WO2002034251A1 - Treatment of asthma with lfm analogues - Google Patents

Abstract

A method for treatment or prevention of asthma is provided. The method comprises treating a mammal by administering a therapeutically effective amount of a LFM analogue, or a pharmaceutically acceptable salt thereof.

Description

TREATMENT OF ASTHMA WITH LFM ANALOGUES

Field of the Invention

This invention relates to methods for the treatment of asthma in mammals with LFM analogues, particularly, with the LFM analogue A8 (LFM-A8 .

Background of the Invention

Asthma, which affects the respiratory tract, is characterized by bronchoconstriction and hyperresponsiveness of the airway passages and is brought about by various stimulating agents. Physiological effects of asthma include smooth muscle contraction of the airway, increased bronchial mucus secretion, and inflammation. These effects lead to symptoms commonly associated with asthma. Over 10 million people in the United States have asthma and the medically-related costs associated with asthma are estimated at over $5 billion annually.

The immune system, cued by environmental allergens, is likely to play a role in generating asthmatic conditions. Such conditions involve the transient enhancement of airway hyperresponsiveness triggered by inhaled allergens. The hyperresponsiveness is associated with airway inflammation brought about by smooth muscle contractions in the bronchioles.

Inhaled allergens can initiate the inflammatory sequence in an allergic response. Leukocytes displaying IgE receptors, particularly mast cells and basophils, are present in the epithelium and bronchiolar smooth muscle. These cells are activated by binding specific inhaled antigens to the IgE receptors. Activated mast cells release a number of preformed or primary chemical mediators of the inflammatory response, such as leukotrienes. The airway (bronchiolar) constriction that occurs soon after allergen exposure is likely to be a result of this chemical release from mast cells. Later in the asthmatic reaction, a substantial increase in the number of inflammatory cells which infiltrate bronchiolar smooth muscle and epithelial tissues is observed. Lymphocytes, neutrophils and eosinophils are attracted to the bronchioles by chemicals released from activated mast cells.

Historically, bronchodilator drugs have been used to relieve the bronchoconstriction associated with asthma. However, more recently, anti- inflammatory drugs, which target many of the cellular types involved in asthmatic reactions, as listed above, have begun to replace bronchodilators as first-line treatments for asthma.

Summary of the Invention

The invention provides a method to alter leukotriene production from mast cells, comprising contacting mast cells with an effective leukotriene-altering amount of a LFM analogue.

The invention provides a method for treating or preventing asthma in a mammal comprising administering to a mammal a therapeutically effective amount of a LFM analogue. The invention also provides a method for altering the number of eosinophils in the bronchioles and for reducing bronchial hyperresponsiveness comprising administering to a mammal a therapeutically effective amount of a LFM analogue. The LFM analogues are compounds of formula I:

where:

R, is (C,-Cj)alkyl, (C C₆)cycloalkyl, phenyl, or NR_aR_b;

R₂ is hydroxy, (C,-C₀)alkoxy (C,-C₆)alkanoyloxy amino (C,- C₅)alkoxy; hydroxy (C₂-C₅)alkoxy amino (C,-C₃)alkanoxy; or hydroxy (C,-C₅) alkanoxy;

R₃ is cyano or (C,~C₃-alkanoyl;

R₄ is hydrogen, (C,-C₃)alkyl; hydroxy (C\-C₅)alkyl; or amino (CV C₅)alkyl; R_s is aryl, or heteroaryl;

R_a and R_b are each independently hydrogen, or (C,-C₃)alkyl; or R. and R_b together with the nitrogen to which they are attached are pyrrolidino, piperidino, moi holino, or thiomorpholino; wherein any aryl, or heteroaryl of R, and R₅ is optionally substituted with one or more (e.g. 1, 2, or 3) substituents independently selected from halo, nitro, cyano, hydroxy, trifluoromethyl, trifluoromethoxy, (C,-C₃)alkoxy, (C,- C₃)alkyl, (C C₃)alkanoyl, -S(O)₂R or NR R_b, wherein R._. is (C,-C₃)alkyl, or aryl or a pharmaceutically acceptable salt thereof; provided that if R₅ is phenyl, the phenyl is substituted by -S(O)_;R_L, or is substituted by halo and at least one other substituent.

Brief Description of the Figures

Figure 1. Effect of LFM and LFM-A8 on IgE receptor/Fc epsilon Rl-mediated human mast cell leukotriene C₄ release. IgE sensitized fetal liver derived human mast cells were stimulated by challenging with anti-IgE. To study the effect of the test compounds, mast cells were incubated with vehicle or 100 μM LFM or LFM-A8 prior to challenge with anti-IgE. Leukotriene C₄ levels were measured in cell-free supernatants by ELISA. Vehicle treated 10⁶ mast cells released 12.5 ± 2.2 ng leukotriene C₄. The results are expressed as percent of vehicle-treated controls (N=4). The data points represent the mean ± SEM values. *P 0.05 compared to vehicle-treated control, as determined by Student's t test.

Figure 2. Inhibition of airway hyperresponsiveness by LFM-A8 in mice. Mice were sensitized with ovalbumin (OVA) on days 0 and 14 mtraperitoneally. On days 20, 21 and 23, mice were challenged for 15 min with 2% OVA via airways using a untrasonic nebulizer. After 24 h of the last OVA challenge airway responsiveness to increasing concentrations of methacholine was measured. To study the effect of LFM-A8 on airway hyperresponsiveness, mice were injected mtraperitoneally with indicated concentrations of LFM-A8 or vehicle on day 20, 21 , and 23 lh prior and

2h post OVA challenge and airway responsiveness to increasing concentrations of methacholine was measured. The data points represent the mean ± SEM values

(N=5- 6 mice).

Figure 3: Inhibition of eosinophil infiltration in bronchoalveolar lavage of

OVA challenged mice by LFM-A8. The lungs of the mice were lavaged after methacholine challenge and eosinophil numbers were counted. The results of eosinophils are expressed as percent of total cell counts. The data points represent the mean ± SEM values obtained from 5-6^" mice. *P<0.05 compared to vehicle- treated control, as determined by Student's t test.

Detailed Description of the Invention

Compounds of the Invention:

Compounds of the invention include compounds of formula I:

where:

R, is (C,-C₃)alkyl, (C₃-C₆)cycloalkyl, phenyl, or NR_aR_b; R₂ is hydroxy, (C,-C₆)alkoxy (C,-C,,)alkanoyloxy amino (C₂- C₅)alkoxy; hydroxy (C,-C₅)alkoxy amino (C₂-C₅)alkanoxy; or hydroxy (C\-C₅) alkanoxy;

R₃ is cyano or (C,-C )alkanoyl;

R₄ is hydrogen, (C,-C₃)alkyl; hydroxy (C₂-C₅)alkyl; or amino (C₂- C₅)alkyl; R₅ is aryl, or heteroaryl;

R_a and R_b are each independently hydrogen, or (C,-C₃)alkyl; or R_a and R_b together with the nitrogen to which they are attached are pyrrolidino, piperidino, moipholino, or thiomorpholino;

wherein any aryl, or heteroaryl of R, and R₅ is optionally substituted with one or more (e.g. 1 , 2, or 3) substituents independently selected from halo, nitro, cyano, hydroxy, trifluoromethyl, trifluoromethoxy, (C,-C₃)alkoxy, (C,- C₃)alkyl, (C,-C₃)alkanoyl, -S(O)₂R,, or NR_aR_b; wherein R, is (C,-C₃)alkyl, or aryl or a pharmaceutically acceptable salt thereof; provided that if R₅ is phenyl, the phenyl is substituted by -S(O)₂R_t, or is substituted by halo and at least one other substituent.

Definitions: The following definitions are used herein, unless otherwise described: halo is fluoro, chloro, bromo, or iodo. Alkyl, alkoxy, alkenyl, alkynyl, etc. denote both straight and branched groups; but reference to an individual isomer such as "propyl" embraces only the straight chain isomer, a branched chain isomer such as "isopropyl" being specifically referred to. Aryl denotes a phenyl group or a bicyclic or tri-cyclic carbocyclic group having about nine to twelve ring atoms in which at least one ring is aromatic. Heteroaryl encompasses a group attached via a ring carbon of a monocyclic aromatic ring containing five or six ring atoms consisting of carbon and one to four heteroatoms each selected from the group consisting of non- peroxide oxygen, sulfur, and N(X) wherein X is absent or is H, O, (C,-C₄)alkyl, phenyl or benzyl, as well as a group of an ortho-fused bicyclic heterocycle of about eight to ten ring atoms derived therefrom, particularly a benz-derivative or one derived by fusing a propylene, trimethylene, or tetramethylene group thereto.

