Patent Application
20110230477
The present invention relates to novel compounds including their pharmaceutically acceptable salts and solvates, which are agonists or partial agonists of G-protein coupled receptor 43 (GPR43) and are useful as therapeutic compounds, particularly in the treatment and/or prevention of Type 2 diabetes mellitus and conditions that are often associated with this disease including, lipid disorders such as dyslipidemia, hypertension, obesity, atherosclerosis and its sequelae.
Under normal conditions, Free Fatty Acids (FFAs) are implicated in numerous physiological processes by serving as fuel in various metabolic pathways and/or acting as signaling molecules in different tissues such as the heart, liver, skeletal muscle, adipocytes and the pancreas (Newsholme et al., Biochem. J., 80 pp 655-662, 1961; Prentki et al., Endocrine Reviews, PubMed print ahead, 2008). Among FFAs, the short-chain fatty acids (SCFAs, carbon length C2-C6) are generated during anaerobic bacterial fermentation of fiber in the gut (Sellin et al., News. Physiol. Sci., 14, pp 58-64, 1999). Long-chain fatty acids (LCFAs, carbon length C14-C24) are products of dietary intake from adipose tissues and liver (McArthur et al., J. Lipid. Res., 40, pp 1371-1383, 1999).
Obesity is an increasing, worldwide public health problem associated with devastating pathologies such as type 2 diabetes (T2D) and dyslipidemia (Wild et al., Diabetes Care 27, pp 1047-1053, 2004). Dyslipidemia is characterized by high levels of triglycerides and/or LDL (bad cholesterol) or low levels of HDL (good cholesterol). Dyslipidemia is a key independent risk factor for cardiovascular diseases. It has long been suggested that FFAs are implicated in the regulation and/or genesis of these diseases (Fraze et al., J. Clin. Endocrinol. Metab., 61, pp 807-811, 1985). It is well established that regular intake of dietary fiber has several beneficial metabolic effects such as lowering of plasma cholesterol and triglyceride levels (Anderson et al., J. Am. Coll. Nutr., 23, pp 5-17, 2004). Specifically, dietary fiber has been shown to increase endogenous levels of SCFAs, leading to the suppression of cholesterol synthesis and improvement in glucose tolerance in rat (Berggren et al., Br. J. Nutr., 76, pp 287-294, 1996), as well as the reduction of hyperglycemia in a diabetic mice model (Sakakibara et al., Biochem. Biophys. Res. Com., 344, pp 597-604, 2006).
Drug therapies are available to address both T2D and dyslipidemia. Specifically, statins, fibrates and nicotinic acid or combinations thereof are often considered as a first line therapy in dyslipidemia whereas metformin, sulphonylureas and thiazolidinediones are three, widely-used classes of oral anti-diabetic drugs (Tenenbaum et al., Cardiovascular Diabetology, 5, pp 20-23, 2006). Although theses therapies are widespread in their use, the common appearance of adverse effects or lack of efficacy after long-term use causes concern. Moreover, the growing patient population suffering from T2D, dyslipidemia and associated metabolic diseases creates a demand for new entrants into this therapeutic market.
GPR43 (also named FFA2R) belongs to a subfamily of G-Protein-Coupled Receptors (GPCRs), including GPR40 and GPR41 that have been identified as receptor for FFAs (Le Poul et al., J. Biol Chem. 278, 25481-489, 2003; Covington et al., Biochemical Society transaction 34, 770-773, 2006). The 3 family members share 30 to 40% sequences identity with specificity toward different fatty acids carbon chain lengths, with SCFAs (short chain fatty acids: six carbons molecules or shorter) activating GPR41 and GPR43 and medium and long chain fatty acids (MCFA, LCFA) activating GPR40 (Rayasam et al., Expert Opinion on therapeutic targets, 11 661-671, 2007). C2 acetate and C3 propionate are the most potent activators of GPR43. GPR43 is mainly coupled with Gq-proteins, with some evidence for its possible coupling with Gi/o pathways as well.
GPR43 is strongly expressed in adipocytes. Also there is evidence suggesting that GPR43 is overexpressed in pancreatic β-cells in prediabetic states as shown in WO2006/036688A2. Recent papers confirmed the GPR43 expression in pancreatic islets (Ahrén, Nature Reviews, 8 pp 396-385; 2009; Regard et al., J; Clin. Invst., 117 pp 4034-4043, 2007). In adipocyte cells, GPR43 is induced during the differentiation process and increased during the high fat feeding in rodents, suggesting that GPR43 may affect adipocyte functions (Hong et al., Endrocrinology, 146 pp 5092-5099, 2005). Indeed, it has been reported that acetate and propionate may stimulate adipogenesis via GPR43. In addition siRNA results hinted that acetate and propionate may inhibit lipolysis in adipocytes via GPR43 activation (Hong et al., Endocrinology, 146 pp 5092-5099, 2005). It is interesting to note that the effect of acetate on reducing plasma free fatty acids level has been documented in humans (Suokas et al., Alcoholism, clinical and experimental research, 12 pp 52-58, 1988; Laurent et al., European journal of clinical nutrition, 49 pp 484-491, 1995). In addition, it has been shown that (i) adipocytes treated with GPR43 endogenous SCFA ligands exhibit a reduction in lipolytic activity and such inhibition of lypolysis is the result of GPR43 activation and (ii) GPR43 activation by acetate results in the reduction of plasma free fatty acids level in vivo (Ge et al., Endocrinology, 149 pp 4519-26, 2008). Recently two GPR43 positive allosteric modulator molecules have been shown able to inhibit the lipolysis in adipocytes similarly to that of GPR43 endogenous SCFA ligands (Lee et al., Mol Pharmacol, 74(6) pp 1599-1609, 2008). Such results suggest a potential role of GPR43 in regulating plasma lipid profiles and aspects of metabolic syndrome.
