CH641441A5 - THERAPEUTICALLY ACTIVE PSEUDOPEPTIDES AND COMPOSITIONS CONTAINING THEM. - Google Patents

Description

The present invention now enables the deleterious effects of such disorders to be mitigated by providing therapeutic agents that are capable of simulating one of the participants in metabolic proteolysis reactions, i. can pretend to be that participant. These simulating molecules act as non-natural substrates that bind the natural catalysts, or as non-natural catalysts that bind the natural substrates, and thus mitigate the disturbances caused by high concentration or hyperactivity of the natural substrate.

In other words, the therapeutic agents according to the invention simulate one of the partners in a proteo-40 lyrical pair. A certain agent can act as a catalyst, i.e. occur as an enzyme or hormone, or as a substrate for an enzyme. In any case, the pseudopeptide or the non-natural partner binds the natural partner and reduces their participation in the natural metabolism. Is the pseudopeptide e.g. part of a natural substrate for a specific enzyme, this enzyme, since it is bound to the non-natural substrate, cannot perform its usual function. The formation of dangerously high amounts of plasmin can thus e.g. can be inhibited by introducing a plasminogen surrogate that binds urokinase.

The present invention is illustrated by the accompanying drawings, in which Fig. 1 shows the similarity in spatial form between glycylglycine and the corresponding counterpart, in which the peptide bond has been replaced by a thiomethylene group. 2 to 10 show different processes for producing the products according to the invention. 11 and 12 illustrate the preparation of pseudophenylalanylglycine and pseudoleucyl-60 glycine using combinations of the methods shown in some of the previous figures.

The compounds in question are pseudopeptides containing up to 10 or more bonds connecting 65 amino acid segments and wherein at least one link between amino acid segments has been replaced by a thiomethylene group so that a pseudopeptide of which at least one is obtained

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Part can be represented by the following substructure formula:

HR Ri Q

il l 1 il

N CHCH2SCH C

in which R and Ri are those parts of a-amino acids which are bonded to the a-carbon atom in addition to the amino group, and in particular R is a radical with at least one carbon atom and Rx stands for the same or a different radical. The remaining bonds between the amino acid segments are peptide bonds. In this structure, the free valence on the nitrogen atom is occupied by a hydrogen atom or the carbon atom of a peptide bond that binds another amino acid to the structure. The free valence on the carbon atom is occupied by a hydroxyl group or a peptide bond by the nitrogen atom.

The above structural formula could e.g. are completed by the amino end group being bonded to the carboxyl group of alanine and the carboxyl end group being bound to the alanine group. The pseudopeptide obtained in this way corresponds to the following structural formula:

CH, 0 H R R.O H CH,

l 3 li I I I I I l '

H2N-CH - c - N - CHCH2SCHC - N - CHCOOH

Q-amino, carboxyl or guanadino groups, such as lysine, glutamic acid or arginine, or an imino group of proline or hydroxyproline could also be bound. It is only important that the structure obtained in this way corresponds chemically in virtually every respect to the one partner of a proteolytic pair, except that it contains at least one thiomethylene group.

The products according to the invention are not only chemically closely related to the naturally occurring materials, but are also practically isosteric with their natural counterparts and practically equivalent in terms of their basicity.

The main reason for the exact mimicking of these important physical and chemical parameters of the natural product is the close relationship between the thiomethylene and peptide groups. This relationship is illustrated by Figure 1 of the accompanying drawings, from which it can be seen that the carbon, hydrogen, nitrogen and oxygen bonds on both sides of the peptide and thiomethylene groups have practically the same spatial relationship and that the thiomethylene group is about the same size as the peptide group. The infrastructure of the peptide group and the thiomethylene group are also very similar, so that the exchange of a thiomethylene group for the peptide group of a natural product does not affect such factors as charge distribution, dielectric constants and the ability to hydrate. A pseudopeptide according to the invention can therefore mimic the corresponding natural substance and influence the reactions which are otherwise triggered by the natural product in a controllable manner.

Two of the most important results, which result from the strong similarity between the products according to the invention and the corresponding natural products, are: (1) The products according to the invention can be used as therapeutic agents for regulating the speed of naturally occurring metabolic reactions taking place under proteolysis, because they can take on the role of the corresponding natural substances; and (2) they are neither toxic nor mutagenic.

The pseudopeptides described above are preferred 5 pseudotetrapeptides that contain only one thiomethylene bond. Smaller pseudopeptides may be advantageous for certain purposes, and pseudopeptides containing up to 8 or more amino acid segments may be preferred for other purposes. The reason for these variations becomes apparent when one starts from the simple "lock and key" analogy that is often used to describe protein-protein interactions, e.g. the effect of a protein enzyme on a protein or peptide substrate. If one considers the price stone or peptide substrate as a key and the enzyme as a lock, the geometric shape of the two structures must match, or the key does not fit in the lock, i.e. there is no reaction.

If the pseudopeptide now takes the place of the lock, 20 its geometric shape must be suitable to make the key fit. This condition can be met by a pseudodipeptide, but often a higher molecular weight pseudopeptide can be more satisfactory.

As is clear to any person skilled in the art, the compounds according to the invention are amphoteric and can be processed into pharmaceutically acceptable acid addition salts and metallic salts. The present invention also includes these salts. In the course of the description it becomes clear that a large number of derivatives of the therapeutically active substances can be produced. Simple derivatives are e.g. Esters and amides. More complex derivatives are products that are formed by reaction with free functional groups on the amino acid residues, 35 e.g. with the hydroxyl group of tyrosine. These derivatives are also encompassed by the present invention.

The compounds according to the invention are normally based on L-amino acids. In certain cases, however, it may be appropriate to use the D-form of the acid. Both forms are thus encompassed by the present invention.

Some of the terms used in this specification are defined below:

Pseudopeptide - a peptide containing amino acid residues 45 in which at least one of the normal peptide bonds has been replaced by a thiomethylene group.

Amino acid residue - the part of an a-amino acid molecule that remains after a hydroxyl part of the carboxyl group and a hydrogen atom of the amino group have been formed to form a peptide bond. The description of the reaction sequences which lead to the formation of the compounds according to the invention clearly shows that an a-amino acid does not necessarily have to be used as the starting material. The person skilled in the art can easily imagine what is to be understood by the term “amino acid residue”.

Amino acid side chain - the part of an a-amino acid attached to the a-carbon atom, in addition to the amino group or, in the case of proline or 60 hydroxyproline, in addition to the imino group. The designation includes e.g. the hydrogen of glycine, the benzyl group of phenylalanine or the isobutyl group of Leu-cin.

For the sake of simplicity, the Greek letter * p (psi) is used instead of the hyphen in the 65 pseudopeptides according to the invention to indicate that a peptide bond has been replaced by a thiomethylene bond. In addition, the well-known abbreviations for

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usual amino acids used. Leu - Leu and Leu * P Leu therefore mean leucyl leucine and pseudoleucyl leucine, respectively.

A typical pseudotetrapeptide according to the invention which contains only one thiomethylene bond is:

Leu W Leu Val - Tyr.

This compound, as well as its pharmaceutically acceptable salts and derivatives, are suitable for the treatment of high blood pressure because they inhibit the conversion of angiotensin into angiotensin I and angiotensin II by the enzyme renin. Treatment of a patient with a pharmaceutical preparation containing this pseudopeptide provides a mimicking substrate that, in competition with the natural substrate angiotensin, binds the available renin. To the extent that renin is bound to the pseudopeptide, it is no longer available for natural conversion. The pseudopeptide is therefore a suitable therapeutic agent for combating high blood pressure.

Some pathological conditions that can be treated with the pseudopeptides according to the invention, the pseudopeptides used and the metabolic reactions to be interrupted are described below:

high blood pressure

Hypertension is a common medical problem. An important step in the regulation of blood pressure is the enzymatic cleavage of the a2-globulin in angiotensin I, which in turn is broken down into angiotensin II. Angiotensin II is a powerful antihypertensive agent, and blocking its formation is an effective therapy. During the conversions, the Phe-His and Leu-Leu bonds are split. By introducing Phe ip His and Leu> p Leu into selected pseudopeptides and by administering these pseudopeptides, the conversion of the a2 globulin to angiotensin II is blocked. Pseudopeptides and their derivatives suitable for such blocking are, for example: Ac-Phe \ P His-Leu-Leu-Val - Tyr-Ser-OH; Ac-Phe-His-Leu ip Leu-Val-Tyr-Ser-OH; and Ac-Phe \ P His-Leu ip Leu-Val-Tyr-Ser-OH. The pseudopeptides can be administered parenterally, intravenously, through nasal sprays or suppositories.

