Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
  • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
  • C07K5/06—Dipeptides
  • C07K5/06086—Dipeptides with the first amino acid being basic
  • C07K5/06095—Arg-amino acid
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P11/00—Drugs for disorders of the respiratory system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P27/00—Drugs for disorders of the senses
  • A61P27/16—Otologicals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/08—Antiallergic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
  • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
  • C07K5/06—Dipeptides
  • C07K5/06008—Dipeptides with the first amino acid being neutral
  • C07K5/06078—Dipeptides with the first amino acid being neutral and aromatic or cycloaliphatic
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
  • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
  • C07K5/06—Dipeptides
  • C07K5/06086—Dipeptides with the first amino acid being basic
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• This invention relates generally to compositions and methods for the prevention and treatment of immunomediated inflammatory disorders. More particularly, the invention relates to the prevention and treatment of inflammatory diseases associated with the respiratory tract, such as asthma and allergic rhinitis.
• the compositions and methods of the present invention are especially useful for preventing or treating the late phase bronchoconstriction and airway hyperresponsiveness associated with chronic asthma.
• Asthma is a complex disease involving multiple biochemical mediators for both its acute and chronic manifestations. Asthma frequently is characterized by progressive development of hyperresponsiveness of the trachea and bronchi to both immunospecific allergens and generalized chemical or physical stimuli. The hyperresponsiveness of asthmatic bronchiolar tissue is believed to result from chronic inflammation reactions, which irritate and damage the epithelium lining the airway wall and promote pathological thickening of the underlying tissue. Bronchial biopsy studies have indicated that even patients with mild asthma have features of inflammation in the airway wall.
• One initiator of the inflammatory sequence is an allergic response to inhaled allergens.
• Leukocytes carrying IgE receptors notably mast cells and basophils, but also including monocytes, acrophages, and eosinophils, are present in the epithelium and underlying smooth muscle tissues of bronchi, where they are activated initially by binding of specific inhaled antigens to the IgE receptors.
• Activated mast cells release a number of preformed or primary chemical mediators of the inflammatory response and enzymes.
• numerous secondary mediators of inflammation are generated in situ by enzymatic reactions of activated mast cells, including superoxide and lipid derived mediators.
• mast cells proteoglycans, peroxidase, arylsulfatase B, and notably the proteases tryptase and chymotryptic proteinase (chymase) .
• proteoglycans peroxidase
• arylsulfatase B notably the proteases tryptase and chymotryptic proteinase (chymase) .
• chymase chymase
• the early asthmatic reaction is followed by a further decline in respiratory function which begins within a few hours and is maximal between six and twelve hours post-exposure.
• This late asthmatic reaction is accompanied by a marked increase in the number of inflammatory cells infiltrating bronchiolar smooth muscle and epithelial tissues, and spilling into the airways. These cells include eosinophils, neutrophils, and lymphocytes, all of which are attracted to the site by release of mast cell derived ⁇ hemotactic agents. The infiltrating cells themselves become activated during the late reaction phase.
• the late asthmatic response is believed to be a secondary inflammatory reaction mediated in part by the secretory activity of macrophages.
• a related set of inflammatory reactions occurs in the upper respiratory tract mucosa, usually in response to airborne allergens.
• mast cells are activated by crosslinking of IgE molecules to particular antigens.
• mast cells may be activated in the absence of discernible exposure to a particular antigen. In either case, activated mast cells release primary and secondary mediators of inflammation upon degranulation. Eosinophils and macrophages are attracted to the site to perpetuate the inflammation reaction. Nasal epithelial tissue destruction often occurs in late-phase reactions.
• Tryptase is the major secretory protease of human mast cells and is proposed to be involved in neuropeptide processing and tissue inflammation.
• Mature human tryptase is a glycosylated, heparin-associated tetramer of heterogenous, catalytically active subunits.
• the tryptase monomer's amino acid sequence like its gene structure, has no close counterpart among the numerous other serine proteinases that have been characterized. See, e . g. , Vanderslice et al . (1990) Proc. Natl. Acad. Sci. USA 87:3811-3815; Miller et al . (1990) J. Clin. Invest.
• Tryptase is stored in mast cell secretory granules.
• tryptase After mast cell activation, human tryptase can be measured readily in a variety of biologic fluids. For example, after anaphylaxis, tryptase appears in the bloodstream, where it remains detectable for several hours. See Schwartz et al . (1987) N. Encrl. J. Med. 316:1622-1626. Its appearance has been detected in samples of nasal and lung lavage fluid from atopic subjects challenged with specific antigen. See Castells and Schwartz 1988) J. Allercr. Clin. Immunol. .821:348-355 and Wenzel et al . (1988) Am. Rev. Resp. Pis. 141:563-568.
• Tryptase levels in lung lavage fluid obtained from atopic asthmatics increase after endobronchial allergen challenge. Id .
• Some smokers of cigarettes have striking elevations of bronchoalveolar lavage fluid tryptase levels compared to nonsmoking controls, a finding that provides some support for the hypothesis that release of proteinases from activated mast cells could contribute to lung destruction in smoker's emphysema.
