Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
  • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
  • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
  • A61K31/4439—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. omeprazole
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
  • A61K31/433—Thidiazoles
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
  • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
  • A61P35/02—Antineoplastic agents specific for leukemia
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D285/00—Heterocyclic compounds containing rings having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by groups C07D275/00 - C07D283/00
  • C07D285/01—Five-membered rings
  • C07D285/02—Thiadiazoles; Hydrogenated thiadiazoles
  • C07D285/04—Thiadiazoles; Hydrogenated thiadiazoles not condensed with other rings
  • C07D285/08—1,2,4-Thiadiazoles; Hydrogenated 1,2,4-thiadiazoles
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
  • C07D417/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings
  • C07D417/10—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings linked by a carbon chain containing aromatic rings
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
  • C07D417/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings
  • C07D417/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings linked by a chain containing hetero atoms as chain links

Definitions

• This invention relates to pharmaceutically-useful compounds.
• the invention also relates to the use of such compounds in the treatment of cancer.
• AMP-activated protein kinase represents a new target for the treatment of several diseases, including cancer.
• Excess adiposity is associated to different degrees with an increased risk of developing cancers, such as colorectal adenomas, breast cancer (postmenopausal), endometrial cancer, kidney cancer, oesophageal adenocarcinoma, ovarian cancer, prostate cancer, pancreatic cancer, gallbladder cancer, liver cancer and cervical cancer (CaIIe and Kaaks (2004), Nature Reviews Cancer, 4, 579-591).
• cancers such as colorectal adenomas, breast cancer (postmenopausal), endometrial cancer, kidney cancer, oesophageal adenocarcinoma, ovarian cancer, prostate cancer, pancreatic cancer, gallbladder cancer, liver cancer and cervical cancer.
• cancer cells require high rates of fatty acid and protein synthesis for their invasive growth and survival.
• AMPK activators also suppress lipid synthesis in tumour cells. It has also been shown that it is a link between AMPK and other anti-cancer targets such as LKB1 and caspase-3 activation.
• Cancer cells use glucose at a higher rate compared to normal cells (Warburg O, 1956). Instead of mitochondrial oxidative phosphorylation to produce ATP, cancer cells metabolise glucose via hydrolysis.
• Elevated plasma free fatty acids stimulate pancreatic ⁇ -cells and is one cause of hyperinsulinemia.
• hyperinsulinemia has been shown to be prospective risk factor for death and data support that the insulin level could be used as a marker of prostate cancer prognosis (Hammarsten and H ⁇ gstedt (2005) European Journal of Cancer, 41 ,
• hyperinsulinemia Several mechanisms may link hyperinsulinemia to the incidence and outcome of breast cancer. Firstly, chronic hyperinsulinemia results in increased production of ovarian testosterone and oestrogen and inhibition of hepatic production of sex hormone binding globulin, a sex-hormonal profile that is associated with breast cancer. Secondly, hyperinsulinemia suppresses hepatic production of insulin-like growth factor binding protein-1 (IGFBP-1), and thus increases circulating levels of IGF-1 , which has potent mitogenic effect on breast tissue. Thirdly, insulin itself may have a direct mitogenic effect on breast cancer cells.
• IGFBP-1 insulin-like growth factor binding protein-1
• Neoplastic cells synthesise lipids to a much larger extent than their normal counterparts and metabolise glucose differently. It has been suggested that this aberrant metabolism constitutes a therapeutic target.
• pathways/targets include glycolysis interfering agents, lipid synthesis pathway, AMPK activating agents and agents affecting mitochondrial function.
• AMP-activated protein kinase is a protein kinase enzyme that consists of three protein sub-units and is activated by hormones, cytokines, exercise, and stresses that diminish cellular energy state (e.g. glucose deprivation). Activation of AMPK increases processes that generate adenosine 5'-triphosphate (ATP) (e.g., fatty-acid oxidation) and restrains others such as fatty acid-, glycerolipid- and protein-synthesis that consume ATP, but are not acutely necessary for survival. Conversely, when cells are presented with a sustained excess of glucose, AMPK activity diminishes and fatty acid-, glycerolipid- and protein-synthesis are enhanced.
• ATP adenosine 5'-triphosphate
• AMPK activity diminishes and fatty acid-, glycerolipid- and protein-synthesis are enhanced.
• AMPK thus is a protein kinase enzyme that plays an important role in cellular energy homeostasis. Therefore, the activation of AMPK is coupled to glucose lowering effects and triggers several other biological effects, including the inhibition of cholesterol synthesis, lipogenesis, triglyceride synthesis, and the reduction of hyperinsulinemia.
• AMPK is a preferred target for the treatment of the metabolic syndrome and especially type 2 diabetes.
• AMPK is also involved in a number of pathways that are important for many different diseases (e.g. AMPK is also involved in a number of pathways that are important in CNS disorders, fibrosis, osteoporosis, heart failure and sexual dysfunction).
• AMPK is also involved in a number of pathways that are important in cancer.
• tumour suppressors are part of the AMP pathway.
• AMPK acts as a negative regulator of the mammalian TOR (mTOR) and EF2 pathway, which are key regulators of cell growth and proliferation. The deregulation may therefore be linked to diseases such as cancer (as well as diabetes).
• AMPK activators may therefore be of utility as anti-cancer drugs.
• AMPK activators e.g. metformin, glitazones
• compounds that are AMPK activators, and preferably direct activators of AMPK may find utility as anti-cancer drugs, as well as for the treatment of many other diseases.
• Kaugars et al., J. Org. Chem. 1979, 44(22), 3840-3843 describe 5-phenyl- and 5-methyl- substituted phenyl urea derivatives of 1 ,2,4, thiadiazol-3-one that are substituted in the 2- position with a methyl group. There is no mention of any biological effects associated with the disclosed compounds.
• US patent 4,093,624 describes 1 ,2,4 thiadiazolidin-3-one compounds, described as having antimicrobial activity, which are substituted by a -NH 2 or -NHAc in the 5-position and H or ribofuranosyls in the 2-position. There is no disclosure of 1,2,4-thiadiazol-3- ones in which the 2-position and 5-position contain substituents bearing an aromatic ring.
• Martinez et al. Bioorg. Med. Chem. 1997, 7, 1275-1283 describes arylimino-1 ,2,4- thiadiazolidinone derivatives as potassium channel openers that are potentially useful for the treatment of diseases involving smooth muscle contraction (e.g. hypertension).
• smooth muscle contraction e.g. hypertension
• thiadiazolidinones substituted at the 2-position with an aromatic group there is no disclosure of such thiadiazolidinones substituted at the 2-position with an aromatic group.
• US patent application publication number 2003/0195238 describes thiadiazolidine derivates as GSK-3 ⁇ inhibitors that are potentially useful for the treatment of Alzheimer's disease.
• this document mainly relates to thiadiazolidines substituted by two carbonyl/thiocarbonyl groups (thereby forming e.g. a 3,5-dioxo-thiadiazolidine or a 3- thioxo-5-oxo-thiadiazolidine). Further, it mainly relates to compounds in which both nitrogen atoms of the thiadiazolidine are substituted.
• the document does not relate to thiadiazolidines substituted at the 2-position with a group bearing an aromatic group and at the 5-position with an amino or amido derivative bearing an aromatic group.
• B represents S or C(-NH-W-D);
• A represents S and B represents C(-NH-W-D) then the bond between B and the NH atom is a double bond;
• X represents -Q-[CR x R y ] n -;
• W represents -[CR x R y ] m - or -C(O)-[CR X RV;
• Q represents a bond, -N(R a )-, -S-, or -O-;
• a 1 to A 5 respectively represent C(R 1 ), C(R 2 ), C(R 3 ), C(R 4 ) and C(R 5 ), or, alternatively, up to two Of A 1 to A 5 may independently represent N;
• D represents phenyl, pyridyl or pyrimidinyl optionally substituted by one or more R 6 groups;
• R x and R y are independently selected from H 1 halo, C 1 ⁇ alkyl (optionally substituted by one or more halo atoms), aryl (optionally substituted by one or more halo atoms) or R x and R y are linked to form, along with the carbon atom to which they are attached, a non-aromatic 3- to 8-membered ring, optionally containing 1 to 3 heteroatoms selected from O 1 S and N, which ring is itself optionally substituted by one or more substituents selected from halo or C 1-6 alkyl (optionally substituted by one or more halo atoms);
• R 1 to R 5 independently represent H, halo, -R 7 , -CF 3 , -CN, -NO 2 , -C(O)R 7 , -C(O)OR 7 , -C(O)-N(R 7a )R 7b , -N(R 7a )R 7b , -N(R 7 J 3 + , -SR 7 , -OR 7 , -NH(O)R 7 , -SO 3 R 7 , aryl or heteroaryl (which aryl and heteroaryl groups are themselves optionally and independently substituted by one or more groups selected from halo and R 16 ), or any two of R 1 to R 5 which are adjacent to each other are optionally linked to form, along with two atoms of the essential benzene ring in the compound of formula I, an aromatic or non-aromatic 3- to 8-membered ring, optionally containing 1 to 3 heteroatoms selected from O, S and N, which ring is itself
• R 6 independently represents, on each occasion when used herein, cyano, -NO 2 , halo, -R 8 , -OR 8 , -N(R 8 )C(O)R 8 , -NR 9 R 10 , -SR 11 , -Si(R 12 ) 3) -OC(O)R 13 , -C(O)OR 13 , -C(O)R 14 , -C(O)NR 153 R 15 ", -S(O) 2 NR 15c R 15d , aryl or heteroaryl (which aryl and heteroaryl groups are themselves optionally and independently substituted by one or more groups selected from halo and R 16 ), or any two R 6 groups which are adjacent to each other are optionally linked to form, along with two atoms of the essential benzene ring in the compound of formula I, an aromatic or non-aromatic 3- to 8-membered ring, optionally containing 1 to 3 heteroatoms selected from O, S and
• R 7 on each occasion when used herein, is selected from H or C 1 -C 6 alkyl, C 1 -C 6 cycloalkyl, aryl and heteroaryl (wherein the latter four groups are optionally substituted by one or more halo atoms);
• R 16 represents, on each occasion when used herein, C 1-6 alkyl optionally substituted by one or more halo atoms;
• n 0 or, more preferably, 1 or 2;
• n 2 or, more preferably, 1 or 0;
• p 2 or, more preferably, 1 or 0;
• salts that may be mentioned include acid addition salts and base addition salts.
