DE2900926C2 - - Google Patents

Description

Most physiological functions of the mammalian body are controlled by enzymes or hormones, which may be called physiological catalysts. In many of these functions metabolic reactions occur in which a hydrolytic cleavage of a protein takes place in one or more stages. In the reaction sequence that leads to blood clotting, z. For example, the conversion of prothrombin into thrombin is a critical step. This transformation is catalyzed by the enzyme factor X _a , which stimulates the hydrolysis of arginylthreonine and arginylleucine dipeptide units in the prothrombin molecule.

Are these proteolytically controlled cleavage steps wrong? this can lead to unwanted pathological conditions. The body protects itself z. B. even before the danger of circulating Blood clots by dissolving these clots. A part the sequence of reactions that ultimately causes this dissolution is the conversion of plasminogen into plasmin by the enzyme Urokinase is triggered. An excessive urokinase activity can lead to the formation of too much plasmin, and this in turn can cause a complete inability to form clots. The body is then no longer able to prevent bleeding to protect. There are many other examples of such metabolic dysfunctions.

The present invention now gives the possibility of damaging Mitigate the effects of such disruptions by:  provides therapeutic agents or pseudopeptides which are one of the participants metabolic reactions involving proteolysis simulate, d. H. posing as this participant. These simulating molecules act as non-natural substrates, which bind the natural catalysts, or as non-natural ones Catalysts that bind the natural substrates, and weaken that way by high concentration or Hyperactivity of natural substance induced disorders from.

In other words, those in the therapeutic agents of the invention contained pseudopeptides simulate one of the partners in a proteolytic pair. A particular pseudopeptide may act as a catalyst, i. H. as an enzyme or Hormone, or even as a substrate for an enzyme to occur. In each Trap binds the pseudopeptide or the non-natural Partner the natural partner and continues its participation in the natural metabolism down. Is the pseudopeptide z. B. a Part of a natural substrate for a specific enzyme, so can this enzyme, as it attaches to the non-natural substrate is bound to not fulfill its usual function. The education of dangerously high levels of plasmin can thus z. B. be inhibited by introducing a plasminogen surrogate, the urokinase binds.

In Biochemical and Biophysical Research Communications, 74 (1977), pp. 273-278, the pseudodipeptide Gly ψ Leu is described. Other pseudodipeptides, in particular those with a glycine residue at the N-terminus will also be considered, but without this document is a suggestion would suggest certain other (especially different from pseudodipeptides) pseudopeptides to provide as pharmaceutical agents.  

The object of the present invention is defined by the claims. The present invention is further illustrated by the accompanying drawings, in which: Fig. 1 shows the similarity in the spatial shape between glycylglycine and the corresponding counterpart in which the peptide bond has been replaced by a thiomethylene group. FIGS. 2 to 10 show various processes which can be used for the production of the products according to the invention. Figures 11 and 12 illustrate the preparation of pseudophenylalanylglycine (not in accordance with the invention) and pseudoleucylglycine (not in accordance with the present invention) using combinations of the methods illustrated in any of the preceding Figures.

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

The main reason for accurately mimicking 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 Fig. 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 virtually the same spatial relationship, and that the thiomethylene group about the same size as the peptide group. The infrastructure of the peptide group and the thiomethylene group are also very similar, so that replacement of a thiomethylene group with the peptide group of a natural product does not affect such factors as charge distribution, dielectric constant and hydration ability. A pseudopeptide of the invention can therefore mimic the corresponding natural substance and controllably affect the responses otherwise elicited by the natural product.

Two of the main results, from the strong similarity between the products according to the invention and the corresponding ones natural products, are: (1) The invention Products can be used as therapeutic means of regulation the speed of naturally occurring, under Proteolytic metabolic reactions used because they play the role of the corresponding natural substances can take over; and (2) they are neither toxic nor mutagenic.

For specific purposes Both small pseudopeptides may be beneficial and others For purposes of use, pseudopeptides of up to 8 or 8 can be preferred contain more amino acid segments. The reason for these variations becomes obvious when one thinks of the simple "lock and key "analogy that is commonly used  to describe protein-protein interactions, such as B. the effect of a protein enzyme on a protein or peptide substrate. Considering the protein or peptide substrate as Key and the enzyme as a lock, so must the geometric Shape of the two structures match, or the key does not fit in the lock, d. H. there is no reaction.

If you take the pseudopeptide now the place of the castle, so Its geometric shape must be suitable to the key to be fit. This condition may be due to a pseudodipeptide can be met, but often a pseudopeptide with higher Molecular weight be more satisfactory.

As one skilled in the art will appreciate, the pseudopeptides of the invention are amphoteric and can lead to pharmaceutically breakable acid Addition salts and metallic salts are processed. The present invention also encompasses these salts. In the course of The description will make it clear that others too Derivatives of therapeutically active substances produced can be. Such derivatives are z. B. Products by reaction formed with free functional groups on the amino acid residues be, for. With the hydroxyl group of tyrosine. Also these derivatives are encompassed by the present invention.

Normally, the compounds of the invention are based on L-amino acids. In certain cases, however, it may be appropriate be to use the D-form of the acid. Both forms become of the present invention.  

Hereinafter, some of those used in the present specification will be used Designations defined in more detail:

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

Amino acid residue - a part of an α-amino acid molecule that after removal of the hydroxyl portion of the carboxyl group and a Hydrogen atom of the amino group in the formation of a peptide bond remains.

Amino acid side chain - the part of an α-amino acid that is bonded to the α-carbon atom, in addition to the amino group or, in the case of proline, in addition to the imino group. The term includes z. B. the Hydrogen of glycine, the benzyl group of phenylalanine or the isobutyl group of leucine.

For the sake of simplicity, pseudopeptides according to the invention are used the Greek letter ψ (psi) instead of the hyphen used to indicate that a peptide bond was replaced by a thiomethylene bond. In addition, will  the known abbreviations used for common amino acids. Leu-Leu and Leu ψ Leu are therefore Leucylleucin or Pseudoleucylleucin.

A typical pseudotetrapeptide of the invention containing only one Containing thiomethylene bond is:

Leu ψ Leu-Val-Tyr.

This compound and its pharmaceutically acceptable salts and derivatives are useful in the treatment of hypertension since the conversion of angiotensin into angiotensin I and angiotensin II inhibited by the enzyme renin. The treatment of a Patients with a pharmaceutical preparation containing this pseudopeptide contains a mimicking substrate that in Competition with the natural substrate angiotensin available Renin binds. To the extent that renin binds to the pseudopeptide it is bound for natural transformation unavailable. The pseudopeptide is therefore a suitable one therapeutic agent for the treatment of hypertension.

Below are some pathological conditions associated with the Pseudopeptides according to the invention can be treated, and the used pseudopeptides and the metabolic to be disrupted Reactions described:

high blood pressure

Hypertension is a common medical problem. A important level in the regulation of blood pressure is the  enzymatic cleavage of the α₂-globulin in angiotensin I, the in turn is split into angiotensin II. Angiotensin II is a strong, blood pressure-enhancing agent and blocking his education is an effective therapy option. Both Transformations is a split in the Phe-His and Leu-Leu Bindings take place. By introducing Phe ψ His and Leu ψ Leu in selected pseudopeptides and by administration of these Pseudopeptides will transform the α₂-globulin into angiotensin II blocked. Suitable for such blocking Pseudopeptides and their derivatives are e.g. Eg: Ac-Phe ψ His-Leu-Leu Val-Tyr-Ser-OH; Ac-Phe-His-Leu ψ Leu-Val-Tyr-Ser-OH; and Ac- Phe ψ His-Leu ψ Leu-Val-Tyr-Ser-OH. The pseudopeptides can parenterally, intravenously, by nasal sprays or suppositories be administered.

