Patent Application
20090221639
The present invention is directed to G-protein coupled receptor (GPCR) agonists. In particular, the present invention is directed to agonists of GPR119 that are useful for the treatment of obesity, e.g. as regulators of satiety, metabolic syndrome and for the treatment of diabetes.
Obesity is characterized by an excessive adipose tissue mass relative to body size. Clinically, body fat mass is estimated by the body mass index (BMI; weight(kg)/height(m)2), or waist circumference. Individuals are considered obese when the BMI is greater than 30 and there are established medical consequences of being overweight. It has been an accepted medical view for some time that an increased body weight, especially as a result of abdominal body fat, is associated with an increased risk for diabetes, hypertension, heart disease, and numerous other health complications, such as arthritis, stroke, gallbladder disease, muscular and respiratory problems, back pain and even certain cancers.
Pharmacological approaches to the treatment of obesity have been mainly concerned with reducing fat mass by altering the balance between energy intake and expenditure. Many studies have clearly established the link between adiposity and the brain circuitry involved in the regulation of energy homeostasis. Direct and indirect evidence suggest that serotonergic, dopaminergic, adrenergic, cholinergic, endocannabinoid, opioid, and histaminergic pathways in addition to many neuropeptide pathways (e.g. neuropeptide Y and melanocortins) are implicated in the central control of energy intake and expenditure. Hypothalamic centres are also able to sense peripheral hormones involved in the maintenance of body weight and degree of adiposity, such as insulin and leptin, and fat tissue derived peptides.
Drugs aimed at the pathophysiology associated with insulin dependent Type I diabetes and non-insulin dependent Type II diabetes have many potential side effects and do not adequately address the dyslipidaemia and hyperglycaemia in a high proportion of patients. Treatment is often focused at individual patient needs using diet, exercise, hypoglycaemic agents and insulin, but there is a continuing need for novel antidiabetic agents, particularly ones that may be better tolerated with fewer adverse effects.
Similarly, metabolic syndrome (syndrome X) places people at high risk of coronary artery disease, and is characterized by a cluster of risk factors including central obesity (excessive fat tissue in the abdominal region), glucose intolerance, high triglycerides and low HDL cholesterol, and high blood pressure. Myocardial ischemia and microvascular disease is an established morbidity associated with untreated or poorly controlled metabolic syndrome.
There is a continuing need for novel antiobesity and antidiabetic agents, particularly ones that are well tolerated with few adverse effects.
GPR119 (previously referred to as GPR116) is a GPCR identified as SNORF25 in WO00/50562 which discloses both the human and rat receptors, U.S. Pat. No. 6,468.756 also discloses the mouse receptor (accession numbers: AAN95194 (human), AAN95195 (rat) and ANN95196 (mouse)).
In humans, GPR119 is expressed in the pancreas, small intestine, colon and adipose tissue. The expression profile of the human GPR119 receptor indicates its potential utility as a target for the treatment of obesity and diabetes.
International patent applications WO2005/061489, WO2006/070208, WO2006/067532 disclose heterocyclic derivatives as GPR119 receptor agonists. International patent application WO2006/067531 discloses GPR119 receptor agonists. International patent applications WO2007/003960, WO2007/003961, WO2007/003962 and WO2007/003964, published after the priority date of the present application, also disclose GPR119 receptor agonists.
The present invention relates to agonists of GPR119 which are useful for the treatment of diabetes and as peripheral regulators of satiety, e.g. for the treatment of obesity and metabolic syndrome.
or pharmaceutically acceptable salts thereof, are agonists of GPR119 and are useful for the treatment of diabetes and as peripheral regulators of satiety, e.g. for the treatment of obesity and metabolic syndrome.
The present invention is directed to a compound of formula (I), or a pharmaceutically acceptable salt thereof:
wherein V is a 5-membered heteroaryl ring containing up to four heteroatoms selected from O, N and S, which is optionally substituted by C1-4 alkyl;
A is —CH═CH— or (CH2)n;
B is (CH2)p—CH(C1-3 alkyl)-(CH2)q, where one of the CH2 groups may be replaced by O, NR5, S(O)m, C(O), C(O)NR5, CH(NR5R55), C(O)O, C(O)S, SC(O) or OC(O);
n is independently 0, 1, 2 or 3;
m is independently 0, 1 or 2;
p+q equals 0, 1 or 2;
x is 0, 1, 2 or 3;
y is 1, 2, 3, 4 or 5;
with the proviso that x+y is 2, 3, 4 or 5;
G is CHR12 or NR2;
R1 is phenyl or a 5- or 6-membered heteroaryl group containing up to four heteroatoms selected from O, N and S, any of which may be optionally substituted by one or more substituents selected from halo, C1-4 alkyl, C1-4 fluoroalkyl, C1-4 hydroxyalkyl, C2-4 alkenyl, C2-4 alkynyl, C3-7cycloalkyl, aryl, OR6, CN, NO2, S(O)mR6, C(O)NR6R66. NR6R66, NR10C(O)R6, NR10SO2R6, SO2NR6R66, COR10, C(O)OR10, a 4- to 7-membered heterocyclyl group or a 5- or 6-membered heteroaryl group;
R2 is C(O)OR3, C(O)NR3R13, C1-4alkylene-C(O)OR3, C(O)C(O)OR3, S(O)2R3, C(O)R3 or P(O)(O-Ph)2; or heterocyclyl or heteroaryl, either of which may optionally be substituted by one or two groups selected from C1-4alkyl, C1-4alkoxy and halogen;
R3 is C1-8 alkyl, C2-8 alkenyl or C2-8 alkynyl, any of which may be optionally substituted by one or more halo atoms, NR4R44, OR4, C(O)OR4, OC(O)R4 or CN groups, and may contain a CH2 group that is replaced by O or S; or C3-7 cycloalkyl, aryl, heterocyclyl, heteroaryl, C1-4 alkyleneC3-7 cycloalkyl, C1-4 alkylenearyl, C1-4 alkyleneheterocyclyl or C1-4 alkyleneheteroaryl, any of which may be substituted with one or more substituents selected from halo, C1-4 alkyl, C1-4 fluoroalkyl, OR4, CN, NR4R44, SO2Me, NO2 or C(O)OR4;
R4 and R44 are independently hydrogen or C1-4 alkyl; or, taken together, R4 and R44 may form a 5- or 6-membered heterocyclic ring;
R5 and R55 independently represent hydrogen or C1-4 alkyl;
R6 and R66 are independently hydrogen or C1-4 alkyl, which may optionally be substituted by halo (e.g. fluoro), hydroxy, C1-4 alkyloxy-, C1-4 alkylthio-, C3-7 heterocyclyl or N(R10)2; or C3-7 cycloalkyl, aryl, heterocyclyl or heteroaryl, wherein the cyclic groups may be substituted with one or more substituents selected from halo, C1-4 alkyl, C1-4 fluoroalkyl, OR9, CN, SO2CH3, N(R10)2 and NO2; or, taken together, R6 and R66 may form a 5- or 6-membered heterocyclic ring optionally substituted by hydroxy, C1-4 alkyl or C1-4 hydroxyalkyl; or R66 is C1-4 alkyloxy-;
R9 is hydrogen, C1-2 alkyl or C1-2 fluoroalkyl;
R10 are independently hydrogen or C1-4 alkyl; or a group N(R10)2 may form a 4- to 7-membered heterocyclic ring optionally containing a further heteroatom selected from O and NR10;
R12 is C3-6 alkyl; and
R13 is hydrogen or C1-4 alkyl.
The molecular Weight of the compounds of formula (I) is preferably less than 800, more preferably less than 600, especially less than 500.
In the compounds of formula (I) V is preferably a 5-membered heteroaryl ring containing up to three heteroatoms selected from O, N and S of the formula:
wherein W, X and Y represent the positions of the heteroatom(s) or otherwise represent CH.
Particular heterocyclic rings which V may represent include oxadiazole, oxazole, isoxazole, thiadiazole, thiazole and pyrazole.
Preferably two of W, X and Y are N, and the other is O.
W is preferably N.
V is preferably:
In A, n is preferably 0.1 or 2, more preferably 0.
In B, p+q is preferably 1 or 2, especially 1.
p is preferably 0 and q is preferably 1.
When one of the CH2 groups in B is replaced, it is preferably replaced by O or NR5, more preferably by O. In one embodiment of the invention a CH2 group in B is replaced. In a second embodiment of the invention a CH2 group in B is not replaced.
In one embodiment of the invention, the —CH(C1-3 alkyl)- group in B is —CH(CH3)—. In a further embodiment B is —CH(CH3)—O—. In compounds where B is —CH(CH3)—O— the absolute configuration at the chiral carbon atom is preferably (R).
R1 is preferably phenyl or a 6-membered heteroaryl group containing up to two N atoms either of which rings may optionally be substituted, especially optionally substituted phenyl. Examples of R1 heteroaryl groups include oxazolyl, isoxazolyl, thienyl, pyrazolyl, imidazolyl, furanyl, pyridazinyl or pyridyl. Preferred substituent groups for R1 are halo, C1-4 alkyl, C1-4 fluoroalkyl, C2-4 alkenyl, C2-4 alkynyl, CN, S(O)mR6, C(O)NR6R66, SO2NR6R66, COR10, C(O)OR10 or a 5- or 6-membered heteroaryl group; especially halo, e.g. fluoro or chloro, C1-4 alkyl, C1-4 fluoroalkyl, C2-4 alkenyl, C2-4 alkynyl, CN, S(O)mR6, C(O)NR6R66 or SO2NR6R66 or a 5- or 6-membered heteroaryl group; in particular fluoro, chloro, methyl, S(O)mR6, e.g. where m is 1 or 2, C(O)NR6R66 or SO2NR6R66.
G is preferably NR2.
In one embodiment of the invention x+y is 2, 3, or 4. In a preferred embodiment of the invention x and y each represent 1. In a more preferred embodiment of the invention x and y each represent 2.
R2 is preferably C(O)OR3, C(O)NR3R13, C1-4alkylene-C(O)OR3, C(O)C(O)OR3, heterocyclyl, heteroaryl, S(O)2R3, C(O)R3 or P(O)(O-Ph)2; especially C(O)OR3, C(O)NR3R13. C1-4alkylene-C(O)OR3, heteroaryl, S(O)2R3 or C(O)R3; in particular C(O)OR3, C(O)NR3R13, heteroaryl, S(O)2R3 or C(O)R3. More preferably, R2 is C(O)OR3, C(O)NR3R13 or heteroaryl. R2 is most preferably C(O)OR3. When R2 is heteroaryl the heteroaryl ring is preferably pyrimidinyl, especially pyrimidin-2-yl. R2 is more preferably oxadiazole.