It will be appreciated by those skilled in the art that compounds of the invention having a chiral center may exist in and be isolated in optically active and racemic forms. Some compounds may exhibit polymorphism. It is to be understood that the present invention encompasses any racemic, optically-active, polymorphic, or stereoisomeric form, or mixtures thereof, of a compound of the invention, which possess the useful properties described herein, it being well known in the art how to prepare optically active forms (for example, by resolution of the racemic form by recrystallization techniques, by synthesis from optically-active starting materials, by chiral synthesis, or by chromatographic separation using a chiral stationary phase). Specific and preferred values listed below for substituents and ranges, are for illustration only; they do not exclude other defined values or other values within defined ranges for the radicals and substituents

Specifically, (C,-C₃)alkyl can be methyl, ethyl, propyl, or isopropyl; (C,- C₄)alkyl can be methyl, ethyl, propyl, isopropyl, butyl, iso-butyl, or sec-butyl; (C₃- C₆)cycloalkyl can be cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl; (C₃- C₀)cycloalkyl(C,-C₆)alkyl can be cyclopropylmethyl, cyclobutylmethyl, cyclopentylm ethyl, cyclohexylmethyl, 2-cyclopropylethyl, 2-cyclobutylethyl, 2- cyclopentylethyl, or 2-cyclohexylethyl; (C C )alkoxy can be methoxy, ethoxy, propoxy, isopropoxy; (C,-C₀)alkoxy can be methoxy, ethoxy, propoxy, isopropoxy, butoxy, iso-butoxy, sec-butoxy, pentoxy, 3-pentoxy, or hexyloxy; (C₂- C₆)alkanoyloxy can be acetoxy, propanoyloxy, butanoyloxy, isobutanoyloxy, pentanoyloxy, or hexanoyloxy; (C₂-C₄)alkenyl can be vinyl, allyl, 1-propenyl, 2- propenyl, 1-butenyl, 2-butenyl, or 3-butenyl; (C₂-C₄)alkynyl can be ethynyl, 1- propynyl, 2-propynyl, 1-butynyl, 2-butynyl, or 3-butynyl; hydroxy(C₎-C₄)alkyl can be hydroxymethyl, 1-hydroxyethyl, 2-hydroxyethyl, 1-hydroxypropyl, 2- hydroxypropyl, 3-hydroxypropyl, 1-hydroxybutyl, or 4-hydroxybutyl; hydroxy(C₂- C₄)alkenyl can be 3-hydroxy-l-propenyl, 4-hydroxy-l-butenyl, or 4-hydroxy-2- butenyl; hydroxy(C₂-C₄)alkynyl can be 3-hydroxy-l-propynyl, l-hydroxy-2- propynyl, 3-hydroxy-l-butynyl, 4-hydroxy-l-butynyl, l-hydroxy-2-butynyl, 4- hydroxy-2-butynyl, l-hydroxy-3-butynyl, or 2-hydroxy-3-butynyl; (C,-C₄)alkylthio can be methylthio, ethylthio, propylthio, isopropylthio, butylthio, or isobutylthio; aryl can be phenyl, indenyl, or naphthyl; and heteroaryl can be furyl, imidazolyl, triazolyl, triazinyl, oxazoyl, isoxazoyl, thiazolyl, isothiazoyl, pyrazolyl, pyrrolyl, pyrazinyl, tetrazolyl, pyridyl, (or its N-oxide), thienyl, pyrimidinyl (or its N-oxide), indolyl, isoquinolyl (or its N-oxide) or quinolyl (or its N-oxide). Specific and preferred values

A specific value for R, is (C,-C₃)alkyl, or (C₃-C₀)cycloalkyl.

A specific value for R, is hydroxy.

A specific value for R₃ is cyano. A specific value for R₄ is hydrogen.

A specific value for R₅ is phenyl substituted with halo, and substituted with 1, 2, or 3 other substituents independently selected from halo, nitro, cyano, trifjuoromefhyl, trifluoromethoxy, (C,-C₃)alkoxy, (C,-C₃)alkyl, (C,-C )alkanoyl and NR_aR_b A specific value for R₆ is methyl, trifluoromethyl, methoxymethyl, ethyl, isopropyl, tert-butyl, or propyl.

A specific value for R₇ is hydrogen, methyl, or ethyl.

A specific value for R,, is acetyl, trifluoroacetyl, propanoyl, crclopropylcarbonyl, vinylcarbonyl, 2-propenoyl, methoxycarbonyl, methylthiocarbonyl, ethoxycarbonyl, or ethylthiocarbonyl.

A more specific value for R, is (C,-C₃)alkyl.

A more specific value for R_s is phenyl substituted with halo, and substituted with 1, 2, or 3 other substituents independently selected from halo, trifluoromethyl, trifluoromethoxy, and (C,-C₃)alkoxy. A more specific value for R₅ is phenyl substituted with 2 or 3 halo.

A more specific value for R₅ is phenyl substituted with two bromo.

A preferred compound of formula I is α-Cyano-β-hydroxy-β-methyl-N-[3- (trifluoromethyl)phenylj-propenamide (LFM-A8); or a pharmaceutically acceptable salt thereof.

Therapeutic Use

LFM appears to exert effects on cells involved in immune system regulation, such as mast cells. Mast cells are known as the primary effector cells involved in the pathogenesis of allergy and early phase of asthma by virtue of their intrinsic capacity to release large amounts of inflammatory mediators following IgE/FcεRI-receptor ligation (Socha, B.,(1977) Pol. Tyg. Lek. 32, 1295-1297). Mast cells release preformed granule-associated proinflammatory mediators [e.g. histamine], and newly synthesized arachidonic acid metabolites (e.g. leukotrienes) upon crosslinking of their IgE/FcεRI receptor (Galli, S.J., (1993) N Engl. J Med. 328, 257-265; Metcalfe, D.D. et al, (1997) Physiol. Rev. 77, 1033-1079).

Leukotrienes (LTC₄, D₄, E₄, and B₄) are produced in a multistep process triggered by activation of the 5-lipoxygenase pathway (Malaviya, R., et al., (2000) J Pharmacol. Exp. Ther., in press; Malaviya, R., et al., (1993) J Biol Chem, 268, 4939-4944; Peters-Golden, M. and McΝish, R.W., (1993) Biochem Biophys Res Commun, 196, 147-153; Peters-Golden, M., et al, (1996) Biochem J, 318, 797- 803.) Because of their ability to produce smooth muscle contraction, stimulate mucous production, enhance vascular permeability, and recruit neutrophils and eosinophils, leukotrienes play an important role in the pathophysiology of asthma (Arm, IP. and Lee T.H., (1992) Adv Immunol, 51, 323-382; Smith, L.J., (1996) Arch Intern Med,156, 2181-2189.)

Agents that block the release of leukotrienes may be useful for treatment of allergy and asthma. In recent years a number of chemical compounds that inhibit the synthesis of leukotrienes (leukotriene synthesis inhbitors) or the action of leukotrienes on target organ (leukotriene receptor antagonists) have been explored as an effective method for the treatment of allergy and asthma (Reiss, T.F. (1997) Thorax, 52, 45-48; Rouzer, CA. et al., (1990) J Biol Chem, 265, 1436-1442). Leflunomide, N-(4-trifluoromethylphenyl)-5-methylisoxazole-4-carboxamide), is a novel immunomodulatory and anti-inflammatory drug that is shown to be effective in prevention and treatment of several immunologic disorders including rheumatoid arthritis (Bruyn, G.A., et al., (1999) Lancet, 353, 1883-1884; Dimitrijevic, M., et al., (1996) Inflamm Res, 45, 550-556; Fox, R.I., (1998) J Rheumatol Suppl, 53, 20-26), systemic lupus erythematosis (Strand, V., (1997) Curr Opin Rheumatol, 9, 410-420), and reactions of organ graft rejection by modulating T and B cell responses of the host (Salomon, S., et al, (1996) Transplant Proc, 28, 698-699; Bartlett, R.R., et al., (1991) Agents Actions, 32, 10- 21). After systemic administration, leflunomide is rapidly converted into an active metabolite α-Cyano-/i-hydroxy- ?-methyl-N-[4-

(trifluoromethyl)phenyljpropenamide (LFM) (Bruneau, J.M., et al., (1998) Biochem J, 336, 299-303). LFM administered to rats has been shown to significantly reduce IgE and IgG levels and therefore prevent allergic sensitization (Eber, E., et al. (1998) Clin Exp Allergy, 28, 376-84). In addition, LFM has also been shown to inhibit histamine release from isolated human basophils, and rat peritoneal mast cell (Bartlett, R.R., et al., (1991) Agents Actions, 32, 10-21). As disclosed herein, 13 different analogues of LFM were synthesized and examined for their in vitro effect on IgE/FcεRI receptor mediated mast cell leukotriene release. Experimental data described below indicates that the novel LFM analogue, «-Cyano- i-hydroxy- ?-methyl-N-[3-

(trifluoromethyl)phenyl]propenamide (LFM-A8) is a more potent inhibitor of IgE/FcεRI receptor mediated leukotriene C release from RBL-2H3 rat mast cells as well as fetal liver-derived human mast cells than LFM. LFM-A8 effectively inhibited airway hyper-responsiveness and eosinophil infiltration in a mouse model of allergic asthma.

