WO2011028740A1

WO2011028740A1 - ENaC BLOCKERS

Info

Publication number: WO2011028740A1
Application number: PCT/US2010/047422
Authority: WO
Inventors: Dramane Ibrahim Laine; Hongxing Yan
Original assignee: Glaxo Group Ltd
Current assignee: Glaxo Group Ltd
Priority date: 2009-09-03
Filing date: 2010-09-01
Publication date: 2011-03-10
Anticipated expiration: 2012-03-03

Abstract

The present invention relates to quaternary amide analogs, pharmaceutical compositions containing them and their use as ENaC blockers.

Description

ENaC BLOCKERS

FIELD OF THE INVENTION The present invention relates to quaternary amide analogs, pharmaceutical compositions containing them and their use as ENaC blockers.

BACKGROUND OF THE INVENTION

The epithelial sodium channel (ENaC) is a membrane-bound ion-channel that is permeable for Li⁺, protons and especially Na⁺. It is a 'constitutively active' channel, i.e. does not require a gating stimulus and is open at rest. ENaC is a heteromeric protein comprised of three different subunits a SCNN1A), β (SCNN1 B), and γ SCNN1 G).

(Pitkanen, OM, Smith, D, O'Brodovich, H, Otulakowski, G. (2001 ) Expression of α, β, and γ-hENaC mRNA in the human nasal, bronchial and distal lung epithelium. Am J Respir Crit Care Med 163: 273-276.) ENaC is located in the apical membrane of polarized epithelial cells particularly in the kidney, the lung and the colon. It is involved in the transepithelial Na⁺-ion transport which it accomplishes together with the Na+/K+-ATPase. It plays a major role in the Na⁺- and K⁺-ion homeostasis of blood, epithelia and

extraepithelial fluids by resorption of Na⁺-ions. The activity of ENaC in colon and kidney is modulated by the mineralcorticoid aldosterone. ENaC can furthermore be found in taste receptor cells, where it plays an important role in salt taste perception.

The airways are lined with a film of liquid about 10 microm deep that is in two layers. Around the cilia is the watery periciliary sol. Over this is a mucous blanket that traps inhaled particles. The mucus layer itself traps inhaled pathogens/particles, allowing their removal via ongoing mucociliary clearance, without the need to trigger a potentially injurious inflammatory response. The low viscosity of the periciliary sol allows the cilia to beat and propel the mucous blanket along airways to the mouth. In large airways, mucus comes predominantly from the mucous glands but also from goblet cells in the surface epithelium. Water is added to the airway surface by gland secretion that is driven by active CI secretion by serous cells. Water is removed by Na transport via ENaC across the surface epithelium. In airway diseases, the balance is shifted from water secretion to mucus secretion. (Widdicombe, JH and Widdicombe JG (2002) Regulation of airway surface liquid. Respir. Physiol. 93: 3-12; Randell, SH and Boucher, RC (2006) Effective Mucus Clearance is Essential for Respiratory Health. Am. J Resp. Cell Mol. Biol. 35: 20- 28, Kunzelmann, K, Schreiber, R, Boucherot, A. (2001 ) Mechanisms of the inhibition of epithelial Na+ channels by CFTR and purinergic stimulation. Kidney Internat. 60:455-461 ) Both periciliary sol volume depletion and mucus layer dehydration contribute to reductions in mucociliary and cough clearance, as cilial movement is restricted as the height of the sol layer becomes more shallow than the height of the cilia themselves, and because the mucus layer is less transportable when dehydrated it ultimately adheres to the airway surface. Mucus stasis results, causing obstruction. Adherent mucus is the nidus for the onset of first intermittent, and then chronic bacterial airway infection.

Blockade of ENaC would reverse this imbalance, thus restoring mucociliary clearance. Blockers of ENaC would be useful in the treatment of cystic fibrosis, COPD, and (non- cystic fibrosis) bronchiectasis. (Sood, N. et al (2003) Increasing Concentration of Inhaled Saline with or without Amiloride. Am J Resp Crit. Care Med. 167:158-163, Brown MA, Leman RJ. Bronchiectasis. In: Chernick V, Boat T, eds. Kendig's disorders of the respiratory tract in children. 6th Edn. Philadelphia, W.B. Saunders, 1998; pp. 538-560, Livraghi, A and Randell, SH (2007) Cystic fibrosis and other respiratory diseases of impaired mucus clearance. Toxicol. Pathol. 35: 1 16-129, Boucher, RC (2007) Evidence for airway surface dehydration as the initiating event in CF airway disease J Int. Med. 261 :5-16, Knowles, MR et al. (1990) A pilot study of aerosolized amiloride for the treatment of lung disease in cystic fibrosis. New Eng. J Med. 322:1 189-1 194, Donaldson, S. (2008) Hydrator therapies for cystic fibrosis lung disease. Ped. Pulmonol. 43:S18-S23, Mall, MA et al. (2008) Development of chronic bronchitis and emphysema in β-epithelial Na+ channel-everexpressing mice. Am J Resp Crit Care Med. 177:730-742, Li, W, XU, YJ, Zhang, ZX. (2004) Detection of the mRNA level of the subunits of amiloride-sensitive Na_ channel in human bronchial epithelium cells from patients with chronic obstructive pulmonary disease. Chinese J Tuberculosis Resp Dis. 27:533-536)

SUMMARY OF THE INVENTION

In one aspect this invention provides for quaternary amide analogs,

pharmaceutically acceptable salts thereof, and pharmaceutical compositions containing them.