Preferably R3 represents C1-8 alkyl, C2-8 alkenyl or C2-8 alkynyl, optionally substituted by one or more halo atoms, NR4R44, OR4, C(O)OR4, OC(O)R4 or CN groups, and may contain a CH2 group that is replaced by O or S; or a C3-7 cycloalkyl, aryl or C1-4 alkylC3-7 cycloalkyl, any of which may be substituted with one or more substituents selected from halo, C1-4 alkyl, C1-4 fluoroalklyl, OR4, CN, NR4R44, NO2 or C(O)OC1-4 alkyl. More preferably R3 represents C1-4 alkyl, C2-8 alkenyl or C2-8 alkynyl optionally substituted by one or more halo atoms or CN, and may contain a CH2 group that may be replaced by O or S; or a C3-7 cycloalkyl or aryl, either of which may be substituted with one or more substituents selected from halo, C1-4 alkyl, C1-4 fluoroalkyl, OR4, CN, NR4R44, NO2 or C(O)OC1-4 alkyl. Most preferred R3 groups are C3-5 alkyl optionally substituted by one or more halo atoms or CN, and may contain a CH2 group that is replaced by O or S, or C3-5cycloalkyl optionally substituted by C1-4 alkyl. In one embodiment of the invention the group represented by R3 is unsubstituted.
R6 and R66 are preferably optionally substituted C1-4 alkyl or optionally substituted C3-7 cycloalkyl.
When the group R6 is attached to a sulfinyl or sulfonyl group, R6 is preferably optionally substituted C1-4 alkyl or optionally substituted C3-7 cycloalkyl, more preferably optionally substituted C1-4 alkyl, e.g. methyl or ethyl. When the group R6 is attached to C(O)N, R6 is preferably hydrogen, optionally substituted C1-4 alkyl or optionally substituted C3-7 cycloalkyl, more preferably optionally substituted C1-4 alkyl, e.g. methyl or ethyl.
R9 is preferably C1-2 alkyl or C1-2 fluoroalkyl.
While the preferred groups for each variable have generally been listed above separately
for each variable, preferred compounds of this invention include those in which several or each variable in formula (I) is selected from the preferred, more preferred or particularly listed groups for each variable. Therefore, this invention is intended to include all combinations of preferred, more preferred and particularly listed groups.
Specific compounds of the invention which may be mentioned are those included in the Examples and pharmaceutically acceptable salts thereof.
As used herein, unless stated otherwise, “alkyl” as well as other groups having the prefix “alk” such as, for example, alkenyl, alkynyl, and the like, means carbon chains which may be linear or branched or combinations thereof. Examples of alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, sec- and tert-butyl, pentyl, hexyl, heptyl and the like. “Alkenyl”, “alkynyl” and other like terms include carbon chains having at least one unsaturated carbon-carbon bond.
The term “fluoroalkyl” includes alkyl groups substituted by one or more fluorine atoms, e.g. CH2F, CHF2 and CF3.
The term “cycloalkyl” means carbocycles containing no heteroatoms, and includes monocyclic and bicyclic saturated and partially saturated carbocycles. Examples of cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl. Examples of partially saturated cycloalkyl groups include cyclohexene and indane. Cycloalkyl groups will typically contain 3 to 10 ring carbon atoms in total, e.g. 3 to 6, or 8 to 10.
The term “halo” includes fluorine, chlorine, bromine, and iodine atoms.
The term “aryl” includes phenyl and naphthyl, in particular phenyl.
Unless otherwise indicated the term “heterocyclyl” and “heterocyclic ring” includes 4- to 10-membered monocyclic and bicyclic saturated rings, e.g. 4- to 7-membered monocyclic saturated rings, containing up to three heteroatoms selected from N, O and S. Examples of heterocyclic rings include oxetane, tetrahydrofuran, tetrahydropyran, oxepane, oxocane, thietane, tetrahydrothiophene, tetrahydrothiopyran, thiepane, thiocane, azetidine, pyrrolidine, piperidine, azepane, azocane, [1,3]dioxane, oxazolidine, piperazine, and the like. Other examples of heterocyclic rings include the oxidised forms of the sulfur-containing rings. Thus, tetrahydrothiophene 1-oxide, tetrahydrothiophene 1,1-dioxide, tetrahydrothiopyran 1-oxide, and tetrahydrothiopyran 1,1-dioxide are also considered to be heterocyclic rings.
Examples of heterocyclic rings that R2 may represent include azetidine, pyrrolidine, piperidine and piperazine. R2 heterocyclyl groups may also contain additional heteroatoms, e.g. morpholine.
Unless otherwise stated, the term “heteroaryl” includes mono- and bicyclic 5- to 10-membered, e.g. monocyclic 5- or 6-membered, heteroaryl rings containing up to 4 heteroatoms selected from N, O and S. Examples of such heteroaryl rings are furyl, thienyl, pyrrolyl, pyrazolyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, triazolyl, oxadiazolyl, thiadiazolyl, tetrazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl and triazinyl. Bicyclic heteroaryl groups include bicyclic heteroaromatic groups where a 5- or 6-membered heteroaryl ring is fused to a phenyl or another heteroaromatic group. Examples of such bicyclic heteroaromatic rings are benzofuran, benzothiophene, indole, benzoxazole, benzothiazole, indazole, benzimidazole, benzotriazole, quinoline, isoquinoline, quinazoline, quinoxaline and purine.
Compounds described herein may contain one or more asymmetric centers and may thus give rise to diastereomers and optical isomers. The present invention includes all such possible diastereomers as well as their racemic mixtures, their substantially pure resolved enantiomers, all possible geometric isomers, and pharmaceutically acceptable salts thereof. The above formula (I) is shown without a definitive stereochemistry at certain positions. The present invention includes all stereoisomers of formula (II) and pharmaceutically acceptable salts thereof. Further, mixtures of stereoisomers as well as isolated specific stereoisomers are also included. During the course of the synthetic procedures used to prepare such compounds, or in using racemization or epimerization procedures known to those skilled in the art, the products of such procedures can be a mixture of stereoisomers.
When a tautomer of the compound of formula (I) exists, the present invention includes any possible tautomers and pharmaceutically acceptable salts thereof, and mixtures thereof, except where specifically drawn or stated otherwise.
When the compound of formula (I) and pharmaceutically acceptable salts thereof exist in the form of solvates or polymorphic forms, the present invention includes any possible solvates and polymorphic forms. A type of a solvent that forms the solvate is not particularly limited so long as the solvent is pharmacologically acceptable. For example, water, ethanol, propanol, acetone or the like can be used.
The term “pharmaceutically acceptable salts” refers to salts prepared from pharmaceutically acceptable non-toxic bases or acids. When the compound of the present invention is acidic, its corresponding salt can be conveniently prepared from pharmaceutically acceptable non-toxic bases, including inorganic bases and organic bases. Salts derived from such inorganic bases include aluminum, ammonium, calcium, copper (ic and ous), ferric, ferrous, lithium, magnesium, potassium, sodium, zinc and the like salts. Particularly preferred are the ammonium, calcium, magnesium, potassium and sodium salts. Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary, and tertiary amines, as well as cyclic amines and substituted amines such as naturally occurring and synthesized substituted amines. Other pharmaceutically acceptable organic non-toxic bases from which salts can be formed include arginine, betaine, caffeine, choline, N′,N′-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purines, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine and the like.
When the compound of the present invention is basic, its corresponding salt can be conveniently prepared from pharmaceutically acceptable non-toxic acids, including inorganic and organic acids. Such acids include, for example, acetic, benzenesulfonic, benzoic, camphorsulfonic, citric, ethanesulfonic, fumaric, gluconic, glutamic, hydrobromic, hydrochloric, isethionic, lactic, maleic, malic, mandelic, methanesulfonic, mucic, nitric, pamoic, pantothenic, phosphoric, succinic, sulfuric, tartaric, p-toluenesulfonic acid and the like
Since the compounds of formula (I) are intended for pharmaceutical use they are preferably provided in substantially pure form, for example at least 60% pure, more suitably at least 75% pure, especially at least 98% pure (% are on a weight for weight basis).
The compounds of formula (I) can be prepared as described below, in which, for illustrative purposes, —V— is shown as a group of the formula:
and R1, A, B, x, y, G, W, X and Y are as defined above.
The compounds of formula (I), in which X═N, Y═O and W═N, may be prepared according to the method illustrated in Scheme 1. The nitriles of formula 2 are either commercially available or can be synthesised using known techniques. Compounds of formula 2 are treated with hydroxylamine in a suitable solvent, such as ethanol-water, at elevated temperature, to afford amidoximes of formula 3 (synthesis of amidoximes is further described by A. R. Martin et al, J. Med. Chem., 2001, 44, 1560). Compounds of formula 3 are subsequently condensed with acids of formula 4, which are themselves either commercially available or can be readily synthesised using known techniques. The condensation firstly entails activation of compounds of formula 4 by, for example, formation of the mixed anhydride, in which the acid is treated with a chloroformate, such as isobutylchloroformate, in the presence of a suitable base, such as triethylamine, in a suitable solvent, such as THF or toluene, followed by addition of compounds of formula 3. Alternatively, compounds of formula 4 may be activated by conversion to the acid halide, generated by treatment of the acid with, for example, oxalyl chloride in a suitable solvent, such as CH2Cl2-DMF. The intermediates arising from the condensation of amidoximes of formula 3 and acids of formula 4 are dissolved in an appropriate solvent, such as toluene or xylene, and heated under reflux, with concomitant removal of water by Dean-Stark apparatus or by molecular sieves, to form oxadiazoles of formula (I). Alternatively, amidoximes of formula 3 can firstly be treated with a suitable base, for example sodium hydride, in an appropriate solvent, such as THF, and subsequently esters of formula 5. Heating of this mixture also generates oxadiazoles of formula (I) (this process is further illustrated by R. H. Mach et al, Bioorg. Med. Chem., 2001, 9, 3113).
Compounds of formula (I) in which X═O, Y═N and W═N may be prepared according to the method outlined in Scheme 2. The nitriles of formula 6 are either commercially available or can be synthesised using known techniques. These are converted to the corresponding amidoximes of formula 7, as described above, and subsequently condensed with acids of formula 8, which are commercially available or can readily be synthesised by those skilled in the art. This condensation is performed in a fashion analogous to that described in Scheme 1, to afford the corresponding oxadiazoles of formula (I).
