Patent Application
20080214638
The present invention relates to therapeutically active compounds which are anthranilic acid derivatives, processes for the manufacture of said derivatives, pharmaceutical formulations containing the active compounds and the use of the compounds in therapy, particularly in the treatment of diseases in which under-activation of the HM74A receptor contributes to the disease or in which activation of the receptor will be beneficial.
Dyslipidaemia is a general term used to describe individuals with aberrant lipoprotein profiles. Clinically, the main classes of compounds used for the treatment of patients with dyslipidaemia, and therefore at risk of cardiovascular disease are the statins, fibrates, bile-acid binding resins and nicotinic acid. Nicotinic acid (Niacin, a B vitamin) has been used clinically for over 40 years in patients with various forms of dyslipidaemia. The primary mode of action of nicotinic acid is via inhibition of hormone-sensitive triglyceride lipase (HSL), which results in a lowering of plasma non-esterified fatty acids (NEFA) which in turn alters hepatic fat metabolism to reduce the output of LDL and VLDL (low and very low density lipoprotein). Reduced VLDL levels are thought to lower cholesterol ester transfer protein (CETP) activity to result in increased HDL (high density lipoprotein) levels which may be the cause of the observed cardiovascular benefits. Thus, nicotinic acid produces a very desirable alteration in lipoprotein profiles; reducing levels of VLDL and LDL whilst increasing HDL. Nicotinic acid has also been demonstrated to have disease modifying benefits, reducing the progression and increasing the regression of atherosclerotic lesions and reducing the number of cardiovascular events in several trials.
The observed inhibition of HSL by nicotinic acid treatment is mediated by a decrease in cellular cyclic adenosine monophosphate (cAMP) caused by the G-protein-mediated inhibition of adenylyl cyclase. Recently, the G-protein coupled receptors HM74 and HM74A have been identified as receptors for nicotinic acid (PCT patent application WO02/84298; Wise et. al. J Biol Chem. 2003 278 (11) 9869-9874). The DNA sequence of human HM74A may be found in Genbank; accession number AY148884. Two other papers support this discovery, (Tunaru et. al. Nature Medicine 2003 (3) 352-255 and Soga et. al. Biochem Biophys Res Commun. 2003 303 (1) 364-369), however the nomenclature differs slightly. In the Tunaru paper what they term human HM74 is in fact HM74A and in the Soga paper HM74b is identical to HM74A. Cells transfected to express HM74A and/or HM74 gain the ability to elicit Gi G-protein mediated responses following exposure to nicotinic acid. In mice lacking the homologue of HM74A (m-PUMA-G) nicotinic acid fails to reduce plasma NEFA levels.
We now present a group of anthranilic acid derivatives which are selective agonists of the nicotinic acid receptor HM74A and are thus of benefit in the treatment, prophylaxis and suppression of diseases where under-activation of this receptor either contributes to the disease or where activation of the receptor will be beneficial.
The present invention provides therapeutically active anthranilic acid derivatives and the use of these derivatives in therapy, particularly in the treatment of diseases in which under-activation of the HM74A receptor contributes to the disease or in which activation of the receptor will be beneficial, in particular diseases of lipid metabolism including dislipidaemia or hyperlipoproteinaemia such as diabetic dyslipidaemia and mixed dyslipidaemia, heart failure, hypercholesterolaemia, cardiovascular disease including atherosclerosis, arteriosclerosis, and hypertriglyceridaemia. As such, the compounds may also find favour as therapeutics for coronary artery disease, thrombosis, angina, chronic renal failure, peripheral vascular disease and stroke, as well as the cardiovascular indications associated with type II diabetes mellitus, type I diabetes, insulin resistance, hyperlipidaemia, anorexia nervosa, obesity. The compounds may also be of use in the treatment of inflammatory diseases or conditions, as set out further below.
Intermediates, formulations, methods and processes described herein form further aspects of the invention.
The present invention provides a compound of Formula (I)
and salts, solvates and physiologically functional derivatives thereof, wherein:
R represents (CH2)qCO2H or a 5 or 6-member aryl, heteroaryl, heterocyclic or alicyclic ring;
R1 represents H, halogen, C1-5alkyl, C1-C5alkoxy, C1-5haloalkyl, or C1-5haloakloxy;
R2 represents a 5 or 6-member aryl, heteroaryl, heterocyclic or alicyclic ring;
Z represents —(CH2)q—; —CH═CH—; —(CH2)pNHC(O)—; —(CH2)pNHC(O)NH—; —(CH2)pNHC(O)O—; —(CH2)pSO2NR3—; —(CH2)pNR3SO2—; —(CH2)nO—; —C(R4R5)O— or —Y—W—X—;
W represents a 5 or 6-member aryl, heteroaryl, heterocyclic or alicyclic ring;
X and Y, which may independently be present or absent, where present independently represent —(CH2)q—; —CH═CH—; —(CH2)pNHC(O)—; —(CH2)pNHC(O)O—; —(CH2)pNHC(O)NH—; —(CH2)pSO2NR3—; —(CH2)pNR3SO2—; —(CH2)pC(O)—; —(CH2)pNH—; —(CH2)pO—; —(CH2)pS— or —(CH2)pO—CH2—;
n represents an integer selected from 2, 3 and 4;
p represents an integer selected from 0, 1 and 2;
q represents an integer selected from 1, 2, 3 and 4;
R3 represents hydrogen or methyl; and
R4 and R5, which may be the same or different, independently represent C1-C3alkyl.
Also provided is a compound of Formula (Ia)
and salts, solvates and physiologically functional derivatives thereof, wherein:
R represents CO2H;
R1 represents C1-C5alkoxy, C1-5haloalkyl, or C1-5haloakloxy;
Z and R2 are as set out above.
In compounds of the present invention, the R2 ring system may be joined to the Z linker unit via either a ring carbon atom or via a ring heteroatom, where present. Similarly, where R represents a ring system, this may be joined to the phenyl moiety via either a ring carbon atom or via a ring heteroatom, where present.