Leukotrienes play a central role in the pathogenesis of allergic airway inflammation. Because of the strategic location of mast cells in the linings of the airways (Malaviya, R. and Abraham, S.Ν., (1998) J Mol Med, 76, 617-623) and their reported ability to release large amounts of leukotrienes in response to antigen challenge (Malaviya, R. and Jakschik, B.A., (1993) J Biol Chem, 268, 4939-4944), it is imperative to control mast cell leukotrienes release in order to limit the peφetuation of the inflammatory reaction in the airways. A number of leukotrienes synthesis inhibitors have been developed and are available for treating asthma (Reiss, T.F. (1997) Thorax, 52, 45-48, Drazen, J.M., et al, (1999) N Engl J Med, 340, 197-206). We examined the effects of the active leflunomide metabolite (LFM) and 13 analogues of LFM on IgE/FcεRI -receptor mediated leukotriene release by mast cells in vitro. The data presented herein show that the lead compound LFM-A8 is a potent inhibitor of IgE/FcεRI receptor mediated mast cell leukotriene C₄ release.

A number of studies have previously shown that leukotriene synthesis inhibitors such as MK886 and Zilutron attenuate allergen induced bronchial hyperresponsiveness in human (Smith, L.J., (1996) Arch Intern Med,\ 5b, 2181- 2189; Henderson, W.R., et al., {1996) J Exp Med, 184, 1483-1494; Nagase, T., et al., (1997) Am J Respir Crit Care Med, 156, 1621-1627). Because LFM-A8 inhibited IgE/FcεRI receptor-mediated leukotriene release, it was reasoned that pretreatment of mice with LFM-A8 would prevent allergen-induced hyperresponsiveness in vivo. To study in vivo airway responsiveness in conscious and spontaneously breathing mice, a well characterized mouse model of allergic asthma was utilized. The results described herein show that airway hyperresponsiveness to methacholine in ovalbumin-sensitized and challenged mice was significantly attenuated by LFM-A8.

Eosinophil infiltration of airways is a cardinal feature of allergic asthma.

Leukotrienes are chemotactic molecules for leukocytes (Zhang, Y., et al., (1992)

Science, 258, 1957-1959). Leukotriene B₄ is one of the most potent chemoattractant for leukocytes Zhang, Y., et al., (1992) Science, 258, 1957-1959; Spada, C.S., et al., (1997) Adv Exp Med Biol, 400B, 699-706). Mast cells are situated in the nasal and the airway mucosa and readily activated upon allergen challenge. Therefore, mast cell-derived leukotrienes might be critical for the initiation of the inflammatory response in asthma. The results shown herein provide unprecedented experimental evidence that LFM-A8 is a potent inhibitor of eosinophil infiltration of airways. As disclosed herein, it has been discovered that an analogue of active metabolite of leflunomide, LFM-A8 is a potent inhibitor IgE/FcεRI receptor-mediated mast cell leukotriene C₄ release. In addition, LFM-A8 showed biologic activity in a mouse model of allergic asthma. Treatment of OVA-sensitized mice with increasing doses of LFM-A8 prevented the development of airway hyper-responsiveness in a dose dependent fashion. Furthermore, LFM-A8 also resulted in decreased eosinophil recruitment to the airway lumen after the OVA challenge in a dose-dependent fashion. This demonstrates that that LFM-A8 is useful as an agent that affects leukotriene release in mast cells. LFM-A8 is therefore useful for the treatment of asthma and can be used to prevent the development of airway hyperresponsiveness and eosinophill recruitment to the airway lumem, conditions associated with asthma. LFM-A8 provides the basis for new and effective treatments as well as prevention programs for allergic asthma in clinical settings.

Conjugation to a Targeting Moiety The compounds of the invention can be targeted for specific delivery to a cell type to be treated by conjugation of the LFM analogue to a targeting moiety. Targeting moieties useful for conjugation to LFM analogues include antibodies, cytokines, and receptor ligands that are specific to the cell to be treated.

The term "conjugate" means a compound formed as a composite between two or more molecules. More specifically, in the present invention, the quinazoline derivative is bonded, for example, covalently bonded, to cell-specific targeting moieties forming a conjugate compound for efficient and specific delivery of the agent to a cell of interest.

The phrase "targeting moiety" means a molecule that serves to deliver the compound of the invention to a specific site for the desired activity. Targeting moieties include, for example, molecules that specifically bind molecules on a specific cell surface. Such targeting moieties useful in the invention include anti- cell surface antigen antibodies. Cytokines, including interleukins and factors such as granulocyte/macrophage stimulating factor (GMCSF) are also specific targeting moieties, known to bind to specific cells expressing high levels of their receptors. Particularly useful targeting moieties for targeting the LFM analogue compounds of the invention to cells for therapeutic activity include those ligands present on mast cells or other cells involved in generating asthmatic conditions. Mast cells can be targeted via the CD48 antigen with anti-CD48 antibodies. Cytokines are also useful targeting moieties. For example, mast cells can be targeted with C-KIT, MGF, GMCSF, and IL-3. These and other cell surface antigen antibodies are commercially available, for example, from Pharmingen; cytokines are commercially available, for example, from R&D Systems.

Compounds as Salts In cases where an agent ("compound") is sufficiently basic or acidic to form stable nontoxic acid or base salts, administration of the compound as a salt may be appropriate. Examples of pharmaceutically acceptable salts are organic acid addition salts formed with acids which form a physiologically acceptable anion, for example, tosylate, methanesulfonate, acetate, citrate, malonate, tartarate, succinate, benzoate, ascorbate, α-ketoglutarate, and α-glycerophosphate. Suitable inorganic salts may also be formed, including hydrochloride, sulfate, nitrate, bicarbonate, and carbonate salts. Pharmaceutically acceptable salts may be obtained using standard procedures well known in the art, for example by reacting a sufficiently basic compound such as an amine with a suitable acid affording a physiologically acceptable anion. Alkali metal (for example, sodium, potassium or lithium) or alkaline earth metal (for example calcium) salts of carboxylic acids can also be made.

Prodrug Derivatives

The compounds of the invention may have attached thereto functional groups to provide a prodrug derivative. The prodrug deriviative facilitates use of the drug in the body, for example, by facilitating entry into cells. The term "prodrug moiety" is a substitution group that facilitates use of a compound of the invention, for example by facilitating entry of the drug into cells or administration of the compound. The prodrug moiety may be cleaved from the compound, for example by cleavage enzymes in vivo. Examples of prodrug moieties include phosphate groups, peptide linkers, and sugars, which moieties can be hydrolized in vivo.

Pharmaceutical formulations

A compound can be formulated as pharmaceutical compositions and administered to a mammalian host, such as a human patient in a variety of forms adapted to the chosen route of administration, i.e., orally or parenterally, by intravenous, intramuscular, topical, subcutaneous routes, or by inhalation.

Thus, compounds may be systemically administered, e.g., orally, in combination with a pharmaceutically acceptable vehicle such as an inert diluent or an assimilable edible carrier. They may be enclosed in hard or soft shell gelatin capsules, may be compressed into tablets, or may be incoφorated directly with the food of the patient's diet. For oral therapeutic administration, the compound may be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 0.1% of active compound. The percentage of the compositions and preparations may be varied and may conveniently be between about 2 to about 60° o of the weight of a given unit dosage form. The amount of active compound in such therapeutically useful compositions is such that an effective dosage level will be obtained.