Compounds of formula (I) in which X═N, Y═N and W═O can be synthesised as outlined in Scheme 3. The acyl chlorides of formula 9 are either commercially available or may be synthesised using known methods. The acid hydrazides of formula 10 can be readily obtained by, for example, treating an ethanolic solution of the corresponding ester with hydrazine (for further details see K. M. Kahn et al, Bioorg. Med. Chem., 2003, 11, 1381). Treating the acyl chlorides of formula 9 with the acid hydrazides of formula 10 in a suitable solvent, such as pyridine, affords compounds of formula 11 (further illustrated by V. N. Kerr et al, J. Am. Chem. Soc., 1960, 82, 186), which are then-converted by POCl3 at elevated temperature to compounds of formula (I) (this process is further described by S-A. Chen et al, J. Am. Chem. Soc., 2001, 123, 2296). Similarly, compounds of formula (I) where X═Y═W═N can be prepared via the condensation of the amidrazone analogue of 10 with the appropriate activated carboxylic acid derivative, such as 9. The reactive groups in this reaction may be exchanged, i.e. an amidrazone of formula R1-A-C(═NH)NHNH2 can form a compound of formula (I) by condensation with an activated carboxylic acid derivative LG-C(═O)—B-cycle where LG is halogen or oxycarbonyl (P. H. Olesen et al, J. Med. Chem., 2003, 46, 3333-3341).
Compounds of formula (I) where X═N, Y═N, and W═S can also be prepared from compounds of formula 11 by heating with Lawesson's reagent in a suitable solvent, such as toluene or acetonitrile (D. Alker et al, J. Med. Chem., 1989, 32, 2381-2388). Compounds of formula (I) where X═S, Y═N and W═N can be formed from compounds of formula 12 (Scheme 4) which are commercially available, or can be readily synthesised from the corresponding carbonyl compound and Lawesson's reagent under standard conditions. Treating a compound of formula 12 with a compound of formula 13 in a suitable solvent such as dichloromethane at about 20° C. gives compounds of formula 14. Compounds of formula 13 can be obtained by treating the corresponding dimethylamide with Meerwein's reagent (for details see M. Brown U.S. Pat. No. 3,092,637). Compounds of formula 14 are then cyclised using hydroxylamine-O-sulfonic acid in the presence of a base, such as pyridine, in a suitable solvent such as methanol (for further details, see A. MacLeod et al, J. Med. Chem., 1990, 33, 2052).
The regioisomeric derivatives of formula (I), where X═N, Y═S and W═N, can be formed in a similar manner by reversing the functionality of the reactants so the R1 fragment contains the acetal moiety and the G containing cycle fragment contains the thiocarbonyl.
Compounds of formula (I) where W═O, X═N and Y═CH can be formed from compounds of formula 15 (Scheme 5). Compounds of formula 15 are commercially available or synthesised using known techniques. Chlorides of formula 16 are commercially available, or can readily be formed by chlorinating the corresponding ketone using standard conditions, for example, bubbling chlorine gas through a methanol solution of the ketone (for further details see R. Gallucci & R. Going, J. Org. Chem., 1981, 46, 2532). Mixing a compound of formula 15 with a chloride of formula 16 in a suitable solvent, such as toluene, with heating, for instance at about 100° C. gives compounds of formula (I) (for further information, see A. Hassner et al, Tetrahedron, 1989, 45, 6249). Compounds of formula (I) where W═O, X═CH and Y═N can be formed is a similar fashion by reversing the functionality of the reactants so the R1 fragment contains the haloketone moiety and the G containing cycle fragment contains the C(O)NH2.
Alternatively, compounds of formula (I) where X═S, W═N and Y═CH can also be formed from compounds of formula 16. Heating an compound of formula 15 with phosphorus pentasulfide, followed by the addition of a compound of formula 16 followed by further heating gives compounds of formula (I) (for further details, see R. Kurkjy & E. Brown, J. Am. Chem. Soc., 1952, 74, 5778). The regioisomeric compounds where X═CH, W═N and Y═S can be formed is a similar fashion by reversing the functionality of the reactants, so the R1 fragment contains the haloketone moiety and the G containing cycle fragment contains the C(O)NH2.
Compounds of formula I where W═N, X═O and Y═CH can be formed from compounds of formula 15 and formula 17 (Scheme 6) under similar conditions to those outlined for Scheme 5. Compounds of formula 1 where W═S, X═N and Y═CH can also be formed from compounds of formula 15 and formula 17 using the conditions involving phosphorus pentasulfide described above.
Compounds of formula (I) where X═O, Y═N and W═CH, and where X═N, Y═O and W═CH and can be formed from compounds of formula 20 (Scheme 7). Acylation of compounds of formula 18 with a compound of formula 19, where Q is alkoxide or chloride, can occur under standard conditions, for example, deprotonation of ketone 18 with a suitable base, such as lithium diisopropylamide or potassium ethoxide, in a suitable solvent, such as tetrahydrofuran, generally at low temperature. Treatment of compounds of formula 20 with hydroxylamine, in a suitable solvent, such as ethanol, at elevated temperature, for example 75° C., yields compounds of formula (I) as a mixture of both regioisomers of the isoxazole. Using standard separation techniques, such as chromatography on silica gel, the individual isomers can be isolated (for further details, see M. Rowley et al, J. Med. Chem., 1997, 40, 2374).
Compounds of formula (I) where X═S, Y═N and W═CH can be formed by hydrogenation of a compound of formula (I) where X═O, Y═N and W═CH, with platinum oxide in a suitable solvent such as ethanol, followed by heating with phosphorus pentasulfide to give compounds of formula (I) where X═S, Y═N and W═CH (for further details, see G. Wiegand et al, J. Med. Chem., 1971, 14, 1015). For details of the synthesis of the regioisomer where X═N, Y═S and W═CH also see G. Wiegand ibid.
Compounds of formula (I) where X═N, Y═N and W═CH can be formed from compounds of formula 20. Treatment of compounds of formula 20 with hydrazine in a suitable solvent, such as methanol, would give rise to compounds of formula (I) where X═N, Y═N and W═CH (this process is further illustrated by R. Baker et al, J. Med. Chem., 1997, 40, 2374).
Compounds of formula (I) in which X═CH, Y═N and W═N can be synthesised as described in Scheme 8. Bromides of formula 23 are either commercially available or may be synthesised from the corresponding ketone by, for example, treating an aqueous solution of the ketone with Br2 and HBr (as described by J. Y. Becker et al, Tetrahedron Lett., 2001, 42, 1571). The amidines of formula 22 may be synthesised by known methods, for example by treatment of the corresponding alkyl imidates of formula 21 with ammonia in a suitable solvent, such as ethanol (as detailed by D. A. Pearson et al, J. Med. Chem., 1996, 39, 1372). The imidates of formula 21 may in turn be generated by, for example, treatment of the corresponding nitrile with HCl in a suitable solvent, such as methanol (for further details see J. P. Lokensgard et al, J. Org. Chem., 1985, 50, 5609). Reaction of amidines of formula 22 with bromides of formula 23 in a suitable solvent, such as DMF, affords compounds of formula (I) (illustrated by N. J. Liverton et al, J. Med. Chem., 1999, 42, 2180).
The regioisomeric compounds where X═N, Y═CH and W═N can be formed in a similar fashion by reversing the functionality of the reactants, so the R1 fragment contains the amidine moiety and the R2 fragment contains the bromide.
Compounds of formula (I) in which X═CH, Y═CH and W═N can be synthesised as illustrated in Scheme 9. Diketones of formula 25 are readily accessible by, for example, the condensation of ketones of formula 24, which are commercially available or are readily synthesised using known techniques, with bromides of formula 23 in a suitable solvent, such as benzene using an appropriate catalyst. Illustrative examples are described by O. G. Kulinkovich et al, Synthesis, 2000, 9, 1259. Using a Paal-Knorr reaction, diketones of formula 25 may be treated with, for example, ammonium carbonate in a suitable solvent, such as ethanol at elevated temperature (for further details see R. A. Jones et al, Tetrahedron, 1996, 52, 8707) to afford compounds of formula (I).
Compounds of formula (I) in which R2 contains either a carbamate or a sulfonamide group may be synthesised as described in Scheme 10. Compounds of formula 26, in which P represents a suitable protecting group, for example tert-butoxycarbonyl (Boc), may be synthesised as outlined in Schemes 1-9 above. The protecting group is firstly removed under suitable conditions to afford compounds of formula 27. In the case of the Boc group this can be achieved by treatment of compounds of formula 26 with a suitable acid, such as trifluoroacetic acid, in an appropriate solvent, such as CH2Cl2. Treatment of compounds of formula 27 with chloroformates of formula 28, which are generally commercially available or can be readily synthesised, in a suitable solvent, such as CH2Cl2, in the presence of a suitable base, such as triethylamine, affords compounds of formula (I). Similarly, compounds of formula 27 may be reacted with sulfonyl chlorides of formula 29, which are generally commercially available or can readily be synthesised, in a suitable solvent, such as CH2Cl2, in the presence of a suitable base, such as triethylamine, to afford compounds of formula (I). Compounds of formula (I) in which R2 contains a urea moiety may be prepared by reacting a compound of formula 27 with an isocyanate of formula O≡C═N—R3. Furthermore, compounds of formula (I) in which R2 a heteroaryl group may be prepared by reacting the amine 27 with the appropriate heteroaryl chloride or bromide under Pd(0) catalysis in the presence of a suitable ligand and base (Urgaonkar, S.; Hu, J.-H.; Verkade. J. G. J. Org. Chem., 2003, 68, 8416-8423).
Compounds of formula (I) in which R2 contains an amide group may be synthesised from compounds of formula 27 and a suitable acid (R3COOH), or activated derivative thereof, in an amide bond forming reaction.
Compounds of formula (I) where B contains a NR5 group where R5 is hydrogen can be further transformed into compounds of formula (I) where R5 is C1-4 alkyl group using standard techniques known to those with skill in the art.
Compounds of the formula (I) where R1 is pyridyl optionally substituted with CN can be prepared from the corresponding unsubstituted pyridine by the Reissert reaction (Fife, W. K. J. Org. Chem., 1983, 48, 1375-1377). Similar reactions can be used to prepare the compounds where R1 is pyridyl optionally substituted with halogen (Walters, M. A.; Shay, J. J. Tetrahedron Lett., 1995, 36, 7575-7578). The compounds where R1 is pyridyl optionally substituted with halogen can be transformed into the corresponding compounds where R1 is pyridyl optionally substituted with C1-4 alkyl by transition metal-catalysed cross-coupling reactions (Fürstner, A., et al, J. Am. Chem. Soc., 2002, 124, 13856-13863).