• tryptase has been shown to be a potent mitogen for fibroblasts, suggesting its involvement in pulmonary fibrosis and interstitial lung diseases. See Ruoss et al . (1991) J. Clin. Invest. 88:493-499.
• Tryptase has been implicated in a variety of biological processes, including degradation of vasodilating and bronchorelaxing 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 bronchial responsiveness to histamine (see Sekizawa et al. (1989) J.
• tryptase has been shown to cleave fibrinogen ⁇ -chains, as well as high molecular weight kininogen with a possible release of kinins and thus, may play a role with 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 also may be involved in tissue inflammation and remodeling. This finding also intimates that tryptase may play a role in joint destruction in rheumatoid arthritis.
• tryptase has been shown to cleave calcitonin gene-related peptide.
• tryptase could be a factor in the regulation of flare reaction in cutaneous neurogenic inflammation. See Caughey (1991) Am. J. Respir. Cell Mol. Biol. 1:387-394.
• Ar is hydroxyl substituted aryl or hydroxyl substituted heteroaryl, wherein the hydroxyl is positioned ortho to the amide side chain and wherein if Ar is hydroxyl substituted aryl, the aromatic ring bearing the amide side chain is not substituted with halogen and does not bear a lower alkyl group on the position ortho to the hydroxyl;
• R 1 is hydrogen, lower alkyl, arylalkyl, or heteroarylalkyl;
• R 2 is hydrogen or lower alkyl
• R 3 is selected from the group consisting of:
• m is an integer from 3-6, n is an integer from 0-3, p is an integer from 0-2, q is an integer from 0-2; r is an integer from 0-5; s is an integer from 0-2; t is an integer from 1-3; u is 1 or 2; v is an integer from 3-6; and w is an integer from 0-3;
• X is -NH- or -CH 2 -;
• R 4 is lower alkyl, substituted arylalkyl or substituted heteroarylalkyl and R 5 and R 6 are independently selected from the group consisting of hydrogen, lower alkyl, substituted arylalkyl, and substituted heteroarylalkyl; or R 4 and R 5 together with the nitrogen and carbon to which they are attached, form a substituted 4-membered, 5-membered, or 6-membered heterocycle and R 6 is hydrogen; or R 4 and R 6 together with the nitrogen and
• R 7 is -OR 8 or -NR 8 R 9 , wherein R 8 and R 9 are independently selected from the group consisting of hydrogen, lower alkyl, aryl, arylalkyl, or heteroarylalkyl or R 8 and R 9 together with the nitrogen to which they are attached, form a substituted 5-membered or 6-membered heterocycle.
• Ar is l-hydroxy-2- naphthyl, 2-hydroxyl-l-naphthyl, 3-hydroxy-2-pyridyl, or 2- hydroxy-3-quinoxalyl;
• R 1 is hydrogen;
• R 2 is hydrogen;
• R 3 is selected from the group consisting of:
• m is an integer from 3-6, n is an integer from 0-3, p is an integer from 0-2, q is an integer from 0-2; and w is an integer from 0-3;
• X is - NH- or -CH 2 -; either R 4 is lower alkyl and R 5 and R 6 are hydrogen; or R 4 and R 5 , together with the nitrogen and carbon to which they are attached form a substituted 4-membered, 5-membered, or 6-membered heterocycle and R 6 is hydrogen; and R 7 is -OH, - CH 3 , -NH 2 , 3'-aminocarboxy-l'-piperidyl, or -N(CH 3 ) 2 .
• a particularly preferred tryptase inhibitor of Formula I is Compound 3 of Tables I and II:
• Another preferred tryptase inhibitor of Formula I is Compound 15 of Tables I and II:
• the compounds described herein are useful for the prevention and treatment of immunomediated inflammatory disorders, and particularly those associated with the respiratory tract, including asthma, and particularly the hyperresponsiveness phase associated with chronic asthma, and allergic rhinitis.
• the present invention also provides a method for treating immunomediated inflammatory disorders wherein a patient having an immunomediated inflammatory disorder that is susceptible to treatment with a tryptase inhibitor receives, or is administered, a therapeutically effective dose or amount of a compound of the present invention.
• the invention also provides for pharmaceutical compositions of the compounds described herein. These pharmaceutical compositions can be in a variety of forms including oral dosage forms, as well as injectable and infusible solutions.
• these pharmaceutical compositions are in an aerosol form of powders or solutions.
• the compounds of the instant invention are used in combination with a non-toxic, pharmaceutically acceptable topical carrier.
• the compounds of the instant invention can be used in combination with antiinflammatories or other asthma therapies, such as ⁇ - adrenergic agonists, antiinflammatory corticosteroids, anticholinergics, and the like.
• Figure 1 is a graph showing specific lung resistance in sheep as a function of time in hours post-antigen challenge. Open squares indicate control values, and filled circles indicate values for the same animal after administration of Compound 3 of Tables I and II.