On this basis, new agonists or partial agonists of GPR43 may be of therapeutic value for T2D mellitus and conditions that are associated with this disease including, lipid disorders such as dyslipidemia, hypertension, obesity, atherosclerosis and its sequelae.
The invention encompasses compounds of general Formula I, their pharmaceutically acceptable salts and solvates as well as methods of use of such compounds or compositions comprising such compounds as modulators of GPR43 activity.
In a general aspect, the invention provides compounds of general formula I:
wherein
wherein R is H or linear or branched alkyl, aryl, acyloxyalkyl, dioxolene, R3 is H, methyl or ethyl, and R4 is hydroxyl —SO2CH3, —SO2cyclopropyl or —SO2CF3;
In another aspect, the present invention provides a pharmaceutical composition comprising at least one compound according to the invention or a pharmaceutically acceptable salt or solvate thereof.
The invention also relates to the use of the above compounds or their pharmaceutically acceptable salts and solvates as modulators of GPR43, preferably as agonists or partial agonists of GPR43.
The invention further provides methods of treatment and/or prevention of type II diabetes, obesity, dyslipidemia such as mixed or diabetic dyslipidemia, hypercholesterolemia, low HDL cholesterol, high LDL cholesterol, hyperlipidemia, hypertriglyceridemia, hypoglycemia, hyperglycemia, glucose intolerance, insulin resistance, hyperinsulinemia hypertension, hyperlipoproteinemia, metabolic syndrome, syndrome X, thrombotic disorders, cardiovascular disease, atherosclerosis and its sequelae including angina, claudication, heart attack, stroke and others, kidney diseases, ketoacidosis, nephropathy, diabetic neuropathy, diabetic retinopathy, nonalcoholic fatty liver diseases such as steatosis or nonalcoholic steatohepatitis (NASH) comprising the administration of a therapeutically effective amount of a compound or pharmaceutically acceptable salt or solvate of formula (I), to a patient in need thereof. Preferably the patient is a warm-blooded animal, more preferably a human.
The invention also provides the use of a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof as a medicament. Preferably, the medicament is used for the treatment and/or prevention of type II diabetes, obesity, dyslipidemia such as mixed or diabetic dyslipidemia, hypercholesterolemia, low HDL cholesterol, high LDL cholesterol, hyperlipidemia, hypertriglyceridemia, hypoglycemia, hyperglycemia, glucose intolerance, insulin resistance, hyperinsulinemia hypertension, hyperlipoproteinemia, metabolic syndrome, syndrome X, thrombotic disorders, cardiovascular disease, atherosclerosis and its sequelae including angina, claudication, heart attack, stroke and others, kidney diseases, ketoacidosis, nephropathy, diabetic neuropathy, diabetic retinopathy, nonalcoholic fatty liver diseases such as steatosis or nonalcoholic steatohepatitis (NASH).
In a preferred embodiment the disease is type II diabetes, a lipid disorder such as dyslipidemia, hypertension, obesity, or atherosclerosis and its sequelae.
As noted above, the invention relates to compounds of formula I, as well as their pharmaceutically acceptable salts and solvates.
Preferred compounds of formula I and pharmaceutically acceptable salts and solvates thereof are those wherein
Still other preferred compounds of formula I are those wherein D is SO2 and Ar1, Ar2, Ar3, R1, R2, L1, L2, L3, and Z are as defined above in respect to formula I.
In one embodiment, preferred compounds of Formula I are those of formula Ia:
and pharmaceutically acceptable salts, and solvates thereof, wherein
Preferred compounds of formula Ia are those wherein
In another embodiment, preferred compounds of Formula I are those of formula Ib:
and pharmaceutically acceptable salts, and solvates thereof, wherein
either in position 4 or 5, preferably in position 4; and
Preferred compounds of formula Ib are those wherein Z is —COOR, preferably COOH, and R, Ar1, Ar2, Ar3, R1, R2, L1, L2 and L3 are as defined above in respect to formula I.
Particularly preferred compounds of formula Ib are those of formula Ib-1
wherein L1, L2, L3, Ar3, X, Y, Z, R1, R2, and R5 are as defined above in respect to formula Ib, preferably L1 is methylene, optionally substituted by C1-C2 alkyl or halo, preferably by methyl or fluoro, even more preferably L1 is methylene; and
Preferred compounds of formula Ib-1 are those of formula Ib-1a
wherein L2, L3, Ar3, X, Y, R2, R5, R6, R7, R′6, R′7 and R8 are as defined above in respect to formula Ib-1.