In certain compounds of Formula (I) in which R is heteroaryl, R may be pyridinyl, pyrimidinyl, pyridazinyl, pyrazinyl, pyrazolyl, imidazolyl, tetrazolyl, oxazolyl and isoxazolyl, for example tetrazolyl. Where R represents (CH2)qCO2H, in certain embodiments q represents 1 or 2, for example 1. In certain embodiments in which R is heterocyclic, R is selected from pyrrolidinyl, imidazolidinyl, piperidinyl and morpholinyl.
In further compounds of Formula (I), R is selected from cyclohexyl, phenyl, pyridinyl, pyrimidinyl, pyridazinyl, tetrazolyl and isoxazolyl. As defined below, the 5 or 6-member aryl, heteroaryl, heterocyclic or alicyclic R groups may be substituted and thus include substituted cyclohexyl, substituted phenyl, substituted pyridine, substituted pyrimidine, substituted pyridazine, substituted tetrazole or substituted isoxazole, in which the substituents are as defined further below.
In certain compounds of Formula (I), R1 groups are H, fluoro or C1-C3alkoxy, for example methoxy.
In certain compounds of Formula (Ia), R1 groups are C1-C3alkoxy, for example methoxy.
In certain embodiments of the present invention in which R2 is heteroaryl, R2 is selected from pyridinyl, pyrimidinyl, pyridazinyl, pyrazinyl, pyrazolyl, imidazolyl, oxazolyl and isoxazolyl. In certain embodiments in which R2 is heterocyclic, R2 is selected from pyrrolidinyl, imidazolidinyl, piperidinyl and morpholinyl.
In further embodiments, R2 is selected from cyclohexyl, phenyl, pyridinyl, pyrimidinyl, pyridazinyl and isoxazolyl. As defined below, the 5 or 6-member aryl, heteroaryl, heterocyclic or alicyclic R2 groups may be substituted and thus include substituted cyclohexyl, substituted phenyl, substituted pyridine, substituted pyrimidine, substituted pyridazine or substituted isoxazole, in which the substituents are as defined further below.
Thus, where R2 is substituted phenyl, the substituents will be those defined for “aryl” substituents below. In some embodiments the substituted phenyl bears one or two substituents selected from halogen C1-3alkyl (for example methylphenyl), C1-3haloalkyl (for example trifluoroalkyl including trifluoromethylphenyl), C1-3alkoxy (for example methoxyphenyl) and C1-3haloakloxy (for example trifluoroalkoxy including trifluoromethoxyphenyl).
In certain embodiments in which R2 represents singly substituted phenyl, the substituent is at the meta or para position, for example para. In certain embodiments in which R2 represents doubly substituted phenyl, the substituents are at the para and meta, or at both meta positions.
In certain embodiments, R2 is selected from the group consisting of:
In certain embodiments of the present invention, Z represents —Y—W—X—, —(CH2)q—, —(CH2)nO— or —(CH2)pNHC(O)—.
In certain embodiments Y represents —O—, —CH2— or —CH2O—. In particular embodiments, X is absent or represents —(CH2)pSO2NR3—, —(CH2)pNHC(O)— or —(CH2)pNHC(O)NH—. In certain embodiments in which Y represents —CH2—, X represents —(CH2)pSO2NR3—. In certain embodiments in which Y represents —O— or —CH2O—, X is absent.
Particular W groups are 5 or 6 member aryl or heteroaryl rings. In certain embodiments in which W is aryl, for example C6 aryl (e.g. phenyl), W is linked through the 1 and 4 or the 1 and 3 positions. In certain embodiments in which W is heteroaryl, for example a 5 member heteroaryl ring (e.g. 1,2,4 oxadiazolyl), W may be linked through the 3 and 5 positions. In other embodiments in which W is heteroaryl, for example a 6 member heteroaryl ring (e.g. pyridinyl), W may be linked through the 2 and 5 positions. When X is —(CH2)pSO2NR3—, p is 0 and W is unsubstituted phenyl, W may for example be linked through the 1 and 4 (para) positions.
In certain embodiments, n represents 2.
In certain embodiments, p represents an integer selected from 0 or 1.
In certain embodiments W and R2 each represent unsubstituted phenyl, whilst in other embodiments W represents unsubstituted phenyl and R2 represents substituted phenyl.
It is to be understood that the present invention includes any combination of particular embodiments and covers all combinations of particular substituents described hereinabove.
Throughout the present specification and the accompanying claims the words “comprise” and “include” and variations such as “comprises”, “comprising”, “includes” and “including” are to be interpreted inclusively. That is, these words are intended to convey the possible inclusion of other elements or integers not specifically recited, where the context allows
As used herein, the terms “halogen” or “halo” refer to fluorine, chlorine, bromine and iodine.
As used herein, the term “alkyl” (when used as a group or as part of a group) refers to an optionally substituted straight or branched hydrocarbon chain containing the specified number of carbon atoms. For example, C1-C3alkyl means a straight or branched hydrocarbon chain containing at least 1 and at most 3 carbon atoms.
Examples of alkyl as used herein include, but are not limited to; methyl (Me), ethyl (Et), n-propyl, i-propyl and the like. Unless otherwise stated, optional substituents include hydroxy, halogen, ═S and ═O.
As used herein, the term “alkoxy” (when used as a group or as part of a group) refers to an alkyl ether radical, wherein the term “alkyl” is defined above. Examples of alkoxy as used herein include, but are not limited to; methoxy, ethoxy, n-propoxy, i-propoxy and the like.
As used herein, the term “alicyclic” (when used as a group or as part of a group) refers to a cyclic hydrocarbon ring containing the specified number of carbon atoms. Examples of alicyclic as used herein include, but are not limited to cyclohexyl, cyclopropyl and the like. Said alicyclic groups may be optionally substituted with one or more, for example 1 to 3, groups selected from hydroxy, halogen, ═S, ═O, C1-C3alkyl (which may be further substituted with one or more hydroxy, ═O or halo groups), optionally halogenated C1-C3alkoxy, C1-C3alkoxyC1-C3alkyl, NR32, —NHC(O)C1-C3alkyl, —C(O)NR32, and —S(O)2C1-C3alkyl, wherein R3 is as defined above.