Ulcer formation on the cornea

Corneal ulceration is a common ophthalmological problem with a wide range of etiologies, such as Burns or burns to the eye, rheumatoid arthritis and infections. Ulceration is caused by the excessive formation of collagenase during the healing process. The cornea consists of 70% collagen. The cleavage of collagen by collagenase is highly specific and only involves Gly-Ile and Gly-Leu bonds. The pseudopeptide required to avoid these destructive cleavages therefore contains Gly ip Ile and Gly \ P Leu. Examples of therapeutic agents according to the invention which contain the desired structures are:

Ac-Pro-Gln-Gly Vff Ile-Ala-Gly-Gln-Arg-Gly-OEt, Ac-Pro-Cln-Gly * P Leu-Ala-Gly-Gln-Arg-Gly-OEt, Ac-Pro-Leu -Gly ip Ile-Ala-Gly-Leu-Arg-Gly-OEt, Ac-Pro-Leu-Gly ip Leu-AIa-Gly-Leu-Arg-Gly-OEt, the amide analogues thereof and the basic compounds in which the carboxyl group of the clycine molecule is a free one. In the case of ulceration, the cornea is treated by applying the pseudopeptide externally several times a day in an isotonic, aqueous solution with a pH of about 7.4.

Since, as is known, the terminal C group is removed from the Arg when the peptide comes into contact with plasma, Gin ip Arg or Leu Y Arg is expediently introduced into the same peptide or a D-Arg into the terminal C group. The usable preudopeptides thus obtained are:

Ac-Pro-Gln-Gly \ p Ile-Ala-Gly-Gln ip Arg, Ac-Pro-Gln-Gly ip Ile-Ala-Gly-Leu ip Arg or the corresponding esters or amides. The ile in the pseudo binding can be replaced by leu.

Rheumatic arthritis

Rheumatoid arthritis is a syndrome that affects millions of people. This disease has a destructive effect, among other things, because the tissue collagenase acts hydrolytically on the collagen matrix of the affected joint. Since the same enzyme that also causes ulcer formation on the cornea plays a role in this syndrome, the therapeutically suitable pseudopeptides are identical to the pseudopeptides used for this ulcer formation. In clinical treatment, the excess synovial fluid is usually removed from the inflamed joints in rheumatic patients. The patient can then be injected with a sterile saline solution or suspension of the pseudopeptide in a volume of 1 cc or less that replaces only a small portion of the withdrawn liquid.

Anticoagulant

Many cardiac patients are given anticoagulant medication to prevent the risk of thrombosis. These agents are often vitamin K antagonists and therefore act on all known and unknown effects of vitamin K. A more specific effect on blood coagulation is achieved if the conversion of prothrombin to thrombin is blocked by the proteolytic enzyme factor Xa. This enzyme causes a specific cleavage at Arg-Thr (residues 274-275) and Arg-Ue (residues 323-324) of the prothrombin sequence. Intervention in these cleavages by synthesis and administration of suitable pseudopeptides leads to the fact that highly specific blocking agents become effective already at the beginning of blood clotting. These pseudopeptides contain Arg * P Thr and Arg * P Ile and are e.g.

Ac-Ile-Glu-Arg ip Thr-Ser-Glu-OEt as well

Ac-Ile-Glu-Gly-Arg * P Ile-Val-Glu-OEt. The pseudopeptides can be administered in tablet form, intravenously, by nasal sprays or dabbing and in suppositories.

Reduction of the urokinase effect

The enzyme urokinase activates plasminogen to plasmin. This transformation is an important step in preventing thrombosis. Urokinase is therefore a commonly used therapeutic. Bleeding is an undesirable side effect of urokinase therapy. The present invention now makes it possible to prevent such bleeding by weakening the action of urokinase by a specific inhibitor. If bleeding occurs during treatment and the urokinase proves to be unsuitable, the corresponding pseudodipeptides can be administered. These pseudo-dipeptides contain Arg ip Met and Lys * P Ser. "

The complete pseudodipeptides suitable for such blockings include:

Ac-Ser-Ile-Arg W Met-Arg-Asp-Val-OEt and

Ac-Glu-Asn-Arg-Lys * P Ser-Ser-Ue-Ile-OEt.

They can be given parenterally, intravenously, through nasal sprays

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15

20th

25th

30th

35

40

45

50

55

60

65

or dabbing and administered in suppositories. The preparations are also suitable for the treatment of hemophilia.

Prevention of destruction by elastin in emphysema

Pulmonary emphysema is a great risk for people who have a genetic deficiency of antitrypsin. The a2-antitrypsin in the bloodstream of the lungs normally protects the lung tissue from exposure to elastase. Without this protection, the lungs are severely damaged by elastase. The peptide bond most commonly cleaved by Elastase is an Ala-Ala bond. Therefore, the pseudodipeptide to be incorporated into the inhibitor is Ala ip Ala. The therapeutic peptides obtained are e.g. Ac-Ala - Ala ip Ala-OEt and Ac-Ala-Ala-Ala * p Ala-OEt. They are administered by inhaling an aqueous aerosol.

LHRH *

A hormone that releases luteinizing hormone (pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2) is used to treat various endocrine disorders, e.g. Loss or lack of fertility, menstrual and ovulation disorders, and male infertility. This hormone has a short biological half-life (2 minutes) because it is quickly destroyed by proteolysis. A long-lasting hormone is therefore a very important therapeutic agent. The bonds subject to proteolysis are e.g. Gly-Leu, Tyr-Gly and Pro-Gly-NH2. The following pseudodipeptide units are required: Gly IF Leu, Try * P Gly and Pro ip Gly-NH2. The corresponding long-lived hormones are e.g.

pGlu-His-Trp-Ser-Tyr * P Gly-Leu-Arg-Pro-Gly-NH2, pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro ip Gly-NH2, pGlu-His-Trp -Ser-Tyr ^ p Gly-Leu-Arg-Pro * p Gly-NH2, pGlu-His-Trp-Ser-Tyr-Gly ip Leu-Arg-Pro-Gly-NH2. They can be administered in nasal sprays or by dabbing, in suppositories, parenterally and intravenously in a carrier made of normal saline.

Anticoagulant blocking of the action of thrombin or fibrinogen

As previously stated, anticoagulants are important therapeutic agents. Blood clotting involves a complicated sequence of activations by proteolytic enzymes, which culminate in the cleavage of fibrinogen with thrombin to form fibrin, which then spontaneously clumps into a soft lump. Thrombin cleavage in the A-a chain starts at the Arg - Gly bond. As a result, the p vseudodipeptide unit Arg vp Gly is required for an inhibitor. The complete peptide inhibitor for thrombin activity is: Ac-Gly-Gly-Gly-Val-Arg> P Gly-Prop-Arg-Val-NH2. He can e.g. in tablets, intravenously, by nasal sprays or dabbing and in suppositories.

Protection against stomach ulcers

Pepsin is the predominant proteolytic enzyme in gastric juices. People who secrete excessive gastric juice tend to develop ulceration through proteolysis. Inhibition of pepsin-induced proteolysis protects against enzymatic damage. Phe-Phe binding in particular is subject to proteolysis by pepsin. The pseudodipeptide unit required for the synthesis is therefore Phe \ P Phe. The therapeutic inhibitor is e.g.

Ac-Ala-Ala-Phe ip Phe-NH2.

* Luteinizing hormone - releasing hormone

5 641441

This substance can be given orally as a tablet or as a liquid in combination with acid antagonists.

Tuftsin 5 pseudopeptides Medical syndromes are known in which the patient's cells are practically incapable of phagocytosis or pinocytosis. A therapeutically useful class of peptides stimulating phagocytosis contains basic and hydroxyamino acids according to certain schemes. These means correspond to the structure H-B-Pro-B ', where H stands for a position occupied by the hydroxyamino acid (Thr or Ser), and B and B' stand for basic amino acids, such as e.g. Arg, Orn or Lys. Plasma is known to show carboxypeptidase B activity and the kidney to show aminopeptidase activity. Pseudotuftsine, e.g. Pro * P Lys, Pro ip Arg, Ser * p Lys or Ser ip Arg therefore have a longer biological half-life than the normal peptide. Examples of such pseudotuftsins are: Thr-Lys-Pro ip Arg, Thr ip Lys-Pro-Arg and Thr * P Lys-20 -Pro vp Arg. Arg can also be replaced by Orn. The pseudotuftsins show different resistance to proteolysis.

They can be administered as tablets, injections or orally in the form of an aqueous solution.

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Insulin with a longer biological half-life

Insulin is a well-known treatment for diabetes. It is inactivated by removing the terminal C group from octapeptides by a trypsin-like effect. Therefore, pseudoinsulins resistant to proteolysis can be made by introducing Cys ip Asp to position 20-21 of the A chain of human insulin and / or residues 22 and 23 in the B chain of the insulin by Arg ip Gly replaced. 35 Coupling (oxidation) of the chains produced by synthesis in the solid phase provides an insulin which is resistant to proteolysis. The pseudoinsulins thus obtained can be administered in the same way as natural or synthetic human insulin.

40

Enkephalins, endorphins

The structures of the endogenous opiate peptides have recently been discovered and researched. These substances (enkephalins and ß-endorphins) show a pain-relieving effect similar to that of opium. With Leus-enkephalin, which has the structure Tyr-Gly-Gly-Phe-Leu, there is a loss of biological effectiveness through cleavage of the Tyr-Gly bond. If you add Tyr * P Gly instead, you get an analog substance with a longer biological half-life and better therapeutic value. The finished pseudoenkephalin is Thr XF Gly-Gly-Phe-Leu. Leu can also be replaced by mead.

