-mentioned Biological Psychiatry state that administration of alpha-helix-S protein to rats reduces self-stimulation for pleasure responses in rats. Using the method established by Caldwell and co-workers, different peptides according to the invention have been examined to find their ability to reverse or suppress the action of alpha-helix-S protein. In this way, the activity of constituent parts according to the invention for the attenuation of schizophrenia symptoms in humans is illustrated. It has also been found that peptides that counteract the activity of alpha-helix-S protein show inhibition against tryptophan uptake and, as mentioned in example 17, tryptophan uptake analysis can be used as an aid in isolating peptide fractions that are active against alpha -helix-S protein.

Example 18

The procedure for intracranial self-stimulation in rats is described on pages 237 - 239 of Caldwell et al.: Biological Psychiatry, referred to earlier (to which reference is hereby made in its entirety), and this procedure is repeated except for the conditions below. The rats have stimulation electrodes planted in the middle forebrain bundle and undergo tests to find the number of times one can wait for the rod to be pressed down by an animal within a given period of time. Alpha-helix-S-protein is administered intercisternally and one can then observe that the number of times the rod is pressed down falls to approx. 77% of the original number. Various polypeptides are given intramuscularly to rats in an amount of 0.2 mg/kg body weight to determine whether the polypeptides counteract the effect of alpha-helix-S protein. Tryptophan uptake is also carried out for the relevant polypeptides. The inhibition of tryptophan uptake is determined after 10 minutes. The results of the trials appear in the table:

The following polypeptides undergo trials to find inhibition of tryptophan uptake. The inhibition of tryptophan uptake is determined after 10 minutes.

The following polypeptides are tested for inhibition of tryptophan uptake after 10, 15 and 30 minutes.

The aforementioned data illustrate the use of amino acid groups

with D configuration for inhibition of tryptophan uptake. As shown for D-alanyl-L-threonyl-L-valyl-L-leucine, inhibition of tryptophan uptake can be reduced after some inhibition-

time. However, when the essential polypeptide with the T-V-L sequence had a D-configuration lucine residue, the peptide showed a shortened activity time.

The following polypeptides are tested for comparison purposes. The inhibition of tryptophan uptake be-

vote after 10 minutes.

As can be seen from the above figures after pro-

ing of L-threonyl-L-valyl-L-leucyl-D-alanine, the presence of substituents that are relatively difficult to hydrolyse from the carboxylic acid function of the amino acid on the (L) residue in the essential T-V-L sequence will prevent the peptide from -

equal activity on tryptophan uptake.

In essentially the same way as described above, N-acetyl-tyrosylthreonylvalylleucine, propylthreonylvalyl-leucylprolylglycine are tested to determine the effect of these compounds on rats that have been given alpha-helix-S protein. Both of these polypeptide-containing compounds are found to restore rod depressor activity in rats. Other polypeptides that attenuate or suppress symptoms of schizophrenia are threonyl-valylleucylarginine.

Claims (14)

1. Method for producing a polypeptide, characterized by converting amino acid residues with structure: in sequence by reacting the amine function on one amino acid residue and the carboxylic acid group on another amino acid residue, during which groups on the amino acid residues that cause unwanted side reactions are protected, to produce a polypeptide compound of formula where A denotes an oc-monoiminoacyl group with 2-10 C atoms, B is a hydrolysable a-monoiminoacyl group with 2-10 C- atoms, d is -H, or an α-amino protecting group where P denotes the alkylene with 1-20 C atoms and p equals 0 or 1, E equals R1"*, a terminal carboxylic acid protecting group, a metal hydroxide complex or an acid addition salt, where denotes 0Re or -NRCR^, where Re is hydrogen or alkyl with 1-20 C atoms, or aryl or aralkyl with 6-24 cd C atoms, and R and R are the same or different and denote hydrogen or lower alkyl or denote the alkylene or together form a cyclic structure comprising the nitrogen atom to which they are attached; R equals H, alkyl with 1-20 C atoms, acyl with 1-20 C atoms, tetrahydropyranyl or monovalent metal, t is equal to 0-5, x is equal to 0-5, and such that the sum of t and x is 0-5, as well as their pharmaceutical salts. 2. Method as stated in claim 1, characterized in that the compound produced has the formula 3. Method as specified in claim 1 or 2, characterized in that in the compound produced 0 is the -NHCHC group threonyl and B has L configuration if it IN CHOH CH, 3 is optically active. 4. Method as specified in claim 1, 2 or 3, characterized in that in the manufactured compound the leucyl group. 5. Method as stated in claim 1, 2, 3 or 4, characterized in that in the compound produced, A and B denote the same or different peptide residues selected from prolyl, threonyl, allotreonyl, leucyl, isoleucyl, glycyl, seryl, arginyl, phenylalanyl, glutamyl, asparagyl and alanyl. 6. Method as stated in claims 1-5, characterized in that in the compound denotes 0 ii HR <a> C acetyl. 7. Method as stated in claims 1-6, characterized in that in the peptide R denotes methoxy. 8. Method as stated in claims 1-6, characterized in that in the peptide R denotes -NPL,. 9. Method as stated in claims 1-6, characterized in that in the peptide R denotes -OH. 10. Method as stated in claims 1-9, characterized in that in the peptide compound denotes the D-leucyl group. 11. Method as stated in claim 1 or 2, characterized in that the compound is L-threonyl-L-valyl-D-leucine. 12. Method as stated in claims 1 or 2, characterized in that the compound is phenylalanyl-prolylthreonylvalylleucylprolyl-phenylalanine. 13. Method as stated in claims 1 or 2, characterized in that the compound is N-acetyl-threonyl-ylvalyl leucinamide. 14. Method as stated in claims 1-9, characterized in that the peptide is the remainder L-threonyl-L-valyl-L-leucine.