PL176941B1 - Agents and methods used in treating immunological inflammatory states - Google Patents

Abstract

Compositions and methods for the prevention and treatment of immunomediated inflammatory disorders, especially for those disorders associated with the respiratory tract, are provided. More particularly, a tryptase inhibitor, typically a hydroxyaroyl or hydroxyheteroaroyl substituted dipeptide, is administered. Also provided by this invention are pharmaceutical compositions, typically aerosol or topical, as well as aerosol devices for administering these compositions intranasally.

Description

The present invention relates to novel peptides and a pharmaceutical for the treatment of immune inflammation, in particular inflammatory diseases of the airways such as asthma and allergic rhinitis. The agents of the invention are particularly useful in the prevention and treatment of late phase bronchoconstriction and airway hyperresponsiveness due to chronic asthma.

Low molecular weight molecules that inhibit enzymes, especially proteases, have found wide application in the treatment of various disease pathologies. Many of these compounds are called peptidomimetics because, as their name implies, they mimic different parts of peptide inhibitors that are composed entirely of condensed amino acids. These low molecular weight peptidomimetic protease inhibitors have a distinct advantage over natural peptides and proteins in that they may be more stable and thus exhibit the desired pharmacokinetic and pharmaceutical properties.

For example, a zinc metalloprotease called angiotensin converting enzyme (ACE) is responsible for the cleavage of angiotensin t into angiotensin tt. Inhibiting ACE is a measure for reducing high blood pressure. ACE is effectively inhibited by mercaptoacyl proline derivatives, in particular the peptide mimetic called captopril, (1- (22S-33-cell capto-2-methyropropionyl) -L-prlCna, and its analogs, disclosed in U.S. Patent No. 4,456,595.

Serine protease, thrombin is a key enzyme in the blood clotting cascade process. Drug inhibition of thrombin is an agent that reduces the tendency of the blood to clot and therefore reduces the possibility of a blood clot forming that could lead to a heart attack or stroke. Thrombin is effectively inhibited by a number of peptidomimetics, especially derivatives and substitutes for the amino acid arginine. One of the mimetic groups

176,941 inhibitors are the group of N (2) -arylsulfonyl-L-argininamides, especially argipidine (argatroban), disclosed in U.S. Patent No. 4,201,863.

Asthma is a complex disease that involves a number of biochemical mediators, both acute and chronic. Asthma is often characterized by the gradual development of tracheal and bronchial hyperresponsiveness to immunospecific allergens and generalized chemical or physical stimuli. The hyperresponsiveness of bronchiolar tissue in asthma is believed to result from chronic inflammatory reactions that irritate and damage the epithelium lining the airway walls and promote pathological thickening of the underlying tissue. Bronchial biopsy studies have shown that even patients with mild asthma show signs of inflammation of the airway walls.

One of the initiators of the inflammatory sequence is the allergic response to inhalation allergens. IgE receptor-containing leukocytes, especially mast cells and basophils, but also monocytes, macrophages and eosinophils, are present in the epithelium and the underlying bronchial smooth muscle tissues where they are activated initially by the binding of specific inhalation antigens to IgE receptors. When activated, mast cells release a number of preformed mediators, i.e. primary chemical mediators of the inflammatory response, and enzymes ¹ . In addition, numerous secondary mediators of inflammation arise in situ as a result of enzymatic / activated mast cell reactions, including peroxide mediators and lipid-derived mediators. In addition, several large molecules are released as a result of mast cell degranulation, namely proteoglycans, peroxidase, arylsulfatase B, and especially the proteases tryptase and chymotryphal proteinase (chymase) (see Drug Therapy of Asthma, Ch. 62, 1054-54).

This release of chemicals from mast cells is likely to be the cause of the immediate bronchiolar contraction response that occurs in susceptible individuals upon exposure to inhaled allergens. The maximum immediate asthmatic reaction occurs approximately 15 minutes after exposure to the allergen and continues for the next 1-2 hours. In 25-35% of individuals, an immediate asthmatic reaction is followed by a further deterioration in the respiratory function which begins after a few hours and reaches its maximum 6-12 hours after exposure to the allergen. This late asthmatic reaction is accompanied by a marked increase in the number of inflammatory cells infiltrating the smooth muscle tissues of the bronchioles and epithelium and then into the respiratory tract. These cells include eosinophils, neutrophils, and lymphocytes, all of which are attracted to this site by chemotactic substances secreted by mast cells. Infiltrated cells themselves become activated in a late reaction. The asthmatic delay reaction is believed to be a secondary inflammatory reaction mediated by substances secreted by macrophages.

The associated complex of inflammatory reactions occurs in the mucosa of the upper respiratory tract, usually as a result of the action of inhaled allergens. As in asthma, mast cells are activated by the binding of IgE molecules to specific antigens. In allergic, chronic, or vasomotor rhinitis, mast cells may be activated despite no discernible exposure to the antigen. In each case, activated mast cells secrete primary and secondary mediators of the inflammatory process after degranulation. Eosinophils and macrophages are attracted to this site to perpetuate the inflammatory response. Destruction of the epithelial tissue in the nose often occurs during the late reaction.

Tryptase is the main protease secreted by human mast cells and is believed to be involved in neuropeptide metabolism and the inflammatory process in tissues. Adult human tryptase is a glycosylated, heparin-linked tetramer of heterogeneous, catalytically active subunits. The amino acid sequence of the tryptase monomer, like its gene structure, lacks a close correspondence between the numerous serine proteinases that have been characterized (see, e.g., Yanderslice et al.

176 941 (1990) Proc. Natl. Acad. Sci. USA, 87: 3811-3815; Miller et al. (1990) J. Clin. Invest. 86: 864-870; Miller et al. (1989) J. Clin. Invest. 84: 1188-1195, Vanderslice et al. (1989) Biochemistry 28: 4148-4155 and Katunuma et al (1990) Monographs in Allergy 27: 51-66).

Tryptase is stored in the grains of mast cells. After mast cell activation, human tryptase levels can be easily measured in a variety of biological fluids. For example, after anaphylaxis, tryptase appears in the bloodstream where it is detectable for several hours (see Schwartz et al. (1987) N. Engl. J. Med. 316: 1622-1626). Its presence is detectable in nasal and lung fluid samples of atopic patients after challenge with specific antigen (see Castells and Schwartz (1988) J. Allerg. Clin. Immunol. 82: 348-355 and Wenzel et al. (1988) Am. Rev. Resp. Dis. 141: 563-568). The level of tryptase in the lung fluid of patients with atopic asthma increases after exposure to an endobronchial allergen (ibid.). Some cigarette smokers have unusually high levels of bronchoalveolar fluid tryptase compared to nonsmokers, and this finding supports the hypothesis that the release of proteinases from activated mast cells could contribute to lung damage in smokers' emphysema (see Kalenderian et al. (1988) Chest 94: 119-123). Furthermore, tryptase has been shown to be a potent mitogen against fibroblasts, suggesting its involvement in pulmonary fibrosis and interstitial lung disease (see Ruoss et al. (1991) J. Clin. Invest. 88: 493-499).

Tryptase has been implicated in many biological processes, including the breakdown of vasodilating and bronchodilator neuropeptides (see Caughey et al. (1988) J. Pharmacol. Exp. Ther. 244: 133-137; Franconi et al. (1988) J. Pharmacol. Exp. Ther. 248: 947-951 and Tam et al. (1990) Am. J. Respir. Cell. Mol. Biol. 3: 27-32) and modulation of the bronchial response to histamine (see Sekizawa et al. (1989) J. Clin. Invest. 83: 175-179). These studies suggest that tryptase probably increases bronchospasm in asthma by destroying bronchodilator peptides.