Ulcus formation on the cornea

Ulcus formation on the cornea is a common ophthalmic Problem with a wide range of etiologies, such as B. Burns or cauterization of the eye, rheumatic Arthritis and infections. Ulcus formation is caused by excessive formation of collagenase during the healing process caused. The cornea consists of 70% collagen. The split Collagenase is highly specific and collagenase involves only Gly-Ile and Gly-Leu bonds. That to avoid contains these destructive cleavages required pseudopeptide therefore Gly ψ Ile and Gly ψ Leu. Examples of inventive therapeutic agents containing the desired structures included are:

Ac-Pro-Gln-Gly ψ Ile-Ala-Gly-Gln-Arg-Gly-OEt,
Ac-Pro-Cln-Gly ψ Leu-Ala-Gly-Gln-Arg-Gly-OEt,
Ac-Pro-Leu-Gly ψ Ile-Ala-Gly-Leu-Arg-Gly-OEt,
Ac-Pro-Leu-Gly ψ Leu-Ala-Gly-Leu-Arg-Gly-OEt,

the amide analogs thereof and the parent compounds in which the carboxyl group of the glycine molecule is a free one. In ulceration The cornea is treated by passing the pseudopeptide several times daily in an isotonic, aqueous solution with a pH Value of about 7.4 externally.

Since it is known that the terminal C group is removed from the Arg, if the peptide comes into contact with plasma, is suitably Gln ψ Arg or Leu ψ Arg in the same peptide or a D-Arg in introduced the terminal C group. The thus obtained, usable Pseudopeptides are:

Ac-Pro-Gln-Gly ψ Ile-Ala-Gly-Gln ψ Arg,
Ac-Pro-Gln-Gly ψ Ile-Ala-Gly-Leu ψ Arg

or the corresponding esters or amides. The Ile in the pseudo connection can be replaced by Leu.

Rheumatoid arthritis

Rheumatoid arthritis is a syndrome of which millions of People are infested. This disease works among other things by destroying the tissue collagenase hydrolytically acting on the collagen matrix of the affected joint. There in this syndrome, the same enzyme plays a role, the also causes the formation of ulceration on the cornea, which are therapeutic suitable pseudopeptides identical to, for  this ulceration used pseudopeptides. In the clinical Treatment is common to rheumatic patients excess synovial fluid from the inflamed joints away. Then the patient is given a sterile saline solution or suspension of the pseudopeptide in a volume of Injected 1 ml or less, which is only a small part of replaced withdrawn liquid.

Anticoagulants

Many heart patients are given anticoagulants for prophylaxis in order to reduce 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 by blocking the conversion of prothrombin to thrombin by the proteolytic enzyme factor X _a . This enzyme causes a specific cleavage at Arg-Thr (residues 274-275) and Arg-Ile (residues 323-324) of the prothrombin sequence. Intervention in these cleavages by synthesis and administration of suitable pseudopeptides results in highly specific blocking agents becoming effective at the beginning of blood clotting. These pseudopeptides contain Arg ψ Thr and Arg ψ Ile and are z. B .:

Ac-Ile-Glu-Gly-Arg ψ Thr-Ser-Glu-OEt
such as
Ac-Ile-Glu-Gly-Arg ψ Ile-Val-Glu-OEt.

The pseudopeptides can be administered in tablet form, intravenously, through Nasal sprays or spotting and administered in suppositories become.  

Mitigation of urokinase activity

The enzyme urokinase activates plasminogen to plasmin. These Transformation is an important step in preventing Thromboses. Urokinase is therefore a commonly used therapeutic. An undesirable side effect of urokinase therapy however, are bleeding. The present invention now provides the ability to prevent such bleeding by removing the Effect of urokinase attenuated by a specific inhibitor. Occur during treatment bleeding and proves the urokinase thus unsuitable, so can the corresponding Pseudodipeptides are administered. These pseudodipeptides contain Arg ψ Met and Lys ψ Ser.

The complete pseudopeptides suitable for such blocking are z. B .:

Ac-Ser-Ile-Arg ψ Met-Arg-Asp-Val-OEt
and
Ac-Glu-Asn-Arg-Lys ψ Ser-Ser-Ile-Ile-OEt.

They may be parenteral, intravenous, by nasal sprays or spotting and in suppositories. Drugs are also suitable for the treatment of haemophilia.

Prevention of destruction by elastin in emphysema

For people who have a genetic deficiency of α₂-antitrypsin, pulmonary emphysema is a great risk. The α₂- Antitrypsin in the bloodstream of the lung protects the lung tissue usually before the action of elastase. Without this Protection is the lung heavily damaged by elastase. The of  Elastase most frequently cleaved peptide bond is an alfa Ala bond. Therefore, that is to be incorporated into the inhibitor Pseudodipeptide Ala ψ Ala. The obtained therapeutic peptides are z. Aca Ala Ala OEt and Ac Ala Ala Ala Ala OEt. administered They are made 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 used, for. B. loss or Lack of fertility, menstrual disorders and ovulation as well as infertility of the man. This hormone has a short biological half-life (2 minutes), as it is due to proteolysis is quickly destroyed. A long-lasting hormone is therefore a very good one important therapeutic. The bonds undergoing proteolysis are z. B. Gly-Leu, Tyr-Gly and Pro-Gly-NH₂. Needed thus the following pseudodipeptide units: Gly ψ Leu, Tyr ψ Gly and Pro ψ Gly-NH₂. The corresponding long-lived hormones are z. B .:

PGlu-His-Trp-Ser-Tyr ψ Gly-Leu-Arg-Pro-Gly-NH₂,
PGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro ψ Gly-NH₂,
PGlu-His-Trp-Ser-Tyr ψ Gly-Leu-Arg-Pro ψ Gly-NH₂,
PGlu-His-Trp-Ser-Tyr-Gly ψ Leu-Arg-Pro-Gly-NH₂.

They can be administered in nasal sprays or by dabbing, in suppositories, parenterally and intravenously in one Carrier of normal saline.  

Anticoagulant blocking of the action of thrombin or fibrinogen

As already stated, are anticoagulants important therapeutics. When clotting takes a complicated Sequence of activations by proteolytic enzymes instead, taking in the cleavage of fibrinogen with thrombin below Formation of fibrin culminates, which then spontaneously becomes one soft clumps. Thrombin cleavage in the A-α- Chain attaches to the Arg-Gly bond. As a result, for an inhibitor requires the pseudodipeptide unit Arg ψ Gly. The complete peptide inhibitor for the trombinant action is:

Ac-Gly-Gly-Gly-Val-Arg ψ Gly-Pro-Arg-Val-NH₂.

The z. In tablets, intravenously, by nasal sprays or spotting and be given in suppositories.

Protection against gastric ulcers

Pepsin is the predominant proteolytic enzyme in gastric juices. People who secrete excess gastric juice are prone to ulceration by proteolysis. An inhibition of Pepsin-induced proteolysis protects against enzymatic Damage. Especially the Phe-Phe bond is subject to the Proteolysis by pepsin. The pseudodipeptide required for the synthesis Unity is as Phe ψ Phe.

The therapeutic inhibitor is z. B .:

Ac-Ala-Ala-Phe ψ Phe-NH₂.

The substance can be taken orally as a tablet or as a liquid in it Be given combination with acid antagonists.  

Pseudopeptides of tuftsin

There are known medical syndromes in which the cells of the Patients virtually unable to phagocytosis or pinocytosis are. A therapeutically useful, the phagocytosis stimulating Class of peptides contains basic and hydroxy amino acids according to certain schemes. These funds correspond to the Structure H-B-Pro-B ', where H is one, from the hydroxyamino acid (Thr or Ser) position, and B and B stand for basic amino acids, such as. Arg, Orn or Lys. Plasma is known to exhibit carboxypeptidase B activity, and the kidney has an aminopeptidase activity. Pseudotuftsine, the z. Pro ψ Lys, Pro ψ Arg, Ser ψ Lys or Ser ψ Arg, therefore have a longer biological half life than that normal peptide. Examples of such pseudotune lines are: Thr-Lys-Pro ψ Arg, Thr ψ Lys-Pro-Arg and Thr ψ Lys-Pro ψ Arg. Arg can also be replaced by Orn. The pseudotuntsine show different resistance to proteolysis.