In a second aspect, this invention provides for the use of the compounds of Formula (I) as ENaC blockers.

In another aspect, this invention provides for the use of the compounds of Formula (I) for treating and preventing conditions associated with ENaC imbalance.

In yet another aspect, this invention provides for the use of the compounds of Formula (I) for the treatment or prevention of cystic fibrosis, COPD, and non-cystic fibrosis bronchiectasis. The ENaC blocker may be administered alone or in conjunction with one or more other therapeutic agents, eg. agents being selected from the group consisting of: advair, tiotropium, tobi, pulmozyme, azithromycin, hypertonic saline, albuterol, pancreatic enzymes, oral antibiotics, and oral steroids.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides for compounds of Formula (I):

(I)

wherein:

Y is CI or Br;

R-i, R₂, and R₃ are independently Ci_-3 alkyl; wherein R-i is substituted with phenyl;

wherein the phenyl is further substituted with -SCF₃ ; and

X^" is a physiologically acceptable anion selected from the group consisting of: chloride, bromide, iodide, hydroxide, sulfate, nitrate, phosphate, acetate, trifluoroacetate, fumarate, citrate, tartrate, oxalate, succinate, mandelate, methanesulfonate and p-toluenesulfonate.

"Alkyl" refers to a monovalent saturated hydrocarbon chain having the specified number of member atoms. For example, Ci_-3 alkyl refers to an alkyl group having from 1 to 3 member atoms. Alkyl groups may be straight or branched. Representative branched alkyl groups have one, two, or three branches. Alkyl includes methyl, ethyl, and propyl (n- propyl and isopropyl).

"Substituted" in reference to a group indicates that one or more hydrogen atom attached to a member atom within the group is replaced with a substituent selected from the group of defined substituents. It should be understood that the term "substituted" includes the implicit provision that such substitution be in accordance with the permitted valence of the substituted atom and the substituent and that the substitution results in a stable compound (i.e. one that does not spontaneously undergo transformation such as by rearrangement, cyclization, or elimination and that is sufficiently robust to survive isolation from a reaction mixture).

With regard to stereoisomers, the compounds of Formula (I) may have one or more asymmetric carbon atom and may occur as racemates, racemic mixtures and as individual enantiomers or diastereomers. All such isomeric forms are included within the present invention, including mixtures thereof.

As used herein, "pharmaceutically acceptable" refers to those compounds, materials, compositions, and dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

As used herein, the term "a compound of Formula (I)" or "the compound of Formula (I)" refers to one or more compounds according to Formula (I). The compound of Formula (I) may exist in solid or liquid form. In the solid state, it may exist in crystalline or noncrystalline form, or as a mixture thereof. The skilled artisan will appreciate that pharmaceutically acceptable solvates may be formed for crystalline compounds wherein solvent molecules are incorporated into the crystalline lattice during crystallization.

Solvates may involve non-aqueous solvents such as, but not limited to, ethanol, isopropanol, DMSO, acetic acid, ethanolamine, or ethyl acetate, or they may involve water as the solvent that is incorporated into the crystalline lattice. Solvates wherein water is the solvent incorporated into the crystalline lattice are typically referred to as "hydrates." Hydrates include stoichiometric hydrates as well as compositions containing variable amounts of water. The invention includes all such solvates.

The skilled artisan will further appreciate that certain compounds of the invention that exist in crystalline form, including the various solvates thereof, may exhibit polymorphism (i.e. the capacity to occur in different crystalline structures). These different crystalline forms are typically known as "polymorphs." The invention includes all such polymorphs. Polymorphs have the same chemical composition but differ in packing, geometrical arrangement, and other descriptive properties of the crystalline solid state. Polymorphs, therefore, may have different physical properties such as shape, density, hardness, deformability, stability, and dissolution properties. Polymorphs typically exhibit different melting points, IR spectra, and X-ray powder diffraction patterns, which may be used for identification. The skilled artisan will appreciate that different polymorphs may be produced, for example, by changing or adjusting the reaction conditions or reagents, used in making the compound. For example, changes in temperature, pressure, or solvent may result in polymorphs. In addition, one polymorph may spontaneously convert to another polymorph under certain conditions.

The subject invention also includes isotopically-labelled compounds, which are identical to those recited in Formula (I) and following, but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that can be incorporated into compounds of the invention and pharmaceutically acceptable salts thereof include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, sulphur, fluorine, iodine, and chlorine, such as 2H, 3H, 1 1 C, 13C, 14C, 15N, 170, 180, 31 P, 32P, 35S, 18F, 36CI, 1231 and 1251.

Compounds of the present invention and pharmaceutically acceptable salts of said compounds that contain the aforementioned isotopes and/or other isotopes of other atoms are within the scope of the present invention. Isotopically-labelled compounds of the present invention, for example those into which radioactive isotopes such as 3H, 14C are incorporated, are useful in drug and/or substrate tissue distribution assays. Tritiated, i.e., 3H, and carbon-14, i.e., 14C, isotopes are particularly preferred for their ease of preparation and detectability. 1 1 C and 18F isotopes are particularly useful in PET

(positron emission tomography), and 1251 isotopes are particularly useful in SPECT (single photon emission computerized tomography), all useful in brain imaging. Further, substitution with heavier isotopes such as deuterium, i.e., 2H, can afford certain therapeutic advantages resulting from greater metabolic stability, for example increased in vivo half-life or reduced dosage requirements and, hence, may be preferred in some circumstances. Isotopically labelled compounds of formula I and following of this invention can generally be prepared by carrying out the procedures disclosed in the Schemes and/or in the Examples below, by substituting a readily available isotopically labelled reagent for a non-isotopically labelled reagent.

