WO2011086347A1 - Pyrrolidine iminosugars used in the treatment of cystic fibrosis - Google Patents

Abstract

A compound of formula (I) wherein: R¹ is selected from H; linear or branched, substituted or unsubstituted alkyl, alkenyl, alkynyl and aralkyl and wherein the optional substitution may be with one or more groups independently selected from: -OH; -F; -Cl; -Br; -I; -NH₂; alkylamino; dialkylamino; linear or branched alkyl, alkenyl, alkynyl and aralkyl; aryl; heteroaryl; linear or branched alkoxy; aryloxy; aralkoxy; -(alkylene)oxy(alkyl); -CN; -NO₂; - COOH; -COO(alkyl); -COO(aryl); -C(O)NH(alkyl); -C(O)NH(aryl); sulfonyl; alkylsulfonyl; arylsulfonyl; sulfamoyl; alkylsulfamoyl; alkylthio; alkylsulfonamide; arylsulfonamide; -NHNH₂; and -NHOH; or a bioisostere, pharmaceutically acceptable salt or derivative thereof, finds application in the treatment of cystic fibrosis.

Description

PYRROLIDINE IMINOSUGARS USED IN THE TREATMENT OF CYSTIC FIBROSIS

Field of the Invention

The present invention relates to novel compounds, to compositions (including

pharmaceutical compositions) containing these compounds and to methods of treating cystic fibrosis using the compounds and compositions.

Background to the Invention

Cystic fibrosis (referred to herein as "CF", and also known as mucoviscidosis) is a common hereditary disease which affects the entire body, causing progressive disability and often early death. Respiratory dysfunction is the most serious symptom and results from frequent lung infections. Most individuals die in their 20s and 30s from lung failure and lung transplantation is often necessary as CF worsens. A multitude of other symptoms, including sinus infections, poor growth, diarrhoea and infertility result from the effects of CF on other parts of the body.

CF is an autosomal recessive disease caused by mutation of the cystic fibrosis

transmembrane conductance regulator (CFTR) gene. CFTR is a chloride ion channel important in creating sweat, digestive juices and mucus and cystic fibrosis occurs when the mutation leads to reduced ion channel activity (via increased clearance of the misfolded CFTR proteins). This leads to sodium hyperabsorption by the airways, profound lung inflammation and dysregulation of calcium homeostasis.

The most common mutation, comprising over 60% of all mutant alleles, results in the deletion of phenylalanine in the ATP binding cassette at position 508 of the CFTR protein. The resultant AF508 CFTR polypeptide is expressed as a large, misfolded nascent polypeptide which is prematurely destroyed via the ubiquitin pathway but which aggregates following defective processing in the translocation machinery.

Recently, it has been reported that several iminosugars, including N-butyldeoxynojirimycin (a.k.a. Miglustat, Λ/Β-DNJ and Zavesca®), bind to nascent mutant CFTR protein during cotranslational processing. This appears to restore CFTR function via a postulated pharmacological chaperone mechanism and/or glucosidase inhibition (see Zhang et al. (2003) J. Biol. Chem. 278: 51232-51242; Norez et al. (2006) FEBS Lett. 580: 2081-2086); Dechecchi ef al. (2008) J. Cystic Fibrosis 7(6): 555-565; Antigny et al. (2008) Cell Calcium 43(2): 175-193; Norez et al. (2008) J. Pharm. Exp. Ther. 325(1 ): 89-99; Cox et al. (2007) Medicinal use of iminosugars, in Iminosugars From Synthesis to Therapeutic Applications: Compain, Philippe / Martin, Olivier R. (eds.) ISBN-13: 978-0-470-03391-3 - John Wiley & Sons, pages 318-319); Asano, N. (2009) Cell. Mol. Life Sci. 66: 1479-1492.

More recently, Lubamba et al. (2009) Am. J. Resp. Crit. Care 179 (11): 1022-1028 have shown that nasal delivery of Λ Β-DNJ, at picomolar doses, normalizes sodium and CFTR- dependent chloride transport in AF508 transgenic mice, while Norez et al. (2009) Am. J. Resp. Cell Mol. Biol. 41 (2): 217-225 have shown that a human respiratory epithelial cell line of the AF508 CFTR phenotype can acquire a non-CF-like phenotype when chronically treated with low concentrations of Λ/Β-DNJ.

WO2005/046672 describes the use of various DNJ derivatives having glucosidase inhibitory activity in the treatment of CF.

WO2007/ 23403 describes the use of various iminosugars (and in particular DNJ derivatives) having non-lysosomal glucosylceramidase (glucosidase beta (bile acid) 2) inhibitory activity in the treatment of CF.

In October 2007, Actelion Pharmaceuticals Limited initiated a Phase 11 a proof-of-concept clinical trial with Λ/Β-DNJ for CF involving 25 patients affected by the AF508 CFTR mutation.

Notwithstanding the promising findings and clinical research summarized above, there is at present no cure for CF. Current treatments are merely palliative: they include antibiotic treatment (for lung infections), chest physiotherapy/mechanical expecoration (for mucus accumulation), surgery and mechanical ventilation. There is therefore a need for improved treatments for CF which at least partially reverse the underlying proteostatic dysfunction.

Summary of the Invention

The present inventors have now unexpectedly discovered that the carbon-branched pyrrolidine iminosugar (3S, 4S)-3-(hydroxymethyl)pyrrolidine-3,4-diol (1 ,4-dideoxy-2- hydroxymethyl-1 ,4-imino-L-threitol, referred to herein as isoLAB) exhibits superior activity to Λ/Β-DNJ in assays for the restoration of AF508 CFTR function. Moreover, its

glycosidase inhibitory profile is strikingly different to Λ/Β-DNJ: no inhibitory activity has been detected against a range of different glycosidases (see infra), whereas Λ/Β-D J exhibits potent inhibition of a wide range of a-glucosidases (including those involved in digestive and ER processing functions). Thus, isoLAB and its derivatives is likely to exhibit an improved protective/therapeutic index compared with Λ/Β-DNJ.

IsoLAB and its bioisosteres, pharmaceutically acceptable salts and pharmaceutically acceptable derivatives therefore find application in improved treatments for CF, as described herein.

Therefore, in a first aspect of the present invention, there is provided a compound of Formula I:

wherein R¹ is selected from H; linear or branched, substituted or unsubstituted alkyl, alkenyl, alkynyl and aralkyl and wherein the optional substitution may be with one or more groups independently selected from: -OH; -F; -CI; -Br; -I; -NH₂; alkylamino; dialkylamino; linear or branched alkyl, alkenyl, alkynyl and aralkyl; aryl; heteroaryl; linear or branched alkoxy; aryloxy; aralkoxy; -(alkylene)oxy(alkyl); -CN; -N0₂; -COOH; -COO(alkyl); - COO(aryl); -C(0)NH(alkyl); -C(0)NH(aryl); sulfonyl; alkylsulfonyl; arylsulfonyl; sulfamoyl; alkylsulfamoyl; alkylthio; alkylsulfonamide; arylsulfonamide; -NHNH₂; and -NHOH; or a bioisostere, pharmaceutically acceptable salt or derivative thereof.