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

Insulin with a longer biological half-life

Insulin is a well known treatment for diabetes. It is inactivated by the terminal C group of octapeptides removed by trypsin-like action. Therefore, you can Pseudoinsulins resistant to proteolysis, be prepared by placing Cys ψ Asp in position 20-21  introduces the A chain of human insulin and / or the Residues 22 and 23 in the B chain of insulin by Arg ψ Gly replaced. Coupling (oxidation) by synthesis in solid Phase-produced chains provide an insulin opposite Proteolysis is resistant. The pseudoinsulins thus obtained can be administered in the same way as natural or synthetic human insulin.

Enkephalins, endorphins

Recently, the structures of the endogenous opiate peptides discovered and explored. These substances (enkephalins and β- Endorphins) show a similar analgesic effect as Opium. For leu-enkephalin, which has the structure Tyr-Gly- Gly-Phe-Leu possesses a loss of biological activity by cleavage of the Tyr-Gly bond. If one takes place whose Tyr ψ Gly, we obtain an analogous substance with longer biological half-life and better therapeutic Value. The finished pseudoenkephalin is Tyr ψ Gly-Gly-Phe-Leu. Leu can also be replaced by Met.

Similarly, the β-endorphins can be stabilized by introducing a Met ψ Thr bond into the β-endorphin. Leu ψ Thr can be similarly effective.

Preferably, the products thus obtained are administered intravenously; However, administration by nose or rectum is also possible.  

Inhibition of acrosin

Acrosin is the key enzyme in spermatozoa. This trypsin similar enzyme is said to penetrate the egg during the Facilitate fertilization. Interrupting the action of the acrosin, this prevents fertilization. The inhibitory Substance in this case is benzoyl-Arg ψ Gly-OEt. The pseudodipeptide Unit is Arg ψ Gly.

This inhibitor is similar in an intravaginal foam Dolphin, applied.

Long-lasting oxytocin

Oxytocin causes uterine contractions and is used in obstetrics used to initiate labor. It owns following structure:

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

As pseudodipeptides serve Tyr ψ Ile and Leu ψ Gly-NH₂.

Long-lasting vasopressin

Vasopressin is used in medicine for surgery shock treatment as a remedy used to increase blood pressure. It also serves It is used to treat delayed postpartum bleeding and for deliveries to repair uterine weaknesses ("inertia").

Vasopressin has the following structure:

The invention suitable pseudodipeptides are z. B. Arg-Gly NH₂, Tyr ψ Phe, Pro ψ Arg. A pseudovasopressin containing this Contains combinations is:  

The product can be given intravenously or intranasally.

For the sake of simplicity, all the above-described physiological States referred to as metabolic disorders. Some however, there are no metabolic abnormalities, though they include metabolic reactions. In pain relief, z. For example, one can not speak of 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 each state leads, a substrate is split, the has a natural peptide bond. By the present Invention now reduces the total amount of cleavage products, by introducing a pseudopeptide at the reaction site that is either in competition with the substrate or with the The reaction-affecting catalyst occurs by itself for one of these participants spends or takes his place.

Since the development of the genetic code, it has become clear that Polymers, such as. As DNA, RNA and proteins and peptides Information containing products are. In the case of nucleic acids  is the information in the order in which purine and pyrimidine bases occur. "Translated" this information now for protein and peptide structures, it's the amino acid Side chains that are the distinguishing features of the protein or peptides. The function of the peptide backbone corresponds essentially that of a connecting structure. So missing z. As in polyglycine virtually all information. It is the appearance of these unique side chains by the genetic code were brought into play that sort of and Way in which a peptide or protein is folded, and with it also the spatial relationship between the different atoms and determine molecular segments, in particular the side chains at the basic structure. The resulting, three-dimensional Structure allows a cooperative interaction between various proteins, e.g. As enzymes and substrates, and determined by their biological effect.

The aim of the present invention is now the construction of a protein- or peptide-like structure that is virtually the same same molecular geometry as the corresponding natural Substance. This goal is achieved by using a peptide bond replaced by a thiomethylene bond. Products, which have been built around such Thiomethylenbindungen show essentially the same molecular geometry as the natural one Products. You can participate in the same kind of reactions take part. However, the thiomethylene bond is resistant to Hydrolysis is by increasing the concentration Pseudopeptide in the presence of the natural product and its  natural partner in the reaction causes the Concentration of the normal reaction product decreases, as the Pseudopeptide has the correct geometric configuration, to bind the natural partner. Because of the resilience the pseudo-peptide against hydrolysis, the so bound materials are then not easily split.

A particular advantage of the present invention is that the results can be predicted practically exactly. Is the essential feature of a natural reaction known, so a pseudopeptide can be constructed with certainty is effective. If z. B. is known that in the reaction between an enzyme and a substrate binding certain Peptide bonds of each reactant involved, so This reaction can be controlled by using one Pseudopeptide which is the part of the enzyme or substrate, which exactly reproduces the peptide bond, in which, however the peptide bond was replaced by a thiomethylene group. To determine the best mode of administration or other parameters, can simple, carried out in a known manner experiments to be required. The basic reaction, however, is certain predictable.

The products of the invention are good therapeutic agents for humans and mammals and can hydrolytic cleavage of protein or peptide substrates even at extraordinary inhibit low doses. The for each administration  The most suitable dosage can be given by the doctor or Vet to be determined. It can vary from patient to patient and depends on the size of the patient, the one to be treated Disruption and other easily detectable factors. Should patients with more or less chronic metabolic disorders over be treated continuously longer periods of time, so be the products are often initially in relatively large dosage units given to immediately build up a certain concentration in the blood, and then in relatively small dosage units to maintain an effective concentration. For temporary Treatment of acute or chronic conditions may be different Dosage forms are provided. The products can be given in very large amounts, even up to 2 g or more per day. Usually they are in dosage units prepared containing about 250 mg of active ingredient, and the doctor may prescribe how many dosage units needed per day for the disorder to be treated.

The products according to the invention can be administered alone, in general, however, together with a pharmaceutically acceptable, non-toxic carrier, its amount of suitability and chemical nature of the chosen carrier, the mode of administration and usual pharmaceutical practices. to Treatment of certain disorders or maintenance therapeutically effective concentration in the blood or tissues can the products z. B. orally in the form of tablets or Capsules containing such carriers as starch, Lactose, certain types of clay and the like. you  can be provided with a colon-soluble coating for them more resistant to acid and digestive enzymes of the stomach are. For intravenous and intramuscular injections they can be made in the form of a sterile solution, containing other solutes, eg. B. sufficient amounts Saline or glucose to make the solution isotonic.

A particular advantage of the products according to the invention is that they - unlike many others, a peptide bond containing therapeutic agents - be administered orally because they are significantly more resistant to enzymatic hydrolysis by the enzymes of the lower digestive tract are. Because they are amphoteric, they can be given by mouth adsorbed on non-toxic ion exchange resins which may be either anionic or cationic; on This way, the active ingredient in the stomach and / or intestines becomes slow released. In addition, the resistance of the products towards enzymatic degradation by adsorption on such Resins still improved.

Another, by the amphoteric nature of the invention Products advantage is that the products, as already listed above, in the form of their pharmacologically acceptable salts can be applied, 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 alkali salts, are relatively stable and are therefore preferred over the acid addition salts. Especially  sodium salts are preferred since they are very easily prepared can be.

The acids used to prepare the invention, pharmacologically useful acid addition salts can be used, include non-toxic anions and are e.g. Hydrochloric acid, Sulfuric acid, phosphoric acid, acetic acid, lactic acid, citric acid, Tartaric acid, oxalic acid, succinic acid, maleic acid, Gluconic acid or sugar acid.