Compounds of formula (I) and where X═N, Y═N, U═N and W═CH can be synthesised as shown in Scheme 11 below. Illustrative examples are described by M. Meldal et al, J. Org. Chem., 2002, 67(9), 3057-3064. Azides of formula 30 are either commercially available or may be synthesised, for example, from the displacement of the corresponding halides with azide ion using known techniques; or synthesised from the corresponding amine derivative via reaction with sodium nitrite in acidic media. The alkynes of formula 31 may be commercial or synthesised by known methods, for example by reaction of acetylide ions with boranes (see J. Org. Chem., 1981. 46(11) 2311-2314) or aldehydes or ketones.
Compounds of formula (I) and where X═N, Y═CH, U═N and W═CH can be synthesised as shown in Scheme 12 by reaction of 1,3-dicarbonyl compounds of formula 33 (or their equivalents, such as enol ethers) with hydrazines of formula 32. The hydrazines of formula 32 may be commercial or synthesised by known methods, for example by reaction of the corresponding amine with sodium nitrite and reacting the resulting diazonium salt with a reducing agent such as sodium sulfite.
Other compounds of formula (I) may be prepared by methods analogous to those described above or by methods known per se.
Further details for the preparation of the compounds of formula (I) are found in the examples.
The compounds of formula (I) may be prepared singly or as compound libraries comprising at least 2, for example 5 to 1,000, compounds and more preferably 10 to 100 compounds of formula (I). Compound libraries may be prepared by a combinatorial “split and mix” approach or by multiple parallel synthesis using either solution or solid phase chemistry, using procedures known to those skilled in the art.
During the synthesis of the compounds of formula (I), labile functional groups in the intermediate compounds, e.g. hydroxy, carboxy and amino groups, may be protected. The protecting groups may be removed at any stage in the synthesis of the compounds of formula (I) or may be present on the final compound of formula (I). A comprehensive discussion of the ways in which various labile functional groups may be protected and methods for cleaving the resulting protected derivatives is given in, for example, Protective Groups in Organic Chemistry, T. W. Greene and P. G. M. Wuts, (1991) Wiley-Interscience, New York, 2nd edition.
Any novel intermediates as defined above are of use in the synthesis of compounds of formula (I) and are therefore also included within the scope of the invention. For example, compounds of formula (XXVII):
wherein the groups R1, A, V, B, x and y are as defined above for compounds of formula (I), provided that R1 is not 4-fluoroalkylpyrid-3-yl or 4-fluoroalkylpyrimidin-5-yl.
US 2003/0162812 discloses certain 4-fluoroalkylpyrid-3-yl and 4-fluoroalkylpyrimidin-5-yl derivatives as pesticides.
As indicated above the compounds of formula (I) are useful as GPR119 agonists, e.g. for the treatment and/or prophylaxis of obesity and diabetes. For such use the compounds of formula (I) will generally be administered in the form of a pharmaceutical composition.
The invention also provides a compound of formula (I), or a pharmaceutically acceptable salt thereof, for use as a pharmaceutical.
The invention also provides a pharmaceutical composition comprising a compound of formula (I), in combination with a pharmaceutically acceptable carrier.
Preferably the composition is comprised of a pharmaceutically acceptable carrier and a non-toxic therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof.
Moreover, the invention also provides a pharmaceutical composition for the treatment of disease by modulating GPR119, resulting in the prophylactic or therapeutic treatment of obesity, e.g. by regulating satiety, or for the treatment of diabetes, comprising a pharmaceutically acceptable carrier and a non-toxic therapeutically effective amount of compound of formula (I), or a pharmaceutically acceptable salt thereof.
The pharmaceutical compositions may optionally comprise other therapeutic ingredients or adjuvants. The compositions include compositions suitable for oral, rectal, topical, and parenteral (including subcutaneous, intramuscular, and intravenous) administration, although the most suitable route in any given case will depend on the particular host, and nature and severity of the conditions for which the active ingredient is being administered. The pharmaceutical compositions may be conveniently presented in unit dosage form and prepared by any of the methods well known in the art of pharmacy.
In practice, the compounds of formula (I), or pharmaceutically acceptable salts thereof, can be combined as the active ingredient in intimate admixture with a pharmaceutical carrier according to conventional pharmaceutical compounding techniques. The carrier may take a wide variety of forms depending on the form of preparation desired for administration, e.g. oral or parenteral (including intravenous).
Thus, the pharmaceutical compositions can be presented as discrete units suitable for oral administration such as capsules, cachets or tablets each containing a predetermined amount of the active ingredient. Further, the compositions can be presented as a powder, as granules, as a solution, as a suspension in an aqueous liquid, as a non-aqueous liquid, as an oil-in-water emulsion, or as a water-in-oil liquid emulsion. In addition to the common dosage forms set out above, the compound of formula (I), or a pharmaceutically acceptable salt thereof, may also be administered by controlled release means and/or delivery devices. The compositions may be prepared by any of the methods of pharmacy. In general, such methods include a step of bringing into association the active ingredient with the carrier that constitutes one or more necessary ingredients. In general, the compositions are prepared by uniformly and intimately admixing the active ingredient with liquid carriers or finely divided solid carriers or both. The product can then be conveniently shaped into the desired presentation.
The compounds of formula (I), or pharmaceutically acceptable salts thereof, can also be included in pharmaceutical compositions in combination with one or more other therapeutically active compounds.
The pharmaceutical carrier employed can be, for example, a solid, liquid, or gas. Examples of solid carriers include lactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, and stearic acid. Examples of liquid carriers are sugar syrup, peanut oil, olive oil, and water. Examples of gaseous carriers include carbon dioxide and nitrogen.
In preparing the compositions for oral dosage form, any convenient pharmaceutical media may be employed. For example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents, and the like may be used to form oral liquid preparations such as suspensions, elixirs and solutions; while carriers such as starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents, and the like may be used to form oral solid preparations such as powders, capsules and tablets. Because of their ease of administration, tablets and capsules are the preferred oral dosage units whereby solid pharmaceutical carriers are employed. Optionally, tablets may be coated by standard aqueous or nonaqueous techniques.
A tablet containing the composition of this invention may be prepared by compression or molding, optionally with one or more accessory ingredients or adjuvants. Compressed tablets may be prepared by compressing, in a suitable machine, the active ingredient in a free-flowing form such as powder or granules, optionally mixed with a binder, lubricant, inert diluent, surface active or dispersing agent. Molded tablets may be made by molding in a suitable machine, a mixture of the powdered compound moistened with an inert liquid diluent. Each tablet preferably contains from about 0.05 mg to about 5 g of the active ingredient and each cachet or capsule preferably containing from about 0.05 mg to about 5 g of the active ingredient.
For example, a formulation intended for the oral administration to humans may contain from about 0.5 mg to about 5 g of active agent, compounded with an appropriate and convenient amount of carrier material which may vary from about 5 to about 95 percent of the total composition. Unit dosage forms will generally contain between from about 1 mg to about 2 g of the active ingredient, typically 25 mg, 50 mg, 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 800 mg, or 1000 mg.
Pharmaceutical compositions of the present invention suitable for parenteral administration may be prepared as solutions or suspensions of the active compounds in water. A suitable surfactant can be included such as, for example, hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof in oils. Further, a preservative can be included to prevent the detrimental growth of microorganisms.
Pharmaceutical compositions of the present invention suitable for injectable use include sterile aqueous solutions or dispersions. Furthermore, the compositions can be in the form of sterile powders for the extemporaneous preparation of such sterile injectable solutions or dispersions. In all cases, the final injectable form must be sterile and must be effectively fluid for easy syringability. The pharmaceutical compositions must be stable under the conditions of manufacture and storage; thus, preferably should be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g. glycerol, propylene glycol and liquid polyethylene glycol), vegetable oils, and suitable mixtures thereof.
Pharmaceutical compositions of the present invention can be in a form suitable for topical use such as, for example, an aerosol, cream, ointment, lotion, dusting powder, or the like. Further, the compositions can be in a form suitable for use in transdermal devices. These formulations may be prepared, using a compound of formula (I), or a pharmaceutically acceptable salt thereof, via conventional processing methods. As an example, a cream or ointment is prepared by admixing hydrophilic material and water, together with about 5 wt % to about 10 wt % of the compound, to produce a cream or ointment having a desired consistency.
Pharmaceutical compositions of this invention can be in a form suitable for rectal administration wherein the carrier is a solid. It is preferable that the mixture forms unit dose suppositories. Suitable carriers include cocoa butter and other materials commonly used in the art. The suppositories may be conveniently formed by first admixing the composition with the softened or melted carrier(s) followed by chilling and shaping in molds.
In addition to the aforementioned carrier ingredients, the pharmaceutical formulations described above may include, as appropriate, one or more additional carrier ingredients such as diluents, buffers, flavoring agents, binders, surface-active agents, thickeners, lubricants, preservatives (including anti-oxidants) and the like. Furthermore, other adjuvants can be included to render the formulation isotonic with the blood of the intended recipient. Compositions containing a compound of formula (I), or pharmaceutically acceptable salts thereof, may also be prepared in powder or liquid concentrate form.
Generally, dosage levels on the order of 0.01 mg/kg to about 150 mg/kg of body weight per day are useful in the treatment of the above-indicated conditions, or alternatively about 0.5 mg to about 7 g per patient per day. For example, obesity may be effectively treated by the administration of from about 0.01 to 50 mg of the compound per kilogram of body weight per day, or alternatively about 0.5 mg to about 3.5 g per patient per day.
It is understood, however, that the specific dose level for any particular patient will depend upon a variety of factors including the age, body weight, general health, sex, diet, time of administration, route of administration, rate of excretion, drug combination and the severity of the particular disease undergoing therapy.
The compounds of formula (I) may be used in the treatment of diseases or conditions in which GPR119 plays a role.
Thus the invention also provides a method for the treatment of a disease or condition in which GPR119 plays a role comprising a step of administering to a subject in need thereof an effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof. Diseases or conditions in which GPR119 plays a role include obesity and diabetes. In the context of the present application the treatment of obesity is intended to encompass the treatment of diseases or conditions such as obesity and other eating disorders associated with excessive food intake e.g. by reduction of appetite and body weight, maintenance of weight reduction and prevention of rebound and diabetes (including Type 1 and Type 2 diabetes, impaired glucose tolerance, insulin resistance and diabetic complications such as neuropathy, nephropathy, retinopathy, cataracts, cardiovascular complications and dyslipidaemia). And the treatment of patients who have an abnormal sensitivity to ingested fats leading to functional dyspepsia. The compounds of the invention may also be used for treating metabolic diseases such as metabolic syndrome (syndrome X), impaired glucose tolerance, hyperlipidemia, hypertriglyceridemia, hypercholesterolemia, low HDL levels and hypertension.