As used herein, the term “aryl” (when used as a group or as part of a group) refers to an aromatic hydrocarbon ring of the specified number of carbons. Examples of aryl as used herein include, but are not limited to, phenyl and benzyl. Said aryl groups may be optionally substituted with one or more, for example 1 to 3 groups selected from hydroxy, halogen, ═S, ═O, C1-C3alkyl (which may be further substituted with one or more hydroxy, ═O or halo groups), optionally halogenated C1-C3alkoxy, C1-C3alkoxyC1-C3alkyl, NR32, —NHC(O)C1-C3alkyl, —C(O)NR32, and —S(O)2C1-C3alkyl, wherein R3 is as defined above.
As used herein, the term “heteroaryl” (when used as a group or as part of a group) refers to an aryl group, as defined above, which contains one or more nitrogen or oxygen heteroatoms. Examples of heteroaryl as used herein include, but are not limited to, pyridine, pyrimidine, pyridazine, imidazole, isoxazole, oxadiazoles, tetrazoles and the like. Said heteroaryl groups may be optionally substituted with one or more, for example 1 to 3 groups selected from hydroxy, halogen, ═S, ═O, C1-C3alkyl (which may be further substituted with one or more hydroxy, ═O or halo groups), optionally halogenated C1-C3alkoxy, C1-C3alkoxyC1-C3alkyl, NR32, —NHC(O)C1-C3alkyl, —C(O)NR32, and —S(O)2C1-C3alkyl, wherein R3 is as defined above.
As used herein, the term “heterocyclic” (when used as a group or as part of a group) refers to an alicyclic group, as defined above, which contains one or more nitrogen or oxygen heteroatoms. Said heterocyclic groups may be optionally substituted with one or more, for example 1 to 3 groups selected from hydroxy, halogen, ═S, ═O, C1-C3alkyl (which may be further substituted with one or more hydroxy, ═O or halo groups), optionally halogenated C1-C3alkoxy, C1-C3alkoxyC1-C3alkyl, NR32, —NHC(O)C1-C3alkyl, —C(O)NR32, and —S(O)2C1-C3alkyl, wherein R3 is as defined above.
As used herein, the term “physiologically functional derivative” refers to any pharmaceutically acceptable derivative of a compound of the present invention, for example an ester or an amide thereof, and includes any pharmaceutically acceptable salt, ester, or salt of such ester of a compound of Formula (I) or of Formula (Ia) which, upon administration to a mammal, such as a human, is capable of providing (directly or indirectly) a compound of Formula (I) or of Formula (Ia) or an active metabolite or residue thereof. It will be appreciated by those skilled in the art that the compounds of Formula (I) and of Formula (Ia) may be modified to provide physiologically functional derivatives thereof at any of the functional groups in the compounds, and that the compounds of Formula (I) and (Ia) may be so modified at more than one position.
As used herein, the term “pharmaceutically acceptable” used in relation to an ingredient (active ingredient or excipient) which may be included in a pharmaceutical formulation for administration to a patient, refers to that ingredient being acceptable in the sense of being compatible with any other ingredients present in the pharmaceutical formulation and not being deleterious to the recipient thereof.
As used herein, the term “solvate” refers to a complex of variable stoichiometry formed by a solute (in this invention, a compound of Formula (I), a salt thereof or a physiologically functional derivative thereof, or a compound of Formula (Ia), a salt thereof or a physiologically functional derivative thereof,) and a solvent. Such solvents for the purposes of the present invention may not interfere with the biological activity of the solute. Examples of suitable solvents include water, methanol, ethanol and acetic acid. Preferably the solvent used is a pharmaceutically acceptable solvent. Examples of suitable pharmaceutically acceptable solvents include water, ethanol and acetic acid. Most preferably the solvent used is water, in which case the solvate may be referred to as a hydrate of the solute in question.
It will be appreciated that, for pharmaceutical use, the “salt or solvate” referred to above will be a pharmaceutically acceptable salt or solvate. However, other salts or solvates may find use, for example, in the preparation of a compound of Formula (I) or of Formula (Ia) or in the preparation of a pharmaceutically acceptable salt or solvate thereof.
Pharmaceutically acceptable salts include those described by Berge, Bighley and Monkhouse, J. Pharm. Sci., 1977, 66, 1-19. Suitable pharmaceutically acceptable salts include acid addition salts formed from the addition of inorganic acids or organic acids, preferably inorganic acids. Examples of suitable acid addition salts include hydrochlorides, hydrobromides, sulphates and acetates. Further representative examples of pharmaceutically acceptable salts include those formed from maleic, fumaric, benzoic, ascorbic, pamoic, succinic, bismethylenesalicylic, methanesulfonic, ethanedisulfonic, propionic, tartaric, salicylic, citric, gluconic, aspartic, stearic, palmitic, itaconic, glycolic, p-aminobenzoic, glutamic, benzenesulfonic, cyclohexylsulfamic, phosphoric and nitric acids. Suitable pharmaceutically acceptable salts also include alkali metal salts formed from the addition of alkali metal bases such as alkali metal hydroxides. An example of a suitable alkali metal salt is a sodium salt.
In a further aspect, the present invention provides the use of a compound of Formula (I)
and salts, solvates and physiologically functional derivatives thereof as define above in the manufacture of a medicament for the treatment of disorders of lipid metabolism, including dislipidaemia or hyperlipoproteinaemia, or of inflammatory diseases or conditions. It is to be understood that this aspect of the present invention includes, with respect to the use of compounds of Formula (I) in the manufacture of a medicament, any combination of particular embodiments and covers all combinations of particular substituents of compounds of Formula (I) described hereinabove.
This aspect of the invention also provides the use of a compound of Formula (Ia)
and salts, solvates and physiologically functional derivatives thereof as define above in the manufacture of a medicament for the treatment of disorders of lipid metabolism, including dislipidaemia or hyperlipoproteinaemia, or of inflammatory diseases or conditions. It is to be understood that this aspect of the present invention includes, with respect to the use of compounds of Formula (Ia) in the manufacture of a medicament, any combination of particular embodiments and covers all combinations of particular substituents of compounds of Formula (Ia) described hereinabove.