In preferred embodiments, R¹ is selected from H, C,.₁₈ alkyl (for example, C_1-9 alkyl, e.g. Ci. 6 alkyl), C₂.i₈ alkenyl (for example, C₂.₉ alkenyl, e.g. C_2-6 alkenyl) and C₂.i₈ alkynyl (for example, C₂.₉ alkynyl, e.g. C_2-6 alkynyl). For example, R¹ may be -H and R^1' selected from C_1-18 alkyl (for example, C_1-9 alkyl, e.g. C e alkyl), C_2-i₈ alkenyl (for example, C₂.₉ alkenyl, e.g. C₂.₆ alkenyl) and C_2-i₈ alkynyl (for example, C₂.₉ alkynyl, e.g. C₂.₆ alkynyl). In preferred embodiments, R represents H; C1-15 alkyl, C1-15 alkenyl or C1 -15 alkynyl, optionally substituted with one or more R²; oxygen or an oxygen containing group such that the compound is an N-oxide; C(0)OR³; C(0)NR³R⁴; S0₂NR³; OH, OR³, or formyl.

In other embodiments, R¹ may represent C1 -9 alkyl, optionally substituted with up to 6 OH, NR³R⁴, aryl, 0-C1 -3 alkyl, 0-C1-3 alkenyl, C0₂H, NH(NH)NH₂, CONR³R⁴; C(0)OR³;

C(0)NR³R⁴; or S0₂NR³.

Preferably, the compound of formula I is (3S, 4S)-3-(hydroxymethyl)pyrrolidine-3,4-diol (isoLAB).

Other aspects of the invention are as defined in the claims attached hereto.

Detailed Description of the Invention Definitions and general preferences

Where used herein and unless specifically indicated otherwise, the following terms are intended to have the following meanings in addition to any broader (or narrower) meanings the terms might enjoy in the art:

Unless otherwise required by context, the use herein of the singular is to be read to include the plural and wee versa. The term "a" or "an" used in relation to an entity is to be read to refer to one or more of that entity. As such, the terms "a" (or "an"), "one or more," and "at least one" are used interchangeably herein.

As used herein, the term "comprise," or variations thereof such as "comprises" or

"comprising," are to be read to indicate the inclusion of any recited integer (e.g. a feature, element, characteristic, property, method/process step or limitation) or group of integers (e.g. features, element, characteristics, properties, method/process steps or limitations) but not the exclusion of any other integer or group of integers. Thus, as used herein the term "comprising" is inclusive or open-ended and does not exclude additional, unrecited integers or method/process steps. As used herein, the term "treatment" or "treating" refers to an intervention (e.g. the administration of an agent to a subject) which cures, ameliorates or lessens the symptoms of a disease or removes (or lessens the impact of) its cause(s). In this case, the term is used synonymously with the term "therapy".

Additionally, the terms "treatment" or "treating" refers to an intervention (e.g. the administration of an agent to a subject) which prevents or delays the onset or progression of a disease or reduces (or eradicates) its incidence within a treated population. In this case, the term treatment is used synonymously with the term "prophylaxis".

The term "subject" (which is to be read to include "individual", "animal", "patient" or "mammal" where context permits) defines any subject, particularly a mammalian subject, for whom treatment is indicated. Mammalian subjects include, but are not limited to, humans, primates, domestic animals, farm animals, pet animals and rodents such as mice, rats, hamsters and guinea pigs. In preferred embodiments, the subject is a human.

The term pharmacoperone is a term of art (from "pharmacological chaperone") used to define a class of biologically active small molecules (sometimes also referred to in the art as "chemical chaperones") that serve as molecular scaffolds, causing otherwise misfolded mutant proteins to fold and route correctly within the cell.

As used herein, an effective amount of a compound or composition defines an amount that can be administered to a subject without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio, but one that is sufficient to provide the desired effect, e.g. the treatment or prophylaxis manifested by a permanent or temporary improvement in the subject's condition. The amount will vary from subject to subject, depending on the age and general condition of the individual, mode of administration and other factors. Thus, while it is not possible to specify an exact effective amount, those skilled in the art will be able to determine an appropriate "effective" amount in any individual case using routine experimentation and background general knowledge. A therapeutic result in this context includes eradication or lessening of symptoms, reduced pain or discomfort, prolonged survival, improved mobility and other markers of clinical improvement. A therapeutic result need not be a complete cure. The term bioisostere (or simply isostere) is a term of art used to define drug analogues in which one or more atoms (or groups of atoms) have been substituted with replacement atoms (or groups of atoms) having similar steric and/or electronic features to those atoms which they replace. The substitution of a hydrogen atom or a hydroxyl group with a fluorine atom is a commonly employed bioisosteric replacement. Sila-substitution (C/Si-exchange) is a relatively recent technique for producing isosteres. This approach involves the replacement of one or more specific carbon atoms in a compound with silicon (for a review, see Tacke and Zilch (1986) Endeavour, New Series 10: 191-197). The sila-substituted isosteres (silicon isosteres) may exhibit improved pharmacological properties, and may for example be better tolerated, have a longer half-life or exhibit increased potency (see for example Englebienne (2005) Med. Chem., 1 (3): 215-226). Similarly, replacement of an atom by one of its isotopes, for example hydrogen by deuterium, may also lead to improved pharmacological properties, for example leading to longer half-life (see for example Kushner et al (1999) Can J Physiol Pharmacol. 77(2):79-88). In its broadest aspect, the present invention contemplates all bioisosteres (and specifically, all silicon bioisosteres) of the compounds of the invention.

The term pharmaceutically acceptable derivative as applied to the compounds of the invention define compounds which are obtained (or obtainable) by chemical derivatization of the parent compounds of the invention. The pharmaceutically acceptable derivatives are therefore suitable for administration to or use in contact with mammalian tissues without undue toxicity, irritation or allergic response (i.e. commensurate with a reasonable benefit/risk ratio). Preferred derivatives are those obtained (or obtainable) by alkylation, esterification or acylation of the parent compounds of the invention. The derivatives may be active per se, or may be inactive until processed in vivo. In the latter case, the derivatives of the invention act as prodrugs. Particularly preferred prodrugs are ester derivatives which are esterified at one or more of the free hydroxyls and which are activated by hydrolysis in vivo. Other preferred prodrugs are covalently bonded

compounds which release the active parent drug according to general formula (I) after cleavage of the covalent bond(s) in vivo.

The term pharmaceutically acceptable salt as applied to the inhibitors of the invention defines any non-toxic organic or inorganic acid addition salt of the free base which are suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and which are commensurate with a reasonable benefit/risk ratio. Suitable pharmaceutically acceptable salts are well known in the art. Examples are the salts with inorganic acids (for example hydrochloric, hydrobromic, sulphuric and phosphoric acids), organic carboxylic acids (for example acetic, propionic, glycolic, lactic, pyruvic, malonic, succinic, fumaric, malic, tartaric, citric, ascorbic, maleic, hydroxymaleic, dihydroxymaleic, benzoic, phenylacetic, 4-aminobenzoic, 4- hydroxybenzoic, anthranilic, cinnamic, salicylic, 2-phenoxybenzoic, 2-acetoxybenzoic and mandelic acid) and organic sulfonic acids (for example methanesulfonic acid and p- toluenesulfonic acid).