Other preferred compounds of formula Ib are selected form the group consisting of formulae Ib-2a, Ib-2b, Ib-2c, Ib-2d, Ib-2e and Ib-2f:
wherein L1, L2, L3, Ar3, X, Y, Z, R1, R2 and R5 are as defined above in respect to formula Ib, preferably L1 is methylene;
Particularly preferred compounds of formula Ib-2a are
wherein A is —(CH2)n—O—, —(CH2)n—NRa—, —(CH2)n—SO2—, or —(CH2)m—, wherein n is equal to 0 or 1, m is equal to 1 or 2, and Ra is as defined above in respect to formula Ib-2b, preferably Ra is H or alkyl, preferably linear or branched C1-C4 alkyl; C1-C4 alkylcarbonyl, C1-C4 alkylsulfonyl, more preferably linear or branched C1-C4 alkyl; and
Even more preferred compounds of formula Ib-2a are selected from
wherein A is —(CH2)n—O—, —(CH2)n—NRa—, —(CH2)n—SO2—, or —(CH2)m—, wherein n is equal to 0 or 1, m is equal to 1 or 2, and Ra is as defined above in respect to formula Ib-2b, preferably Ra is H or alkyl, preferably linear or branched C1-C4 alkyl; C1-C4 alkylcarbonyl, C1-C4 alkylsulfonyl, more preferably linear or branched C1-C4 alkyl; and
Further preferred compounds of formula Ib are those of formula Ib-3
preferably
wherein L2, L3, Ar3, X, Y, R, R1, R2 and R5 are as defined above in respect to formula Ib; and
Further preferred compounds of formula Ib are those of formula Ib-4
wherein
wherein
wherein
wherein
wherein,
wherein,
wherein
wherein
wherein
wherein
wherein
wherein,
wherein
wherein
wherein
wherein
wherein
wherein,
wherein,
wherein
wherein,
wherein,
In yet another embodiment, preferred compounds of formula I are those of formula Ic
and pharmaceutically acceptable salts, and solvates thereof, wherein
Preferred compounds of formula Ic are those wherein
Particularly preferred compounds of formula Ic are those of formula Ic-1
and pharmaceutically acceptable salts, and solvates thereof, wherein
Preferred compounds of formula Ic-1 are those wherein
In yet another embodiment, preferred compounds of formula I are those of formula Id
and pharmaceutically acceptable salts, esters, esters, amides, phosphates, and solvates thereof, wherein
In one variant of the compounds of formula Id the dotted line is present.
Preferred compounds of formula Id are those of formula Id-1
wherein
R2 is as defined above in respect to formula I, preferably R2 is H, linear or branched C1-C4 alkyl, C1-C2 hydroxyalkyl, allyl, propargyl, cyclopropyl, cyclopentyl, cyclopentylmethyl, cyclopropylmethyl, benzyl, benzyloxyethyl, methoxyethyl, 1,1,1-trifluoroethyl, —C2H4CO2CH3, —CH2CO2CH3, or —CH2CONH2, more preferably R2 is H, methyl, ethyl, allyl, cyclopropyl, hydroxyethyl, —C2H4CO2CH3, —CH2CO2CH3, or —CH2CONH2, more preferably R2 is methyl or cyclopropyl.
In still another embodiment, preferred compounds of Formula I are those of formula Ie:
wherein
wherein
In still another embodiment, preferred compounds of Formula I are those of formula Ig:
wherein
In still another embodiment, preferred compounds of Formula I are those of formula Ih:
wherein
In still another embodiment, preferred compounds of Formula I are those of formula Ii
wherein
In still another embodiment, preferred compounds of Formula I are those of formula Ij:
wherein
In still another embodiment, preferred compounds of Formula I are those of formula Ik:
wherein
In still another embodiment, preferred compounds of Formula I are those of formula Il:
wherein
Particularly preferred compounds of the invention are those listed in Table 1 hereafter:
The compounds of formula I can be prepared by different ways with reactions known by the person skilled in the art. Reaction schemes as described in the example section illustrate by way of example different possible approaches.
The invention further provides the use of the compounds of the invention or pharmaceutically acceptable salts, or solvates thereof as agonists or partial agonists of G-protein coupled receptor 43 (GPR43).
Accordingly, in a particularly preferred embodiment, the invention relates to the use of compounds of formula I and subformulae in particular those of table 1 above, or pharmaceutically acceptable salts and solvates thereof, as GPR43 agonists or partial agonists.
The compounds of the invention are therefore useful in the prevention or in the prevention and/or treatment of type II diabetes, obesity, dyslipidemia such as mixed or diabetic dyslipidemia, hypercholesterolemia, low HDL cholesterol, high LDL cholesterol, hyperlipidemia, hypertriglyceridemia, hypoglycemia, hyperglycemia, glucose intolerance, insulin resistance, hyperinsulinemia hypertension, hyperlipoproteinemia, metabolic syndrome, syndrome X, thrombotic disorders, cardiovascular disease, atherosclerosis and its sequelae including angina, claudication, heart attack, stroke and others, kidney diseases, ketoacidosis, nephropathy, diabetic neuropathy, diabetic retinopathy, nonalcoholic fatty liver diseases such as steatosis or nonalcoholic steatohepatitis (NASH).
Preferred diseases are type II diabetes, lipid disorders such as dyslipidemia, hypertension, obesity, atherosclerosis and its sequelae.
In a particular preferred embodiment the diseases are type II diabetes and a lipid disorder such as dyslipidemia.
The invention also provides for a method for delaying in patient the onset of type II diabetes, obesity, dyslipidemia such as mixed or diabetic dyslipidemia, hypercholesterolemia, low HDL cholesterol, high LDL cholesterol, hyperlipidemia, hypertriglyceridemia, hypoglycemia, hyperglycemia, glucose intolerance, insulin resistance, hyperinsulinemia hypertension, hyperlipoproteinemia, metabolic syndrome, syndrome X, thrombotic disorders, cardiovascular disease, atherosclerosis and its sequelae including angina, claudication, heart attack, stroke and others, kidney diseases, ketoacidosis, nephropathy, diabetic neuropathy, diabetic retinopathy, nonalcoholic fatty liver diseases such as steatosis or nonalcoholic steatohepatitis (NASH)comprising the administration of a pharmaceutically effective amount of a compound of formula (I) or pharmaceutically acceptable salt thereof to a patient in need thereof.
Preferably, the patient is a warm-blooded animal, more preferably a human.