The invention also provides a method for the regulation of satiety comprising a step of administering to a subject in need thereof an effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof.
The invention also provides a method for the treatment of obesity comprising a step of administering to a subject in need thereof an effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof.
The invention also provides a method for the treatment of diabetes, including type I and type 2 diabetes, particularly type 2 diabetes, comprising a step of administering to a patient in need thereof an effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof.
The invention also provides a method for the treatment of metabolic syndrome (syndrome X), impaired glucose tolerance, hyperlipidemia, hypertriglyceridemia, hypercholesterolemia, low HDL levels or hypertension comprising a step of administering to a patient in need thereof an effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof.
The invention also provides a compound of formula (I), or a pharmaceutically acceptable salt thereof, for use in the treatment of a condition as defined above.
The invention also provides the use of a compound of formula (I), or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment of a condition as defined above.
In the methods of the invention the term “treatment” includes both therapeutic and prophylactic treatment.
The compounds of formula (I) may exhibit advantageous properties compared to known GPR 119 agonists, for example, the compounds may exhibit improved potency, for example when compared to compounds wherein group B is unbranched, or improved solubility thus improving absorption properties and bioavailability, or other advantageous properties for compounds to be used as pharmaceuticals.
The compounds of formula (I), or pharmaceutically acceptable salts thereof, may be administered alone or in combination with one or more other therapeutically active compounds. The other therapeutically active compounds may be for the treatment of the same disease or condition as the compounds of formula (I) or a different disease or condition. The therapeutically active compounds may be administered simultaneously, sequentially or separately.
The compounds of formula (I) may be administered with other active compounds for the treatment of obesity and/or diabetes, for example insulin and insulin analogs, gastric lipase inhibitors, pancreatic lipase inhibitors, sulfonyl ureas and analogs, biguanides, α2 agonists, glitazones, PPAR-γ agonists, mixed PPAR-α/γ agonists, RXR agonists, fatty acid oxidation inhibitors, α-glucosidase inhibitors, β-agonists, phosphodiesterase inhibitors, lipid lowering agents, glycogen phosphorylase inhibitors, antiobesity agents e.g. pancreatic lipase inhibitors, MCH-1 antagonists and CB-1 antagonists (or inverse agonists), amylin antagonists, lipoxygenase inhibitors, somostatin analogs, glucokinase activators, glucagon antagonists, insulin signalling agonists, PTP1B inhibitors, gluconeogenesis inhibitors, antilypolitic agents, GSK inhibitors, galanin receptor agonists, anorectic agents, CCK receptor agonists, leptin, serotonergic/dopaminergic antiobesity drugs, reuptake inhibitors e.g. sibutramine, CRF antagonists, CRF binding proteins, thyromimetic compounds, aldose reductase inhibitors, glucocorticoid receptor antagonists, NHE-1 inhibitors or sorbitol dehydrogenase inhibitors.
Combination therapy comprising the administration of a compound of formula (I), or a pharmaceutically acceptable salt thereof, and at least one other antiobesity agent represents a further aspect of the invention.
The present invention also provides a method for the treatment of obesity in a mammal, such as a human, which method comprises administering an effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof, and another antiobesity agent, to a mammal in need thereof.
The invention also provides the use of a compound of formula (I), or a pharmaceutically acceptable salt thereof, and another antiobesity agent for the treatment of obesity.
The invention also provides the use of a compound of formula (I), or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for use in combination with another antiobesity agent, for the treatment of obesity.
The compound of formula (I), or a pharmaceutically acceptable salt thereof, and the other antiobesity agent(s) may be co-administered or administered sequentially or separately.
Co-administration includes administration of a formulation which includes both the compound of formula (I), or a pharmaceutically acceptable salt thereof, and the other antiobesity agent(s); or the simultaneous or separate administration of different formulations of each agent. Where the pharmacological profiles of the compound of formula (I), or a pharmaceutically acceptable salt thereof, and the other antiobesity agent(s) allow it, coadministration of the two agents may be preferred.
The invention also provides the use of a compound of formula (I), or a pharmaceutically acceptable salt thereof, and another antiobesity agent in the manufacture of a medicament for the treatment of obesity.
The invention also provides a pharmaceutical composition comprising a compound of formula (I), or a pharmaceutically acceptable salt thereof, and another antiobesity agent, and a pharmaceutically acceptable carrier. The invention also encompasses the use of such compositions in the methods described above.
GPR119 agonists are of particular use in combination with centrally acting antobesity agents.
The other antiobesity agent for use in the combination therapies according to this aspect of the invention is preferably a CB-1 modulator, e.g. a CB-1 antagonist or inverse agonist. Examples of CB-1 modulators include SR141716 (rimonabant) and SLV-319 ((4S)-(−)-3-(4-chlorophenyl)-N-methyl-N-[(4-chlorophenyl)sulfonyl]-4-phenyl-4,5-dihydro-1H-pyrazole-1-carboxamide); as well as those compounds disclosed in EP576357, EP656354, WO 03/018060, WO 03/020217, WO 03/020314, WO 03/026647, WO 03/026648, WO 03/027076, WO 03/040105, WO 03/051850, WO 03/051851, WO 03/053431, WO 03/063781, WO 03/075660, WO 03/077847, WO 03/078413, WO 03/082190, WO 03/082191, WO 03/082833, WO 03/084930, WO 03/084943, WO 03/086288, WO 03/087037, WO 03/088968, WO 04/012671, WO 04/013120, WO 04/026301, WO 04/029204, WO 04/034968, WO 04/035566, WO 04/037823 WO 04/052864, WO 04/058145, WO 04/058255, WO 04/060870, WO 04/060888, WO 04/069837, WO 04/069837, WO 04/072076, WO 04/072077. WO 04/078261 and WO 04/108728, and the references disclosed therein.
Other diseases or conditions in which GPR119 has been suggested to play a role include those described in WO 00/50562 and U.S. Pat. No. 6,468,756, for example cardiovascular disorders, hypertension, respiratory disorders, gestational abnormalities, gastrointestinal disorders, immune disorders, musculoskeletal disorders, depression, phobias, anxiety, mood disorders and Alzheimer's disease.
All publications, including, but not limited to, patents and patent application cited in this specification, are herein incorporated by reference as if each individual publication were specifically and individually indicated to be incorporated by reference herein as fully set forth.
The invention will now be described by reference to the following examples which are for illustrative purposes and are not to be construed as a limitation of the scope of the present invention.
t-Bu: tert-Butyl; DCM: Dichloromethane; DMAP: 4-Dimethylaminopyridine; DMF: N,N-Dimethylformamide; DMSO: Dimethylsulfoxide; EDCI: 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; EtOAc: Ethyl acetate; h: hour; HOBt: 1-Hydroxybenzotriazole hydrate; HPLC: High performance liquid chromatography; mCPBA: 3-Chloroperoxybenzoic acid; IH: Isohexane; Me: Methyl; min: Minutes; RP-HPLC: Reverse phase high performance liquid chromatography; TFA: Trifluoroacetic acid; THF: Tetrahydrofuran.
LCMS data were obtained as follows: Waters Atlantis C18, 3 μl (3.0×20 mm, flow rate 0.85 ml/min) eluting with a H2O-MeCN gradient containing 0.1% v/v HCO2H over 6.5 min with UV detection at 220 nm. Gradient information: 0.0-0.3 min 100% H2O; 0.3-4.25 min: Ramp to 10% H2O-90% CH3CN; 4.25 min-4.4 min: Ramp to 100% CH3CN; 4.4-4.9 min: Hold at 100% MeCN; 4.9-5.0 min: Return to 100% H2O; 5.00-6.50 min: Hold at 100% H2O. The mass spectra were obtained using an electrospray ionisation source in either the positive (ESI−) ion or negative ion (ESI−) mode. 1H nmr spectra were recorded on a Varian Mercury 400 spectrometer, operating at 400 MHz. Chemical shifts are reported as ppm relative to tetramethylsilane (δ=0).
HPLC was performed using a Phenomenex™ C18 column (210×21 mm) eluting with a H2O—CH3CN solution at 20 mL/min, with UV detection at 220 nm. Typical gradient: 0-0.5 min, 10% CH3CN-90% H2O; 0.5 min-10 min, ramp to 90% CH3CN-10% H2O and hold at 90% CH3CN-10% H2O for 5 min; 15 min-16 min, return to 10% CH3CN-90% H2O.
3-tert-Butyl-5-chloro-[1,2,4]oxadiazole: WO95/05368; 3,5-difluoro-4-methylsulfanylbenzaldehyde: EP 1251126; 4-(2-ethoxycarbonyl-1-methylethyl)piperidine-1-carboxylic acid tert-butyl ester: WO04/092124; N-hydroxy-4-methylsulfanylbenzamidine: Buu-Hoi; Lecocq J, Bull Soc. Chim. Fr. 1946, 139; 1-(3-isopropyl-[1,2,4]oxadiazol-5-yl)piperidin-4-ol: WO05/121121; 4-(2-methoxycarbonylpropyl)piperidine-1-carboxylic acid tert-butyl ester: WO98/07620; N-Hydroxyisonicotinamidine: Martin A. R. et al, J. Med. Chem., 2001, 44, 1560; 2-Methylisonicotinonitrile: Ashimori A. et al, Chem. Pharm. Bull., 1990, 38, 2446.
A solution of 4-(2-methoxycarbonylethyl)piperidine-1-carboxylic acid tert-butyl ester (780 mg, 2.88 mmol) in anhydrous THF (3 mL) was added dropwise to a stirred solution of lithium diisopropylamide (3.16 mL of a 1M solution in THF, 3.16 mmol) at −78° C. under argon. After 1 h the mixture was added slowly via cannula to a solution of methyliodide (360 μL, 5.76 mmol) and N,N′-dimethylpropyleneurea (1 mL) in anhydrous THF (6 mL) at −78° C. The stirred mixture was warmed to 0° C. and, after 4 h, quenched by the addition of saturated aqueous NH4Cl (5 mL). Ether (100 mL) was added and the organic phase washed with brine (10 mL), dried (MgSO4) and evaporated to afford an oil that was then purified by column chromatography (IH-EtOAc 7:3) to give the title ester: δH (CDCl3) 1.01-1.13 (m, 2H), 1.15 (d, 3H), 1.25-1.32 (m, 1H), 1.35-1.44 (m, 1H), 1.45 (s, 9H), 1.58-1.71 (m, 3H), 2.52-2.60 (m, 1H), 2.66 (br t, 2H), 3.68 (s, 3H), 4.01-4.13 (m, 2H).