The term substantially pure as applied to compositions containing the compounds of the invention defines compositions in which the compound of the invention is at least 90% pure, preferably at least 95% pure and most preferably at least 99% pure.

In the present specification the term "alkyl" defines a straight or branched saturated hydrocarbon chain. The term "d-C₆ alkyl" refers to a straight or branched saturated hydrocarbon chain having one to six carbon atoms. Examples include methyl, ethyl, n- propyl, isopropyl, t-butyl, n-hexyl. The term "CrCg alkyl" refers to a straight or branched saturated hydrocarbon chain having one to nine carbon atoms. The term "Ο,-Ο^ alkyl" refers to a straight or branched saturated hydrocarbon chain having one to fifteen carbon atoms. The alkyl groups of the invention may be optionally substituted by one or more halogen atoms. Thus, the term alkyl may define the radical of saturated aliphatic groups, including straight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl groups, alkyl substituted cycloalkyl groups and cycloalkyl substituted alkyl groups. In preferred embodiments, a straight chain or branched chain alkyl has 30 or fewer carbon atoms in its backbone (e.g., Ci-C₃₀ for straight chain, C₃-C₃₀ for branched chain), and more preferably up to 20, 15, 12, 10, 8 or 6. Likewise, preferred cycloalkyls have from 3-10 carbon atoms in their ring structure, and more preferably have 5, 6 or 7 carbons in the ring structure.

The term aralkyl defines an alkyl group substituted with an aryl group (e.g., an aromatic or heteroaromatic group).

"C₁-C4 alkyl" has a similar meaning except that it contains from one to four carbon atoms. "C₂-C₆ alkenyl" refers to a straight or branched hydrocarbon chain having from two to six carbon atoms and containing at least one carbon-carbon double bond. Examples include ethenyl, 2-propenyl, and 3-hexenyl.

The term "C -C_e haloalkyl" refers to a C^ alkyl group as defined above substituted by one or more halogen atoms.

In the present specification the term "alkenyl" defines a straight or branched hydrocarbon chain having containing at least one carbon-carbon double bond. The term "Ci-C₆ alkenyl" refers to a straight or branched unsaturated hydrocarbon chain having one to six carbon atoms. The term "( Cg alkenyl" refers to a straight or branched unsaturated hydrocarbon chain having one to nine carbon atoms. The term "Ci-C ₅ alkenyl" refers to a straight or branched unsaturated hydrocarbon chain having one to fifteen carbon atoms. Preferred is C C₆ alkenyl. Examples include ethenyl, 2-propenyl, and 3-hexenyl. The alkenyl groups of the invention may be optionally substituted by one or more halogen atoms.

In the present specification the term "alkynyl" defines a straight or branched hydrocarbon chain having containing at least one carbon-carbon triple bond. The term "Ci-C₆ alkynyl" refers to a straight or branched unsaturated hydrocarbon chain having one to six carbon atoms. The term

alkynyl" refers to a straight or branched unsaturated hydrocarbon chain having one to nine carbon atoms. The term "C_rC ₅ alkynyl" refers to a straight or branched unsaturated hydrocarbon chain having one to fifteen carbon atoms. Preferred is C C₆ alkynyl. Examples include ethynyl, 2-propynyl, and 3-hexynyl. The alkynyl groups of the invention may be optionally substituted by one or more halogen atoms.

The term "heterocyclyl" defines a saturated or partially saturated 3 to 14 membered ring system (except when alternative numbers of ring atoms are specified) similar to cycloalkyl but in which at least one of the carbon atoms has been replaced by N, O, S, SO or S0₂. Examples include piperidine, piperazine, morpholine, tetrahydrofuran and pyrrolidine.

As used herein, the term "carbocyclyl" means a mono- or polycyclic residue containing 3 or more (e.g. 3-14, 3-10 or 3-8) carbon atoms. The carbocyclyl residues of the invention may be optionally substituted by one or more halogen atoms. Mono- and bicyclic carbocyclyl residues are preferred. The carbocyclyl residues can be saturated or partially unsaturated and include fused bicyclic or tricyclic systems. Examples of such groups include g cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexenyl and also bridged systems such as norbornyl and adamantyl.

Saturated carbocyclyl residues are preferred and are referred to herein as "cycloalkyls" and the term "cycloalkyl" is used herein to define a saturated 3 to 14 membered carbocyclic ring including fused bicyclic or tricyclic systems. Examples of such groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and also bridged systems such as norbornyl and adamantyl. The cycloalkyl residues of the invention may be optionally substituted by one or more halogen atoms.

In the present specification the term "aryl" defines a 5-14 (e.g. 5-10) membered aromatic mono-, bi- or tricyclic group at least one ring of which is aromatic. Thus, bicyclic aryl groups may contain only one aromatic ring. Examples of aromatic moieties are benzene, naphthalene, imidazole and pyridine. The term also includes bicyclic or tricyclic systems in which one or more of the rings has aromatic character. Indane is an example of this type of system.

As used herein, the term "heteroaryl" are aryl moieties as defined above which contain heteroatoms (e.g. nitrogen, sulphur and/or oxygen). The term also includes systems in which a ring having aromatic character is fused to a saturated or partially saturated ring. Examples include pyridine, pyrimidine, furan, thiophene, indole, isoindole, indoline, benzofuran, benzimidazole, benzimidazoline quinoline, isoquinoline,

tetrahydroisoquinoline, quinazoline, thiazole, benzthiazole, benzoxazole, indazole and imidazole ring systems. Unless otherwise indicated, the term "aryl" is to be interpreted to include heteroaryl groups as defined above.

The aryl and heteroaryl groups of the invention may optionally be substituted by one or more halogen atoms. In the present specification, "halo" refers to fluoro, chloro, bromo or iodo.

N-alkylation

N-alkyl derivates of compounds of formula (I) may be prepared by techniques known to those skilled in the art. Typical approaches involve: (a) reductive amination by

hydrogenantion of the amine in the presence of palladium with an aldehyde; (b) reductive amination by sodium cyanoborohydride of the amine with an aldehyde; (c) reductive amination by sodium triacetoxyborohydride of the amine with an aldehyde; and (d) N- alkylation of the amine with an akyl halide or other leaving group such as a tosylate, etc. (all of the preceding in water, alcohols or other suitable solvents).

Exemplary techniques and reaction schemes which may be adapted for use in the synthesis of compounds of formula (I) are described for example in: (a) Rawlings et al. (2009) Chembiochem 10: 1 101-1105; (b) Mellor et al. (2002) Biochem. J. 366: 225-233; (c) WO2001/010429; and (d) Butters et al. (2000) Tetrahedron Asymm. 1 1 : 1 13-125.

CFTR protein rescue

The ability of the compounds of the invention to rescue mutant CFTR activity may be determined by routine assays known to those skilled in the art (an example of which is described in the Exemplification section (Example 3), below). Preferred are compounds which can rescue the activity of the mutant AF508 CFTR polypeptide.

Without wishing to be bound by any theory, it is thought that the compounds of the invention act as CFTR pharmacoperones, restoring mutant CFTR function via a

pharmacological chaperone mechanism.

