Patent Grant
9067922
The invention relates to sulfonamide derivatives, to their use in medicine, to compositions containing them, to processes for their preparation and to intermediates used in such processes.
Uric acid is the final product of purine metabolism in humans. In humans, unlike many other animals, uric acid is not further broken down, but is predominantly (70%) excreted into the urine with the remaining 30% excreted in faeces. Hyperuricemia is defined as an excessive production or decreased excretion of uric acid and can occur as an overproduction or under excretion of serum uric acid (sUA), or a combination of the both. Renal under excretion of uric acid is the primary cause of hyperuricemia in about 90% of cases, while overproduction is the cause in less than 10%. Increased sUA concentration above 6.8 mg/dL results in crystallisation of uric acid in the form of salts, such as monosodium urate, and to precipitation of these crystals in joints, on tendons and in the surrounding tissues. These crystals (known as tophi) trigger a local immune-mediated inflammatory reaction, leading to gout. The risk of gout increases with increased sUA levels.
Gout is a painful condition that can present in a number of ways, although the most usual is a recurrent attack of acute inflammatory arthritis (a red, tender, hot, swollen joint) often occurring in big toes, heels, knees, wrists and fingers.
Gout is treated by agents to both decrease the cause and effects of uric acid crystal inflammation and pain.
The pain associated with gout is commonly treated with pain and anti-inflammatory drugs such as nonsteroidal anti-inflammatory drugs (NSAIDs), colchicine and steroids. Agents that decrease sUA levels may be used to treat the cause of gout. These include agents that: inhibit the enzymes that result in uric acid production, such as xanthine oxidase inhibitors (e.g. allopurinol, febuxostat or tisopurine), or purine nucleoside phosphorylase (PNP) inhibitors (e.g. ulodesine); metabolise uric acid, such as urate oxidases—also known as uricases (e.g. pegloticase); or increase the excretion of uric acid in the urine (uricosurics), Uricosurics include agents that inhibit the transporters responsible for renal reabsorption of uric acid back into the blood, such as benziodarone, isobromindione, probenecid and sulphinpyrazone, and URAT-1 inhibitors (e.g. benzbromarone).
URAT-1 is also known as solute carrier family 22 (organic anion/cation transporter), member 12, and is encoded by the gene SLC22A12. Human genetic analysis has demonstrated that polymorphisms in the SLC22A12 gene are directly associated with changes in serum uric acid. Inhibitors of uric acid transport, such as URAT-1, are therefore effective in the treatment of gout.
There is a continuing need to provide new treatments for gout that are more effective and/or are better tolerated.
Certain URAT-1 inhibitors for the treatment of gout are known. WO2011/159840 discloses phenylthioacetate URAT-1 inhibitors. Additionally, WO2008/118758, WO2009/012242, WO2010/079443, WO2012/004706, WO2012/004714 and WO2012/004743 disclose sulphonamides.
There is, however, an ongoing need to provide new URAT-1 inhibitors that are good drug candidates.
Furthermore, preferred compounds should have one or more of the following properties: be well absorbed from the gastrointestinal tract; be metabolically stable; have a good metabolic profile, in particular with respect to the toxicity or allergenicity of any metabolites formed; or possess favourable pharmacokinetic properties whilst still retaining their activity profile as URAT-1 inhibitors. They should be non-toxic and demonstrate few side-effects. Ideal drug candidates should exist in a physical form that is stable, non-hygroscopic and easily formulated.
We have now found new sulphonamide URAT-1 inhibitors.
According to a first aspect of the invention there is provided a compound of formula (I)
or a pharmaceutically acceptable salt thereof, wherein:
R1 is a ‘C-linked’ 6-membered heteroaryl containing one, two or three nitrogen atoms wherein said heteroaryl is optionally substituted by one, two or three, valency permitting, X1;
each X1 is independently selected from: F; Cl; CN; (C1-C4)alkyl optionally substituted by one, two or three F; and (C1-C4)alkyloxy optionally substituted by one two or three F;
R2, R3 and R5 are independently selected from: H; halogen; CN; (C1-C4)alkyl optionally substituted by one, two or three F; and (C1-C4)alkyloxy optionally substituted by one, two or three F;
R4 is selected from: halogen; CN; (C1-C4)alkyl optionally substituted by one, two or three F; and (C1-C4)alkyloxy optionally substituted by one, two or three F;
R6 is phenyl substituted by one, two or three X2; or a ‘C-linked’ 6-membered heteroaryl containing one or two nitrogen atoms wherein said heteroaryl is optionally substituted by one, two or three X2;
each X2 is independently selected from: F; Cl; CN; —S(C1-C4)alkyl; —NR7R8; (C1-C6)alkyloxy optionally substituted by one, two or three F; (C3-C6)cycloalkyloxy; (C1-C6)alkyl optionally substituted by one, two or three F; and (C1-C6)alkyl substituted by OH; and
R7 and R8 are independently H or (C1-C4)alkyl or, together with the nitrogen atom to which they are attached, form a saturated 4- to 6-membered nitrogen containing monocycle.
Described below are a number of embodiments (E) of this first aspect of the invention, where for convenience E1 is identical thereto.
Alkyl and alkoxy groups, containing the requisite number of carbon atoms, can be unbranched or branched. Examples of alkyl include methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, sec-butyl and t-butyl. Examples of alkoxy include methoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy, i-butoxy, sec-butoxy and t-butoxy.
Examples of cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.
Halo means fluoro, chloro, bromo or iodo.
The term ‘C-linked’ used in the definitions of formula (I) means that the group in question is joined via a ring carbon.
Specific examples of ‘C-linked’ 6-membered heteroaryl containing one, two or three nitrogen atoms include pyridinyl, pyridazinyl, pyrimidinyl and pyrazinyl.
Hereinafter, all references to compounds of the invention include compounds of formula (I) or pharmaceutically acceptable salts, solvates, or multi-component complexes thereof, or pharmaceutically acceptable solvates or multi-component complexes of pharmaceutically acceptable salts of compounds of formula (I), as discussed in more detail below.
Preferred compounds of the invention are compounds of formula (I) or pharmaceutically acceptable salts thereof.
Suitable acid addition salts are formed from acids which form non-toxic salts. Examples include the acetate, adipate, aspartate, benzoate, besylate, bicarbonate/carbonate, bisulphate/sulphate, borate, camsylate, citrate, cyclamate, edisylate, esylate, formate, fumarate, gluceptate, gluconate, glucuronate, hexafluorophosphate, hibenzate, hydrochloride/chloride, hydrobromide/bromide, hydroiodide/iodide, isethionate, lactate, malate, maleate, malonate, mesylate, methylsulphate, naphthylate, 2-napsylate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate/hydrogen phosphate/dihydrogen phosphate, pyroglutamate, saccharate, stearate, succinate, tannate, tartrate, tosylate, trifluoroacetate and xinofoate salts.
Suitable base salts are formed from bases which form non-toxic salts. Examples include the aluminium, arginine, benzathine, calcium, choline, diethylamine, diolamine, glycine, lysine, magnesium, meglumine, olamine, potassium, sodium, tromethamine and zinc salts.
Hemisalts of acids and bases may also be formed, for example, hemisulphate and hemicalcium salts.
The skilled person will appreciate that the aforementioned salts include ones wherein the counterion is optically active, for example d-lactate or l-lysine, or racemic, for example dl-tartrate or dl-arginine.
For a review on suitable salts, see “Handbook of Pharmaceutical Salts: Properties, Selection, and Use” by Stahl and Wermuth (Wiley-VCH, Weinheim, Germany, 2002).
Pharmaceutically acceptable salts of compounds of formula (I) may be prepared by one or more of three methods:
All three reactions are typically carried out in solution. The resulting salt may precipitate out and be collected by filtration or may be recovered by evaporation of the solvent. The degree of ionisation in the resulting salt may vary from completely ionised to almost non-ionised.
The compounds of formula (I) or pharmaceutically acceptable salts thereof may exist in both unsolvated and solvated forms. The term ‘solvate’ is used herein to describe a molecular complex comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof and one or more pharmaceutically acceptable solvent molecules, for example, ethanol. The term ‘hydrate’ is employed when said solvent is water. Pharmaceutically acceptable solvates in accordance with the invention include those wherein the solvent of crystallization may be isotopically substituted, e.g. D2O, d6-acetone and d6-DMSO.
A currently accepted classification system for organic hydrates is one that defines isolated site, channel, or metal-ion coordinated hydrates—see Polymorphism in Pharmaceutical Solids by K. R. Morris (Ed. H. G. Brittain, Marcel Dekker, 1995), incorporated herein by reference. Isolated site hydrates are ones in which the water molecules are isolated from direct contact with each other by intervening organic molecules. In channel hydrates, the water molecules lie in lattice channels where they are next to other water molecules. In metal-ion coordinated hydrates, the water molecules are bonded to the metal ion.
When the solvent or water is tightly bound, the complex will have a well-defined stoichiometry independent of humidity. When, however, the solvent or water is weakly bound, as in channel solvates and hygroscopic compounds, the water/solvent content will be dependent on humidity and drying conditions. In such cases, non-stoichiometry will be the norm.
The compounds of the invention may exist in a continuum of solid states ranging from fully amorphous to fully crystalline. The term ‘amorphous’ refers to a state in which the material lacks long range order at the molecular level and, depending upon temperature, may exhibit the physical properties of a solid or a liquid. Typically such materials do not give distinctive X-ray diffraction patterns and, while exhibiting the properties of a solid, are more formally described as a liquid. Upon heating, a change from solid to liquid properties occurs which is characterised by a change of state, typically second order (‘glass transition’). The term ‘crystalline’ refers to a solid phase in which the material has a regular ordered internal structure at the molecular level and gives a distinctive X-ray diffraction pattern with defined peaks. Such materials when heated sufficiently will also exhibit the properties of a liquid, but the change from solid to liquid is characterised by a phase change, typically first order (‘melting point’).
Also included within the scope of the invention are multi-component complexes (other than salts and solvates) of compounds of formula (I) or pharmaceutically acceptable salts thereof wherein the drug and at least one other component are present in stoichiometric or non-stoichiometric amounts. Complexes of this type include clathrates (drug-host inclusion complexes) and co-crystals. The latter are typically defined as crystalline complexes of neutral molecular constituents which are bound together through non-covalent interactions, but could also be a complex of a neutral molecule with a salt. Co-crystals may be prepared by melt crystallisation, by recrystallisation from solvents, or by physically grinding the components together—see Chem Commun, 17, 1889-1896, by O. Almarsson and M. J. Zaworotko (2004), incorporated herein by reference. For a general review of multi-component complexes, see J Pharm Sci, 64 (8), 1269-1288, by Haleblian (August 1975), incorporated herein by reference.
The compounds of the invention may also exist in a mesomorphic state (mesophase or liquid crystal) when subjected to suitable conditions. The mesomorphic state is intermediate between the true crystalline state and the true liquid state (either melt or solution). Mesomorphism arising as the result of a change in temperature is described as ‘thermotropic’ and that resulting from the addition of a second component, such as water or another solvent, is described as ‘lyotropic’. Compounds that have the potential to form lyotropic mesophases are described as ‘amphiphilic’ and consist of molecules which possess an ionic (such as —COO−Na+, —COO−K+, or —SO3−Na+) or non-ionic (such as —N−N+(CH3)3) polar head group. For more information, see Crystals and the Polarizing Microscope by N. H. Hartshorne and A. Stuart, 4th Edition (Edward Arnold, 1970), incorporated herein by reference.
The compounds of the invention may be administered as prodrugs. Thus certain derivatives of compounds of formula (I) which may have little or no pharmacological activity themselves can, when administered into or onto the body, be converted into compounds of formula (I) having the desired activity, for example, by hydrolytic cleavage. Such derivatives are referred to as ‘prodrugs’. Further information on the use of prodrugs may be found in ‘Pro-drugs as Novel Delivery Systems, Vol. 14, ACS Symposium Series (T Higuchi and W Stella) and ‘Bioreversible Carriers in Drug Design’, Pergamon Press, 1987 (ed. E B Roche, American Pharmaceutical Association).
Prodrugs can, for example, be produced by replacing appropriate functionalities present in a compound of formula (I) with certain moieties known to those skilled in the art as ‘pro-moieties’ as described, for example, in “Design of Prodrugs” by H Bundgaard (Elsevier, 1985).
Examples of prodrugs include phosphate prodrugs, such as dihydrogen or dialkyl (e.g. di-tert-butyl) phosphate prodrugs. Further examples of replacement groups in accordance with the foregoing examples and examples of other prodrug types may be found in the aforementioned references.
Also included within the scope of the invention are metabolites of compounds of formula (I), that is, compounds formed in vivo upon administration of the drug. Some examples of metabolites in accordance with the invention include, where the compound of formula (I) contains a phenyl (Ph) moiety, a phenol derivative thereof (-Ph>-PhOH);
Compounds of the invention containing one or more asymmetric carbon atoms can exist as two or more stereoisomers. Included within the scope of the invention are all stereoisomers of the compounds of the invention and mixtures of one or more thereof.