A stirred solution of 4-hydroxypiperidine-1-carboxylic acid tert-butyl ester (300 mg, 1.5 mmol) in 1,4-dioxane (4 mL) under an argon atmosphere was treated portionwise with sodium hydride (239 mg of a 60% dispersion in oil, 6 mmol). After 30 min a solution of (R)-2-bromopropionic acid (135 μL, 1.5 mmol) in 1,4-dioxane (2 mL) was added and stirring continued for 22 h. Water (15 mL) was added and the aqueous phase washed with ether (15 mL) then acidified to pH 1 using 1M HCl. The suspension was extracted into EtOAc (50 mL), which was dried (MgSO4) and evaporated to afford the title acid: δH (CDCl3) 1.46 (s, 9H), 1.46 (d, 3H), 1.57 (m, 2H), 1.86 (m, 2H), 3.09 (ddd, 2H), 3.61 (sept, 1H), 3.82 (m, 2H), 4.16 (q, 1H).
The acids listed in Table 1 were prepared using the same method as that described in Preparation 2:
A stirred solution of 3,5-difluoro-4-methylsulfanylbenzaldehyde (1.0 g, 5.32 mmol) in ethanol (12 mL) was treated with NH2OH.HCl (778 mg, 11.2 mmol) in water (5 mL) followed immediately by K2CO3 (780 mg, 5.64 mmol) in water (10 mL). After 1 h at room temperature the ethanol was largely removed in vacuo and water (30 mL) added. The mixture was extracted into EtOAc (150 mL), the organic phase separated, dried (MgSO4) and evaporated. The residue was purified by column chromatography (IH-EtOAc-CH2Cl2 88:10:2) to afford 3,5-difluoro-4-methylsulfanylbenzaldehyde oxime: δH (CDCl3) 2.50 (s, 3H), 7.14 (d, 2H), 7.47 (s, 1H), 8.03 (s, 1H). Acetic anhydride (45 mL) was added to a sample of this oxime (1.139 g, 5.6 mmol) followed by p-toluenesulfonic acid monohydrate (1.066 g, 5.6 mmol). The mixture was heated under reflux for 20 h, cooled and the excess acetic anhydride removed in vacuo. Water (50 mL) was added and adjusted to pH 8 using saturated aqueous Na2CO3, then extracted with CH2Cl2 (100 mL). The solvent was removed and the residue purified by column chromatography (IH-EtOAc 9:1) to give the title nitrile: δH (CDCl3) 2.58 (s, 3H), 7.20 (d, 2H).
Sodium thiomethoxide (1.59 g, 22.6 mmol) was suspended in anhydrous DMF (35 mL) and cooled to 5° C. A solution of 3,4-difluorobenzonitrile (3.0 g, 21.6 mmol) dissolved in cold DMF (25 mL) was added via cannula and the mixture stirred for 1 h. The DMF was evaporated and the residual solid recrystallised from ether triturated with hexane, giving the title nitrile: δH (CDCl3) 2.53 (s, 3H), 7.26 (t, 1H), 7.29 (dd, 1H), 7.43 (dd, 1H).
Using the procedure outlined in Preparation 9,4-fluoro-3-methylbenzonitrile was converted to the title compound: δH (CDCl3) 2.33 (3H, s), 2.52 (3H, s), 7.16 (1H, d), 7.38 (1H, s) 7.47 (1H, d).
A stirred slurry of Na2CO3 (9.8 g, 117 mmol) in water (15 mL) was cooled on ice-water and 4-hydroxypiperidine.HCl (4.0 g, 29.2 mmol) was added. A solution of cyanogen bromide (3.7 g, 35 mmol) in CH2Cl2 (90 mL) was added portionwise over 5 min and the stirring continued for 1 h. The reaction was diluted with CH2Cl2 (100 mL), the organic phase separated and dried (MgSO4) and the solvent evaporated to afford the title cyanamide: δH (CDCl3) 1.65 (ddt, 2H), 1.91 (ddt, 2H), 2.17 (br s, 1H), 3.08 (ddd, 2H), 3.45 (ddd, 2H), 3.86 (sept, 1H).
3-Fluoro-4-methylsulfanylbenzonitrile (1.5 g, 8.9 mmol) was dissolved in CH2Cl2 (75 mL) and water (15 mL) added. Chlorine gas was bubbled gently through the vigorously-stirred mixture for 30 min and the organic component then separated and evaporated to dryness. The residue was dissolved in thionyl chloride (30 mL) and heated under reflux for 6.5 h. The mixture was evaporated to dryness to afford near-pure 4-cyano-2-fluorobenzenesulfonyl chloride: δ11 (CDCl3) 7.67 (d, 1H), 7.70 (d, 1H), 8.14 (dd, 1H). A sample of the sulfonyl chloride (1.53 g, 7 mmol) was dissolved in CH2Cl2 and ammonia was bubbled gently through the stirred solution. After 10 min the mixture was filtered, the filtrate evaporated and the residue purified by column chromatography (IH-EtOAc 2:1) to give the title sulfonamide: δH (d6 DMSO) 7.88 (dd, 1H), 7.96 (t, 1H), 7.96 (br s, 2H), 8.12 (dd, 1H).
Water (10 mL) was added to a mixture of K2CO3 (491 mg, 3.55 mmol) and NH2OH.HCl (492 mg, 7.1 mmol) and the resulting solution added to a stirred solution of 4-cyano-2-fluorobenzenesulfonamide (710 mg, 3.55 mmol) in ethanol (60 mL). The mixture was heated under reflux for 18 h, cooled and partitioned between EtOAc (250 mL) and water (100 mL). The organic phase was separated, dried (MgSO4) and evaporated and the residue purified by column chromatography (EtOAc) to afford the title compound: δH (d6DMSO) 6.00 (s, 2H), 7.63 (dd, 1H), 7.66 (dd, 1H), 7.67 (s, 2H), 7.77 (t, 1H), 10.01 (s, 1H).
The amidoximes listed in Table 2 were prepared using a similar method to that of Preparation 13.
A stirred solution of 4-hydroxypiperidine-1-carboxylic acid tert-butyl ester (2 g, 9.94 mmol) in anhydrous dioxane (20 mL) was treated portionwise with NaH (828 mg of a 60% dispersion in oil, 20 mmol). The temperature was raised to 65° C. and, after 5 min, a solution of 2-bromopropionic acid (0.89 mL, 9.94 mmol) in dry dioxane (20 mL) was added in a dropwise manner. After 3 h at this temperature further NaH (828 mg) was added and heating continued for 18 h. The cooled reaction was quenched by the careful addition of water (50 mL) the mixture was washed with EtOAc (50 mL). This organic layer was extracted with 1M aqueous NaOH (20 mL) and the combined aqueous phases acidified to pH 2 using conc HCl. After extraction with EtOAc (3×50 mL) the combined organic extracts were dried (MgSO4) and evaporated to afford the title compound: δH (CDCl3) 1.46 (9H, s), 1.48 (3H, s), 1.57 (2H, m), 1.86 (2H, m), 3.09 (2H, ddd), 3.61 (1H, tt), 3.80 (2H, m), 4.15 (1H, q).
Sufficient methanol (ca. 10 mL) was added to a stirred suspension of 4-cyano-2-fluorobenzoic acid (2 g, 12.11 mmol) in toluene (5 mL) to give a clear solution. Trimethylsilyldiazomethane (7.87 mL of a 2 M solution in hexane, 15.75 mmol) was added dropwise, until the persistence of a yellow colour, after which the solution was stirred for a further 10 min and AcOH then added until a colourless solution was again obtained. The reaction mixture was diluted with EtOAc (50 mL) and washed with saturated aqueous Na2CO3 (2×20 mL) and brine (20 mL) and dried (MgSO4). Evaporation of the solvent afforded the title ester: δH (CDCl3) 3.98 (3H, s), 7.47 (1H, dd), 7.53 (1H, dd), 8.06 (1H, t).
Isobutyric acid (360 μL, 3.9 mmol), 4-N-dihydroxypiperidine-1-carboxamidine (620 mg, 3.9 mmol) and HOBt (656 mg, 4.3 mmol) were dissolved in dry DMF (5 mL). Diisopropylethylamine (2.2 mL, 12.9 mmol) was added followed by EDCI (900 mg, 4.7 mmol) in one portion and the mixture stirred for 18 h. Water (20 mL) was added and the mixture extracted with EtOAc (150 mL). The organic phase was washed with water (15 mL) saturated aqueous Na2CO3 (15 mL) and brine (10 mL) then dried (MgSO4) and evaporated to give the title oxadiazole: RT=2.45 min; m/z (ES+)=212.0 [M+H]+.
A stirred solution of 3-fluoro-N-hydroxy-4-methylsulfanylbenzamidine (200 mg, 1 mmol) in anhydrous THF (10 mL) was treated with sodium hydride (33.3 mg, 832 μmol). After min a solution of 4-(2-ethoxycarbonyl-1-methylethyl)piperidine-1-carboxylic acid tert-butyl ester (249 mg, 832 μmol) in dry THF (3 mL) was added and the mixture heated under reflux for 18 h. The solvent was removed and the residue purified by column chromatography (IH-EtOAc 4:1) to afford the title oxadiazole: δ11 (CDCl3) 0.99 (d, 3H), 1.22-1.31 (m, 2H), 1.39-1.45 (m, 1H), 1.47 (s, 9H), 1.68 (br d, 2H), 2.08 (m, 1H), 2.53 (s, 3H), 2.66 (m, 2H), 2.80 (dd, 1H), 3.00 (dd, 1H), 4.18 (m, 2H), 7.31 (t, 1H), 7.74 (dd, 1H), 7.84 (dd, 1H).
Using a similar procedure to that in Example 1, 3-fluoro-N-hydroxy-4-methylsulfanylbenzamidine was reacted with 4-(2-methoxycarbonylpropyl)piperidine-1-carboxylic acid tert-butyl ester to give the title oxadiazole: RT=4.45 min; m/z (ES+)=436.1 [M+H]+.
4-(1-Carboxypropoxy)piperidine-1-carboxylic acid tert-butyl ester (167 mg, 580 μmol). EDCI (111 mg, 580 μmol) and HOBt (78 mg, 580 μmol) were dissolved in dry THF (10 mL), diisopropylethylamine (300 μL, 1.75 mmol) added and the mixture stirred for 15 min. Solid 3-fluoro-N-hydroxy-4-methylsulfanylbenzamidine (106 mg, 530 mol) was added in one portion and stirring continued for 4 h. The reaction mixture was diluted with EtOAc (50 mL), washed with saturated aqueous Na2CO3 (10 mL) and brine (10 mL) then dried (MgSO4) and evaporated. The residue was taken up in toluene (15 mL), the solution heated under reflux for 18 h. Removal of the solvent and purification of the residue by column chromatography (IH-EtOAc 4:1) afforded the title oxadiazole: RT=4.42 min; m/z (ES−)=452.1 [M+H]−.