In addition, tryptase has been shown to cleave fibrinogen? Chains as well as high molecular weight kininogen with optional release of kinins, and thus may play a role relative to heparin as a local anticoagulant. The ability of tryptase to activate prostromelysin (pro-MMP-3) and procollagenase (pro-MMP-1) via MMP-3 suggests that tryptase may also be involved in tissue inflammation and tissue remodeling. This finding could also mean that tryptase plays a role in the destruction of joints in rheumatoid arthritis. Additionally, it has been shown that tryptase cleaves the peptide associated with the calcitonin gene. Since this peptide has been postulated to be involved in the neurogenic inflammatory process, tryptase could be a regulator of the skin redness phenomenon in neurogenic skin inflammation (see Caughey (1991) Am. J. Respir. Cell. Mol. Biol. 4: 387-394). Recently, various forms of tryptase have been reported to cleave prothrombin into thrombin and participate in recognition of the HIV envelope protein gpl20 by CD4 + T cells (see Katunuma et al. (1990), Monographs in Allergy 27: 51-66).

Asthma has become the most common chronic disease in industrialized countries. To date, known treatments and therapeutics have not been effective in treating asthma and other immune inflammation. For these reasons, there is a need for improved means and methods which overcome the drawbacks of these known means and methods and provide effective treatment of the diseases mentioned.

It has now surprisingly been found that the novel peptides of the invention are effective serine protease inhibitors, and in particular tryptase inhibitors, i.e. substances that slow or prevent tryptase activity. They are useful in reducing immune inflammation, especially bronchospasm induced by exposure of an asthmatic subject to an allergen.

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Thus, the invention relates to novel peptides of formula I

wherein

Ar is a group selected from among

3 2 3

Rc is methyl, R is hydrogen, or R and R together with the nitrogen and carbon atom to which they are attached and with the group C (- = O) R'4 form 5-nbound heterocycles! about the formula

and R ⁴ is -OCH 3, -NH 2 or -N (CH 3) 2, or is selected from the group consisting of

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and their pharmaceutically acceptable salts.

Compounds of formula I are preferred in which Ar is t1-hydroxy-21-yl, 2-hydroxy-t-naphthyl, 3-hydroxy-21-pyridyl or 2-hydroxy-3-hinoxalyl. R 1, R ² and R ³ are as defined above and R ⁴ is -OCH ;, -NH ;, -3'-carboxamido-l'l-pipdridyl or -N (CH3) 2.

Especially preferred are compounds of formula I wherein Ar is tlhydroksy-2-on-to-back or two-hydrolłsy-1-naphthyl, R ¹ is - (CH ^ NH / CNHjNH2, R 2 and R 3 together with the nitrogen and a carbon atom, to which they are attached and with the group C (= O) R4 form the above-described eight-membered heterocyclyl, and R4 is -NH2.

Thus, a particularly preferred tryptase inhibitor is a compound of the formula below

Another particularly preferred tryptase inhibitor is the compound of the formula below

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Yet another particularly preferred tryptase inhibitor is a compound of the formula below

MN

The compounds described herein are useful in the prophylaxis and treatment of immune inflammation, in particular airway related conditions including asthma, and in particular the overactive phase associated with chronic asthma and allergic rhinitis. Thus, the compounds of the invention are used in a method of treating immunological inflammation, in which a patient with an immune inflammation amenable to treatment with tryptase inhibitors takes or is administered a therapeutically effective amount of a compound of the invention. These compounds are typically formulated into pharmaceutical compositions suitable for administration by a variety of routes.

The pharmaceutical composition according to the invention comprises the active ingredient and a pharmaceutically acceptable carrier and / or auxiliaries, and its feature is that it contains an active ingredient of 0.001-95% of a compound of formula I or a pharmaceutically acceptable salt thereof.

These pharmaceutical compositions can take various forms, for example, they can be dosage forms for oral administration, as well as solutions for injection and infusion. Typically, when these pharmaceuticals are used for the prophylaxis or treatment of asthma, and especially hyperresponsiveness associated with chronic asthma, they are powders or aerosol solutions. When used in the treatment of immune inflammatory skin conditions, the compounds of the invention are used in combination with a non-toxic pharmaceutically acceptable topical carrier. The compounds of the invention can be used in combination with therapeutic agents for the treatment of inflammation and asthma such as β-adrenergic agonists, anti-inflammatory corticosteroids, anticholinergics, etc.

The following definitions are provided to illustrate and define the meaning and scope of the various terms used to describe the present invention.

The term "immune inflammation" generally includes diseases associated with the secretion of mediators from mast cells amenable to treatment with tryptase inhibitors. Examples of such conditions include early-type hypersensitivity diseases such as asthma, allergic rhinitis, urticaria and angioedema, and eczema12

176 941 dermatitis (atopic dermatitis) and skin disease associated with increased cell proliferation, peptic ulcer, enteritis, skin inflammation, etc.

"Hyperresponsiveness" is the late phase of bronchospasm and hyperreactivity of the airways associated with chronic asthma. The hyperreactivity of bronchiolar tissues in asthmatics is believed to be the result of chronic inflammatory reactions that irritate and destroy the epithelium lining the airway walls and promote pathological thickening of the underlying tissue.

A "pharmacologically acceptable salt" is a salt that retains the biological effectiveness and properties of the parent compound, and is not biologically or otherwise undesirable.

A "therapeutically or pharmaceutically acceptable carrier" is a carrier that does not interfere with the efficacy of the biological activity of the chemical substance and is not toxic to the patient.

A "stereoisomer" is a chemical compound that has the same molecular weight, chemical composition, and chemical structure as another compound, but with differently grouped atoms. Certain identical chemical molecules have different orientations in space and therefore, in their pure state, have the ability to rotate the plane of polarized light. Some pure stereoizomets can have optical rotation so poor that it cannot be detected with currently available instruments. The compounds of the invention may contain one or more asymmetric carbon atoms and may therefore have different stereoisomers. All these stereoisomers are within the scope of the invention.

"Treatment" includes any in vitro and in vivo use of a tryptase inhibitor, including: (i) inhibiting disease symptoms; (ii) diminishing or inhibiting the long-term effects of the disease and / or (iii) alleviating the symptoms of the disease.

The compounds of the invention can be prepared by known methods from readily available starting materials as described in more detail below.

The compounds of the invention may, depending on the nature of the functional groups, form salts with various organic and inorganic acids and bases. These salts can be formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, and with organic acids such as acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfenic acid, p-toluenesulfonic acid, salicylic acid, etc.

Salts can also be formed with the carboxylic acid moiety by treatment with alkali metals or alkali metal containing bases such as alkali metal hydroxides and alkoxides, or with alkaline earth metals or alkaline earth metal bases such as alkaline earth metal hydroxides and alkoxides. In addition, salts can be formed from carboxylic acid and an organic base such as trimethylamine, diethylamine, ethanolamine, piperidine, isopropylamine, choline, caffeine, etc.

Salts may be prepared by known methods, e.g. by reacting the free acid or base product with one or more equivalents of the appropriate base or the appropriate acid in a solvent or medium in which the salt is insoluble, or in a solvent such as water, and then is removed in vacuo or lyophilized, it is also possible to replace the cations of the given salts with other cations using a suitable ion exchange resin.