Other useful derivatives are obtained by using the free functional groups on the pseudopeptide backbone modified, d. H. the free hydroxyl or free amino groups. A very suitable class of derivatives are derivatives which is a free hydroxyl group, for. B. the free hydroxyl group of threonine or tyrosine, with an alkanoyl or alkenoyl group was esterified with up to about 18 carbon atoms or more. It can also be an amino group, such as. B. the amino group of Threonine or lysine, an alkanoyl or alkenoyl group be acylated with up to about 18 carbon atoms. In both Cases, the derivatives are preferred in which the derivative groups contain about 11 to 18 carbon atoms, because  the longer hydrocarbon chains give the molecules a better lipid solubility and improve their transport through the cell dividing walls.

Both types of derivatives can be obtained directly from the Pseudopeptide are produced; preferably, however, they will produced during synthesis by adding an amino acid with the selected group, eg. B. the already existing alkanoyl group, implements.

The compounds of the invention have a number of particular Characteristics. One for her therapeutic value especially important feature is the close analogy with regard to configuration and chemical composition between the invention Products and the natural compounds that they to replace. In their use, therefore, hardly any antigenic To count reactions. Besides, they seem little or not to be toxic at all. Finally they can Introduced without risk in the metabolism and excreted again become.

The natural substrates by the invention Pseudopeptides are to be replaced when exercising their normal physiological function on a given, natural Peptide binding site hydrolyzed. The amino acid segments on both sides of this passage are known. The pseudopeptide is usually constructed so that it is a substructure contains, in which the natural place by a  Thiomethylene group has been replaced, and this substructure has another substructure on at least one side, which resembles the corresponding structure of the natural substrate or the same.

An example of the low toxicity of the invention Compounds are the results obtained by the treatment of Mice were obtained with the known pseudodipeptide Gly ψ Leu. In these experiments received Mice (Swiss Wistar) intraparenteral injections with Gly ψ Leu in the dosages given below. For each dosage amount five mice were used. In no case the mice showed signs of acute toxicity. Behaviour The mice were maintained for 7 days after injection observed. In no case were harmful effects observed.

Weight gain of Swiss Wistar mice (N = 5) injected with Gly ψ Leu dose % Gain in weight after 3 days 44 mg / kg 17 94 mg / kg 19 208 mg / kg 20 432 mg / kg 23 Saline as a comparison 18

Potential mutagenic effects of the pseudodipeptides were limited to two studied different ways. A possibility for metabolic Complications is the effect of methionine adenosyltransferase  (MAT). Ethionine (which contains a thiomethylene bond) is known to be carcinogenic as well as a substrate for MAT. Although the specificity requirements for MAT are very high Strictly, there was the possibility that Gly ψ Leu either a substrate or a competing inhibitor. The below listed by the method of Hoffman "Arch. Biochem. and Biophys. 179, 136 (1977), however, show results obtained that neither Gly ψ Leu nor Gly ψ D-Leu have a noticeable Influences the MAT activity.

Table II

Influence of Gly ψ Leu and Gly ψ D-Leu on MAT

The mutagenicity of Gly ψ Leu and Gly ψ Ile was investigated based on a modification of the Ames experiment in which "repair-deficient" strains used by Bacillus subtilis were.

The results of Table IIa show that neither Gly ψ Leu Gly ψ Ile still acts as a mutagen.  

Table IIa

Results of the mutagenic test

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

• 1. Laumbach, 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.

There are already a number of methods for the synthesis of the invention Pseudopeptides known. These procedures are in the figures of the accompanying drawings. In these Figures R and R₁ represent the corresponding amino acid Side chains, ALK is an alkyl group with up to 5 carbon atoms, X is halogen, Y is an amino blocking group and TOS the tosyl group.  

The skilled artisan will recognize that various of the in the drawings shown reaction sequences on the formation of a Thiomethylene linking group by reaction of a primary Halides with a secondary thiol or a secondary Halides are based on a primary thiol. The individual syntheses differ from each other through the procedures, produced by the crucial reactants become.

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

The starting materials for this synthesis are amino acids that were reduced to amino alcohols. A series of such amino alcohols is already known and commercially available. Other may be prepared by reduction with borane according to the method of Yoon et al. in Journal of Organic Chemistry, Vol. 38, No. 16, p 2786 (1973).

In general, one uses a method in which the to be reduced Amino acid is contacted with borane, usually with a molar excess of borane in one Solvent of the ether type. The preferred solvent is Tetrahydrofuran. The reaction temperature may be about 15 ° to 40 ° C and the reaction time from ½ hour to 24 hours.  

The reaction product can be prepared by any isolation method be won. A suitable method is to that the excess reducing agent by adding cold water diluted with a small amount of solvent can be decomposed.

The conversion of the aminoalcohol to the desired mercaptothiazoline takes place in a reaction-inert, organic solvent in the presence of a strongly alkaline reagent, wherein carbon disulfide used as the cyclizing agent becomes. Typically, one works with a molar excess of carbon disulfide, e.g. B. a molar excess up to about 30% to ensure the fullest possible reaction. The most suitable alkaline reagents are alkali hydroxides, z. For example, sodium or potassium hydroxide. It can be one Variety of reaction-inert, polar organic solvents be used. At present, the lower aliphatic are preferred Alcohols, especially methyl and ethyl alcohol.

The reaction temperature can be between about 30 ° C and 45 ° C at vary in normal atmospheric conditions. The reaction is usually over after about 6 to 12 hours.

The desired product can be obtained by simply evaporating the Carbon disulfide and the solvent can be recovered. The residue is washed with cold dilute acid, e.g. B. with 2% aqueous hydrochloric acid. He may if necessary to Purification recrystallized, usually a 1: 1 mixture made from water and alcohol.  

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

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

The mercaptothiazoline can easily be replaced by a ring-opening Reaction with a halogen acid, suitably aqueous Hydrochloric acid, convert to a primary thiol. The reaction temperature can be about 60 ° to 160 ° C and will take 8 to 20 hours maintained. A very simple procedure exists in that the reaction mixture is a normal excess to 6N hydrochloric acid for 10 to 72 hours to Reflux heated.

At the end of the reaction period, the desired product can be used as Hydrochloride salt by evaporation at 50 ° to 80 ° C under reduced Pressure to be gained. Isolation of the product in the form The salt is particularly useful because the salt is direct can be used in the following reactions. But it is for some reason the free base is desired, so she can be obtained by z. B. dilute potassium carbonate titrated to the isoelectric point and then with a extracted water-immiscible organic solvent.  

The α-halocarboxylic acids, d. H. the secondary halides, the can be used by known methods manufactured.

The reaction of a primary thiol with a secondary halide or the reaction of a primary halide with a secondary thiol can be stopped by at least Equimolar amounts of the reactants in dilute, aqueous Media under an inert atmosphere at a temperature used from about 20 ° to 60 ° C and about 10 to 30 hours implements. To ensure a complete reaction and especially disorders due to competing reactions restrict, for. B. the reaction in which an amine group instead of a mercapto group, the halide is replaced recommends it is to use a molar excess of thiol. Even the use of an excess of 2 to 3 moles of this reactant is not unusual.

The currently preferred method for the isolation of the resulting Pseudodipeptide is desalting using an ion exchange resin; see the method of Dreze et al. in Anal. Chin. Acta 11, page 554 (1954). As resin becomes generally "Dowex®2-X8" preferred.

Fig. 3 shows a reaction sequence in which an amino-substituted, primary halide is reacted with an α-mercaptocarboxylic acid, ie a secondary thiol.

The starting amino alcohol can be found in the above prepared and then into a primary halide being transformed.

To prepare the primary halides, the amino alcohols prepared as described above are reacted with halogen substituents. As the halogen-substituting agent, any known means can be used; However, preferred are those indicated in Fig. 3 means, ie thionyl chloride, thionyl bromide or 48% aqueous hydrogen bromide.