The invention further provides the use of a compound of formula (I) or a pharmaceutically acceptable salt or solvates thereof for the manufacture of a medicament for use in treating a patient and/or preventing a patient from developing a disease selected from the group consisting of type II diabetes, obesity, dyslipidemia such as mixed or diabetic dyslipidemia, hypercholesterolemia, low HDL cholesterol, high LDL cholesterol, hyperlipidemia, hypertriglyceridemia, hypoglycemia, hyperglycemia, glucose intolerance, insulin resistance, hyperinsulinemia hypertension, hyperlipoproteinemia, metabolic syndrome, syndrome X, thrombotic disorders, cardiovascular disease, atherosclerosis and its sequelae including angina, claudication, heart attack, stroke and others, kidney diseases, ketoacidosis, nephropathy, diabetic neuropathy, diabetic retinopathy, nonalcoholic fatty liver diseases such as steatosis or nonalcoholic steatohepatitis (NASH).
Preferably, the patient is a warm-blooded animal, more preferably a human.
According to a further feature of the present invention there is provided a method for modulating GPR43 receptor activity, in a patient, preferably a warm blooded animal, and even more preferably a human, in need of such treatment, which comprises administering to said animal an effective amount of compound of the present invention, or a pharmaceutically acceptable salt or solvate thereof.
According to one embodiment, the compounds of the invention, their pharmaceutical acceptable salts or solvates may be administered as part of a combination therapy. Thus, are included within the scope of the present invention embodiments comprising coadministration of, and compositions and medicaments which contain, in addition to a compound of the present invention, a pharmaceutically acceptable salt or solvate thereof as active ingredient, additional therapeutic agents and/or active ingredients. Such multiple drug regimens, often referred to as combination therapy, may be used in the treatment and/or prevention of any of the diseases or conditions mediated by or associated with GPR43 receptor modulation, particularly type II diabetes, obesity, dyslipidemia such as mixed or diabetic dyslipidemia, hypercholesterolemia, low HDL cholesterol, high LDL cholesterol, hyperlipidemia, hypertriglyceridemia, hypoglycemia, hyperglycemia, glucose intolerance, insulin resistance, hyperinsulinemia hypertension, hyperlipoproteinemia, metabolic syndrome, syndrome X, thrombotic disorders, cardiovascular disease, atherosclerosis and its sequelae including angina, claudication, heart attack, stroke and others, kidney diseases, ketoacidosis, nephropathy, diabetic neuropathy, diabetic retinopathy, nonalcoholic fatty liver diseases such as steatosis or nonalcoholic steatohepatitis (NASH). The use of such combinations of therapeutic agents is especially pertinent with respect to the treatment of the above-mentioned list of diseases within a patient in need of treatment or one at risk of becoming such a patient.
In addition to the requirement of therapeutic efficacy, which may necessitate the use of active agents in addition to the GPR43 agonist or partial agonist compounds of Formula I or their pharmaceutical acceptable salts or solvates thereof, there may be additional rationales which compel or highly recommend the use of combinations of drugs involving active ingredients which represent adjunct therapy, i.e., which complement and supplement the function performed by the GPR43 receptor agonist or partial agonist compounds of the present invention. Suitable supplementary therapeutic agents used for the purpose of auxiliary treatment include drugs which, instead of directly treating or preventing a disease or condition mediated by or associated with GPR43 receptor modulation, treat diseases or conditions which directly result from or indirectly accompany the basic or underlying GPR43 receptor modulated disease or condition.
Thus, the methods of treatment and pharmaceutical compositions of the present invention may employ the compounds of Formula I or their pharmaceutical acceptable salts or solvates thereof in the form of monotherapy, but said methods and compositions may also be used in the form of multiple therapy in which one or more compounds of Formula I or their pharmaceutically acceptable salts or solvates are coadministered in combination with one or more other therapeutic agents such as those described in detail further herein.
Examples of other active ingredients that may be administered in combination with a compound of Formula I or a pharmaceutically acceptable salt or solvate thereof, and either administered separately or in the same pharmaceutical composition, include but are not limited to:
The above combinations include combinations of a compound of the present invention or a pharmaceutically acceptable salt or solvate not only with one other active compound but also with two or more active compounds. Non limiting examples include combinations of compounds having Formula I with two or more active compounds selected from biguanides, sulfonylureas, HMG-CoA reductase inhibitors, other PPAR agonists, PTP-1B inhibitors, DP-IV inhibitors and anti-obesity compounds.
In the above-described embodiment combinations of the present invention, the compound of Formula I, a pharmaceutically acceptable salt or solvate thereof and other therapeutic active agents may be administered in terms of dosage forms either separately or in conjunction with each other, and in terms of their time of administration, either serially or simultaneously. Thus, the administration of one component agent may be prior to, concurrent with, or subsequent to the administration of the other component agent(s).
The invention also provides pharmaceutical compositions comprising a compound of formula I or a pharmaceutically acceptable salt or solvate thereof and at least one pharmaceutically acceptable carrier, diluent, excipient and/or adjuvant. As indicated above, the invention also covers pharmaceutical compositions which contain, in addition to a compound of the present invention, a pharmaceutically acceptable salt or solvate thereof as active ingredient, additional therapeutic agents and/or active ingredients.
Another object of this invention is a medicament comprising at least one compound of the invention, or a pharmaceutically acceptable salt or solvate thereof, as active ingredient.