Compounds of the present invention are of potential therapeutic benefit in the treatment and amelioration of the symptoms of many diseases of lipid metabolism including dislipidaemia or hyperlipoproteinaemia such as diabetic dyslipidaemia and mixed dyslipidaemia, heart failure, hypercholesterolaemia, cardiovascular disease including atherosclerosis, arteriosclerosis, and hypertriglyceridaemia. As such, the compounds may also find favour as therapeutics for coronary artery disease, thrombosis, angina, chronic renal failure, peripheral vascular disease and stroke, as well as the cardiovascular indications associated with type II diabetes mellitus, type I diabetes, insulin resistance, hyperlipidaemia, anorexia nervosa, obesity. The use of a compound of Formula (I) or a compound of Formula (Ia) in the treatment of one or more of these diseases is a further aspect of the present invention.
Furthermore, it is also believed that the HM74 and HM74A receptors are involved in inflammation. Inflammation represents a group of vascular, cellular and neurological responses to trauma. Inflammation can be characterised as the movement of inflammatory cells such as monocytes, neutrophils and granulocytes into the tissues. This is usually associated with reduced endothelial barrier function and oedema into the tissues. Inflammation with regards to disease typically is referred to as chronic inflammation and can last up to a lifetime. Such chronic inflammation may manifest itself through disease symptoms. The aim of anti-inflammatory therapy is therefore to reduce this chronic inflammation and allow for the physiological process of healing and tissue repair to progress.
Thus, a further aspect of the present invention resides in the use of a compound of Formula (I) or a salt, solvate or physiologically functional derivative thereof or a compound of Formula (Ia) or a salt, solvate or physiologically functional derivative thereof as defined above in the treatment of inflammatory diseases or conditions of the joint, particularly arthritis (e.g. rheumatoid arthritis, osteoarthritis, prosthetic joint failure), or the gastrointestinal tract (e.g. ulcerative colitis, Crohn's disease, and other inflammatory bowel and gastrointestinal diseases, gastritis and mucosal inflammation resulting from infection, the enteropathy provoked by non-steroidal anti-inflammatory drugs), of the lung (e.g. adult respiratory distress syndrome, asthma, cystic fibrosis, or chronic obstructive pulmonary disease), of the heart (e.g. myocarditis), of nervous tissue (e.g. multiple sclerosis), of the pancreas, (e.g. inflammation associated with diabetes melitus and complications thereof), of the kidney (e.g. glomerulonephritis), of the skin (e.g. dermatitis, psoriasis, eczema, urticaria, burn injury), of the eye (e.g. glaucoma) as well as of transplanted organs (e.g. rejection) and multi-organ diseases (e.g. systemic lupus erythematosis, sepsis) and inflammatory sequelae of viral or bacterial infections and inflammatory conditions associated with atherosclerosis and following hypoxic or ischaemic insults (with or without reperfusion), for example in the brain or in ischaemic heart disease.
In particular, the compounds of Formula (I) and (Ia) are useful in the treatment and prevention of inflammation, and cardiovascular diseases or conditions including atherosclerosis, arteriosclerosis, hypertriglyceridemia, and mixed dyslipidaemia.
Thus, there is also provided the use of a compound of Formula (I) or a pharmaceutically acceptable salt, solvate or physiologically functional derivative thereof, or a compound of Formula (Ia) or a salt, solvate or physiologically functional derivative thereof, in the manufacture of a medicament for the treatment of disorders of lipid metabolism including dislipidaemia or hyperlipoproteinaemia such as diabetic dyslipidaemia and mixed dyslipidaemia, heart failure, hypercholesterolaemia, cardiovascular disease including atherosclerosis, arteriosclerosis, and hypertriglyceridaemia. The compounds are also provided for use in the treatment of coronary artery disease, thrombosis, angina, chronic renal failure, peripheral vascular disease and stroke, as well as the cardiovascular indications associated with type II diabetes mellitus, type I diabetes, insulin resistance, hyperlipidaemia, anorexia nervosa, obesity.
Nicotinic acid has a significant side effect profile, possibly because it is dosed at high level (gram quantities daily). The most common side effect is an intense cutaneous flushing. The compounds of the present invention preferably exhibit reduced side effects compared to nicotinic acid.
HM74A has been identified as a high affinity receptor for nicotinic acid whilst HM74 is a lower affinity receptor. Desirably, the compounds of the present invention are selective for HM74A by which is meant that they show greater affinity for HM74A than for HM74.
The potential for compounds of Formula (I) and Formula (Ia) to activate HM74A may be demonstrated, for example, using the following in vitro and in vivo assays:
For transient transfections, HEK293T cells (HEK293 cells stably expressing the SV40 large T-antigen) are maintained in DMEM containing 10% foetal calf serum and 2 mM glutamine. Cells are seeded in 90 mm culture dishes and grown to 60-80% confluence (18-24 h) prior to transfection. Human HM74A (GenBank™ accession number AY148884) is subcloned in to a mammalian expression vector (pcDNA3; Invitrogen) and transfected using Lipofectamine reagent. For transfection, 9 μg of DNA is mixed with 30 μl Lipofectamine in 0.6 ml of Opti-MEM (Life Technologies Inc.) and incubated at room temperature for 30 min prior to the addition of 1.6 ml of Opti-MEM. Cells are exposed to the Lipofectamine/DNA mixture for 5 h and 6 ml of 20% (v/v) foetal calf serum in DMEM is then added. Cells are harvested 48 h after transfection. Pertussis toxin treatment is carried out by supplementation into media at 50 ngml−1 for 16 h. All transient transfection studies involve co-transfection of receptor together with the Gi/o G protein, Go1α.
For generation of stable cell lines the above method is used to transfect CHO-K1 cells seeded in six well dishes grown to 30% confluence. These cells are maintained in DMEM F-12 HAM media containing 10% foetal calf serum and 2 mM glutamine. 48 h post-transfection the media is supplemented with 400 μg/ml Geneticin (G418, Gibco) for selection of antibiotic resistant cells. Clonal CHO-K1 cell lines stably expressing HM74A are confirmed by [35S]-GTPγS binding measurements, following the addition of nicotinic acid.