The reaction with the thionyl halide can be carried out without solvent be performed; for better regulation finds the reaction but generally in a reaction-inert, polar solvent instead, preferably in anhydrous dimethylformamide or dimethyl sulfoxide. The reaction is normal after about 1 to 3 hours under reflux conditions, wherein at least a molar amount of thionyl halide, suitably but a molar excess of at least 10% of the reagent is used.

After completion of the reaction, the excess thionyl halide decomposed by adding water. The thus obtained Mixture is neutralized with dilute, aqueous sodium bicarbonate and with a water-immiscible, organic solvent extracted, z. B. with diethyl ether. The non-aqueous Layer is dried, expediently over anhydrous Magnesium sulfate, and filtered, whereupon the desired  Product obtained by evaporation of the solvent in vacuo becomes.

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 are carried out. The desired reaction product can be obtained in the manner described above, wherein but of course no excess reagent decomposes must become.

The secondary thiol is made from a secondary halide, the Z. B. by a known Diazotierungsverfahren was obtained.

The secondary halide can be converted to the desired thiol by mixing 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% ethanol and takes about 1 up to 4 hours. It can be equimolar amounts of the reactants be used; in general, however, will be an excess to thiourea preferred to complete as complete To ensure reaction. The preferred alkaline reagent is sodium hydroxide, and it becomes practically catalytic Quantities applied, eg. B. up to about 5 mol%.  

An isothiourea halide is obtained as an intermediate, but that does not have to be isolated. Instead, it is additionally aqueous Alkali added to the reaction medium and the reaction mixture thus obtained another 1 to 4 hours at about 70 ° to 100 ° C heated.

The product thus obtained is conveniently isolated by: after neutralization with dilute acid, e.g. B. diluted Hydrochloric acid, with a water-immiscible organic Solvents, such as. As ether, extracted. The product is out The organic solution obtained after passing over an anhydrous Dried reagent, filtered and in vacuo from the solvent was freed. This method is described in Org. Syn., Coll. Vol. 4, page 401 (1963), described in more detail.

Subsequently, the primary halide in the above-described Were reacted with the secondary thiol.

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

The trithiocarbonate is normally produced by reaction of Carbon disulfide and sodium sulfide in aqueous alkali, e.g. B.  10% sodium hydroxide formed. The primary halide is at a temperature of about 20 ° to 60 ° C to the trithiocarbonate given. Then, the reaction mixture becomes about 5 to 15 Hours kept at about 20 ° to 60 ° C to the thioester too form.

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

The method of reacting the primary thiol with the secondary Halide has already been described above.

Fig. 6 shows more specifically an example of a reaction sequence which may be the last stage of 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 will be explained.

The key reaction in the synthesis of Figure 7 is the conversion of a primary halide to a primary thiol using thiourea. When the primary thiol is prepared in this way, the reaction conditions are virtually the same as in the preparation of a secondary thiol.

Figure 6 illustrates a conclusion from the reaction sequence of Figure 4. It illustrates the preparation of a secondary thiol by the same method used in Figure 4 to make a primary thiol.

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

Fig. 9 shows a method in which an aminoalcohol whose amino group is blocked is reacted with tosyl chloride, followed by reacting the product thus obtained with the dialkali salt of an α-mercaptocarboxylic acid. As a blocking group can serve a variety of known amino blocking groups, for. B. the tert-butoxy group or the carbobenzoxy. These groups are preferred, but you can also work with other groups. The choice of the blocking group is carried out in a known manner and in dependence on the subsequent reactions in which the dipeptide thus obtained should participate.

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

Suitably, a molar excess of tosyl compound used to ensure the fullest possible reaction. The tosyl compound can therefore be in equimolar amount  to an excess of at least 2 moles, based on the Number of moles of aminoalcohol used.

In the reaction, a halo acid is generated and it becomes therefore useful in the presence of a basic reagent performed, which immediately neutralizes the resulting acid. In general, an alkaline reagent is used which in soluble in the chosen solvent. The skilled person is clear that the chosen solvent must be reaction-inert. Thus can neither water nor ethyl alcohol or other, active hydrogen containing solvents are used as they would react with tosyl chloride.

The most preferred solvent is pyridine, as these Neutralize both the hydrogen halide and liquid also the reactants can solve. In addition, can be Easily remove pyridine after completion of the reaction as it is is soluble in water. The isolation of the reaction product is very simple because the solvent is water-soluble, while the reaction product is soluble in organic solvents is.

The tosyl derivative is converted to a pseudodipeptide by it is z. With the disodium or dipotassium salt of glycine, Alanine, leucine or isolucine in a reaction-inert solvent under alkaline conditions at a temperature of about 20 ° to 75 ° and reacted for about 2 to 12 hours. expedient if a molar excess of mercapto compound is used,  so that the reaction proceeds as completely as possible. is however, the mercapto compound is the more expensive of the two reactants, so can also with an excess of tosyl derivative to be worked. Therefore, based on the number of moles Tosylderivat, the tosyl compound in an amount of 0.5 to 3 moles per mole of mercapto compound be present. examples for 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 chosen aminoalcohol is cyclized to a cyclic imine. This cyclization is carried out in an aqueous medium under strongly alkaline conditions, ie a pH of at least 11 to 13, at a temperature of 25 ° to 75 ° C and takes 1 to 4 hours. Alkali hydroxides are suitable alkaline reagents.

The substituted imine is added to the selected α-mercapto acid bound by placing the reaction in aqueous for 0.5 to 3 hours Alkali at a temperature of 20 ° to 50 ° C. The Mixture is acidified at the end of the reaction time, conveniently with a mineral acid. The pseudopeptide can pass through Extraction with an organic solvent from the aqueous Medium are obtained.

Figures 11 and 12 illustrate the application of the above-described reactions to certain pseudopeptides (not according to the invention). In Fig. 11, the preparation of Boc-Phe ψ Leu and in Fig. 12, the production of Leu ψ Gly is shown.

In the synthesis of Boc-Phe ψ Leu is first phenylalanine with Borane reduced to the corresponding aminoalcohol, and the Amino group is blocked with a t-Boc residue. The blocked Aminoalcohol is then converted to the O-tosyl derivative and with the disodium salt of leucine reacted.

In the process shown in Fig. 12, leucine 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 acid-opened to form the thioleucric acid salt, and this compound is reacted with α-iodoacetic acid to give the desired product.

It will be appreciated by those skilled in the art that in some of the reactions described in connection with the synthetic methods of Figures 2 to 12, inversion may occur. The course of the individual reactions can be monitored in a known manner. Therefore, if a D-pseudopeptide of the present invention is to be produced, it may be necessary to use an L-configuration starting compound. Similarly, production of an L-pseudopeptide may require a starting compound of D configuration.

With the help of the methods described above, numerous Produced pseudopeptides. In Table III are some of these Pseudopeptides and their physical constants are listed.  

Table III

Characterization of Gly ψ Leu, Gly ψ Ile and their stereoisomers

A special feature of the present invention is that a Pseudodipeptide, once it has been made as normal Peptide can be treated. So can the chain length both at the amino and at the carboxyl end group by means of the usual method used in peptide synthesis increase.

The easiest is probably the Merrifield method, at an amino acid, a peptide or a pseudopeptide via a Ester bond is bound to a resin particle. Other amino acids, Peptides or pseudopeptides are then in a known manner attached to the growing molecule. Once the desired molecule has been obtained, it is split off from the resin. On suitable cleavage agent is anhydrous, liquid hydrogen fluoride in a reaction-inert solvent at 0 ° C.

The following examples illustrate the present invention.  