The invention also provides the use of a compound of formula I or a pharmaceutically acceptable salt or solvate thereof for the manufacture of a medicament. Preferably, the medicament is used for the treatment and/or prevention of type II diabetes, obesity, dyslipidemia such as mixed or diabetic dyslipidemia, hypercholesterolemia, low HDL cholesterol, high LDL cholesterol, hyperlipidemia, hypertriglyceridemia, hypoglycemia, hyperglycemia, glucose intolerance, insulin resistance, hyperinsulinemia hypertension, hyperlipoproteinemia, metabolic syndrome, syndrome X, thrombotic disorders, cardiovascular disease, atherosclerosis and its sequelae including angina, claudication, heart attack, stroke and others, kidney diseases, ketoacidosis, nephropathy, diabetic neuropathy, diabetic retinopathy, nonalcoholic fatty liver diseases such as steatosis or nonalcoholic steatohepatitis (NASH).
Preferred diseases are type II diabetes, lipid disorders such as dyslipidemia, hypertension, obesity, atherosclerosis and its sequelae.
In a particular preferred embodiment the disease are type II diabetes and a lipid disorder such as dyslipidemia.
According to a further feature of the present invention there is provided the use of a compound of formula I or a pharmaceutically acceptable salt or solvate thereof for the manufacture of a medicament for modulating GPR43 receptor activity, in a patient, in need of such treatment, which comprises administering to said patient an effective amount of compound of the present invention, or a pharmaceutically acceptable salt or solvate thereof.
Preferably, the patient is a warm-blooded animal, more preferably a human.
As set forth above, the compounds of the invention, their pharmaceutically acceptable salts or solvates may be used in monotherapy or in combination therapy. Thus, according to one embodiment, the invention provides the use of a compound of the invention for the manufacture of a medicament for at least one of the purposes described above, wherein said medicament is administered to a patient in need thereof, preferably a warm-blooded animal, and even more preferably a human, in combination with at least one additional therapeutic agent and/or active ingredient. The benefits and advantages of such a multiple drug regimen, possible administration regimens as well as suitable additional therapeutic agents and/or active ingredients are those described above.
Generally, for pharmaceutical use, the compounds of the inventions may be formulated as a pharmaceutical preparation comprising at least one compound of the invention and at least one pharmaceutically acceptable carrier, diluent, excipient and/or adjuvant, and optionally one or more further pharmaceutically active compounds.
By means of non-limiting examples, such a formulation may be in a form suitable for oral administration, for parenteral administration (such as by intravenous, intramuscular or subcutaneous injection or intravenous infusion), for topical administration (including ocular), for administration by inhalation, by a skin patch, by an implant, by a suppository, etc. Such suitable administration forms—which may be solid, semi-solid or liquid, depending on the manner of administration—as well as methods and carriers, diluents and excipients for use in the preparation thereof, will be clear to the skilled person; reference is made to the latest edition of Remington's Pharmaceutical Sciences.
Some preferred, but non-limiting examples of such preparations include tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols, ointments, creams, lotions, soft and hard gelatin capsules, suppositories, drops, sterile injectable solutions and sterile packaged powders (which are usually reconstituted prior to use) for administration as a bolus and/or for continuous administration, which may be formulated with carriers, excipients, and diluents that are suitable per se for such formulations, such as lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, polyethylene glycol, cellulose, (sterile) water, methylcellulose, methyl- and propylhydroxybenzoates, talc, magnesium stearate, edible oils, vegetable oils and mineral oils or suitable mixtures thereof. The formulations can optionally contain other substances that are commonly used in pharmaceutical formulations, such as lubricating agents, wetting agents, emulsifying and suspending agents, dispersing agents, desintegrants, bulking agents, fillers, preserving agents, sweetening agents, flavoring agents, flow regulators, release agents, etc. The compositions may also be formulated so as to provide rapid, sustained or delayed release of the active compound(s) contained therein.
The pharmaceutical preparations of the invention are preferably in a unit dosage form, and may be suitably packaged, for example in a box, blister, vial, bottle, sachet, ampoule or in any other suitable single-dose or multi-dose holder or container (which may be properly labeled); optionally with one or more leaflets containing product information and/or instructions for use. Generally, such unit dosages will contain between 0.05 and 1000 mg, and usually between 1 and 500 mg, of the at least one compound of the invention, e.g. about 10, 25, 50, 100, 200, 300 or 400 mg per unit dosage.
Usually, depending on the condition to be prevented or treated and the route of administration, the active compound of the invention will usually be administered between 0.01 to 100 mg per kilogram, more often between 0.1 and 50 mg, such as between 1 and 25 mg, for example about 0.5, 1, 5, 10, 15, 20 or 25 mg, per kilogram body weight day of the patient per day, which may be administered as a single daily dose, divided over one or more daily doses, or essentially continuously, e.g. using a drip infusion.
The definitions and explanations below are for the terms as used throughout the entire application, including both the specification and the claims.
When describing the compounds of the invention, the terms used are to be construed in accordance with the following definitions, unless indicated otherwise.
Where groups may be substituted, such groups may be substituted with one or more substituents, and preferably with one, two or three substituents. Substituents may be selected from but not limited to, for example, the group comprising halogen, hydroxyl, oxo, nitro, amido, carboxy, amino, cyano haloalkoxy, and haloalkyl.
As used herein the terms such as “alkyl, aryl, or cycloalkyl, each being optionally substituted with . . . ” or “alkyl, aryl, or cycloalkyl, optionally substituted with . . . ” encompasses “alkyl optionally substituted with . . . ”, “aryl optionally substituted with . . . ” and “cycloalkyl optionally substituted with . . . ”.
The term “halo” or “halogen” means fluoro, chloro, bromo, or iodo. Preferred halo groups are fluoro and chloro.
The term “alkyl” by itself or as part of another substituent refers to a hydrocarbyl radical of Formula CnH2n+1 wherein n is a number greater than or equal to 1. Generally, alkyl groups of this invention comprise from 1 to 6 carbon atoms, preferably from 1 to 4 carbon atoms, more preferably from 1 to 3 carbon atoms, still more preferably 1 to 2 carbon atoms. Alkyl groups may be linear or branched and may be substituted as indicated herein.