Representative Embodiments

In one embodiment:

Y is CI or Br;

wherein the phenyl is further substituted with -SCF₃ ; and

In another embodiment:

Y is CI;

Ri, R₂, and R₃ are independently Ci alkyl;

The phenyl is substituted in the ortho position; and X^" is a physiologically acceptable anion selected from the group consisting of: chloride, bromide, iodide, hydroxide, sulfate, nitrate, phosphate, acetate, trifluoroacetate, fumarate, citrate, tartrate, oxalate, succinate, mandelate, methanesulfonate and p-toluenesulfonate.

It is to be understood that the present invention covers all combinations of particular groups described hereinabove.

Specific examples of compounds of the present invention include the following: 2-{[(3,5-Diamino-6-chloro-2-pyrazinyl)carbonyl]amino}-N,N-dimethyl-N-({2- [(trifluoromethyl)thio]phenyl}methyl)ethanaminium bromide; and

2-{[(3,5-Diamino-6-chloro-2-pyrazinyl)carbonyl]amino}-N,N-dimethyl-N-({2- [(trifluoromethyl)thio]phenyl}methyl)ethanaminium iodide.

Compound Preparation

The skilled artisan will appreciate that if a substituent described herein is not compatible with the synthetic methods described herein, the substituent may be protected with a suitable protecting group that is stable to the reaction conditions. The protecting group may be removed at a suitable point in the reaction sequence to provide a desired intermediate or target compound. Suitable protecting groups and the methods for protecting and de-protecting different substituents using such suitable protecting groups are well known to those skilled in the art; examples of which may be found in T. Greene and P. Wuts, Protecting Groups in Chemical Synthesis (3rd ed.), John Wiley & Sons, NY (1999). In some instances, a substituent may be specifically selected to be reactive under the reaction conditions used. Under these circumstances, the reaction conditions convert the selected substituent into another substituent that is either useful as an intermediate compound or is a desired substituent in a target compound.

The synthesis of the compounds of the general Formula (I) and pharmaceutically acceptable derivatives and salts thereof may be accomplished as outlined below in Schemes 1- 2. In the following description, the groups are as defined above for compounds of Formula (I) unless otherwise indicated. Starting materials are commercially available or are made from commercially available starting materials using methods known to those skilled in the art.

The compounds of Formula (I) can be prepared as depicted in Scheme 1.

Treatment of methyl 3,5-diamino-6-chloro-2-pyrazinecarboxylate 1 with a monosubstituted ehylenediamine derivative 2 gives the substituted amide 3, which can be further elaborated to the tertiary amine 4 by reductive amination with a suitably substituted aldehyde or ketone. Compound 4 may also be prepared by direct treatment of 1 with a suitably disubstituted ethylenediamine derivative 5. Quaternization of the tertiary amine 4 with a suitable alkylating agent R₃X affords the desired compound 5 of Formula (I).

Scheme 1

Reagents and conditions: a) 2, MeOH, microwave reactor; b) Ketone or aldehyde,

NaBH(OAC)₃, DMSO; c)) 5_± MeOH, microwave reactor; d) R₃X Biological Activity

As stated above, the compounds according to Formula (I) are ENaC blockers, and are useful in the treatment or prevention of cystic fibrosis, COPD, (non-cystic fibrosis) bronchiectasis., and obliterative bronchiolitis.

The biological activity of the compounds according to Formula (I) can be determined using any suitable assay for determining the activity of a candidate compound as an ENaC blockers, as well as tissue and in vivo models.

The biological activity of the compounds of Formula (I) are demonstrated by the following tests.

ENaC lonWorks Assay

Cellular Reagent

The potency of ENaC inhibitors were assessed in recombinant cells expressing the three ENaC subunits required to give functional ENaC sodium currents, the a-subunit (SCNN 1 A, NCBI Reference Sequence Accession NM_001 159576), the β-subunit

(SCNN 1 B, NCBI Reference Sequence Accession NM_000336) and the γ-subunit

(SCNN 1 G, NCBI Reference Sequence Accession NM_001039). A HEK293 cell line was generated which stably co-expressed variants of the SCN N 1 B and SCN N 1 G subunits, SCNN 1 B-(P618A,Y620L) and SCNN 1 G-(P624stop). HEK293 cells were grown in DMEM- F12 with 10%FBS and 2mM glutamine. The stable HEK293- SCNN 1 B-(P618A,Y620L)/ SCNN 1 G-(P624stop) cell line was generated following transfection (by Amaxa

Nucleofection according to manufacturer's protocols) of CMV expression vectors pBacMIRE-SCNN1 B-(P618A,Y620L) and pCIHGW- SCNN1 G-(P624stop) and co- selection in 500micrograms/ml Geneticin-G418 and 200micrograms/ml hygromycin-B. The clonal cell line was isolated by single cell dilution cloning. In order to reconstitute the functional ENaC channel in this cell line the wild-type SCNN1 A subunit was introduced by BacMam virus transduction. The SCNNIA-BacMam virus was prepared according to the protocols described in Condreay et al (1999, Proc. Natl. Acad. Sci. USA 96:127-132). Transduction of the HEK293- SCNN1 B-(P618A,Y620L)/ SCNN1 G-(P624stop) cell line with the SCNNIA-BacMam virus was carried out 24 hours prior to assay by adding 10% virus (at approximately 1 .37 x10⁸ pfu/ml) to 1 x10⁷ cells, and continuing to incubate at 37°C, in the presence of the selection agents (G418 & hygromycin).