Thus, preferred compounds of the invention are CFTR pharmacoperones.

Some or all of the observed CFTR rescue activity may arise from other mechanisms. For example, the compounds may act as an indirect chaperone of CFTR via a chaperone effect attendant on binding to a protein (e.g. enzyme) which itself acts as a chaperone or co- chaperone of CFTR. For example, the compounds of the invention may bind to (or otherwise inhibit) chaperone proteins such as calnexin and so influence protein trafficking through the Golgi apparatus. Thus, compounds of the invention may prevent the interaction of mutant CFTR polypeptides (e.g. the AF508 CFTR polypeptide) to the chaperone calnexin.

Thus, preferred compounds of the invention inhibit the interaction of calnexin with CFTR polypeptide. Posologv

The compounds of the present invention can be administered by oral or parenteral routes, including intravenous, intramuscular, intraperitoneal, subcutaneous, transdermal, airway (aerosol), nasal, rectal, vaginal and topical (including buccal and sublingual) administration. Preferred is oral administration.

The amount of the compound administered can vary widely according to the particular dosage unit employed, the period of treatment, the age and sex of the patient treated, the nature and extent of the disorder treated, and the particular compound selected.

The desired dose is preferably presented as a single dose for daily administration.

However, two, three, four, five or six or more sub-doses administered at appropriate intervals throughout the day may also be employed. These sub-doses may be

administered in unit dosage forms, for example, containing 0.001 to 100 mg, preferably 0.01 to 10 mg, and most preferably 0.5 to 1.0 mg of active ingredient per unit dosage form.

In determining an effective amount or dose, a number of factors are considered by the attending physician, including, but not limited to, the potency and duration of action of the inhibitors used, the nature and severity of the illness to be treated, as well as the sex, age, weight, general health and individual responsiveness of the patient to be treated, and other relevant circumstances. Those skilled in the art will appreciate that dosages can also be determined with guidance from Goodman & Goldman's The Pharmacological Basis of Therapeutics, Ninth Edition (1996), Appendix II, pp. 1707-1711.

The effectiveness of a particular dosage of the compound of the invention can be determined by monitoring the effect of a given dosage on the progression of the disease or its prevention.

Compositions of the invention may be delivered to the respiratory tract and lungs by inhalation. They may be delivered systemically by oral administration.

Formulation Illustrative pharmaceutically acceptable salts are prepared from formic, acetic, propionic, succinic, glycolic, gluconic, lactic, malic, tartaric, citric, ascorbic, glucuronic, maleic, fumaric, pyruvic, aspartic, glutamic, benzoic, anthranilic, mesylic, stearic, salicylic, p- hydroxybenzoic, phenylacetic, mandelic, embonic (pamoic), methanesulfonic,

ethanesulfonic, benzenesulfonic, pantothenic, toluenesulfonic, 2-hydroxyethanesulfonic, sulfanilic, cyclohexylaminosulfonic, algenic, b-hydroxybutyric, galactaric and galacturonic acids.

Suitable pharmaceutically-acceptable base addition salts include metallic ion salts and organic ion salts. Metallic ion salts include, but are not limited to, appropriate alkali metal (group la) salts, alkaline earth metal (group lla) salts and other physiologically acceptable metal ions. Such salts can be made from the ions of aluminium, calcium, lithium, magnesium, potassium, sodium and zinc. Organic salts can be made from tertiary amines and quaternary ammonium salts, including in part, trimethylamine, diethylamine, N, N'- dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine) and procaine. All of the above salts can be prepared by those skilled in the art by conventional means from the corresponding compound.

Pharmaceutical compositions can include stabilizers, antioxidants, colorants and diluents. Pharmaceutically acceptable carriers and additives are chosen such that side effects from the pharmaceutical compound are minimized and the performance of the compound is not compromised to such an extent that treatment is ineffective.

The compound of the invention can be administered parenterally, for example

subcutaneously, intravenously, or intramuscularly, or by infusion techniques, in the form of sterile injectable aqueous or oleaginous suspensions. Such suspensions can be formulated according to known art using suitable dispersing or wetting agents and suspending agents such as those mentioned above or other acceptable agents. A sterile injectable preparation can be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example a solution in 1 ,3- butanediol. Among acceptable vehicles and solvents that can be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed, including synthetic mono-or diglycerides. In addition, omega-3 polyunsaturated fatty acids can find use in preparation of injectables. Administration can also be by inhalation, in the form of aerosols or solutions for nebulizers, or rectally, in the form of suppositories prepared by mixing the drug with a suitable non-irritating excipient which is solid at ordinary

temperature, but liquid at rectal temperature and will therefore, melt in the rectum to release the drug. Such materials are cocoa butter and polyethylene glycols. Also encompassed by the present invention is buccal and sub-lingual administration, including administration in the form of lozenges, pastilles or a chewable gum comprising the inhibitors set forth herein. The inhibitors can be deposited in a flavoured base, usually sucrose, and acacia or tragacanth.

Preservatives are optionally employed to prevent microbial growth prior to or during use. Suitable preservatives include polyquaternium-1 , benzalkonium chloride, thimerosal, chlorobutanol, methylparaben, propylparaben, phenylethyl alcohol, edetate disodium, sorbic acid, or other agents known to those skilled in the art. Typically, such preservatives are employed at a level of about 0.001 % to about 1.0% by weight of a pharmaceutical composition.

Solubility of components of the present compositions can be enhanced by a surfactant or other appropriate cosolvent in the composition. Such cosolvents include polysorbates 20,60 and 80, polyoxyethylene/polyoxypropylene surfactants (e. g., Pluronic F-68, F-84 and P-103), cyclodextrin, or other agents known to those skilled in the art. Typically, such cosolvents are employed at a level of about 0.01 % to about 2% by weight of a

pharmaceutical composition.

Pharmaceutically acceptable excipients and carriers encompass all the foregoing and the like. The above considerations concerning effective formulations and administration procedures are well known in the art and are described in standard textbooks. See for example Remington: The Science and Practice of Pharmacy, 20th Edition (Lippincott, Williams and Wilkins), 2000; Lieberman et al., ed. , Pharmaceutical Dosage Forms, Marcel Decker, New York, N. Y. (1980) and Kibbe ef a/., ed. , Handbook of Pharmaceutical Excipients (3rd Edition), American Pharmaceutical Association, Washington (1999).

The compounds and compositions of the invention are preferably formulated for oral delivery in tablet form. Formulations for delivery to the respiratory tract

The compounds of the invention can be formulated into a solution and/or a suspension of particles in a carrier appropriate for inhalation into the respiratory tract and the lungs.

A wide range of suitable carriers are known to those skilled in the art. In general, powders, mists or aerosols with particle sizes of 0.5 to 1 micron may be delivered to the respiratory tract. Such particle size ranges are commonly achieved by micronisation or spray drying and such delivery methods are described for example in Remington: The Science and Practice of Pharmacy, 20th Edition (Lippincott, Williams and Wilkins), 2000; Lieberman et al., ed. , Pharmaceutical Dosage Forms, Marcel Decker, New York, N. Y. (1980) and Kibbe er a/., ed. , Handbook of Pharmaceutical Excipients (3rd Edition), American Pharmaceutical Association, Washington (1999).