Suitable alkyl groups include methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl and t-butyl, pentyl and its isomers (e.g. n-pentyl, iso-pentyl), and hexyl and its isomers (e.g. n-hexyl, iso-hexyl). Preferred alkyl groups include methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl and t-butyl.
When the suffix “ene” (“alkylene”) is used in conjunction with an alkyl group, this is intended to mean the alkyl group as defined herein having two single bonds as points of attachment to other groups. The term “alkylene” includes methylene, ethylene, methylmethylene, propylene, ethylethylene, and 1,2-dimethylethylene.
The term “alkenyl” as used herein refers to an unsaturated hydrocarbyl group, which may be linear or branched, comprising one or more carbon-carbon double bonds. Suitable alkenyl groups comprise between 2 and 6 carbon atoms, preferably between 2 and 4 carbon atoms, still more preferably between 2 and 3 carbon atoms. Examples of alkenyl groups are ethenyl, 2-propenyl, 2-butenyl, 3-butenyl, 2-pentenyl and its isomers, 2-hexenyl and its isomers, 2,4-pentadienyl and the like.
The term “alkynyl” as used herein refers to a class of monovalent unsaturated hydrocarbyl groups, wherein the unsaturation arises from the presence of one or more carbon-carbon triple bonds. Alkynyl groups typically, and preferably, have the same number of carbon atoms as described above in relation to alkenyl groups. Non limiting examples of alkynyl groups are ethynyl, 2-propynyl, 2-butynyl, 3-butynyl, 2-pentynyl and its isomers, 2-hexynyl and its isomers-and the like. The terms “alkenylene” and “alkynylene” respectively mean an alkenyl group or an alkinyl group as defined above having two single bonds as points of attachment to other groups.
The term “haloalkyl” alone or in combination, refers to an alkyl radical having the meaning as defined above wherein one or more hydrogens are replaced with a halogen as defined above. Non-limiting examples of such haloalkyl radicals include chloromethyl, 1-bromoethyl, fluoromethyl, difluoromethyl, trifluoromethyl, 1,1,1-trifluoroethyl and the like.
The term “cycloalkyl” as used herein is a cyclic alkyl group, that is to say, a monovalent, saturated, or unsaturated hydrocarbyl group having 1 or 2 cyclic structures. Cycloalkyl includes monocyclic or bicyclic hydrocarbyl groups. Cycloalkyl groups may comprise 3 or more carbon atoms in the ring and generally, according to this invention comprise from 3 to 10, more preferably from 3 to 8 carbon atoms still more preferably from 3 to 6 carbon atoms. Examples of cycloalkyl groups include but are not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, with cyclopropyl being particularly preferred.
When the suffix “ene” is used in conjunction with a cyclic group, this is intended to mean the cyclic group as defined herein having two single bonds as points of attachment to other groups.
Therefore, “cycloalkylene” herein refers to a saturated homocyclic hydrocarbyl biradical of Formula CnH2n−2. Suitable cycloalkylene groups are C3-6 cycloalkylene group, preferably a C3-5 cycloalkylene (i.e. 1,3-cyclopropylene, 1,1-cyclopropylene, 1,1-cyclobutylene, 1,2-cyclobutylene, 1,3-cyclopentylene, or 1,1-cyclopentylene), more preferably a C3-4 cycloalkylene (i.e. 1,3-cyclopropylene, 1,1-cyclopropylene, 1,1-cyclobutylene, 1,2-cyclobutylene).
Where at least one carbon atom in a cycloalkyl group is replaced with a heteroatom, the resultant ring is referred to herein as “heterocycloalkyl” or “heterocyclyl”.
The terms “heterocyclyl”, “heterocycloalkyl” or “heterocyclo” as used herein by itself or as part of another group refer to non-aromatic, fully saturated or partially unsaturated cyclic groups (for example, 3 to 7 member monocyclic, 7 to 11 member bicyclic, or containing a total of 3 to 10 ring atoms) which have at least one heteroatom in at least one carbon atom-containing ring. Each ring of the heterocyclic group containing a heteroatom may have 1, 2, 3 or 4 heteroatoms selected from nitrogen, oxygen and/or sulfur atoms, where the nitrogen and sulfur heteroatoms may optionally be oxidized and the nitrogen heteroatoms may optionally be quaternized. Any of the carbon atoms of the heterocyclic group may be substituted by oxo (for example piperidone, pyrrolidinone).The heterocyclic group may be attached at any heteroatom or carbon atom of the ring or ring system, where valence allows. The rings of multi-ring heterocycles may be fused, bridged and/or joined through one or more spiro atoms. Non limiting exemplary heterocyclic groups include oxetanyl, piperidinyl, azetidinyl, 2-imidazolinyl, pyrazolidinyl imidazolidinyl, isoxazolinyl, oxazolidinyl, isoxazolidinyl, thiazolidinyl, isothiazolidinyl, piperidinyl, 3H-indolyl, indolinyl, isoindolinyl, 2-oxopiperazinyl, piperazinyl, homopiperazinyl, 2-pyrazolinyl, 3-pyrazolinyl, tetrahydro-2H-pyranyl, 2H-pyranyl, 4H-pyranyl, 3,4-dihydro-2H-pyranyl, 3-dioxolanyl, 1,4-dioxanyl, 2,5-dioximidazolidinyl, 2-oxopiperidinyl, 2-oxopyrrolodinyl, indolinyl, tetrahydropyranyl, tetrahydrofuranyl, tetrahydroquinolinyl, tetrahydroisoquinolin-1-yl, tetrahydroisoquinolin-2-yl, tetrahydroisoquinolin-3-yl, tetrahydroisoquinolin-4-yl, thiomorpholin-4-yl, thiomorpholin-4-ylsulfoxide, thiomorpholin-4-ylsulfone, 1,3-dioxolanyl, 1,4-oxathianyl, 1H-pyrrolizinyl, tetrahydro-1,1-dioxothiophenyl, N-formylpiperazinyl, and morpholin-4-yl.