P2 membrane preparation—Plasma membrane-containing P2 particulate fractions are prepared from cell pastes frozen at −80° C. after harvest. All procedures are carried out at 4° C. Cell pellets are resuspended in 1 ml of 10 mM Tris-HCl and 0.1 mM EDTA, pH 7.5 (buffer A) and by homogenisation for 20s with a Ultra Turrax followed by passage (5 times) through a 25-gauge needle. Cell lysates are centrifuged at 1,000 g for 10 min in a microcentrifuge to pellet the nuclei and unbroken cells and P2 particulate fractions are recovered by microcentrifugation at 16,000 g for 30 min. P2 particulate fractions are resuspended in buffer A and stored at −80° C. until required.
[35S]-GTPγS binding—Assays are performed at room temperature either in 96-well format as described previously (Wieland, T. and Jakobs, K. H. (1994) Methods Enzymol. 237, 3-13) or in an adapted protocol carried out in 384-well format.
96-well format: Briefly, membranes (10 μg per point) are diluted to 0.083 mg/ml in assay buffer (20 mM HEPES, 100 mM NaCl, 10 mM MgCl2, pH7.4) supplemented with saponin (10 mg/l) and pre-incubated with 10 μM GDP. Various concentrations of nicotinic acid or related molecules are added, followed by [35S]-GTPγS (1170 Ci/mmol, Amersham) at 0.3 nM (total vol. of 100 μl) and binding is allowed to proceed at room temperature for 30 min. Non-specific binding is determined by the inclusion of 0.6 mM GTP. Wheatgerm agglutinin SPA beads (Amersham) (0.5 mg) in 25 μl assay buffer are added and the whole is incubated at room temperature for 30 min with agitation. Plates are centrifuged at 1500 g for 5 min and bound [35S]-GTPγS is determined by scintillation counting on a Wallac 1450 microbeta Trilux scintillation counter.
384-well format: Briefly, the dilution of standard or test compounds are prepared and added to a 384-well plate in a volume of 10 μl. Membranes (HM74A or HM74) are diluted in assay buffer (20 mM HEPES, 100 mM NaCl, 10 mM MgCl2, pH7.4) supplemented with saponin (60 μg/ml), Leadseeker WGA beads (Amersham; 250 μg/well) and 10 μM GDP, so that the 20 μl volume added to each well contains 5 μg of membranes. [35S]-GTPγS (1170 Ci/mmol, Amersham) is diluted (1:1500) in assay buffer and 20 μl added to each well. Following the addition of the radioligand, the plates are sealed, pulse spun and incubated for 4 hours at room temperature. At the end of the incubation period the plates are read on a Leadseeker machine (VIEWLUX PLUS; Perkin-Elmer) to determine the levels of specific binding.
HM74A agonists are tested in male Spague-Dawley rats (200-250 grams) which have been fasted for at least 12 hours prior to the study. The compounds are dosed intravenously (5 ml/kg) or by oral gavage (10 ml/kg). Blood samples (0.3 ml tail vein bleed) are taken pre-dose and at three times post-dose (times ranging from 15 minutes to 8 hours post-dose). Each blood sample is transferred to a heparin tube (Becton Dickinson Microtainer, PST LH) and centrifuged (10,000 g for 5 minutes) to produce a plasma sample. The plasma samples are assayed for levels of non-esterified fatty acids (NEFA) using a commercially available kit (Randox). Inhibition of plasma NEFA levels, relative to pre-dose levels, is used as a surrogate for HM74A agonist activity.
In order to determine whether compounds of the invention exhibit the flushing response associated with nicotinic acid, they are dosed to anaesthetised guinea-pigs. Nicotinic acid is used as positive control. Male Dunkin Hartley guinea pigs (300-800 g) are fasted for 12 hours prior to being anaesthetised with a mixture of Ketamine hydrochloride (Vetalar, 40 mg/kg i.m.), Xylazine (Rompun, 8 mg/kg i.m.) and sodium pentobarbitone (Sagatal, 30 mg/kg i.p.). Following anaesthesia a tracheostomy is performed and the animals are mechanically ventilated with room air (10-12 mL/kg, 60 breaths/min). A jugular vein, and a carotid artery, are cannulated for intravenous administration of test compound and collection of blood respectively. An infra-red temperature probe (Extech Instruments) is placed 3-5 mm from the tip of the left ear. Temperature measurements are recorded every minute from 5 minutes prior to test compound or nicotinic acid and up to 40 minutes post-administration of test compound or nicotinic acid. Data is automatically collected on a Psion computer before being transferred for data analysis within an Excel spreadsheet. Prior to, and at frequent time points after compound administration, blood samples (0.3 ml) are taken via the carotid arterial cannula and transferred to Microtainer (BD) tubes containing lithium heparin. The samples are mixed thoroughly on a blood roller and then stored on ice prior to centrifugation at 1200 g for 5 minutes.
Compounds according to Formulae (I) and (Ia) have been synthesised (see synthetic examples below).
As indicated above, compounds of Formula (I) or of Formula (Ia) are useful in human or veterinary medicine, in particular as activators of HM74A, in the management of dyslipidaemia and hyperlipoproteinaemia.
Thus, there is provided as a further aspect of the present invention a compound of Formula (I) or a pharmaceutically acceptable salt, solvate or physiologically functional derivative thereof, or a compound of Formula (Ia) or a pharmaceutically acceptable salt, solvate or physiologically functional derivative thereof, for use in human or veterinary medicine, particularly in the treatment of disorders of lipid metabolism including dislipidaemia or hyperlipoproteinaemia such as diabetic dyslipidaemia and mixed dyslipidaemia, heart failure, hypercholesterolaemia, cardiovascular disease including atherosclerosis, arteriosclerosis, and hypertriglyceridaemia. As such, the compounds may also find favour as therapeutics for coronary artery disease, thrombosis, angina, chronic renal failure, peripheral vascular disease and stroke, as well as the cardiovascular indications associated with type II diabetes mellitus, type I diabetes, insulin resistance, hyperlipidaemia, anorexia nervosa, obesity.
It will be appreciated that references herein to treatment extend to prophylaxis, prevention of recurrence and suppression of symptoms as well as the treatment of established conditions.