The tablets, troches, pills, capsules, and the like may also contain the following: binders such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, fructose, lactose or aspartame or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring may be added. When the unit dosage form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier, such as a vegetable oil or a polyethylene glycol. Various other materials may be present as coatings or to otherwise modify the physical form of the solid unit dosage form. For instance, tablets, pills, or capsules may be coated with gelatin, wax, shellac or sugar and the like. A syrup or elixir may contain the active compound, sucrose or fructose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor. Any material used in preparing any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts employed. In addition, the active compound may be incoφorated into sustained-release preparations and devices.

The compound may also be administered intravenously or intraperitoneally by infusion or injection. Solutions of the compound or its salt can be prepared in water, optionally mixed with a nontoxic surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, triacetin, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. The compound can also be administered by inhalation, for example, with an insufflator, in combination with a pharmaceutically acceptable vehicle.

The pharmaceutical dosage forms suitable for injection or infusion can include sterile aqueous solutions or dispersions or sterile powders comprising the active compound which are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes. In all cases, the ultimate dosage form should be sterile, fluid and stable under the conditions of manufacture and storage. The liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions or by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, buffers or sodium chloride. Prolonged absoφtion of the injectable compositions can be brought about by the use in the compositions of agents delaying absoφtion, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incoφorating the active compound in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filter sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze drying techniques, which yield a powder of the active compound plus any additional desired ingredient present in the previously sterile-filtered solutions. For topical administration, the compounds may be applied in pure form, i.e., when they are liquids. However, it will generally be desirable to administer them to the skin as compositions or formulations, in combination with a dermatologically acceptable carrier, which may be a solid or a liquid.

Useful solid carriers include finely divided solids such as talc, clay, microcrystalline cellulose, silica, alumina and the like. Useful liquid carriers include water, alcohols or glycols or water-alcohol/glycol blends, in which the present compounds can be dissolved or dispersed at effective levels, optionally with the aid of non-toxic surfactants. Adjuvants such as fragrances and additional antimicrobial agents can be added to optimize the properties for a given use. The resultant liquid compositions can be applied from absorbent pads, used to impregnate bandages and other dressings, or sprayed onto the affected area using pump-type or aerosol sprayers. Thickeners such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified celluloses or modified mineral materials can also be employed with liquid carriers to form spreadable pastes, gels, ointments, soaps, and the like, for application directly to the skin of the user. Examples of useful dermatological compositions that can be used to deliver the compounds of the invention to the skin are known to the art; for example, see Jacquet et al. (U.S. Pat. No. 4,608,392), Geria (U.S. Pat. No. 4,992,478), Smith et al. (U.S. Pat. No. 4,559, 157) and Wortzman (U.S. Pat. No. 4,820,508).

Useful dosages of the compounds of the invention can be determined by comparing their in vitro activity, and in vivo activity in animal models. Methods for the extrapolation of effective dosages in mice, and other animals, to humans are known to the art; for example, see U.S. Pat. No. 4,938,949.

Generally, the concentration of the compound(s) of the invention in a liquid composition, such as a lotion, will be from about 0.1-25 wt-%, preferably from about 0.5-10 wt-%. The concentration in a semi-solid or solid composition such as a gel or a powder will be about 0.1-5 wt-%, preferably about 0.5-2.5 wt-%.

The amount of the compound, or an active salt or derivative thereof, required for use in treatment will vary not only with the particular salt selected but also with the route of administration, the nature of the condition being treated and the age and condition of the patient and will be ultimately at the discretion of the attendant physician or clinician.

In general, however, a suitable dose will be in the range of from about 0.5 to about 100 mg/kg, e.g., from about 10 to about 75 mg/kg of body weight per day, such as 3 to about 50 mg per kilogram body weight of the recipient per day, preferably in the range of 6 to 90 mg/kg/day, most preferably in the range of 15 to

60 mg/kg/day.

The compound is conveniently administered in unit dosage form; for example, containing 5 to 1000 mg, conveniently 10 to 750 mg, most conveniently, 50 to 500 mg of active compound per unit dosage form. Ideally, the active compound should be administered to achieve peak plasma concentrations of the active compound of from about 0.5 to about 75 μM, preferably, about 1 to 50 μM, most preferably, about 2 to about 30 μM. This may be achieved, for example, by the intravenous injection of a 0.05 to 5% solution of the active ingredient, optionally in saline, or orally administered as a bolus containing about 1- 100 mg of the active ingredient. Desirable blood levels may be maintained by continuous infusion to provide about 0.01-5.0 mg/kg/hr or by intermittent infusions containing about 0.4-15 mg/kg of the active ingredient(s).

The desired dose may conveniently be presented in a single dose or as divided doses administered at appropriate intervals, for example, as two, three, four or more sub-doses per day. The sub-dose itself may be further divided, e.g., into a number of discrete loosely spaced administrations; such as multiple inhalations from an insufflator or by application of a plurality of drops into the eye.

As disclosed herein, it has been discovered that LFM analogues, specifically LFM-A8, are useful as agents to alter leukotriene release from mast cells and to inhibit allergic asthma in mammals. As such, LFM analogues, specifically LFM- A8, can conveniently be administered in combination with other agents that alleviate asthmatic conditions which may be provided in a pharmaceutical composition, if preferred.

The invention will now be illustrated by the following non-limiting Examples.

EXAMPLES

Example 1. Synthesis of specific leflunomide metabolite analogs.

Chemistry. All chemicals were purchased from Aldrich (Milwaukee, WI) and were used without further purification. Except where noted, each reaction vessel was secured with a rubber septum, and the reaction was performed under nitrogen atmosphere. 'H and ^UC NMR spectra were obtained on a Varian Mercury 300 instrument spectrometer (Palo Alto, CA) at ambient temperature in the solvent specified. Melting points were determined using a Fisher-Johns melting point apparatus and are uncorrected. FT-IR spectra were recorded on a Nicolet Protege 460 spectrometer (Madison, WI). GC/MS spectra were obtained on a HP 6890 GC System (Palo Alto, CA) equipped with a HP 5973 Mass Selective Detector. MS (El) spectra were obtained on an HP Series 1100 LL/MSD. The general synthetic scheme for the preparations of LFM, and LFM-A1 - LFM-A12 is diagramed below. Cyanoacetic acid 1 was coupled with the desired aniline 2 in the presence of diisopropylcarbodiimide (DIC) to form 3. Compound 3 was treated with NaH and then acylated with acetyl chloride to afford LFM or LFM- Al to LFM-A12.

General Synthetic Procedure

1,3-diisopropylcarbodiimide (1.75 g; 13.9 mmol) was added to a solution of cyanoacetic acid 1 (1.70 g; 20.0 mmol) and the desired substituted-aniline 2 (12.6 mmol) in tetrahydrofuran (25 mL) at 0°C. The mixture was stirred for 12 hours at room temperature. The resulting urea precipitate (reaction side product) was removed by filtration and the filtrate was partitioned between ethyl acetate and 0.5 N HCl. The organic layer was sequentially washed with brine twice, dried over anhydrous Na SO and concentrated by rotary-evaporation. The crude solid product was recrystallized from ethyl alcohol to give pure 3. See Kuo, E. A., et al. (1996) J. Med. Chem. 39(23), 4608-21 ; and Sjogren, E. R., et al. (1991) J. Med. Chem. 34, 3295-3301.

Sodium hydride (0.93 g; 60% in mineral oil; 23.2 mmol) was added slowly to the solution of 3 (12.0 mmol) in tetrahydrofuran (40 mL) at 0°C. After stirring for 30 minutes at 0°C, acetyl chloride (1.04g; 13.2 mmol) was added to the reaction mixture. The reaction was continued for another hour at room temperature and was quenched by the addition of acetic acid (2 mL). The mixture was poured into ice water (100 mL) containing 2.5 mL of hydrochloric acid to precipitate the crude product, which was collected by filtration and washed with water. The final pure product was obtained by recrystallization.