I on Works Assay

Recombinant cell cultures were prepared according to the platform manufacturers protocol (MDC Corp, Sunnyvale, CA / Finkel et al, J. Biomol. Screen. 2006; 1 1 : 488-496) using TrypLE™ Express (Gibco / Invitrogen, Paisley, Renfrewshire, UK, Cat # 12604) in place of the specified Versene solution. Sodium-ion flux across the cell membrane via ENaC generates a measurable current (l_ENac) that is assessed by the lonWorks platform (lonWorks Quattro™ model, MDC Corp, Sunnyvale, CA). Inhibition of the l_ENac with ENaC blockers was used to assess their potency. l_ENac was measured (lonWorks PPC-mode) under voltage clamp conditions from a mean of up to 64 cells in each of up to 384 wells both before and after addition of compounds. The effect of the compound is calculated by normalizing the post compound addition data to the naive state and comparing this against high (100% block by 10μΜ amiloride gold standard) and low ("zero"-effect; 1 % v/v DMSO) control data.

Stock test compounds were prepared as 10mM solutions in DMSO. Concentration se curves were constructed with appropriate serial dilutions of the stock compound in DMSO. These serial dilutions were further diluted 1 :30 times in to External buffer

(composition, milli-molar: sodium gluconate, 120; sodium chloride, 20; potassium chloride, 5; 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid, N-(2-Hydroxyethyl)piperazine-N'- (2-ethanesulfonic acid) (HEPES), 10; calcium chloride, 2; magnesium chloride, 1 , and pluronic F-127 0.03% w/v, pH 7.35 - 7.40), and added to a 384 well disposable plate (compound plate), which was loaded in to the lonWorks platform. The lonWorks was programmed to transfer an appropriate volume from the compound plate to the assay well to give a further 1 :3 diltution, at approximately 3 mins prior to the post-compound reading. The data corresponding to the series of dilutions of test compounds were then used to estimate the concentration of compound required to produce a 50 % decrease of the ENaC-mediated current response (IC₅₀). Potency was reported as plC₅₀ (-log IC₅₀).

In each experiment curves for amiloride and phenamil standards were included, and used to monitor data quality. Internal buffer composition was (milli-molar): potassium gluconate (120); potassium chloride (20); HEPES (10); sodium chloride (5); magnesium chloride (2) & calcium chloride (1 ), pH 7.35 -7.40. All chemical reagents, including standards were purchased from Sigma Chemical Co.

Compounds of the formula I have plC50's > 4.5. Ussing Chamber Assay

The potency of ENaC inhibitors was assessed in commercially available (MatTek Corporation, 200 Homer Ave., Ashland, MA 01721 ) human bronchial epithelial cells grown in Air-Liquid Interface (Cat # AIR-1 12-SNP). Epithelial cell cultures were mounted in Ussing Chambers filled with Krebs bicarbonate buffer solution (KBS) continuously bubbled with 95% oxygen/5% carbon dioxide. Sodium transport across the epithelium via ENaC generates a measurable current that is assessed my measuring short-circuit current (Isc). Inhibition of the Isc with ENaC blockers was used to assess their potency. Isc was measured (Physiologic Instruments, Model VCC MC6) under voltage clamp conditions so that active ion transport was the dominant ion transport process generating Isc.

Test compounds were prepared as 10mM solutions in DMSO and working solutions were prepared by serial 10 fold dilutions in KBS. Cumulatively increasing concentrations (0.5 logs) of test compound are added to the chamber bathing the apical surface of the epithelium. Concentration response curves were used to calculate the concentration of compound that causes a 50 % decrease of the IsC (EC 50). Potency was reported as plC50 (-log EC50).

In each experiment 1 culture was used to asses the potency of the gold-standard ENaC blocker Amiloride as a positive control.

Methods of Use

The compounds of the invention are ENac blockers, and are useful in the treatment or prevention of cystic fibrosis, COPD, non-cystic fibrosis bronchiectasis, and obliterative bronchiolitis. Accordingly, in another aspect the invention is directed to methods of treating such conditions.

The methods of treatment of the invention comprise administering a safe and effective amount of a compound according to Formula I or a pharmaceutically-acceptable salt thereof to a patient in need thereof. As used herein, "treat" in reference to a condition means: (1 ) to ameliorate or prevent the condition or one or more of the biological manifestations of the condition, (2) to interfere with (a) one or more points in the biological cascade that leads to or is responsible for the condition or (b) one or more of the biological manifestations of the condition, (3) to alleviate one or more of the symptoms or effects associated with the condition, or (4) to slow the progression of the condition or one or more of the biological manifestations of the condition.