Similarly, the ß-endorphins can be stabilized by introducing a Met * P Thr bond into the ß-en-55 dorphine. Leu ip Thr can be similarly effective. The products thus obtained are preferably administered intravenously; However, administration through the nose or rectum is also possible.

6o inhibition of acrosine

Acrosine is the key enzyme in spermatozoa. This trypsin-like enzyme is said to facilitate entry into the egg cell during fertilization. If the action of the acrosine is interrupted, fertilization is prevented. 65 The inhibiting substance in this case is Benzoyl-Arg ip Gly-OEt. The pseudodipeptide unit is Arg ip Gly.

This inhibitor is used in an intravaginal foam, similar to dolphin.

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Long-lived oxytocin

Oxytocin causes uterine contractions and is used in obstetrics to induce labor. It. has the following structure:

NH0

I.

Cys-Tyr-Ile

S I

S

I.

Cys-Asn-Gln

I.

Pro-Leù-Gly-NH2

The structure of the corresponding pseudo-oxytocin according to the invention, which has a longer biological half-life, is:

nh9

I *

Cys-Try ^ Ile

S

I.

S I

Cys-Asn - Gin I

Pro-Leu Y Gly-NH ^

Tyr ^ Ile and Leu M * Gly-NH2 serve as pseudodipeptides.

Long-lived vasopressin

Vasopressin is used in medicine for surgical shock treatment as an aid to increase blood pressure. In addition, it is used to treat delayed postpartum bleeding and, in the case of childbirth, to remedy uterine weakness (“inertia”).

Vasopressin has the following structure:

Cys-Tyr-Phe

!

S

Cys-Asn-Gln I

Pro-Arg-Gly-NH2

The pseudodipeptides suitable according to the invention are e.g. Arg Gly-NH2, Tyr Phe, Pro Arg. A pseudo-vasopressin that contains these combinations is:

Cys-Tyr Y Phe I

S

5 I

S I

Cys-Asn - Gin io-I

10 per V Arg y Gly-NH2

The product can be given intravenously or intranasally.

15 For the sake of simplicity, all of the physiological conditions described above are referred to as metabolic disorders. However, some of them are not metabolic abnormalities, although they involve metabolic reactions. When it comes to pain relief, for example, it cannot be said that a metabolic reaction has gone wrong.

A common feature of all the states described above is that at some point in the sequence of reactions leading to the respective state, a substrate 25 which has a natural peptide bond is cleaved. The compounds according to the invention can now be used to reduce the total amount of cleavage products by introducing a pseudopeptide at the reaction site, which either competes with the substrate or with the catalyst influencing the reaction by pretending to be one of these participants or takes its place.

Since the development of the genetic code, it has become clear that polymers such as DNA, RNA and proteins as well as 35 peptides are products containing information. In the case of nucleic acids, the information is in the order in which purine and pyrmidine bases occur. “Translating” this information for protein and peptide structures, it is the amino acid side chains that provide the distinguishing features of the protein or peptide. The function of the main peptide chain corresponds essentially to that of a connecting structure. For example, in poly-glycine practically all information. It is the appearance of these unique side chains, brought into play by the genetic code, that determine the way in which a peptide or protein is folded, and hence the spatial relationship between the different atoms and molecular segments, especially the one Side chains on the basic structure. The resulting three-dimensional structure allows a cooperative interaction between different proteins, e.g. Enzymes and substrates, and thereby determines their biological effect.

The aim of the present invention is now a protein-55 or peptide-like structure which has practically the same molecular geometry as the corresponding natural substance. This goal is achieved by replacing a peptide bond with a thiomethylene bond. Products built around such thiomethylene bonds 60 show essentially the same molecular geometry as the natural products. You can participate in the same type of reactions. However, since the thiomethylene bond is resistant to hydrolysis, increasing the concentration of pseudopeptide in the presence of the natural product and its natural partner in the reaction causes the concentration of the normal reaction product to decrease because the pseudopeptide has the correct geometric configuration, to the

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to bind natural partners. Because of the resistance of the pseudopeptide to hydrolysis, the materials bound in this way can then no longer be cleaved so easily.

A particular advantage of the present invention is that the results can be predicted practically precisely. If the essential characteristic of a natural reaction is known, a pseudopeptide can also be constructed which is certainly effective. If e.g. it is known that the reaction between an enzyme and a substrate involves binding of certain peptide bonds of each reaction participant, this reaction can be controlled by treating with a pseudopeptide which contains the part of the enzyme or substrate which has the peptide bond, reproduces exactly, but in which the peptide bond was replaced by a thiomethylene group. In order to determine the best mode of administration or other parameters, simple tests performed in a known manner may be required. The basic reaction is, however, predictable with certainty.

The products according to the invention are good therapeutic agents for humans and mammals and can inhibit the hydrolytic cleavage of protein or peptide substrates even at extremely low doses. The most suitable dosage for the respective administration can be determined by the doctor or veterinarian. It can vary from patient to patient and depends on the patient's weight, the disorder to be treated and other factors that are easy to determine. If patients with more or less chronic metabolic disorders are to be treated continuously over longer periods of time, the products are often given initially in relatively large dosage units in order to immediately build up a certain concentration in the blood, and then in relatively small dosage units in order to maintain an effective concentration. Various dosage forms can be provided for the temporary treatment of acute or chronic conditions. The products can be administered in very large quantities, even up to 2 g or more per day. They are usually made in dosage units containing about 250 mg of active ingredient and the doctor can prescribe how many dosage units a day are needed for the disorder to be treated.

The products of the invention can be administered alone, but generally together with a pharmaceutically acceptable, non-toxic carrier, the amount of which depends on the suitability and chemical nature of the carrier selected, the mode of administration and common pharmaceutical practices. For the treatment of certain disorders or to maintain therapeutically effective concentration in the blood or in the tissues, e.g. to be taken orally in the form of tablets or capsules containing such carriers as starch, lactose, certain types of clay and the like. They can be coated with a gut-soluble coating to make them more resistant to the acid and digestive enzymes in the stomach. For intravenous and intramuscular injections, they can be prepared in the form of a sterile solution containing other solutes, e.g. sufficient amounts of saline or glucose to make the solution isotonic.

A particular advantage of the products according to the invention is that, in contrast to many other therapeutic agents which have a peptide bond, they can be administered orally, since they are considerably more resistant to enzymatic hydrolysis by the enzymes of the lower digestive tract. Because they are amphoteric, they can be adsorbed for oral administration on non-toxic ion exchange resins, which can be either anionic or cationic; in this way the active ingredient is slowly released in the stomach and / or intestine. In addition, the resistance of the products to enzymatic degradation is further improved by adsorption on such resins.

Another advantage brought about by the amphoteric nature of the products according to the invention is that the products, as already stated above, can be used in the form of their pharmacologically acceptable salts, either as metal salts or as acid addition salts. These salts are water soluble and are particularly suitable for parenteral administration. The metal salts, especially the 15 alkali salts, are relatively stable and are therefore preferred to the acid addition salts. Sodium salts are particularly preferred since they can be prepared very easily.

The acids which can be used to prepare the pharmacologically acceptable acid addition salts according to the invention comprise non-toxic anions and are, for example, hydrochloric acid, sulfuric acid, phosphoric acid, acetic acid, lactic acid, citric acid, tartaric acid, oxalic acid, succinic acid, maleic acid, gluconic acid, sugar acid and the like

A large number of non-toxic derivatives of the pseudopeptides according to the invention are also suitable. The pharmacologically acceptable salts have already been described above. Amides, esters, acylated derivatives and the like can also be used.

Other useful derivatives are obtained by modifying the free functional groups on the main pseudopeptide chain, e.g. the free hydroxyl or free amino groups. A very suitable class of derivatives are derivatives in which a free hydroxyl group, e.g. the free hydroxyl group of threonine or tyrosine has been esterified with an alkanoyl or alkenoyl group of up to about 18 carbon atoms or more. An amino group, e.g. the amino group of threonine or lysine can be acylated with an alkanoyl or alkenoyl group having up to about 18 carbon atoms. In both cases, the derivatives in which the derivative groups contain about 11 to 18 carbon atoms are preferred, since the longer hydrocarbon chains give the molecules better lipoid solubility and improve their transport through the cell dividing walls. 45 Both types of derivatives can either be prepared directly from the pseudopeptide; however, they are preferably prepared during synthesis by adding an amino acid with the selected group, e.g. the existing alkanoyl group. 50 The compounds according to the invention have a number of special features. A feature which is particularly important for its therapeutic value is the close analogy with regard to configuration and chemical composition between the products according to the invention and the natural compounds which they are intended to replace. When used, antigen reactions can therefore hardly be expected. They also appear to be little or not toxic at all. Finally, they can be introduced into the metabolism and excreted without any risk. 60 The natural substrates which are to be replaced by the pseudopeptides according to the invention are hydrolyzed at a certain natural peptide binding site when they perform their normal physiological function. The amino acid segments on either side of this site are known 65. The pseudopeptide is normally constructed so that it contains a partial structure in which the natural site has been replaced by a thiomethylene group, and this partial structure has a white on at least one side

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tere substructure that is similar to the corresponding structure of the natural substrate.