• Such salts may be formed by conventional means, for example by reaction of a free acid or a free base form of a compound of formula I with one or more equivalents of an appropriate acid or base, optionally in a solvent, or in a medium in which the salt is insoluble, followed by removal of said solvent, or said medium, using standard techniques (e.g. in vacuo, by freeze-drying or by filtration). Salts may also be prepared by exchanging a counter-ion of a compound of formula I in the form of a salt with another counter-ion, for example using a suitable ion exchange resin.
• Examples of pharmaceutically acceptable addition salts include those derived from mineral acids, such as hydrochloric, hydrobromic, phosphoric, metaphosphoric, nitric and sulphuric acids; from organic acids, such as tartaric, acetic, citric, malic, lactic, fumaric, benzoic, glycolic, gluconic, succinic, arylsulphonic acids; and from metals such as sodium, magnesium, or preferably, potassium and calcium.
• mineral acids such as hydrochloric, hydrobromic, phosphoric, metaphosphoric, nitric and sulphuric acids
• organic acids such as tartaric, acetic, citric, malic, lactic, fumaric, benzoic, glycolic, gluconic, succinic, arylsulphonic acids
• metals such as sodium, magnesium, or preferably, potassium and calcium.
• “Pharmaceutically functional derivatives” of compounds of formula I as defined herein includes ester derivatives and/or derivatives that have, or provide for, the same biological function and/or activity as any relevant compound. Thus, for the purposes of this invention, the term also includes prodrugs of compounds of formula I.
• prodrug of a relevant compound of formula I includes any compound that, following oral or parenteral administration, is metabolised in vivo to form that compound in an experimentally-detectable amount, and within a predetermined time (e.g. within a dosing interval of between 6 and 24 hours (i.e. once to four times daily)).
• parenteral administration includes all forms of administration other than oral administration.
• Prodrugs of compounds of formula I may be prepared by modifying functional groups present on the compound in such a way that the modifications are cleaved, in vivo when such prodrug is administered to a mammalian subject. The modifications typically are achieved by synthesizing the parent compound with a prodrug substituent.
• Prodrugs include compounds of formula I wherein a hydroxyl, amino, sulfhydryl, carboxy or carbonyl group in a compound of formula I is bonded to any group that may be cleaved in vivo to regenerate the free hydroxyl, amino, sulfhydryl, carboxy or carbonyl group, respectively.
• prodrugs include, but are not limited to, esters and carbamates of hydroxy functional groups, esters groups of carboxyl functional groups, N-acyl derivatives and N- Mannich bases. General information on prodrugs may be found e.g. in Bundegaard, H. "Design of Prodrugs” p. I-92, Elsevier, New York-Oxford (1985).
• Compounds of formula I may contain double bonds and may thus exist as E (entadel) and Z (zusammen) geometric isomers about each individual double bond. All such isomers and mixtures thereof are included within the scope of the invention.
• Compounds of formula I contain one or more asymmetric carbon atoms and may therefore exhibit optical and/or diastereoisomerism.
• Diastereoisomers may be separated using conventional techniques, e.g. chromatography or fractional crystallisation. The various stereoisomers may be isolated by separation of a racemic or other mixture of the compounds using conventional, e.g. fractional crystallisation or HPLC, techniques.
• the desired optical isomers may be made by reaction of the appropriate optically active starting materials under conditions which will not cause racemisation or epimerisation (i.e. a 'chiral pool 1 method), by reaction of the appropriate starting material with a 'chiral auxiliary' which can subsequently be removed at a suitable stage, by derivatisation (i.e.
• a resolution for example with a homochiral acid followed by separation of the diastereomeric derivatives by conventional means such as chromatography, or by reaction with an appropriate chiral reagent or chiral catalyst all under conditions known to the skilled person. All stereoisomers and mixtures thereof are included within the scope of the invention.
• alkyl refers to an unbranched or branched, cyclic, saturated or unsaturated (so forming, for example, an alkenyl or alkynyl) hydrocarbyl radical, which may be substituted or unsubstituted (with, for example, one or more halo atoms).
• alkyl refers to an acyclic group, it is preferably C 1-10 alkyl and, more preferably, C 1-6 alkyl (such as ethyl, propyl, (e.g. n-propyl or isopropyl), butyl (e.g. branched or unbranched butyl), pentyl or, more preferably, methyl).
• alkyl is a cyclic group (which may be where the group “cycloalkyl” is specified), it is preferably C 3 - I2 cycloalkyl and, more preferably, C 5-10 (e.g. C 5 . 7 ) cycloalkyl.
• alkylene refers to C 1-10 (e.g. C 1-5 ) alkylene and, preferably C 1-3 alkylene, such as pentylene, butylene (branched or unbranched), preferably, propylene (/7-propylene or isopropylene), ethylene or, more preferably, methylene (i.e. -CH 2 -).
• halogen when used herein, includes fluorine, chlorine, bromine and iodine.
• aryl when used herein includes C 6 . 14 (such as C 6-13 (e.g. C 6-10 )) aryl groups. Such groups may be monocyclic, bicyclic or tricyclic and have between 6 and 14 ring carbon atoms, in which at least one ring is aromatic. The point of attachment of aryl groups may be via any atom of the ring system. However, when aryl groups are bicyclic or tricyclic, they are linked to the rest of the molecule via an aromatic ring.
• C 6-14 aryl groups include phenyl, naphthyl and the like, such as 1 ,2,3,4-tetrahydronaphthyl, indanyl, indenyl and fluorenyl. Most preferred aryl groups include phenyl.
• heteroaryl when used herein refers to an aromatic group containing one or more heteroatom(s) (e.g. one to four heteroatoms) preferably selected from N 1 O and S (so forming, for example, a mono-, bi-, or tricyclic heteroaromatic group).
• Heteroaryl groups include those which have between 5 and 14 (e.g. 10) members and may be monocyclic, bicyclic or tricyclic, provided that at least one of the rings is aromatic. However, when heteroaryl groups are bicyclic or tricyclic, they are linked to the rest of the molecule via an aromatic ring.
• Heterocyclic groups that may be mentioned include benzothiadiazolyl (including 2,1 ,3-benzothiadiazolyl), isothiochromanyl and, more preferably, acridinyl, benzimidazolyl, benzodioxanyl, benzodioxepinyl, benzodioxolyl (including 1 ,3-benzodioxolyl), benzofuranyl, benzofurazanyl, benzothiazolyl, benzoxadiazolyl (including 2,1,3-benzoxadiazolyl), benzoxazinyl (including 3,4-dihydro- 2H-1 ,4-benzoxazinyl), benzoxazolyl, benzomorpholinyl, benzoselenadiazolyl (including 2,1,3-benzoselenadiazolyl), benzothienyl, carbazolyl, chromanyl, cinnolinyl, furanyl, imid
• heteroaryl groups may, where appropriate, be located on any atom in the ring system including a heteroatom.
• the point of attachment of heteroaryl groups may be via any atom in the ring system including (where appropriate) a heteroatom (such as a nitrogen atom), or an atom on any fused carbocyclic ring that may be present as part of the ring system.
• Heteroaryl groups may also be in the N- or S- oxidised form.
• heteroaryl groups include pyridyl, pyrrolyl, quinolinyl, furanyl, thienyl, oxadiazolyl, thiadiazolyl, thiazolyl, oxazolyl, pyrazolyl, triazolyl, tetrazolyl, isoxazolyl, isothiazolyl, imidazolyl, pyrimidinyl, indolyl, pyrazinyl, indazolyl, pyrimidinyl, thiophenetyl, thiophenyl, pyranyl, carbazolyl, acridinyl, quinolinyl, benzoimidazolyl, benzthiazolyl, purinyl, cinnolinyl and pterdinyl.