The ring atoms of heterocyclyl and heterocyclylene moieties are numbered based on scheme below
The term “aryl” as used herein refers to a polyunsaturated, aromatic hydrocarbyl group having a single ring (i.e. phenyl) or multiple aromatic rings fused together (e.g. naphthyl) or linked covalently, typically containing 5 to 12 atoms; preferably 6 to 10, wherein at least one ring is aromatic. The aromatic ring may optionally include one to two additional rings (either cycloalkyl, heterocyclyl or heteroaryl) fused thereto. Aryl is also intended to include the partially hydrogenated derivatives of the carbocyclic systems enumerated herein. Non-limiting examples of aryl comprise phenyl, biphenylyl, biphenylenyl, 5- or 6-tetralinyl, naphthalen-1- or -2-yl, 4-, 5-, 6 or 7-indenyl, 1- 2-, 3-, 4- or 5-acenaphtylenyl, 3-, 4- or 5-acenaphtenyl, 1- or 2-pentalenyl, 4- or 5-indanyl, 5-, 6-, 7- or 8-tetrahydronaphthyl, 1,2,3,4-tetrahydronaphthyl, 1,4-dihydronaphthyl, 1-, 2-, 3-, 4- or 5-pyrenyl.
The term “arylene” as used herein is intended to include divalent carbocyclic aromatic ring systems such as phenylene, biphenylylene, naphthylene, indenylene, pentalenylene, azulenylene and the like. Arylene is also intended to include the partially hydrogenated derivatives of the carbocyclic systems enumerated above. Non-limiting examples of such partially hydrogenated derivatives are 1,2,3,4-tetrahydronaphthylene, 1,4-dihydronaphthylene and the like.
Where at least one carbon atom in an aryl group is replaced with a heteroatom, the resultant ring is referred to herein as a heteroaryl ring.
The term “heteroaryl” as used herein by itself or as part of another group refers but is not limited to 5 to 12 carbon-atom aromatic rings or ring systems containing 1 to 2 rings which are fused together or linked covalently, typically containing 5 to 6 atoms; at least one of which is aromatic, in which one or more carbon atoms in one or more of these rings is replaced by oxygen, nitrogen and/or sulfur atoms where the nitrogen and sulfur heteroatoms may optionally be oxidized and the nitrogen heteroatoms may optionally be quaternized. Such rings may be fused to an aryl, cycloalkyl, heteroaryl or heterocyclyl ring. Non-limiting examples of such heteroaryl, include: furanyl, thiophenyl, pyrazolyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, triazolyl, oxadiazolyl, thiadiazolyl, tetrazolyl, oxatriazolyl, thiatriazolyl, pyridinyl, pyrimidyl, pyrazinyl, pyridazinyl, oxazinyl, dioxinyl, thiazinyl, triazinyl, imidazo[2,1-b][1,3]thiazolyl, thieno[3,2-b]furanyl, thieno[3,2-b]thiophenyl, thieno[2,3-d][1,3]thiazolyl, thieno[2,3-d]imidazolyl, tetrazolo[1,5-a]pyridinyl, indolyl, indolizinyl, isoindolyl, benzofuranyl, isobenzofuranyl, benzothiophenyl, isobenzothiophenyl, indazolyl, benzimidazolyl, 1,3-benzoxazolyl, 1,2-benzisoxazolyl, 2,1-benzisoxazolyl, 1,3-benzothiazolyl, 1,2-benzoisothiazolyl, 2,1-benzoisothiazolyl, benzotriazolyl, 1,2,3-benzoxadiazolyl, 2,1,3-benzoxadiazolyl, 1,2,3-benzothiadiazolyl, 2,1,3-benzothiadiazolyl, thienopyridinyl, purinyl, imidazo[1,2-a]pyridinyl, 6-oxo-pyridazin-1(6H)-yl, 2-oxopyridin-1(2H)-yl, 6-oxo-pyridazin-1(6H)-yl, 2-oxopyridin-1(2H)-yl, 1,3-benzodioxolyl, quinolinyl, isoquinolinyl, cinnolinyl, quinazolinyl, quinoxalinyl.
The term “heteroarylene” as used herein means divalent carbocyclic aromatic ring systems including pyridinylene and the like.
The ring atoms of heteroaryl or heteroarylene moieties are numbered on scheme below:
The term “biaryl” as used herein designates two aryl moieties as defined herein linked via a single bond. Non-limiting examples of such biaryl moieties include biphenyl.
The term “heterobiaryl” as used herein designates two heteroaryl moieties as defined herein or a heteroaryl moiety and an aryl moiety as defined herein linked via a single bond. Non-limiting examples of such heterobiaryl moieties include pyridinylphenyl which is meant to include (2-pyridinyl)phenyl, (3-pyridinyl)phenyl and (4-pyridinyl)phenyl, bipyridinyl.
The term “alkylamino” as used herein means an amino group substituted with one or two alkyl groups. This includes monoalkylamino and dialkylamino groups.