Example 1 Gly ψ Leu (reference example) 2-bromo-4-methylpentanoic acid

A total of 50 g of D-Leu in 1200 ml of 2.5 N sulfuric acid which contained 250 g of potassium bromide. The mixture was cooled to 0 ° C and stirred, taking within 1½ Hours 65.5 g of sodium nitrite added in small portions were. The solution was stirred for a further hour at 0 ° C, heated to 25 ° C and stirred for another hour. Then it became the mixture is extracted with ether; the ether layer was separated, washed with water and anhydrous sodium sulfate dried to the desired product.

2-Mercaptor-4-methylpentanoic acid

There were added 1.25 g (6.5 mmol) of the above product in 2 ml of water. Then, a sufficient amount of 1N sodium hydroxide solution was added to dissolve the bromic acid. Into this mixture was added 5 ml of 40% sodium thiocarbonate, whereupon the vessel was sealed and allowed to stand at room temperature for 24 hours. After this time, the solution was acidified with 18N sulfuric acid and extracted twice with 10 ml of ethyl ether. The combined extracts were dried over anhydrous magnesium sulfate and filtered. The filtrate was concentrated to about half of its volume and chromatographed on a silica gel column (2.2 x 30 cm). The original solution was eluted with 150 ml of ethyl ether and the product was recovered by evaporation of the solvent from the eluate. The yield was about 34%. The R _f 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 ψ Leu

30 mmol of ethylenimine was added to 2.2 mmol of the above product in 10 ml of 0.5 N 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 according to the method of Dreze et al in a Dowex® column (2 x 8). The acetic acid fractions were recovered and the product isolated by evaporation. The residue was recrystallized from 50% ethanol to give a product having an R _f value of 0.82 by TLC on cellulose in the butanol / acetic acid / water mixture described above. The specific rotation was -16.3 °.

Gly ψ Leu

There were a total of 5.3 g (27 mmol) of the above bromic acid in 530 ml of nitrogen-purged 0.5 N sodium bicarbonate dissolved and with 9.2 g (81 mmol) of 2-mercaptoethylamine hydrochloride added. The reaction vessel was purged with nitrogen for 1 hour and then closed. It was at room temperature for 24 hours let stand, and in that time formed the desired product. The solution was acidified with 6N HCl and extracted twice with ether. The ether extracts were discarded and the aqueous portion is neutralized with 2N sodium hydroxide  and diluted with 2 L of deionized water. The solution was in a 5.5 x 30 cm column with Dowex® 2 X8 resin according to desalted according to the method of Dreze et al. The acetic acid fractions were combined and reduced by evaporation under reduced pressure Pressure dried. Then the residue was triturated with 20 ml 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 ψ Ile, wherein D-alloisoleucine is used in place of D-leucine has been.

It has been found that in the course of the above reactions Halogen the original amino group without reversing the configuration replaced. The conversion took place during the exchange of the Broms instead of. The amino acid residues of the pseudopeptides are therefore in L-shape before. Analog pseudopeptides whose components are in D-form are obtained when the appropriate L-form the starting amino acids used.

example 2 Phe ψ His 2-mercapto-4-benzylthiazolin

There were 75 mmol of Phe in 225 mmol of ethanol containing 300 mmol of carbon disulfide stirred with 75 mmol solid potassium hydroxide, while the temperature was kept below 40 ° C. Then the temperature was raised to 40 ° C and the reaction mixture 12 hours in a closed container. To  This time, alcohol and carbon disulfide were on a rotary evaporator away. The residue was in a small amount suspended in cold water and the product from a water Recrystallized ethanol mixture.

2-mercapto-1-benzylethylamine

A total of 4 g of mercaptothiazoline was dissolved in 25 ml of 12N HCl taken and heated at 155 ° C for 5 hours under reflux. Then the excess acid was removed on a rotary evaporator. The desired product was recovered as the hydrochloride and recrystallized from aqueous ethanol.

Phe ψ His

This product was prepared from α-bromo-β- (5-imidazolyl) -propionic acid and 2-mercapto-1-benzylethylamine by reaction in the presence made of sodium carbonate, which in Example 1 for described the analogous reaction of 2-mercaptoethylamine Procedure was applied.

The pseudopeptide Leu ψ Leu is obtained in a similar manner wherein as reactants an isobutyl-substituted ethanolamine and an isobutyl-substituted 2-mercaptoethylamine serve.

Ala ψ Ala is also prepared from 4-methyl-2 in the same way. mercaptothiazoline (Gabriel and Ohle, Ber., 50, p. 804, 1917) and α-bromopropionic acid recovered.  

According to the method of this example, the following pseudodipeptides became manufactured:

Trp ψ Gly *) Arg ψ Val *) Phe ψ Tyr *) Met ψ Lys *)
Gln ψ Lys *) Lys ψ His *) Arg ψ Gly Gly ψ Lys *)
Gln ψ Arg *) Lys ψ Tyr *) Lys ψ Arg *)
Arg ψ Thr *) Arg ψ Lys *) Tyr ψ Leu *) Phe ψ Arg *)
Arg ψ Leu *) Arg ψ Pro *)
Tyr ψ Gly Lys ψ Lys *) Phe ψ Ser *)
Phe ψ Met *) Lys ψ Leu *) Pro ψ Arg *) Tyr ψ Glu *)
Phe ψ Leu *) Phe ψ Trp *) Tyr ψ Phe *) Tyr ψ Gly
*) = not according to the invention.

example 3 Solid-phase synthesis of Ac-Pro-Gln-Gly ψ Leu-Ala Gly-Leu ψ Orn-Gly-NH₂ (reference example)

The synthesis of these pseudopeptides is carried out with the aid of the peptide Shaking device with medium reaction vessel performed, which was described by Steward and Young (Solid Phase Peptides Synthesis, W.H. Freeman and Company, 1969).

The starting material used was 4 g of Boc-Gly resin, which was 0.24 mmol Gly per gram of resin contained. The following scheme was included any sequence of Boc-amino acid or Boc-pseudodipeptide additions applied to the resin.

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

Before Boc-Leu ψ Orn and Boc-Gly ψ Leu were coupled, were these substances from their corresponding Dicyclohexylaminsalzen released by adding 2.5 g of the salt in 10 ml of water suspended. Citric acid (1 mole) was added slowly, until the powder was liquefied. Then it became an equal volume added to ether. After thorough mixing, the ether layer became separated, washed once with water and over sodium sulfate dried. The ether was removed under reduced pressure and the sample dried over after; she delivered that free pseudodipeptide. Boc pseudodipeptides are in the same Wrapped up as the amino acids.  

With addition of Gln, the Boc-Gln-p-nitrophenyl ester became 4-fold applied molar excess. DMF was used instead of methylene chloride used in the coupling, washing and acetylation stages. The coupling time for each nitrophenyl ester cycle was 19 hours. After Pro was added, the Schutzgruppeabspaltungs- and acetylation carried out, and the final resin-coupled peptide was treated with MeOH and triethylamine freed from the resin and provided the methyl ester, then by treatment with anhydrous ammonia in dry Methanol was converted to the corresponding amide.

Conversion of Ac-Pro-Gln-Gly ψ Leu-Ala-Gly-Leu ψ Orn-Gly-NH₂ in Ac-Pro-Gln-Gly ψ Leu-Ala-Gly-Leu ψ Arg-Gly-NH₂

The Leu ψ Orn-containing peptide amide was completed in vacuo dried and then at 0 ° C for 30 minutes with stirring anhydrous, liquid hydrogen fluoride in the presence of Treated 100 equivalents of methyl ethyl sulfide. The peptide was on a Sephadex® G-100 column in 10% aqueous acetic acid Chromatograph. The peptide containing the no longer protected Fractions were pooled and lyophilized and provided Ac-Pro-Gln-Gly ψ Leu-Ala-Gly-Leu ψ Orn-Gly-NH₂.