• Particularly preferred heteroaryl groups include monocylic heteroaryl groups.
• R 15c and R 15d independently represent R 16 then those R 16 groups may be the same or different.
• provisos (a) to (c) above will become redundant (for example, where it is stated that at least one (or both) of the A 1 to As- containing ring and/or ring D carries a substituent other than hydrogen, then all of the provisos (a) to (c) above are redundant).
• B represents C(-NH-W-D) and W represents -[CR x R y ] m -
• Preferred compounds of formula I include those in which at least one of A 1 to A 5 is not (C-H) and D is substituted by one or more -R 6 groups. It is preferred that either the Ai to A 5 -containing ring or ring D is substituted with a substituent other than H.
• one of A 1 to A 5 represents N or, preferably, one of Ai to A 5 represents C(R 1 ), C(R 2 ),
• R 1 , R 2 , R 3 , R 4 or R 5 represents a substituent other than H (i.e. at least one of R 1 to R 5 substituent is present that represents halo, -R 7 , -CF 3 , -CN, -NO 2 , -C(O)R 7 , -C(O)OR 7 , -C(O)-N(R 7a )R 7b ,
• ring D represents pyridyl or pyrimidinyl or, preferably, ring D is substituted by one or more R 6 groups.
• More preferred compounds of formula I include those in which: one of Ai to A 5 represents N or, preferably, one of Ai to A 5 represents C(R 1 ), C(R 2 ), C(R 3 ), C(R 4 ) or C(R 5 ) (as appropriate) in which at least one of R 1 , R 2 R 3 , R 4 or R 5 represents a substituent other than H (e.g. a substituent as defined herein); and ring D represents pyridyl or pyrimidyl or, preferably, ring D is phenyl substituted by one or more R 6 groups.
• Preferred compounds of formula I that may be mentioned include those in which:
• A represents S.
• More preferred compounds of formula I that may be mentioned include those in which: B represents S.
• each -[CR x R y ]- unit may be independently selected from:
• R x and R y are independently selected from H, halo, Ci -6 alkyl (optionally substituted by one or more halo atoms);
• each -[CR x R y ]- unit may be independently selected from:
• R x and R y are independently selected from H, halo, Ci_ 3 alkyl (optionally substituted by one or more halo atoms) (e.g. at least one of R* and R y is H);
• R x and R y are linked to form, along with the carbon atom to which they are attached, a non-aromatic ring selected from cyclobutyl, cyclopentyl, cyclohexyl or, more particularly, cyclopropyl, which ring is itself optionally substituted by one or more substituents selected from halo or C 1-6 alkyl (optionally substituted by one or more halo atoms),
• R x and R y are linked to form, along with the carbon atom to which they are attached, a non-aromatic 3- to 8-membered ring, optionally containing 1 to 3 heteroatoms selected from O, S and N, which ring is itself optionally substituted by one or more substituents selected from halo and/or C 1-6 alkyl (optionally substituted by one or more halo atoms).
• a non-aromatic 3- to 8-membered ring optionally containing 1 to 3 heteroatoms selected from O, S and N, which ring is itself optionally substituted by one or more substituents selected from halo and/or C 1-6 alkyl (optionally substituted by one or more halo atoms).
• R x and R y are linked to form, along with the carbon atom to which they are attached, a cyclobutyl, cyclopentyl, cyclohexyl or, more preferably, cyclopropyl ring which ring is itself optionally substituted by one or more substituents selected from halo and/or C 1-6 alkyl (optionally substituted by one or more halo atoms, or more preferably unsubstituted).
• one -[CR x R y ] ⁇ unit forms a non-aromatic 3- to 8-membered ring, optionally containing 1 to 3 heteroatoms selected from O, S and N, which ring is itself optionally substituted by one or more substituents selected from halo or C 1-6 alkyl (optionally substituted by one or more halo atoms) and, if other -[CR x R y ]- units are present, then the additional R x and R y groups are independently selected from H, halo, C 1-6 alkyl (optionally substituted by one or more halo atoms) or aryl (optionally substituted by one or more halo atoms).
• one -[CR x R y ]- unit is linked to form a cyclobutyl, cyclopentyl, cyclohexyl or, more preferably, cyclopropyl ring which ring is itself optionally substituted by one or more substituents selected from halo or C 1-6 alkyl (optionally substituted by one or more halo atoms, or more preferably unsubstituted) and, if other -[CR x R y ]- units are present, then the additional R x and R y groups are independently selected from phenyl (optionally substituted by one or more halo atoms) or, more preferably, H, halo, C 1-6 alkyl (optionally substituted by one or more halo atoms).
• R x and R y are independently selected from phenyl (optionally substituted by one or more halo atoms) or, more preferably, H, halo, C 1-6 alkyl (optionally substituted by one or more halo atoms).
• R x and R y are independently selected from phenyl (optionally substituted by one or more halo atoms) or, more preferably, H, halo, C 1-6 alkyl (optionally substituted by one or more halo atoms).
• Q represents -S-, preferably, -N(CH 3 )-, -O- or, more preferably, a bond.
• the -[CR x R y ] n - moiety preferably represents -CR x R y - (e.g. -CH 2 -, -C(-CH 2 CH 2 -)-, i.e. -C(cyclopropyl)-, or -C(H)(aryl)-) or -[CR x R y ] 2 -
• R x and R y independently represent C 1-6 (e.g. C 1-3 ) alkyl or, preferably, hydrogen or aryl (e.g. phenyl; optionally substituted by one or more halo, e.g. chloro, atoms, so forming e.g. a chlorophenyl group), or, R x and R y are linked together to form a non-aromatic carbocyclic 3- to 6-membered spirocycle (preferably cyclopropyl), which ring is preferably unsubstituted; both R x and R y , when attached to the same carbon atom, preferably do not represent optionally substituted aryl;
• R x and R y independently represent Ci -6 (e.g. C 1-3 ) alkyl or, preferably, hydrogen;
• n 1 or 2.
• X may represent include -CH 2 -, -CH 2 CH 2 -, -0-CH 2 CH 2 -, -N(CHs)-CH 2 CH 2 -, -S-CH 2 CH 2 -, 1 ,1-cyclopropyl and -C(H)(4-chlorophenyl)- (i.e. it is preferred that X is not a direct bond, but represents a group containing at least one linking atom).
• n and p independently represent 0 or 1;
• R x and R y independently represent Ci -6 alkyl or, preferably hydrogen;
• W represents a direct bond (i.e. m represents 0), -CH 2 -, -C(O)- (i.e. p represents 0) or -C(O)CH 2 -.
• the Ai to A 5 -containing ring may be pyridyl (e.g. 2-pyridyl, 3-pyridyl, 4-pyridyl, 5-pyridyl or 6-pyridyl), but is preferably phenyl.
• the D ring is preferably pyridyl (e.g. 2-pyridyl, 3- pyridyl, 4-pyridyl, 5-pyridyl or 6-pyridyl) or, more preferably, phenyl.
• Each ring may be unsubstituted or substituted with one or two substituents defined herein (by R 1 to R 6 , as appropriate). As stated herein, in a preferred embodiment of the invention, one of these rings is substituted with at least one substituent other than H as defined herein (by R 1 to R 6 , as appropriate).
• a 1 to A 5 respectively represent C(R 1 ), C(R 2 ), C(R 3 ), C(R 4 ) and C(R 5 );
• D represents phenyl, pyridyl or pyrimidinyl optionally substituted by one or more R 6 groups;
• R x and R y are independently selected from fluoro, preferably, H, C 1-6 alkyl (optionally substituted by one or more fluoro atoms), aryl (optionally substituted by one or more halo, e.g. chloro atoms) or R x and R y are linked to form, along with the carbon atom to which they are attached, a non-aromatic 3- to 8- membered ring (e.g. 3- to 6-membered ring), which ring is itself optionally substituted by one or more substituents selected from fluoro and/or C 1-6 (e.g.
