WO2000004003A1

WO2000004003A1 - Ccr-3 receptor antagonists

Info

Publication number: WO2000004003A1
Application number: PCT/US1999/015865
Authority: WO
Inventors: Dashyant Dhanak
Original assignee: Smithkline Beecham Corporation
Priority date: 1998-07-14
Filing date: 1999-07-13
Publication date: 2000-01-27

Abstract

A CCR-3 receptor antagonist and methods for its use are provided.

Description

CCR-3 RECEPTOR ANTAGONISTS FIELD OF THE INVENTION

The present invention relates to the use of (S)-N-(l-(4,5-Dihydro-oxazol-2-yl)-2- (4-nitrophenyl)ethyl)-l-naphtharnide as a CKJ36/CCR-3 receptor antagonist. Chemokines are a superfamily of small secreted proteins. There are approximately

30 distinct chemokines known with many others being characterized. See Oppenheim et al., Properties of the Novel Proinflammatory Supergene "Intercrine" Cytokine Family, Ann. Rev. Immun. , 9, 617-648 (1991); and Baggiolini, et al, Interleukin-8 and Related Chemotactic Cytokines-CXC and CC Chemokines, Adv. Immun., 55, 97-179 (1994). The properties of the chemokines suggest that they are essential for leukocyte trafficking and inflammatory processes, and are thus important components in a number of disease states. See Kita et al., Chemokines Active on Eosinophils: Potential Roles in Allergic Inflammation, EXΓJ, Med., 183, 2421-2426 (1996); Strieter, et al., "The Good, the Bad and the Ugly" The Role of Chemokines in Models of Human Diseases, J. Immun., 157, 3583-3586 (1996); and Baggiolini, Eotaxin: a VIC (Very Important Chemokine) of Allergic Inflammation, Clin. Invest., 97, 587 (1996).

Chemokines mediate their effects via interactions with 7TM-G-protein coupled receptors on the surface of immune and inflammatory cells. Eosinophils are proinflammatory granulocytes that play a major role in allergic diseases, such as bronchial asthma, allergic rhinitis, pruritis and atopic dermatitis. Upon activation, eosinophils release lipid mediators, cytotoxic proteins, oxygen metabolites and cytokines, all of which have the potential to produce pathophysiology. Numerous studies have demonstrated the presence of eosinophils or eosinophil-specific products in inflamed tissues in human diseases.

The mechanisms responsible for the selective infiltration of eosinophils in allergic diseases have yet to be clarified. Recently, a CC chemokine, Eotaxin, was identified in guinea pigs and demonstrated to be present in a guinea pig model of allergic airway inflammation. See Jose, et al., Eotaxin: A Potent Eosinophil Chemoattractant Cytokine Detected in Guinea Pig Model of Allergic Airways Inflammation, J. Exp. Med., 179, 881- 887 (1994); and Jose, et al., Eotaxin: Cloning of an Eosinophil Chemoattractant Cytokine and Increased mRNA Expression in Allergen-challenged Guinea-pig Lungs, Biochem.

Biophvs. Res. Comm., 205, 788-794 (1994). The human homologue of Guinea-pig eotaxin has been expressed and has been shown to induce eosinophil infiltration when injected into the skin of the rhesus monkey. See Ponath, et al., Cloning of the Human Eosinophil

Chemoattractant, Eotaxin: Expression, Receptor Binding, and Functional Properties Suggest a Mechanism for Selective Recruitment of Eosinophils, J. Clin. Invest., 97, 604- 612 (1996).

The cloning, expression and characterization of a novel C-C chemokine receptor, designated CCR-3 from peripheral blood eosinophils and from an eosinophil cDNA library have also been reported. See Kitaura, et al., Molecular Cloning of Human Eotaxin, an Eosinophil-selective CC Chemokine, and Identification of a Specific Eosinophil Eotaxin Receptor, CC Chemokine Receptor 3, Biol. Chem., 271, 7725-7730 (1996); Ahuja, et al., Cloning and Functional Expression of a Human Eosinophil CC Chemokine Receptor, J. Biol. Chem., 270, 16491-16494 (1995); Daugherty, et al., Cloning, Expression and Characterization of the Human Eosinophil Eotaxin Receptor, J. Exp. Med. 183, 2349-2354 (1996); and Ponath, et al., Molecular Cloning and Characterization of a Human Eotaxin Receptor Expressed Selectively on Eosinophils, J, Exp. Med., 183, 2437-2448 (1996).