As indicated above, "treatment" of a condition includes prevention of the condition. The skilled artisan will appreciate that "prevention" is not an absolute term. In medicine, "prevention" is understood to refer to the prophylactic administration of a drug to substantially diminish the likelihood or severity of a condition or biological manifestation thereof, or to delay the onset of such condition or biological manifestation thereof.

As used herein, "safe and effective amount" in reference to a compound of the invention or other pharmaceutically-active agent means an amount of the compound sufficient to treat the patient's condition but low enough to avoid serious side effects (at a reasonable benefit/risk ratio) within the scope of sound medical judgment. A safe and effective amount of a compound will vary with the particular compound chosen (e.g.

consider the potency, efficacy, and half-life of the compound); the route of administration chosen; the condition being treated; the severity of the condition being treated; the age, size, weight, and physical condition of the patient being treated; the medical history of the patient to be treated; the duration of the treatment; the nature of concurrent therapy; the desired therapeutic effect; and like factors, but can nevertheless be routinely determined by the skilled artisan.

As used herein, "patient" refers to a human or other animal.

The compounds of the invention may be administered by any suitable route of administration, including both systemic administration and topical administration.

Systemic administration includes oral administration, parenteral administration, transdermal administration, rectal administration, and administration by inhalation.

Parenteral administration refers to routes of administration other than enteral,

transdermal, or by inhalation, and is typically by injection or infusion. Parenteral administration includes intravenous, intramuscular, and subcutaneous injection or infusion. Inhalation refers to administration into the patient's lungs whether inhaled through the mouth or through the nasal passages. Topical administration includes application to the skin as well as intraocular, otic, intravaginal, and intranasal

administration. The compounds of the invention may be administered once or according to a dosing regimen wherein a number of doses are administered at varying intervals of time for a given period of time. For example, doses may be administered one, two, three, or four times per day. Doses may be administered until the desired therapeutic effect is achieved or indefinitely to maintain the desired therapeutic effect. Suitable dosing regimens for a compound of the invention depend on the pharmacokinetic properties of that compound, such as absorption, distribution, and half-life, which can be determined by the skilled artisan. In addition, suitable dosing regimens, including the duration such regimens are administered, for a compound of the invention depend on the condition being treated, the severity of the condition being treated, the age and physical condition of the patient being treated, the medical history of the patient to be treated, the nature of concurrent therapy, the desired therapeutic effect, and like factors within the knowledge and expertise of the skilled artisan. It will be further understood by such skilled artisans that suitable dosing regimens may require adjustment given an individual patient's response to the dosing regimen or over time as individual patient needs change.

Typical daily dosages may vary depending upon the particular route of

administration chosen. Typical dosages for oral administration range from 1 mg to 1000 mg per person per dose.

Additionally, the compounds of the invention may be administered as prodrugs. As used herein, a "prodrug" of a compound of the invention is a functional derivative of the compound which, upon administration to a patient, eventually liberates the compound of the invention in vivo. Administration of a compound of the invention as a prodrug may enable the skilled artisan to do one or more of the following: (a) modify the onset of the compound in vivo; (b) modify the duration of action of the compound in vivo; (C) modify the transportation or distribution of the compound in vivo; (d) modify the solubility of the compound in vivo; and (e) overcome or overcome a side effect or other difficulty encountered with the compound. Typical functional derivatives used to prepare prodrugs include modifications of the compound that are chemically or enzymatically cleaved in vivo. Such modifications, which include the preparation of phosphates, amides, esters, thioesters, carbonates, and carbamates, are well known to those skilled in the art.

Compositions

The compounds of the invention will normally, but not necessarily, be formulated into pharmaceutical compositions prior to administration to a patient. Accordingly, in another aspect the invention is directed to pharmaceutical compositions comprising a compound of the invention and one or more pharmaceutically-acceptable excipient. The pharmaceutical compositions of the invention may be prepared and packaged in bulk form wherein a safe and effective amount of a compound of the invention can be extracted and then given to the patient such as with powders or syrups. Alternatively, the pharmaceutical compositions of the invention may be prepared and packaged in unit dosage form wherein each physically discrete unit contains a safe and effective amount of a compound of the invention. When prepared in unit dosage form, the pharmaceutical compositions of the invention typically contain from 1 mg to 1000 mg.

The pharmaceutical compositions of the invention typically contain one compound of the invention. However, in certain embodiments, the pharmaceutical compositions of the invention contain more than one compound of the invention. For example, in certain embodiments the pharmaceutical compositions of the invention contain two compounds of the invention. In addition, the pharmaceutical compositions of the invention may optionally further comprise one or more additional pharmaceutically active compounds.

As used herein, "pharmaceutically-acceptable excipient" means a

pharmaceutically acceptable material, composition or vehicle involved in giving form or consistency to the pharmaceutical composition. Each excipient must be compatible with the other ingredients of the pharmaceutical composition when commingled such that interactions which would substantially reduce the efficacy of the compound of the invention when administered to a patient and interactions which would result in

pharmaceutical compositions that are not pharmaceutically acceptable are avoided. In addition, each excipient must of course be of sufficiently high purity to render it pharmaceutically-acceptable.