By treating this peptide with O-methylisourea according to the method of Merrifield (Cosand and Merrifield, Proc. Natl. Acad. Sci. USA 74, pages 2771-2775, 1977) became the desired Ac-Pro-Gln-Gly ψ Leu-Ala-Gly-Leu ψ Arg-Gly-NH₂ obtained.  

example 4 Ac-Pro-Gln-Gly ψ Leu-Ala-Gly-Leu-Arg-NH₂

The table below summarizes the reactions for preparation the above compound together. The values in the right Columns show the amino acid composition in each stage of the Synthesis. The values for the pseudodipeptide shown in the phe- Position is eluted when a 4.5 hour citrate buffer elution program was used after the HCl-propionic acid Hydrolysis determined using cysteamine as a scavenger. For comparison, appropriate analyzes were made in the absence of pseudopeptide carried out.

After the table the different synthesis steps are described.

In the table and the description "Aoc" stands for a tert. Amyloxycarbonyl.

The collagenase activity is characterized by the presence of this Pseudooctapeptids inhibits the Semgent of the natural Collagen molecule replaces when using the enzyme of the octapeptide as a substrate in the natural sequence becomes.  

Table IV

Preparation of Boc-Gly ψ Leu

There were 5.4 g (22 mmol) of 2- (tert-butyloxycarbonyloximino) - 2-phenylacetonitrile at room temperature to a mixture from 3.81 g (20 mmol) of Gly ψ Leu, 4.1 ml (30 mmol) of triethylamine, Added 12 ml of dioxane and 12 ml of water. The mixture became homogeneous with stirring within 1 hour, and stirring was continued for 10 hours. Then, 30 ml of water and Added 30 ml of ethyl acetate. The aqueous layer was separated and washed with ethyl acetate (2 x 40 ml). The watery Layer was acidified with 5% aqueous citric acid solution and extracted with ethyl acetate. The ethyl acetate extract was washed twice with saturated aqueous sodium chloride and dried over anhydrous sodium sulfate. By removal of desiccant and solvent was 7.7 g of an oil. This oil was dissolved in 40 ml of ethyl ether and mixed with 7.4 g (40 mmol) of dicyclohexylamine. The thus obtained Suspension was stored at 40 ° C for 4 hours before the desired salt was isolated. The salt was washed with ether and dried in vacuo over phosphorus pentoxide; it 10 g of white crystals were obtained (80% yield, based on Gly ψ Leu). The purified product was recrystallized of the dicyclohexylamine salt of Boc-Gly ψ Leu Methanol / ether (2: 1 v / v); F = 143-144 ° C.

Analysis C₂₅H₄₈N₂O₄S:
Calc .: C 63.5%; H 10.23%; N 5.93%; S 6.78%
Found: C 63.73%; H 10.48%; 5.66%; S 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 mixing them with 50 ml of 25% triethylamine for 10 minutes stirred in methylene chloride. The resin was filtered off and washed with additional methylene chloride (25 ml per g of resin). Then, the resin was suspended in 40 ml of methylene chloride and 10 hours in the presence of a 2.5-fold molar Shaken excess of Aoc-Arg (Tos). After washing with Methylene chloride was the remaining, unreacted Amino groups of the resin with a mixture of acetic anhydride and N-methylmorpholine in dimethylformamide acetylated. The amino acid analyzes of a weighed sample of the hydrolyzed Aoc-Arg (Tos) resin indicated that 0.24 mmol arginine were released per gram of resin.

Chain extension by double attachment and acetylation

After the first amino acid had been coupled to the resin, the remaining amino acids were sequentially in the following The resin was washed three times with methylene chloride and then with trifluoroacetic acid / anisole / methylene chloride (40: 2: 60. Vol./vol.) Prewashed. The amino groups were released by shaking with a TFA solution for 20 minutes same composition. After five washes with methylene chloride was twice with 7% (v / v) N, N-diisopropylethylamine washed in methylene chloride. Excess amine was washed another five times with methylene chloride away. Then, tert-butyloxycarbonyl-amino acid (2.5-fold molar excess) in methylene chloride in the reaction vessel  given. Dicyclohexylcarbodiimide (2.3-fold molar Excess) was added in methylene chloride (final volume 40 ml). The reaction vessel was shaken for 2 hours. To 2 washes with methylene chloride were washed twice with ethanol. The five methylene chloride pre-washes as well as the Coupling stages were repeated. After washing three times with dimethylformamide were the remaining, non-acylated Amino groups of the resin for 20 minutes by shaking with a Mixture of 4 ml of acetic anhydride, 2 ml of N-methylmorpholine and washed 34 ml of dimethylformamide. The double clutch was after three more washes with dimethylformamide and three further washes with ethanol. The one described above double acetylation sequence was added to all amino acid residues, including acetylproline, but with the exception from Gln, applied. Gln was introduced (twice) in shape of the tert-Boc-p-nitrophenyl ester in dimethylformamide (under Avoidance of dicyclohexylcarbodiimide), and the reaction time was 10 hours instead of just 2 hours. The progression The synthesis was characterized by frequent hydrolysis and analysis of the growing Peptide resin tracked.

Addition of Boc-Gly ψ Leu

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

Release of the peptide from the benzhydrylamine resin product and cleavage of the protecting groups of the peptide

The tosylated benzhydrylamine resin peptide (2.0 g) became 1 hour at 0 ° C with 20 ml of CoF₃ dried HF in the presence treated with 2.0 ml anisole. After HF under reduced Pressure was removed, the resin was in vacuo overnight dried. The freed, free from protective groups Peptide was extracted from the resin with 60 ml of 7% acetic acid. The filtered extract was taken consecutively with each Washed 30 ml of ethyl ether and ethyl acetate to the anisole remove. The solution was lyophilized and the residue in Dissolved 0.2m-acetic acid and lyophilized again. The crude peptide amide was chromatographed on Sephadex® G-15 in 0.5 M acetic acid. The main component determined by the Sakaguchi method was lyophilized to yield 334 mg of peptide amide (Yield 80%). The product was separated by distribution and / or Purified carboxymethyl cellulose chromatography.

Distribution chromatography on Sephadex® G-25

A column (73 x 2.5 cm) was washed with Sephadex® G-25 (fine Quality) packed in water. The column was with the lower phase of a solvent mixture of n-butanol, pyridine and 0.1% aqueous acetic acid (5: 3: 11, v / v). (15)  and then with the upper phase of the same solvent system (16) at a flow rate of about 1 cm / Min. Balanced. There were 55 g of the peptide in Dissolve 5 ml of the upper phase and elute with the upper one Phase chromatographed. The fractions containing the peptide were determined by the quantitative Sakaguchi method (17). Fractions of the main component have been merged diluted with an equal volume of water and under reduced pressure Concentrated pressure in a rotary evaporator. The concentrate after lyophilization yielded 32 mg of a white powder (Yield 58%).

example 5 Boc-Phe ψ gly-dicyclohexylamine salt (Reference Example)

This compound can be freed from the dicyclohexylamine moiety and with the help of the described methods into a larger one Be incorporated peptide. If desired, the Boc Group also by, in the last paragraph of Example 4 described Treatment with HF can be removed. The thus obtained Pseudodipeptide can serve as a surrogate for Phe-Gly.

Preparation of Boc-phenylalaninol

In a mixture of 10 ml of water and 10 ml of dioxane were 3 g (20 mmol) of phenylalaninol and 20 mmol of tert-butyloxycarbonylazide and a sufficient amount of 1N sodium hydroxide solution to bring the pH to 8.0. The reaction mixture was stirred for 24 hours, mixed with 50 ml of ethyl ether and on Cooled to 0 ° C. Solid citric acid was added until the  pH had dropped to 3.5. The aqueous layer was washed twice extracted with 25 ml of ether. The combined ether extracts were twice with 25 ml of 5% citric acid and then washed three times with saturated sodium chloride solution. The organic layer was over anhydrous sodium sulfate dried. After removal of the solvent under reduced Pressure was obtained 5.4 g of oil. The oil was made with hot hexane treated and provided 4.1 g of a white solid (needles; 81%). The crude product was recrystallized from 100 ml of hot hexane, containing 10 drops of ether. It became a white one Obtained solid; Weight - 2.8 g, F = 93-94 ° C.