In therapeutic applications, a therapeutically or pharmaceutically effective amount of a tryptase inhibitor, i.e. a compound of Formula I, optionally in the form of a pharmaceutical composition containing it, is administered to a patient suffering from immune inflammation. The agents of the invention are useful in the prevention or alleviation of asthma. When using the agents of the invention in the treatment of asthma, the compounds can be administered prophylactically prior to or after exposure to an allergen or other causative agent. The agents of the invention are particularly useful for ameliorating the late phase tissue damage associated with both seasonal and chronic rhinitis. They are also useful

176 941 in the prevention and treatment of other mast cell-related immune inflammation such as urticaria and angioedema, eczema dermatitis (atopic dermatitis) and anaphylaxis, as well as skin disease associated with increased cell proliferation, peptic ulcer, etc.

Pharmaceutical compositions containing these compounds can be used prophylactically and / or therapeutically. In therapeutic applications, these agents are administered to a patient already suffering from the disease described above in an amount sufficient to cure or at least partially arrest the symptoms of the disease and its complications. Sufficient volume to achieve this result is defined as "therapeutically effective amount or dose". The effective amount for this use will depend on the severity of the disease, previous therapy, the patient's health and response to medications, and the judgment of the attending physician.

In prophylactic applications, the pharmaceuticals of the invention are administered to a patient susceptible to the disease in question or belonging to a risk group. Such an amount is defined as "prophylactically effective amount or dose". In this application, the specific amount also depends on the patient's health, weight, etc.

After the patient's condition improves, a maintenance dose is given as needed. Thereafter, the dose or frequency of administration may be reduced, depending on the symptoms, to a level where the improved health is maintained. Treatment may be discontinued once symptoms have improved to the desired level. However, patients will require periodic long-term treatment in case of any recurrence of disease symptoms.

In general, a suitable effective dose of a tryptase inhibitor will be 0.1-1000 mg per day, preferably 1-100 mg per day. The desired dose is preferably administered in a single dose or in two, three, four or more sub-doses administered at appropriate intervals throughout the day. These sub-doses may be administered in the form of a dosage form, for example, 5-1000 mg, preferably 10-100 mg, of the active ingredient in a dosage form.

The agent used in such therapy can be given various forms. These include, for example, solid, semi-solid and liquid dosage forms, such as tablets, pills, powders, liquid solutions or suspensions, liposomes, and injection and infusion solutions. The preferred form depends on the intended route of administration and therapeutic application.

While it is possible to administer the compounds of the invention as such, they are preferably a component of a pharmaceutical composition. Agents of the invention contain at least one compound of formula t of the invention, i.e. a tryptase inhibitor, in a therapeutically or pharmaceutically effective amount together with one or more pharmaceutically or therapeutically acceptable carriers and may be combined with additional therapeutically active substances. Various possibilities are described e.g. in the work edited by Gilman et al. (1990) Goodman and Gilman ': The Pharmacological Bases of Therapeutics, ed. 8, Pergamon Press, and in Remington 'mentioned below. Methods of use, e.g., oral, intravenous, intraperitoneal or intramuscular, and others are discussed there. Pharmaceutically acceptable carriers include water, saline, buffers, and other compounds described, e.g., in The Merck Index, Merck & Co., Rahway, NJ.

Typically, when the compounds of the invention are to be used in the treatment of asthma or allergic rhinitis, they are formulated as aerosols. The term "aerosol" includes any gas suspended phase of the compound of the invention which is inhalable into the bronchioles or nasal passages. In particular, the aerosol contains a droplet size of the droplets of a compound of the invention in a gas which can be generated in an inhaler or dosing nebulizer or in a nebulizer for producing a mist of the formulation. Aerosols may also be compositions of a compound of the invention in the form of a dry powder suspended in air or other carrier gas which can be delivered by, for example, insufflation from an inhaler.

For the aerosolizing solutions of the invention, the preferred concentration range of the compounds of the invention is 0.1-100 mg / ml, more preferably 0.1-30 mg / ml, and most preferably 1-10 mg / ml. Typically solutions are physiologically buffered

A compatible buffer, such as a phosphate or bicarbonate buffer. Typically the range of pH values is 5-9, preferably 6.5-7.8, and more preferably 7.0-7.6. In general, sodium chloride is added to bring the osmolarity within the physiological range, preferably with an isotonic difference of no more than 10%. The preparation of such solutions for the preparation of aerosols for inhalation is discussed in Remington 'Pharmaceutical Sciences as well as in Ganderton and Jones Drug Delivery to the Respiratory Tract, Ellis Horwood (1987); Gonda (1990) Critical Reviews in Therapeutic Drug Carrier Systems 5: 273-313 and Raeburn et al. (1992) "). Pharmacol. Toxicol. Methods 27: 143-159.

Solutions of the compounds of the invention may be aerosolized by any means known in the art for the preparation of pharmaceuticals intended for aerosol inhalation. Generally, these methods involve applying an elevated pressure in a solution-containing container, which may optionally contain equipment to generate such pressure, typically using an inert gaseous carrier, and then passing the pressurized gas through a small nozzle so that droplets of solution penetrate into the subject's mouth and trachea. receiving the drug Typically, the mouth of the nozzle is equipped with a mouthpiece that facilitates the delivery of medication to the oral cavity and trachea.

Such device containing solutions of the compounds of the invention may be combined with or contained in any device for generating aerosols used in the treatment of asthma, such as metered dose inhalers, jet nebulizers or ultrasonic nebulizers. Such a device may optionally include a mouthpiece fitted to the nozzle.

A device for use in the treatment of allergic rhinitis may be a nasal spray containing a solution of the compound of the invention.

Another form of an agent of the invention is a dry powder containing a compound of the invention, optionally together with an excipient. It can be administered using a dry powder inhaler containing such powder.

It should of course be understood that the compounds of the invention may be used in conjunction with the use of other agents intended for the treatment of immunological inflammation, especially asthma. Β-adrenergic agonists are particularly useful in such combinations because they provide relief from the immediate asthmatic response, while the compounds of the invention provide relief from late asthmatic response. Preferred adrenergic agonists. The β in these solutions are β-agonists usually used to relieve asthma, e.g., albuterol, tetrabutalin, formoterol, fanoterol or prenalin.

Other agents useful in combination with the compounds of the invention are anti-cholinergic agents such as ipratropium bromide, as well as anti-inflammatory corticosteroids (cortical adrenal steroids) such as beclomethasone, triamcinolone, flurisolide or dexamethasone.

The compounds of the invention may also be used in mammals for the treatment of immune inflammatory skin conditions such as urticaria and angioedema, eczema dermatitis and a skin disease associated with increased cell proliferation, e.g., psoriasis. As a result of topical application of the compound of formula I, remission of symptoms is expected. Thus, a person suffering from immune dermatitis can expect to reduce flaking, erythema, lesion size, itching, and other symptoms associated with a skin condition. The dosage of the drug and the length of time needed to effectively treat an individual patient may vary, but one skilled in the art will be able to determine these changes and conduct therapy accordingly.

Also included within the scope of the invention are formulations for topical application to the skin, containing a compound of formula I, typically in a concentration of about 0.001-10%, and a non-toxic pharmaceutically acceptable topical carrier. Such topical formulations can be prepared by combining the active ingredient of the invention with known pharmaceutical diluents or carriers commonly used in dry, liquid, cream and aerosol formulations. Ointments and creams may, for example, be formulated with a water or oily base with the addition of a suitable thickener and / or gelling agent. Such bases may contain water and / or an oil such as liquid paraffin or a vegetable oil such as arachis or castor oil. Thickening agents that can be used depending on the nature of the base are soft paraffin, aluminum stearate, cetostearyl alcohol, propylene glycol, polyethylene glycols, lanolin, hydrogenated lanolin, beeswax, etc.