• Figure 2 is a graph showing production of hyperresponsiveness of sheep to carbachol-induced bronchoconstriction at 24 hours post-allergen challenge when Compound 3 of Tables I and II was administered before carbachol.
• the dark, solid bar corresponds to the control, baseline.
• the lighter, solid bar corresponds to the drug. baseline.
• the hatched bar corresponds to control, post- antigen.
• the white bar corresponds to the drug, post-antigen.
• Immunomediated inflammatory disorder includes generally those diseases associated with mast cell mediator release and susceptible to treatment with a tryptase inhibitor.
• diseases of immediate type hypersensitivity such as asthma, allergic rhinitis, urticaria and angioedema, and eczematous dermatitis (atopic dermatitis) , and anaphylaxis, as well as hyperproliferative skin disease, peptic ulcers, inflammatory bowel disorder, inflammatory skin conditions, and the like.
• “Hyperresponsiveness” refers to late phase bronchoconstriction and airway hyperreactivity associated with chronic asthma. Hyperresponsiveness of asthmatic bronchiolar tissue is believed to result from chronic inflammation reactions, which irritate and damage the epithelium lining the airway wall and promote pathological thickening of the underlying tissue. "Halogen” refers to fluorine, bromine, chlorine, and iodine atoms.
• Hydroxyl refers to the group -OH.
• Lower alkyl refers to a cyclic, branched or straight chain alkyl group of one to six carbon atoms. This term is further exemplified by such groups as methyl, ethyl, n-propyl, i-propyl, n-butyl, t-butyl, i-butyl (or 2- methylpropyl) , cyclopropylmethyl, i-amyl, n-amyl, and hexyl.
• Aryl or “Ar” refers to an aromatic carbocyclic group having a single ring (e.g., phenyl), multiple rings (e.g., biphenyl) or multiple condensed rings in which at least one ring is aromatic, (e.g., 1,2,3,4-tetrahydronaphthyl, naphthyl, anthryl, or ph ⁇ nanthryl) , which can optionally be unsubstituted or substituted with, e.g., halogen, lower alkyl. lower alkoxy, lower alkylthio, trifluoromethyl, lower acyloxy, aryl, heteroaryl and hydroxy.
• the aromatic ring bearing the amide side chain cannot be further substituted with halogen.
• the aromatic ring bearing the amide side chain cannot possess a lower alkyl group ortho to the hydroxyl group (i.e., meta to the amide side chain) .
• Heterocycle refers to a saturated, unsaturated, or aromatic carbocyclic group having a single ring (e.g., morpholino, pyridyl or furyl) or multiple condensed rings
• heteroaryl or “HetAr” refers to a heterocycle in which at least one heterocyclic ring is aromatic.
• Arylalkyl refers to the group -R-Ar where Ar is an aryl group and R is straight-chain or branched-chain aliphatic group. Arylalkyl groups can optionally be unsubstituted or substituted with, e.g., halogen, lower alkyl, lower alkoxy, lower alkylthio, trifluoromethyl, lower acyloxy, and hydroxy.
• Heteroarylalkyl refers to the group -R-HetAr where HetAr is an heteroaryl group and R is straight-chain or branched-chain aliphatic group.
• Heteroarylalkyl groups can optionally be unsubstituted or substituted with, e.g., halogen, lower alkyl, lower alkoxy, lower alkylthio, trifluoromethyl, lower acyloxy, and hydroxy.
• “Pharmaceutically acceptable salt” refers to those salts which retain the biological effectiveness and properties of the parent compound and which are not biologically or otherwise undesirable.
• “Pharmaceutically or therapeutically acceptable carrier” refers to a carrier medium which does not interfere with the effectiveness of the biological activity of the active ingredients and which is not toxic to the host or patient.
• “Stereoisomer” refers to a chemical compound having the same molecular weight, chemical composition, and constitution as another, but with the atoms grouped differently. That is, certain identical chemical moieties are at different orientations in space and, therefore, when pure, has the ability to rotate the plane of polarized light. However, some pure stereoisomers may have an optical rotation that is so slight that it is undetectable with present instrumentation.
• the compounds of the instant invention may have one or more asymmetrical carbon atoms and therefore include various stereoisomers. All stereoisomers are included within the scope of the invention.
• Treatment refers to any administration of a tryptase inhibitor in vitro or in vivo and includes:
• compositions comprising an effective serine protease inhibitor, and more particularly a tryptase inhibitor, that is useful for reducing immunomediated-inflammatory disorders, and particularly bronchoconstriction induced by allergenic challenge in an asthmatic animal.
• Tryptase inhibitors are substances which slow down or prevent tryptase activity.