Conventional techniques for the preparation/isolation of individual enantiomers include chiral synthesis from a suitable optically pure precursor or resolution of the racemate (or the racemate of a salt or derivative) using, for example, chiral high pressure liquid chromatography (HPLC).
Alternatively, the racemate (or a racemic precursor) may be reacted with a suitable optically active compound, for example, an alcohol, or, in the case where the compound of formula (I) contains an acidic or basic moiety, a base or acid such as 1-phenylethylamine or tartaric acid. The resulting diastereomeric mixture may be separated by chromatography and/or fractional crystallization and one or both of the diastereoisomers converted to the corresponding pure enantiomer(s) by means well known to a skilled person.
Chiral compounds of the invention (and chiral precursors thereof) may be obtained in enantiomerically-enriched form using chromatography, typically HPLC, on an asymmetric resin with a mobile phase consisting of a hydrocarbon, typically heptane or hexane, containing from 0 to 50% by volume of isopropanol, typically from 2% to 20%, and from 0 to 5% by volume of an alkylamine, typically 0.1% diethylamine. Concentration of the eluate affords the enriched mixture.
Mixtures of stereoisomers may be separated by conventional techniques known to those skilled in the art; see, for example, “Stereochemistry of Organic Compounds” by E. L. Eliel and S. H. Wilen (Wiley, New York, 1994.
The scope of the invention includes all crystal forms of the compounds of the invention, including racemates and racemic mixtures (conglomerates) thereof. Stereoisomeric conglomerates may also be separated by the conventional techniques described herein just above.
The scope of the invention includes all pharmaceutically acceptable isotopically-labelled compounds of the invention wherein one or more atoms are replaced by atoms having the same atomic number, but an atomic mass or mass number different from the atomic mass or mass number which predominates in nature.
Examples of isotopes suitable for inclusion in the compounds of the invention include isotopes of hydrogen, such as 2H and 3H, carbon, such as 11C, 13C and 14C, chlorine, such as 36Cl, fluorine, such as 18F, iodine, such as 123I and 125I, nitrogen, such as 13N and 15N, oxygen, such as 15O, 17O and 18O, phosphorus, such as 32P, and sulphur, such as 35S.
Certain isotopically-labelled compounds of the invention, for example, those incorporating a radioactive isotope, are useful in drug and/or substrate tissue distribution studies. The radioactive isotopes tritium, i.e. 3H, and carbon-14, i.e. 14C, are particularly useful for this purpose in view of their ease of incorporation and ready means of detection. Substitution with heavier isotopes such as deuterium, i.e. 2H, may 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. Substitution with positron emitting isotopes, such as 11C, 18F, 15O and 13N, can be useful in Positron Emission Topography (PET) studies for examining substrate receptor occupancy.
Isotopically-labeled compounds of formula (I) can generally be prepared by conventional techniques known to those skilled in the art or by processes analogous to those described in the accompanying Examples and Preparations using an appropriate isotopically-labeled reagent in place of the non-labeled reagent previously employed.
Also within the scope of the invention are intermediate compounds as hereinafter defined, all salts, solvates and complexes thereof and all solvates and complexes of salts thereof as defined hereinbefore for compounds of formula (I). The invention includes all polymorphs of the aforementioned species and crystal habits thereof.
When preparing a compound of formula (I) in accordance with the invention, a person skilled in the art may routinely select the form of intermediate which provides the best combination of features for this purpose. Such features include the melting point, solubility, processability and yield of the intermediate form and the resulting ease with which the product may be purified on isolation.
The compounds of the invention may be prepared by any method known in the art for the preparation of compounds of analogous structure. In particular, the compounds of the invention can be prepared by the procedures described by reference to the Schemes that follow, or by the specific methods described in the Examples, or by similar processes to either.
The skilled person will appreciate that the experimental conditions set forth in the schemes that follow are illustrative of suitable conditions for effecting the transformations shown, and that it may be necessary or desirable to vary the precise conditions employed for the preparation of compounds of formula (I). It will be further appreciated that it may be necessary or desirable to carry out the transformations in a different order from that described in the schemes, or to modify one or more of the transformations, to provide the desired compound of the invention.
In addition, the skilled person will appreciate that it may be necessary or desirable at any stage in the synthesis of compounds of the invention to protect one or more sensitive groups, so as to prevent undesirable side reactions. In particular, it may be necessary or desirable to protect amino or carboxylic acid groups. The protecting groups used in the preparation of the compounds of the invention may be used in conventional manner. See, for example, those described in ‘Greene's Protective Groups in Organic Synthesis’ by Theodora W Greene and Peter G M Wuts, fourth edition, (John Wiley and Sons, 2006), in particular chapters 7 (“Protection for the Amino Group”) and 5 (“Protection for the Carboxyl Group”), incorporated herein by reference, which also describes methods for the removal of such groups.
In the following general processes R1, R2, R3, R4, R5 and R6 are as previously defined for a compound of formula (I) unless otherwise stated. PG is a suitable amino protecting group, such as methoxymethyl or dimethoxybenzyl. Hal is a suitable halogen, such as F or Cl. Where ratios of solvents are given, the ratios are by volume.
According to a first process, compounds of formula (I) may be prepared from compounds of formula (II) and (III), as illustrated by Scheme 1.
Compounds of formula (I) may be prepared from compounds of formula (II) according to reaction step (ii) by nucleophilic aromatic substitution reaction with compounds of formula (IV) under basic reaction conditions. Convenient conditions are potassium carbonate in DMF or DMSO; cesium carbonate in DMSO; or potassium phosphate in DMSO; and at from room temperature to elevated temperature. Typical conditions comprise potassium carbonate in DMSO at 80-100° C. for 18 hours.
Compounds of formula (II) may be prepared from compounds of formula (III) according to reaction step (i) by displacement of a sulfonyl chloride with compounds of formula (V) under basic reaction conditions. Convenient conditions are pyridine in DCM; 1,4-diazabicyclo[2.2.2]octane in acetonitrile; N-methylmorpholine in THF; or an excess of compound of formula (V). Preferred conditions comprise pyridine in DCM at room temperature.
According to a second process, compounds of formula (I) may be prepared from compounds of formulae (II) and (VIII), as illustrated by Scheme 2.
Compounds of formula (I) may be prepared from compound of formula (VI) according to process step (ii) according to the conditions described in Scheme 1 step (ii), followed by deprotection step (iii), typically mediated by an inorganic or organic acid. Preferred conditions comprise potassium carbonate in DMSO at room temperature, followed by trifluoroacetic acid in DCM or HCl in 1,4-dioxane. It is also possible that deprotection step (iii) may occur under the conditions for effecting the nucleophilic aromatic substitution of step (ii).
Compounds of formula (VI) may be prepared from compounds of formula (II) according to process step (iv) by introduction of a suitable protecting group, such as methoxymethyl or dimethoxybenzyl, under basic reaction conditions or Mitsunobu reaction conditions. Typical conditions comprise diisopropylethylamine in DCM with chloromethoxymethane.
Alternatively, compounds of formula (VI) may be prepared from compounds of formula (III) according to process step (i) according to the conditions described in Scheme 1 step (i), or by using sodium or lithium hexamethyldisilazane in THF at from −78° C. to room temperature.
Compounds of formulae (II) and (III) may be prepared as described in Scheme 1.
Compounds of formula (VII) may be prepared from compounds of formula (VIII) according to reaction step (v) by a nucleophilic aromatic substitution reaction with compounds of formula (IX) under basic reaction conditions. Preferred conditions comprise diisopropylethylamine in n-butanol at 100° C. or potassium carbonate in DMSO at 110° C.
According to a third process, compounds of formula (I) may be prepared from compounds of formula (XIII) as illustrated by Scheme 3.
Compounds of formula (I) may be prepared from compounds of formula (X) according to the conditions described in Scheme 2, step(i).
Compounds of formula (X) may be prepared from compounds of formula (XI) according to process step (vii), an oxidation reaction in the presence of trichloroisocyanuric acid. Preferred conditions comprise trichloroisocyanuric acid with benzyltrimethylammonium chloride and sodium carbonate in acetonitrile and water.
Compounds of formula (XI) may be prepared from compounds of formula (XII) according to process step (ii), a nucleophilic aromatic substitution reaction with compounds of formula (IV) as described in Scheme 1, step (i).
Compounds of formula (XII) may be prepared from compounds of formula (XIII) according to process step (vi), a cross-coupling reaction with benzylmercaptan in the presence of a suitable catalyst. Conveniently the catalyst is a palladium catalyst. Preferred conditions comprise diisopropylethyamine with [1,1-bis(di-tert-butylphosphino)]ferrocene palladium (II) in toluene at 60° C.
The skilled person will appreciate that a compound of formula (I) wherein R2, R3, R4 or R5 is Cl, Br or I may be converted into the corresponding compound of formula (I) wherein the group in question is H, by dehalogenation in the presence of a suitable catalyst. Typical conditions comprise zinc dust in acetic acid at room temperature, or triethylsilane with tetrakis(triphenylphosphine)palladium(0) in THF at reflux.
The skilled person will further appreciate that a compound of formula (I) wherein R6 is 2- or 4-halo substituted ‘C-linked’ 6-membered heteroaryl containing one or two nitrogen atoms may be converted into the corresponding compound of formula (I) substituted by —NR7R8, by reaction with an appropriate amine. Where halo is fluoro, typical conditions comprise heating the compound of formula (I) and the amine of formula HNR7R8 in a solvent such as DMSO, in the presence of an inorganic base such as potassium carbonate, to a temperature of between 50-70° C.
The skilled person will further appreciate that in the aforementioned conversion, when R7 and R8 are both H, it may be necessary or desirable to employ an amino protecting group, such as dimethoxybenzyl, to introduce a protected amine; the protecting group is then removed under conventional conditions, such as in the presence of an organic acid. Preferred conditions comprise dimethoxybenzylamine with potassium carbonate in THF at 70° C., followed by stirring in TFA at room temperature.
Compounds of formula (III), (IV), (V), (VIII), (IX) and (XIII) are commercially available, known from the literature, easily prepared by methods well known to those skilled in the art, or can be made according to preparations described herein.
All new processes for preparing compounds of formula (I), and corresponding new intermediates employed in such processes, form further aspects of the present invention.
Compounds of the invention intended for pharmaceutical use may be administered as crystalline or amorphous products or may exist in a continuum of solid states ranging from fully amorphous to fully crystalline. They may be obtained, for example, as solid plugs, powders, or films by methods such as precipitation, crystallization, freeze drying, spray drying, or evaporative drying. Microwave or radio frequency drying may be used for this purpose.
They may be administered alone or in combination with one or more other compounds of the invention or in combination with one or more other drugs (or as any combination thereof). Generally, they will be administered as a formulation in association with one or more pharmaceutically acceptable excipients. The term ‘excipient’ is used herein to describe any ingredient other than the compound(s) of the invention. The choice of excipient will to a large extent depend on factors such as the particular mode of administration, the effect of the excipient on solubility and stability, and the nature of the dosage form.
In another aspect the invention provides a pharmaceutical composition comprising a compound of the invention together with one or more pharmaceutically acceptable excipients.
Pharmaceutical compositions suitable for the delivery of compounds of the present invention and methods for their preparation will be readily apparent to those skilled in the art. Such compositions and methods for their preparation may be found, for example, in “Remington's Pharmaceutical Sciences”, 19th Edition (Mack Publishing Company, 1995).
Suitable modes of administration include oral, parenteral, topical, inhaled/intranasal, rectal/intravaginal, and ocular/aural administration.
Formulations suitable for the aforementioned modes of administration may be formulated to be immediate and/or modified release. Modified release formulations include delayed-, sustained-, pulsed-, controlled-, targeted and programmed release.
The compounds of the invention may be administered orally. Oral administration may involve swallowing, so that the compound enters the gastrointestinal tract, or buccal or sublingual administration may be employed by which the compound enters the blood stream directly from the mouth. Formulations suitable for oral administration include solid formulations such as tablets, capsules containing particulates, liquids, or powders, lozenges (including liquid-filled), chews, multi- and nano-particulates, gels, solid solution, liposome, films, ovules, sprays, liquid formulations and buccal/mucoadhesive patches.
Liquid formulations include suspensions, solutions, syrups and elixirs. Such formulations may be employed as fillers in soft or hard capsules and typically comprise a carrier, for example, water, ethanol, polyethylene glycol, propylene glycol, methylcellulose, or a suitable oil, and one or more emulsifying agents and/or suspending agents. Liquid formulations may also be prepared by the reconstitution of a solid, for example, from a sachet.