Medicinal lotions may be formulated with an aqueous and oily base and will generally contain one or more additives such as stabilizers, emulsifiers, dispersants, suspending agents, thickeners, dyes, fragrances, etc.

Powders may be prepared with any powdery base, e.g. talc, lactose, starch, etc. The drops may be prepared in an aqueous or non-aqueous base and may contain one or more additives such as dispersants, suspending agents, solubilizers, etc.

According to the invention, the topical pharmaceutical compositions may also contain one or more preservatives and bacteriostats, e.g. methyl hydroxybenzoate, propyl hydroxybenzoate, chloro-1, benzalkonium chlorides, etc. Topical pharmaceutical compositions may also contain other active ingredients, such as microbicides, especially antibiotics, anesthetics, analgesics and anti-itching agents.

The compounds of the invention are also useful in the treatment of peptic ulcers. In particular, they exhibit chemotherapeutic activity. allowing the relief of the symptoms of peptic ulcer and stress ulceration and promoting the healing of gastric and / or duodenal ulcers. These compounds can be used in conjunction with other therapeutic agents such as anti-inflammatory and / or analgesic agents such as aspirin, inarmetacin, phenylbutazone, ibuprofen, naproxen, tolemtim, etc.

The pharmaceutical compositions can be administered parenterally or orally, prophylactically and / or therapeutically. The pharmaceutical compositions can take a wide variety of dosage forms, depending on the route of administration. For example, dosage forms suitable for oral administration include powders, tablets, pills, capsules, and dragees.

The pharmaceuticals can be administered intravenously. Agents for intravenous use are a solution or suspension of a compound in an acceptable carrier, preferably an aqueous carrier. Various aqueous carriers can be used, e.g., buffered water. water, 0.4% saline, etc. These agents will sometimes be sterilized using known sterilization methods, or may be sterile filtered. The resulting aqueous solutions may be packaged as is or lyophilized, the lyophilized preparation being combined with a sterile aqueous solution prior to administration. These agents may contain pharmaceutically acceptable excipients that enable the adjustment of parameters similar to physiological conditions, such as pH and buffering agents, tonicity agents, wetting agents, etc., e.g. sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monrlauate, triethanolamine oleate, etc.

The solids may contain known non-toxic solid carriers such as, for example, mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, magnesium carbonate and the like, all of which are pharmaceutical grade. Oral non-toxic pharmaceutically acceptable agents are prepared using known excipients such as the aforementioned carriers, the active ingredient concentration being generally from 0.1% to 95% and preferably from about 20%.

The compounds of the invention were tested in in vitro and in vivo tests according to the procedures described below.

In vitro test procedures are known for screening potential inhibitors for their tryptase inhibition capacity (see, e.g., Sturzebecher et al. (1992) Biol. Chem. Hoppe-Seyler 373: 1025-1030). Typically, these tests measure the tryptase-induced hydrolysis of the peptide lawns. Details of an example procedure are described below.

In addition, the activity of the compounds of the invention can be assessed in vivo in one of a number of animal models of asthma (see Larson, "Experimental Models of Reversible

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Airway Obstruction ”, The Lung: Scientific Foundation, edited by Crystal, West et al. Reven Press, New York, 1991; Warner et al. (1990) Am. Rev. Respir. Dis. 141: 253-257). An ideal animal model will exhibit the major clinical and physiological features of human asthma, including airway hyperactivity to chemical mediators and physical stimuli, reversibility of airway obstruction by drugs useful in human asthma (beta-adrenergic drugs, methylxanthines, corticosteroids, etc. .), inflammation of the airways with pressure from activated leukocytes, and chronic inflammatory degenerative changes such as basement membrane thickening, smooth muscle hypertrophy and epithelial damage. Species used as animal models include mice, rats, guinea pigs, rabbits, dogs, and sheep. All of them have some limitations, and the correct selection of an animal model depends on the problem that you want to solve.

Immediate asthmatic response can be assessed in guinea pigs and dogs, especially Basenji and Greyhound hybrids, who have nonspecific airway hyperreactivity to numerous non-allergenic substances such as methacholine and citric acid. Certain selected sheep show a dual response to an antigenic challenge with Ascaris proteins. Dual Immediate Asthmatic Reaction (IAR) animals exhibit a Late Asthmatic Reaction (LAR) 6-8 hours post-exposure. Hypersensitivity to the cholinergic antagonist carbachol increases 24 hours after antigen challenge in LAR-bearing animals.

An allergic sheep model was used to evaluate the potential anti-asthma effects of the compounds of the invention. Administration of agents in the form of aerosol solutions of the compounds of this invention to allergic sheep following or prior to exposure to specific allergens has been found to significantly reduce or abolish late asthmatic response and associated hyperactivity.

The compounds of the invention are also useful in the treatment of other immune inflammatory conditions in which the pathological condition is promoted by tryptase activity. Such diseases include inflammatory mast cell diseases such as rheumatoid arthritis, ankylosing spondylitis, osteoarthritis, gouty arthritis and other conditions associated with arthritis, enteritis, peptic ulcers, and various skin conditions.

The effectiveness of the compounds of the invention in treating a wide variety of immune inflammation can be assessed using in vivo and in vitro procedures. Thus, the anti-inflammatory efficacy of the compounds of the invention can be demonstrated by known trials, e.g., by the Arthus Reverse Passive Reaction (RPAR) -PAW technique (see, e.g., U.S. Patent No. 5126352). Attempts to determine the therapeutic value of compounds in the treatment of various skin disorders, such as skin disease associated with increased cell proliferation, are well known, such as, for example, the mice's ear test with ara, la, and, honic acid (ibid.). The compounds of the invention can be evaluated for their anti-ulcer activity according to the procedures described in Chiu et al. (1984) Archives Internationales de Pharmacodynamie et de Therapie 270: 128-140).

The biological activity of the compounds according to the invention was demonstrated in the following tests.

A. In Vitro Tryptase Inhibition Test

Compounds (approximately 1 mg) to be tested in the tryptase test were dissolved in dimethylsulfoxide (DMSO) and diluted 1:10 with a buffer containing 50 MM Tris-HCl pH 8.2 and 100 mM NaCl and 0.05% Tween-20 . Seven additional 3-fold dilutions from the starting dilution were made using the same buffer but containing 10% DMSO. Aliquots (50 µl) of each dilution in this series were transferred to separate wells of a 96-well U-bottom microtiter plate. Tryptase (25 μl, final concentration 0.5 nM) was added to each well and the samples were mixed and then incubated for 1 hour at room temperature under ambient light (i.e. the room light during the experiment or under controlled "light" and "light" conditions). "Darkness" refers to an illuminance of 4304-4842 lx using a fluorescent bulb. "Darkness" was obtained by wrapping the sample containers with aluminum foil. The enzymatic reaction was initiated by adding the synthetic tosyl-GlyProLysp-nitroanilide tripeptide ( 25 µl, final concentration 0.5 mM) The microtiter plates were transferred immediately to a UV / MAX Kinetic Microplate Reader (Molecular Devices) and the hydrolysis of the dye forming substrate was followed by spectrophotometry (405 nm) for 5 minutes. linear progress curves of the reaction Initial velocity values calculated from the post curves pu using kinetic analysis with a program called "BatchKi" (available from Biokin Ltd., Madison, WI), was used to determine apparent inhibition constants for each inhibitor. The program allowed to calculate regression and fit to curves of nonlinear data.