In a preferred formulation for inhalation, the compound of the invention forms part of a powdered composition within a gelatin capsule, blister pack and a multi-dose metering device. The capsule or blister is ruptured within the device enabling the powder to be inhaled.

Powdered compositions typically comprise the compounds of the invention blended or mixed with an inert carrier. Usually the inert carrier has a mean particle size substantially larger than that of the drug. This provides, among other advantages, an improvement in the flow properties and dispensing accuracy of the composition. Suitable carriers include calcium carbonate and sugars.

In other embodiments, the compound of the invention is formulated as an aerosol, for example by preparing a suspension of the compound as a finely divided powder in a liquefied propellant gas. Alternatively, a solution can be prepared which may contain solubilizers and co-solvents. Pressurized metered dose inhalers (pMDI) are normally used to dispense such formulations to a patient. Suitable propellants include

chlorofluorocarbons, fluorocarbons and hydrofluoroalkanes.

Inhalation devices, such as inhalers (including dry powder inhaler and metered dose inhalers (MDIs)) and nebulizers (also known as atomizers) may be used to deliver the compounds of the invention to the respiratory tract and/or lungs. Metered dose inhalers are designed to deliver a fixed unit dosage of medicament per actuation. Exemplary nebulizers for delivering an aerosolized solution include the AERx™ (Aradigm), the Ultravent® (Mallinkrodt), the Pari LC Plus™ or the Pari LC Star™ (Pari GmbH, Germany), the DeVilbiss Pulmo-Aide, and the Acorn II® (Marquest Medical Products).

Exemplification

The invention will now be described with reference to specific Examples. These are merely exemplary and for illustrative purposes only: they are not intended to be limiting in any way to the scope of the monopoly claimed or to the invention described. These examples constitute the best mode currently contemplated for practicing the invention.

(a) Example 1 : Preparation of isoLAB

Reaction scheme summary

Scheme 1

IsoLAB was synthesized from D-tagatose [Scheme 1]. The C2 hydroxymethyl group in the diacetonide 17 was introduced in D-tagatose by a Kiliani synthesis, followed by

acetonation. The diacetonide 17 was converted into the corresponding triflate on reaction with triflic anhydride in dichloromethane in the presence of pyridine; subsequent reaction of the triflate with sodium azide in DMF gave the D-fa/ono-azidolactone 18 [mp. 81-83 °C;

[ot]_D ²⁵ -82.9 (c 1.0)] in 94% yield. Two-step reduction of the lactone 18 by DIBALH in dichloromethane, followed by sodium borohydride in methanol, afforded the diol 19 [oil;

[OC]D²⁵ -52.2 (c 1.0)] in 90% yield. Selective hydrolysis of the terminal acetonide in 19 formed the tetraol 20; [a]_D ²⁵ -98.1 (c 1.0, CHCI₃)] (83% yield) which on oxidation with sodium periodate cleaved the C4-C5 and C5-C6 bonds to give the isopropylidene 3-C-azidomethyl- D-erythrose 15D [oil, [oc]_D ²⁵ -102.1 (c 1.0)] in 91% yield. Hydrolysis of the acetonide 15D with acid ion exchange resin afforded the deprotected azidolactol 16D [oil; [a]_D ²⁵ +1.7 (c 1.3, MeOH)] which on hydrogenation gave isoLAB 1 L [oil, [a]_D +35.6 (c 0.27, H₂0)] in 71% yield. The overall yield of isoLAB 1L from the /a/o/?o-diacetonide 17 was 45%.

2-C-Azidomethyl-2,3:5,6-di-0-isopropylidene-D-talono-1,4-lactone

Trifluoromethanesulfonic anhydride (1 .9 mL, 1 1.3 mmol) was added dropwise to a stirred solution of 2,3:5,6-di-0-isopropylidene-2-C-hydroxymethyl-D-talono-1 ,4-lactone (2.07g, 7.19 mmol) and pyridine (1.75 mL, 21.7 mmol) in DCM (40 mL) at -30°C. After 2.5 h TLC analysis (1 :1 EtOAc/cyclohexane) indicated the conversion of starting material (R_f 0.41) into one major product (R_f 0.77). The reaction mixture was diluted with DCM (70 mL) and washed with aqueous hydrochloric acid (1 M, 60 mL). The organic residue was dried (MgS0₄), filtered and concentrated in vacuo to afford the triflate, which was used without further purification. Sodium azide (701 mg, 10.78 mmol) was added to a stirred solution of the crude triflate in dry DMF (35 mL) at RT. The reaction mixture was stirred for 3 h after which TLC analysis (1 : 1 EtOAc/cyclohexane) showed the conversion of starting material to one major product (R_f 0.73). The reaction mixture was concentrated in vacuo and the residue was partitioned between EtOAc (90 mL) and brine (50 mL). The aqueous layer was extracted with EtOAc (40 mL) and the combined organic layers were dried (MgS0₄), filtered and concentrated in vacuo. The resulting residue was purified by flash column chromatography (1 :6 - 1 :2 EtOAc/cyclohexane) to afford the azide (2.12 g, 94% over 2 steps).

HRMS (ESI +ve): found 336.1 162 (M+Na)⁺; C₁₃H₁₉N₃Na0₆ requires 336.1166; [a]_D ²⁵ -82.9 (c 1.0, CHCI₃); m.p. 81 -83°C; v_max (thin film): 2107 (s, N₃), 1786 (s, C=0); δ_Η (CDCI₃, 400 MHz): 1.32, 1.36, 1.47, 1.54 (4 x 3H, s, Me), 3.57 (1 H, d, H2'a, J_gern 13.3), 3.86 (1 H, d, H2'b, gem 13.3), 3.97 (1 H, a-t, H6a, 7.9), 4.14 (1 H, dd, H6b, J_gem 8.3, J₆b_,5 6.9), 4.29-4.34 (1 H, m, H5), 4.53 (1 H, s, H4), 4.76 (1 H, s, H3); 5_C (CHCI₃, 100 MHz): 25.5, 25.5, 26.4, 27.2 (C(CH₃)₂), 49.9 (C2'), 65.3 (C6), 75.2 (C5), 79.8 (C3), 81.4 (C4), 85.2 (C2), 1 10.7, 114.3 (C(CH₃)₂), 173.8 (C1); m/z (ESI +ve): 336 (75% M+Na⁺), 649 (100% 2M+Na⁺);

2-C-Azidomethyl-2,3:5,6-di-0-isopropylidene-D-talitol

Diisobutylaluminium hydride solution (1.5M in toluene, 6.2 mL, 9.3 mmol) was added dropwise to a stirred solution of the lactone (2.09 g, 6.68 mmol) in DCM (25 mL) at -78°C. The reaction mixture was stirred for 2 h at -78°C after which time TLC analysis (1 :2 EtOAc/cyclohexane) revealed the conversion of starting material (R_f 0.54) into one major product (R_f 0.42). The reaction was quenched with methanol (5.7 mL) and allowed to warm to RT. Saturated aqueous potassium sodium tartrate solution (48 mL) was added and the reaction was stirred for 16 h at RT. The aqueous phase was extracted with DC (3 ^χ 30 mL), dried (MgS0₄), filtered and concentrated in vacuo. The crude lactol was dissolved in methanol (25 mL) and stirred with sodium borohydride (244 mg, 6.68 mmol) at 0°C for 30 min and allowed to warm to RT for a further 2 h, after which time TLC analysis (1 :2 EtOAc/cyclohexane) revealed the formation of one major product (R_f 0.21 ). The reaction was neutralised with Dowex^® (50W-X8, H⁺), filtered and concentrated in vacuo. The resulting residue was purified by flash column chromatography (1 :5 - 1 : 1 EtOAc/cyclohexane) to afford the diol as a colourless oil (1.91 g, 90% over 2 steps).