• C L3 such as C 1-2 ) alkyl (optionally substituted by one or more fluoro atoms), but which 3- to 8- (e.g. 3- to 6-) membered ring is preferably unsubstituted (e.g. unsubstituted cyclopropyl);
• R 1 to R 5 independently represent -CN, -N(R 7a )R 7b , -N(R 7 ) 3 + . -SR 7 or, preferably, H, halo (e.g. chloro or fluoro), -R 7 , -CF 3 , -C(O)-N(R 7a )R 7b , -OR 7 or heteroaryl (e.g. a 5- or 6- membered heteroaryl group preferably containing one to three heteroatoms (preferably nitrogen heteroatoms), and which heteroaryl group is optionally substituted by one or more groups selected from R 16 and, preferably halo, e.g. chloro);
• R 6 independently represents, on each occasion when used herein, -NO 2 , -NR 9 R 10 , -SR 11 , or, preferably, cyano, halo, -R 8 or -OR 8 ;
• R 7 on each occasion when used herein, is selected from H and Ci -6 (e.g. C 1-3 ) alkyl (e.g. methyl) optionally substituted by one or more fluoro atoms (so forming for example a -CHF 2 or, preferably a -CF 3 group);
• Ci -6 e.g. C 1-3 alkyl (e.g. methyl) optionally substituted by one or more fluoro atoms (so forming for example a -CHF 2 or, preferably a -CF 3 group);
• Further compounds of formula I that may be mentioned include those in which: at least one of R 1 to R 5 , when present, represents heteroaryl, -OR 7
• R 1 to R 5 which are adjacent to each other are optionally linked to form, along with two atoms of the essential benzene ring in the compound of formula I, an aromatic or non-aromatic 3- to 8-membered ring selected from 2,3-dihydrobenzo[1 ,4]dioxinyl or tetrahydroquinolinyl, which may optionally be substituted by one or more halo atoms.
• R 1 to R 5 when present, represents heteroaryl, -OR 7 , halo, -CF 3 , -CN, -C(O)R 7 , -C(O)OR 7 , -C(O)-N(R 7a )R 7b , -N(R 7 ) 3 + or -NH(O)R 7 .
• R 1 to R 5 when present, represents 4H-[i,2,4]-triazolyl, -OR 7 (e.g. -OCH 3 , or more preferably, -OCHF 2 or -OCF 3 ), or, more preferably, -Cl, -F, -CF 3 , -CN or -C(O)- N(R 7a )R 7b .
• -OR 7 e.g. -OCH 3 , or more preferably, -OCHF 2 or -OCF 3
• -Cl, -F, -CF 3 e.g., -CF 3
• -CN e.g. -C(O)- N(R 7a )R 7b
• R 1 to R 5 when present, represents -OR 7 or, more preferably, -Cl, -F,
• R 6 independently represents -C(O)NR 15a R 15b or, more preferably, cyano, -NO 2 , -Br, -Cl, -F, -R 8 , -OR 8 , -NR 9 R 10 , -SR 11 , -C(O)OR 13 , -C(O)R 14 , -S(O) 2 NR 15c R 15d , aryl or heteroaryl (which aryl and heteroaryl groups are themselves optionally and independently substituted by one or more groups selected from halo and R 16 ), or any two R 6 groups which are adjacent to each other are optionally linked to form, along with two atoms of the essential benzene ring in the compound of formula I, quinoline, tetrahydroquinoline, isoquinoline or tetrahydroisoquinoline, wherein the additional ring system of the quinoline, tetrahydroquinoline, isoquinoline or tetrahydroisoquino
• R 6 independently represents -C(O)NR 15a R 15b or, more preferably, -R 8 or yet more preferably, cyano, -NO 2 , -Br, -Cl, -F, -OR 8 , -NR 9 R 10 , -SR 11 , -C(O)OR 13 , -C(O)R 14 , -S(O) 2 NR 15c R 15d , aryl or heteroaryl (which aryl and heteroaryl groups are themselves optionally and independently substituted by one or more groups selected from halo and
• R 6 independently represents -C(O)NR 15a R 15b , -R 8 , or, more preferably, -CN, -NO 2 , -Br, -Cl, -F, -OR 8 , -NR 9 R 10 , -SR 11 , -C(O)OR 13 or -C(O)R 14 .
• R 6 independently represents -R 8 or, more preferably, -CN, -OCF 3 , -NO 2 , -Br, -Cl, -F, -OR 8 , -NR 9 R 10 or -SR 11 .
• R 6 independently represents -CN, -CF 3 , -OCF 3 , -F or, most preferably -Cl.
• Compounds of formula I that may be mentioned include those in which:
• n 2 or, more preferably, 1.
• n 1 or, more preferably, O
• p 1 or, more preferably, O.
• a 5 represents N or, more preferably, C(CI) or C(H).
• Further compounds of formula I that may be mentioned include those in which:
• a 1 and A 3 independently represent N or, more preferably, C(H).
• a 2 represents C(R 2 );
• a 1 and A 3 to A 5 independently represent C(H) or N.
• a 2 represents C(R 2 );
• R 2 represents -CF 3 ;
• a 1 and A 3 to A 5 independently represent C(H).
• Further compounds of formula I that may be mentioned include those in which:
• a 5 represents C(R 5 );
• a 1 to A 4 independently represent C(H) or N.
• Yet further compounds of formula I that may be mentioned include those in which:
• a 5 represents C(R 5 );
• R 5 represents -Cl
• a 1 to A 4 independently represent C(H).
• Compounds of formula I that may be mentioned include those in which:
• D represents (ort/7o-,para-)dichlorophenyl.
• D represents para-chlorophenyl
• More preferred compounds of formula I include those of the examples described hereinafter.
• Preferred compounds of formula I include:
• Ai to A 5 , X, W and D are as hereinbefore defined, under reaction conditions known to those skilled in the art, for example in the presence of a suitable bromine source (e.g. N-bromo succinimide or bromine) and a suitable solvent (e.g. methanol, ethanol, ethyl acetate) and at a suitable temperature (e.g. -10 * C to 80"C) as described in Castro et al. (Bioorganic. Med. Chem. 2008, 16, 495-510) or Kaugars et al. (J. Org. Chem. 1979, 44(22), 3840-3843), or in the presence of a suitable base (e.g.
• a suitable bromine source e.g. N-bromo succinimide or bromine
• a suitable solvent e.g. methanol, ethanol, ethyl acetate
• a suitable temperature e.g. -10 * C to 80"C
• a suitable solvent e.g. water containing hydrogen peroxide (e.g. a 30% H 2 O 2 solution in water)
• a suitable temperature e.g. -10 ' C to 100 ° C
• a 1 to A 5 and X are as hereinbefore defined, with a compound of formula IV, L 2 -W 1 -D IV wherein L 2 represents a suitable leaving group such as halo (e.g. chloro), W 1 represents
• a suitable base e.g. NaH, NaOH, triethylamine, pyridine, another suitable base mentioned at process step or mixtures thereof
• solvent e.g. pyridine (which may serve as the base and solvent), DMF or dichloromethane (e.g. further in the presence of water and, optionally, a phase transfer catalyst)
• room temperature e.g. as described in Hurst, D. T.; Stacey, A. D., Nethercleft, M., Rahim, A., Harnden, M. R. Aust. J. Chem. 1998, 41, 1221;
• L 3 is a suitable leaving group (e.g. halo) and D is as hereinbefore defined, under reaction conditions known to those skilled in the art, for example in the presence of a suitable base (e.g. a tributyltin amine or cyclohexylamine and lithium bis(trimethylsilyl)amide), a suitable catalyst (e.g. PdCI 2 (P(o-toluyl) 3 ) 2 ), a suitable solvent (e.g. toluene) and at a suitable temperature (e.g. from room temperature to 105°C), e.g. as described in Harwig et al. J. Am. Chem. Soc.
• a suitable base e.g. a tributyltin amine or cyclohexylamine and lithium bis(trimethylsilyl)amide
• a suitable catalyst e.g. PdCI 2 (P(o-toluyl) 3 ) 2
• a suitable solvent e
• L 4 is a suitable leaving group (e.g. halo) or -OH 1 W 2 represents -C(O)-[CR x R y ] p -, and D is as hereinbefore defined, when L 4 represents a suitable leaving group, under reaction conditions known to those skilled in the art, for example in an appropriate solvent (e.g. toluene, xylenes, DCM, chloroform), optionally in the presence of an base (e.g. pyridine, Hunig's base, triethylamine) and at reduced to elevated temperatures (e.g.
• an appropriate solvent e.g. toluene, xylenes, DCM, chloroform
• an base e.g. pyridine, Hunig's base, triethylamine
• elevated temperatures e.g.
• L 4 represents OH 1 under standard coupling reaction conditions, for example, in the presence of a suitable coupling reagent (e.g. 1,1'- carbonyldiimidazole, ⁇ /./V-dicyclohexylcarbodiimide, 1-(3-dimethylamino-propyl)-3- ethylcarbodiimide (or hydrochloride thereof), ⁇ /. ⁇ /'-disuccinimidyl carbonate, benzotriazol- 1-yloxytris(dimethylamino)-phosphonium hexafluorophosphate, 2-(1H-benzotriazol-1-yl)-
• a suitable coupling reagent e.g. 1,1'- carbonyldiimidazole, ⁇ /./V-dicyclohexylcarbodiimide, 1-(3-dimethylamino-propyl)-3- ethylcarbodiimide (or hydrochloride thereof
• L 5 is a suitable leaving group (e.g. bromo, chloro, iodo) and either Q is a bond and n is 0, 1 or 2, or Q is -O- or -S- and n is 1 or 2, under reaction conditions known to those skilled in the art, for example in the presence of a suitable base (e.g. NaH, NaOH, triethylamine, pyridine) and solvent (e.g. pyridine (which may serve as the base and solvent) DMF or dichloromethane (e.g. further in the presence of water and, optionally, a phase transfer catalyst)), for example at room temperature e.g. as described in Hurst, D. T.; Stacey, A. D., Nethercleft, M.,
• a suitable base e.g. NaH, NaOH, triethylamine, pyridine
• solvent e.g. pyridine (which may serve as the base and solvent)
• DMF or dichloromethane e.