Eotaxin, and, to a lesser extent, RANTES and MCP-3 activate this receptor. The CCR-3 receptor is expressed at high levels on eosinophils, typically 40,000- 400,000 receptors per cell are present. This is 10-100 fold more than the other chemokine receptor (CCR-1) expressed in eosinophils. Monoclonal antibodies raised to the CCR-3 receptor demonstrate that the receptor is primarily restricted to eosinophils. This restricted expression on eosinophils may be responsible for the selective recruitment of eosinophils in allergic inflammation. Additionally, CCR-3 is potently activated by Eotaxin. In contrast, other known chemokines appear to activate more than one chemokine receptor, e.g. RANTES binds to CCR-1, CCR-3, CCR-4 and CCR-5 receptors.

The foregoing research advances have provided the impetus to investigate the inhibition of eosinophil-specific chemokines in order to examine its role in blocking cellular infiltration in inflamed tissues. CCR-3 receptor antagonists thus offer a unique approach toward decreasing the pathophysiology associated with allergic diseases.

Antagonism of this receptor may be useful in the treatment of allergic disorders, including but not limited to bronchial asthma, allergic rhinitis, nasal polyposis, atopic dermatitis and pruritis.

SUMMARY OF THE INVENTION The present invention involves the CCR-3 receptor antagonist (S)-N-(l-(4,5-

Dihydro-oxazol-2-yl)-2-(4-nitrophenyl)ethyl)-l-naphthamide and compositions containing the present compound. The present compound is useful in the treatment of a variety of diseases associated with allergic disorders, including but not limited to bronchial asthma, allergic rhinitis, nasal polyposis, atopic dermatitis and pruritis. DETAILED DESCRIPTION OF THE INVENTION

As used herein "present compound" signifies (S)-N-(l-(4,5-Dihydro- oxazol-2-yl)-2-(4-nitrophenyl)ethyl)- 1 -naphthamide.

As used herein "present method" signifies the use of the present compound as a CCR-3 receptor antagonist.

As used herein "present composition" signifies a composition comprising the present compound and a pharmaceutically acceptable carrier.

Also included in the present invention are pharmaceutically acceptable salt complexes of the present compound. Preferred are the ethylene diamine, sodium, potassium, calcium and ethanolamine salts.

The present compound was prepared as follows: a) (S)-2-π-Naphthoylamino)-3-(4-nitrophenyl)propionic acid

L-4-Nitrophenylalanine (5.33 g, 23.4 mmol) was dissolved in 2.0M NaOH (12.9 mL) and cooled to 5 °C. 1-Naphthoyl chloride (4.90 g, 25.7 mmol) and 2.0M NaOH (14.2 mL) were added in several portions over 15 minutes. The mixture was warmed to room temperature over 30 minutes and washed with Et2θ. The aqueous portion was acidified using 6N HC1 and the precipitated off-white solid was filtered and dried (7.47 g, 88%). MS (ES-) m/e 363 [M-H], 727 b) (S)-N-( 1 -(2-hydroxyethylcarbamoyl)-2-('4-nitrophenyl)ethyl)- 1 -naphthamide (S)-2-(l-Naphthoylamino)-3-(4-nitrophenyl)propionic acid (1.5 g, 4.1 mmol) was dissolved in DMF (10 mL). Ethanolamine (0.25 g, 4.1 mmol) was added, followed by HOBT (0.56 g, 4.1 mmol) and EDCI (0.79 g, 4.1 mmol). The yellow mixture was allowed to stir at room temperature overnight. The solvent was removed in vacuo, and water was added to the residue. Upon stirring, the crude product precipitated as a yellow solid. The solid was sequentially stirred in Et2θ and 1 : 1 EtOAc:Et2θ. Filtration gave the product as a pale yellow solid (1.3 g, 79%). MS (ES+) m/e 408 [M+H]+, 430 c) (S)-N-( 1 -(4,5-Dihydro-oxazol-2-yl)-2-(4-nitrophenyl)ethyl)- 1 -naphthamide (S)-N-(l-(2-hydroxyethylcarbamoyl)-2-(4-nitrophenyl)ethyl)-l -naphthamide (0.50 g, 1.2 mmol) was dissolved in N,N-dimethylacetamide (6 mL). Methoxy-carbonylsulfamoyl- triethylammonium hydroxide (0.32 g, 1.4 mmol) was added slowly. The mixture was stirred at room temperature for 10 minutes and heated to 70 °C for one hour. The mixture was cooled to room temperature and the solvent was removed in vacuo. Water was added to the residue and the pH adjusted to 5-6 using saturated aqueous NH4CI. Upon stirring, the crude product precipitated as a yellow solid. The product was filtered and purified by flash chromatography on silica gel ( 100% EtOAc as eluant) to furnish the title compound (0.18 g, 38%).