The compounds of the invention may also be used in fast-dissolving, fast-disintegrating dosage forms such as those described in Expert Opinion in Therapeutic Patents, 11 (6), 981-986, by Liang and Chen (2001).
For tablet dosage forms, depending on dose, the drug may make up from 1 weight % to 80 weight % of the dosage form, more typically from 5 weight % to 60 weight % of the dosage form. In addition to the drug, tablets generally contain a disintegrant. Examples of disintegrants include sodium starch glycolate, sodium carboxymethyl cellulose, calcium carboxymethyl cellulose, croscarmellose sodium, crospovidone, polyvinylpyrrolidone, methyl cellulose, microcrystalline cellulose, lower alkyl-substituted hydroxypropyl cellulose, starch, pregelatinised starch and sodium alginate. Generally, the disintegrant will comprise from 1 weight % to 25 weight %, preferably from 5 weight % to 20 weight % of the dosage form.
Binders are generally used to impart cohesive qualities to a tablet formulation. Suitable binders include microcrystalline cellulose, gelatin, sugars, polyethylene glycol, natural and synthetic gums, polyvinylpyrrolidone, pregelatinised starch, hydroxypropyl cellulose and hydroxypropyl methylcellulose. Tablets may also contain diluents, such as lactose (monohydrate, spray-dried monohydrate, anhydrous and the like), mannitol, xylitol, dextrose, sucrose, sorbitol, microcrystalline cellulose, starch and dibasic calcium phosphate dihydrate.
Tablets may also optionally comprise surface active agents, such as sodium lauryl sulfate and polysorbate 80, and glidants such as silicon dioxide and talc. When present, surface active agents may comprise from 0.2 weight % to 5 weight % of the tablet, and glidants may comprise from 0.2 weight % to 1 weight % of the tablet.
Tablets also generally contain lubricants such as magnesium stearate, calcium stearate, zinc stearate, sodium stearyl fumarate, and mixtures of magnesium stearate with sodium lauryl sulphate. Lubricants generally comprise from 0.25 weight % to 10 weight %, preferably from 0.5 weight % to 3 weight % of the tablet. Other possible ingredients include anti-oxidants, colourants, flavouring agents, preservatives and taste-masking agents.
Exemplary tablets contain up to about 80% drug, from about 10 weight % to about 90 weight % binder, from about 0 weight % to about 85 weight % diluent, from about 2 weight % to about 10 weight % disintegrant, and from about 0.25 weight % to about 10 weight % lubricant. Tablet blends may be compressed directly or by roller to form tablets. Tablet blends or portions of blends may alternatively be wet-, dry-, or melt-granulated, melt congealed, or extruded before tabletting. The final formulation may comprise one or more layers and may be coated or uncoated; it may even be encapsulated. The formulation of tablets is discussed in “Pharmaceutical Dosage Forms: Tablets”, Vol. 1, by H. Lieberman and L. Lachman (Marcel Dekker, New York, 1980).
Suitable modified release formulations for the purposes of the invention are described in U.S. Pat. No. 6,106,864. Details of other suitable release technologies such as high energy dispersions and osmotic and coated particles are to be found in “Pharmaceutical Technology On-line”, 25(2), 1-14, by Verma et al (2001). The use of chewing gum to achieve controlled release is described in WO 00/35298.
The compounds of the invention may also be administered directly into the blood stream, into muscle, or into an internal organ. Suitable means for parenteral administration include intravenous, intraarterial, intraperitoneal, intrathecal, intraventricular, intraurethral, intrasternal, intracranial, intramuscular and subcutaneous. Suitable devices for parenteral administration include needle (including microneedle) injectors, needle-free injectors and infusion techniques.
Parenteral formulations are typically aqueous solutions which may contain excipients such as salts, carbohydrates and buffering agents (preferably to a pH of from 3 to 9), but, for some applications, they may be more suitably formulated as a sterile non-aqueous solution or as a dried form to be used in conjunction with a suitable vehicle such as sterile, pyrogen-free water.
The preparation of parenteral formulations under sterile conditions, for example, by lyophilisation, may readily be accomplished using standard pharmaceutical techniques well known to those skilled in the art.
The solubility of compounds of formula (I) used in the preparation of parenteral solutions may be increased by the use of appropriate formulation techniques, such as the incorporation of solubility-enhancing agents. Formulations for parenteral administration may be formulated to be immediate and/or modified release. Modified release formulations include delayed-, sustained-, pulsed-, controlled-, targeted and programmed release. Thus compounds of the invention may be formulated as a solid, semi-solid, or thixotropic liquid for administration as an implanted depot providing modified release of the active compound. Examples of such formulations include drug-coated stents and poly(dl-lactic-coglycolic)acid (PGLA) microspheres.
The compounds of the invention may also be administered topically to the skin or mucosa, that is, dermally or transdermally. Typical formulations for this purpose include gels, hydrogels, lotions, solutions, creams, ointments, dusting powders, dressings, foams, films, skin patches, wafers, implants, sponges, fibres, bandages and microemulsions. Liposomes may also be used. Typical carriers include alcohol, water, mineral oil, liquid petrolatum, white petrolatum, glycerin, polyethylene glycol and propylene glycol. Penetration enhancers may be incorporated—see, for example, J Pharm Sci, 88 (10), 955-958, by Finnin and Morgan (October 1999).
Other means of topical administration include delivery by electroporation, iontophoresis, phonophoresis, sonophoresis and microneedle or needle-free (e.g. Powderject™ Bioject™, etc.) injection.
The compounds of the invention can also be administered intranasally or by inhalation, typically in the form of a dry powder (either alone, as a mixture, for example, in a dry blend with lactose, or as a mixed component particle, for example, mixed with phospholipids, such as phosphatidylcholine) from a dry powder inhaler or as an aerosol spray from a pressurised container, pump, spray, atomiser (preferably an atomiser using electrohydrodynamics to produce a fine mist), or nebuliser, with or without the use of a suitable propellant, such as 1,1,1,2-tetrafluoroethane or 1,1,1,2,3,3,3-heptafluoropropane. For intranasal use, the powder may comprise a bioadhesive agent, for example, chitosan or cyclodextrin.
The pressurised container, pump, spray, atomizer, or nebuliser contains a solution or suspension of the compound(s) of the invention comprising, for example, ethanol, aqueous ethanol, or a suitable alternative agent for dispersing, solubilising, or extending release of the active, a propellant(s) as solvent and an optional surfactant, such as sorbitan trioleate, oleic acid, or an oligolactic acid.
Prior to use in a dry powder or suspension formulation, the drug product is micronised to a size suitable for delivery by inhalation (typically less than 5 microns). This may be achieved by any appropriate comminuting method, such as spiral jet milling, fluid bed jet milling, supercritical fluid processing to form nanoparticles, high pressure homogenisation, or spray drying.
Capsules (made, for example, from gelatin or hydroxypropylmethylcellulose), blisters and cartridges for use in an inhaler or insufflator may be formulated to contain a powder mix of the compound of the invention, a suitable powder base such as lactose or starch and a performance modifier such as l-leucine, mannitol, or magnesium stearate. The lactose may be anhydrous or in the form of the monohydrate, preferably the latter. Other suitable excipients include dextran, glucose, maltose, sorbitol, xylitol, fructose, sucrose and trehalose.
A suitable solution formulation for use in an atomiser using electrohydrodynamics to produce a fine mist may contain from 1 μg to 20 mg of the compound of the invention per actuation and the actuation volume may vary from 1 μl to 100 μl. A typical formulation may comprise a compound of formula (I), propylene glycol, sterile water, ethanol and sodium chloride. Alternative solvents which may be used instead of propylene glycol include glycerol and polyethylene glycol.
Suitable flavours, such as menthol and levomenthol, or sweeteners, such as saccharin or saccharin sodium, may be added to those formulations of the invention intended for inhaled/intranasal administration.
In the case of dry powder inhalers and aerosols, the dosage unit is determined by means of a valve which delivers a metered amount. Units in accordance with the invention are typically arranged to administer a metered dose or “puff” containing from 1 μg to 100 mg of the compound of formula (I). The overall daily dose will typically be in the range 1 μg to 200 mg which may be administered in a single dose or, more usually, as divided doses throughout the day.
The compounds of the invention may be administered rectally or vaginally, for example, in the form of a suppository, pessary, microbicide, vaginal ring or enema. Cocoa butter is a traditional suppository base, but various alternatives may be used as appropriate.
The compounds of the invention may also be administered directly to the eye or ear, typically in the form of drops of a micronised suspension or solution in isotonic, pH-adjusted, sterile saline. Other formulations suitable for ocular and aural administration include ointments, biodegradable (e.g. absorbable gel sponges, collagen) and non-biodegradable (e.g. silicone) implants, wafers, lenses and particulate or vesicular systems, such as niosomes or liposomes. A polymer such as crossed-linked polyacrylic acid, polyvinylalcohol, hyaluronic acid, a cellulosic polymer, for example, hydroxypropylmethylcellulose, hydroxyethylcellulose, or methyl cellulose, or a heteropolysaccharide polymer, for example, gelan gum, may be incorporated together with a preservative, such as benzalkonium chloride. Such formulations may also be delivered by iontophoresis.
The compounds of the invention may be combined with soluble macromolecular entities, such as cyclodextrin and suitable derivatives thereof or polyethylene glycol-containing polymers, in order to improve their solubility, dissolution rate, taste-masking, bioavailability and/or stability for use in any of the aforementioned modes of administration.
Drug-cyclodextrin complexes, for example, are found to be generally useful for most dosage forms and administration routes. Both inclusion and non-inclusion complexes may be used. As an alternative to direct complexation with the drug, the cyclodextrin may be used as an auxiliary additive, i.e. as a carrier, diluent, or solubiliser. Most commonly used for these purposes are alpha-, beta- and gamma-cyclodextrins, examples of which may be found in International Patent Applications Nos. WO 91/11172, WO 94/02518 and WO 98/55148.
For administration to human patients, the total daily dose of the compounds of the invention is typically in the range 1 mg to 10 g, such as 10 mg to 1 g, for example 25 mg to 500 mg depending, of course, on the mode of administration and efficacy. For example, oral administration may require a total daily dose of from 50 mg to 100 mg. The total daily dose may be administered in single or divided doses and may, at the physician's discretion, fall outside of the typical range given herein. These dosages are based on an average human subject having a weight of about 60 kg to 70 kg. The physician will readily be able to determine doses for subjects whose weight falls outside this range, such as infants and the elderly.
As noted above, the compounds of the invention are useful because they exhibit pharmacological activity in animals, i.e., URAT-1 inhibition. More particularly, the compounds of the invention are of use in the treatment of disorders for which a URAT-1 inhibitor is indicated. Preferably the animal is a mammal, more preferably a human.
In a further aspect of the invention there is provided a compound of the invention for use as a medicament.
In a further aspect of the invention there is provided a compound of the invention for the treatment of a disorder for which a URAT-1 inhibitor is indicated.
In a further aspect of the invention there is provided use of a compound of the invention for the preparation of a medicament for the treatment of a disorder for which a URAT-1 inhibitor is indicated.
In a further aspect of the invention there is provided a method of treating a disorder in an animal (preferably a mammal, more preferably a human) for which a URAT-1 inhibitor is indicated, comprising administering to said animal a therapeutically effective amount of a compound of the invention.
Disorders for which a URAT-1 inhibitor is indicated include diseases associated with high levels of uric acid in humans and other mammals including (but not limited to) hyperuricemia, asymptomatic hyperuricemia, gout (including juvenile forms), gouty arthritis, inflammatory arthritis, joint inflammation, deposition of urate crystals in the joint, tophaceous gout, chronic kidney disease, nephrolithiasis (kidney stones), Lesch-Nyhan syndrome and Kelley-Seegmiller syndrome.
Hyperuricemia may be defined by blood uric acid levels over 6.8 mg/dL. Guidelines for the management of hyperuricemia recommend that therapies aimed at lowering blood uric acid levels should be maintained until such blood uric acid levels are lowered to below 6.0 mg/dL, such as below 5.0 mg/dL.
The skilled person will appreciate that while by definition without symptoms, asymptomatic hyperuricemia may nevertheless lead to the onset of diseases associated with high levels of uric acid.
The skilled person will also appreciate that the compounds of formula (I) may be used in the treatment of hyperuricemia where this is present together with one or more other diseases, such as kidney failure, type 2 diabetes, cardiovascular disease (e.g. hypertension, myocardial infarction, heart failure, coronary artery disease, cerebrovascular disease, atherosclerosis, angina, aneurism, hyperlipidemia and stroke), obesity, metabolic syndrome, myeloproliferative disorders, lymphoproliferative disorders and disorders associated with certain medications, such as a diuretic (e.g. a thiazide), an immunosuppressant (e.g. a cyclosporine therapy), a chemotherapeutic agent (e.g. cisplatin) or aspirin.