• L 6 represents a suitable leaving group (e.g. halo) and A 1 to A 5 and X are as hereinbefore defined, with a compound of formula X,
• a suitable solvent e.g. chloroform, methylenechloride
• a suitable base e.g. Hunig's base, triethyl amine
• a suitable temperature e.g. room temperature to 15O 0 C, such as less than
• W and D 1 to D 5 are as hereinbefore defined, under reaction conditions known to those skilled in the art, for example in a suitable solvent (e.g. acetone, dimethylformamide or 20% dimethylformamide in acetonitrile) at a suitable temperature (e.g. from -10 ' C to 50 ° C) and in the absence of a base, e.g. as described in Castro et al. ⁇ Bioorganic. Med. Chem. 2008, 16, 495-510) or Kaugars et al. (J. Org. Chem. 1979, 44(22), 3840-3843).
• a compound of formula I may be formed directly by allowing any product so formed to be directly treated under reaction conditions such as those described hereinbefore (e.g. process step (i) above).
• Compounds of formula lib may be prepared by reaction of a compound of formula Xl as hereinbefore described, with a compound of formula XII under reaction conditions known to those skilled in the art, for example in a suitable solvent (e.g. acetone, dimethylformamide) at a suitable temperature (e.g. from -10'C to 50°C) and in the presence of a suitable base (e.g. n-butyl lithium), e.g. as described in Castro et al. (Bioorganic. Med. Chem. 2008, 16, 495-510) or Kaugars et al. ⁇ J. Org. Chem. 1979, 44(22), 3840-3843).
• a compound of formula I may be formed directly by allowing any product so formed to be directly treated under reaction conditions such as those described hereinbefore (e.g. process step (vii) above).
• compounds of formula lib may be formed by the selective N-alkylation of N-(3- oxo-1 ,2,4-thiadiazolidin-5-ylidene) amide derivates as described by Castro et al (Bioorganic. Med. Chem. 2008, 16, 495-510).
• Compounds of formula IX may be prepared by reaction of a compound of formula III with
• halogen source e.g. hydrochloric acid
• reaction conditions known to those skilled in the art, for example such as those described in Foroumadi et al. (1999) Arzneim. Forsch. 49, 1035-1038 or Foroumadi et al. (2005) Arch. Pharm. Chem. Life Sci. 338, 112-116, for example in the presence of a suitable metal (e.g. copper powder).
• a suitable metal e.g. copper powder
• acyl halide e.g. acyl chloride
• Di to D 5 are as hereinbefore defined, with a thiocyanate (e.g. an alkali metal thiocyanate, such as potassium thiocyanate), under reaction conditions known to those skilled in the art, for example in the presence of a suitable solvent (such as acetone), as described in Cho et al, J. Heterocyclic Chem. 1991, 28, 1645-1649).
• a suitable solvent such as acetone
• Substituents such as R 2 , R 3 and R 4 in final compounds of formula I (or precursors thereto and other relevant intermediates) may be modified one or more times, after or during the processes described above by way of methods that are well known to those skilled in the art. Examples of such methods include substitutions, reductions (e.g. carbonyl bond reductions in the presence of suitable and, if necessary, chemoselective, reducing agents such as LiBH 4 or NaBH 4 ), oxidations, alkylations, acylations, hydrolyses, esterifications, and etherifications.
• the precursor groups can be changed to a different such group, or to the groups defined in formula I, at any time during the reaction sequence.
• the protection and deprotection of functional groups may take place before or after a reaction in the above-mentioned schemes.
• Protecting groups may be removed in accordance with techniques that are well known to those skilled in the art and as described hereinafter. For example, protected compounds/intermediates described herein may be converted chemically to unprotected compounds using standard deprotection techniques.
• the term "functional groups” means, in the case of unprotected functional groups, hydroxy-, thiolo-, aminofunction, carboxylic acid and, in the case of protected functional groups, lower alkoxy, N-, O-, S- acetyl, carboxylic acid ester.
• compounds of formula I may be AMPK agonists, i.e. they may activate AMPK.
• AMPK' we mean that the steady state level of phosphorylation of the Thr-172 moiety of the AMPK- ⁇ subunit is increased compared to the steady state level of phosphorylation in the absence of the agonist.
• ACC acetyl-CoA carboxylase
• the compounds of formula I may be AMPK activators, they may therefore be useful in the treatment of diseases such as those described herein, especially cancer.
• Compounds of formula I may reduce the rate of cell proliferation when tested in an assay using a human breast cancer cell line (e.g. MDA-MB-231). The compounds may thus possess a beneficial inhibitory effect on the ability of tumors of this type, and of cancers generally, to survive. Compounds of formula I may also reduce the rate of cell proliferation when tested in other cancer cells lines (e.g.
• any p53 mutant or p53 null cell line such as, but not limited to, MCF-7, PC-3, Jurkat, SK-OV-3, HL60, MV4-11 , HT-29, K562, MDA-MB-231 , HCT116wt, A-549, DU-145, LOVO, HCT-116 and PANC-1 , independent of p53 status.
• Compounds of formula I are therefore indicated for the inhibition of cell proliferation. Compounds of formula I are therefore indicated for use in the treatment of cancer.
• a compound of formula I or a pharmaceutically-acceptable salt or solvate, or a pharmaceutically functional derivative thereof for the manufacture of a medicament for the treatment of cancer.
• the compounds of formula I may be useful in the treatment of both primary and metastatic cancers.
• cancer will be understood by those skilled in the art to include one or more diseases in the class of disorders that is characterized by uncontrolled division of cells and the ability of these cells to invade other tissues, either by direct growth into adjacent tissue through invasion, proliferation or by implantation into distant sites by metastasis.
• compounds of formula I are capable of inhibiting the proliferation of cancer cells.
• proliferation we include an increase in the number and/or size of cancer cells.
• compounds of formula I are capable of inhibiting metastasis of cancer cells.
• metastasis we mean the movement or migration (e.g. invasiveness) of cancer cells from a primary tumor site in the body of a subject to one or more other areas within the subject's body (where the cells can then form secondary tumors).
• the invention provides compounds and methods for inhibiting, in whole or in part, the formation of secondary tumors in a subject with cancer. It will be appreciated by skilled persons that the effect of a compound of formula I on "metastasis" is distinct from any effect such a compound may or may not have on cancer cell proliferation.
• compounds of formula I may be capable of inhibiting the proliferation and/or metastasis of cancer cells selectively.
• the combination product inhibits the proliferation and/or metastasis of cancer cells to a greater extent than it modulates the function (e.g. proliferation) of non-cancer cells.
• the compound inhibits the proliferation and/or metastasis of cancer cells only.
• cancer cells may be selected from the group consisting of cancer cells of the breast, bile duct, brain, colon, stomach, reproductive organs, thyroid, hematopoietic system, lung and airways, skin, gallbladder, liver, nasopharynx, nerve cells, kidney, prostate, lymph glands and gastrointestinal tract.
• the cancer is selected from the group of colon cancer (including colorectal adenomas), breast cancer (e.g.
• the cancer is selected from the group of colon, prostate and, particularly, breast cancer.
• the cancer is a non-solid tumor, it is preferably a hematopoietic tumor such as a leukemia (e.g. Acute Myelogenous Leukemia (AML), Chronic Myelogenous Leukemia (CML), Acute Lymphocytic Leukemia (ALL), Chronic Lymphocytic Leukemia (CLL).
• AML Acute Myelogenous Leukemia
• CML Chronic Myelogenous Leukemia
• ALL Chronic Lymphocytic Leukemia
• CLL Chronic Lymphocytic Leukemia
• the cancer cells are breast cancer cells.
• a method of treatment of cancer which method comprises the administration of an effective amount of a compound of formula I, or a pharmaceutically-acceptable salt or solvate, or a pharmaceutically functional derivative thereof, to a patient in need of such treatment.
• Compounds of formula I may also be of use in the treatment of a disorder or condition ameliorated by the activation of AMPK.
• Compounds of formula I may be suitable for use in the treatment of side-effects caused by cancer (e.g. cachexia).
• a compound of formula I or a pharmaceutically-acceptable salt or solvate, or a pharmaceutically functional derivative thereof, for the manufacture of a medicament for the treatment of a disorder or condition ameliorated by the activation of AMPK.
• disorder or condition ameliorated by the activation of AMPK will be understood by those skilled in the art to include, in addition to cancer, diabetes, hyperinsulinemia and associated conditions, a condition/disorder where fibrosis plays a role, sexual dysfunction, osteoporosis and neurodegenerative diseases.