1H NMR (DMSO) - 8.96 (IH, d, J = 9.0 Hz), 8.19 (2H, d, J = 8.7 Hz), 7.95 (2H, dd, J = 14.3 Hz, J = 8.2 Hz), 7.69-7.61 (3H, m), 7.54-7.49 (2H, m), 7.41-7.34 (2H, m), 5.14-5.11 (IH, m), 4.34 (2H, t, J = 9.5 Hz), 3.83 (2H, t, J = 9.4 Hz), 3.43-3.32 (IH, m), 3.14 (IH, dd, J = 13.5 Hz, J = 11.0 Hz) MS (ES+) m/e 390 [M+H]⁺, 779 IC50 (binding assay) = 0.56uM In order to use the present compound or a pharmaceutically acceptable salt thereof for the treatment of humans and other mammals it is normally formulated in accordance with standard pharmaceutical practice as a pharmaceutical composition.

As used herein, "treatment" of a disease includes, but is not limited to prevention, retardation and prophylaxis of the disease.

The present compound is useful for the treatment of diseases including but not limited to bronchial asthma, allergic rhinitis, nasal polyposis, eczema, conjunctivitis, atopic dermatitis, pruritis and inflammatory bowel disease.

The present compound and its pharmaceutically acceptable salts may be administered in a standard manner for the treatment of the indicated diseases, for example orally, parenterally, sub-lingually, dermally, transdermally, rectally, via inhalation or via buccal administration.

The present compositions and their pharmaceutically acceptable salts which are active when given orally can be formulated as syrups, tablets, capsules and lozenges. A syrup formulation will generally consist of a suspension or solution of the compound or salt in a liquid carrier for example, ethanol, peanut oil. olive oil, glycerine or water with a flavoring or coloring agent. Where the composition is in the form of a tablet, any pharmaceutical carrier routinely used for preparing solid formulations may be used. Examples of such carriers include magnesium stearate, terra alba, talc, gelatin, acacia, stearic acid, starch, lactose and sucrose. Where the composition is in the form of a capsule, any routine encapsulation is suitable, for example using the aforementioned carriers in a hard gelatin capsule shell. Where the composition is in the form of a soft gelatin shell capsule any pharmaceutical carrier routinely used for preparing dispersions or suspensions may be considered, for example aqueous gums, celluloses, silicates or oils, and are incorporated in a soft gelatin capsule shell.

Typical parenteral compositions consist of a solution or suspension of a compound or salt in a sterile aqueous or non-aqueous carrier optionally containing a parenterally acceptable oil, for example polyethylene glycol, polyvinylpyrrolidone, lecithin, arachis oil or sesame oil.

Typical compositions for inhalation are in the form of a solution, suspension or emulsion that may be administered as a dry powder or in the form of an aerosol using a conventional propellant such as dichlorodifluoromethane or trichlorofluoromethane.

A typical suppository formulation comprises the present compound, or a pharmaceutically acceptable salt thereof, which is active when administered in this way, with a binding and/or lubricating agent, for example polymeric glycols, gelatins, cocoa-butter or other low melting vegetable waxes or fats or their synthetic analogs.

Typical dermal and transdermal formulations comprise a conventional aqueous or non-aqueous vehicle, for example a cream, ointment, lotion or paste or are in the form of a medicated plaster, patch or membrane.

Preferably the composition is in unit dosage form, for example a tablet, capsule or metered aerosol dose, so that the patient may administer a single dose.