The skilled person will also appreciate that the compounds of formula (I) may be used in the treatment of hyperuricemia where this is present following organ transplant.
A URAT-1 inhibitor may be usefully combined with another pharmacologically active compound, or with two or more other pharmacologically active compounds, particularly in the treatment of a disease associated with elevated blood uric acid levels. Such combinations offer the possibility of significant advantages, including patient compliance, ease of dosing and synergistic activity.
In the combinations that follow the compound of the invention may be administered simultaneously, sequentially or separately in combination with the other therapeutic agent or agents.
The compounds of formula (I) may be administered in combination with one or more additional therapeutic agents. Agents of interest include those that also lower blood uric acid levels. Other agents of interest include those that reduce inflammation or pain. The one or more additional therapeutic agents may be selected from any of the agents or types of agent that follow:
There is also included within the scope the present invention combinations of a compound of the invention together with one or more additional therapeutic agents which slow down the rate of metabolism of the compound of the invention, thereby leading to increased exposure in patients. Increasing the exposure in such a manner is known as boosting. This has the benefit of increasing the efficacy of the compound of the invention or reducing the dose required to achieve the same efficacy as an unboosted dose. The metabolism of the compounds of the invention includes oxidative processes carried out by P450 (CYP450) enzymes, particularly CYP 3A4 and conjugation by UDP glucuronosyl transferase and sulphating enzymes. Thus, among the agents that may be used to increase the exposure of a patient to a compound of the present invention are those that can act as inhibitors of at least one isoform of the cytochrome P450 (CYP450) enzymes. The isoforms of CYP450 that may be beneficially inhibited include, but are not limited to, CYP1A2, CYP2D6, CYP2C9, CYP2C19 and CYP3A4. Suitable agents that may be used to inhibit CYP 3A4 include ritonavir, saquinavir, ketoconazole, N-(3,4-difluorobenzyl)-N-methyl-2-{[(4-methoxypyridin-3-yl)amino]sulfonyl}benzamide and N-(1-(2-(5-(4-fluorobenzyl)-3-(pyridin-4-yl)-1H-pyrazol-1-yl)acetyl)piperidin-4-yl)methanesulfonamide.
It is within the scope of the invention that two or more pharmaceutical compositions, at least one of which contains a compound of the invention, may conveniently be combined in the form of a kit suitable for coadministration of the compositions. Thus the kit of the invention comprises two or more separate pharmaceutical compositions, at least one of which contains a compound of the invention, and means for separately retaining said compositions, such as a container, divided bottle, or divided foil packet. An example of such a kit is the familiar blister pack used for the packaging of tablets, capsules and the like. The kit of the invention is particularly suitable for administering different dosage forms, for example, oral and parenteral, for administering the separate compositions at different dosage intervals, or for titrating the separate compositions against one another. To assist compliance, the kit typically comprises directions for administration and may be provided with a so-called memory aid.
In another aspect the invention provides a pharmaceutical product (such as in the form of a kit) comprising a compound of the invention together with one or more additional therapeutically active agents as a combined preparation for simultaneous, separate or sequential use in the treatment of a disorder for which a URAT-1 inhibitor is indicated.
It is to be appreciated that all references herein to treatment include curative, palliative and prophylactic treatment.
The invention is further illustrated by the non-limiting Examples and Preparations that follow (for the avoidance of doubt, those compounds marked as Reference Examples do not fall within formula (I)).
In these non-limiting Examples and Preparations, and in the aforementioned Schemes, the following abbreviations, definitions and analytical procedures may be referred to:
AcOH is acetic acid;
nBuOH is n-butanol
Cu(acac)2 is copper (II) acetylacetonate;
Cu(OAc)2 is copper (II) acetate;
DABCO is 1,4-diazabicyclo[2,2,2]octane
DAD is diode array detector;
DCM is dichloromethane; methylene chloride;
DEA is diethylamine
DIP-Cl is chlorodiisopinocampheylborane;
DIPEA is N-ethyldiisopropylamine, N,N-diisopropylethylamine;
DMAP is 4-dimethylaminopyridine;
DMF is N,N-dimethylformamide;
DMSO is dimethyl sulphoxide;
EDCI is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride;
EDTA is ethylenediaminetetraacetic acid;
ELSD is evaporative light scattering detection;
Et2O is diethyl ether;
EtOAc is ethyl acetate;
EtOH is ethanol;
HPLC is high-performance liquid chromatography
IPA is isopropanol;
Ir2(OMe)2COD2 is bis(1,5-cyclooctadiene)di-μ-methoxydiiridium (I);
KOAc is potassium acetate;
K3PO4 is potassium phosphate tribasic;
LCMS is liquid chromatography mass spectrometry (Rt=retention time)
Me is methyl
MeOH is methanol;
MS is mass spectrometry
NMM is N-methylmorpholine
NMP is N-Methyl-2-pyrrolidone;
Pd/C is palladium on carbon;
Pd(PPh3)4 is palladium tetrakis;
Pd(dppf)2Cl2 is [1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II), complex with dichloromethane;
TBAF is tetra-n-butylammonium fluoride
TBME is tert-butyl methyl ether;
TFA is trifluoroacetate;
THF is tetrahydrofuran;
THP is tetrahydropyran;
TLC is thin layer chromatography;
UV is ultraviolet; and
WSCDI is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride.
1H and 19F Nuclear magnetic resonance (NMR) spectra were in all cases consistent with the proposed structures. Characteristic chemical shifts (δ) are given in parts-per-million downfield from tetramethylsilane (for 1H-NMR) and upfield from trichloro-fluoro-methane (for 19F NMR) using conventional abbreviations for designation of major peaks: e.g. s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br, broad. The following abbreviations have been used for common solvents: CDCl3, deuterochloroform; d6-DMSO, deuterodimethylsulphoxide; and CD3OD, deuteromethanol.
Mass spectra, MS (m/z), were recorded using either electrospray ionisation (ESI) or atmospheric pressure chemical ionisation (APCI). Where relevant and unless otherwise stated the m/z data provided are for isotopes 19F, 35Cl, 79Br and 127I.
LCMS Conditions:
System 1
A: 0.1% formic acid in water
B: 0.1% formic acid in acetonitrile
Column: C18 phase Phenomenex 20×4.0 mm with 3 micron particle size
Gradient: 98-2% or 98-10% A over 1.5 min, 0.3 min hold, 0.2 re-equilibration, 1.8 mL/min flow rate
UV: 210 nm-450 nm DAD
Temperature: 75° C.
System 2
A: 0.1% formic acid in water
B: 0.1% formic acid in acetonitrile
Using either:
Column: Agilent Extend C18 phase 50×3 mm with 3 micron particle size
Gradient: 95-0% A over 3.5 min, 1 min hold, 0.4 min re-equilibration, 1.2 mL/min flow rate
3-Cyano-4-fluoro-N-(5-fluoropyridin-2-yl)benzenesulfonamide (Preparation 2, 10.5 g, 35.6 mmol) was added to a solution of 2-chloro-4-hydroxybenzonitrile (8.19 g, 53.3 mmol) and potassium carbonate (14.74 g, 106.7 mmol) in dimethylsulfoxide (100 mL) at room temperature. The resulting mixture was stirred at 80° C. for 44 hours. On cooling, the reaction mixture was poured into saturated aqueous sodium hydrogen carbonate solution (200 mL) and ethyl acetate (1000 mL) was added. The aqueous layer was separated and the organic layer was washed with saturated aqueous sodium hydrogen carbonate solution (3×200 mL), water (2×200 mL) and brine (2×200 mL). The organic layer was dried over sodium sulphate, filtered and concentrated in vacuo. The residue was purified by reverse phase column chromatography eluting with acetonitrile (containing 0.1% HCO2H): water (containing 0.1% HCO2H) from 0 to 100% to afford the title compound as a white solid (6.982 g, 46%).
1H NMR (400 MHz, DMSO-d6): δ ppm 7.09 (dd, 1H), 7.28 (d, 1H), 7.44 (dd, 1H), 7.68 (td, 1H), 7.81 (d, 1H), 8.09-8.15 (m, 2H), 8.19 (d, 1H), 8.41 (d, 1H), 11.30 (br s, 1H).
19F NMR (400 MHz, DMSO-d6): δ ppm −134
MS m/z 427 [M−H]−
General Method:
Wherein R1, R2, R3, R4, R5 and R6 are as previously defined for a compound of formula (I), unless otherwise stated, and PG (where present) is a suitable amino protecting group, such as methoxymethyl or dimethoxybenzyl.
For compounds of the Examples prepared as singletons (i.e. other than via the Library Protocols described hereinafter), one of two preparative HPLC methods was used, as shown below:
Acidic Conditions
Basic Conditions
Purification Method B: Silica gel column chromatography eluting with:
i) 95:5 DCM:EtOAc;
ii) 12-75% EtOAc in heptanes;
iii) 1:1 TBME:Heptanes; or
iv) 0-1% MeOH in DCM.
Purification Method C: Reverse phase column chromatography using:
Column: Phenomenex Luna C18 5 u 110A 21.2×150 mm
Detection @ 254 nm, threshold 25 mV
Solvent system:
Unless stated otherwise, the compounds of the Examples in the table below were prepared from 3-cyano-4-fluoro-N-(5-fluoropyridin-2-yl)benzenesulfonamide (Preparation 2) and the appropriate phenol according to Method Variation 1 (MV1), and then purified according to Purification Method A (PM A).
The compounds of the Examples in the table below were prepared from appropriate compounds of formulae (II) and (IV) according to the specified Method Variation (MV) and, as necessary, purified according to the specified Purification Method (PM).
To a solution of 5-bromo-4-(4-cyano-3-fluorophenoxy)-N-(2,4-dimethoxybenzyl)-2-fluoro-N-(5-fluoropyridin-2-yl)benzenesulfonamide (Preparation 1, 220 mg, 0.35 mmol) in THF (8 mL), AcOH (10 mL) and water (2.5 mL) was added zinc dust (800 mg, 12.2 mmol). The reaction mixture was left to stir at room temperature for 70 hours. To the reaction mixture was added EtOAc (25 mL) and the mixture filtered through celite and washed with EtOAc (50 mL). The filtrate was retained and to this was added saturated aqueous NaHCO3 (80 mL). The organic layer was retained, dried over MgSO4, and the solvent removed in vacuo. The residue was purified using reverse phase column chromatography eluting with acetonitrile and water (acidic). The residue was dissolved in DCM and treated with TFA (0.3 mL) and stirred at room temperature for 1 hour. The reaction was concentrated in vacuo azeotroping with MeOH. The residue was purified using preparative HPLC to afford the title compound as a colourless powder (16 mg, 26%).
MS m/z 404 [M−H]−
The title compound was prepared according to the method described for Example 101 using 4-aminopyridine.
MS m/z 411 [M+H]+
To a solution of 4-(3-chloro-4-cyanophenoxy)-3-cyanobenzene-1-sulfonyl chloride (Preparation 21, 240 mg, 0.65 mmol) in dry dichloromethane (3 ml) was added 3-methylpyridin-2-amine (176 mg, 1.63 mmol). The reaction mixture was stirred at room temperature for 16 hours. The reaction mixture was evaporated to dryness under vacuum to leave a brown gum (150 mg). The crude material was dissolved in dimethylsulfoxide and purified by preparative HPLC to afford the title compound as a colourless solid (44 mg, 16%).
1H NMR (400 MHz, DMSO-d6): δ ppm 2.17 (s, 3H), 6.81 (t, 1H), 7.39 (d, 1H), 7.42 (d, 1H), 7.63 (s, 1H), 7.71 (d, 1H), 7.90 (d, 1H), 8.01 (d, 1H), 8.22 (d, 1H), 8.40 (s, 1H).
MS m/z 425 [M+H]+
The title compound was prepared according to the Method described for Example 101 using 3-methoxypyridine-2-amine. The reaction mixture was diluted with dichloromethane and washed with 2 M HCl (100 mL). The organic phase was dried with MgSO4, filtered, and evaporated to dryness under vacuum to leave a light brown solid. The residue was dissolved in dichloromethane and purified using silica gel column chromatography eluting with 0% to 40% ethyl acetate in heptanes.
MS m/z 441 [M+H]+
The title compound was prepared according to the Method described for Example 101 using 3-fluoropyridine-2-amine.
MS m/z 429 [M+H]+
The title compound was prepared according to the Method described for Example 101 using 3-fluoropyridine-2-amine in pyridine.
MS m/z 429 [M+H]+
Library Protocol 1
To a 0.2M solution of 3-cyano-4-fluoro-N-(5-fluoropyridin-2-yl)benzenesulfonamide in DMSO (Preparation 2, 500 uL, 100 umol) was added a 0.2M solution in DMSO of the appropriate phenol/hydroxypyridine (compound of formula (IV), 500 uL, 100 umol) followed by anhydrous K3PO4 (64 mg, 300 umol) and the reaction mixtures were stirred at 80° C. for 18 hours. The reaction mixtures were cooled and purified using one of the two preparative HPLC described below to afford the desired compounds of formula (Ia).