• Compounds of formula I may thus also be indicated for use in the treatment of a disorder or a condition caused by, linked to, or contributed to by, hyperinsulinemia.
• disorder or condition caused by, linked to, or contributed to by, hyperinsulinemia or “treatment of hyperinsulinemia or an associated condition” will be understood by those skilled in the art to include hyperinsulinemia and associated conditions, such as type 2 diabetes, glucose intolerance, insulin resistance, metabolic syndrome, dyslipidemia, hyperinsulinism in childhood, hypercholesterolemia, high blood pressure, obesity, fatty liver conditions, diabetic nephropathy, diabetic neuropathy, diabetic retinopathy, cardiovascular disease, atherosclerosis, cerebrovascular conditions such as stroke, systemic lupus erythematosus, neurodegenerative diseases such as Alzheimer's disease, and polycystic ovary syndrome.
• Other disease states include progressive renal disease such as chronic renal failure.
• Preferred disorders include hyperinsulinemia and, particularly, type 2 diabetes.
• Certain compounds of formula I may also have the additional advantage that they exhibit partial agonist activity and may therefore be useful in conditions, such as late type 2 diabetes, in which stimulation of the production of insulin is required.
• agonist activity we include direct and indirect-acting agonists.
• a method of treatment of a disorder or condition ameliorated by the activation of AMPK comprises the administration of an effective amount of a compound of formula I, or a pharmaceutically-acceptable salt or solvate, or a pharmaceutically functional derivative thereof, to a patient in need of such treatment.
• An effective amount can be determined by a physician and will be determined for a specific patient by assessment of the patient's clinical parameters including, but not limited to the stage of disease, age, gender and histology.
• Compounds of formula I may thus also be of use in the treatment of a condition/disorder where fibrosis plays a role.
• Compounds of formula I may also be useful in the treatment of sexual dysfunction (e.g. the treatment of erectile dysfunction).
• a condition/disorder where fibrosis plays a role includes (but is not limited to) scar healing, keloids, scleroderma, pulmonary fibrosis (including idiopathic pulmonary fibrosis), nephrogenic systemic fibrosis, and cardiovascular fibrosis (including endomyocardial fibrosis), systemic sclerosis, liver cirrhosis, eye macular degeneration, retinal and vitreal retinopathy, Crohn's/inflammatory bowel disease, post surgical scar tissue formation, radiation and chemotherapeutic-drug induced fibrosis, and cardiovascular fibrosis.
• Compounds of formula I may thus also be of use in the treatment of osteoporosis.
• Compounds of formula I may thus also be of use in the treatment of inflammation.
• Compounds of formula I may thus also be of use in the treatment of sexual dysfunction.
• Compounds of formula I may thus also be of use in the treatment of heart failure.
• Compounds of formula I may thus also be of use in the treatment of neurodegenerative diseases (e.g. Alzheimer ' s disease, Parkinson ' s disease and Huntington ' s disease, amyotrophic lateral sclerosis, polyglutamine disorders, such as spinal and bulbar muscular atrophy (SBMA), dentatorubral and pallidoluysian atrophy (DRPLA), and a number of spinocerebellar ataxias (SCA)).
• neurodegenerative diseases e.g. Alzheimer ' s disease, Parkinson ' s disease and Huntington ' s disease, amyotrophic lateral sclerosis, polyglutamine disorders, such as spinal and bulbar muscular atrophy (SBMA), dentatorubral and pallidoluysian atrophy (DRPLA), and a number of spinocerebellar ataxias (SCA)
• SBMA spinal and bulbar muscular atrophy
• DRPLA dentatorubral and pallidoluy
• the term "effective amount” refers to an amount of a compound, which confers a therapeutic effect on the treated patient (e.g. sufficient to treat or prevent the disease).
• the effect may be objective (i.e. measurable by some test or marker) or subjective (i.e. the subject gives an indication of or feels an effect).
• compounds of formula I may be administered alone, but are preferably administered orally, intravenously, intramuscularly, cutaneously, subcutaneously, transmucosally (e.g. sublingually or buccally), rectally, transdermal ⁇ , nasally, pulmonarily (e.g. tracheally or bronchially), topically, by any other parenteral route, in the form of a pharmaceutical preparation comprising the compound in a pharmaceutically acceptable dosage form.
• Preferred modes of delivery include oral, intravenous, cutaneous or subcutaneous, nasal, intramuscular, or intraperitoneal delivery.
• Compounds of formula I will generally be administered as a pharmaceutical formulation in admixture with a pharmaceutically acceptable adjuvant, diluent or carrier, which may be selected with due regard to the intended route of administration and standard pharmaceutical practice.
• a pharmaceutically acceptable adjuvant diluent or carrier
• Such pharmaceutically acceptable carriers may be chemically inert to the active compounds and may have no detrimental side effects or toxicity under the conditions of use.
• Suitable pharmaceutical formulations may be found in, for example, Remington The Science and Practice of Pharmacy, 19th ed., Mack Printing Company, Easton, Pennsylvania (1995).
• a parenterally acceptable aqueous solution may be employed, which is pyrogen free and has requisite pH, isotonicity, and stability. Suitable solutions will be well known to the skilled person, with numerous methods being described in the literature. A brief review of methods of drug delivery may also be found in e.g. Langer, Science 249, 1527 (1990).
• Another aspect of the present invention includes a pharmaceutical composition
• a pharmaceutical composition comprising a therapeutically effective amount of a compound of formula I, or a pharmaceutically-acceptable salt or solvate, or a pharmaceutically functional derivative thereof, in combination with a pharmaceutically acceptable excipient, such as an adjuvant, diluent or carrier.
• the amount of compound of formula I in the formulation will depend on the severity of the condition, and on the patient, to be treated, as well as the compound(s) which is/are employed, but may be determined non-inventively by the skilled person.
• compounds of formula I may be administered at varying therapeutically effective doses to a patient in need thereof.
• the dose administered to a mammal, particularly a human, in the context of the present invention should be sufficient to effect a therapeutic response in the mammal over a reasonable timeframe.
• the selection of the exact dose and composition and the most appropriate delivery regimen will also be influenced by inter alia the pharmacological properties of the formulation, the nature and severity of the condition being treated, and the physical condition and mental acuity of the recipient, as well as the potency of the specific compound, the age, condition, body weight, sex and response of the patient to be treated, and the stage/severity of the disease.
• Administration may be continuous or intermittent (e.g. by bolus injection).
• the dosage may also be determined by the timing and frequency of administration.
• the dosage can vary from about 0.01 mg to about 1000 mg per day of a compound of formula I (or, if employed, a corresponding amount of a pharmaceutically acceptable salt or prodrug thereof).
• the medical practitioner or other skilled person, will be able to determine routinely the actual dosage, which will be most suitable for an individual patient.
• the above-mentioned dosages are exemplary of the average case; there can, of course, be individual instances where higher or lower dosage ranges are merited, and such are within the scope of this invention.
• the compounds of formula I may be used or administered in combination with one or more additional drugs useful in the treatment of cancer, in combination therapy.
• a combination product comprising:
• each of components (A) and (B) is formulated in admixture with a pharmaceutically-acceptable adjuvant, diluent or carrier.
• cancer therapies such as cytostatica, irradiation and photodynamic therapy, among others known to the physician.
• the other therapeutic agent is a cytostatic (such as a taxane (e.g. docetaxel and, particularly, paclitaxel) or preferably, a platin (e.g. cisplatin and carboplatin) or an anthracycline (e.g. doxorubicin)) or an angiogenesis inhibitor, or a pharmaceutically-acceptable salt, solvate or pharmaceutically functional derivative of either of these.
• the other therapeutic agent may also be selected from: (i) tamoxifen, or a pharmaceutically-acceptable salt, solvate or pharmaceutically functional derivative thereof;
• an aromatase inhibitor i.e. a compound that blocks the production of estrogen from adrenal androgens via the aromatase pathway in peripheral tissues
• a pharmaceutically-acceptable salt, solvate or pharmaceutically functional derivative thereof i.e. anastrozole, letrozole and exemastane
• trastuzumab Herceptin
• another antibody that is useful in the treatment of cancer such as bevacizumab, cetuximab or panitumumab;
• a tyrosine kinase inhibitor i.e. a compound that blocks (or is capable of blocking), to a measurable degree, the autophosphorylation of tyrosine residues, thereby preventing activation of the intracellular signalling pathways in tumor cells
• a pharmaceutically-acceptable salt, solvate or pharmaceutically functional derivative thereof include inhibitors of the vascular endothelial growth factor (VEGF) family, and/or the HER-family of TKs 1 such as HER- 1/Human Epidermal Growth Factor (EGFR; erbB1), HER3 (erbB3), HER4 (erbB4) and, more particularly, HER2 (erbB2).