Table 1 shows inhibition constants (K /, in micromoles (pM)) determined for several compounds of the formula I, wherein R ¹ is - (CH 2) 3 NH (C = NH) -NH 2, and R ² and R ³ together with the nitrogen and carbon to which they are attached and with the group C (= O) R ⁴ form a substituted pyrrolidinyl. According to the invention, a compound was considered "active" or effective as a tryptase inhibitor when its Kf value was less than 1000 pM. In contrast to the K i, the K f value may not be the actual dissociation constant of the enzyme inhibitor complex; K i 'is equal to K, for a non-competing inhibitor and is directly proportional to the Kj for both competing and non-competing inhibitors.

Exposure of test compound solutions to light in a text environment may lower the Kj 'value. General test conditions are light dependent and variations in ambient light intensity may degrade the reproducibility of the test results. Due to this potential source of error, some tests were conducted in the dark. The values in Tables 1 and 4 without parentheses are the values determined under ambient light conditions. The Kj 'values in parentheses were determined under the conditions of "dark" as defined above. Kj 'values with an asterisk (*) were determined under the conditions of "light" as defined above (that is, at an illuminance of 4304-4842 lux).

Table 1

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Table 2 lists the inhibition constants (Ki ', μΜ) determined for several compounds of the invention. According to the invention, a compound was considered to be "active" or effective as a tryptase inhibitor when its K, 'value was less than 1000 µ 1000.

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Table 2

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B. Additional Cquractorism ą © / tab d 1 and 4 under ovzglgclem Id ^ imovama tryptase

and). Effect of pH value

Compound # 3 was tested for tryptase inhibition at four different pH values in the following buffer system: 120 mM NaCl, 2.7 mM KCl, 0.13 mM NaH2PO) 4, 0.896 mM Na2HPO4. The last buffer system used contained 0.05% Tween 20. Table 3 shows the K, '(pM) value for compound 3 at various pH values.

Table 3

pH 6.5 7.0 7.5 8.0 k; (pM) 14 8.0 5.4 6.1

b). The optic test was carried out at pH 7.5

Compound # 3 was found to be less sensitive to light in the following buffer system: 120 mM NaCl, 2.7 mM KCl, 0.13 mM NalEP0) 4, 0.896 mM Na2HPO4, and 0.05% BH2N2 at pH 7. 5. Thus, the above-described test procedure was performed by diluting compound 3 and adding tryptase under ambient light conditions and at room temperature (22-26 ° C). The test plate was wrapped in aluminum foil for a one-hour incubation period before adding the substrate, which was also added under ambient light conditions. Dilution and tryptase addition steps must be completed within 5 minutes. In addition, Compound # 3 must be taken up in DMSO.

C. In vivo tests

A sheep model with allergies was used in these studies. Research methods have been previously published (Abraham et al. (1983) Am. Rev. Respir. Dis. 128: 839-844, Allegra et al. (1983) J. Appl. Physiol. 55: 726-730, Russi et al. (1985) J. Appl. Physiol. 59: 1416-1422, Soler et al. (1989) J. Appl. Physiol. 67: 406-413. Each sheep served as its own control The animals weighed 20-50 kg.

In these trials, 9 mg of Compound # 3 of Tables 1 and 4 were dissolved in 3 mL of buffered saline and the entire solution was aerosolized 0.5 hours before, 4 hours after, and 24 hours after antigen challenge (total dose = 27; n = 6). Compound 3 tended to reduce the immediate reaction and attenuated the late reaction as illustrated in Figure 1. In the control (vehicle alone), antigen challenge increased specific lung resistance (SRL) above baseline in the immediate and late reaction to 374 ± 104% and 212 U + U 21% (mean ± standard error). In contrast, when sheep were administered Compound # 3 of Tables 1 and 4, the immediate and late increases in SRL were 280 + 39% and 72 + 9% (p <0.05 vs control, Wilcoxon rank labeled test). The immediate maximum response was taken as the mean of the maximum values occurring immediately after challenge. Maximum late responses were calculated by averaging the maximum abdominal responses of a given animal over a period of 6-8 hours. This is a conservative approach, eliminating possible late response reductions simply due to averaging.

At 24 gadrine after antigen challenge in the controls and drug trials in sheep, airway hyperresponsiveness occurred expressed as PC4000, that is, the concentration of carbaehol resulting in a 400% increase in SRL; a decrease in PC400 indicates that the airways have become ΠίκΕεΐΑΐν ^ Ίϋ). Compound No. 3 of Tables 1 and 4 blocked the overactivity that occurred after 24 goezinrch (see Fig. 2). The PC4000 baseline corresponded to 22.0 ± 3.7 breath units in the control and decreased to 9.1 ± 1.3 breath units after antigen challenge (p <0.05 from baseline). In contrast, in the drug trial, the PC4000 value was unchanged from baseline (16.0 ± 3.8 breath units and 17.6 ± 3.5 breath units after antigen challenge). Thus, the use of Compound No. 3 of Tables 1 and 4 resulted in a statistically significant improvement in airway function in sheep following allergen challenge. The results obtained in these tests were graphically analyzed in the figure.

176 941

Figure 1 is a graph of sheep lung specific resistance as a function of time in hours from the time of antigen challenge. Open squares correspond to control values and full circles to values from the same animal but after administration of Compound No. 3 in Tables 1 and 4.

Figure 2 is a graph of the development of hyperresponsiveness in sheep with carbachol induced bronchospasm at 24 hours post allergen challenge, after administration of compound No. 3 of Tables 1 and 4 prior to carbachol. The shaded rectangle corresponds to the control sample, the baseline. The second, lighter rectangle corresponds to the baseline for the drug. The oblique rectangle represents the antigen challenge control. The white box corresponds to the drug assay following antigen challenge.

The following examples are provided to better understand the invention described herein. It should be understood that they are illustrative only and not in any way limiting to the invention.

Examples.

Starting materials which are not commercially described herein are known or can be prepared by known methods. The compounds of formula I can be prepared by known methods using solid phase or solution phase synthesis techniques. The starting compounds used in the preparation of compounds of formula I are known or can be prepared by methods well known to those skilled in the art. For example, a technique for solid phase polypeptide synthesis is described in Solid Phase Peptide Synthesis: A Practical Approach (edited by R. C. Sheppard), IRL Press at Oxford University Press (1989) and in Marrifield et al., J. Am. Chem. Soc. 85: 2149-2156 (1963). Peptide coupling chemistry is also described in The Peptides, Vol. 1 (edited by E. Gross and J. Meienhofer), Academic Press, Orlando (1979). Other techniques are that described in Geysen et al., J. Imm. Meth. (1987) 102: 259-274 and Houghten et al., Nature, (1991) 354: 84-86.

For the preparation of the compounds of the invention as described herein, conditions for protecting groups will be well known to those skilled in the art of organic chemistry. Thus, the use of appropriate protecting groups is necessary in these processes, although they are not always illustrated.

The isolation and purification of the final and intermediate compounds described herein may be carried out, if desired, by any isolation and purification methods, such as, for example, filtration, extraction, crystallization, column chromatography, thin layer chromatography, thick layer chromatography, high performance liquid chromatography, and combinations of these procedures. An illustration of the isolation and purification methods can be found in the examples below. Of course, other equivalent isolation and purification methods may be used.