Physical data. α-Cyano- β-hydroxy-β-methyl-N-[4-(trifluoromethyI)phenyI]-propenamide

(LFM). mp: 230 - 233°C; IR (KBr): 3303, 2218, 1600 and 1555 cm^'1; 'H NMR (DMSO-J₆): δ 1 1.01 (s, IH, NH), 7.75 (d, J= 8.4 Hz, 2H, ArH), 7.64 (d, J= 8.4 Hz, 2H, ArH), 2.22 (s, 3H, CH₃); GC/MS m/z 270 (M⁺), 161, 142, 111. α-Cyano-β-hydroxy-β-methyl-/V-(4-bromophenyl)propenamide (LFM-A1). mp: 213 - 214°C; IR (KBr): 3288, 2228, 1615, 1555 cm^'1; Η NMR (DMSO-J₆): δ 10.51 (s, IH, NH), 7.49 (s, 4H, ArH), 2.25 (s, 3H, CH₃); MS (El) m/z 280 (M^~). α-Cyano-β-hydroxy-β-methyl-N-(4-chIorophenyl)propenamide (LFM-A2). mp: 209 - 211°C; IR (KBr): 3298, 2223, 1598 and 1552 cm^"1; Η NMR (DMSO-J₆): δ 10.48 (s, IH, NH), 7.54 (d, J= 8.7 Hz, 2H, ArH), 7.45 (s br, IH, OH), 7.36 (d, J= 8.1 Hz, 2H, ArH), 2.25 (s, 3H, CH₃); MS (CI) m/z 236 {MX 129, 127. α-Cyano-β-hydroxy-β-methyl-N-(4-fluorophenyl)propenamide (LFM-A3). mp: 165 - 166°C; IR (IvBr): 3298, 2218, 1610 and 1560 cm^"'; Η NMR (DMSO--/,.): δ 10.33 (s, IH, NH), 7.80 (s br, IH, OH), 7.53 (m, 2H, ArH), 7.16 (m, 2H, ArH), 2.26 (s, 3H, CH₃); GC/MS m/z 220 (M⁺), 1 11. α-Cyano-β-hydroxy-β-methyl-N-[2-(trifluoromethyl)phenyl]-propenamide (LFM-A4). mp: 61 - 63°C; IR (KBr): 3435, 2209, 1619, 1952 and 1548 cm^"1; 'H NMR (DMSO--7₀): δ 10.99 (s, I H, NH), 8.03 (d, J = 7.5 Hz, IH, ArH), 7.67 (d, J = 7.5 Hz, IH, ArH), 7.60 (dd, -/= 7.5, 7.5 Hz, IH, ArH), 7.29 (dd, J= 7.5, 7.5 Hz, IH, ArH) 5.71 (s br, IH, OH), 2.20 (s, 3H, CH₃); GC/MS m/z 270 {MA), 161 , 141 , 114. α-Cyano-β-hydroxy-β-methyl-/V-(2-bromophenyl)propenamide (LFM-A5). mp: 98 - 100°C; IR (KBr): 3351 , 2214, 1609, 1585 and 1536 cm^'1; Η NMR (DMSO-J₀): δ 10.76 (s, IH, NH), 8.06 (dd, J= 8.1, 1.5 Hz, IH, ArH), 7.62 (dd, J- 8.1 , 1.5 Hz, IH, ArH), 7.33 (m, IH, ArH), 7.03 (m, IH, ArH), 6.60 (s br, IH, OH), 2.22 (s, 3H, CH ); ); MS (El) m/z 280 (M⁺), 173, 171. α-Cyano-β-hydroxy-β-methyl-N-(2-chlorophenyI)propenamide (LFM-A6). mp: 93 - 94°C; IR (KBr): 3372, 2208, 1644, 1621 and 1587 cm^'1; Η NMR (DMSO-J_b): δ 10.96 (s, IH, NH), 8.16 (d, J - 8.1 Hz, IH, ArH), 7.46 (dd, J = 1.5, 1.5 Hz, IH, ArH), 7.29 (m, IH, ArH), 7.08 (m, IH, ArH), 2.22 (s, 3H, CH₃); MS (CI) m/z 236 (M⁺), 129, 127.

α-Cyano-β-hydroxy-β-methyl-N-(2-fluorophenyI)propenamide (LFM-A7). mp: 1 18 - 119°C; IR (KBr): 3409, 2212, 1613, 1591 and 1532 cm^'1; 'H NMR (DMSO- J₅): δ 10.70 (s, I H, NH), 7.91 (m, IH, ArH), 7.23 (m, IH, ArH), 7.13 (m, 2H, ArH), 7.10 (s br, IH, OH), 2.22 (s, 3H, CH₃); GC/MS m/z 220 (M⁺), 111.

α-Cyano-β-hydroxy-β-methyl-N-[3-(trifluoromethyI)phenyl]-propenamide (LFM-A8). mp: 182 - 184°C; IR (KBr): 3303, 2221, 1619 and 1572 cm^'1; 'H NMR (DMSO-J₆): δ 10.79 (s, IH, NH), 8.05 (s br, IH, OH) 8.04 (s, IH, ArH), 7.75 (d, J = 8.1 Hz, IH, ArH), 7.53 (dd, J = 8.1, 7.5 Hz, IH, ArH), 7.42 (d, J = 7.5 Hz, IH, ArH), 2.24 (s, 3H, CH₃); GC/MS m/z 270 (M⁺), 161.

α-Cyano-β-hydroxy-β-methyl-/V-(3-bromophenyl)propenamide (LFM-A9). mp: 184 - 185°C; IR (KBr): 3303, 2228, 1610, 1595 and 1550 cm^'1; 'H NMR (DMSO-J₀): δ 10.56 (s, I H, NH), 7.89 (m, IH, ArH), 7.47 (m, IH, ArH), 7.28 (m, 2H, ArH), 6.37 (s br, IH, OH), 2.26 (s, 3H, CH₃); MS (El) m/z 282 (NT + H, ⁸¹Br), 280 (M^* + H, ⁷⁹Br), 173, 171. α-Cyano-β-hydroxy-β-methyl-N-(3-chIorophenyI)propenamide (LFM-A10). mp: 184 - 187°C; IR (KBr): 3293, 2221, 1610, 1595 and 1557 cm^'1; Η NMR (DMSO-Jtf): δ 10.61 (s, IH, NH), 776 (m, IH, ArH), 7.42 (m, I H, ArH), 7.33 (dd, J = 8.1 , 8.1 Hz, IH, ArH), 7.16 (m, IH, ArH), 6.84 (s br, IH, OH), 2.25 (s, 3H, CH₃); MS (CI) m/z 239 (M⁺ + H, ³⁷C1), 237 (M⁺ + H ³⁵C1), 129, 127.

α-Cyano-β-hydroxy-β-methyl-N-(3-fluorophenyl)propenamide (LFM-A11). mp: 136 - 138°C; IR (KBr): 3297, 2221, 1613, 1597 and 1567 cm^'1; 'H NMR (DMSO-J : δ 10.54 (s, IH, NH), 7.54 (m, IH, ArH), 7.33 (m, 2H, ArH), 6.93 (m, IH, ArH), 2.27 (s, 3H, CH₃); GC/MS m/z 220 (M⁺), 111.

α-Cyano-β-hydroxy-β-methyl-N-[4-(trifluoromethoxy)phenyI]-propenamide

(LFM-A12). mp: 182 - 183°C; IR (KBr): 3308, 2213, 1625 and 1580 cm^'1; 'H NMR (DMSO-J_&): δ 10.57 (s, IH, NH), 7.90 (s br, IH, OH), 7.64 (d, J= 8.7 Hz, 2H, ArH), 7.32 (d, J= 8.7 Hz, 2H, ArH), 2.25 (s, 3H, CH₃); GC/MS m/z 286 (M⁺), 177, 108.