The compound of the invention and the pharmaceutically-acceptable excipient or excipients will typically be formulated into a dosage form adapted for administration to the patient by the desired route of administration. For example, dosage forms include those adapted for (1 ) oral administration such as tablets, capsules, caplets, pills, troches, powders, syrups, elixers, suspensions, solutions, emulsions, sachets, and cachets; (2) parenteral administration such as sterile solutions, suspensions, and powders for reconstitution; (3) transdermal administration such as transdermal patches; (4) rectal administration such as suppositories; (5) inhalation such as dry powders, aerosols, suspensions, and solutions; and (6) topical administration such as creams, ointments, lotions, solutions, pastes, sprays, foams, and gels.

Suitable pharmaceutically-acceptable excipients will vary depending upon the particular dosage form chosen. In addition, suitable pharmaceutically-acceptable excipients may be chosen for a particular function that they may serve in the composition. For example, certain pharmaceutically-acceptable excipients may be chosen for their ability to facilitate the production of uniform dosage forms. Certain pharmaceutically- acceptable excipients may be chosen for their ability to facilitate the production of stable dosage forms. Certain pharmaceutically-acceptable excipients may be chosen for their ability to facilitate the carrying or transporting of the compound or compounds of the invention once administered to the patient from one organ, or portion of the body, to another organ, or portion of the body. Certain pharmaceutically-acceptable excipients may be chosen for their ability to enhance patient compliance.

Suitable pharmaceutically-acceptable excipients include the following types of excipients: Diluents, fillers, binders, disintegrants, lubricants, glidants, granulating agents, coating agents, wetting agents, solvents, co-solvents, suspending agents, emulsifiers, sweetners, flavoring agents, flavor masking agents, coloring agents, anticaking agents, hemectants, chelating agents, plasticizers, viscosity increasing agents, antioxidants, preservatives, stabilizers, surfactants, and buffering agents. The skilled artisan will appreciate that certain pharmaceutically-acceptable excipients may serve more than one function and may serve alternative functions depending on how much of the excipient is present in the formulation and what other ingredients are present in the formulation.

Skilled artisans possess the knowledge and skill in the art to enable them to select suitable pharmaceutically-acceptable excipients in appropriate amounts for use in the invention. In addition, there are a number of resources that are available to the skilled artisan which describe pharmaceutically-acceptable excipients and may be useful in selecting suitable pharmaceutically-acceptable excipients. Examples include Remington's Pharmaceutical Sciences (Mack Publishing Company), The Handbook of Pharmaceutical Additives (Gower Publishing Limited), and The Handbook of Pharmaceutical Excipients (the American Pharmaceutical Association and the Pharmaceutical Press).

The pharmaceutical compositions of the invention are prepared using techniques and methods known to those skilled in the art. Some of the methods commonly used in the art are described in Remington's Pharmaceutical Sciences (Mack Publishing

Company).

In one aspect, the invention is directed to a solid oral dosage form such as a tablet or capsule comprising a safe and effective amount of a compound of the invention and a diluent or filler. Suitable diluents and fillers include lactose, sucrose, dextrose, mannitol, sorbitol, starch (e.g. corn starch, potato starch, and pre-gelatinized starch), cellulose and its derivatives (e.g. microcrystalline cellulose), calcium sulfate, and dibasic calcium phosphate. The oral solid dosage form may further comprise a binder. Suitable binders include starch (e.g. corn starch, potato starch, and pre-gelatinized starch), gelatin, acacia, sodium alginate, alginic acid, tragacanth, guar gum, povidone, and cellulose and its derivatives (e.g. microcrystalline cellulose). The oral solid dosage form may further comprise a disintegrant. Suitable disintegrants include crospovidone, sodium starch glycolate, croscarmelose, alginic acid, and sodium carboxymethyl cellulose. The oral solid dosage form may further comprise a lubricant. Suitable lubricants include stearic acid, magnesuim stearate, calcium stearate, and talc.

Compounds of Formula (I) may be administered parenterally, that is by

intravenous, intramuscular, subcutaneous intranasal, intrarectal, intravaginal or intraperitoneal administration. The subcutaneous and intramuscular forms of parenteral administration are generally preferred. Appropriate dosage forms for such administration may be prepared by conventional techniques. Compounds of Formula (I) may also be administered by inhalation, that is by intranasal and oral inhalation administration.

Appropriate dosage forms for such administration, such as an aerosol formulation or a metered dose inhaler, may be prepared by conventional techniques.

In one embodiment of the present invention, the agents of the present invention are delivered via oral inhalation or intranasal administration. Appropriate dosage forms for such administration, such as an aerosol formulation or a metered dose inhaler, may be prepared by conventional techniques.

For administration by inhalation the compounds may be delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g. dichlorodifluoromethane, trichlorofluoromethane,

dichlorotetrafluoroethane, a hydrofluoroalkane such as tetrafluoroethane or

heptafluoropropane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of e.g. gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of a compound of the invention and a suitable powder base such as lactose or starch.

Dry powder compositions for topical delivery to the lung by inhalation may, for example, be presented in capsules and cartridges of for example gelatine or blisters of for example laminated aluminium foil, for use in an inhaler or insufflator. Powder blend formulations generally contain a powder mix for inhalation of the compound of the invention and a suitable powder base (carrier/diluent/excipient substance) such as mono-, di or poly-saccharides (e.g. lactose or starch). Use of lactose is preferred.