Preparative HPLC Method 1:
Mobile phase A: 10 mM ammonium acetate in water; Mobile phase B: MeCN
Column: Gemini NX C18 (20×100 mm, 5 u)
Gradient: Initial 10% B; 2 mins 40% B; 10 mins 70% B, 11-12 mins 95% B, 13-15 mins 10% B
Flow rate: 20 mL/min.
Preparative HPLC Method 2:
Mobile phase A: 10 mM ammonium acetate in water; Mobile phase B: MeCN
Column: Xbridge C18 (19×50 mm, 5 u)
Gradient: Initial 10% B; 2 mins 20% B; 7 mins 80% B, 7.5-8.5 mins 95% B, 9-10 mins 10% B,
Flow rate: 20 mL/min
LCMS Conditions:
Mobile phase A: 0.05% formic acid in water; Mobile phase B: MeCN
Column: RESTEK C18 2.1×30 mm×3μ
Gradient: From 98% A and 2% B to 90% A and 10% B in 1 min, further to 2% A and 98% B in 2.0 min and finally back to initial condition in 3 min
Flow rate: 1.5 mL/min
The compounds of the Examples in the table below were prepared from 3-cyano-4-fluoro-N-(5-fluoropyridin-2-yl)benzenesulfonamide in DMSO (Preparation 2) and the appropriate phenol or hydroxypyridine according to Library protocol 1.
Library Protocol 2
To a 0.2M solution of 3-cyano-4-fluoro-N-(5-fluoropyridin-2-yl)benzenesulfonamide in DMSO (Preparation 2, 500 uL, 100 umol) was added a 0.2M solution in DMSO of the appropriate phenol/hydroxypyridine (compound of formula (IV), 500 uL, 100 umol) followed by anhydrous K3PO4 (64 mg, 300 umol) and the reaction mixtures were stirred at 80° C. for 18 hours. The reaction mixtures were cooled and purified using one of the two preparative HPLC described below to afford the desired compounds of formula (Ia).
Preparative HPLC Method 1:
Mobile phase A: 10 mM ammonium acetate in water; Mobile phase B: MeCN
Column: Gemini NX C18 (20×100 mm, 5 u)
Gradient: Initial 10% B; 2 mins 40% B; 10 mins 70% B, 11-12 mins 95% B, 13-15 mins 10% B
Flow rate: 20 mL/min.
Preparative HPLC Method 2:
Mobile phase A: 0.1% formic acid in water; Mobile phase B: MeCN
Column: Zorbax SB C18 921×250 mm, 7 u)
Gradient: Initial 10% B; 3 mins 20% B; 18 mins 80% B, 19-20 mins 95% B, 22-25 mins 10% B,
Flow rate: 20 mL/min
LCMS Conditions 1:
Mobile phase A: 0.05% formic acid in water; Mobile phase B: MeCN
Column: RESTEK C18 2.1×30 mm×3μ
Gradient: From 98% A and 2% B to 90% A and 10% B in 1 min, further to 2% A and 98% B in 2.0 min and finally back to initial condition in 3 min
Flow rate: 1.5 mL/min
LCMS Conditions 2:
Mobile phase A: 10 mM ammonium acetate in water; Mobile phase B: MeCN
Column: Zorbax Extend C18 (50×4.6 mm, 5 u)
Gradient: From 95% A and 5% B to 85% A and 15% B in 1.5 min, further to 10% A and 90% B in 3-4 min and finally back to initial condition in 5 min
Flow rate: 1.5 mL/min
The compounds of the Examples in the table below were prepared from 3-cyano-4-fluoro-N-(5-fluoropyridin-2-yl)benzenesulfonamide in DMSO (Preparation 2) and the appropriate phenol or hydroxypyridine according to Library protocol 2.
Library Protocol 3
To a 0.2M solution in DMSO of the appropriate sulphonamide (compound of formula (VIa), 500 uL, 100 umol) was added a 0.2M solution in DMSO of the appropriate phenol/hydroxypyridine (compound of formula (IV), 500 uL, 100 umol) followed by anhydrous K3PO4 (64 mg, 300 umol) and the reaction mixtures were stirred at 80° C. for 18 hours. The reaction mixtures were concentrated in vacuo and treated with 4M HCl in dioxane (1 mL). The reaction mixtures were concentrated in vacuo and purified using preparative HPLC as described below to afford the desired compounds of formula (I).
Preparative HPLC Method:
Mobile phase A: 10 mM ammonium acetate in water; Mobile phase B: MeCN
Column: Sunfire C18 (19×150 mm, 5 u)
Gradient: Initial 10% B; 2 mins 30% B; 10 mins 60% B, 12-13 mins 95% B, 14-15 mins 10% B
Flow rate: 16 mL/min.
The compounds of the Examples in the table below were prepared from the appropriate sulphonamide of formula (VIa) and the appropriate phenol of formula (IV) according to Library Protocol 3.
Library Protocol 4
To a stock solution of the appropriate amine of formula (V) (1 mmol) in THF (5 mL) was added NMM (111 uL, 1 mmol).
An aliquot of this solution (50 uL, 10 umol) was added to a solution of 4-(3-chloro-2-cyanophenoxy)benzene-1-sulfonyl chloride (3.6 mg, 12 umol) in DCM (120 uL) and the reaction mixture stirred at room temperature for 24 hours. The reaction mixture was concentrated in vacuo to afford the desired compound of formula (Ib).
The compounds of the Reference Examples in the table below were prepared from 4-(3-chloro-2-cyanophenoxy)benzene-1-sulfonyl chloride (commercially available) and the appropriate amine of formula (V) according to Library Protocol 4. These compounds do not fall within the scope of formula (I).
The title compound was prepared according to the method described for Library Protocol 2 using 3-cyano-4-fluoro-N-(5-fluoropyridin-2-yl)benzenesulfonamide (Preparation 2), 4-aminophenol.
MS m/z 385 [M+H]+
The title compound was prepared according to the method described for Library Protocol 2 using 5-chloro-N-(2,4-dimethoxybenzyl)-2,4-difluoro-N-(pyrimidin-2-yl)benzenesulfonamide (WO2012004743) and 3-cyano-4-fluorophenol.
MS m/z 423 [M+H]+
The compounds of the Examples in the table below were prepared from appropriate compounds of formulae (II) and (IV) according to the specified Method Variation (MV) and, as necessary, purified according to the specified Purification Method (PM).
The title compound was prepared according to the method described for Method Variation 5 using 3-cyano-N-(2,4-dimethoxybenzyl)-4-fluoro-N-(5-fluoropyridin-2-yl)benzenesulfonamide (Preparation 18) and 2,6-dichloro-4-hydroxybenzonitrile, and then purified using preparative HPLC.
1H NMR (400 MHz, DMSO-d6): δ ppm 6.58 (d, 1H), 7.13-7.21 (m, 2H), 7.58-7.59 (m, 2H), 7.78 (s, 1H), 7.94 (m, 1H), 8.12 (s, 1H).
LCMS Rt=3.29 minutes MS m/z no mass ion
The title compound was prepared according to the method described for Method Variation 1 using 3-cyano-4-fluoro-N-(5-fluoropyridin-2-yl)benzenesulfonamide (Preparation 2) and 3-chloro-2-fluoro-5-hydroxypyridine and then purified using silica gel column chromatography eluting with 30-35% EtOAc in heptanes.
MS m/z 423 [M+H]+
To a solution of 4-((5-chloro-6-fluoropyridin-3-yl)oxy)-3-cyano-N-(5-fluoropyridin-2-yl)benzenesulfonamide (Example 242, 200 mg, 0.47 mmol) in dimethylsulfoxide (2 mL) was added azetidine (54 mg, 0.94 mmol) followed by potassium carbonate (130 mg, 0.94 mmol) and the reaction was heated to 50° C. for 17 hours. The mixture was filtered and purified using preparative HPLC to afford the title compound.
1H NMR (400 MHz, DMSO-d6): δ ppm 2.08 (quintet, 2H), 3.98 (t, 4H), 6.84 (d, 1H), 6.90 (dd, 1H), 7.49 (dd, 1H), 7.65 (d, 1H), 7.88 (dd, 1H), 7.93 (d, 1H), 8.01 (d, 1H), 8.15 (d, 1H), 11.08 (br s, 1H).
MS m/z 459 [M+H]+
The title compound was prepared according to the method described for Example 243 using methylamine.
1H NMR (400 MHz, DMSO-d6): δ ppm 2.68 (d, 3H), 6.54 (q, 1H), 6.83 (d, 1H), 6.90 (dd, 1H), 7.49 (dt, 1H), 7.62 (d, 1H), 7.85-7.88 (m, 2H), 8.01 (d, 1H), 8.14 (d, 1H), 11.07 (br s, 1H).
MS m/z 433 [M+H]+
The title compound was prepared according to the method described for Example 243 using dimethylamine.
1H NMR (400 MHz, d4-MeOH): δ ppm 3.00 (s, 6H), 6.99 (d, 1H), 7.16 (dd, 1H), 7.52 (dt, 1H), 7.68 (d, 1H), 8.06-8.11 (m, 3H), 8.31 (d, 1H).
MS m/z 447 [M+H]+
The method described by Example 243 was followed, but using dimethoxybenzylamine instead of azetidine and with heating to 70° C. followed by stirring the residue in TFA (12 mL) for 1 hour. The resulting mixture was concentrated in vacuo and purified using reverse phase column chromatography eluting from 5% to 80% MeCN (0.1% HCO2H) in water (0.1% HCO2H) to afford the title compound.
1H NMR (400 MHz, d4-MeOH): δ ppm 6.97 (d, 1H), 7.16 (dd, 1H), 7.50-7.55 (m, 1H), 7.61 (d, 1H), 7.86 (d, 1H), 8.07 (d, 1H), 8.09 (dd, 1H), 8.29 (d, 1H).
MS m/z 419 [M+H]+
To a stirred solution of 3-chloro-4-(hydroxymethyl)phenol (120 mg, 0.54 mmol) and 3-cyano-N-(2,4-di methoxybenzyl)-4-fluoro-N-(5-fluoropyridin-2-yl)benzenesulfonamide (Preparation 18, 100 mg, 0.22 mmol) in DMSO (1.5 mL) was added potassium carbonate (78 mg, 0.56 mmol) and the reaction stirred at room temperature for 72 hours. Water (10 mL) and EtOAc (15 mL) were added and the layers separated. The aqueous phase was extracted with EtOAc. The combined organic extracts were washed with brine, dried over magnesium sulphate and concentrated in vacuo. The crude product was purified by silica gel column chromatography eluting with 3:2 EtOAc:heptanes. The residue (76 mg, 0.13 mmol) was dissolved in DCM (2 mL) and treated with TFA (2 mL). The reaction was stirred at room temperature for 18 hours. MeOH (1 mL) was added and the reaction concentrated in vacuo. The crude product was purified by silica gel column chromatography eluting with 10-30% EtOAc in DCM to afford the title compound (25 mg, 44%) as a colourless solid (25 mg, 44%).
1H NMR (400 MHz, CDCl3): δ ppm 4.56 (d, 2H), 5.48 (t, 1H), 7.02 (d, 1H), 7.08 (dd, 1H), 7.28 (d, 1H), 7.46 (s, 1H), 7.62-7.72 (m, 2H), 8.08 (d, 1H), 8.20 (s, 1H), 8.36 (s, 1H), 11.24 (br s, 1H).
MS m/z 434 [M+H]+
To a solution of 3-cyano-4-(4-cyano-3-fluorophenoxy)-5-fluorobenzene-1-sulfonyl chloride (Preparation 43, 27 mg, 0.153 mmol) in DCM (5 mL) was added 5-fluoro-2-aminopyridine (26 mg, 0.230 mmol) followed by pyridine (36 mg, 0.459 mmol, 0.037 mL) and the reaction was stirred at room temperature for 18 hours. The reaction was diluted with DCM (20 mL) and water (30 mL). The organic layer was separated and the aqueous re-extracted with DCM (2×30 mL). The combined organic layers were dried over sodium sulfate, filtered and concentrated in vacuo. The residue was purified by reverse phase chromatography eluting with (acetonitrile/0.1% formic acid) and (water/0.1% formic acid), gradient 5% to 60% to afford the title compound as colourless solid (21 mg, 31%).
1H NMR (400 MHz, Acetone-d6): δ ppm 7.29-7.34 (m, 2H), 7.53 (d, 1H), 7.60-7.65 (m, 1H), 7.95 (d, 1H), 8.18 (d, 1H), 8.29 (dd, 1H), 8.34 (m, 1H).