Preparation of Boc-phenylalaninol tosylate

In a cooled from -10 ° C solution of 2.5 g (0.01 mol) Boc Phenylalaninol in 10 ml of dry pyridine, 3.8 g (0.02 mol) Given tosyl chloride. The solution was overnight in a Freezer left until no white solid (Pyridinium hydrochloride) more precipitated. Then the Mixture poured into 100 ml of ice and ether. The organic Layer was separated and three times with 5% Citric acid and saturated sodium chloride solution. Then The solution was dried over anhydrous sodium sulfate and the solvent removed; There was 4.1 g of white solid receive; F = 92-93 ° C. By recrystallization from ether / Hexane could not be further improved in melting point.  

Preparation of Boc-Phe ψ Gly / DCHA salt

To 0.24 g (1.8 mmol) of the disodium salt of mercaptoacetic acid in 2 ml of dry methanol and 5 ml of dimethylformamide 0.6 g (2.4 mmol) Boc-phenylalanine-tosylate. The solution was kept at room temperature for 5 hours and then became the methanol is removed under reduced pressure. The solution was cooled to -5 ° C and treated with 25 ml of cooled ethyl acetate. A 25% citric acid solution was added to to lower the pH to 2. The aqueous layer was thrice extracted with 25 ml of ethyl acetate. The combined organic Extracts were once with 5% citric acid solution and washed three times with saturated sodium chloride solution. The ethyl acetate layer was dried over anhydrous sodium sulfate and the solvent removed under reduced pressure. The oily residue was added in ethyl acetate and an excess Dicyclohexylamine dissolved. The addition of anhydrous diethyl ether led to the precipitation of Boc-Phe ψ Gly / DCHA salt in in an amount of 0.54 g (yield 58%). This salt was made Recrystallized from chloroform / ether (4: 1) to give 0.4 g white solid; F = 140-141 ° C (total yield 43%).

example 6 Preparation of tablets (reference example)

1000 g Gly ψ Leu and 2000 g lactose were thoroughly mixed and passed through a 30 mesh screen.

At the same time a paste of 80 g of corn starch and 350 ml made of distilled water.  

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

After drying, the beads were first placed in a granulator crushed and then passed through a 16-mesh sieve. The grains were mixed with a powdery mixture of 30 g Calcium stearate, 200 g cornstarch and 80 g talcum covered and sieved through a 40-mesh sieve.

From the grains thus obtained, tablets were prepared in the usual way each containing 250 mg Gly ψ Leu.

example 7 Production of injection solutions

There were 100 g of the sodium salt of Leu ψ Leu in a special prepared for this purpose, distilled pyrogen-free Dissolved water and made up to 5 l. The solution became isotonic made by giving a predetermined amount of one aqueous, physiological saline added, and then by a microporous bacteria filter is filtered.

example 8 Preparation of an aqueous solution for oral administration

A mix of

Gly ψ Ile | 20.0 g cane sugar 100.0 g glycerin 100 ml Ethyl p-ethoxybenzoate 1.5 g artificial orange essence 0.2 ml essential orange oil 1.0 ml

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

Claims (2)

1. Pseudopeptides having the following amino acid sequences:
Phe ψ His;
Gly ψ Ile;
Leu ψ Leu;
Ala ψ Ala;
Tyr ψ Gly;
Arg ψ Gly;
Pro ψ Gly-NH₂;
Lys ψ Ser;
Arg ψ Met;
Leu ψ Leu-Val-Tyr;
Ac-Phe ψ His-Leu-Leu-Val-Tyr-Ser-OH;
Ac-Phe-His-Leu ψ Leu-Val-Tyr-Ser-OH;
Ac-Phe ψ His-Leu ψ Leu-Val-Tyr-Ser-OH;
Ac-Pro-Gln-Gly ψ Ile-Ala-Gly-Gln-Arg-Gly;
Ac-Pro-Gln-Gly ψ Leu-Ala-Gly-Gln-Arg-Gly;
Ac-Pro-Leu-Gly ψ Ile-Ala-Gly-Leu-Arg-Gly;
Ac-Pro-Leu-Gly ψ Leu-Ala-Gly-Leu-Arg-Gly;
Ac-Pro-Gln-Gly ψ Ile-Ala-Gly-Gln-Arg-Gly-OEt;
Ac-Pro-Gln-Gly ψ Leu-Ala-Gly-Gln-Arg-Gly-OEt;
Ac-Pro-Leu-Gly ψ Ile-Ala-Gly-Leu-Arg-Gly-OEt;
Ac-Pro-Leu-Gly ψ Leu-Ala-Gly-Leu-Arg-Gly-OEt;
Ac-Pro-Gln-Gly ψ Ile-Ala-Gly-Gln-Arg-Gly-NH₂;
Ac-Pro-Gln-Gly ψ Leu-Ala-Gly-Gln-Arg-Gly-NH₂;
Ac-Pro-Leu-Gly ψ Ile-Ala-Gly-Leu-Arg-Gly-NH₂;
Ac-Pro-Leu-Gly ψ Leu-Ala-Gly-Leu-Arg-Gly-NH₂;
Ac-Pro-Gln-Gly ψ Ile-Ala-Gly-Gln ψ Arg;
Ac-Pro-Gln-Gly ψ Ile-Ala-Gly-Leu ψ Arg;
Ac-Pro-Gln-Gly ψ Leu-Ala-Gly-Gln ψ Arg;
Ac-Pro-Gln-Gly ψ Leu-Ala-Gly-Leu ψ Arg;
Ac-Ile-Glu-Gly-Arg ψ Thr-Ser-Glu-OEt;
Ac-Ile-Glu-Gly-Arg ψ Ile-Val-Glu-OEt;
Ac-Ser-Ile-Arg ψ Met-Arg-Asp-Val-OEt;
Ac-Glu-Asn-Arg-Lys ψ Ser-Ser-Ile-Ile-OEt;
Ac-Ala-Ala ψ Ala-OEt;
Ac Ala Ala Ala Ala OEt;
pGlu-His-Trp-Ser-Tyr ψ Gly-Leu-Arg-Pro-Gly-NH₂;
pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro ψ Gly-NH₂;
pGlu-His-Trp-Ser-Tyr ψ Gly-Leu-Arg-Pro ψ Gly-NH₂;
pGlu-His-Trp-Ser-Tyr-Gly ψ Leu-Arg-Pro-Gly-NH₂;
Ac-Gly-Gly-Gly-Val-Arg ψ Gly-Pro-Arg-Val-NH₂;
Ac-Ala-Ala-Phe ψ Phe-NH₂;
Thr-Lys-Pro ψ Arg;
Thr ψ Lys-Pro-Arg;
Thr ψ Lys-Pro ψ Arg;
Thr-Lys-Pro ψ Orn;
Thr ψ Lys-Pro-Orn;
Thr ψ Lys-Pro ψ Orn;
Thr ψ Gly-Gly-Phe-Leu;
Tyr ψ Gly-Gly-Phe-Met;
Benzoyl-Arg ψ Gly-OEt;
Ac-Pro-Gln-Gly ψ Leu-Ala-Gly-Leu-Arg-NH₂; Human insulins in which the Cys ψ Asp moiety is inserted into the A chain positions 20-21 and / or the amino acids 22 and 23 in the B chain are replaced by Arg ψ Gly;
their derivatives acylated at the free hydroxyl or amino group with an alkanoyl or alkenoyl group containing up to 18 carbon atoms;
and pharmaceutically acceptable salts of the above pseudopeptides and derivatives;
where ψ is the replacement of a peptide bond by a CH₂-S group, Ac acetyl and OEt ethoxy. 2. Pharmaceutical preparations containing a compound according to Claim 1 and a pharmaceutically acceptable carrier.