• the tryptase inhibitors will comprise a compound of Formula I:
• Ar is hydroxyl substituted aryl or hydroxyl substituted heteroaryl, wherein the hydroxyl is positioned ortho to the amide side chain and wherein if Ar is hydroxyl substituted aryl, the aromatic ring bearing the amide side chain is not substituted with halogen and does not bear a lower alkyl group on the position ortho to the hydroxyl;
• R 1 is hydrogen, lower alkyl, arylalkyl, or heteroarylalkyl
• R 2 is hydrogen or lower alkyl
• R 3 is selected from the group consisting of:
• m is an integer from 3-6, n is an integer from 0-3, p is an integer from 0-2, q is an integer from 0-2; r is an integer from 0-5; s is an integer from 0-2; t is an integer from 1-3; u is 1 or 2; v is an integer from 3-6; and w is an integer from 0-3;
• X is -NH- or -CH 2 -;
• R 4 is lower alkyl, substituted arylalkyl, or substituted heteroarylalkyl and R 5 , and R 6 are independently selected from the group consisting of hydrogen, lower alkyl, substituted arylalkyl, and substituted heteroarylalkyl; or R 4 and R 5 together with the nitrogen and carbon to which they are attached form a substituted 4-membered, 5-membered, or 6-membered heterocycle and R 6 is hydrogen; or R 4 and R 6 together with the
• R 7 is -OR 8 or -NR 8 R 9 , wherein R 8 and R 9 are independently selected from the group consisting of hydrogen, lower alkyl, aryl, arylalkyl, or heteroarylalkyl or R 8 and R 9 together with the nitrogen to which they are attached, form a substituted 5-membered or 6-membered heterocycle.
• Ar is l-hydroxy-2- naphthyl, 2-hydroxy1-1-naphthyl, 3-hydroxy-2-pyridyl, or 2- hydroxy-3-quinoxalyl;
• R 1 is hydrogen;
• R 2 is hydrogen;
• R 3 is selected from the group consisting of:
• m is an integer from 3-6, n is an integer from 0-3, p is an integer from 0-2, q is an integer from 0-2; and w is an integer from 0-3;
• X is - NH- or -CH 2 -; either R 4 is lower alkyl and R 5 and R 6 are hydrogen; or R 4 and R 5 together with the nitrogen and carbon to which they are attached form a substituted 4-membered, 5-membered, or 6-membered heterocycle and R 6 is hydrogen; and R 7 is -OH, -OCH 3 , -NH 2 , -3'-aminocarboxy-1'-piperidyl, or -N(CH 3 ) 2 .
• a preferred tryptase inhibitor is Compound 3 of Tables I and II:
• An additional preferred tryptase inhibitor is Compound 15 of Tables I and II:
• Another preferred tryptase inhibitor is Compound 21 of Tables I and II:
• the tryptase inhibitors of the present invention can be obtained by known techniques from readily available starting materials, as described in greater detail below.
• Compounds of this invention can, depending on the nature of the functional groups, form addition salts with various inorganic and organic 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 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, andelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and the like.
• inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like
• organic acids such as acetic acid, propionic acid, glycolic acid,
• Salts can also be formed from a carboxylic acid residue via treatment with alkali metals or alkali metal bases, such as alkali metal hydroxides and alkali metal alkoxides, or alkaline earth metals or alkaline earth metal bases, such as alkaline earth metal hydroxides and alkaline earth metal alkoxides.
• alkali metals or alkali metal bases such as alkali metal hydroxides and alkali metal alkoxides, or alkaline earth metals or alkaline earth metal bases, such as alkaline earth metal hydroxides and alkaline earth metal alkoxides.
• salts can be formed from a carboxylic acid and an organic base, such as trimethylamine, diethylamine, ethanolamine, piperidine, isopropylamine, choline, caffeine, and the like.
• the salts can be formed by conventional means, as by reacting the free acid or base forms of the product with one or more equivalents of the appropriate base or acid in a solvent or medium in which the salt is insoluble, or in a solvent such as water, which is then removed in vacuo or by freeze-drying or by exchanging the cations of an existing salt for another cation on a suitable ion exchange resin.
• the activity of the compounds of the present invention can be evaluated in vivo in one of the numerous animal models of asthma. See Larson, "Experimental Models of Reversible Airway Obstruction", in The Lung: Scientific Foundations. Crystal, West et al . , eds. , Raven Press, New York, 1991; Warner et al. (1990) Am. Rev. Respir. Pis. 141:253-257.
• An ideal animal model would duplicate the chief clinical and physiological features of human asthma, including: airway hyperresponsiveness to chemical mediators and physical stimuli; reversal of airway obstruction by drugs useful in human asthma (/3-adr energies, ethylxanthines, corticosteroids, and the like) ; airway inflammation with infiltration of 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 have some limitations, and the proper choice of animal model depends upon the question which is to be addressed.
• the initial asthmatic response can be evaluated in guinea pigs, and dogs, and particularly, with a basenji- greyhound cross strain which develops nonspecific airway hyperresponsiveness to numerous nonallergenic substances, such as methacholine and citric acid.
• Certain selected sheep exhibit a dual response after antigen challenge with Ascaris proteins.
• the initial asthmatic response (IAR) is followed by a late asthmatic response (LAR) at 6-8 hours post-exposure.
• Hypersensitivity to the cholinergic agonist carbachol increases at 24 hours after antigen challenge in those animals which exhibit LAR.