An example of the low toxicity of the compounds according to the invention are the results obtained by treating mice with Gly Leu. In these trials, mice (Swiss Wistar) received intraparenteral injections with Gly * P Leu in the dosages given below. Five mice were used for each dose amount. In no case did the mice show signs of acute toxicity. The behavior of the mice was observed for 7 days after the injection. In no case were there any harmful effects.

TABLE I

Weight gains of Swiss-Wistar mice (N ~ 5) injected with Gly ip Leu

dose

44 mg / kg 94 mg / kg 208 mg / kg 432 mg / kg 23

Saline as a comparison 18

Possible mutagenic effects of the pseudodipeptides were investigated in two different ways. One possibility for metabolic complications is the effect of methionine adenosyl transferase (MAT). Ethionine (which contains a thiomethylene bond) is known to be both carcinogenic and a substrate for MAT. Although the 5 specificity requirements for MAT are very strict, there was a possibility that Gly ip Leu would be either a substrate or a competing inhibitor. The ones listed below, using the Hoffman method from «Arch. Biochem. and Biophys. » 179, 136 (1977), however, results show that neither Gly ^ P Leu nor Gly ip D-Leu exert a noticeable influence on the MAT activity.

TABLE II Influence of Gly \ P Leu and Gly * p D-Leu on MAT

15

Substance concentration number / min.

none - 15 760

Gly ^ P Leu 12 mM 16 620

Gly ^ D-Leu 12 mM 16 760

none - 7 380

Gly ip Leu 80mM 7,990

Gly ip D-Leu 80mM 7 710

The mutagenic effects of Gly ^ Leu and Gly \ P Ile were investigated using a modification of the Ames-30 experiment, in which "repair-deficient" strains of Bacillus subtilis were used.

The results in Table IIa show that neither Gly * P Leu nor Gly "* P Ile acts as mutagen.

% Weight gain trial I after 3 days

20th

17th

Trial II

20 25

TABLE IIa Results of the mutagen experiment

Growth inhibition of the bacterial strains in mm compound concentration «Wild» type MC-1 Hcr-9 FB-13 Pol A

Gly xp Leu

0.1M

0

0

0

0

0

Gly D-Leu

0.1M

0

0

0

0

0

Gly W Ile

0.1M

0

0

0

0

0

Gly W D-Ile

0.1M

0

0

0

0

0

Chloroacetaldehyde

0.6 m

8th

16

4th

1

5

The methods by which the above experiments were carried out are described in the following publications:

55 1. Launibach, A.D., Lee, S., Wong, J., and Streips, U.N., Studies on the Mutagenicity of Vinyl Chloride Metabolites and Related Chemicals. In: The Prevention and Detection of Cancer, Part 1, Prevention, Vol. 1, Etiology, Edited by H.E. Nieburgs, Marcel-Dekker, Inc., New York (1977).

2. Kada, T., Tutikawa, K., and Sadaie, Y., Mutation ^ Research 16 (1972), 165.

A number of methods for the synthesis of pseudopeptides are already known. These methods are illustrated in the figures of the accompanying drawings. In these figures, R and Rj represent the amino acid side chains described above, ALK is an alkyl group of up to

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5 carbon atoms, X is halogen, Y is an amino blocking group and TOS is the tosyl group.

Those skilled in the art will recognize that various of the reaction sequences shown in the drawings are based on the formation of a thiomethylene linking group by reacting a primary halide with a secondary thiol or a secondary halide with a primary thiol. The individual syntheses differ from one another in the processes by which the decisive reaction participants are produced.

Fig. 2 shows a synthesis in which a primary thiol, prepared via a mercaptothiazoline intermediate, is reacted with an a-halocarboxylic acid, which is a secondary halide.

The starting materials for this synthesis are amino acids that have been reduced to amino alcohols. A number of such amino alcohols are already known and commercially available. Others can be made by reduction with borane according to the method of Yoon et al in Journal of Organic Chemistry, Volume 38, No. 16, page 2786 (1973).

In general, a method is used in which the amino acid to be reduced is brought into contact with borane, usually with a molar excess of borane in an ether-type solvent. The preferred solvent is tetrahydrofuran. The reaction temperature can be about 15 ° to 40 ° C and the reaction time from H hour to 24 hours.

The reaction product can be obtained using any isolation method. A suitable method is to decompose the excess reducing agent by adding cold water, which can be diluted with a small amount of solvent.

The amino alcohol is converted into the desired mercaptothiazoline in a reaction-inert organic solvent in the presence of a strongly alkaline reagent, carbon disulfide being used as the cyclizing agent. A molar excess of carbon disulfide, e.g. a molar excess of up to about 30% to ensure the most complete reaction possible. The most convenient alkaline reagents are alkali hydroxides, e.g. Sodium or potassium hydroxide. A variety of reaction-inert, polar organic solvents can be used. The lower aliphatic alcohols, in particular methyl and ethyl alcohol, are currently preferred.

The reaction temperature can vary between about 30 ° C and 45 ° C under normal atmospheric conditions. The reaction usually ends after about 6 to 12 hours.

The desired product can be obtained by simply evaporating the carbon disulfide and the solvent. The residue is washed with cold dilute acid, e.g. with 2% aqueous hydrochloric acid. If necessary, it can be recrystallized for purification, usually using a 1: 1 mixture of water and alcohol.

Although borane has been the preferred reducing agent to date, other reducing agents can also be used. As can be seen from FIG. 2, it is also possible to work with alkali boron tetrahydrides, sodium borohydride being generally preferred.

The mercaptothiazoline can, as indicated in Fig. 2, be hydrolyzed to a primary thiol. In the reaction sequence described so far, the carboxyl group of an amino acid starting material is therefore replaced by a mercapto group.

The mercaptothiazoline can easily be converted into a primary thiol by a ring-opening reaction with a halogen acid, expediently aqueous hydrochloric acid. The reaction temperature can be about 60 ° to 160 ° C and is maintained for 8 to 20 hours. A very simple procedure is to reflux the reaction mixture, which contains a normal excess of 6N hydrochloric acid, for 10 to 72 hours.

At the end of the reaction period, the desired product can be obtained as the hydrochloride salt by evaporation at 50 ° to 80 ° C under reduced pressure. Isolation of the product in the form of the salt is particularly expedient since the salt can be used directly in the subsequent reactions. However, if for some reason the free base is desired it can be obtained by using e.g. diluted potassium carbonate titrated to the isoelectric point and then extracted with a water-immiscible organic solvent.

The a-halocarboxylic acids, i.e. the secondary halides used in the present invention are made by known methods.

The reaction of a primary thiol with a secondary halide or the reaction of a primary halide with a secondary thiol can be terminated by at least equimolar amounts of the reactants in dilute aqueous media under an inert atmosphere at a temperature of about 20 ° to 60 ° C used and reacted for about 10 to 30 hours. In order to ensure the most complete possible reaction and in particular to limit disturbances caused by competing reactions, e.g. the reaction in which an amine group replaces the halide instead of a mercapto group, it is advisable to use a molar excess of thiol. Even using an excess of 2 to 3 moles of this reactant is not uncommon.

The currently preferred method for isolating the resulting pseudodipeptide is desalination using an ion exchange resin; see the Dreze et al method in Anal. Chin. Acta 11, page 554 (1954). "Dowex 2-X8" is generally preferred as the resin.

Fig. 3 shows a reaction sequence in which an amino-substituted primary halide with an a-mercaptocarboxylic acid, i.e. a secondary thiol.

The starting amino alcohol can be prepared in the manner described above and then converted to a primary halide.

In order to produce the primary halides, the amino alcohols prepared as described above are reacted with halogen-substituting agents. Any known agents can be used as halogen substitution agents; however, the means given in Fig. 3, i.e. Thionyl chloride, thionyl bromide or 48% aqueous hydrogen bromide.

The reaction with the thionyl halide can be carried out without a solvent; for better control, however, the reaction generally takes place in an inert polar solvent, preferably in anhydrous dimethylformamide or dimethyl sulfoxide. The reaction is normally complete after about 1 to 3 hours under reflux conditions, using at least a molar amount of thionyl halide, but advantageously a molar excess of at least 10% of the reagent.

After the reaction has ended, the excess thionyl halide is decomposed by adding water. The mixture thus obtained is neutralized with dilute aqueous sodium bicarbonate and extracted with a water-immiscible, organic solvent, e.g. with di

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

641441

10th

ethyl ether. The non-aqueous layer is dried, advantageously over anhydrous magnesium sulfate, and filtered, whereupon the desired product is obtained by evaporating the solvent in vacuo.