HRMS (ESI -ve): found 340.1484 (M+Na)⁺; C₁₃H₂₃N₃Na0₆ requires 340.1479; [a]_D ²⁵ -52.2 (c 1.0, CHCI₃); v_max (thin film): 3417 (OH), 21 10 (N₃); D_H (400 MHz, CDCI₃): 1.39 (3 H, s, CH₃), 1.43 (3 H, s, CH₃), 1.46 (3 H, s, CH₃), 1.47 (3 H, s, CH₃), 2.89 (1 H, s, OH1), 2.97 (1 H, s, OH4), 3.38 (1 H, d, H2'a, J_gem 13.2), 3.64-3.70 (2H, m, H1 a, H4), 3.74 (1 H, m, M b), 3.77 (1 H, d, H2'b, Jg_em 13.2), 3.92 (1 H, dd, H6a, J_gem 8.6, J_6a,5 6.4), 4.03 (1 H, d, H3, J_3,4 9.5), 4.08 (1 H, dd, H6b, J_gem 8.6, J_6b,₅ 6.6), 4.24 (1 H, dt, H5, J_s,urJs,eb 6.4, J₅,₄ 4.7); D_c (100 MHz, CDCI₃): 25.1 (CH₃), 26.1 (CH₃), 26.5 (CH₃), 28.5 (CH₃), 54.2 (C2'), 62.6 (C1), 66.2 (C6), 69.7 (C4), 76.7 (C5), 77.7 (C3), 84.7 (C2), 109.3 (CMe₂), 109.5 (CMe₂); m/z (ESI +ve): 340 (100% M+Na⁺), 657 (100% 2M+Na⁺);

2-C-Azidomethyl-2,3-0-isopropylidene-D-talitol

A solution of 2-C-azidomethyl-2,3:5,6-di-0-isopropylidene-D-talitol (337 mg, 1.06 mmol) in AcOH:H₂0 (1 :1 , 7 mL) was stirred for 16 h at RT. After this time TLC analysis (EtOAc) showed complete conversion of starting material (R_f 0.78) to a single product (R_f 0.22). The reaction mixture was concentrated in vacuo, coevaporated with toluene (3 x 15 mL) and purified by flash column chromatography (EtOAc) to afford the tetraol as white crystals (244 mg, 83%).

HRMS (ESI -ve): found 300.1 168 (M+Na)⁺; C₁₀H_1sN₃NaO₆ requires 300.1 166; [a]_D ²⁵ -98.1 (c 1.0, CHCI₃); max (thin film): 3424 (w, br, OH), 2107 (s, N₃); D_H (100 MHz, (CD₃)₂CO): 3.40 (1 H,d, H2'a, J_gem 12.9), 3.57-3.68 (3H, m, H1a, H6a, H1 b or H6b), 3.70-3.80 (5H, m, H5, H2'b, H1 b or H6b, OH1 or OH6, OH5), 3.91 (1 H, ddd, H4, _4,3 9.6, J_4,o_H4 6.2, J₄,₅ .5, H4), 4.18 (1 H, d, OH4, J_QHA 6.3), 4.27 (1 H, d, H3, J₃ 9.6), 4.40 (1 H, t, OH1 or OH6, J 5.4); Dc (100 MHz, (CD₃)₂CO): 26.8, 29.2 (2 x 3H, s, C(CH₃)₂), 55.6 (C2¹) , 63.2, 64.7 (C1 , C6), 70.2 (C4), 72.3 (C5), 78.0 (C3), 85.8 (C2), 109.0 (C(CH₃)₂); m/z (ESI +ve): 300 (98% M+Na⁺), 577 (100% 2M+Na⁺).

3-C-Azidomethyl-2,3-0-isopropylidene-D-erythrose Sodium periodate (1.83 g, 8.57 mmol) was added to a solution of 2-C-azidomethyl-2,3-0- isopropylidene-D-talitol (1.19 g, 0.74 mmol) in water (30 mL) at RT and stirred for 3 h after which time TLC analysis (EtOAc) showed the complete conversion of the starting material (R( 0.22) to one major product (R_f 0.88). The reaction mixture was extracted with EtOAc (3 x 15 mL), the organic layers were combined, dried (MgS0₄) and concentrated in vacuo. The resulting residue was purified by flash column chromatography (1 :4 - 1 :2 EtOAc/cyclohexane) to afford 3-C-azidomethyl-2,3-0-isopropylidene-D-erythrose (829 mg, 90%).

HRMS (ESI -ve): found 238.0798 (M+Na)⁺; C₈H₁₃N₃Na0₄ requires 238.0798; [a]_D ²⁵ -102.1 (c 1.0, CHCI₃); (thin film): 3425 (w, br, OH), 2106 (s, N₃); D_H (400 MHz, CDCI₃) major anomer only: 1.46, 1.49 (2 x 3H, s, CH₃), 3.00 (1 H, s, OH), 3.53 (1 H, d, H3'a, J_GEM 12.9), 3.61 (1 H, d, H3'b, J_GEM 12.9), 3.97 (1 H, d, H4a, J_GEM 10.1), 4.03 (1 H, d, H4b, J_GEM 10.1), 4.34 (1H, s, H2), 5.43 (1H, s, H1); rj_c (100 MHz, CDCI₃) major anomer only: 27.2, 27.3 (C(CH₃)₂), 54.7 (C3'), 74.6 (C4), 86.7 (C2), 97.3 (C3), 101.4 (C1), 113.8 (C(CH₃)₂); m/z (ESI +ve): 238 (70% M+Na⁺), (100% M+CH₃CN+H⁺).

3-C-Azidomethyl-D-erythrose

Dowex^® (50W-X8, H⁺) (1.73 g) was added to a solution of 3-C-azidomethyl-2,3-0- isopropylidene-D-erythrose (770 mg, 3.58 mmol) in 1 :1 water: 1 ,4-dioxane (18 mL) and stirred at 60°C. After 18 h TLC analysis (1 :1 EtOAc/cyclohexane) showed conversion of starting material (R_f 0.64) to a single product (R_f 0.12). The crude mixture was filtered and concentrated under reduced pressure to afford 3-C-azidomethyl-D-erythrose (572 mg, 91%) as a pale brown oil, in a 3:2 ratio of anomers.