• the allergic sheep model was used to evaluate the potential antiasthmatic effects of the compounds of the present invention.
• Administration of compositions comprising aerosolized solutions of the compounds of the instant invention to allergic sheep prior to or following exposure to specific allergens demonstrates that such compositions substantially lessen or abolish the late asthmatic response and consequent hyperresponsiveness.
• the compounds of this invention are also useful for the treatment of other immunomediated inflammatory disorders in which tryptase activity contributes to the pathological condition.
• diseases include inflammatory diseases associated with mast cells, such as rheumatoid arthritis, rheumatoid spondylitis, osteoarthritis, gouty arthritis and other arthritic conditions, inflammatory bowel disease, peptic ulcer and various skin conditions.
• the efficacy of the compounds of the instant invention for the treatment of the vast majority of immunomediated inflammatory disorders can be evaluated by either in vitro or in vivo procedures.
• the anti- inflammatory efficacy of the compounds of the instant invention can be demonstrated by assays well known in the art, for example, the Reversed Passive Arthus Reaction (RPAR)-PAW technique (see, e.g., Ganguly et al . (1992) U.S. Patent No. 5,126,352).
• Assays for determining the therapeutic value of compounds in the treatment of various skin conditions, such as hyperproliferative skin disease are well known in the art, for example, the Arachidonic Acid Mouse Ear Test (id) .
• the compounds of the instant invention can be evaluated for their antiulcer activity according to the procedures described in
• a therapeutically or pharmaceutically effective amount of a tryptase inhibitor and particularly, a compound of Formula I is administered to a patient suffering from an immunomediated inflammatory disorder.
• the compositions of the present invention are useful for preventing or ameliorating asthma.
• the compounds may be administered prophylactically prior to exposure to allergen or other precipitating factor, or after such exposure.
• the compounds of the instant invention are particularly useful in ameliorating the late-phase tissue destruction seen in both seasonal and perennial rhinitis.
• compositions containing the compounds can be administered for prophylactic and/or therapeutic treatments.
• compositions are administered to a patient already suffering from a disease, as described above, in an amount sufficient to cure or at least partially arrest the symptoms of the disease and its complications.
• An amount adequate to accomplish this is defined as "therapeutically effective amount or dose.” Amounts effective for this use will depend on the severity and course of the disease, previous therapy, the patient's health status and response to the drugs, and the judgment of the treating physician.
• compositions containing the compounds of the invention are administered to a patient susceptible to or otherwise at risk of a particular disease.
• a patient susceptible to or otherwise at risk of a particular disease is defined to be a "prophylactically effective amount or dose.”
• prophylactically effective amount or dose the precise amounts again depend on the patient's state of health, weight, and the like.
• a maintenance dose is administered if necessary. Subsequently, the dosage or the frequency of administration, or both, can be reduced, as a function of the symptoms, to a level at which the improved condition is retained. When the symptoms have been alleviated to the desired level, treatment can cease. Patients can, however, require intermittent treatment on a long-term basis upon any recurrence of the disease symptoms.
• a suitable effective dose of the tryptase inhibitor will be in the range of 0.1 to 1000 milligram (mg) per recipient per day, preferably in the range of 1 to 100 mg per day.
• the desired dosage is preferably presented in one, two, three, four or more subdoses administered at appropriate intervals throughout the day. These subdoses can be administered as unit dosage forms, for example, containing 5 to 1000 mg, preferably 10 to 100 mg of active ingredient per unit dosage form.
• composition used in these therapies can be in a variety of forms. These include, for example, solid, semi- solid and liquid dosage forms, such as tablets, pills, powders, liquid solutions or suspensions, liposomes, injectable and infusible solutions.
• solid, semi- solid and liquid dosage forms such as tablets, pills, powders, liquid solutions or suspensions, liposomes, injectable and infusible solutions.
• the preferred form depends on the intended mode of administration and therapeutic application.
• the formulations of the present invention comprise at least one compound or inhibitor of this invention in a therapeutically or pharmaceutically effective dose together with one or more pharmaceutically or therapeutically acceptable carriers and optionally other therapeutic ingredients.
• a pharmaceutical formulation comprise at least one compound or inhibitor of this invention in a therapeutically or pharmaceutically effective dose together with one or more pharmaceutically or therapeutically acceptable carriers and optionally other therapeutic ingredients.
• Various considerations are described, e.g., in Gilman et al. (eds) (1990) Goodman and Gilman's: The Pharmacological Bases of Therapeutics, 8th Ed., Pergamon Press; and Remington's supra . Methods for administration are discussed therein, e.g., for oral, intravenous, intraperitoneal, or intramuscular administration, and others.
• Pharmaceutically acceptable carriers will include water, saline, buffers, and other compounds described, e.g., in the Merck Index, Merck & Co. , Rahway, NJ.
• aerosol includes any gas-borne suspended phase of the compounds of the instant invention which is capable of being inhaled into the bronchioles or nasal passages.