The reaction with hydrogen bromide can be carried out in an aqueous medium using a molar excess of HBr at reflux conditions within a period of about 1 to 3 hours. The desired reaction product can be obtained in the manner described above, but of course no excess reagent has to be decomposed.

The secondary thiol is made from a secondary halide, e.g. was obtained by a known diazotization method.

The secondary halide can be converted to the desired thiol by reacting it with thiourea in an aqueous alkanol medium and in the presence of dilute base. Typically, the reaction takes place at a temperature of about 70 ° to 100 ° C in 95% aqueous ethanol and takes about 1 to 4 hours. Equimolar amounts of the reactants can be used; however, an excess of thiourea is generally preferred to ensure the most complete reaction possible. The preferred alkaline reagent is sodium hydroxide and is used in practical catalytic amounts, e.g. up to about 5 mole%.

An isothiourea halide is obtained as an intermediate, but does not have to be isolated. Instead, additional aqueous alkali is added to the reaction medium and the reaction mixture thus obtained is heated at about 70 ° to 100 ° C. for a further 1 to 4 hours.

The product so obtained is conveniently isolated by, after neutralization with dilute acid, e.g. dilute hydrochloric acid, with a water-immiscible organic solvent, e.g. Ether, extracted. The product is obtained from the organic solution after it has been dried over an anhydrous reagent, filtered and freed from solvent in vacuo. This procedure is described in Org. Syn. Coli. Vol. 4, page 401 (1963).

The primary halide is then reacted with the secondary thiol in the manner described above.

Fig. 4 shows another process for producing a primary thiol which is reacted with a secondary halide. Preferably, a halogen atom of a primary halide is replaced by trithiocarbonate and the thioester obtained is subsequently decomposed with acid. This very expedient method is described by Martin et al in J. Org. Chem. 33, page 1275 (1968).

The trithiocarbonate is normally made by reacting carbon disulfide and sodium sulfide in aqueous alkali, e.g. 10% sodium hydroxide. The primary halide is added to the trithiocarbonate at a temperature of about 20 ° to 60 ° C. Then the reaction mixture was held at about 20 to 60 ° C for about 5 to 15 hours to form the thioester.

The thioester does not need to be isolated. It is even advisable not to isolate it. Instead, the pH of the reaction medium is adjusted by adding an acid, suitably a mineral acid, e.g. Hydrochloric acid, reduced to about 2 to 3. The resulting primary thiol can be isolated by mixing it with a water-immiscible organic solvent such as e.g. Ether, extracted, dried over an anhydrous reagent, filtered and freed from solvent in vacuo.

The process for reacting the primary thiol with the secondary halide has already been described above.

FIG. 6 shows, more specifically, an example of a reaction sequence that may be the last step in the synthetic route of FIG. 2. The preparation of a secondary halide by diazotization and conversion to a pseudodipeptide by reaction with a primary thiol is explained.

The key reaction in the synthesis of Figure 5 is the conversion of a primary halide to a primary thiol using thiourea. If the primary thiol is prepared in this way, the reaction conditions are practically the same as for the preparation of a secondary thiol.

FIG. 7 represents a conclusion from the reaction sequence of FIG. 4. It explains the production of a secondary thiol using the same method that was used according to FIG. 4 for the production of a primary thiol.

The synthesis according to FIG. 8 was developed as a conclusion from the synthesis according to FIG. 5; 5 shows that the 20 thiourea process for the production of primary thiols can also be applied to the production of secondary thiols.

Fig. 9 shows a method in which an amino alcohol whose amino group is blocked is reacted with tosyl chloride, whereupon the product obtained in this way is reacted with the dialkali salt of an a-mercaptocarboxylic acid. A variety of known amino blocking groups can serve as the blocking group, e.g. the tert-butoxy group or the carbobenzoxy group. These groups are preferred, 30 but you can also work with other groups. The blocking group is selected in a known manner and depending on the subsequent reactions in which the dipeptide thus obtained is to participate.

To prepare the tosyl ester, the blocked amino alcohol is reacted with a tosyl halide, preferably tosyl chloride, in a suitable solvent for 1 to 100 hours at a temperature of from -20 ° C. to 10 ° C.

A molar excess of tosyl compound is expediently used in order to ensure that the reaction is as complete as possible. The tosyl compound can therefore be used in an equimolar amount up to an excess of at least 2 moles, based on the number of moles of amino alcohol.

A halogen acid is generated in the reaction and it is therefore expedient to carry it out in the presence of a basic reagent which immediately neutralizes the acid formed. Generally an alkaline reagent is used which is soluble in the chosen solvent. It is clear to the person skilled in the art that the solvent selected must be inert to the reaction. Thus, water, ethyl alcohol, or other active hydrogen-containing solvents cannot be used since they would react with the tosyl chloride.

The most preferred solvent is pyridine, since this liquid can both neutralize the hydrogen halide and dissolve the reactants. In addition, pyridine can be easily removed after the reaction has ended, since it is soluble in water. The isolation of the reaction product is therefore very simple, since the solvent is water-soluble, while the reaction product is soluble in organic solvents.

The tosyl derivative is converted to a pseudodipeptide by e.g. with the disodium or potassium salt of glycine, alanine, leucine or isoleucine in 65 a reaction inert solvent under alkaline conditions at a temperature of about 20 ° C to 75 ° C and for about 2 to 12 hours. A molar excess of mercapto compound is expediently used,

11

641441

FIG. 11 shows the production of Boc-Phe ip Leu and FIG. 12 shows the production of Leu * P Gly.

In the synthesis of Boc-Phe ^ P Leu, phenylalanine is first reduced with borane to the corresponding amino alcohol 5, and the amino group is blocked with a t-Boc residue. The blocked amino alcohol is then converted to the O-tosyl derivative and reacted with the disodium salt of Des-NH2-a-mercapto-leucine.

In the process shown in Figure 12, leucine io is converted to 4-isobutyl-2-mercaptothiazoline by reacting with carbon disulfide in KOH-EtOH at 40 ° C for 24 hours. The thiazoline ring is opened with acid to form the thioleucinoleic acid salt, and this compound is reacted with a-iodoacetic acid to the desired product.

It will be clear to those skilled in the art that some of the reactions described in connection with the synthetic methods of FIGS. 2 to 12 can involve inversion. The course of the individual reactions can be followed in a known manner. Therefore, if a D-pseudopeptide according to the invention is to be produced, it may be necessary to use a starting compound with an L configuration. Similarly, preparation of an L-pseudopeptide may require a starting compound 25 with a D configuration.

A large number of pseudopeptides were produced using the methods described above. Table III lists some of these pseudopeptides and their physical constants.

TABLE III

Characterization of Gly * P Leu, Gly * p Ile and their stereo isomers

Dipeptide analog

Mp ° C

(under decomposition)

[a] D22

(c = 2, H20)

C.

Analysis C8H17NO2S *, Found • H N

■%

S

Gly ip Leu

214-216

-23.2 ± 1.2 °

50.10

9.08

7.20

16.60

Gly tpR Leu

221-224

+24.1 ± 1.3 °

50.56

9.13

7.37

16.45

Gly ip ile

218-220

-57.2 ± 1.0 °

50.57

9.04

7.35

16.65

Gly \ PR all

210-213

+37.9 ± 1.2 °

50.40

8.96

7.36

16.81

* Calculated: C 50.23; H 8.96; -N 7.32; S 16.76%. 45

A particular feature of the present invention is that once a pseudodipeptide is made it can be treated like a normal peptide. The chain length at the amino as well as at the carboxyl end group can thus be increased by means of the customary methods used in peptide synthesis.

The simplest is probably the Merrifield method, in which an amino acid, a peptide or pseudopeptide 55 is bound to a resin particle via an ester bond. Further amino acids, peptides or pseudopeptides are then added to the growing molecule in a known manner. Once the desired molecule is obtained, it is cleaved from the resin. A suitable release agent is anhydrous, liquid hydrogen fluoride in an inert solvent at 0 ° C.

The following examples illustrate the present invention.

Example 1: Gly \ P Leu 2-bromo-4-methylpentanoic acid

A total of 50 g of D-Leu was taken up in 1200 cc of 2.5N sulfuric acid, which contained 250 g of potassium bromide.

so that the reaction is as complete as possible. However, if the mercapto compound is the more expensive of the two reactants, an excess of tosyl derivative can also be used. Therefore, based on the number of moles of tosyl derivative, the tosyl compound may be present in an amount of 0.5 to 3 moles per mole of mercapto compound. Examples of suitable organic solvents are polar solvents such as dimethylformamide and lower alkanols, including methanol and ethanol.

Fig. 10 shows a process in which the tosyl derivative of the selected aminoalkanol is cyclized to a cyclic imine. This cyclization takes place in an aqueous medium under strongly alkaline conditions, i.e. a pH of at least 11 to 13, at a temperature of 25 ° to 75 ° C and lasts 1 to 4 hours. Alkali hydroxides are suitable alkaline reagents.

The substituted imine is bound to the selected a-mercapto acid by carrying out the reaction in aqueous alkali at a temperature of 20 ° to 50 ° C. for 0.5 to 3 hours. The mixture is acidified at the end of the reaction time, advantageously with a mineral acid. The pseudopeptide can be obtained from the aqueous medium by extraction with an organic solvent.