The compounds listed in Table 3 were synthesised using the same procedure as that outlined in Example 3.
4-{(R)-1-[3-(3-Fluoro-4-methylsulfanylphenyl)-[1,2,4]oxadiazol-5-yl]ethoxy}-piperidine-1-carboxylic acid tert-butyl ester (133 mg, 304 μmol) was dissolved in 4M HCl in dioxane (10 mL). After stirring for 30 min the solvent was removed to afford the HCl salt of 4-{(R)-1-[3-(3-fluoro-4-methylsulfanylphenyl)-[1,2,4]oxadiazol-5-yl]ethoxy}piperidine: RT=2.69 min; m/z (ES+)=338.0 [M+H]+. A sample of this salt (114 mg, 304 μmol) and 3-tert-butyl-5-chloro-[1,2,4]oxadiazole (73 mg, 456 μmol) were dissolved in anhydrous DMF (10 mL) and K2CO3 (63 mg, 456 μmol) added. After stirring for 18 h the solvent was removed and the residue dissolved in EtOAc (20 mL). The organic phase was washed with 2M NaOH (5 mL) and brine and dried (MgSO4). Removal of the solvent afforded the title oxadiazole: RT=4.29 min; m/z (ES+)=462.1 [M+H]+.
Solid mCPBA (69 mg of 78% w/w, 310 μmol) was added to a stirred solution of 4-{1-[3-(3-fluoro-4-methylsulfanylphenyl)-[1,2,4]oxadiazol-5-yl]propoxy}piperidine-1-carboxylic acid tert-butyl ester (140 mg, 310 μmol) in CH2Cl2 (5 mL). After 1 h the solvent was removed and EtOAc (10 mL) added. This solution was washed with saturated aqueous Na2CO3 (5 mL), water (5 mL) and brine (5 mL) then dried (MgSO4) and evaporated. The residue was purified by column chromatography (IH-EtOAc 1:1 the 1:2) to afford the title sulfoxide: RT=4.42 min; m/z (ES+)=452.1 [M+H]+. Using two equivalents of mCPBA affords the corresponding sulfone.
The compounds in Table 4 were produced by oxidising the corresponding sulfide using the method of Example 18.
The compounds listed in Table 5 were synthesised by reaction of 3-fluoro-N-hydroxy-4-sulfamoylbenzamidine with the corresponding acid and cyclising to the oxadiazole using the method described in Example 18.
4-{(R)-1-[3-(3-Fluoro-4-methanesulfonylphenyl)-[1,2,4]oxadiazol-5-yl]ethoxy}piperidine-1-carboxylic acid tert-butyl ester (89 mg, 190 μmol) was dissolved in 4M HCl in dioxane (5 mL) and stirred for 1 h. The solvent was removed to afford the HCl salt of 4-{(R)-1-[3-(3-fluoro-4-methanesulfonylphenyl)-[1,2,4]oxadiazol-5-yl]ethoxy}piperidine: RT=2.20 min; m/z (ES−)=370.0 [M+H]−. This material was dissolved in CH2Cl2 (15 mL) and triethylamine (65 μL, 460 μmol) and isopropylchloroformate (239 μmol, of a 1M solution in CH2Cl2, 239 mmol) added. After stirring for 30 min the solvent was removed and the residue purified by column chromatography (IH-EtOAc 7:3) to give the title carbamate: RT=3.70 min; m/z (ES+)=456.0 [M+H]+.
EDC (107 mg, 560 μmol) and HOBt (76 mg, 560 μmol) were added quickly to a solution of 4-(1-carboxyethoxy)piperidine-1-carboxylic acid tert-butyl ester (153 mg, 56 μmol) in anhydrous THF. The resulting mixture was stirred under argon for 10 min then solid N-hydroxy-4-methanesulfonylbenzamidine was added in one portion and the stirring continued for 18 h. The reaction mixture was diluted with EtOAc (20 mL) and washed with 2M aqueous HCl (2×10 mL) and saturated aqueous Na2CO3 (3×10 mL) then dried (MgSO4). The solvent was removed and the residue dissolved in toluene (3 mL) and heated under gentle reflux for 14 h. The cooled mixture was filtered through celite, the filtrate evaporated to dryness and the residue purified by flash chromatography (IH-EtOAc 3:2) to give the title compound: δH (CDCl3) 1.46 (9H, s), 1.61 (2H, m), 1.69 (3H, d), 1.79 (1H, m), 1.90 (1H, m), 3.11 (3H, s), 3.13 (2H, m), 3.67 (1H, tt), 3.77 (2H, m), 4.96 (1H, q), 8.09 (2H, d), 8.32 (2H, d): RT=3.76 min, m/z (ES+)=452.1 [M+H]+.
3-Fluoro-N-hydroxy-4-methylsulfanylbenzamidine was reacted with 4-(1-carboxyethoxy)piperidine-1-carboxylic acid tert-butyl ester using the same procedure as that described in Example 40 to give the title compound: δ11 (CDCl3) 1.46 (9H, s), 1.60 (2H, m), 1.67 (3H, d), 1.78 (1H, m), 1.90 (1H, m), 2.54 (3H, s), 3.12 (2H, m), 3.65 (1H, tt), 3.77 (2H, m), 4.92 (1H, q), 7.32 (1H, t), 7.76 (1H, dd), 7.86 (1H, dd); RT=4.27 min, m/z (ES+)=438.1 [M+H]+.
2-Fluoro-4-(N-hydroxycarbamimidoyl)benzoic acid methyl ester was reacted with 4-(1-carboxyethoxy)piperidine-1-carboxylic acid tert-butyl ester using the same procedure as that described in Example 40 to give the title compound: δH (CDCl3) 1.46 (9H, s), 1.60 (2H, m), 1.68 (3H, d), 1.79 (1H, m), 1.90 (1H, m), 3.12 (2H, m), 3.66 (1H, tt), 3.77 (2H, m), 3.98 (3H, s), 4.95 (1H, q), 7.90 (1H, dd), 7.95 (1H, dd), 8.07 (1H, t); RT=4.17 min, ml (ES+)=450.0 [M+H]+.
A solution of 4-{1-[3-(3-fluoro-4-methoxycarbonylphenyl)-[1,2,4]oxadiazol-5-yl]ethoxy}piperidine-1-carboxylic acid tert-butyl ester (Example 42, 430 mg, 958 μmol) in MeOH (3 mL) was treated with 1M aqueous NaOH (2 mL, 2 mmol). After stirring for 3 h, the mixture was acidified with citric acid and diluted with CH2Cl2 (20 mL). The organic component was separated and dried (MgSO4) and evaporated to give 4-{1-[3-(4-carboxy-3-fluorophenyl)-[1,2,4]oxadiazol-5-yl]ethoxy}piperidine-1-carboxylic acid tert-butyl ester: RT=3.69 min, m/z (ES+)=436.1 [M+H]+. A sample of this acid (137 mg, 315 μmol), EDC and HOBt were dissolved in anhydrous THF (4 mL) and stirred for 10 min. A solution of ammonia (1.89 mL of a 0.5M solution in dioxane, 945 μmol) was introduced, the reaction mixture stirred at room temperature for 4 h then partitioned between 1M aqueous NaOH (5 mL) and CH2Cl2 (20 mL), the organic phase was separated, washed with 1M aqueous HCl (5 mL), water (5 mL) and dried (MgSO4). The solvent was removed and the residue recrystallised (EtOAc-1H) to afford the title compound: δH (CDCl3) 1.46 (9H, s), 1.61 (2H, m), 1.68 (3H, d), 1.79 (1H, m), 1.90 (1H, m), 3.13 (2H, m), 3.67 (1H, tt), 3.77 (2H, m), 4.95 (1H, q), 5.84 (1H, br s), 6.71 (1H, br d), 7.91 (1H, d), 8.03 (1H, d), 8.27 (1H, t); RT=3.29 min, ml: (ES−)=435.0 [M+H]−.
4-{1-[3-(3-Fluoro-4-methoxycarbonylphenyl)-[1,2,4]oxadiazol-5-yl]ethoxy}piperidine-1-carboxylic acid tert-butyl ester was saponified and reacted with ethylamine using the procedure described in Example 43 to afford the title compound: δH (CDCl3) 1.30 (3H, t), 1.46 (9H, s), 1.60 (2H, m), 1.68 (3H, d), 1.79 (1H, m), 1.90 (1H, m), 3.13 (2H, m), 3.56 (2H, pentet), 3.66 (1H, tt), 3.76 (2H, m), 4.95 (1H, q), 6.74 (1H, br t), 7.87 (1H, d), 8.00 (1H, d), 8.24 (1H, t); RT=3.42 min, m/z (ES+)=463.0 [M+H]+.
Solid mCPBA (140 mg, of 77% purity, 63 μmol) was added in one portion to a stirred solution of 4-{1-[3-(3-fluoro-4-methylsulfanylphenyl)-[1,2,4]oxadiazol-5-yl]ethoxy}piperidine-1-carboxylic acid tert-butyl ester (Example 41, 183 mg, 420 μmol) in CH2Cl2 (3 mL). After 2 h saturated aqueous Na2CO3 (15 mL) was added and the mixture extracted with CH2Cl2 (3×20 mL). The combined extracts were dried (MgSO4), evaporated and the residue purified by flash chromatography (IH-EtOAc 3:2) to afford firstly the sulfone: δH (CDCl3) 1.46 (9H, s), 1.60 (2H, m), 1.68 (3H, d), 1.79 (1H, m), 1.90 (1H, m), 3.13 (2H, m), 3.28 (3H, s), 3.67 (1H, tt), 3.77 (2H, m), 4.95 (1H, q), 8.01 (1H, d), 8.06-8.16 (2H, m); RT=3.90 min, m/z (ES+)=470.0 [M+H]+; then the sulfoxide: δH (CDCl3) 1.46 (9H, s), 1.60 (2H, m), 1.68 (3H, d), 1.79 (1H, m), 1.90 (1H, m), 2.89 (3H, s), 3.13 (2H, m), 3.67 (1H, tt), 3.77 (2H, m), 4.95 (1H, q), 7.88 (1H, dd), 8.01 (1H, t), 8.15 (1H, dd); RT=3.67 min, m/z (ES+)=454.0 [M+H]+.