• aerosol includes a gas-borne suspension of droplets of the compounds of the instant invention, as may be produced in a etered dose inhaler or nebulizer, or in a mist sprayer.
• Aerosol also includes a dry powder composition of a compound of the instant invention suspended in air or other carrier gas, which may be delivered by insufflation from an inhaler device, for example.
• the preferred range of concentration of the compounds of the instant invention is 0.1-100 milligrams (mg)/ milliliter (mL) , more preferably 0.1-30 mg/mL, and most preferably, 1-10 mg/mL.
• a physiologically compatible buffer such as phosphate or bicarbonate.
• the usual pH range is 5 to 9, preferably 6.5 to 7.8, and more preferably 7.0 to 7.6.
• sodium chloride is added to adjust the osmolarity to the physiological range, preferably within 10% of isotonic.
• such methods comprise pressurizing or providing a means of pressurizing a container of the solution, usually with an inert carrier gas, and passing the pressurized gas through a small orifice, thereby pulling droplets of the solution into the mouth and trachea of the animal to which the drug is to be administered.
• a mouthpiece is fitted to the outlet of the orifice to facilitate delivery into the mouth and trachea.
• devices of the present invention comprise solutions of the compounds of the instant invention connected to or contained within any of the conventional means for creating aerosols in asthma medication, such as metered dose inhalers, jet nebulizers, or ultrasonic nebulizers.
• a device may include a mouthpiece fitted around the orifice.
• a device may comprise a solution of a compound of the instant invention in a nasal sprayer.
• a dry powder comprising a compound of the instant invention, optionally with an excipient, is another embodiment of the present invention. This may be administered by a drug powder inhaler containing the above described powder.
• /3-Adrenergic agonists are especially useful in these combinations, because they provide symptomatic relief of the initial asthmatic response, whereas the compounds of the present invention provide relief for the late asthmatic response.
• Preferred /3-adrenergic agonists in these solutions include any of the usual jS-agonists employed for the relief of asthma, such as albuterol, terbutaline, formoterol, fanoterol, or prenaline.
• agents useful in combination with the compounds of the instant invention include anticholinergics, such as ipratropium bromide, and antiinflammatory corticosteroids
• (adrenocortical steroids) such as beclomethasone, triamcinolone, flurisolide, or dexamethasone.
• the compounds of the inventions can also be used in the treatment of immunomediated inflammatory skin conditions, such as urticaria and angioedema, eczematous dermatitis, and hyperproliferative skin disease, e.g., psoriasis, in mammals.
• immunomediated inflammatory skin conditions such as urticaria and angioedema, eczematous dermatitis, and hyperproliferative skin disease, e.g., psoriasis, in mammals.
• a remission of the symptoms can be expected.
• one affected by an immunomediated inflammatory skin condition can expect a decrease in scaling, erythema, size of the plaques, pruritus, and other symptoms associated with the skin condition.
• the dosage of medicament and the length of time required for successfully treating each individual patient may vary, but those skilled in the art will be able to recognize these variations and adjust the course of therapy accordingly.
• preparations for topical application to the skin comprising a compound of Formula I, typically in concentrations in the range of from about 0.001% to 10%, together with a non-toxic, pharmaceutically acceptable topical carrier.
• Topical preparations can be prepared by combining an active ingredient according to this invention with conventional pharmaceutical diluents and carriers commonly used in topical dry, liquid, cream and aerosol formulations.
• Ointment and creams may, for example, be formulated with an aqueous or oily base with the addition of suitable thickening and/or gelling agents.
• bases may include water and/or an oil such as liquid paraffin or a vegetable oil such as peanut oil or castor oil.
• Thickening agents which may be used according to the nature of the base include soft paraffin, aluminum stearate, cetostearyl alcohol, propylene glycol, polyethylene glycols, woolfat, hydrogenated lanolin, beeswax, and the like.
• Lotions may be formulated with an aqueous or oily base and will, in general, also include one or more of the following: stabilizing agents, emulsifying agents, dispersing agents, suspending agents, thickening agents, coloring agents, perfumes, and the like.
• Powders may be formed with the aid of any suitable powder base, e.g., talc, lactose, starch, and the like.
• Drops may be formulated with an aqueous base or non-aqueous base also comprising one or more dispersing agents, suspending agents, solubilizing agents, and the like.
• the topical pharmaceutical compositions according to this invention may also include one or more preservatives or bacteriostatic agents, e.g., methyl hydroxybenzoate, propyl hydroxybenzoate, chlorocresol, benzalkonium chlorides, and the like.
• the topical pharmaceutical compositions also can contain other active ingredients such as antimicrobial agents, particularly antibiotics, anesthetics, analgesics, and antipruritic agents.
• the compounds of this invention are also useful in the treatment of peptic ulcers.
• chemotherapeutic activity which enables them to relieve the symptoms of peptic ulcer disease and stress ulceration, and to promote the healing of gastric and/or duodenal ulcers.
• the compounds can be used in conjunction with other therapeutic agents, such as anti-inflammatory and/or analgesic agents, such as aspirin, indomethacin, phenylbutazone, ibuprofen, naproxen, tolemtim, and the like.