The following synthetic schemes were used to generate the compounds:

Scheme 1

LFM, LFM-AO - LFM-A12

LFM: X ψara-CF₃ LFM-A4 X =ortho-CF₃ LFM-A9: X =metα-Br

LFM-AO X = H LFM-A5 X =ortho-Br LFM-A10: X =meta-C\

LFM-A1 X =ρara-Br LFM-A6 X =ortho-C\ LFM-A11 : X =meta~V

LFM-A2 X =para-C\ LFM-A7 X =ortho-Y LFM-A12: X =ψara-OCF_i

LFM-A3 X =para-F LFM-A8 X =ιneta-CF₃

Synthetic Procedure A. 1,3-diisopropylcarbodiimide (1.75 g; 13.9 mmol) was added to a solution of cyanoacetic acid 1 (1.70 g; 20.0 mmol) and the desired substituted -aniline 2 (12.6 mmol) in tetrahydrofuran (25 mL) at 0°C. The mixture was stirred for 12 hours at room temperature. The urea precipitate (reaction side product) was removed by filtration and partitioned between ethyl acetate and 0.5 N HCl. The organic layer was sequentially washed with brine twice, dried over anhydrous Na₂SO₄ and concentrated by rotary-evaporation. Finally, the crude solid product was recrystallized from ethyl alcohol to give pure 3. Sodium hydride (0.93 g; 60% in mineral oil; 23.2 mmol) was added slowly to the solution of 3 (12.0 mmol) in tetrahydrofuran (40 mL) at 0°C. After stirring for 30 minutes at 0°C, acetyl chloride (1.04g; 13.2 mmol) was added to the reaction mixture. The reaction was continued for another hour and then was quenched by the addition of acetic acid (2 mL). The mixture was poured into ice water (100 mL) containing 2.5 mL of hydrochloric acid to precipitate the crude product, which was collected by filtration and washed with water. The pure product was obtained by recrystallization. Synthetic Procedure B. α-Cyano-β-hydroxy-β-methyl-N-

[(methylthio)phenyl]propenamide (2.48g, 10.0 mmol) was dissolved in acetic acid (150 mL), and peracetic acid (8.6 mL of 32% wt solution in acetic acid) was added. The mixture was stirred overnight at room temperature, and water (75 mL) was added. The precipitate was filtered and washed with water. The pure product was obtained by recrystallization.

Example 2: LFM-A8 inhibits leukotriene synthesis in mast cells

The following example provides biochemical evidence that the LFM analogue LFM-A8 is a potent inhibitor of IgE/FcεRI receptor-mediated leukotriene C₄ release in RBL-2H3 rat mast cells as well as fetal liver-derived human mast cells.

Reagents. Fetal bovine serum (FBS) was obtained from Hyclone (Logan,

UT). Bovine serum albumin (BSA), dimethyl sulphoxide (DMSO), methacholine and formamide were purchased from Sigma (St. Louis, MO). Leukotriene C₄,

ELISA kits were from Cayman Company (An l Arbor, MI). The preparations of dinitrophenyl (DNP)-BSA (Wei, Y.F., et al., (1986) J Immunol, 137, 1993-2000) and monoclonal anti-DNP-IgE (Liu, F.T., et al., (1980) J Immunol, 124, 2728-2737) were previously described. Recombinant hSCF and IL-4 were purchased from Genzyme (Cambridge, MA). Human IgE was purchased from Calbiochem (San Diego, CA). Mouse anti- human IgE was from Serotec (UK). Mast Cell Cultures. RBL-2H3 mast cells were a gift from Dr. Reuben P.

Siraganian (Laboratory of Microbiology and Immunology, National Institute of Dental Research, National Institute of Health). The cells were maintained as monolayer cultures in 75- or 150-cm² flask in Eagle's essential medium supplemented with 20% fetal calf serum. Human fetal livers (16 to 21 weeks of gestational age) were obtained from prostaglandin-induced human abortuses. Subsequently, single cell suspensions were prepared and mononuclear cells were isolated by centrifugation on Ficoll-Hypaque gradients as described (Malaviya, R., et al., (1999) J Biol Chem, 21 A, 27028-27038). Isolated cells were cultured for 8 weeks in the presence of 100 ng/ml rhSCF, 2 ng/ml rhIL-4 (Malaviya, R., et al., (1999) J Biol Chem, 274, 27028-27038; Xia, H.Z., et al., ( 1997) J Immunol, 159, 291 1-2921). Culture medium was replaced with fresh medium once a week for the first 2 weeks and twice a week thereafter. All human tissue specimens were used following the guidelines of the Parker Hughes Institute Institutional Review Board on the Use of Human Subjects in Research for secondary use of pathologic or surgical tissue. At the end of the 8 weeks the fetal liver derived human mast cell culture contained 70-90 percent mast cells, based on toluidine blue and tryptase staining (Xia, H.Z., et al., {1991) J Immunol, 159, 2911-2921).

Stimulation of Mast Cells. RBL-2H3 rat mast were sensitized with monoclonal anti-DNP IgE antibody (0.24 mg/ml) for lh at 37 °C in a 48-well tissue culture plate. Unbound IgE was removed by washing the cells with PIPES-buffered saline. After washing, PIPES-buffered saline containing 1 mM calcium chloride was added to the monolayers of the RBL-2H3 cells. The cells were challenged with 20 ng/ml DNP-BSA for 30 min at 37 °C. The plate was centrifuged at 200 for 10 min at 4 °C. Supernatants were removed and saved. Fetal liver-derived human mast cells were resuspended in culture medium at a cell density of 5 x lOVml and sensitized with human IgE (150 μg/ml) for 3 h at 4°C. After sensitization the cells were washed with tyrode buffer containing 1 mM calcium and 1 mM magnesium and challenged with mouse monoclonal anti-human IgE (40 μg/ml) for 30 min at 37°C. To study the effect of test drugs, RBL-2H3 rat mast cells or human mast cells were incubated with the drugs at the indicated concentrations or vehicle for 30 min prior to antigen challenge. Mediator Release Assay. Leukotriene C₄ levels were estimated in cell free supernatants of mast cells by immunoassay (Malaviya, R. and Abraham, S.N., (1995) Methods Enzymol, 253, 27-43).

Mast cells release large amounts of leukotrienes upon IgE receptor ligation. Because of the reported ability of LFM as a mast cell inhibitor, we utilized 13 analogues of LFM (Table 1) and examined their effect on IgE/FcεRI receptor mediated mast cell leukotriene C₄ release. RBL-2H3 cells, a mucosal mast cell line, were treated with 5 different concentrations of the compounds ranging from l μM to 100 μM or vehicle for 30 min before challenge with antigen (DNP-BSA) at 37° C. Leukotriene C₄ release was quantitated in the extracellular medium employing a previously described ELISA (Malaviya, R. and Abraham, S.N., (1995) Methods Enzvmol, 253, 27-43). Stimulation of vehicle-treated RBL-2H3 mast cells with IgE/antigen resulted in the release of 14.6 to 48 ng (mean ± SEM; 20 ± 4.7) leukotriene C₄/10⁶ cells in 24 independent experiments.

TABLE 1 : Inhibition of IgE/FcεRI Receptor Mediated RBL-2H3 Mast Cell Leukotriene C₄ Release by LFM and its Analogues.

Compounds Substitution ECso (μM) (mean+SEM)

LFM P-CF3 34+14.5 LFM-AO unsubstituted >100 LFM-A1 p-Bτ 64±13.9 LFM-A2 p-Cl 58+13 LFM-A3 p-Ε >100 LFM-A4 0-CF3 >100 LFM-A5 o-Br 46+10 LFM-A6 o-Cl >100 LFM-A7 ø-F >100

LFM-A9 m- τ >100 LFM-A 10 m-Cl 51±12 LFM-A1 1 m-F >100 LFM-A12 p-OC¥3 30±5.6

All the compounds were tested at 1 , 3, 10, 30 and 100 μM concentrations; The data points represent the mean ± SEM values (N=3-4.. As shown in Table 1 , all the 13 derivatives of LFM (LFM-AO to LFM-A12) inhibited IgE/FcεRI receptor-mediated leukotriene C₄ release. However, marked differences were noted in their potency. The inhibition of leukotriene release was not due to reduced cell viability since >95% of mast cells remained capable of trypan blue dye exclusion after treatment with 100 μM of the test compounds (data not shown). Table 1 shows the EC₅₀ values of the test compounds calculated from their concentration-effect curves. LFM which has CF3 group at the para position of the phenyl ring, inhibited IgE/antigen induced leukotriene C₄ release in a concentration dependent manner with an EC₅₀ value of 34+ 14.5 μM (Table 1). A substitution of the para CF3 group with a para OCF3 group did not result in improved activity. Among the 13 analogues of LFM, LFM-A8 [a -Cynno-β- hydroxy- ?-methyl-N-[3-(trifluoromethyl)phenyl]propenamide] with a CF3 group at the meta position of the phenyl ring was found to be most active with an average EC₅₀ value of 28 + 1.0 μM (Table 1). By comparison, the unsubstituted compound, LFM-AO and p- and o- F, o- CI and o- CF3 substituted compounds were inactive (EC₅₀>100μM) and the bromo-substituted compounds (LFM-A5, and -A9) exhibited mild to moderate activity (Table 1).