The Sakaguchi assay was used to detect arginine and arginine-containing peptides (see Stewart and Young, Solid Phase Peptide Synthesis, 2nd ed., Pierce Chemical Company, p. 114. Solution I was prepared containing 0.015 α-naphthol and 5% urea in 95% ethanol Solution II was prepared by dissolving 2 g of bromine in 100 ml of 8% w / w sodium hydroxide solution. 5 pellets of sodium hydroxide were added to solution I. The sample for analysis was spotted onto a thin layer chromatography (TLC) plate. The TLC plate was sprayed with solution I, dried in air and sprayed with solution II A red spot indicated the presence of amineiminomethane.

Example 1. Solid phase synthesis of compounds of formula I

A. Preparation of compound No. 3 from Tables 1 and 4

In 30 ml of a 1: 1 (v / v) HN-dimethylformamide (DMF) and toluene solution containing 30% v / v piperidine, a suspension of 4- (2 ', 4'''-methoxyphenylyfluorenylmethexecarbonyl (Fmec) -amamiomethyl) -phenoxype> listen'ecic resin was prepared (Rink, Bachem, CA resin, 3.5 g with a load of 0.289 meq / g, 1.01 mmol). The reaction mixture was stirred for 5 minutes and then it was filtered. Another 30 ml of piperidine solution was added and the mixture was stirred for a further 5 minutes. After filtration, the resin beads were washed sequentially with DMF (6 ×) and methylene chloride (6 ×).

176 941

Fmoc-L-proline (Milligen, 6.06 mmol), benzotriazol-1-yloxy-tris (dimethylamino) phosphonium hexafluorophosphate (BOP, Novabiochem, 6.06 mmol) and 1-hydroxybenzotriazole (HOBT, Aldrich, 6.06 mmol) dissolved in about 30 ml of DMF. The resulting clear solution was added to the Rink resin and the suspension was stirred (with nitrogen bubbling through it) for 4 hours. The reaction mixture was filtered and the resin beads were washed as described above. The Fmoc group was removed with piperidine as described above.

A solution of Fmoc-L-arginine (PMC) (Milligen, 6.06 mmol), BOP (6.06 mmol) and HOBT (6.06 mmol) in DMF (30 mL) was added to the resin beads and the suspension was stirred for 4 hours. then it was filtered, washed, treated with piperidine and washed again as described above.

A solution of 1-hydroxy-2-naffoic acid (Aldrich, 1.14 g, 6.06 mmol), 1,3-diisopropylcarbodiimide (DIPCDi, Aldrich, 0.949 ml, 6.06 mmol) and HOBT (0.819 g, 6.06 ml) were added. ) in DMF (45 ml) and the reaction mixture was stirred (mechanical frame stirrer) for 7 hours. After filtration, washing and treatment with piperidine (as described above), the resin was washed sequentially with DMF (6 ×), methylene chloride (6 ×), and methanol (6 ×). The resin was suspended in methanol and stirred (mechanical frame stirrer) overnight (15 hours). The resin was filtered and washed twice with methanol.

The white free flowing solid was suspended in trifluoroacetic acid (TFA): anisole: water (90: 5: 5, 30 ml). The resin immediately turned pink and then darkened to a deep red color within 1-2 minutes. After stirring for 5 minutes, the reaction mixture was filtered. This procedure was repeated two more times with fresh cleavage reagent except that the contact time was increased to 10 minutes for each replicate.

The TFA solutions were combined and the resulting clear orange solution was allowed to stand at room temperature for 3.5 hours. After concentration in vacuo, a pale yellow oil was obtained which was kept under vacuum (<133 Pa) for 3 hours. This oil was dissolved in 50% aqueous acetonitrile (100 ml) and the crude product was obtained as an off-white solid (417 mg) after lyophilization.

Analytical HPLC (Polymer Labs 100A PLRP column, 1.0 x 150 mm, linear gradient elution 20 - 45% acetonitrile over 13 minutes at a flow rate of 0.1 ml / min) showed that the crude product was essentially a single compound ( retention time 10.35 minutes, UV absorbance> 98%). The product was purified by HPLC using a C 18 silica gel column (Vydac, 22 x 250 mm. 15-20 (tm, 300 Å, linear gradient elution 20 - 35% in 30 minutes at a flow rate of 10 mL / min). Multiple gushes of 30-35 mg were required Similar fractions were combined (retention time 44-48 minutes) and freeze-dried to give a fluffy white solid (351 mg), which was a single compound by HPLC.

The results of electron beam mass spectroscopy (calculated molecular weight = 440.49, found M ⁺¹ = 441.1) and NMR spectroscopy (proton and ¹³ C) were consistent with the assumed structure.

1 H NMR (CD 3 OD) S 7.89 (d, J = 8 Hz, 1H), 7.78 (d, J = 6 Hz, 1H), 7.76 (d, J = 6 Hz, 1H), 7.57 ( dt, J = 8.1Hz, 1H), 7.48 (dt, J = 8.1Hz, 1H), 7.29 (d, J - 8Hz, 1H), approx 4.95 (masked, 1H), 4 , 49 (dd, J = 8.5Hz, 1H), 3.97 (dt, J = 10.7Hz, 1H), 3.23 (t, J = 7Hz, 2H), 2.29 (m, 1H) , 1.7 - 2.2 (m, 8H).

B. Preparation of Other Compounds of Formula I.

By using the method described in Part A above, and replacing 1-hydroxy-2-naphthoic acid with the following compounds:

2-hydroxy-4-methylbenzoic acid, 3-hydroxy-2-quinoxalinecarboxylic acid, 3-hydroxy-2-naphthoic acid, 2-hydroxy-1-naphthoic acid, 3-hydroxy-2-pyridine carboxylic acid, 4-hydroxy acid -7-methyl-3-naphthyridine carboxylic acid,

176 941 with 2-hydroxy-3-pyridinecarboxylic acid, 2-hydroxybenzoic acid, 2,5-dihydroxybenzoic acid and 2-hydroxy-5-phenylbenzoic acid (see E below), compounds No. 2, 9, 14, 15, 16 were obtained, 17, 18, 19, 20 and 21 of Tables 1 and 4.

C. Preparation of Other Compounds of Formula I.

Compound No. 12 in Table 2 was obtained by using the method described in Part A above and replacing 'FmohlL-proline with Fmoc-D-proline.

D. Preparation of Additional Compounds of Formula I.

Compound No. 13 of Tables 1 and 4 was prepared by first coupling Fmoc-L-phenylanaline to the resin in a manner analogous to the Fmoc-L-proline coupling in Part A above. The rest of the synthesis of Compound No. 13 was performed using the techniques described in Part A above.

Similarly, the preparation of Compounds Nos. 6 and 7 of Tables 1 and 4 required the coupling of Fmoh-pipdridine-3-carboxylic acid or Fmoh-β-pipdridine-4l carboxylic acid, respectively, with a resin, removal of the Fmoc group, and then coupling with Fmoh-Llβroline. The rest of the synthesis was carried out using the methods described in part A above.