The biological activity of the compounds of the invention may be tested in the following assay systems:
The yeast cell-based reporter assays have previously been described in the literature (e.g. see Miret J. J. et al, 2002, J. Biol. Chem., 277:6881-6887; Campbell R. M. et al, 1999, Bioorg. Med. Chem. Lett. 9:2413-2418; King K. et al, 1990, Science, 250:121-123: WO 99/14344; WO 00/12704; and U.S. Pat. No. 6,100,042). Briefly, yeast cells have been engineered such that the endogenous yeast G-alpha (GPA1) has been deleted and replaced with G-protein chimeras constructed using multiple techniques. Additionally, the endogenous yeast alpha-cell GPCR, Step 3 has been deleted to allow for a homologous expression of a mammalian GPCR of choice. In the yeast, elements of the pheromone signaling transduction pathway, which are conserved in eukaryotic cells (for example, the mitogen-activated protein kinase pathway), drive the expression of Fus1. By placing β-galactosidase (LacZ) under the control of the Fus1 promoter (Fus1p), a system has been developed whereby receptor activation leads to an enzymatic read-out.
Yeast cells were transformed by an adaptation of the lithium acetate method described by Agatep et al, (Agatep, R. et al, 1998, Transformation of Saccharomyces cerevisiae by the lithium acetate/single-stranded carrier DNA/polyethylene glycol (LiAc/ss-DNA/PEG) protocol. Technical Tips Online, Trends Journals, Elsevier). Briefly, yeast cells were grown overnight on yeast tryptone plates (YT). Carrier single-stranded DNA (10 μg), 2 μg of each of two Fus1p-LacZ reporter plasmids (one with URA selection marker and one with TRP), 2 μg of GPR119 (human or mouse receptor) in yeast expression vector (2 μg origin of replication) and a lithium acetate/polyethylene glycol/TE buffer was pipetted into an Eppendorf tube. The yeast expression plasmid containing the receptor/no receptor control has a LEU marker. Yeast cells were inoculated into this mixture and the reaction proceeds at 30° C. for 60 min. The yeast cells were then heat-shocked at 42° C. for 15 min. The cells were then washed and spread on selection plates. The selection plates are synthetic defined yeast media minus LEU, URA and TRP (SD-LUT). After incubating at 30° C. for 2-3 days, colonies that grow on the selection plates were then tested in the LacZ assay.
In order to perform fluorimetric enzyme assays for β3-galactosidase, yeast cells carrying the human or mouse GPR119 receptor were grown overnight in liquid SD-LUT medium to an unsaturated concentration (i.e. the cells were still dividing and had not yet reached stationary phase). They were diluted in fresh medium to an optimal assay concentration and 90 μL of yeast cells are added to 96-well black polystyrene plates (Costar). Compounds, dissolved in DMSO and diluted in a 10% DMSO solution to 10× concentration, were added to the plates and the plates placed at 30° C. for 4 h. After 4 h, the substrate for the β-galactosidase was added to each well. In these experiments, Fluorescein di(β-D-galactopyranoside) was used (FDG), a substrate for the enzyme that releases fluorescein, allowing a fluorimetric read-out. 20 μL per well of 500 μM FDG/2.5% Triton X100 was added (the detergent was necessary to render the cells permeable). After incubation of the cells with the substrate for 60 min, 20 μL per well of 1M sodium carbonate was added to terminate the reaction and enhance the fluorescent signal. The plates were then read in a fluorimeter at 485/535 nm.
The compounds of the invention give an increase in fluorescent signal of at least ˜1.5-fold that of the background signal (i.e. the signal obtained in the presence of 1% DMSO without compound). Compounds of the invention which give an increase of at least 5-fold may be preferred.
cAMP Assay
A stable cell line expressing recombinant human GPR119 was established and this cell line was used to investigate the effect of compounds of the invention on intracellular levels of cyclic AMP (cAMP). The cell monolayers were washed with phosphate buffered saline and stimulated at 37° C. for 30 min with various concentrations of compound in stimulation buffer plus 1% DMSO. Cells were then lysed and cAMP content determined using the Perkin Elmer AlphaScreen™ (Amplified Luminescent Proximity Homogeneous Assay) cAMP kit. Buffers and assay conditions were as described in the manufacturer's protocol.
Compounds of the invention produced a concentration-dependent increase in intracellular cAMP level and generally had an EC50 of <10 μM. Compounds showing an EC50 of less than 1 μM in the cAMP assay may be preferred.
In Vivo Feeding Study
The effect of compounds of the invention on body weight and food and water intake was examined in freely-feeding male Sprague-Dawley rats maintained on reverse-phase lighting. Test compounds and reference compounds were dosed by appropriate routes of administration (e.g. intraperitoneally or orally) and measurements made over the following 24 h. Rats were individually housed in polypropylene cages with metal grid floors at a temperature of 21±4° C. and 55±20% humidity. Polypropylene trays with cage pads were placed beneath each cage to detect any food spillage. Animals were maintained on a reverse phase light-dark cycle (lights off for 8 h from 09.30-17.30 h) during which time the room was illuminated by red light. Animals had free access to a standard powdered rat diet and tap water during a two week acclimatization period. The diet was contained in glass feeding jars with aluminum lids. Each lid had a 3-4 cm hole in it to allow access to the food. Animals, feeding jars and water bottles were weighed (to the nearest 0.1 g) at the onset of the dark period. The feeding jars and water bottles were subsequently measured 1, 2, 4, 6 and 24 h after animals were dosed with a compound of the invention and any significant differences between the treatment groups at baseline compared to vehicle-treated controls.
Selected compounds of the invention showed a statistically significant hypophagic effect at one or more time points at a dose of ≦100 mg kg1.
HIT-T15 cells (passage 60) were obtained from ATCC, and were cultured in RPMI1640 medium supplemented with 10% fetal calf serum and 30 nM sodium selenite. All experiments were done with cells at less than passage 70, in accordance with the literature, which describes altered properties of this cell line at passage numbers above 81 (Zhang H J, Walseth T F, Robertson R P. Insulin secretion and cAMP metabolism in HIT cells. Reciprocal and serial passage-dependent relationships. Diabetes. 1989 January; 38(1):44-8).
cAMP Assay
HIT-T15 cells were plated in standard culture medium in 96-well plates at 100,000 cells/0.1 mL/well and cultured for 24 h and the medium was then discarded. Cells were incubated for 15 min at room temperature with 100 μL stimulation buffer (Hanks buffered salt solution, 5 mM HEPES, 0.5 mM IBMX, 0.1% BSA, pH 7.4). This was discarded and replaced with compound dilutions over the range 0.001, 0.003, 0.01, 0.03, 0.1, 0.3, 1, 3, 10, 30 μM in stimulation buffer in the presence of 0.5% DMSO. Cells were incubated at room temperature for min. Then 75 μL lysis buffer (5 mM HEPES, 0.3% Tween-20, 0.1% BSA, pH 7.4) was added per well and the plate was shaken at 900 rpm for 20 min. Particulate matter was removed by centrifugation at 3000 rpm for 5 min, then the samples were transferred in duplicate to 384-well plates, and processed following the Perkin Elmer AlphaScreen cAMP assay kit instructions. Briefly 25 μL reactions were set up containing 8 μL sample, 5 μL acceptor bead mix and 12 μL detection mix, such that the concentration of the final reaction components is the same as stated in the kit instructions. Reactions were incubated at room temperature for 150 min, and the plate was read using a Packard Fusion instrument. Measurements for cAMP were compared to a standard curve of known cAMP amounts (0.01, 0.03, 0.1, 0.3, 1, 3, 10, 30, 100, 300, 1000 nM) to convert the readings to absolute cAMP amounts. Data was analysed using XLfit 3 software.
Representative compounds of the invention were found to increase cAMP at an EC50 of less than 10 μM. Compounds showing an EC50 of less than 1 μM in the cAMP assay may be preferred.
HIT-TI 5 cells were plated in standard culture medium in 12-well plates at 106 cells/1 ml/well and cultured for 3 days and the medium was then discarded. Cells were washed ×2 with supplemented Krebs-Ringer buffer (KRB) containing 119 mM NaCl, 4.74 mM KCl, 2.54 mM CaCl2, 1.19 mM MgSO4, 1.19 mM KH2PO4, 25 mM NaHCO3, 10 mM HEPES at pH 7.4 and 0.1% bovine serum albumin. Cells were incubated with 1 ml KRB at 37° C. for 30 min which was then discarded. This was followed by a second incubation with KRB for 30 min, which was collected and used to measure basal insulin secretion levels for each well. Compound dilutions (0, 0.1, 0.3, 1, 3, 10 μM) were then added to duplicate wells in 1 ml KRB, supplemented with 5.6 mM glucose. After 30 min incubation at 37° C. samples were removed for determination of insulin levels. Measurement of insulin was done using the Mercodia Rat insulin ELISA kit, following the manufacturers instructions, with a standard curve of known insulin concentrations. For each well insulin levels were corrected by subtraction of the basal secretion level from the pre-incubation in the absence of glucose. Data was analysed using XLfit 3 software.
Representative compounds of the invention were found to increase insulin secretion at an EC50 of less than 10 μM. Compounds showing an EC50 of less than 1 μM in the insulin secretion assay may be preferred.
The effects of compounds of the invention on oral glucose (Glc) tolerance were evaluated in male C57Bl/6 or male ob/ob mice. Food was withdrawn 5 h before administration of Glc and remained withdrawn throughout the study. Mice had free access to water during the study. A cut was made to the animals' tails, then blood (20 μL) was removed for measurement of basal Glc levels 45 min before administration of the Glc load. Then, the mice were weighed and dosed orally with test compound or vehicle (20% aqueous hydroxypropyl-β-cyclodextrin or 25% aqueous Gelucire 44/14) 30 min before the removal of an additional blood sample (20 μL) and treatment with the Glc load (2-5 g kg−1 p.o.). Blood samples (20 μL) were then taken 25, 50, 80, 120; and 180 min after Glc administration. The 20 μL blood samples for measurement of Glc levels were taken from the cut tip of the tail into disposable micro-pipettes (Dade Diagnostics Inc., Puerto Rico) and the sample added to 480 μL of haemolysis reagent. Duplicate 20 μL aliquots of the diluted haemolysed blood were then added to 180 μL of Trinders glucose reagent (Sigma enzymatic (Trinder) colorimetric method) in a 96-well assay plate. After mixing; the samples were left at room temperature for 30 min before being read against Glc standards (Sigma glucose/urea nitrogen combined standard set). Representative compounds of the invention statistically reduced the Glc excursion at doses ≦100 mg kg−1.
| Number | Date | Country | Kind |
|---|---|---|---|
| 0606913.2 | Apr 2006 | GB | national |
| 0700112.6 | Jan 2007 | GB | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/GB2007/050183 | 4/5/2007 | WO | 00 | 4/3/2009 |