In a further or alternative aspect there is provided a method for the treatment of a human or animal subject with a condition in which under-activation of the HM74A receptor contributes to the condition or in which activation of the receptor will be beneficial, which method comprises administering to said human or animal subject an effective amount of a compound of Formula (I) or a physiologically acceptable salt or solvate thereof, or a compound of Formula (Ia) or a pharmaceutically acceptable salt, solvate or physiologically functional derivative thereof.
More particularly, the present invention provides a method for the treatment of disorders of lipid metabolism including dislipidaemia or hyperlipoproteinaemia such as diabetic dyslipidaemia and mixed dyslipidaemia, heart failure, hypercholesterolaemia, cardiovascular disease including atherosclerosis, arteriosclerosis, or hypertriglyceridaemia which method comprises administering to said human or animal subject an effective amount of a compound of Formula (I) or a physiologically acceptable salt or solvate thereof or a compound of Formula (Ia) or a pharmaceutically acceptable salt, solvate or physiologically functional derivative thereof. The invention also provides methods for the treatment of coronary artery disease, thrombosis, angina, chronic renal failure, peripheral vascular disease or stroke, as well as the cardiovascular indications associated with type II diabetes mellitus, type I diabetes, insulin resistance, hyperlipidaemia, anorexia nervosa, obesity which methods comprise administering to said human or animal subject an effective amount of a compound of Formula (I) or a physiologically acceptable salt, solvate or derivative thereof or a compound of Formula (Ia) or a pharmaceutically acceptable salt, solvate or physiologically functional derivative thereof.
The amount of a HM74A modulator which is required to achieve the desired biological effect will, of course, depend on a number of factors, for example, the mode of administration and the precise clinical condition of the recipient. In general, the daily dose will be in the range of 0.1 mg-1 g/kg, typically 0.1-100 mg/kg. An intravenous dose may, for example, be in the range of 0.01 mg to 0.1 g/kg, typically 0.01 mg to 10 mg/kg, which may conveniently be administered as an infusion of from 0.1 μg to 1 mg, per minute. Infusion fluids suitable for this purpose may contain, for example, from 0.01 μg to 0.1 mg, per millilitre. Unit doses may contain, for example, from 0.01 μg to 1 g of a HM74A modulator. Thus ampoules for injection may contain, for example, from 0.01 μg to 0.1 g and orally administrable unit dose formulations, such as tablets or capsules, may contain, for example, from 0.1 mg to 1 g. No toxicological effects are indicated/expected when a compound of the invention is administered in the above mentioned dosage range.
A compound of the present invention may be employed as the compound per se in the treatment of a the treatment of diseases where under-activation of the HM74A receptor contributes to the disease or where activation of the receptor will be beneficial, but is preferably presented with an acceptable carrier in the form of a pharmaceutical formulation. The carrier must, of course, be acceptable in the sense of being compatible with the other ingredients of the formulation and must not be deleterious to the recipient. The carrier may be a solid or a liquid, or both, and is preferably formulated with the HM74A modulator as a unit-dose formulation, for example, a tablet, which may contain from 0.05% to 95% by weight of the HM74A modulator.
The formulations include those suitable for oral, rectal, topical, buccal (e.g. sub-lingual) and parenteral (e.g. subcutaneous, intramuscular, intradermal or intravenous) administration.
There is also provided according to the invention a process for preparation of such a pharmaceutical composition which comprises mixing the ingredients.
Formulations suitable for oral administration may be presented in discrete units, such as capsules, cachets, lozenges or tablets, each containing a predetermined amount of a HM74A modulator; as a powder or granules; as a solution or a suspension in an aqueous or non-aqueous liquid; or as an oil-in-water or water-in-oil emulsion. In general, the formulations are prepared by uniformly and intimately admixing the active HM74A modulator with a liquid or finely divided solid carrier, or both, and then, if necessary, shaping the product. For example, a tablet may be prepared by compressing or moulding a powder or granules of the HM74A modulator optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing, in a suitable machine, the compound in a free-flowing form, such as a powder or granules optionally mixed with a binder, lubricant, inert diluent and/or surface active/dispersing agent(s). Moulded tablets may be made by moulding, in a suitable machine, the powdered compound moistened with an inert liquid diluent.
Tablets and capsules for oral administration may contain conventional excipients such as binding agents, for example syrup, acacia, gelatin, sorbitol, tragacanth, mucilage of starch or polyvinyl pyrrolidone; fillers, for example, lactose, microcrystalline cellulose, sugar, maize-starch, calcium phosphate or sorbitol; lubricants, for example, magnesium stearate, stearic acid, talc, polyethylene glycol or silica; disintegrants, for example, potato starch, croscarmellose sodium or sodium starch glycollate; or wetting agents such as sodium lauryl sulphate. The tablets may be coated according to methods well known in the art. Oral liquid preparations may be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may contain conventional additives such as suspending agents, for example, sorbitol syrup, methyl cellulose, glucose/sugar syrup, gelatin, hydroxymethyl cellulose, carboxymethyl cellulose, aluminium stearate gel or hydrogenated edible fats; emulsifying agents, for example, lecithin, sorbitan mono-oleate or acacia; non-aqueous vehicles (which may include edible oils), for example almond oil, fractionated coconut oil, oily esters, propylene glycol or ethyl alcohol; or preservatives, for example, methyl or propyl p-hydroxybenzoates or sorbic acid. The preparations may also contain buffer salts, flavouring, colouring and/or sweetening agents (e.g. mannitol) as appropriate.
Formulations suitable for buccal (sub-lingual) administration include lozenges comprising a HM74A modulator in a flavoured base, usually sucrose and acacia or tragacanth, and pastilles comprising the HM74A modulator in an inert base such as gelatin and glycerin or sucrose and acacia.
Formulations of the present invention suitable for parenteral administration conveniently comprise sterile aqueous preparations of an HM74A modulator, preferably isotonic with the blood of the intended recipient. These preparations are preferably administered intravenously, although administration may also be effected by means of subcutaneous, intramuscular, or intradermal injection. Such preparations may conveniently be prepared by admixing the HM74A modulator with water and rendering the resulting solution sterile and isotonic with the blood. Injectable compositions according to the invention will generally contain from 0.1 to 5% w/w of the HM74A modulator.