HRMS (ESI -ve): found 174.0518 [M-H]^'; C₅H₈N₃0₄ requires 174.0520; [cc]_D ²⁵ +1.7 (c 1.3, MeOH); v_max (thin film, Ge): 3356 (m, br, OH), 2110 (s, N₃); δ_Η ((CD₃)₂C0, 400 MHz): 3.36 (1 H, d, H3'a^B, J_GEM 10.9), 3.41 (1 H, d, H3'b^B, J_GEM 10.9), 3.45 (2H, s, H3^,A), 3.72 (1 H, d, H4a^A, J_GEM 9.6), 3.73-3.78 (1 H, m, H2^A), 3.79 (1 H, d, H4a^B, J_gem 8.6), 3.80-3.84 (1 H, m, H2^B), 3.85 (1 H, d, H4b^B, J_GEM 9.6), 3.94 (1 H, d, H4b^A, J_GEM 9.6), 4.31 (1 H, s, OH3^B), 4.32 (1 H, s, OH3^A), 4.55 (1 H, d, OH2^B, J₀H_2,2 7.3), 4.90 (1H, d, OH2^A, J_OH2,2 5.6), 5.18 (1 H, d, H1^A, JLOHI 2.3), 5.23 (1 H, d, H1^B, J_1i0Hi 4.6), 5.40 (1H, d, OH1^B, ₀m,i 3.5), 5.53 (1H, d, OH1^A, JOHI,I 4.5); 6_c ((CD₃)₂CO, 100 MHz); 56.9 (C3'^B), 57.0 (C3^,A), 73.6 (C2^B), 74.0 (C4^B), 74.6 (C4^A), 78.6 (C3^B), 79.1 (C2^A), 79.8 (C3^A), 97.4 (C1 ^B), 104.1 (C1^A); m/z (ESI -ve): 174 (100% [M-H]-).

1 ,4-Dideoxy-2-C-hydroxymethyl-1 ,4-imino-L-threitol (iso-LAB) Palladium on activated carbon (10%, 1 12 mg, 3.08 mmol) was added to a solution of 3-C- azidomethyl-D-erythrose (545 mg, 3.11 mmol) in 9:1 water/acetic acid (70 mL) and the reaction was purged with argon then hydrogen. The reaction mixture was stirred under hydrogen at RT for 24 h. The reaction was monitored by LRMS until no starting material was detected. The reaction mixture was filtered through Celite® and concentrated under reduced pressure to approximately 5 mL and absorbed onto a column of Dowex^® (50W-X8, H⁺). The resin was washed with water before liberation of the amine with 2M aqueous ammonia. The ammoniacal fractions were concentrated under reduced pressure to afford 1 ,4-dideoxy-2-C-hydroxymethyl-1 ,4-imino-L-threitol (333 mg, 80%) as a brown oil.

HRMS (ESI +ve): found 134.0806 (M+H)⁺; C₅H₁₂N0₃ requires 134.0812; [a]_D ²⁵ +35.6 (c 0.27, H₂0); (thin film, Ge): 3326 (w, br, OH); δ_Η (D₂0, 400 MHz): 2.89 (1 H, d, H4a, _gem 12.9), 2.89 (1 H, d, H1 a, J_gem 12.6), 3.00 (1 H, d, H1 b, _gem 12.6), 3.41 (1 H, dd, H4b, J_gem 12.7, J_4|3 4.5), 3.70 (1H, d, H2'a, _gem 12.0), 3.82 (1 H, d, H2'b, _gem 12.0), 4.10 (1 H, d, H3, . 4.3); 5_C (D₂0, 100 MHz): 53.0 (C4), 53.3 (C1 ), 63.1 (C2'), 76.6 (C3), 83.7 (C2); m/z (ESI +ve): 134 (100% M+H⁺).

The above exemplified synthesis starts from D-tagatose, but those skilled in the art will recognize that isoLAB can also be prepared by routes from other sugars. Examples of the many suitable starting monosaccharides include (but are not restricted to) L-ribose, D- lyxose, L-psicose, D-mannose, D-fructose and L-sorbose, D-mannose, L-ribonolactone, L- gulose, D-lyxonolactone, L-gulonolactone, D-mannonolactone, L-gulonolactone and suitably protected derivatives of any of the foregoing. In the latter case, protection may be of cis-1 ,2-dioIs by acetone to acetonides, or any suitable ketone (such as cyclohexanone, pentan-3-one or other ketones) to its corresponding ketal.

(b) Example 2: CFTR rescue activity

Materials and methods Cell culture

The human tracheal gland serous epithelial cell line CF-KM4 is derived from a CF patient homozygous for the AF508 mutation. The details of the generation, characterization, and routine propagation have been described elsewhere (Kammouni et al. (1999) Resp. Cell Mol. Biol. 20(4): 684-91 ). Functional analysis of CFTR activity

CFTR ion channel functions were assessed by single-cell fluorescence imaging, using the potential-sensitive probe bis-( ,3-diethylthiobarbituric acid)trimethine oxonol (DiSBAC₂(3); Molecular Probes, Eugene, OR), as previously reported (see Norez et al. (2009) Am. J. Respir. Cell Mol. Biol. 41(2): 217-225). Fluorescence intensity was recorded by confocal laser scanning microscopy using Bio-Rad MRC 1024 equipped with 15 mW Ar/Kr gas laser (Hemel Hempstead, UK). Maximal resolution was obtained with Olympus plan apo X60 oil, 1.4 NA, objective lens. Fluorescence signal collection was performed through the control software Lasersharp 3.2 (Hemel Hempstead, UK). The resolution time was 30 s. Bis- oxonol slowly distributes across biological membrane according to the membrane potential and binds to hydrophobic cell components; since the quantum yield of the dye increases impressively upon the binding, the fluorescence of cells incubated in a medium containing bis-oxonol increases upon depolarization and, conversely, decreases with

hyperpolarization (Dall'Asta et al. (1997) Exp. Cell Res. 231 : 260-268). CFTR-dependent current was stimulated by application of Forskolin + Genistein (Fsk+Gst), inducing a depolarization characterized by an increase of the fluorescence, while CFTR-dependent current was inhibited by application of CFTR_inh-1 2 characterized by a decrease of the fluorescence. Results are presented as transformed data to obtain the percentage signal variation (Fx) relative to the time of addition of the stimulus, according to the equation : Fx = ([Ft-FO]/FO]X100 where Ft and F0 are the fluorescent values at the time t and at the time of addition of the stimulus, respectively. For histogram representation, the values correspond to the level of stable variation of fluorescence induced by each drug.

The CF-KM4 cells were treated 2 hours with 100μΜ of test compound and then CFTR proteins were stimulated by a cocktail of forskolin (Fsk) + genistein (Gst).

Results

Fig. 1 shows the functional evaluation of AF508 CFTR by DiSBAC2(3) assay in CF-KM4 cells treated (100μΜ for 2h) or not (negative control) with isoLAB. Λ/Β-DNJ (100μΜ for 2h) served as a positive control. Fig. 1 A shows examples of typical time courses obtained with untreated (NT) or treated with isoLAB. Data represent the mean (± SEM) of the relative fluorescence collected from all the cells of a field (n=12). A mixture of forskolin (Fsk, 10 μΜ) + genistein (Gst, 30 μΜ) is used to activate CFTR. CFTRinh-172 (10 μΜ) is used to inhibit CFTR. Fig. 1 B shows histograms report the mean of the relative fluorescence collected from separate experiments (N=2) with a total of 24 cells with the test compound (isoLAB) and positive (NB-DNJ) and negative (NT) controls.