• the pharmaceutical compositions can be administered by parenteral or oral administration for prophylactic and/or therapeutic treatment.
• the pharmaceutical compositions can be administered in a variety of unit dosage forms depending upon the method of administration.
• unit dosage forms suitable for oral administration include powder, tablets, pills, capsules and dragees.
• compositions for intravenous administration which comprise a solution of the compound dissolved or suspended in an acceptable carrier, preferably an aqueous carrier.
• an acceptable carrier preferably an aqueous carrier.
• aqueous carriers can be used, e.g., water, buffered water, 0.4% saline, and the like.
• These compositions will sometimes be sterilized by conventional, well known sterilization techniques, or can be sterile filtered.
• the resulting aqueous solutions can be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile aqueous solution prior to administration.
• compositions can contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, and the like.
• auxiliary substances such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, and the like.
• conventional nontoxic solid carriers can be used which include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like.
• a pharmaceutically acceptable nontoxic composition is formed by incorporating any of the normally employed excipients, such as those carriers previously listed, and generally 0.1-95% of active ingredient, preferably about 20%.
• active ingredient preferably about 20%.
• Isolation and purification of the compounds and intermediates described herein can be effected, if desired, by any suitable separation or purification procedure such as, for example, filtration, extraction, crystallization, column chromatography, thin-layer chromatography or thick-layer chromatography, high-pressure liquid chromatography, or a combination of these procedures.
• suitable separation and isolation procedures can be had by reference to the examples hereinbelow. However, other equivalent separation or isolation procedures can, of course, also be used.
• the Sakaguchi assay was for the detection of arginine and peptides containing arginine. See Stewart and Young "Solid Phase Peptide Synthesis", 2d Ed., Pierce Chemical Company, p. 114.
• Solution I containing 0.01% ⁇ -naphthol and 5% urea in 95% ethanol was prepared.
• Solution II was prepared by dissolving 2 gram (g) bromine in 100 milliliters (mL) of 8% aqueous sodium hydroxide. To Solution I was added 5 sodium hydroxide pellets. The sample to be analyzed was spotted on a thin layer chromatography (TLC) plate. The TLC plate was then sprayed with Solution I. The TLC plate was dried in air and then sprayed with solution II. A red spot indicated the existence of an aminoimino methane group.
• TLC thin layer chromatography
• Fmoc-L-proline (Milligen; 6.06 mmol), benzotriazole- 1-yl-oxy-tris-(dimethylamino)-phosphonium hexafluorophosphate (BOP, Novabiochem; 6.06 mmol), and 1-hydroxybenzotriazole (HOBT; Aldrich; 6.06 mmol) were dissolved in -30 L of DMF. The resulting clear solution was added to the Rink resin and the slurry was agitated (with nitrogen bubbling) 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.
• Analytical HPLC (Polymer Labs 100A PLRP column, 1.0 x 150 millimeters (mm) , eluting with a linear gradient of 20 to 45% acetonitrile over 13 minutes at a flow of 0.1 mL/min) showed that the crude product was essentially a single compound (retention time 10.35 minutes, >98% by UV absorbance) .
• Purification was accomplished via HPLC using a C 18 silica gel column (Vydac; 22 x 250 mm, 15-20 ⁇ m, 300 A, eluting with a linear gradient of 20 to 35% over 30 minutes at a flow of 10 mL/minute) . Multiple injections of 30-35 mg were required.
• Compound 13 of Tables I and II was prepared by first coupling Fmoc-L-phenylalanine to the resin in an analogous procedure to that used to couple Fmoc-L-proline in part A above. The Fmoc group was then removed and the phenylalanine was coupled to Fmoc-L-proline. The remainder of the synthesis of Compound 13 was performed using the techniques described in Part A above.
• reaction mixture was cooled to -78°C and treated with excess anhydrous C0 2 for several minutes.
• the reaction mixture was stirred and warmed to ambient temperature.
• the reaction mixture was partitioned between ethyl ether and aqueous IN NaOH.
• the aqueous layer was cooled to 0°C with ice and acidified to pH 2 with aqueous IN HC1.
• the aqueous solution was partitioned with methylene chloride.
• the organic layer was dried over magnesium sulfate. Concentration in vacuo yielded crude product as an off-white solid (258 mg, 2-hydroxy-5-phenylbenzoic acid) .
• NMR spectroscopy of the crude material was consistent with the expected structure.
• t-Butyoxylcarbonyl (BOC)-protected amino acids can be prepared using standard techniques. See, e .g. , Greene and Wuts Protective Groups in Organic Synthesis. 2nd Ed. , John Wiley & Sons, Inc.: New York, pp. 327-328 (1991). To a -20°C solution of BOC-protected p- nitrophenylalanine (3.00 g, 9.67 mmol) in anhydrous methylene chloride (5 mL) was added N-methylmorpholine (NMM, 1.07 mL, 9.67 mmol), followed by isobutyl chloroformate (1.26 mL, 9.67 mmol) .