Thus, formulations of the present invention suitable for parenteral administration comprising a compound according to the invention may be formulated for parenteral administration by bolus injection or continuous infusion and may be presented in unit dose form, for instance as ampoules, vials, small volume infusions or pre-filled syringes, or in multi-dose containers with an added preservative. The compositions may take such forms as solutions, suspensions, or emulsions in aqueous or non-aqueous vehicles, and may contain formulatory agents such as anti-oxidants, buffers, antimicrobial agents and/or toxicity adjusting agents. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g. sterile, pyrogen-free water, before use. The dry solid presentation may be prepared by filling a sterile powder aseptically into individual sterile containers or by filling a sterile solution aseptically into each container and freeze-drying.
Formulations suitable for rectal administration are preferably presented as unit-dose suppositories. These may be prepared by admixing a HM74A modulator with one or more conventional solid carriers, for example, cocoa butter or glycerides and then shaping the resulting mixture.
Formulations suitable for topical application to the skin preferably take the form of an ointment, cream, lotion, paste, gel, spray, aerosol, or oil. Carriers which may be used include vaseline, lanolin, polyethylene glycols, alcohols, and combinations of two or more thereof. The HM74A modulator is generally present at a concentration of from 0.1 to 15% w/w of the composition, for example, from 0.5 to 2%.
By topical administration as used herein, we include administration by insufflation and inhalation. Examples of various types of preparation for topical administration include ointments, creams, lotions, powders, pessaries, sprays, aerosols, capsules or cartridges for use in an inhaler or insufflator or drops (e.g. eye or nose drops).
Ointments and creams may, for example, be formulated with an aqueous or oily base with the addition of suitable thickening and/or gelling agents and/or solvents. Such bases may thus, for example, include water and/or an oil such as liquid paraffin or a vegetable oil such as arachis oil or castor oil or a solvent such as a polyethylene glycol. Thickening agents which may be used include soft paraffin, aluminium stearate, cetostearyl alcohol, polyethylene glycols, microcrystalline wax and beeswax.
Lotions may be formulated with an aqueous or oily base and will in general also contain one or more emulsifying agents, stabilising agents, dispersing agents, suspending agents or thickening agents.
Powders for external application may be formed with the aid of any suitable powder base, for example, talc, lactose or starch. Drops may be formulated with an aqueous or non-aqueous base also comprising one or more dispersing agents, solubilising agents or suspending agents.
Spray compositions may be formulated, for example, as aqueous solutions or suspensions or as aerosols delivered from pressurised packs, with the use of a suitable propellant, e.g. dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, 1,1,1,2,3,3,3-heptafluoropropane, 1,1,1,2-tetrafluorethane, carbon dioxide or other suitable gas.
Capsules and cartridges for use in an inhaler or insufflator, of for example gelatin, may be formulated containing a powder mix of a compound of the invention and a suitable powder base such as lactose or starch.
The pharmaceutical compositions according to the invention may also be used in combination with other therapeutic agents, for example in combination with other classes of dyslipidaemic drugs (e.g. statins, fibrates, bile-acid binding resins or nicotinic acid).
The compounds of the instant invention may be used in combination with one or more other therapeutic agents for example in combination with other classes of dyslipidaemic drugs e.g. 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors (statins) or fibrates or bile acid binding resins or nicotinic acid. The invention thus provides, in a further aspect, the use of such a combination in the treatment of diseases in which under-activation of the HM74A receptor contributes to the disease or in which activation of the receptor will be beneficial and the use of a compound of Formula (I) or a pharmaceutically acceptable salt, solvate or physiologically functional derivative thereof, or a compound of Formula (Ia) or a pharmaceutically acceptable salt, solvate or physiologically functional derivative thereof, in the manufacture of a medicament for the combination therapy of disorders of lipid metabolism including dislipidaemia or hyperlipoproteinaemia such as diabetic dyslipidaemia and mixed dyslipidaemia, heart failure, hypercholesterolaemia, cardiovascular disease including atherosclerosis, arteriosclerosis, or hypertriglyceridaemia, coronary artery disease, thrombosis, angina, chronic renal failure, peripheral vascular disease or stroke, as well as the cardiovascular indications associated with type II diabetes mellitus, type I diabetes, insulin resistance, hyperlipidaemia, anorexia nervosa, obesity.
When the compounds of the present invention are used in combination with other therapeutic agents, the compounds may be administered either sequentially or simultaneously by any convenient route.
The combinations referred to above may conveniently be presented for use in the form of a pharmaceutical formulation and thus pharmaceutical formulations comprising a combination as defined above optimally together with a pharmaceutically acceptable carrier or excipient comprise a further aspect of the invention. The individual components of such combinations may be administered either sequentially or simultaneously in separate or combined pharmaceutical formulations.
When combined in the same formulation it will be appreciated that the two components must be stable and compatible with each other and the other components of the formulation and may be formulated for administration. When formulated separately they may be provided in any convenient formulation, conveniently in such a manner as are known for such compounds in the art.
When in combination with a second therapeutic agent active against the same disease, the dose of each component may differ from that when the compound is used alone. Appropriate doses will be readily appreciated by those skilled in the art.
The invention thus provides, in a further aspect, a combination comprising a compound of Formula (I) or a physiologically acceptable salt or solvate thereof together, or a compound of Formula (Ia) or a pharmaceutically acceptable salt, solvate or physiologically functional derivative thereof, with another therapeutically active agent. The combination may conveniently be presented for use in the form of a pharmaceutical formulation and thus pharmaceutical formulations comprising a combination as defined above together with a pharmaceutically acceptable carrier thereof represent a further aspect of the invention.
Compounds of Formula (I) and (Ia), and salts and solvates thereof, may be prepared by various synthetic routes, including the methodology described hereinafter which constitutes a further aspect of the invention.