MS m/z 447 [M+H]+
To a degassed mixture of lithium chloride (42 mg, 1 mmol) in tetrahydrofuran (2 mL) was added tetrakis-(triphenylphosphine)palladium (0) (12 mg, 0.01 mmol) followed by a degassed solution of 5-bromo-4-({4-cyano-3,5-bis[methyl-D3]-2,3-D2-phenyl}oxy)-N-(5-fluoropyridin-2-yl)benzenesulfonamide (Example 236, 100 mg, 0.20 mmol) in tetrahydrofuran (2 mL) and triethylsilane (26 mg, 0.22 mmol). The reaction mixture was heated under reflux for 17 hours. An additional aliquot of tetrakis-(triphenylphosphine)palladium (0) (12 mg, 0.01 mmol), followed by another aliquot of triethylsilane (26 mg, 0.22 mmol), were added and the mixture heated under reflux for a further 24 hours. The reaction was cooled, diluted with ethyl acetate (20 mL), and washed with saturated aqueous ammonium chloride solution (20 mL), water (50 mL) and brine (10 mL). The organic layer was concentrated in vacuo and the residue purified using preparative HPLC to afford the title compound (21 mg, 25%).
1H NMR (400 MHz, DMSO-d6): δ ppm 6.99 (dd, 1H), 7.05 (dd, 1H), 7.14 (dd, 1H), 7.67 (dt, 1H), 7.92 (t, 1H), 8.14 (d, 1H), 11.41 (br s, 1H).
MS m/z 424 [M+H]+
The compounds of formula (I) that follow may be prepared by procedures described in the aforementioned: Schemes; General Methods and Method Variations, as further illustrated by the Examples and corresponding Preparations; or by processes similar thereto.
To a solution of 5-bromo-N-(2,4-dimethoxybenzyl)-2,4-difluoro-N-(5-fluoropyridin-2-yl)benzenesulfonamide (Preparation 12, 425 mg, 0.90 mmol) in DMSO (5 mL) was added K2CO3 (372 g, 2.70 mmol) followed by 2-fluoro-4-hydroxybenzonitrile (123 g, 0.90 mmol). The reaction mixture was left to stir at room temperature for 18 hours. To the reaction mixture was added water (20 mL) and saturated brine (20 mL) and the product was extracted with EtOAc (50 mL). The organic layer was retained and washed with saturated brine (3×40 mL), dried over MgSO4, and the solvent removed under vacuum. The residue was purified using silica gel column chromatography eluting with 15% EtOAc in heptanes followed by trituration with heptane to afford the title compound as a colourless solid (74 mg, 28%).
1H NMR (400 MHz, CDCl3): δ ppm 3.68 (s, 3H), 3.76 (s, 3H), 5.00 (s, 2H), 6.34-6.38 (m, 2H), 6.82-6.86 (m, 3H), 7.17 (d, 1H), 7.30-7.35 (m, 2H), 7.66 (dd, 1H, 8.12 (d, 1H), 8.17 (d, 1H).
19F NMR (376 MHz, CDCl3) δ ppm −101.9 (m, 1F), −104.1 (m, 1F), −128.5 (m, 1F).
3-Cyano-4-fluorobenzene-1-sulfonyl chloride (25 g, 114 mmol) was added to a solution of 5-fluoropyridin-2-amine (16.85 g, 150 mmol) and pyridine (23.7 g, 300 mmol) in dichloromethane (500 mL) at room temperature. The resulting mixture was stirred at room temperature for 3 hours. After evaporating the solvent the solid was stirred in diluted aqueous hydrochloric acid (2N, 400 mL) for 16 hours. The reaction mixture was filtered, washed with water (200 mL) and dried under high vacuum to afford the title compound as an orange solid (30.9 g, 98%).
1H NMR (400 MHz, DMSO-d6): δ ppm 7.09 (dd, 1H), 7.65-7.77 (m, 2H), 8.18 (d, 1H), 8.22-8.29 (m, 1H), 8.44 (dd, 1H)), 10.82 (br s, 1H).
MS m/z 296 [M+H]+
The title compound was prepared according to the method described for Preparation 2 using 2-aminopyridine.
1H NMR (400 MHz, DMSO-d6): δ ppm 6.85 (t, 1H), 7.25 (d, 1H), 7.65 (t, 1H), 7.75-7.83 (m, 1H), 7.93 (d, 1H), 8.19 (ddd, 1H), 8.35 (dd, 1H).
MS m/z 278 [M+H]+
The title compound was prepared according to the method described for Preparation 2 using 2-aminopyrazine.
1H NMR (400 MHz, DMSO-d6): δ ppm 7.75 (t, 1H), 8.23 (m, 1H), 8.26-8.27 (m, 1H), 8.32-8.36 (m, 2H), 8.53 (dd, 1H).
The title compound was prepared according to the method described for Preparation 2 using 2-aminopyrazine.
MS m/z 278 [M+H]+
To a solution of 3-cyano-4-fluorobenzenesulfonyl chloride (11.55 g, 52.63 mmol) in anhydrous acetonitrile (250 mL) was added pyridazin-3-amine (5 g, 52.63 mmol) followed by DABCO (5.9 g, 52.63 mmol) at 0° C. The reaction was stirred at room temperature for 18 hours, and then filtered. The filtrate was concentrated in vacuo and purified using silica gel column chromatography to afford the title compound (4.5 g, 30%)
1H NMR (400 MHz, DMSO-d6): δ ppm 7.61-7.71 (m, 2H), 7.87-7.92 (m, 1H), 8.16 (m, 1H), 8.32-8.37 (m, 2H), 14.61 (s, 1H).
The title compound was prepared according to the method described for Preparation 2 using 5-aminopyrimidine.
MS m/z 277 [M−H]−
The title compound was prepared according to the method described for Preparation 6 using 4-aminopyridazine.
1H NMR (400 MHz, DMSO-d6): δ ppm 7.29 (dd, 1H), 7.66 (t, 1H), 8.19 (ddd, 1H), 8.34 (d, 1H), 8.39 (dd, 1H), 8.57 (d, 1H).
The title compound was prepared according to the method described for Preparation 2 using 4-aminopyridazine.
MS m/s 276 [M−H]−
To a solution of 3-cyano-4-fluoro-N-(pyridazin-3-yl)benzenesulfonamide (Preparation 6, 6.70 g, 24.1 mmol) in DCM (150 mL) at 0° C. was added DIPEA (6.48 mL, 35.18 mmol) followed by chloromethoxymethane (1.46 mL, 25.54 mmol) and the reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was quenched by the addition of 1N NaOH (aq) solution, the organic layer was collected, washed with water, brine, and dried over Na2SO4 before concentrating in vacuo. The residue was purified using silica gel column chromatography eluting with EtOAc:Heptane 1:1 to afford the title compound as a mixture of isomers (3.85 g, 49%) that was used without further purification.
MS m/z 323 [M+H]+
N-(2,4-Dimethoxybenzyl)pyrimidin-4-amine (WO2012004743, 0.70 g, 2.86 mmol), 3-cyano-4-fluorobenzene-1-sulfonyl chloride (0.75 g, 3.43 mmol) and 1,4-diazabicyclo[2.2.2]octane (0.39 g, 3.43 mmol) in acetonitrile (15 mL) were stirred at room temperature for 18 hours. The reaction mixture was diluted with dichloromethane (100 mL), washed with water (100 mL), the organic layer was dried over anhydrous magnesium sulphate and the filtrate was concentrated in vacuo. The residue was purified by silica gel column chromatography eluting with 10% dichloromethane in ethyl acetate to afford the title compound as a yellow glass (0.69 g, 57%).
1HNMR (400 MHz, CDCl3): δ ppm 3.59 (s, 3H), 3.80 (s, 3H), 5.15 (s, 2H), 6.35 (d, 1H), 6.45-6.43 (m, 1H), 7.17-7.10 (m, 2H), 7.30 (t, 1H), 8.00-7.98 (m, 1H), 8.23-8.19 (m, 1H), 8.55 (d, 1H), 8.87 (s, 1H).
19F NMR (376 MHz, CDCl3): δ ppm −98.30
The title compound was prepared according to the method described for Preparation 11 using N-(2,4-dimethoxybenzyl)-5-fluoropyridin-2-amine (Preparation 20).
1H NMR (400 MHz, CDCl3): δ ppm 3.65 (s, 3H), 3.76 (3, 3H), 4.99 (s, 2H), 6.32-6.36 (m, 2H), 6.97 (dd, 1H), 7.16 (dd, 1H), 7.28-7.38 (m, 2H), 8.02 (t, 1H), 8.17 (s, 1H).
19F NMR (376 MHz, CDCl3): δ ppm −128 (s, 1F), −104 (s, 1F), −94 (s, 1F).
The title compound may be prepared according to the method described for Preparation 11 using 2,4,5-trifluorobenzenesulfonyl chloride and N-2,4-dimethoxybenzyl-5-fluoropyridin-2-amine (WO2012004743).
1H NMR (400 MHz, CDCl3): δ ppm 3.65 (s, 3H), 3.76 (3, 3H), 4.99 (s, 2H), 6.32-6.36 (m, 2H), 6.97 (dd, 1H), 7.16 (dd, 1H), 7.28-7.38 (m, 2H), 8.02 (t, 1H), 8.17 (s, 1H).
To a suspension of N-(2,4-dimethoxybenzyl)pyridazin-3-amine (Preparation 19, 1.0 g, 4.00 mmol) in THF (15 mL) at −20° C. was slowly added LiHMDS (1M, 4.0 mL, 4.00 mmol). The reaction mixture was left to stir at −20° C. for 30 minutes and then cooled to −78° C. To the reaction mixture was slowly added 3-cyano-4-fluorobenzene-1-sulfonyl chloride (0.8 g, 3.64 mmol) suspended in THF (15 mL). The reaction mixture was slowly warmed to room temperature and left at room temperature for 18 hours. To the vessel was added saturated aqueous NH4Cl solution (30 mL) and the product was extracted with EtOAc (25 mL). The organic layer was washed with saturated brine (40 mL), dried over MgSO4, and the solvent removed under vacuum. The residue was purified using silica gel column chromatography eluting with 70% EtOAc in heptanes to afford the title compound as an orange oil (561 mg, 36%).
1H NMR (400 MHz, CDCl3): δ ppm 3.54 (s, 3H), 3.76 (s, 3H), 5.01 (s, 2H), 6.28 (d, 1H), 6.36 (dd, 1H), 7.16 (d, 1H), 7.32 (t, 1H), 7.48 (m, 2H), 7.94 (dd, 1H), 8.09 (ddd, 1H), 9.03 (dd, 1H).
19F NMR (376 MHz, MeOD): δ ppm −98.8
The title compound was prepared according to the method described for Preparation 14 using N-(2,4-dimethoxybenzyl)-5-fluoropyrimidin-2-amine (WO2012004706).
1H NMR (400 MHz, CDCl3): δ ppm 3.30 (s, 3H), 3.81 (s, 3H), 5.38 (s, 2H), 6.23 (d, 1H), 6.46 (dd, 1H), 7.15-7.20 (m, 2H), 7.77 (dd, 1H), 8.14 (m, 1H), 8.23 (s, 2H).
The title compound was prepared according to the method described for Preparation 14 using 3,4,-difluorobenzenesulfonyl chloride and N-2,4-dimethoxybenzyl-5-fluoropyridin-2-amine (WO2012004743).
1H NMR (400 MHz, CDCl3): δ ppm 3.64 (s, 3H), 3.75 (s, 3H), 4.82 (s, 2H), 6.36 (m, 2H), 7.22 (d, 1H), 7.27 (m, 1H), 7.38 (m, 2H), 7.46 (m, 1H), 7.53 (m, 1H), 8.18 (d, 1H).
The title compound was prepared according to the method described for Preparation 14 using NaHMDS and N-(2,4-dimethoxybenzyl)-2-pyrimidinamine (WO2012004743) at −50° C. The residue was triturated with EtOAc:Heptane 1:1 followed by acetone.
1H NMR (400 MHz, CDCl3): δ ppm 3.28 (s, 3H), 3.82 (s, 3H), 4.42 (s, 2H), 6.23 (s, 1H), 6.44 (m, 1H), 6.99 (m, 1H), 7.18 (m, 2H), 7.82 (m, 1H), 8.15 (m, 1H), 8.57 (d, 2H).
To a −40° C. solution of N-(2,4-dimethoxybenzyl)-5-fluoropyridin-2-amine (Preparation 20, 2.32 g, 8.84 mmol) in THF (60 ml) was added LiHMDS (9.72 ml, 9.72 mmol, 1M in THF) keeping the temperature below −35° C. The reaction mixture was warmed to 0° C. for 40 minutes before re-cooling to −40° C. 3-Cyano-4-fluorobenzene-1-sulfonyl chloride (1.94 g, 8.84 mmol) was added to the reaction mixture as a solution in THF (10 mL) and the reaction mixture allowed to warm to room temperature for 18 hours. The reaction mixture was quenched with saturated ammonium chloride solution (100 mL) and extracted with EtOAc (2×100 mL). The organic layer was washed with saturated brine (100 mL), dried over MgSO4, filtered and the solvent removed under vacuum. The crude material was purified by silica gel column chromatography eluting with 20-40 EtOAc in heptane followed by reverse phase chromatography eluting with 0-100% MeCN in H2O to afford the title compound as a brown gum (2.48 g, 63%).