• VEGF vascular endothelial growth factor
• HER-family of TKs 1 such as HER- 1/Human Epidermal Growth Factor (EGFR; erbB1), HER3 (erbB3), HER4 (erbB4) and, more particularly, HER2 (erbB
• Preferred TKIs thus include imatinib, gefitinib, erlotinib, canertinib, sunitinib, zactima, vatalanib, sorafenib, leflunomide and, particularly, lapatinib;
• a glitazone such as troglitazone, pioglitazone and rosiglitazone, or a pharmaceutically-acceptable salt, solvate or pharmaceutically functional derivative thereof;
• a biguanide such as phenformin, buformin, or, most preferably, metformin, or a pharmaceutically-acceptable salt, solvate or pharmaceutically functional derivative thereof;
• statin such as fluvastatin, simvastatin, rosuvastatin, pravastatin, atorvastatin and, particularly, lovastatin, or a pharmaceutically-acceptable salt, solvate or pharmaceutically functional derivative thereof;
• rapamycin an inhibitor of activity of the mammalian target of rapamycin (mTOR), such as rapamycin, or a pharmaceutically-acceptable salt, solvate or pharmaceutically functional derivative thereof;
• an oligomycin such as oligomycin A, oligomycin B, oligomycin C, oligomycin D (rutamycin A), oligomycin E 1 oligomycin F, rutamycin B, 44-homooligomycin A and 44-homooligomycin B, or a pharmaceutically-acceptable salt, solvate or pharmaceutically functional derivative thereof;
• AICAR aminoimidazole carboxamide ribonucleotide
• PPAR peroxisome proliferator-activated receptor
• AM251 (a CB 1 receptor antagonist), or a pharmaceutically-acceptable salt, solvate or pharmaceutically functional derivative thereof;
• a SIRT1 activator such as resveratrol and SRT-1720 (N-[2-[3-(piperazin-1- ylmethyl)imidazo[2,1-b][1 ,3]thiazol-6-yl]phenyl]quinoxal-ine-2-carboxamide), or a pharmaceutically-acceptable salt, solvate or pharmaceutically functional derivative thereof; and/or
• agonist we include direct and indirect-acting agonists. It has recently been suggested in the literature (see, for example, MoI. Cancer Then, 5, 430 (2006), Cancer Res., 66, 10269 (2006) and Int. J. Cancer, 118, 773 (2006)) that the above mentioned compound classes (v) to (vii) may be used in the treatment of cancer, as described herein.
• the other therapeutic agent is (particularly) in category (i) or (ii) above, combination products according to embodiments of the invention are particularly useful in the treatment of ER-positive cancers and/or early-stage breast cancers, for example in adjuvant therapy (i.e.
• combination products according to embodiments of the invention are also particularly useful in the treatment of patients at a high risk of breast cancer.
• the other therapeutic agent is (particularly) in category (iii) or (iv) above, combination products according to embodiments of the invention are particularly useful in the treatment of HER2-positive cancers.
• compositions solvates or pharmaceutically functional derivatives of any of the compounds listed in categories (i), (ii) and (iv) to (xvi) above are as described hereinbefore.
• preferred pharmaceutically-acceptable salts include those of citric acid
• preferred pharmaceutically-acceptable salts include mesylate salts
• preferred pharmaceutically-acceptable salts include maleate salts.
• Combination products as described herein provide for the administration of compound of formula I in conjunction with the other therapeutic agent, and may thus be presented either as separate formulations, wherein at least one of those formulations comprises compound of formula I, and at least one comprises the other therapeutic agent, or may be presented (i.e. formulated) as a combined preparation (i.e. presented as a single formulation including compound of formula I and the other therapeutic agent).
• compositions including a compound of formula I; another therapeutic agent useful in the treatment of cancer; and a pharmaceutically-acceptable adjuvant, diluent or carrier; and
• kits of parts comprising components:
• components (a) and (b) are each provided in a form that is suitable for administration in conjunction with the other.
• Components (a) and (b) of the kits of parts described herein may be administered simultaneously or sequentially.
• a method of making a kit of parts as defined above comprises bringing component (a), as defined above, into association with a component (b), as defined above, thus rendering the two components suitable for administration in conjunction with each other.
• components (a) and (b) of the kit of parts may be:
• kit of parts comprising:
• kits of parts described herein may comprise more than one formulation including an appropriate quantity/dose of compound of formula I, and/or more than one formulation including an appropriate quantity/dose of the other therapeutic agent, in order to provide for repeat dosing. If more than one formulation (comprising either active compound) is present, such formulations may be the same, or may be different in terms of the dose of either compound, chemical composition(s) and/or physical form(s).
• kits of parts as described herein by “administration in conjunction with”, we include that respective formulations comprising compound of formula I and the other therapeutic agent are administered, sequentially, separately and/or simultaneously, over the course of treatment of the relevant condition.
• the term "administration in conjunction with” includes that the two components of the combination product (compound of formula I and the other therapeutic agent) are administered (optionally repeatedly), either together, or sufficiently closely in time, to enable a beneficial effect for the patient, that is greater, over the course of the treatment of the relevant condition, than if either a formulation comprising compound of formula I, or a formulation comprising the other therapeutic agent, are administered (optionally repeatedly) alone, in the absence of the other component, over the same course of treatment. Determination of whether a combination provides a greater beneficial effect in respect of, and over the course of treatment of, a particular condition will depend upon the condition to be treated or prevented, but may be achieved routinely by the skilled person.
• the term "in conjunction with” includes that one or other of the two formulations may be administered (optionally repeatedly) prior to, after, and/or at the same time as, administration with the other component.
• the terms “administered simultaneously” and “administered at the same time as” include that individual doses of compound of formula I and the other therapeutic agent are administered within 48 hours (e.g. 24 hours) of each other.
• the compounds/combinations/methods/uses described herein may have the advantage that, in the treatment of the conditions described herein, they may be more convenient for the physician and/or patient than, be more efficacious than, be less toxic than, have better selectivity, have a broader range of activity than, be more potent than, produce fewer side effects than, or may have other useful pharmacological properties over, similar compounds, combinations, methods (treatments) or uses known in the prior art for use in the treatment of those conditions or otherwise, for example over the compounds disclosed in international patent applications WO 2007/010273 and WO
• Figure 1 shows the effect of the compound of Example 1 or the compound of
• Example 2c on AMPK phosphorylation. After starvation of PC3 cells in serum-free medium for 24 h, 0.3, 0.6, 1.2 and 2.5 ⁇ M of the compound of Example 1 or the compound of Example 2c was added and incubated for an additional 4 h.
• the Figure provides representative immunoblots of AMPK phosphorylation by the compound of Example 1 or the compound of Example 2c.
• the compound of Example 1 and the compound of Example 2c stimulates AMPK phosphorylation in PC3 cells.
• Figure 2 which shows the effect of the compound of selected compounds of the examples on AMPK phosphorylation in comparison to total AMPK levels, and on acetyl co-enzyme A (acetyl-CoA) carboxylase phosphorylation (a substrate of AMPK).
• acetyl-CoA acetyl-CoA carboxylase phosphorylation
• PC3 cells and induce the phosphorylation of acetyl-CoA carboxylase.
• FIG 3 which shows that the compound of Example 1 and 2c can reduce PP2C- ⁇ 1 mediated dephosphorylation of AMPK.
• the compounds were tested in the assay at concentrations of 2.5, 5 and 10 ⁇ M (as depicted in the figure).
• the relevant intermediate is commercially available (e.g. from Chemical Diversity, San Diego, CA 1 USA or other available commercial sources).
• LC-MS was performed on a Sciex API 150 LC/ES-MS equipped with an ACE 3 C8 column (30 x 3.0 mm) using a flow of 1 mL/min. Two gradient systems of acetonitrile in water (with 0.1% TFA) are used for elution: A) 5-100% under 10 min, then 2 min 100% isocratic or B) 90-100% under 2 min, then 2 min 100% isocratic.
• Direct inlet ES-MS was also performed on a Bruker Esquire LC/ES-MS. 1 H nuclear magnetic resonance was recorded on a Bruker Avance DRX 400 spectrometer at 400.01 MHz using residual solvent as internal standard.
• the material was purified by preparative HPLC (basic conditions) XTerra Prep MS C18 5mm 19x50mm column, flow 25ml/min, 5OmM pH10 NH4HCO3 / ACN, 5-97%ACN in 6 min, fractions collected based on UV-signal (254nm) to give the product in 21.7 mg (52%).