Figures 11 and 12 illustrate the application of the reactions described above to certain pseudopeptides. In

641441

12

held. The mixture was cooled to 0 ° C. and stirred, 65.5 g of sodium nitrite being added in small portions over the course of 1% hours. The solution was stirred for a further hour at 0 ° C, warmed to 25 ° C and stirred for another hour. Then the mixture was extracted with ether; the ether layer was separated, washed with water and dried over anhydrous sodium sulfate to the desired product.

2-mercapto-4-methylpentanoic acid

1.25 g (6.5 mmol) of the above product were taken up in 2 cc of water. A sufficient amount of sodium hydroxide solution was then added to dissolve the bromic acid. 5 cc of 40% sodium thiocarbonate was added to this mixture, whereupon the vessel was closed and left to stand at room temperature for 24 hours. After this time, the solution was acidified with 18N sulfuric acid and extracted twice with 10 cc of ethyl ether. The combined extracts were dried over anhydrous magnesium sulfate and filtered. The filtrate was concentrated to about half its volume and chromatographed on a silica gel column (2.2 × 30 cm). The original solution was eluted with 150 cc of ethyl ether and the product was obtained from the eluate by evaporating off the solvent. The yield was about 34%. The Rr values (thin layer chromatography - TLC) were 0.88 and 1.0 on silica gel and cellulose plates (n-butanol / acetic acid / water = 12: 3: 5).

Gly ¥ Leu

30 mmoles of ethylene imine were added to 2.2 mmoles of the above product in 10 cc of 0.5N sodium bicarbonate. This solution was kept at about 20 ° C for 2 hours and then acidified with 12N hydrochloric acid. It was extracted several times with ether and the extracts were discarded. The aqueous solution was desalted in a Dowex column (2 × 8) according to the procedure of Dreze et al. The acetic acid fractions were recovered and the product isolated by evaporation. The residue was recrystallized from 50% ethanol and gave a product with an Rr value of 0., 82 by TLC on cellulose in the butanol / acetic acid / water mixture described above. The specific rotation [a] n22 was -16.3 °.

Gly ¥ Leu

A total of 5.3 g (27 mmol) of the above bromic acid were dissolved in 530 ccm, 0.5m sodium bicarbonate flushed with nitrogen and 9.2 g (81 mmol) of 2-mercapto-ethylamine hydrochloride were added. The reaction vessel was flushed with nitrogen for 1 hour and then sealed. It was left to stand at room temperature for 24 hours, during which time the desired product was formed. The solution was acidified with 6N HCl and extracted twice with ether. The ether extracts were discarded and the aqueous portion was neutralized with 2N sodium hydroxide and diluted with 21 deionized water. The solution was desalted in a 5.5 X 30 cm column with Dowex2 X8 resin according to the procedure of Dreze et al. The acetic acid fractions were pooled and dried by evaporation under reduced pressure. Then the residue was triturated with 20 cc of acetone and crystallized from 47.5% ethanol, whereby the desired product was obtained in the form of white needles. Yield: 2.34 g, 44.5%; F = 205-210 ° C (with decomposition).

The same procedures were used to make Gly ¥ Ile using D-alloisoleucine instead of D-leucine.

In the course of the above reactions, it was found that the halogen replaced the original amino group without reversing the configuration. The reversal took place when the bromine was replaced. The amino acid residues of the pseudopeptides are therefore in L form. Analogous pseudopeptides, the components of which are in D-form, are obtained if the corresponding L-form of the starting amino acids is used.

Example 2: Phe ip His

2-Mercapto-4-benzylthiazoline 75 mmoles of Phe in 225 mmoles of ethanol containing 300 mmoles of carbon disulfide were stirred with 75 mmoles of solid potassium hydroxide while the temperature was kept below 40 ° C. The temperature was then raised to 40 ° C. and the reaction mixture was left to stand in a closed vessel for 12 hours. After this time, alcohol and carbon disulfide were removed by rotary evaporation. The residue was suspended in a small amount of cold water and the product was recrystallized from a water-ethanol mixture.

2-Mercapto-l-benzylethylamine A total of 4 g of the mercaptothiazoline were taken up in 25 cc of 12N HCl and heated to 155 ° C. under reflux for 5 hours. Then the excess acid was removed in a rotary evaporator. The desired product was obtained as the hydrochloride and recrystallized from aqueous ethanol.

Phe ip His

This product was prepared from a-bromo-ß- (5-imidazolyl) -propionic acid and 2-mercapto-l-benzylethylamine by reaction in the presence of sodium carbonate, which in Example 1 for the analogous reaction of 2-mer-captoethylamine described procedure was applied.

The pseudopeptide Leu ¥ Leu is obtained in a similar manner, with an isobutyl-substituted ethanolamine and an isobutyl-substituted 2-mercaptoethylamine serving as reactants.

Ala ip Ala is likewise obtained in the same way from 4-methyl-2-mercaptothiazoline (Gabriel and Ohle, Ber., 50, page 804, 1917) and a-bromopropionic acid.

The following pseudodipeptides were produced according to the method of this example:

Trp

¥

Gly

Arg

¥

Val

Phe

¥

Tyr

Mead

¥

Lys

gin

¥

Lys

Lys

¥

His

Arg

¥

Gly

Gly

¥

Lys

gin

¥

Arg

Arg

¥

Val

Lys

¥

Tyr

Lys

¥

Arg

Arg

¥

Thr

Arg

¥

Lys

Tyr

¥

Leu

Phe

¥

Arg

Arg

¥

Leu

Arg

¥

Val

Lys

¥

Tyr

Arg

¥

Per

Tyr

¥

Gly

Lys

¥

Lys

Tyr

¥

Leu

Phe

¥

Ser

Phe

¥

Mead

Lys

¥

Leu

Per

¥

Arg

Tyr

¥

Glu

Phe

¥

Leu

Phe

¥

Trp

Tyr

¥

Phe

Tyr

¥

Gly

Example 3

Solid-phase synthesis of Ac-Pro-Gln-Gly ip Leu-Ala-

Gly-Leu ip Z Orn-Gly-NH2 The synthesis of these pseudopeptides is carried out using the peptide shaker with medium reaction vessel described by Stewart and Young (Solid Phase Peptide Synthesis, W.H. Freeman and Company, 1969).

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

13

641441

4 g of Boc-Gly resin, which contained 0.24 mmol of Gly per gram of resin, served as starting material. The following scheme was applied to each series of Boc amino acid or Boc pseudodipeptide additions to the resin.

step

Reagent or solvent

Number d.

Encores

Volume, ccm

Time, minutes

1

CH2C12

3rd

40

1

2a

40% TFA / CH2C12 / anisole

1

40

20th

2 B

TFA / CH2C12 / anisole

1

40

1

3rd

CH2C12

5

40

1

4th

(i-Pr) 2NEt / CH2Cl2

2nd

40

2nd

5

CH2C12

4th

40

1

6

Boc amino acid (3 eq.)

1

15

20th

7

Boc-amino acid line flushing

1

5

-

8th

DCC (2.7 eq.)

1

15

90

9

CH2C12

2nd

40

1

10th

EtOH

2nd

40

1

11

CH2C12

4th

40

1

12

Boc amino acid +

1

15

-

13

Boc-amino acid line flushing

1

15

14

DCC

1

15

-

15

DCC line flushing and coupling 1

5

90

16

CH2C12

3rd

40

1

17th

EtOH

3rd

40

1

18th

DMF

3rd

40

2nd

19th

Ac20 4 cc

1

50

50

20th

DMF

2nd

40

2nd

21st

EtOH

3rd

40

2nd

The above scheme shows that a double coupling with acetylation of any remaining amino groups was used in this synthesis. In each step, 3 equivalents of amino acid or pseudodipeptide were used per equivalent of Boc-Gly resin.

Before Boc-Leu ¥ Z Orn and Boc-Gly ¥ Leu were coupled, these substances were released from their corresponding dicyclohexylamine salts by suspending 2.5 g of the salt in 10 cc of water. Citric acid (1 mole) was added slowly until the powder was liquefied. Then an equal volume of ether was added. After thorough mixing, the ether layer was separated, washed once with water and dried over sodium sulfate. The ether was removed under reduced pressure and the sample dried overnight; it provided the free pseudodipeptide. Boc pseudodipeptides are coupled in the same way as the amino acids.

When adding gin, the Boc-Gln-p-nitrophenyl ester was used in a 4-fold molar excess. DMF was used in place of methylene chloride in the coupling, washing and acetylation stages. The coupling time for 5 of each nitrophenyl ester cycle was 19 hours. After Pro was added, the deprotection and acetylation steps were performed and the finished resin-coupled peptide was freed from the resin with MeOH and triethylamine to provide the methyl-10 ester, which was then dried by treatment with anhydrous ammonia Methanol was converted into the corresponding amide.