1H NMR (400 MHz, CDCl3): δ ppm 3.62 (s, 3H), 3.76 (s, 3H), 4.82 (s, 2H), 6.31 (d, 1H), 6.35 (dd, 1H), 7.10 (d, 1H), 7.26-7.41 (m, 3H), 7.95-8.01 (m, 2H), 8.19 (d, 1H).
19F NMR (376 MHz, CDCl3): δ ppm −99.36, −127.93
To a solution of 3,6-dichloropyridazine (1 g, 6.71 mmol) in nBuOH (25 mL) was added DIPEA (3.31 mL, 18.6 mmol) and 2,4-dimethoxybenzylamine (1.12 g, 6.71 mmol) and the reaction mixture was heated to 100° C. for 18 hours. The reaction mixture was concentrated in vacuo to afford 6-chloro-N-(2,4-dimethoxybenzyl)pyridazin-3-amine.
To a solution of 6-chloro-N-(2,4-dimethoxybenzyl)pyridazin-3-amine (7.5 g, 26.9 mmol) in ethanol (250 mL) was added ammonium formate (5.94 g, 94.15 mmol) and the mixture was degassed with nitrogen three times. Palladium on charcoal (10 wt %, 2.14 g) was added and the mixture was degassed and recharged with nitrogen and stirred at 80° C. for 90 minutes under a nitrogen atmosphere. The mixture was filtered, concentrated to 15 mL and partitioned between DCM (200 mL) and water (150 mL). The organic layer was washed with brine (150 mL) and concentrated in vacuo. The residue was recrystallised from EtOAc to afford the title compound as cream coloured solid (4.5 g, 68%).
1H NMR (400 MHz, CDCl3): δ ppm 3.79 (s, 3H), 3.83 (s, 3H), 4.51 (d, 2H), 5.19 (br s, 1H), 6.43 (dd, 1H), 6.47 (d, 1H), 6.62 (dd, 1H), 7.12 (dd, 1H), 7.24 (d, 1H), 8.52 (d, 1H).
Potassium carbonate (16.75 g, 128.1 mmol) was added to a solution of 2,5-difluoropyridine (4.91 g, 42.7 mmol) and 2,4-dimethoxybenzylamine (6.85 g, 42.7 mmol) in DMSO (30 mL). The mixture was heated to 110° C. for 16 hours, cooled to room temperature and poured into water (100 mL) and ethyl acetate (100 mL). The organic layer was separated and the aqueous layer was extracted with two further portions of ethyl acetate (25 mL). The combined organic extracts were washed with brine (50 mL), dried over MgSO4 and concentrated in vacuo. The residue was purified using silica gel column chromatography eluting with 10 to 30% ethyl acetate in heptanes to afford the title compound as a white solid (2.43 g, 22%).
1H NMR (400 MHz, CDCl3): δ ppm 3.79 (s, 3H), 3.83 (s, 3H), 4.37 (m, 2H), 4.91 (br s, 1H), 6.34-6.44 (m, 3H), 7.17 (m, 2H) 7.94 (s, 1H).
To a solution of trichloroisocyanuric acid (2.68 g, 11.55 mmol) in acetonitrile (25 ml) was added a solution of benzyltrimethylammonium chloride (6.56 g, 35.28 mmol) in water (11.7 ml) and the mixture was stirred at room temperature for 30 min, then cooled with an ice bath. The mixture was added to an ice cold solution of 4-(4-(benzylthio)-2-cyanophenoxy)-2-chlorobenzonitrile (Preparation 22, 4.61 g, 10.50 mmol) in acetonitrile (50 ml) with 1M sodium carbonate (10.5 ml, 10.5 mmol). The reaction mixture was stirred for 30 minutes at 0° C. The reaction mixture was doubled in volume using ethyl acetate and washed twice with dilute sodium hydrogen carbonate solution (2×200 mL). The organic layer was dried over MgSO4, filtered, and concentrated in vacuo. The residue was purified using silica gel column chromatography eluting with 0% to 20% ethyl acetate in heptanes to afford the title compound as a yellow solid (2.1 g, 46%).
1H NMR (400 MHz, CDCl3): δ ppm 7.13 (d, 1H), 7.19 (d, 1H), 7.36 (s, 1H), 7.83 (d, 1H), 8.22 (d, 1H), 8.41 (s, 1H).
To a solution of 5-(benzylthio)-2-fluorobenzonitrile (Preparation 23, 6.18 g, 25.4 mmol) in dimethylsulfoxide (64 mL) was added potassium carbonate (10.92 g, 79 mmol) and 2-fluoro-4-hydroxybenzonitrile (6.07 g, 39.5 mmol). The reaction mixture was heated at 80° C. for 18 hours. The reaction mixture was diluted to 500 mL volume with diethyl ether and washed with dilute brine (250 mL), then 2N sodium hydroxide (aq) (2×250 mL) and finally dilute brine (250 mL). The combined organic layers were dried over MgSO4, filtered and the solvent removed in vacuo. The crude material was dissolved in 10% methanol in ethyl acetate, silica (25 g) was added and the mixture evaporated to dryness under vacuum. The resulting solid was purified using silica gel column chromatography eluting with 0% to 100% ethyl acetate in heptanes to furnish the title compound as light brown solid (5.61 g, 51%).
1H NMR (400 MHz, CDCl3): δ ppm 4.14 (s, 2H), 6.98 (d, 2H), 7.17 (s, 1H), 7.22 to 7.38 (m, 5H), 7.46 (d, 1H), 7.58 (s, 1H), 7.65 (d, 1H).
To a degassed solution of 5-bromo-2-fluorobenzonitrile (10 g, 50 mmol) in toluene (250 mL) was added N,N-diisopropylethylamine (26 mL, 150 mmol), benzyl mercaptan (5.87 mL, 52 mmol) and dichloro[1,1′ bis(di-tert-butylphosphino)]ferrocene palladium (II) (500 mg, 1 mmol). The reaction mixture was heated at 60° C. for 16 hours. The reaction mixture was diluted to 500 mL volume with ethyl acetate and washed with dilute brine (300 mL), then 2N HCl (2×300 mL) and finally dilute brine (300 mL). The combined organic layers were dried over MgSO4, filtered and the solvent removed in vacuo. The residue was purified using silica gel column chromatography eluting with 10% ethyl acetate in heptane to afford the title compound as light brown solid (6.18 g, 51%)
1H NMR (400 MHz, CDCl3): δ ppm 4.04 (s, 2H), 7.08 (t, 1H), 7.40 to 7.55 (m, 5H), 7.43 (m, 2H).
To a solution of 3-D3-methoxy-4-acetylchlorobenzene (Preparation 25, 200 mg, 1.07 mmol) in DCM (10 mL) was added metachloroperbenzoic acid (276 mg, 1.6 mmol) and TFA (200 uL) and the reaction mixture was stirred at reflux for 18 hours. The cooled reaction mixture was poured onto 10% aqueous sodium metabisulphite (100 mL) and extracted with DCM (3×50 mL). The combined organic extracts were washed with saturated aqueous NaHCO3 (3×100 mL), dried over MgSO4 and concentrated in vacuo. The residue was dissolved in THF/water, sodium hydroxide (40 mg, 1 mmol) was added and the reaction stirred at room temperature for 18 hours. The reaction mixture was concentrated in vacuo then partitioned between EtOAc and water. The aqueous layer was collected and acidified with 2N HCl (aq). The compound was extracted from the aqueous phase with EtOAc, dried over MgSO4 and concentrated in vacuo to afford the title compound as an oil (125 mg, 78%), which was used without further purification.
To a solution of 3-chloro-4-acetylphenol (5 g, 5.88 mmol) in DMF (5 mL) was added potassium carbonate (974 mg, 7.05 mmol) followed by CD3I (1.02 g, 7.05 mmol) and the reaction mixture was stirred at 50° C. for 18 hours. The reaction mixture was poured into diethyl ether and washed with water, dried over MgSO4 and concentrated in vacuo to afford the title compound as a pale yellow solid (1.24 g, 94%).
MS m/z 188 [M+H]+
To a solution of 4-chloro-2-(difluoromethoxy)bromobenzene (Preparation 27, 200 mg, 0.63 mmol) in THF (6 mL) was added bis-neopentylglycolatodiboron (149 mg, 0.660 mmol), potassium acetate (191 mg, 1.88 mmol) and Pd(dppf)Cl2 (23 mg, 0.031 mmol). The reaction mixture was heated to reflux for 4 hours before cooling and diluting with EtOAc and water. The organic layer was collected, washed with brine, dried over Na2SO4 and concentrated in vacuo. The residue was dissolved in acetone (6 mL) and treated with a solution of oxone (1.64 g, 2.54 mmol) in water (6 mL). The reaction mixture was stirred at room temperature for 15 minutes. The reaction mixture was diluted with diethyl ether (10 mL) and water (5 mL). The organic layer was collected, washed with brine, dried over Na2SO4 and concentrated in vacuo. The residue was purified using silica gel column chromatography eluting with 0-15% diethyl ether in DCM to afford the title compound.
MS m/z 193 [M−H]−
To a solution of 2-bromo-5-chlorophenol (1 g, 4.8 mmol) in DMF/water (45 mL/5 mL) was added sodium chlorodifluoroacetate (1.95 g, 12.0 mmol) and cesium carbonate (3.14 g, 9.64 mmol), and the reaction mixture was heated to 100° C. for 4 hours. The reaction mixture was cooled and diluted with TBME (20 mL) and water (20 mL). The organic layer was collected, washed with saturated aqueous NaHCO3 solution, brine, dried over Na2SO4 and concentrated in vacuo to afford the title compound (1.21 g, 98%), which was used without further purification.
To a solution of trimethylsilylethanol (0.953 mL, 6.68 mmol) in THF (20 mL) was added sodium hydride (267 mg, 6.68 mmol) at 0° C. The reaction mixture was stirred at this temperature for 30 minutes before the addition of 4-cyano-2-(difluoromethoxy)fluorobenzene (Preparation 29, 500 mg, 2.67 mmol). The reaction mixture was stirred at room temperature for 5 hours. The reaction mixture was quenched by the addition of methanol and concentrated in vacuo. The residue was purified using silica gel column chromatography eluting with 10-80% EtOAc in heptanes. The residue was dissolved in THF (6 mL) and TBAF (2 mL) was added. The reaction mixture was stirred at room temperature for 5 hours before the addition of silica gel and concentrating in vacuo. The residue was purified using silica gel column chromatography eluting with 10-100% EtOAc in heptanes to afford the title compound (260 mg, 41%).
MS m/z 184 [M−H]−
The title compound was prepared according to the method described for Preparation 27 using 2-fluoro-5-cyanophenol and was used without further purification.
The title compound was prepared according to the method described for the preparation of 5-chloro-6-(2-methylpropoxy)-3-pyridinol (WO2012007869), using 2-ethoxy-3-chloro-5-hydroxypyridine.
The title compound was prepared according to the method described for Preparation 14 using N-[(2,4-dimethoxyphenyl)methyl]pyridazin-3-amine (Preparation 19) and 2,4-difluoro-5-chlorobenzenesulfonyl chloride.
MS m/z 456 [M+H]+
The title compound was prepared according to the method described for Preparation 18 using N-(2,4-dimethoxybenzyl)-5-fluoropyridin-2-amine (Preparation 20) and 4-fluoro-5-chlorobenzenesulfonyl chloride.
1H NMR (400 MHz, CDCl3): δ ppm 3.63 (s, 3H), 3.73 (s, 3H), 4.82 (s, 2H), 6.30-6.34 (m, 2H), 7.11 (d, 1H), 7.20 (t, 1H), 7.29-7.37 (m, 2H), 7.57 (ddd, 1H), 7.75 (dd, 1H), 8.16 (dd, 1H).
19F NMR (400 MHz, CDCl3): δ ppm −128.61 (dd, ArF), −107.49 to −107.44 (m, ArF).
The title compound was prepared according to the method described for Preparation 11 using N-(2,4-dimethoxybenzyl)-5-fluoropyridin-2-amine (Preparation 20) and 4-fluoro-5-bromobenzenesulfonyl chloride at 40° C. The residue was purified by silica gel column chromatography eluting with 10% EtOAc in heptanes.
1H NMR (400 MHz, CDCl3): δ ppm 3.63 (s, 3H), 3.73 (s, 3H), 4.82 (s, 2H), 6.30 (d, 1H), 6.32 (dd, 1H), 7.11 (d, 1H), 7.17 (t, 1H), 7.29-7.37 (m, 2H), 7.60-7.63 (m, 1H), 7.89 (dd, 1H), 8.16 (d, 1H).