• Example 8 The following compounds, were (a to d, f, k, I 1 o to r and w), or may be prepared using the procedures described in Example 1 with the exceptions that in step (i):
• Test A Descriptions of the cancer cell lines including source, tumor type, and morphology may be obtained from the American Type Culture Collection (ATCC) or its website (www.atcc.org). The cell lines are both from primary tumors and metastatic sites (for example, MCF-7, MDA-MB231 , HT-29, SKOV-3 and PC-3 among others tested). Test A
• D-MEM Dulbecco's modified Eagle's medium
• Glucose GlutaMAXTM1 + Pyruvate
• V/V Foetal Bovine Serum (Gibco 10500-064)
• PC-3 cancer cell lines were propagated in D-MEM (Gibco 21885) supplemented with 10% Foetal calf serum. 15000 cells per well were seeded in 96 well plates and incubated overnight. The culture media was changed to serum-free D-MEM for 24 h. The culture media was then changed to serum free D-MEM containing either 0.2 % DMSO as vehicle control or 0.3, 0.6, 1.2 or 2.5 ⁇ M (or 1.25, 2.5, 5, 10 ⁇ M (as indicated in Table 1 below)) of the selected compounds of Example 1 to Example 8 in 0.2% DMSO in quadruplicate. After 18 h incubation, BrdU was added according to manufacturer's recommendations. After 6 h incubation in the presence of BrdU, the culture media was removed and BrdU incorporation was measured using "Cell Proliferation ELISA, BrdU colorimetric" Roche (11647229001) according to manufacturer's recommendations.
• Proliferation rate of PC-3 cells are reduced by relevant concentrations of the test compounds as measured by BrdU incorporation.
• the selected compounds of Example 1 to Example 8 relative to the vehicle control (which display a BrdU incorporation of 1 unit) displayed the following (approximate) units of BrdU incorporations at the indicated concentrations in Table 1 below.
• mice 5 week old Athymic BALB/cA nude mice are delivered from Taconic (Denmark) and kept under barrier conditions for 1 week acclimatisation. At 6 weeks, 17 mice are injected subcutaneously on the flank with 1.8 x 10 6 MDA-MB-231 human breast cancer cells (LGC Promochem-ATCC) in a 50/50 v/v solution of phosphate buffered saline (PBS) (Gibco 10010-015, Invitrogen) Matrigel HC (BD Biosciences).
• PBS phosphate buffered saline
• mice After 11 days, palpable tumors are observed in 16 mice. 2 mice are sacrificed and the tumors dissected and examined. 2 groups of 7 mice each are treated once daily by intraperitoneal injections of 1-10 mg/kg bodyweight of test compound in 79% PBS/20% Solutol HS 15(BASF)/1% DMSO or vehicle control respectively for 5-30 days. The mice are sacrificed by cervical dislocation and tumors are dissected.
• the tumor tissue is fixated overnight in PBS (containing 4% w/v paraformaldehyde (Scharlau PA0095, Sharlau Chemie SA 1 Spain) at +4 0 C.
• the tumor tissue is cryopreserved by 24 hour incubation in PBS containing 30% w/v sucrose (BDH #102745C (www.vwr.com) at +4°C and is embedded in Tissue-Tek embedding media (Sakura Finetek Europa BV, Netherlands). 10 ⁇ m cryosections are generated and stained with Mayers Hematoxylin (Dako) for 5 minutes and destained for 3 x 10 minutes in tap water.
• mice are injected subcutaneously.
• Test B as described above provides one of many potential in vivo xenograft models. Modifications to Test B above may be made (e.g. by changing some or all of: the formulation of the test compounds; the cell line; and the type of mice used).
• Selected compounds of Examples 1 , 4 to 6 and 8 were prepared.
• a stock solution of 10 ⁇ M was prepared by dissolving the compound in 100% DMSO.
• PC3 cells were purchased from LGC Promochem-ATCC (ATCC catalog no CRL- 1435). PC3 cells were maintained in Dulbecco's modified Eagle's medium (Gibco 21885) containing 5% fetal bovine serum (Gibco 10500-064), 25 ⁇ g/ml Gentamicin (Gibco 20 15750) and 1x non essential amino acids (Gibco 11140). The cells were incubated in a humidified atmosphere of 5% CO2 at 37°C and passaged every 3 days by trypsinization.
• Dulbecco's modified Eagle's medium Gibco 21885
• fetal bovine serum Gibco 10500-064
• 25 ⁇ g/ml Gentamicin Gibco 20 15750
• 1x non essential amino acids Gibco 11140
• PC3 cells were cultured in complete medium with 10% fetal bovine serum in 60-mm-diameter dishes, grown to 70-80% confluence and cultured in serumfree Dulbecco's modified Eagle's medium for 5 h. Cells were then treated with 10 ⁇ M of the compound of Example 1 for 24 h. The final concentration of DMSO did not exceed 0.1%, which did not affect AMPK or eEF2 phosphorylation (0.1% DMSO was used as control).
• PC3 cells were lysed in buffer (10OmM TRIS pH 6,8, 2%w/v Sodium dodecylsulfate
• Filters were blocked in 2OmM TRIS pH 7.5, 137mM NaCI, 25%v/v Tween20 and 5%w/v fat free powdered milk for 30 min. Filters were incubated overnight in blocking solution with phospho-AMPK (ThM 72) or phospho-acetyl CoA carboxylase (Cell signalling #2531 and #3661) or with a pan-AMPK antibody (Cell signalling #2532).
• Filters were washed in 2OmM TRIS pH 7.5, 137mM NaCI, 25%v/v Tween20 for 3x5min. Filters were incubated in blocking solution with secondary antibody, peroxidase conjugated Goat anti-rabbit IgG (Jackson immunoResearch #111-035-003) at room temperature for 1h. Filters were washed as above for 3x10 min. Signal was developed with SuperSignal West Dura ECL kit (Pierce #1859024) and exposed to Hyperfilm ECL Amersham #28906837).
• Cells are seeded and grown in the presence of varying concentrations of test compound(s) for a period of 3 days (72 hours). The cells are then fixed to the plate and exposed to the dye sulphorhodamine B (SRB). The varying amounts of inhibition of proliferation produces a standard curve from which the IC 50 value is determined.
• SRB dye sulphorhodamine B
• Section A Seeding the cells into the plate
• Assay uses a sterile 96 well plate cell culture plate (Microtest flat bottom tissue culture plate, Falcon 3072).
• Assay uses a sterile 96 well plate cell culture plate (Microtest flat bottom tissue culture plate, Falcon 3072).
• Section B Adding Test Compound(s) to cells 7. Prepare compound plate for test compound(s) and transfer diluted compound to prepared assay plate in section A.
• test articles Dilute the test articles to 250 ⁇ M in the cell culture medium in a separate tube, which will make start concentration of 50 ⁇ M.
• Stock concentration of test compound(s) is 1OmM, therefore dilute 1:40 to obtain 250 ⁇ M concentration.
• Blank control Pipette 100 ⁇ l_ blank medium to row 1 and 12, 25 ⁇ L form this will be added to assay plate containing cells.
• Section C Fixing and staining the cells
• Adherent cell lines Fix cells to the plate by carefully adding 30 ⁇ L of cold 50% v/v Trichloroacetic acid (TCA BDH 102863H) to the cell culture medium already in the wells so the final concentration of TCA is 10% v/v.
• TCA BDH 102863H 50% v/v Trichloroacetic acid
• Suspension cell lines Fix cells to the plate by carefully adding 30 ⁇ L of cold 80% v/v Trichloroacetic acid (TCA BDH 102863H) to the cell culture medium already in the wells so the final concentration of TCA is 16% v/v.
• TCA BDH 102863H Trichloroacetic acid
• test compound(s) Dilute the test compound(s) to 120 ⁇ M in the cell culture medium in a separate tube, which will make start concentration of 40 ⁇ M.
• Stock concentration of test compound(s) is 1 OmM, therefore dilute 1 :83.3 to obtain 120 ⁇ M concentration.
• DMSO controls same as drug dilute 100% DMSO 1 :83.3 in medium. Pipette 200 ⁇ L to Column 2 on compound plate by quadruplicate., which will give end concentration of 0.4%. 6. Blank control: Pipette 200 ⁇ L blank medium per well to Column 1 by quadruplicate.
• each colony must have 50 cells.
• Hyperphosphosporylated AMPK trimer 10 (ng) ( ⁇ 1/ ⁇ 1/ ⁇ 1) from Invitrogen (PV4672) was incubated in a buffer containing: 4OmM Hepes pH 7.45, 2mM MnCI 2 , 0.5mM DTT and 0.125 ng recombinant PP2C- ⁇ 1 (Abeam Ab51205) in the presence or absence of potential inhibitors of the interaction/enzymatic reaction in at total volume of 10 ⁇ L for 20 minutes in 30 0 C.
• the reaction was terminated by addition of 40 ⁇ L stop solution containing 1 % Bovine Serum Albumin (BSA), 1OmM EDTA and anti Phospho-AMPK ⁇ (ThM 72) Antibody(cell signaling #2531) at a dilution of 1/1000.
• BSA Bovine Serum Albumin
• ThM 72 anti Phospho-AMPK ⁇
• the samples were transferred to glutathione coated 96 well plated (Pierce #15140) and incubated O/N at +4°C.
• the plate was washed 3X200 ⁇ l_ in PBS/0,05% Triton X-100.
• the plate was incubated with PBS/1 %BSA and horse radish peroxidase conjugated goat-anti rabbit antibody (Jackson immunoresearch Laboratories Inc.
• Example 1 and Example 2c have the ability to reduce PP2C- ⁇ 1 mediated dephosphorylation of AMPK.

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