• NMM N-methylmorpholine
• the BOC group was removed by treatment with HCl in dioxane using standard conditions. See, e.g., Greene and Wuts, id at 328-329.
• HCl HCl in dioxane
• 1-hydroxybenzotriazole 159 mg, 1.17 mmol.
• the solution was cooled to 0°C and l-(3- dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EPCI, 161 mg, 0.84 mmol) was added.
• the ethyl acetate solution was washed with 5% aqueous citric acid, saturated aqueous sodium bicarbonate, and saturated aqueous sodium chloride and dried over magnesium sulfate.
• the solution was concentrated in vacuo and the residue was diluted in ethanol (40 mL, containing 10% methanol and 10% acetic acid) and hydrogenated at 52 pounds per square inch (psi) over 10% palladium on carbon (10 mg) until the reaction was complete (TLC on silica gel, eluting with chloroform:methanol 9:1) as indicated by disappearance of the faster moving spot.
• the reaction mixture was filtered through celite, washed with ethanol and methanol.
• a binary gradient system consisting of acetonitrile and water was used in all cases. All eluents contain 0.1% trifluoroacetic acid. A: 20-45% acetonitrile over 13 minutes. B: 10-35% acetonitrile over 13 minutes. C: 5-95% acetonitrile over 45 minutes. P: 2-95% acetonitrile over 10 minutes. E: 25-45% acetonitrile over 13 minutes.
• Tryptase (25 ⁇ l; 0.5 nM final concentration) was added to each well and the samples were mixed and incubated for 1 hr at room temperature under either ambient light (i.e., the light available in the room during the experiment) or under controlled "light” or “dark” conditions.
• ambient light i.e., the light available in the room during the experiment
• light conditions refers herein to a light intensity of 400-450 footcandles from a fluorescent bulb.
• Park refers herein to sample containers wrapped in aluminum foil.
• the enzyme reaction was then initiated with the addition of the synthetic tripeptide substrate, tosyl-GlyProLys-p-nitroanilide (25 ⁇ l; 0.5 mM final concentration) .
• microtiter plates were immediately transferred to a UV/MAX Kinetic Microplate Reader (Molecular Pevices) and hydrolysis of the chromogenic substrate was followed spectrophotometrically at 405 nM for 5 minutes.
• the enzyme assays routinely yielded linear progress curves under these conditions.
• Initial velocity measurements, calculated from progress curves by a kinetic analysis program called "BatchKi" (available commercially from Biokin Ltd. , of Madison WI) , were used to determine apparent inhibition constants for each inhibitor. This program is designed to perform regressions and curve-fitting of non-linear data.
• a compound was termed "active" or effective as a tryptase inhibitor when its ⁇ i ' was less than 1000 ⁇ M.
• K i ' may not be a true dissociation constant of the enzyme-inhibitor complex; K i » is equal to K A for a noncompetitive inhibitor and is directly proportional to K for competitive and uncompetitive inhibitors.
• Exposure of solutions of the test compounds in the assay medium to light may decrease the K ⁇ '.
• the assay conditions are light sensitive and variations in ambient light intensity may affect assay reproducibility. As a result of this potential source of error, some assays were run under dark conditions.
• the ⁇ 's reported in Tables II and III without parentheses were determined under conditions of ambient light.
• K i 's reported with parentheses were determined under "dark" conditions as defined above.
• K j ⁇ 's reported with an asterisk (*) were measured under "light” conditions as defined above (i.e., at a light intensity of 400-450 footcandles).
• Table III lists the inhibition constants (K ⁇ * , micromolar ( ⁇ M) ) which were determined for several of the compounds of the present invention. According to the present invention, a compound was termed "active" or effective as a tryptase inhibitor when its K j _' was less than 1000 ⁇ M. Table III
• Compound 3 was assayed against tryptase at four different pH's in the following buffer system: 120 mM NaCl, 2.7 mM KC1, 0.13 mM NaH 2 P0 4 , 0.896 mM Na 2 HP0 4 .
• the final buffer system used included 0.05% of Tween-20.
• Table IV shows K ⁇ ' ( ⁇ M) for Compound 3 at different pH's.
• Airway hyperresponsiveness is expressed as PC400, i.e., the concentration of carbachol that causes a 400% increase in SRL. Thus a decrease in the PC400 indicates that the airways have become hyperresponsive.
• Compound 3 of Tables I and II blocked the 24 hour hyperresponsiveness. See Figure 2. Baseline PC400 was 22.0 ⁇ 3.7 breath units in the control trial, falling to 9.1 ⁇ 1.3 breath units after antigen challenge (p ⁇ 0.05 vs. baseline). In contrast, in the drug trial, PC400 was unchanged relative to baseline (16.0 ⁇ 3.8 breath units vs. 17.6 ⁇ 3.5 breath units after antigen challenge) . Thus treatment with Compound 3 of Tables I and II resulted in a statistically significant improvement in airway function in allergen challenged sheep.

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