19F NMR (400 MHz, CDCl3): δ ppm −128.63 (dd, ArF), −99.49 to −99.44 (m, ArF).
The title compound was prepared according to the method described for Preparation 11 using N-(2,4-dimethoxybenzyl)-5-fluoropyridin-2-amine (Preparation 20) and 2,4-difluorobenzene-1-sulfonyl chloride.
1H NMR (400 MHz, CDCl3): δ ppm 3.67 (s, 3H), 3.75 (s, 3H), 4.99 (s, 2H), 6.34 (s, 2H), 6.87-6.97 (m, 2H), 7.16 (d, 1H), 7.29-7.36 (m, 2H), 7.80-7.86 (m, 1H), 8.13 (d, 1H).
The title compound was prepared according to the method described for Preparation 20 using 2,4-dimethoxybenzylamine and 2-fluoroisonicotinonitrile.
1H NMR (400 MHz, CDCl3): δ ppm 3.80 (s, 3H), 3.84 (s, 3H), 4.41 (d, 2H), 5.26 (br s, 1H), 6.44 (dd, 1H), 6.48 (d, 1H), 6.70 (dd, 1H), 7.18 (d, 1H), 8.19 (d, 1H).
MS m/z 270 [M+H]+
The title compound was prepared according to the method described for Preparation 18 using 2-((2,4-dimethoxybenzyl)amino)isonicotinonitrile (Preparation 35) and 3-cyano-4-fluorobenzene-1-sulfonyl chloride. The residue was purified by reverse phase chromatography eluting with 0-100% MeCN in water/0.1% formic acid.
1H NMR (400 MHz, CDCl3): δ ppm 3.60 (s, 3H), 3.78 (s, 3H), 4.96 (s, 2H), 6.32 (d, 1H), 6.39 (dd, 1H), 7.13 (d, 1H), 7.29-7.34 (m, 2H), 7.48 (s, 1H), 8.01 (dd, 1H), 8.05 (m, 1H), 8.50 (d, 1H).
19F NMR (376 MHz, CDCl3): δ ppm −98.74.
The title compound was prepared according to the method described for Preparation 20 using 2,4-dimethoxybenzylamine and 6-fluororonicotinonitrile.
1H NMR (400 MHz, CDCl3): δ ppm 3.80 (s, 3H), 3.84 (s, 3H), 4.47 (d, 2H), 5.47 (br s, 1H), 6.38 (d, 1H), 6.44 (dd, 1H), 6.48 (d, 1H), 7.53 (dd, 1H), 8.36 (d, 1H).
MS m/z 270 [M+H]+
The title compound was prepared according to the method described for Preparation 18 using 6-((2,4-dimethoxybenzyl)amino)nicotinonitrile (Preparation 37) and 3-cyano-4-fluorobenzene-1-sulfonyl chloride. The residue was purified by reverse phase chromatography eluting with 0-100% MeCN in water/0.1% formic acid.
1H NMR (400 MHz, d4-MeOH): δ ppm 3.47 (s, 3H), 3.77 (s, 3H), 5.10 (s, 2H), 6.34 (d, 1H), 6.44 (dd, 1H), 7.14 (d, 1H), 7.45-7.51 (m, 2H), 7.98 (dd, 1H), 8.10 (dd, 1H), 8.24 (m, 1H), 8.68 (d, 1H).
19F NMR (376 MHz, CDCl3): δ ppm −102.69.
The title compound was prepared according to the method described for Preparation 2 using 5-chloropyridin-2-amine.
MS m/z 310 [M−H]−
The title compound was prepared according to the method described for Preparation 2 using 5-methylpyridin-2-amine.
1H NMR (400 MHz, DMSO-d6): δ ppm 2.12 (s, 3H), 5.73 (s, 1H), 7.17 (d, 1H), 7.62-7.66 (m, 2H), 7.79 (br s, 1H), 8.14-8.18 (m, 1H), 8.34 (dd, 1H).
MS m/z 292 [M+H]+
The title compound was prepared according to the method described for Preparation at 120° C. for 68 hours using 5-bromo-3-fluoro-2-hydroxybenzonitrile (WO 2006022374) and 2-chloro-4-fluorobenzonitrile.
1H NMR (400 MHz, CDCl3): δ ppm 6.94 (dd, 1H), 7.06 (d, 1H), 7.65-7.70 (m, 3H).
The title compound was prepared according to the method described for Preparation at reflux using 5-bromo-2-(4-cyano-3-chlorophenoxy)-3-fluorobenzonitrile (Preparation 41) and benzyl mercaptan. The reaction was allowed to cool to room temperature and diluted with EtOAc (100 mL), water (50 mL) and saturated aqueous sodium hydrogen carbonate solution (100 mL). The organic was separated and the aqueous was re-extracted with EtOAc (2×100 mL). The combined organic layers were washed with brine (100 mL), dried over sodium sulfate, filtered and concentrated in vacuo. The residue was absorbed onto silica and purified by silica gel column chromatography eluting with 10% EtOAc in heptanes to afford the title compound as a yellow solid (115 mg, 28%).
1H NMR (400 MHz, CDCl3): δ ppm 4.18 (s, 2H), 6.89 (dd, 1H), 7.00 (d, 1H), 7.29-7.35 (m, 7H), 7.63 (d, 1H).
The title compound was prepared according to the method described for Preparation 21 using 5-(benzylthio)-2-(4-cyano-3-chlorophenoxy)-3-fluorobenzonitrile (Preparation 42).
1H NMR (400 MHz, CDCl3): δ ppm 7.01 (dd, 1H), 7.16 (d, 1H), 7.75 (m, 1H), 8.13 (dd, 1H), 8.23 (m, 1H).
A solution of 2-chloro-6-fluoro-4-hydroxybenzaldehyde (WO 2008141249, 90 mg, 0.344 mmol) in formic acid (1 mL) was treated with hydroxylamine hydrochloride (23.9 mg, 0.344 mmol) and sodium formate (23.4 mg, 0.344 mmol) and stirred under nitrogen for 3.5 hours at 105° C. The mixture was concentrated in vacuo and the residue treated with saturated aqueous sodium bicarbonate solution (10 mL). The aqueous solution was extracted with ethyl acetate (2×10 mL), dried over sodium sulphate and concentrated in vacuo. The residue was purified using silica gel column chromatography eluting with 30-50% EtOAc in heptanes to afford the title compound as a beige solid (33 mg, 56%).
1H NMR (400 MHz, MeOH-d4): δ ppm 6.70 (dd, 1H), 6.85 (d, 1H).
To a solution of 4-acetylphenyl acetate (250 mg, 1.4 mmol) in anhydrous THF (5 mL) cooled to −10° C. under nitrogen was added 3M methylmagnesium chloride solution in THF (2.81 mL, 8.42 mmol) over 10 minutes, keeping the internal temperature below −5° C. The resulting pale yellow solution was allowed to warm to room temperature for 5 hours. The reaction mixture was cooled in an ice bath and quenched with saturated aqueous ammonium chloride solution (20 mL). The resulting clear solution was extracted with EtOAc (2×50 mL). The combined organic extracts were washed with brine (50 mL), dried over MgSO4, filtered and concentrated in vacuo. The solid residue was purified by silica gel column chromatography eluting with 10-50% EtOAc in heptanes to afford the title compound as white solid (100 mg, 47%).
1H NMR (400 MHz, DMSO-d6): δ ppm 1.34 (s, 6H), 4.75 (s, 1H), 6.62 (d, 2H), 7.21 (d, 2H), 9.08 (s, 1H).
Biological Assay
1. Generation of a Custom Clonal Cell Line for URAT1 Transporter Activity Assay
The nucleotide sequence for the long isoform of URAT1 (NM—144585) was C-terminally fused to that of enhanced green fluorescent protein (eGFP) (hereinafter referred to as URAT1(L)GFP). The combined sequence was codon-optimised and custom synthesized. The synthesized sequence was generated in pDONR221 Gateway entry vector (Invitrogen Life Technologies) prior to cloning in pLenti6.3/V5 Gateway destination vector (Invitrogen Life Technologies). A schematic of the URAT1(L)GFP construct is set forth in
Lentiviral particles were generated according to ViraPower HiPerform expression system procedure (Invitrogen Life Technologies) and used to transduce CHO cells. Blasticidin selection enabled the generation of a stable clonal pool of cells, confirmed by expression of GFP and V5 epitope. The clonal pools were sorted using fluorescence-activated cell sorting (FACS) on the basis of GFP expression with the gating set at the top 50% of expression into single cells which were subsequently expanded to generate clonal lines. One clone was identified with the best assay performance as determined by maximal separation between complete inhibition of uric acid transport (with 10 μM benzbromarone) and no inhibition (DMSO). This cell line was used for all screening activities and is referred to as CHO-URAT1(L)GFP#8 or CHO#8.
2. URAT-1 Inhibitor Activity
The potency of the compounds of formula (I) as inhibitors of the URAT-1 transporter was determined as follows.
CHO#8 cells were cultured in cell line maintenance flasks in medium consisting of Dulbecco's modified Eagle medium (DMEM) with high glucose and sodium pyruvate (4.5 g of glucose per liter, Invitrogen Life Technologies), supplemented with heat-inactivated foetal bovine serum (FBC, 10% v/v), 1×NEAA (non-essential amino acids) and blasticidin (10 μg/ml). Cultures were grown in 175 cm2 tissue culture flasks in a humidified incubator at approximately 37° C. in approximately 95% air/5% CO2. Near confluent CHO#8 cell cultures were harvested by trypsinisation, re-suspended in culture medium and the process was repeated once or twice weekly to provide sufficient cells for use.
Assay ready flasks were generated by the same method, except the cells were not cultured in blasticidin.
Assay ready frozen cells were generated by freezing 40,000,000 cells in 1 ml of FBS (without blasticidin) containing 10% DMSO per vial. One vial was sufficient for 5 assay plates. Each vial was thawed rapidly to 37° C., washed and re-suspended in pre-warmed culture medium for seeding onto assay plates.
CHO#8 cells were seeded onto Cytostar™ 96-well plates at a density of 5×105 cells per well. The cells were cultured for 1 day at approximately 37° C. in a humidified incubator containing approximately 5% CO2 in air. After approximately 24 hours culture, cells were used for uptake experiments.
On the day of assay, culture medium was removed from the wells and the cells were washed once with 50 μL of chloride-containing buffer (136.7 mM NaCl, 5.36 mM KCl, 0.952 mM CaCl2, 0.441 mM KH2PO4, 0.812 mM MgSO4, 5.6 mM D-glucose, 0.383 mM Na2HPO4.2H2O, 10 mM HEPES, pH 7.4 with NaOH). The cells were pre-incubated with another 50 μL of chloride-containing buffer for one hour at approximately 37° C. in a humidified incubator containing approximately 5% CO2 in air.
Assay compound plates were prepared by diluting the compounds of formula (I) with chloride-free buffer (125 mM Na-gluconate, 4.8 mM K-gluconate, 1.3 mM Ca-gluconate, 1.2 mM KH2PO4, 1.2 mM MgSO4, 5.6 mM D-glucose, 25 mM HEPES, pH 7.4 with NaOH) in 100% DMSO to a final concentration of 1% DMSO. [14C]-Uric Uric acid working stock was made by addition of radiolabeled compound to a final concentration of 120 nM in chloride-free buffer. In all wells, the final assay concentration of solvent (DMSO) was 0.25%; the final assay concentration of [14C]-uric acid was 30 nM in chloride-free buffer and the final compound of formula (I) concentrations ranged from 0 to 10 μM. The vehicle comparator was DMSO (i.e. no inhibition of uric acid transport) and the pharmacological blockade (i.e. 100% inhibition of uric acid transport) was defined by benzbromarone at 10 μM final assay concentration.
After pre-incubation, cells were washed with 50 μL of chloride-free buffer and another 50 μL of chloride-free buffer was added. Thereafter, 25 μL of compound of formula (I) was added from the prepared compound plate and the cells were pre-incubated for 15 minutes prior to the addition of 25 mL of [14C] uric acid. The plate was incubated at room temperature and protected from light for three hours prior to measuring proximity-induced scintillation on a Wallac microbeta at 1 minute/well.
The accumulation of [14C]-uric acid into CHO#8 cells was calculated and the IC50 (μM) values, defined as the concentration of inhibitor required for 50% inhibition of transport, were determined from a 4 parameter logistic fit to generate sigmoid curves from dose response data.
This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Patent Application No. 61/917,532, filed on Dec. 18, 2013, and U.S. Provisional Patent Application No. 61/813,809, filed on Apr. 19, 2013, the disclosures of which are hereby incorporated by reference in their entirety.
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