Each dosage unit for oral administration contains suitably from 0.1 mg to 500 mg/Kg, and preferably from 1 mg to 100 mg/Kg, and each dosage unit for parenteral administration contains suitably from 0.1 mg to 100 mg/Kg, of the present compound or a pharmaceutically acceptable salt thereof calculated as the free acid. Each dosage unit for intranasal administration contains suitably 1-400 mg and preferably 10 to 200 mg per person. A topical formulation contains suitably 0.01 to 5.0% of the present compound.

The daily dosage regimen for oral administration is suitably about 0.01 mg/Kg to 40 mg/Kg, of the present compound, or a pharmaceutically acceptable salt thereof, calculated as the free acid. The daily dosage regimen for parenteral administration is suitably about 0.001 mg/Kg to 40 mg/Kg, of the present compound, or a pharmaceutically acceptable salt thereof, calculated as the free acid. The daily dosage regimen for intranasal administration and oral inhalation is suitably about 10 to about 500 mg/person. The active ingredient may be administered from 1 to 6 times a day, sufficient to exhibit the desired activity.

No unacceptable toxicological effects are expected when the present compound is administered in accordance with the present invention. The biological activity of the present compound is demonstrated by the following tests:

Human eosinophils were purified by standard CD 16 cell depletion using a Miltenyi cell separation column and a magnetic Super Macs magnet. Eosinophils which were >95% pure as assessed by DiffQuick staining and light microscopy were washed in PBS and resuspended in binding buffer (RPMI- 1640 + 25mM Hepes + 0.1% Gelatin + 0.1% sodium azide + 0.008% CHAPS). Into a 96 well plate (Dynatek) 200,000 eosinophils, 0.25 nM 1251-Eotaxin (Amersham Pic), and compound of interest (1 nM to 100 uM) was added. This mixture of cells compound and ligand was allowed to incubate for 60 min at room temperature before harvesting. For harvesting, free ligand from bound ligand was separated over a Packard Unifilter-96 GFC, (cat #6005174) which had been pre-blocked with 1 % polyethylenimine (Sigma Cat # P3143) and 1% Bovine Serum Albumin (BSA) for 2 hours prior to use. After drying, and sealing the plate with Topseal (Packard Topseal A Cat # 6005185) 50 ul of MicroScint (Packard Microscint-20 Cat # 6013621) was added to each well. Bound from free 1251-eotaxin was separated using a Packard Filtermate 196, 96- well plate harvester. To determine total and non-specific binding (NSB) three wells for each condition were set aside. For total binding and NSB, wells received all additions except compound. In addition NSB wells received 200 nM cold eotaxin (PeproTech, Rocky Hill, NJ). Radioactivity associated with the filter was assessed in a Packard Top-count Microplate Scintillation Counter model number 49872V. Percent control binding was assessed by first subtracting the NSB from each well and then expressing the number of counts (CPM) associated with the compound treated sample as a percent of the control binding in the absence of compound addition.

Biological Assay for the determination of the inhibition of intracellular calcium mobilization by the present compound. RBL-2H3 cells expressing the human CCR-3 receptor were grown in cell medium

(EMEM medium with Earl's salts) containing 2mM L-Glutamine, 0.4 mg/ml G418 Sulfate from GIBCO BRL and 10% heat inactivated fetal calf serum from Hyclone Laboratories. The cells were seeded 30,000 cells,/well into 96-well black clear bottom sterile plates from Costar. The seeded plate was incubated overnight at 37^C in 5% CO2* On the day of the assay the cell medium was aspirated before addition of calcium dye loading solution consisting of; 1 mg/mL bovine serum albumin (BSA), 1.5 mM sulfinpyrazone from SIGMA and 4uM Fluo-3 AM dye from Molecular Probes in cell medium, thereafter the 96- well plate was incubated for 1 hour at 37^C. The loading solution containing dye was then aspirated and replaced with fresh solution without dye after which the plate was incubated for a further 10 mins at 37°C.