Conversion of Ac-Pro-Gln-Gly ¥ Leu-Ala-Gly-Leu ¥ Z 15 Orn-Gly-NH2 to Ac-Pro-Gln-Gly ¥ Leu-Ala-Gly-Leu ¥ Arg-Gly-NHZ Das Leu ¥ Peptide amide containing Z orn was dried completely in vacuo and then treated for 30 minutes at 0 ° C. with stirring with anhydrous liquid hydrofluoric acid in the presence of 100 equivalents of methyl ethyl sulfide. The peptide was chromatographed in a Sephadex-G - 100 column in 10% aqueous acetic acid. The fractions containing the no longer protected peptide were pooled and lyophilized 25 and yielded Ac-Pro-Gln-Gly ¥ Leu-Ala-Gly-Leu ¥ Orn - Gly-NHj.

Treatment of this peptide with O-methylisourea according to the method of Merrifield (Cosand and Merrifield, Proc. Nati. Acad. Sci. USA 74, pages 2771-2775, 30 1977) gave the desired Ac-Pro-Gln-Gly ¥ Leu-Ala - Gly-Leu W Arg-Gly-NH2 obtained.

Example 4

35 Ac-Pro-Gln-Gly Leu-Ala-Gly-Leu-Arg-NH2

The table below summarizes the reactions to produce the above compound. The values in the right column show the amino acid composition at each stage of the synthesis. The values for the 40 pseudodipeptide eluted in the Phe position when a 4.5 hour citrate buffer elution program was used were determined after HCl propionic acid hydrolysis using cysteamine as a scavenger. For comparison, suitable analyzes were carried out in the absence of 45 pseudopeptide.

The various synthesis stages are described according to the table.

In the table and the description, “Aoc” stands for a tert-amyloxycarbonyl group. 50 The collagenase activity is inhibited by the presence of this pseudooctapeptide, which replaces the segment of the natural collagen molecule when the enzyme is tested in the natural sequence using the octapeptide as a substrate.

55

60

65

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14

TABLE IV

Benz resin

Aoc-Arg (Tos), DCC Aoc-Arg (Tos) benz resin

Boc-Leu, DCC Boc-Leu-Arg (Tos) -Benz. Resin

•> v

Leu 1.38 Arg 1.00

3 steps

\! /

Boc-Gly ip Leu-Ala-Gly-Leu-Arg (Tos) -benz.-resin TFA 40%, 20 minutes

Gly ¥ Leu-Ala-Gly-Leu-Arg (Tos) -Benz. Resin

(1) Boc-Gln-NPE

(2) 40% TFA

Gln-Gly ip Leu-Ala-Gly-Leu-Arg (Tos) Benz resin (0.276 mmol / g)

Ac-Pro, DCC

Ac-Pro-Gln-Gly ip Leu-Ala-Gly-Leu-Arg (Tos) -Benz. Resin

(0.243 mmol / g) HF / anisole sk

Ac-Pro-Gln-Gly ip Leu-Ala-Gly-Leu-Arg-NH2

Ratio d. Leftovers

Gly

1.0

Ala

0.99

Leu

1.06

Gly ¥ Leu

1.12

Arg

0.92

Gly

1.0

Ala

1.0

Leu

1.01

Gly ^ Leu

1.13

Arg

0.94

gin

0.88

Gly

1.0

Ala

1.0

Leu

0.99

Gly ip Leu

1.10

Arg

0.88

gin

0.86

Per

0.93

Gly

1.0

Ala

1.0

Leu

1.01

Gly ip Leu

1.08

Arg

0.96

gin

0.86

Per

0.87

Gly

1.01

Ala

0.97

Leu

1.08

Gly ip Leu

0.73 *

Arg

1.00

* Based on hydrolysis in the absence of detergent («scavenger»).

Preparation of Boc-Gly ip Leu 5.4 g (Aldrich, 22 mmol) of 2- (tert-butyloxycarbonyloxyimino) -2-phenylacetonitrile were added at room temperature to a mixture of 3.81 g (20 mmol) of Gly ip Leu , 4.1 cc (30 mmol) of triethylamine, 12 cc of dioxane and 12 cc of water. The mixture became homogeneous with stirring within 1 hour and stirring was continued for 10 hours. Then 30 cc of water and 30 ccm of ethyl acetate were added. The aqueous layer was separated and washed with ethyl acetate (2 X 40 ccm). The aqueous layer was acidified with 5% aqueous citric acid solution and extracted with ethyl acetate. The ethyl acetate extract was washed twice with saturated, aqueous

55

like to wash sodium chloride and dry over anhydrous sodium sulfate. Removal of the drying agent and solvent gave 1.1 g of an oil. This oil was dissolved in 40 cc ethyl ether and mixed with 7.4 g 60 (40 mmol) dicyclohexylamine. The suspension thus obtained was stored at 40 ° C for 4 hours before the desired salt was collected. The salt was washed with ether and dried in vacuo over phosphorus pentoxide; 10 g of white crystals were obtained 65 (80% yield, based on Gly ip Leu). The purified product was obtained by recrystallization of the dicyclohexylamine salt of Boc-Gly ip Leu from methanol / ether (2: 1 vol.); F = 143-144 ° C.

15

641441

Analysis C25H43N2O4S:

Calculation,%

Found,%

c

63.5

63.73

H

10.23

10.48

N

5.93

5.66

S

6.78

6.43

Introduction of amino acid with terminal C group into the resin

Benzhydrylamine resin HCl (2.0 g, 0.40 meq / g) was neutralized by stirring for 10 minutes with 50 cc of 25% triethylamine in methylene chloride. The resin was filtered off and washed with additional methylene chloride (25 cc per g resin). The resin was then suspended in 40 cc methylene chloride and shaken for 10 hours in the presence of a 2.5-fold molar excess of Aoc-Arg- (Tos). After washing with methylene chloride, the remaining, unreacted amino groups of the resin were acetylated with a mixture of acetic anhydride and N-methylmorpholine in dimethylformamide. Amino acid analysis of a weighed sample of the hydrolyzed Aoc-Arg (Tos) resin revealed that 0.24 mmol Argin-n per gram of resin was released.

Chain extension through double addition and acetylation

After the first amino acid was coupled to the resin, the remaining amino acids were added sequentially in the following manner: The resin was washed three times with methylene chloride and then with trifluoroacetic acid / anisole / methylene chloride (40: 2:60, v / v. ) pre-washed. The amino groups were released by shaking with a TFA solution of the same composition for 20 minutes. After washing five times with methylene chloride, the mixture was washed twice with 7% (v / v) N, N-diisopropylethylamine in methylene chloride. Excess amine was removed by further washing five times with methylene chloride. Then tert-butyloxycarbonylamino acid (2.5-fold molar excess) in methylene chloride was added to the reaction vessel. Dicyclohexylcarbodimide (2.3-fold molar excess) was added in methylene chloride (final volume 40 ccm). The reaction vessel was shaken for 2 hours. After 2 washes with methylene chloride, it was washed twice with ethanol. The five methylene chloride prewashes as well as the coupling steps were repeated. After washing three times with dimethylformamide, the remaining non-acylated amino groups of the resin were washed for 20 minutes by shaking with a mixture of 4 cc acetic anhydride, 2 ccm N-methylmorpholine and 34 ccm dimethylformamide. The double coupling was complete after three more washes with dimethylformamide and three more washes with ethanol. The double acetylation sequence described above was used to add all amino acid residues, including acetylproline, but with the exception of gin. Gin was introduced (twice) as the tert-Boc-p-nitrophenyl ester in dimethylformamide (avoiding dicyclohexylcarbodiimide) and the reaction time was 10 hours instead of 2 hours. The progress of the synthesis was followed by frequent hydrolysis and analysis of the growing peptide resin.

Addition of Boc-Gly ip Leu

Boc-Gly ip Leu was released from the dicyclohexylamine salt by suspending 2 g of the salt in 30 cc of a mixture of ethyl ether and water (1: 1, v / v). Then 1 mole of citric acid was added until all of the solid was dissolved. The ether layer was separated, washed with water and dried over anhydrous sodium sulfate. The sodium sulfate was filtered off 5 and the ether was removed in a rotary evaporator. The remaining oil was dried to constant weight in a vacuum dryer. The yield was 1.45 g (97%). Boc-Gly ip Leu is added to the peptide-resin in the same way as described above for Boc-amino acids.

10th

Release of the peptide from the benzhydrylamine resin product and deprotection of the peptide. The tosylated benzhydrylamine resin peptide (2.0 g) was HF at 20 ° C. for 1 hour at 20 ° C., dried over CoF3 in the presence of 2.0 cc anisole treated. After the HF was removed under reduced pressure, the resin was dried in vacuo overnight. The released, deprotected peptide was extracted from the resin with 60 cc of 7% acetic acid. The filtered extract was washed successively with 30 cc each of ethyl ether and ethyl acetate to remove the anisole. The solution was lyophilized and the residue dissolved in 0.2m acetic acid and lyophilized again. The crude peptide amide was chromatographed on Sephadex G-15 in 0.5m acetic acid. The main component determined by the Sakaguchi method was lyophilized and gave 334 mg of peptide amide (yield 80%). The product was purified by partition and / or carboxymethyl cellulose chromatography.