The compounds of the invention may be milled using known milling procedures such as wet milling to obtain a particle size appropriate for tablet formation and for other formulation types. Finely divided (nanoparticulate) preparations of the compounds of the invention may be prepared by processes known in the art, for example see International Patent Application No. WO 02/00196 (SmithKline Beecham).

The compounds may be administered alone or in conjunction with one or more other therapeutic agents, said agents being selected from the group consisting of advair, tiotropium, tobi, pulmozyme, azithromycin, hypertonic saline, albuterol, pancreatic enzymes, oral antibiotics, and oral steroids.

EXAMPLES

The following examples illustrate the invention. These examples are not intended to limit the scope of the present invention, but rather to provide guidance to the skilled artisan to prepare and use the compounds, compositions, and methods of the present invention. While particular embodiments of the present invention are described, the skilled artisan will appreciate that various changes and modifications can be made without departing from the spirit and scope of the invention.

In the Examples:

Chemical shifts are expressed in parts per million (ppm) units. Coupling constants (J) are in units of hertz (Hz). Splitting patterns describe apparent multiplicities and are designated as s (singlet), d (doublet), t (triplet), q (quartet), dd (double doublet), dt (double triplet), m (multiplet), br (broad).

Flash column chromatography was performed on silica gel.

The naming program used is ACD Name Pro 6.02.

Purifications and analyses of materials were carried out using the following instruments: LC-MS analysis

The LC/MS of Intermediates and Examples were performed using the following equipment and conditions:

Liquid Chromatoqraph:

System: Shimadzu LC system with SCL-10A Controller and dual UV detector

Autosampler: Leap CTC with a Valco six port injector

Column: Aquasil/Aquasil (C18 40x1 mm)

Inj. Volume: 2.0 μί

Solvent A: H₂0, 0.02% TFA

Solvent B: MeCN, 0.018% TFA

Gradient: linear Channel A: UV 214 nm

Channel B: ELS

Step Time (min) Dura.(min) Flow (μΙ_/Γηίη) Sol.A Sol.B

0 0.00 0.00 300.00 95.00 5.00

1 0.00 0.01 300.00 95.00 5.00

2 0.01 3.20 300.00 10.00 90.00

3 3.21 1.00 300.00 10.00 90.00

4 4.21 0.10 300.00 95.00 5.00

5 4.31 0.40 300.00 95.00 5.00

Mass Spectrometer:

Instrument: PE Sciex Single Quadrupole LC/MS API-150

Polarity: Positive

Acquisition mode: Profile

Preparatory HPLC

Automated preparatory HPLC purifications were conducted using a Gilson® semi- preparative HPLC system under the following conditions:

Column: 75 x 33mm I. D. , S-5um, 12nm

Flow rate: 30mL/min

Injection Volume: 0.800 mL

Room temperature

The eluent was a mixture composed of solvents A and B. Either one of three different solvent combinations were used:

TTFA conditions

Solvent A: 0.1 % trifluoroacetic acid in water

Solvent B: 0.1 % trifluoroacetic acid in acetonitrile

NH₄OH conditions

Solvent A: 0 1 % NH4OH in water

Solvent B: 0 1 % NH4OH in acetonitrile

Neutral conditions

Solvent A: 0.1 % NH₄OH in water

Solvent B: 0.1 % NH₄OH in acetonitrile

The following abbreviations and terms had the indicated meanings throughout: aq. Aqueous DMSO Dimethylsulfoxide

eq. Equivalent

HPLC High pressure liquid chromatography

LC/MS Liquid chromatography-Mass spectrometry

Me Methyl

Rt Retention time

Intermediate 1

3,5-Diamino-6-chloro-N-[2-(methylamino)ethyl]-2-pyrazinecarboxamide

To a solution of methyl 3,5-diamino-6-chloro-2-pyrazinecarboxylate (0.506 g, 2.5 mmol) in methanol (2.5 mL) was added (2-aminoethyl)methylamine (1 .090 mL, 12.50 mmol). The reaction mixture was heated in a microwave reactor at 120 °C for a total of 4 h. The solvent was concentrated under vacuum, and then MeOH (5 mL) and MeCN (10 mL) were added. The mixture was then filtered, washed with MeCN (~2 mL) and dried under high vacuum over oil pump to give the title compound (0.1222 g, 19.98 %). The above filtrate was concentrated under vacuum, and then MeCN (5 mL) was added. The mixture was put in a freezer (~ -20 °C) overnight (-15 h). The resulting crystallized material was filtered, washed with MeCN (~5 mL) then dried under high vacuum to give another batch of the title compound (0.3162 g, 51 .7 %) as an off-white solid. LC/MS: m/z 245.2 (M+H)⁺, Rt 0.37 min.

Intermediate 2

3,5-Diamino-6-chloro-N-[2-(dimethylamino)ethyl]-2-pyrazinecarboxamide

To a solution of methyl 3,5-diamino-6-chloro-2-pyrazinecarboxylate (1 g, 4.94 mmol) in methanol (2.5 mL) was added 2-(dimethylamino)ethylamine (2.70 mL, 24.68 mmol). The reaction mixture was heated in a microwave reactor at 120 °C for a total of 6 h. The solvent was evaporated down and the solid was washed with MeOH (8 + 4 mL) then dried under high vacuum to afford the title compound (0.9879 g, 3.82 mmol, 77 % yield).

LC/MS: m/z 259.0 (M+H)⁺, Rt 0.45 min.