E. Preparation of 2-hydroxy-5-phenylbenzodic acid.

The synthesis of 2-hydroxy-5-phenylbenzoic acid was performed as follows. A solution of tetrahydropyranyl (THP) -protected 4-phenylphenol (474 mg, 2 mmol, prepared from 4-fnrlphenol using standard methods described in Green and Wuts pp. 31-34) in anhydrous THF (5 ml) was cooled to -78 ° C and treated with n-butyllithium (2 mL of a 1.6 M solution in hexanes). The reaction mixture was stirred and warmed to room temperature, during which time a brown suspension formed. After 2 hours, the reaction mixture was cooled to -78 ° C and treated with excess anhydrous CO 2 for several minutes. The reaction mixture was warmed to room temperature with stirring. After 2 hours, the reaction mixture. partitioned between diethyl ether and aqueous IN NaOH solution. The aqueous layer was cooled to 0 ° C with ice and acidified to pH 2 with aq. IN HCl. The organic layer was dried (MgSO4) and concentrated in vacuo to give the crude product as an off-white solid (258 mg of 2-hydroxy 5-phenylbenzoic acid). NMR spectroscopy of the raw material confirmed the assumed structure.

1 H NMR (DMSO-d6) δ 8.04 (d, J = 3 Hz, 1H), 7.76 (dd, J = 9.3 Hz, 1H), 7.62 (d, J = 7 Hz, 2H), 7.44 (t, J = 7 Hz, 2H), 7.32 (t, J = 7 Hz, 1H), 7.00 (d, J = 9 Hz, 1H). Calculated mass 466.5. Observed mass 467.2. LC gradient 25 - 45% acetonitrile in 13 minutes. LC retention time 3.5 minutes. This material was used without further purification.

Example 2. Solution synthesis of a compound of formula I

A. Preparation of compound No. 10 from Table 2

74 ml of concentrated sulfuric acid were added to 20 g of (L) -phenylalanine cooled to -5 ° C in a salt / ice bath. The temperature of the reaction mixture was allowed to equilibrate, and then 9.4 ml of concentrated nitric acid was added dropwise over 5 minutes. The reaction mixture was stirred for 30 minutes and then it was added to 700 ml of crushed ice / water. The solution was adjusted to pH 8-9 with concentrated ammonium hydroxide. The solution was allowed to crystallize at room temperature and then cooled to 4 ° C overnight. The pale yellow crystalline product was filtered on a cured filter, washed with cold water and dried. The product was recrystallized from hot water and the filtrate was evaporated and recrystallized (2x) with a total yield of 55%. M.p. - 218 - 222 ° C (crude). TLC R- (F) 0.18. NMR: (D 2 O / N-OD), y 2.99 (dd, J = 7.2 Hz, 1H), 3.10 (dd, j = 13.4, 7.2 Hz, 1H), 3.57 (dd , J = 7.2, 6.0 Hz, 1H), 7.48 (d, J = 8.6 Hz, 2H), 8.21 (d, J = 8.7 Hz, 2H).

Tbutoxynarbonyldm (BOC) protected amino acids can be prepared by known techniques, see, e.g., Greene and Wuts Protectid Groups in Organic Synteesis, 2nd Ed., John Wiley & Sons, Inc .: New York, pp. 327-328 (1991).

176 941

To a solution of BOC protected p-nitrophenylalanine (3.00 g, 9.67 mmol) in dry methylene chloride was added N-methylmorpholine (NMM, 1.07 mL, 9.67 mmol) at -20 ° C followed by isobutyl chloroformate ( 1.26 mL, 9.67 mmol). The reaction mixture was stirred for 15 minutes at -20 ° C. Methyl-L-proline hydrochloride (L-Pro-OMe, 1.60 g, 9.67 mmol) was added to the mixture as a solid followed by additional NMM (1.07 mL, 9.67 mmol). After stirring at -20 ° C for 1 hour at room temperature for 1 hour, the reaction mixture was concentrated in vacuo then diluted with ethyl acetate. The ethyl acetate solution was washed sequentially with 10% aq. Citric acid, water and brine, dried (MgSO4) and concentrated in vacuo. 2.4 g of product was obtained after column chromatography (yield> 60%).

The BOC group was removed by treatment with HCl in dioxane using standard conditions, see e.g. Greene and Wuts above at 328 - 329. To a solution of 1-hydroxy-2-naphthoic acid (160 mg, 0.84 mmol) in methylene chloride and N, N- dimethylformamide (1: 1, 1.17 mmol) was added 1-hydroxybenzotriazbl (159 mg, 1.17 mmol). The solution was cooled to 0 ° C and 1- (3-dimethylbaminopropyl) -3-ethylcarbodimide hydrochloride (EDCI, 161 mg, 0.84 mmol) was added. The solution was stirred at 0 ° C for 45 minutes then a solution of the dipeptide (300 mg, 0.84 mmol) prepared as above was added followed by NMM (93 µL, 0.84 mmol). The reaction mixture was stirred at 0 ° C for 1 hour and at room temperature for 45 minutes. The reaction mixture was concentrated in vacuo and diluted with ethyl acetate and water. The organic layer was separated, washed (4x) with water then brine, dried and concentrated in vacuo. Chromatography gave the coupled product (300 mg, 73%).

To a solution of this coupled product (410 mg, 0.83 mmol) in ethyl acetate (20 ml) was added glacial acetic acid (10 drops) and 10% palladium on activated carbon (100 mg). This solution was hydrogenated for 5 hours. The reaction mixture was filtered through celite and the solution concentrated in vacuo to give 375 mg (98%) of the product which was used without further purification.

To a solution of the product from the above hydrogenation reaction (200 mg, 0.45 mmol) in anhydrous methanol (10 ml) was added formamidine sulfonic acid (118 g, 0.95 mmol) obtained by oxidation of commercial formamidine sulfinic acid by the method described in Maryanoff et al. (1986 ) J. Org. Chem .. 1882. The reaction mixture was stirred for 3 days. The reaction mixture was concentrated in vacuo and the product was isolated by chromatography (elution with methanol: methylene chloride) to afford 17 mg of the desired product, compound No. 10 in Table 2.

Example 3. Alternative Synthesis of Compounds of Formula I in Solution

A. Preparation of compound No. 3 from Tables 1 and 4

A solution of 1-benzyloxy-2-naphtbesbwegb acid (2.247 g) in thionyl chloride (15 ml) was heated under reflux for 4 hours. The excess reagent was evaporated in vacuo. The residue was diluted with anhydrous DMF (9 mL) and concentrated to about 2 mL. To this solution was added 4-dimethylaminopyridine (1.02 g). The mixture was stirred at room temperature overnight. The resulting pyridinium complex was added to a stirred solution of nitro-L-arginine (1.77 g), tetramethylguanidine (1 ml) and lithium chloride (4.56 g) in DMF (60 ml). The mixture was stirred at temperature for 48 hours. Most of the DMF was evaporated in vacuo and the residue was partitioned between 1.5N aq. HCl and ethyl acetate. The combined organic layers were washed with a saturated aqueous sodium chloride solution and dried over magnesium sulfate. After concentration in vacuo, the crude product (3.5 g) was obtained. TLC: Product Rf 0.22 (silica gel plate, eluting with 10% methanol / acetic acid in chloroform). This product was used without further purification.

A solution of 1-benzyloxy-2-nphitoyl-N-nitro-L-arginma (150 mg) in dry DMF (5 ml) was cooled to -25 ° C and treated with isobutyl chloroformate (0.053 ml). The reaction mixture was warmed to room temperature with stirring. After 20 minutes, crude L-proline amide (as hydrochloride, 65.9 ml) was added. The mixture was stirred for 16 hours and then diluted with ethyl acetate. Ethyl acetate solution

Washed with a 5% aqueous citric acid solution, a saturated aqueous sodium bicarbonate solution and a saturated aqueous sodium chloride solution and then dried over magnesium sulfate. The solution was concentrated in vacuo and the residue was diluted with ethanol (40 ml, containing 10% methanol and 10% acetic acid) and hydrogenated at 365 kPa pressure over 10% palladium on carbon (10 mg) until the reaction was complete (TLC on silica gel, elution with chloroform-methanol 9: 1), as evidenced by the disappearance of the faster moving spot. The reaction mixture was filtered through celite then washed with ethanol and methanol. The mixture was concentrated in vacuo and purified by high performance liquid chromatography to give 1-hydroxy naphthoyl-L-arginyl-L-proline amide (41 mg, electron mass spectroscopy: M ⁺¹ = 441) which was isolated from its salt form with trifluoroacetic acid.