Distribution Chromatography on Sephadex G-25 A column (73 X 2.5 cm) was packed with Sephadex G-25 (fine quality) in water. The column was washed with the lower phase of a solvent mixture of n-buta-nol, pyridine and 0.1% aqueous acetic acid (5: 3: 11, 35 vol./vol.) (15) and then with the upper phase of the same solvent system (16) at a flow rate of about 1 cm / min. brought into balance. 55 g of the peptide were dissolved in 5 ccm of the upper phase and chromatographed by elution with the upper phase. 40 The fractions containing the peptide were determined by the quantitative Sakaguchi method (17). Fractions of the main component were pooled, diluted with an equal volume of water and concentrated in a rotary evaporator under reduced pressure. 45 After lyophilization, the concentrate gave 32 mg of a white powder (yield 58%).

Example 5

15

20th

25th

30th

50

55

Boc-Phe ip Gly-Dicyclohexylamine Salt This compound can be freed from the dicyclohexylamine molecule part and incorporated into a larger peptide using the methods described. If desired, the Boc group can also be removed by the HF treatment described in the last paragraph of Example 4. The pseudodipeptide thus obtained can serve as a surrogate for Phe-Cly.

Preparation of Boc-Phenylalaninol 3 g (20 mmol) of phenylalaninol and 20 mmol of tert-butyloxycarbonyl azide as well as a sufficient amount of in sodium hydroxide solution were added to a mixture of 10 ccm of water and 10 ccm of di-60 oxane, as well as a sufficient amount of sodium hydroxide solution to bring the pH to 8 To bring 0. The reaction mixture was stirred for 24 hours, 50 cc of ethyl ether was added and the mixture was cooled to 0.degree. Solid citric acid was added until the pH dropped to 3.5. The aqueous layer was extracted twice with 25 cc ether each. The combined ether extracts were extracted twice with 25 ccm 5% zi

16

641441

tronic acid and then washed three times with saturated sodium chloride solution. The organic layer was dried over anhydrous sodium sulfate. After removing the solvent under reduced pressure, 5.4 g of oil. receive. The oil was treated with hot hexane and gave 4.1 g of a white solid (needles; 81%). The crude product was recrystallized from 100 cc hot hexane containing 10 drops of ether. A white solid was obtained; Weight -2.8 g, F = 93-94 ° C.

Preparation of Boc-phenylalaninol tosylate 3.8 g (0.02 mol) of tosyl chloride were added to a solution of 2.5 g (0.01 mol) of Boc-phenylalaninol in 10 ccm dry pyridine, cooled to -10 ° C. The solution was left in a freezer overnight until no more white solid (pyridinium hydrochloride) precipitated. Then the mixture was poured into 100 cc of ice and ether. The organic layer was separated and washed three times each with 5% citric acid and saturated sodium chloride solution. The solution was then dried over anhydrous sodium sulfate and the solvent removed; 4.1 g of white solid were obtained; F = 92-93 ° C. The melting point could not be improved further by recrystallization from ether / hexane.

Preparation of Boc-Phe ip Gly / DCHA Salt To 0.24 g (1.8 mmol) of the disodium salt of mercaptoacetic acid in 2 ccm dry methanol and 5 ccm dimethylformamide, 0.6 g (2.4 mmol) Boc- Phenyl alaninol tosylate given. The solution was kept at room temperature for 5 hours and then the methanol was removed under reduced pressure. The solution was cooled to -5 ° C and 25 cc of chilled ethyl acetate was added. A 25% citric acid solution was added to bring the pH down to 2. The aqueous layer was extracted three times with 25 cc each of ethyl acetate. The combined organic extracts were washed once with 5% citric acid and three times with saturated sodium chloride. The ethyl acetate layer was dried over anhydrous sodium sulfate and the solvent was removed under reduced pressure. The oily residue was dissolved in ethyl acetate and an excess of dicyclo-hexylamine. The addition of anhydrous diethyl ether led to the precipitation of the Boc-Phe ip Gly / DCHA salt in an amount of 0.54 g (yield 58%). This salt was recrystallized from chloroform / ether (4: 1) and gave 0.4 g of white solid; F = 140-141 ° C (overall yield 43%).

Example 6

5

Preparation of tablets 1000 g Gly ip Leu and 2000 g lactose were carefully mixed and passed through a 30 mesh sieve. At the same time, a paste was made from 80 g corn starch and io 350 cc distilled water.

The mixture was then carefully kneaded with the paste and the mass passed through a 4-mesh sieve, whereupon the beads thus obtained were dried at 50 ° C. for 15 hours.

15 After drying, the beads were first crushed in a granulator and then passed through a 16 mesh screen. The granules were covered with a powdered mixture of 30 g calcium stearate, 200 g corn starch and 80 g talc and sieved through a 40 mesh 20 sieve.

Tablets, each containing 250 mg Gly ip Leu, were prepared in the usual manner from the granules obtained.

Example 7

25th

Preparation of injection solutions 100 g of the sodium salt Leu ip Leu were dissolved in a distilled, pyrogen-free water specially produced for this purpose and made up to 5 liters. Solution 30 was made isotonic by adding a predetermined amount of an aqueous physiological saline solution and then filtered through a microporous bacteria filter.

Example 8

35

Preparation of an aqueous solution for oral administration A mixture of

Gly ip Ile 40 cane sugar glycerin

Ethyl p-ethoxybenzoate artificial orange essence essential orange oil

45

was made up to a final volume of 1000 ccm with distilled water.

20.0 g 100.0 g 100.0 ccm 1.5 g 0.2 ccm 1.0 ccm v

2 sheets of drawings

Claims (10)

641441 2nd PATENT CLAIMS 1. pseudopeptides which contain peptide bonds between amino acid residues and at least one linkage in which a peptide bond between amino acid residues is replaced by a thiomethylene group, so that a pseudopeptide is present, at least part of which is shown by the following partial structural formula: HR R1 0 II I1 II N CHCH2SCHC in which R and Rt are those parts of a-amino acids which are bonded to the a-carbon atom in addition to the amino group, the free valence of the nitrogen atom is bonded to hydrogen or the carbon atom of a peptide bond and the free valence of the carbon atom to a hydroxyl group or to the nitrogen atom of a peptide bond is bound and its pharmaceutically acceptable salts. 2. Pseudopeptide according to claim 1, characterized in that it contains the structure Phe ip His. 3. Pseudopeptide according to claim 1, characterized in that it contains the structure Leu ^ Leu. 4. Pseudopeptide according to claim 1, characterized in that it contains the structure Ala * [/ Al. 5. Pharmaceutical agent for the treatment of metabolic disorders, characterized in that it contains a pharmaceutically acceptable carrier and a therapeutically effective amount of at least one pseudopeptide which contains peptide bonds between amino acid residues and at least one linkage in which a peptide bond between amino acid residues is replaced by a thiomethylene group that a pseudopeptide is present, of which at least part is represented by the following partial structural formula: HR R, 0 II i1 H N - CHCH2SCHC in which R and Rt are those parts of a-amino acids which are bonded to the a-carbon atom in addition to the amino group, the free valence of the nitrogen atom is bonded to hydrogen or the carbon atom of a peptide bond and the free valence of the carbon atom to a hydroxyl group or is bound to the nitrogen atom of a peptide bond, or contains its pharmaceutically acceptable salts. 6. Pharmaceutical composition according to claim 5, characterized in that the pseudopeptide contains the structure Gly ip Leu. 7. Pharmaceutical composition according to claim 5, characterized in that the pseudopeptide contains the structure Gly Ile. 8. Pharmaceutical composition according to claim 5, characterized in that the pseudopeptide contains the structure Phe His. 9. Pharmaceutical composition according to claim 5, characterized in that the pseudopeptide contains the structure Leu Leu. 10. Pharmaceutical composition according to claim 5, characterized in that the pseudopeptide contains the structure Ala Ala. Most of the physiological functions of the mammalian body are controlled by enzymes or hormones, which can be called physiological catalysts. Metabolic reaction sequences occur in many of these functions, in which hydrolytic cleavage of a protein takes place in one or more stages. In the reaction sequence that leads to blood clotting, for example, the conversion of prothrombin to thrombin is a crucial step. This conversion is catalyzed by the enzyme io factor Xa, which stimulates the hydrolysis of arginylthreonine and arginylleucine dipeptide units in the prothrombin molecule. If these proteolytically regulated cleavage stages go wrong, undesirable pathological conditions can occur. The body protects itself e.g. even from the risk of circulating blood clots by dissolving these clots. Part of the reaction sequence that ultimately causes this dissolution is the conversion of plasminogen to plasmin, which is triggered by the enzyme urokinase. 20 Excessive urokinase activity can lead to the formation of too much plasmin, and this in turn can lead to a complete inability to form clots. The body is then no longer able to protect itself from bleeding. There are many other examples of such 2s metabolic dysfunction.