This solution was aspirated and cells were washed with assay buffer (Kreb's Ringer Henseleit pH 7.4 containing ImM CaC , I mM MgC-2, 1.5 mM sulfinpyrazone and 1.0 mg/mL Gelatin) after aspirating the wash, lOOuLs of fresh assay buffer was added to all the wells and the plate was incubated for five minutes at 37^C before transferring to the Fluorescent Imaging Plate Reader (FLIPR) instrument. The assay and data acquisition were initiated by addition of 50 uLs of sample diluted to a relevant concentration in assay buffer. After 2 mins 75 uLs of human Eotaxin, from PeproTech Inc., diluted to an appropriate concentration in assay buffer with 1 mg mL BSA (no gelatin) was added to the plate and data was acquired for an additional 1.5 mins. Concentration response data for compounds showing inhibition of calcium mobilization were performed in the presence of 33 nM Eotaxin to obtain the IC50 values. IC50 is the concentration of compound needed to inhibit 50% of the Eotaxin response. Animal model for the in vivo evaluation of CCR-3 antagonists (Gonzalo, J.A. et al, Immunity, 1996, 4, 1.) BALs were obtained from Guinea Pigs (+ compound) 24 h after ovalbumin (OA) exposure to eotaxin administered via inhalation. The animals were euthanized by cervical dislocation and exsanguinated. The lungs were lavaged with 50 ml of DulBecco's PBS (5x1 Occ), which was aspirated after a gentle chest massage. The BAL fluid was spun down and the pellet was resuspended in 0.25% NaCl to lyse residual erythrocytes. After centrifugation, the pellet was resuspended again in 0.9% NaCl. After a total cell count, slides were prepared and stained. The cells were differentiated into eosinophils, neutrophils and monocytes by counting a minimum of 200 cells and expressing the results as a percentage of total cells.

Alternatively, OA sensitized Guinea Pigs (+ compound) were exposed to OA via inhalation 24 h after OA exposure and lungs were obtained as described above and assessed for eosinophil infiltration.

The following examples are illustrative but not limiting of the embodiments of the present invention. Formulations for pharmaceutical use incorporating the present compound can be prepared in various forms and with numerous excipients. Examples of such formulations are given below. Example 1 Inhalant Formulation

The present compound, (1 mg to 100 mg) is aerosolized from a metered dose inhaler to deliver the desired amount of drug per use.

Example 2 Tablet Formulation

Tablets/In gredients Per Tablet

1. Present compound 40 mg

2. Corn Starch 20 mg

3. Alginic acid 20 mg 4. Sodium Alginate 20 mg

5. Mg stearate 1.3 mg

Procedure for tablet formulation:

Ingredients 1, 2, 3 and 4 are blended in a suitable mixer/blender. Sufficient water is added portion-wise to the blend with careful mixing after each addition until the mass is of a consistency to permit its conversion to wet granules. The wet mass is converted to granules by passing it through an oscillating granulator using a No. 8 mesh (2.38 mm) screen. The wet granules are then dried in an oven at 140°F (60°C) until dry. The dry granules are lubricated with ingredient No. 5, and the lubricated granules are compressed on a suitable tablet press.

Example 3

Parenteral Formulation

A pharmaceutical composition for parenteral administration is prepared by dissolving an appropriate amount of the present compound in polyethylene glycol with heating. This solution is then diluted with water for injections Ph Eur. (to 100 ml). The solution is then rendered sterile by filtration through a 0.22 micron membrane filter and sealed in sterile containers. All publications, including but not limited to patents and patent applications cited in this specification are herein incorporated by reference as if each individual publication were specifically and individually indicated to be incorporated by reference as though fully set forth.

Claims

What is claimed is:

1. (S)-N-( 1 -(4,5-Dihydro-oxazol-2-yl)-2-(4-nitrophenyl)ethyl)- 1 -naphthamide, or a pharmaceutically acceptable salt thereof.

2. A composition comprising (S)-N-(l-(4,5-Dihydro-oxazol-2-yl)-2-(4- nitrophenyl)ethyl)-l -naphthamide and a pharmaceutically acceptable earner.

3. A method of antagonizing a CCR-3 receptor by administering (S)-N-(l-(4,5- Dihydro-oxazol-2-yl)-2-(4-nitrophenyl)ethyl)-l -naphthamide, or a pharmaceutically acceptable salt thereof.

4. A method of treating an allergic disease comprising administering to a patient in need of treatment a safe and effective amount of (S)-N-(l-(4,5-Dihydro- oxazol-2-yl)-2-(4-nitrophenyl)ethyl)-l -naphthamide, or a pharmaceutically acceptable salt thereof.

5. A method according to claim 4 wherein the disease is selected from the group consisting of bronchial asthma, conjunctivitis, inflammatory bowel disorder, eczema, allergic rhinitis, nasal polyposis, atopic dermatitis and pruritis.