Abbreviations
A process for preparing compounds of Formula (I)
in which R represents tetrazole, R1 represents H, Z represents —Y—W—X—, Y represents —(CH2)pO—, X is absent and W and R2 each represent phenyl is set out in scheme (a). This method is also suitable for preparing compounds of Formula (Ia) in which R represents CO2H, R1 represents C1-C5alkoxy, Z represents —Y—W—X—, Y represents —(CH2)pO—, X is absent and W and R2 each represent phenyl:
Accordingly, the present invention provides a process for preparing a compound of Formula (I) comprising coupling of 4′ substituted anthranilic acid or [2-(1H-tetrazol-5-yl)phenyl]amine with (4-biphenylyloxy)acetic acid using CDI and where desired or necessary converting a resultant free acid or base compound of Formula (I) or Formula (Ia) into a physiologically acceptable salt form, or vice versa, or converting one salt form into another physiologically acceptable salt form.
A process for preparing compounds of Formula (I)
in which R represents (CH2)qCO2H, R1 represents H, Z represents —Y—W—X—, Y represents —(CH2)pO—, X is absent and W and R2 each represent phenyl is set out in scheme (b):
Accordingly, the present invention provides a process for preparing a compound of Formula (I) comprising coupling of an acid-protected amine with (4-biphenylyloxy)acetic acid using CDI followed by hydrolysis of the protecting group e.g. tert-butyl ester using TFA. and where desired or necessary converting a resultant free acid or base compound of Formula (I) into a physiologically acceptable salt form, or vice versa, or converting one salt form into another physiologically acceptable salt form.
The following non-limiting examples illustrate the present invention:
Using method A, a stirred solution of (4-biphenylyloxy)acetic acid (315 mg, 1.38 mmol, 1.0 equiv) and CDI (269 mg, 1.66 mmol, 1.2 equiv) in anhydrous THF (10 ml) was left to react at rt. for 60 minutes. 2-Amino-4-(methyloxy)benzoic acid (Synth. Commun.; 24, 4; 1994; 533-548) (300 mg, 1.79 mmole, 1.3 equiv) and pyridinium p-toluenesulfonate (833 mg, 3.3 mmole, 2.4 equiv) were added and the mixture heated at gentle reflux for 18 hours. The mixture was allowed to cool, filtered and the filtrate concentrated. CH3CN (15 ml) and THF (3 ml) were added to the resulting residue and then heated to near boiling. After cooling the resulting product was collected by filtration. (321 mg, 65%). δH H (400 MHz, d6-DMSO) 3.83 (3H, s), 4.78 (2H, s), 6.77 (1H, dd, J=2.4 and 9.0 Hz), 7.17 (2H, d, J=8.6 Hz), 7.34 (1H, t, J=7.2 Hz), 7.43 (2H, app. t, J=7.6 Hz), 7.64 (4H, app. t, J=8.8 Hz), 7.98 (1H, d, J=8.8 Hz), 8.36 (1H, d, J=2.3 Hz), 12.40 (1H, s), 13.47 (1H, br. s); m/z 378.1 [MH+].
Method A using (4-biphenylyloxy)acetic acid (J. Med. Chem 1976, 19, 1079-1088) (128 mg, 0.56 mmol, 1 equiv) and [2-(1H-tetrazol-5-yl)phenyl]amine (118 mg, 0.73 mmol, 1.3 equiv). Crystallisation from acetonitrile afforded the title compound as a white solid (93.5 mg, 45%); δH (400 MHz, DMSO) 4.83 (2H, s), 7.29-7.40 (4H, m), 7.44 (2H, t, J=7.5 Hz), 7.60-7.72 (5H, m), 8.02 (1H, dd, J=8.0 and 1.0 Hz), 8.71 (1H, d, J=8.0 Hz), 11.93 (1H, s), one NH not observed up to δH 13; m/z 372.1 [MH+].
Method A using (4-biphenylyloxy)acetic acid (76 mg, 0.33 mmol, 1 equiv) and 1,1-dimethylethyl(2-aminophenyl)acetate (Liebigs Annalen der Chemie, 1985, 7, 1398-1412) (90 mg, 0.43 mmol, 1.3 equiv). Purification by Biotage™ chromatography gradient elution with 49-9:1 cyclohexane:ethyl acetate afforded the title compound as a colourless gum (73 mg, 55%); δH (400 MHz, CDCl3) 1.46 (9H, s), 3.44 (2H, s), 4.74 (2H, s), 7.08-7.12 (2H, m), 7.16 (1H, app t, J=7.5 Hz), 7.22 (1H, dd, J=7.5 and 1.5 Hz), 7.32-7.38 (2H, m), 7.45 (2H, t, J=7.5 Hz), 7.55-7.60 (4H, m), 7.94 (1H, d, J=8.0 Hz), 9.81 (1H, s); m/z 416.2 [M-H]−.
To a solution of 1,1-dimethylethyl(2-{[(4-biphenylyloxy)acetyl]amino}phenyl)acetate (30 mg, 0.07 mmol) in DCM (0.5 ml) was added TFA (0.5 ml). The mixture was stirred at room temperature for 3 hours before concentrating in vacuo. The residue was dissolved in DCM (3 ml) and concentrated 3 times to afford the title compound as a pale tan solid (25 mg, 98%); δH (400 MHz, CDCl3) 3.58 (2H, s), 4.73 (2H, s), 7.01 (2H, dd, J=9.0 and 2.5 Hz), 7.20 (1H, app t, J=7.5 Hz), 7.25-7.30 (1H, m), 7.31-7.40 (2H, m), 7.41-7.47 (2H, m), 7.47-7.56 (4H, m), 7.79 (1H, d, J=8.0 Hz), 9.34 (1H, s), one exchangeable proton not observed up to δH 13; m/z 362.1 [MH+].
All publications, including but not limited to patents and patent applications, 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 though fully set forth.
The application of which this description and claims forms part may be used as a basis for priority in respect of any subsequent application. The claims of such subsequent application may be directed to any feature or combination of features described herein. They may take the form of product, composition, process, or use claims and may include, by way of example and without limitation the following claims:
| Number | Date | Country | Kind |
|---|---|---|---|
| 0503054.9 | Feb 2005 | GB | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/GB06/00506 | 2/14/2006 | WO | 00 | 8/2/2007 |