As expected, no variation of the fluorescence was observed after the cocktail stimulation in untreated CF-K 4 bearing the defective AF508 CFTR protein (Fig. 1A). In contrast, treatment of CF-KM4 cells with isoLAB produced a strong increase in the recorded fluorescence signal (greater than that produced by the positive control Λ Β-DNJ) after Fsk+Gst stimulation (Fig. 1A and B). The signal was fully inhibited by the CFTR inhibitor CFTR_inh-172 (Fig. 1A).

In a separate study, the cellular toxicity of isoLAB was evaluated on CF-KM4 cells using the MTT test (Noel et al. (2006) Pharmacol. Expt. Therapeut. 319: 349-359). No apparent cell toxicity (a range of concentration was tested up to 1mM) was detected (data not shown).

(c) Example 3: Glycosidase inhibitory profile of isoLAB

The CFTR-resuing activity of Λ/Β-DNJ has been ascribed (at least in part) to its a- glucosidase inhibitory activity (Norez et al. (2006) FEBS Lett. 580: 2081-2086). Assays of isoLAB with various glycosidases were conducted to evaluate its glycosidase inhibitory profile (see Table 1 , below).

Table 1 : Glycosidase inhibitory profile of isoLAB

Enzyme isoLAB

a-Glucosidase

Rice Nl

Yeast Nl

Rat intestinal maltase Nl

Rat intestinal isomaltase Nl

Rat intestinal sucrase Nl

β-Glucosidase

Almond Nl

Bovine liver Nl

Rat intestinal cellobiase Nl α-Galactosidase

Coffee beans Nl

Human lysosome Nl

β-Galactosidase

Bovine liver Nl

Rat intestinal lactase Nl

a-Mannosidase

Jack beans Nl

β- annosidase

Snail Nl

a-L-Rhamnosidase

P. decumbens Nl

a-L-Fucosidase

Bovine epididymis Nl

Trehalase

Rat intestinal trehalase Ni

Nl : No inhibition (less than 50% inhibition at 1000 )

The results show that isoLAB has no inhibitory activity on any of the glycosidase enzymes listed. In contrast, Λ/Β-DNJ exhibits potent inhibition of a wide range of a-glucosidases (including those involved in digestive and ER processing functions). Since such nonspecific inhibitory activity is implicated in a variety of undesirable side effects (including gastric toxicity), the absence of such inhibitory activity is an unexpected and significant technical advantage of isoLAB versus Λ/Β-DNJ.

Equivalents

The foregoing description details presently preferred embodiments of the present invention. Numerous modifications and variations in practice thereof are expected to occur to those skilled in the art upon consideration of these descriptions. Those modifications and variations are intended to be encompassed within the claims appended hereto.

Claims

CLAIMS:

1. A compound of formula (I):

wherein:

R¹ is selected from H; linear or branched, substituted or unsubstituted alkyl, alkenyl, alkynyl and aralkyl and wherein the optional substitution may be with one or more groups independently selected from: -OH; -F; -CI; -Br; -I; -NH₂; alkylamino;

dialkylamino; linear or branched alkyl, alkenyl, alkynyl and aralkyl; aryl; heteroaryl; linear or branched alkoxy; aryloxy; aralkoxy; -(alkylene)oxy(alkyl); -CN; -N0₂; - COOH; -COO(alkyl); -COO(aryl); -C(0)NH(alkyl); -C(0)NH(aryl); sulfonyl;

alkylsulfonyl; arylsulfonyl; sulfamoyl; alkylsulfamoyl; alkylthio; alkylsulfonamide; arylsulfonamide; -NHNH₂; and -NHOH; or a bioisostere, pharmaceutically acceptable salt or derivative thereof.

2. The compound of claim 1 wherein R¹ is selected from H, C_MS alkyl (for example, C g alkyl, e.g. C_1-6 alkyl), C_2-i₈ alkenyl (for example, C₂.₉ alkenyl, e.g. C_2-6 alkenyl) and C₂.i₈ alkynyl (for example, C_2-9 alkynyl, e.g. C_2-6 alkynyl). For example, R¹ may be -H and R¹ selected from C_M8 alkyl (for example, C_1-g alkyl, e.g. Ci_-6 alkyl), C₂.₁₈ alkenyl (for example, C₂.₉ alkenyl, e.g. C_2-6 alkenyl) and C₂.₁₈ alkynyl (for example, C_2-9 alkynyl, e.g. C₂.₆ alkynyl).

3. The compound of claim 1 wherein R is selected from H; C1-15 alkyl, C1-15 alkenyl or C1-15 alkynyl, optionally substituted with one or more R²; oxygen or an oxygen containing group such that the compound is an N-oxide; C(0)OR³; C(0)NR³R⁴; S0₂NR³; OH, OR³, or formyl.

4. The compound of claim 1 wherein R¹ is selected from C1-9 alkyl, optionally substituted with up to 6 OH, NR³R⁴, aryl, 0-C1-3 alkyl, 0-C1-3 alkenyl, C0₂H, NH(NH)NH₂, CONR³R⁴; C(0)OR³; C(0)NR³R⁴; or S0₂NR³.

5. The compound of claim 1 wherein R¹ is H, being (3S,4S)-3-(hydroxymethyl)pyrrolidine- 3,4-diol (isoLAB).

6. The compound of any one of the preceding claims which is not an ct-glucosidase inhibitor.

7. The compound of any one of the preceding claims which exhibits less than 50% inhibition at 1000 μΜ of each of the a-glucosidase enzymes listed in Table 1 herein.

8. The compound of any one of the preceding claims which exhibits less than 50% inhibition at 1000 μΜ of each of the glycosidase enzymes listed in Table 1 herein.

9. The compound of any one of the preceding claims which rescues mutant CFTR activity.

10. The compound of claim 9 which rescues AF508 CFTR activity.

11. The compound of any one of the preceding claims which is a CFTR pharmacoperone.

12. A composition comprising the compound of any one of the preceding claims.

13. The composition of claim 12 wherein the compound is substantially pure.

14. The composition of claims 12 or claim 13 which is a pharmaceutical composition.

15. The composition of claim 14 which further comprises a pharmaceutically acceptable excipient.

16. The composition of any one of claims 13 to 15 which is formulated for oral delivery.

17. The composition of any one of claims 13 to 15 which is formulated for delivery to the lung.

18. An inhalation device comprising the compound of any one of claims 1 to 11 or the composition of any one of claims 12 to 17.

19. The inhalation device of claim 18 wherein the compound or composition is present in the form of a solution, a suspension or a powder.

20. The inhalation device of claim 18 or 19 which is an MDI or nebulizer.

21. The compound, composition or device of any one of the preceding claims for use in a method of treating cystic fibrosis.

22. A method of treating cystic fibrosis comprising administering an effective amount of the compound as defined in any one of claims 1 to 11 or composition as defined in any one of claims 12 to 17 to a subject in need thereof.