We next sought to determine the effect of LFM-A8 on IgE/FcεRI-mediated human mast cell leukotriene C₄ release and compare its potency with that of LFM. To this end, we cultured fetal liver derived human mast cells in presence of SCF and IL-4 for 8 weeks. IgE-sensitized human mast cells were exposed to vehicle or the test compounds for 30 min. The Fcε receptors of fetal liver-derived human mast cells were crosslin ed with anti-IgE and the resulting mast cell leukotriene C₄ release was quantitated by ELISA (Malaviya, R. and Abraham, S.Ν., (1995) Methods Enzymol, 253, 27-43). As shown in Figure I, LFM-A8 significantly inhibited the leukotriene C₄ release from IgE/anti-IgE-stimulated human mast cells. Example 3: LFM-A8 inhibits allergic asthma

The following example provides cellular and physiological evidence that the LFM analogue LFM-A8 prevented bronchial hyperresponsivess, and inhibited eosinophil influx in a well-characterized murine model of allergic asthma.

Mice. Male Balb/c mice, 6-8 weeks old were purchased from Charles River Laboratories (Wilmington, MA). Animals were caged in groups of five in a pathogen-free environment in accordance with the rules and regulations of the U. S. Animal Welfare Act, and National Institutes of Health (NIH). Mice were allowed free access to autoclaved pellet food and tap water. Animal studies were approved by the Parker Hughes Institute Animal Care and Use Committee and all animal care procedures conformed to Principles of Laboratory Animal Care.

Mouse Model of Allergic Asthma. BALB/c mice were injected intraperitoneally with 20 μg of ovalbumin (OVA) in alum on day 0, and 14. On days 21, 22 and 23 the mice were challenged for 15 min with 2% OVA via their airways by ultrasonic nebulization (Takeda, K, et al., (1997) J Exp Med, 186, 449- 454). In order to study the effect of LFM-A8 on allergic asthma, mice were treated with LFM-A8 or vehicle 2h prior to OVA challenge on day 21 , 22 and 23. Mice were assessed for "airway responsiveness" on day 24, as previously reported (Takeda, K., et al., (1997) J Exp Med, 186, 449-454; Hamelmann, E., et al, (1997) Am JRespir Crit Care Med, 156, 766-775) and described below.

Determination of Airway Responsiveness. Airway responsiveness was measured in unrestrained mice by noninvasive whole body plethysmography using a BUXCO BioSystem plethysmography instrument (BUXCO, Trou, NY) (Hamelmann, E., et al., (1997) Am J Respir Crit Care Med, 156, 766-775). The chamber pressure was measured with a transducer connected to a preamplifier module and analyzed by system XA software) (Hamelmann, E., et al., (1997) Am J Respir Crit Care Med, 156, 766-775). The chamber pressure was used as a measure of the difference between thoracic volume expansion or contraction and air volume removed or added to the chamber during breathing. The differential of this function with respect to time produced a pseudo-flow value that is proportionate to the difference between the rate of the thoracic volume expansion and nasal air flow. The pulmonary airflow obstruction assessed by measuring "Enhanced Pause (Penh)" using the following formula according to the manufacturer's recommendations: Penh = PEP/PIP x Pause. Penh reflects changes in the wave form of the chamber pressure signal from both inspiration (PIP) and expiration (PEP) and combines it with the timing comparison of early and late expiration (Pause). In order to measure the methacholine responses, mice were placed in the chamber and baseline readings were taken and averaged for 3 min. Mice were nebulized with saline or methacholine at increasing doses (1-100 mg/ml) for 3 min and the Penh readings were taken and averaged for 3 min after each nebulization. In order to study the effect of LFM-A8 on allergic asthma, mice were injected intraperitoneally with LFM-A8 or vehicle on day 20, 21, and 23 lh prior and 2h post OVA challenge.

Assessment of Eosinophil Infiltration. After airway responsiveness measurements, lungs were lavaged thoroughly with 1 ml saline. The lavage fluid was centrifuged and the supernatant was removed. The cell pellet was resuspended in saline containing 0.1%) BSA to give a final cell concentration of 0.1 x l OVml. Cytospin smears made from the cell suspension were stained with Wright-Giemsa and the number of eosinophils were counted.

Since mast cells release large amounts of leukotrienes upon allergic stimulation (Malaviya, R., et al., (1999) J Biol Chem, 21 A, 27028-27038; Malaviya, R. and Uckun, F.M., (1999) Biochem Biophys Res Commun, 257, 807-813) and leukotrienes play a central role in allergic asthma by increasing bronchial hyperresponsiveness, mucus secretion and provoking eosinophil recruitment (Henderson, W.R., et al., {1996) J Exp Med, 184, 1483-1494), we next examined the effect of LFM-A8 on bronchial hyperresponsivess, and eosinophil influx in a well- characterized murine model of allergic asthma (Hamelmann, E., et al., (1997) Am J Respir Crit Care Med, 156, 766-775; Henderson, W.R., et al., (1996) J Exp Med, 184, 1483-1494; Hamelmann, E., et al., (1997) Am J Respir Crit Care Med, 155, 819-825). In this model, mice are first sensitized by repeated intraperitoneal injections of ovalbumin to induce ovalbumin-specific IgE response. Mice are then challenged via airway with ovalbumin mimicking a natural mode of allergic sensitization. After 24h of the last ovalbumin challenge, mice are assessed for their bronchial hyperresponsiveness to inhaled methacholine. As shown in Figure 2, mice that were sensitized and challenged with ovalbumin (OVA+OVA) exhibited significantly higher Penh response compared with PBS-sensitized and ovalbumin- challenged mice (PBS+OVA) to aerosolized methacholine. The dose of methacholine required to induce 100% and 200%) increase in Penh response in mice that were sensitized and challenged with ovalbumin was significantly lower than that for PBS-sensitized and ovalbumin-challenged mice (Table 2). Pretreatment of mice with LFM-A8 resulted in a decrease of bronchial hyper-responsiveness (Figure 2). As shown in Table 2, LFM-A8 pretreatment increased the amount of methacholine required to induce 100 and 200% increase in Penh response. These findings demonstrate that LFM-A8 is a potent inhibitor of bronchial hyper- responsiveness in this mouse model of allergic asthma.

We next sought to determine the effect of LFM-A8 on eosinophil recruitment. Broncho-alveolar lavage fluids were obtained after airway responsiveness measurements and the number of eosinophils were quantitated in each group of mice. Examination of cytospin smears of BAL fluids revealed that mice sensitized and challenged with ovalbumin (OVA+OVA) recruit significantly higher numbers of eosinophils to their airway lumen than mice that were sensitized with PBS and challenged with Ovalbumin (PBS+OVA) (Figure 3). Pretreatment of mice with LFM-A8 reduced the numbers of eosinophils in BAL fluid samples of mice that had been sensitized and challenged with Ovalbumin by 80% (Figure 3). These results demonstrate that LFM-A8 is capable of preventing allergen-induced eosinophil recruitment in vivo.

TABLE 2: Effect of LFM-A8 on Bronchial Hyper-responsiveness in Mice.

Treatment Sensitization Challenge Requirement of Methacholine (mg/ml)

Vehicle (negative PBS Ovalbumin 49 5 94 control)

Vehicle (positive control) Ovalbumin Ovalbumin 4.1 25 6

LFM-A8 (15mg /Kg) Ovalbumin Ovalbumin 36 8 82

LFM-A8 (50mg/Kg) Ovalbumin Ovalbumin 67 5 129

Penh, Increase in enhanced pause; PBS, phosphate buffered saline; N= 5-6 mice

Claims

CLAIMSWE CLAIM:

1. A method to alter leukotriene production from mast cells, the method comprising contacting mast cells with an effective leukotriene-altering amount of a LFM analogue of formula I:

where:

R, is (C,-C₃)alkyl, (C₃-C₆)cycloalkyl, phenyl, or R_aR_b;

R, is hydroxy, (C,-C₆)alkoxy (C,-C₆)alkanoyloxy amino (C₂- C₅)alkoxy; hydroxy (C_:-C₅)alkoxy amino (C,-C₅)alkanoxy; or hydroxy {C,-C₅) alkanoxy;

R₃ is cyano or (C,-C₃)alkanoyl;

R₄ is hydrogen, (C,-C₃)alkyl; hydroxy (C,-C₅)alkyl; or amino (C -

C₅)alkyl;

R₅ is phenyl substituted by -8(0)^., or by halo and at least one other substituent;

R_a and R_b are each independently hydrogen, or ( -C^alkyl; or R_a and R_b together with the nitrogen to which they are attached are pyrrolidino, piperidino, morpholino, or thiomorpholino; wherein any aryl or heteroaryl of R, and R₅ is optionally substituted with one or more substituents independently selected from halo, nitro, cyano, hydroxy, trifluoromethyl, trifluoromethoxy, (C_rC₃)alkoxy, (C_rC₃)alkyl, (C,- C₃)alkanoyl, -S(O)_zR. _, or NR_aR_b wherein R_c is (C,-C )alkyl, or aryl or a pharmaceutically acceptable salt thereof.