Intermediate 3

3,5-Diamino-6-chloro-N-{2-[methyl({2-[(trifluoromethyl)thio]phenyl}methyl)amino]ethyl}-2- pyrazinecarboxamide

To a solution of 3,5-diamino-6-chloro-N-[2-(methylamino)ethyl]-2-pyrazinecarboxamide (48.9 mg, 0.200 mmol) in DMSO (1 mL) was added 2-[(trifluoromethyl)thio]benzaldehyde (41.2 mg, 0.200 mmol) and then sodium triacetoxyborohydride (63.6 mg, 0.300 mmol). The resulting mixture was stirred at room temperature for 66 h. After removal of the solvent, the crude residue was purified by preparatory HPLC (basic conditions). The relevant fractions were combined and concentrated under vacuum to afford the title compound (36 mg, 41 %). LC/MS: m/z 433.0 (M+H)⁺ ; Rt 0.74 min.

Example 1

2-{[(3,5-Diamino-6-chloro-2-pyrazinyl)carbonyl]amino}-N,N-dimethyl-N-({2- trifluoromethyl)thio]phenyl}methyl)ethanaminium bromide

To a solution of 3,5-diamino-6-chloro-N-[2-(dimethylamino)ethyl]-2-pyrazinecarboxamide (51.7 mg, 0.2 mmol) in acetone (2 mL) was added 2-(bromomethyl)phenyl trifluoromethyl sulfide (65.1 mg, 0.240 mmol). The resulting reaction mixture was stirred at room temperature for 93 h. The precipitate in the reaction mixture was filtered out, washed with acetone (0.5 mL) and dried under high vacuum to afford the title compound (96.5 mg, 91 % yield). LC/MS: m/z 449 (M)⁺, Rt 0.75 min. ¹H NMR (400 MHz, CD₃OD) δ ppm 8.01 (1 H, d, J=7.53 Hz), 7.90 (1 H, dd, J=7.65, 1 .38 Hz), 7.79 (1 H, td, J=7.53, 1 .51 Hz), 7.69 - 7.76 (1 H, m),4.98 (2 H, s), 3.89 (2 H, t, J=6.40 Hz), 3.60 - 3.69 (2 H, m), 3.16 (6 H, s) Example 2

2-{[(3,5-Diamino-6-chloro-2-pyrazinyl)carbonyl]amino}-N,N-dimethyl-N-({2

[(trifluoromethyl)thio]phenyl}methyl)ethanaminium iodide

To a solution of 3,5-diamino-6-chloro-N-{2-[methyl({2-

[(trifluoromethyl)thio]phenyl}methyl)amino]ethyl}-2-pyrazinecarboxamide (33.3 mg, 0.077 mmol) in acetone (1 .5 mL) was added methyl iodide (9.58 μΙ_, 0.153 mmol). The mixture was stirred at room temperature for 19 h. The precipitate was filtered out and dried to afford the title compound (12 mg, 28 % yield). LC/MS: m/z 448.8 (M)⁺, Rt 0.73 min. ¹H

NMR (400MHz , CD₃OD) δ ppm 7.90 (1 H, d, J=7.5 Hz), 7.81 (1 H, dd, J=1.5, 7.5 Hz),7.73 - 7.60 (2 H, m), 4.89 (2 H, s), 3.79 (2 H, t, J=6.4 Hz), 3.58 - 3.53 (2 H, m), 3.07 (6 H, s)

Claims

What is claimed is:

1. A compound of Formula (I):

(I)

wherein:

Y is CI or Br;

R-i, R₂, and R₃ are independently Ci_-3 alkyl, wherein R-i is substituted with phenyl;

wherein the phenyl is further substituted with -SCF₃ ; and

X^" is a physiologically acceptable anion selected from the group consisting of:

chloride, bromide, iodide, hydroxide, sulfate, nitrate, phosphate, acetate, trifluoroacetate, fumarate, citrate, tartrate, oxalate, succinate, mandelate, methanesulfonate and p-toluenesulfonate.

2. A compound of claim 1 wherein:

Y is CI;

Ri, R₂, and R₃ are independently Ci alkyl;

the phenyl is substituted in the ortho position; and

3. A compound of claim 1 which is 2-{[(3,5-Diamino-6-chloro-2- pyrazinyl)carbonyl]amino}-N,N-dimethyl-N-({2- [(trifluoromethyl)thio]phenyl}methyl)ethanaminium bromide; or

2-{[(3,5-Diamino-6-chloro-2-pyrazinyl)carbonyl]amino}-N,N-dimethyl-N-({2- [(trifluoromethyl)thio]phenyl}methyl)ethanaminium iodide

4. A pharmaceutical composition comprising a compound of any of claims 1 to 3 and a pharmaceutically acceptable carrier or excipient.

5. A method of treating cystic fibrosis, COPD, and (non-cystic fibrosis) bronchiectasis, which comprises administering to a human in need thereof, a compound of any of claims 1 to 3.

6. A method according to claim 5 wherein the compound is administered orally.

7. A method according to claim 5 wherein the compound is inhaled.

8. A method according to claim 5 wherein the compound is administered in conjuction with with one or more other therapeutic agents selected from the group consisting of: advair, tiotropium, tobi, pulmozyme, azithromycin, hypertonic saline, albuterol, pancreatic enzymes, oral antibiotics, and oral steroids.