B. Preparation of Compound No. 1 from Table 2

The procedure in Part A above was used, replacing 1-benzyloxy-2-naphthoic acid with 2-benzylroxy-2-naphthoic acid and L-proline amide with sarcosine amide to afford Compound No. 1 in Table 2.

C. Preparation of Other Compounds of Formula t

The procedure in Part B above was used, the sarcosine amide was replaced with the following:

N, N-almethylamine of proline, L-proline methyl ester and trans-4-hydroxy-L-proline methyl ester to give compounds 5 and 8 of Tables 1 and 4 and Compound No. 4 of Table 2.

The physical data of the compounds of formula 1 are shown in Table 4.

Table 4

Relationship no Calculated mass Found mass (M + ¹ ) ¹ LC gradient ² LC retention time (minutes) 1 2 3 4 5 1 414.24 415.1 D 5.8 2 404.26 405.5 B 11.5 3 440.26 441.1 AND 10.3 4 471.26 472.2 D 6.2 5 468.29 469.1 D 6.8 63 551.34 552.3 D 7.5 7 551.34 552.1 D 7.3 8 455.26 456.0 D 7.0 9 442.26 443.0 D 5.5 11 456.26 456.7 C. 24.4 12 440.26 441.2 C. 25.4 13 587.34 588.0 C. 26.9 14 440.26 440.5 AND 9.0 15 440.26 440.2 ⁴ B 8.4 16 391.5 391.8 ⁴ D 5.6 17 456.49 456.74 B 9.0

176,941 cont. table 4

1 2 3 4 5 18 393.22 391.84 B 5.5 19 390.4 391.1 AND 4.7 twenty 406.4 407.2 C. 20.7 21 466.5 467.2 E. 3.5

¹ All mass values were determined using a Finnigan electron beam spectrometer, unless otherwise stated.

² In all cases a two-component gradient system containing acetonitrile and water was used. All eluents contained 0.1% trifluoroacetic acid. A: 45% acetonitrile for 13 minutes B ': 10 - 35% acetonitrile for 13 minutes. C: 5 - 95% aeetonitrile for 45 minutes. D: 2 - 95% acetonitrile for 10 minutes. E: 25-45% acetonitrile for 13 minutes.

³ The test substance was a mixture of stereoisomers.

Mass values were determined using a Biolon mass spectrometer. The given values correspond to M.

Example 4.

A. Aerosol formulations were prepared by dissolving 9 mg of Compound No. 3 in Tables 1 and 4 in 3 ml of buffered saline, with a phosphide or bicarbonate buffer as the buffer.

B. A topical formulation to the skin was prepared containing 1% of Compound No. 3 of Tables 1 and 4 in the form of an ointment based on paraffin, petroleum jelly and lanolin.

It should be understood that the above description is illustrative only and not limiting. Many characters will become obvious to those skilled in the art after reading this description. The scope of the invention should therefore be established not with reference to the above description, but with reference to the appended claims, taking into account the full range of equivalents arising from these claims.

Publishing Department of the UP RP. Circulation of 70 copies. Price PLN 4.00.

Claims (9)

Patent claims 1. New peptides of general formula I in which Ar is a group selected from among R ² is methyl, R ³ is hydrogen, or R ² and R ³ together with the nitrogen and carbon atom to which they are attached and with the group C (= O) R ⁴ form a 5-membered heterocyclyl of formula 176 941 R ⁴ is -OCH 3, -NH 2 or -N (CH 3) 2, or is selected from the group consisting of and pharmaceutically acceptable salts thereof. 2. Compound according to E2a and ^ tg .., which is chAr oh, nucet 1-hydrohsy-2-naphthyl, a-hydrohsy-1-naphthyl, 3-hydrons-2-pyridyl or 2-hydronsy-3-quinonsalyl; R ¹ is a group or t R ² is methyl, R ³ is hydrogen, or R 2 and R ³ together with the nitrogen atom and the carbon atom to which they are attached and with the group C (= O) R 4 form a heterocyclyl of the formula and R 4 is -OCH 3, -NH 2, -3'-carboxyamido-r-piperidyl or -N (CH3) 2. 3. The compound of claim 1 Is t -hydrol-2-naphthyl. Rt is - (CH2) 3NH (CNH) NH2, ^R2 and R3 together with the nitrogen and carbon atom to which they are attached, and the group C (= 0) R ⁴ form a heterocyclyl of the formula OH and R ⁴ is -NH2 176 941 4. The compound of claim 1 2, wherein Ar is 2-hydroksyU-naphthyl, ^R1 is - (CH2) 3NH (CNH) NH2, ^R2 and ^R3 together with the nitrogen and carbon atom to which they are attached, and with the group. C (= O) R ⁴ forms a heterocyclyl of formula N 4 O 4 or and R 4 is -NH 2. ' 5. A pharmaceutical agent for the treatment and antimicrobial conditions, containing the active ingredient and a pharmaceutically acceptable carrier and / or excipients, characterized in that the active ingredient is a compound of the general formula I used in an amount of 0.001-95%, in which Ar is a group selected from or 176 941 R ² is methyl, R ³ is hydrogen, or R ² and R ³ together with the nitrogen and carbon atom to which they are attached and with the group C (= O) R 4 form a 5-membered heterocyclyl of formula O. 4 and R ⁴ is -OCH 3, -NH 2, or -N (CH 3) 2, or is selected from the group consisting of or a pharmaceutically acceptable salt thereof. 6. The agent in the above mentioned paragraph 5, characterized by the fact that it contains a compound of the vortex I, in which Ar is 1-hydroxy-2-naphthyl, 2-hydro, k5, n-naphthyl, 3-hydroxy - 2-pyridyl or 2-hydroxy-3-quinoxalyl; R ¹ is a group R2 is methyl, R3 is hydrogen or R2 and R3 together with the nitrogen atom and the carbon atom to which they are attached and with the group. C (= O) R ⁴ forms a heterocyclyl of formula and R 4 is -OCH 3, -NH 2, -3'-carboxamido-1'-piperidyl, or -N (CH 3) 2. 7. Measure according to zasm ^ a, rnΗίηΐ ^ ϊην in the fact that aawkm grzazg o groove k in which Ar is 1-hydroxy-2-naphthyl, Ri is - (CH2) 3NH (CNH) NH2, R2 and R3 together with the nitrogen and carbon atom to which they are attached and with the group C (== O) R4 form a heterocyclyl about the formula 176 941 and R ⁴ is -NH 2. 8. The agent according to claim 1 and, znamiznny in that it comprises wwiąa.ek of formula I, wherein Ar is 2-hyeroksy-1-naphthyl, ^R1 is - (CH2) 3NH (CNH) NH2, R2 and ^R3 together with the nitrogen atom and at the carbon to which they are attached and with the group C (= O) R4 form a heterocyclyl of the formula and R4 is -NH2. 9. The agent according to food 5, which is a titanium salt solution containing a peptide of formula t in a concentration of 0.1-100 mg / ml, in particular 0.1-30 mg / ml.