2. The method of claim 1 , wherein the LFM analogue is selected from the group consisting of LFM-AO, LFM-Al , LFM-A2, LFM-A3, LFM-A4, LFM-A5, LFM-A6, LFM-A7, LFM-A8, LFM-A9, LFM-Al 0, LFM-Al 1 and LFM-Al 2 or a pharmaceutically acceptable salt thereof.

3. The method of claim 1 wherein the LFM analogue comprises the structure of LFM-A8 or pharmaceutically acceptable salts thereof.

4. The method of claim 1 wherein leukotriene production results from IgE/FcεRI engagement.

5. The method of claim 1 wherein the mast cells comprise human mast cells

6. The method of claim 1 wherein the leukotriene comprises leukotriene C₄, D₄, E₄, or B₄.

7. The method of claim 4 wherein the leukotriene comprises leukotriene C₄

8. A method for treating or preventing asthma in a mammal, the method comprising administering to a mammal a therapeutically effective amount of a LFM analogue of formula I:

where:

R, is (C,-C₃)alkyl, (C₃-C_b)cycloalkyl, phenyl, or NR_aR_b;

R_: is hydroxy, (C,-C_b)alkoxy (CpC^alkanoyloxy amino (C,- C₅)alkoxy; hydroxy (C₂-C₅)alkoxy amino (C,-C₅)alkanoxy; or hydroxy (C,-C₅) alkanoxy;

R₃ is cyano or (C,-C₃)alkanoyl;

R₄ is hydrogen, (C,-C₃)alkyl; hydroxy (C,-C₅)alkyl; or amino (C, C₅)alkyl; R₅ is phenyl substituted by -S(O)_;R , or by halo and at least one other substituent;

R_a and R_b are each independently hydrogen, or (C_]-C₃)alkyl; or R_a and R_b together with the nitrogen to which they are attached are pyrrolidino, piperidino, morpholino, or thiomorpholino; wherein any aryl or heteroaryl of R, and R₅ is optionally substituted with one or more substituents independently selected from halo, nitro, cyano, hydroxy, trifluoromethyl, trifluoromethoxy, (C,-C₃)alkoxy, (C_rC₃)alkyl, (C,- C )alkanoyl, -S^R. or NR_aR_b, wherein 1^ is (C,-C₃)alkyl, or aryl or a pharmaceutically acceptable salt thereof.

9. The method of claim 8, wherein the LFM analogue is selected from the group consisting of LFM-AO, LFM-Al , LFM- A2, LFM-A3, LFM-A4, LFM-A5, LFM-A6, LFM-A7, LFM-A8, LFM-A9, LFM-A10, LFM-Al 1 and LFM-Al 2 or a pharmaceutically acceptable salt thereof

10. The method of claim 8 wherein the LFM analogue comprises the structure of LFM-A8 or pharmaceutically acceptable salts thereof.

1 1. The method of claim 8 wherein said asthma is associated with an alteration in the number of eosinophils in the bronchioles.

12. The method of claim 8 wherein said asthma is associated with airway hyperresponsiveness.

13. A method of altering the number of eosinophils in the bronchioles, the method comprising administering to a mammal a therapeutically effective amount of a LFM analogue of formula I:

where:

R, is (C,-C₃)alkyl, (C₃-C₆)cycloalkyl, phenyl, or NR_aR_b;

R, is hydroxy, (C,-C₆)alkoxy (C,-C₆)alkanoyloxy amino (C₂- C_s)alkoxy; hydroxy (C₂-C₃)alkoxy amino (C₂-C₅)alkanoxy; or hydroxy (C₂-C₅) alkanoxy;

R₃ is cyano or (C_rC₃)alkanoyl;

R₄ is hydrogen, (C_rC₃)alkyl; hydroxy (C₂-C₅)alkyl; or amino (C₂-

C₅)alkyl;

R₅ is phenyl substituted by -S(O) R^ or by halo and at least one other substituent;

R_a and R_b are each independently hydrogen, or (C_rC₃)alkyl; or R_a and R_b together with the nitrogen to which they are attached are pyrrolidino, piperidino, morpholino, or thiomorpholino; wherein any aryl or heteroaryl of R, and R₅ is optionally substituted with one or more substituents independently selected from halo, nitro, cyano, hydroxy, trifluoromethyl, trifluoromethoxy, (C,-C₃)alkoxy, (C_rC₃)alkyl, (C,- C₃)alkanoyl, -S(O),R,, or NR_aR_b> wherein R, is (C_rC₃)alkyl, or aryl or a pharmaceutically acceptable salt thereof.

14. The method of claim 13, wherein the LFM analogue is selected from the group consisting of LFM-AO, LFM-Al , LFM-A2, LFM-A3, LFM-A4, LFM-A5, LFM-A6, LFM-A7, LFM-A8, LFM-A9, LFM-Al 0, LFM-Al 1 and LFM-Al 2 or a pharmaceutically acceptable salt thereof.

15. The method of claim 13 wherein the LFM analogue comprises the structure of LFM-A8 or pharmaceutically acceptable salts thereof.

16. The method of claim 15 wherein the eosinophil recruitment to the bronchioles is decreased in mammals pretreated with at least 15mg/Kg of LFM-A8 by at least 50% as compared to untreated mammals

17. A method for reducing bronchial hyperresponsiveness the method comprising administering administering to a mammal a therapeutically effective amount of a LFM analogue of formula I:

where:

R, is (C,-C )alkyl, (C₃-C₆)cycloalkyl, phenyl, or NR_aR_b;

R₂ is hydroxy, (C,-C₆)alkoxy (C_rC₆)alkanoyloxy amino (C₂- C₅)alkoxy; hydroxy (C₂-C₃)alkoxy amino (C₂-C₅)alkanoxy; or hydroxy (C₂-C₅) alkanoxy;

R₃ is cyano or (C,-C )alkanoyl;

R₄ is hydrogen, (C,-C₃)alkyl; hydroxy (C₂-C₅)alkyl; or amino (C\-

C₅)alkyl;

R₅ is phenyl substituted by -S(O) R_c, or by halo and at least one other substituent;

R_a and R_b are each independently hydrogen, or (CpC^alkyl; or R_a and R_b together with the nitrogen to which they are attached are pyrrolidino, piperidino, morpholino, or thiomorpholino; wherein any aryl or heteroaryl of R, and R₅ is optionally substituted with one or more substituents independently selected from halo, nitro, cyano, hydroxy, trifluoromethyl, trifluoromethoxy, (C,-C₃)alkoxy, (C,-C )alkyl, (C,- C₃)alkanoyl, -S(O),R. _, or NR_aR_b, wherein R_t is (C,-C₃)alkyl, or aryl or a pharmaceutically acceptable salt thereof.

18. The method of claim 17, wherein the LFM analogue is selected from the group consisting of LFM-AO, LFM-Al, LFM-A2, LFM-A3, LFM-A4, LFM-A5, LFM-A6, LFM-A7, LFM-A8, LFM-A9, LFM-Al 0, LFM-Al 1 and LFM-Al 2 or a pharmaceutically acceptable salt thereof.

19. The method of claim 17 wherein the LFM analogue comprises the structure of LFM-A8 or pharmaceutically acceptable salts thereof.

20. The method of claim 19 wherein the bronchial hyperresponsiveness is decreased in mammals pretreated with at least 15mg/Kg of LFM-A8 so at least 8.9 times as much sensitizing agent is needed to produce a 100% increase in pulmonary airflow obstruction as compared to untreated mammals.

21. The method of claim 8 wherein the treatment comprises administering the LFM analogue with a pharmaceutically acceptable carrier.

22. The method of claim 8 wherein the asthma is allergen induced.

23. The method of claim 8 wherein the LFM analogue is administered by inhalation.

24. The method of claim 8 wherein the LFM analogue is administered systemically.

25. The method of claim 8 wherein the LFM analogue is administered by continuous infusion.

26. The method of claim 8 wherein the LFM analogue is administered by bolus dosage.