The compounds of Formula I and subformulae thereof contain at least one asymmetric center and thus may exist as different stereoisomeric forms. Accordingly, the present invention includes all possible stereoisomers and includes not only racemic compounds but the individual enantiomers and their non racemic mixtures as well. When a compound is desired as a single enantiomer, such may be obtained by stereospecific synthesis, by resolution of the final product or any convenient intermediate, or by chiral chromatographic methods as each are known in the art. Resolution of the final product, an intermediate, or a starting material may be effected by any suitable method known in the art. See, for example, Stereochemistry of Organic Compounds by E. L. Eliel, S. H. Wilen, and L. N. Mander (Wiley-Interscience, 1994), incorporated by reference with regard to stereochemistry.
The bonds from an asymmetric carbon in compounds of the present invention may be depicted herein using a solid line (), a zigzag line (
), a solid wedge (
), or a dotted wedge (
). The use of a solid line to depict bonds from an asymmetric carbon atom is meant to indicate that all possible stereoisomers are meant to be included, unless it is clear from the context that a specific stereoisomer is intended. The use of either a solid or dotted wedge to depict bonds from an asymmetric carbon atom is meant to indicate that only the stereoisomer shown is meant to be included.
The compounds of the invention may also contain more than one asymmetric carbon atom. In those compounds, the use of a solid line to depict bonds from asymmetric carbon atoms is meant to indicate that all possible stereoisomers are meant to be included, unless it is clear from the context that a specific stereoisomer is intended.
The compounds of the invention may be in the form of pharmaceutically acceptable salts. Pharmaceutically acceptable salts of the compounds of formula I include the acid addition and base salts thereof. Suitable acid addition salts are formed from acids which form non-toxic salts. Examples include the acetate, adipate, aspartate, benzoate, besylate, bicarbonate/carbonate, bisulphate/sulphate, borate, camsylate, citrate, cyclamate, edisylate, esylate, formate, fumarate, gluceptate, gluconate, glucuronate, hexafluorophosphate, hibenzate, hydrochloride/chloride, hydrobromide/bromide, hydroiodide/iodide, isethionate, lactate, malate, maleate, malonate, mesylate, methylsulphate, naphthylate, 2-napsylate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate/hydrogen phosphate/dihydrogen phosphate, pyroglutamate, saccharate, stearate, succinate, tannate, tartrate, tosylate, trifluoroacetate and xinofoate salts. Suitable base salts are formed from bases which form non-toxic salts. Examples include the aluminium, arginine, benzathine, calcium, choline, diethylamine, diolamine, glycine, lysine, magnesium, meglumine, olamine, potassium, sodium, tromethamine, 2-(diethylamino)ethanol, ethanolamine, morpholine, 4-(2-hydroxyethyl)morpholine and zinc salts. Hemisalts of acids and bases may also be formed, for example, hemisulphate and hemicalcium salts. Preferred, pharmaceutically acceptable salts include hydrochloride/chloride, hydrobromide/bromide, bisulphate/sulphate, nitrate, citrate, and acetate.
When the compounds of the invention contain an acidic group as well as a basic group the compounds of the invention may also form internal salts, and such compounds are within the scope of the invention. When the compounds of the invention contain a hydrogen-donating heteroatom (e.g. NH), the invention also covers salts and/or isomers formed by transfer of said hydrogen atom to a basic group or atom within the molecule.
Pharmaceutically acceptable salts of compounds of Formula I may be prepared by one or more of these methods:
(i) by reacting the compound of Formula I with the desired acid;
(ii) by reacting the compound of Formula I with the desired base;
(iii) by removing an acid- or base-labile protecting group from a suitable precursor of the compound of Formula I or by ring-opening a suitable cyclic precursor, for example, a lactone or lactam, using the desired acid; or
(iv) by converting one salt of the compound of Formula I to another by reaction with an appropriate acid or by means of a suitable ion exchange column.
All these reactions are typically carried out in solution. The salt, may precipitate from solution and be collected by filtration or may be recovered by evaporation of the solvent. The degree of ionization in the salt may vary from completely ionized to almost non-ionized.
The term “solvate” is used herein to describe a molecular complex comprising the compound of the invention and one or more pharmaceutically acceptable solvent molecules, for example, ethanol. The term ‘hydrate’ is employed when said solvent is water.
All references to compounds of formula I include references to salts, solvates, multi-component complexes and liquid crystals thereof.
The compounds of the invention include compounds of formula I as hereinbefore defined, including all polymorphs and crystal habits thereof, prodrugs and isomers thereof (including optical, geometric and tautomeric isomers) and isotopically-labeled compounds of formula I.
In addition, although generally, with respect to the salts of the compounds of the invention, pharmaceutically acceptable salts are preferred, it should be noted that the invention in its broadest sense also included non-pharmaceutically acceptable salts, which may for example be used in the isolation and/or purification of the compounds of the invention. For example, salts formed with optically active acids or bases may be used to form diastereoisomeric salts that can facilitate the separation of optically active isomers of the compounds of Formula I above.
The invention also generally covers all pharmaceutically acceptable predrugs and prodrugs of the compounds of Formula I.
The term “prodrug” as used herein means the pharmacologically acceptable derivatives of compounds of formula I such as esters whose in vivo biotransformation product is the active drug. Prodrugs are characterized by increased bio-availability and are readily metabolized into the active compounds in vivo. Suitable prodrugs for the purpose of the invention include carboxylic esters, in particular alkyl esters, aryl esters, acyloxyalkyl esters, and dioxolene carboxylic esters; ascorbic acid esters as well as compounds of formula I in which Z is a substituent selected from the table 2 below.
The term “predrug”, as used herein, means any compound that will be modified to form a drug species, wherein the modification may take place either inside or outside of the body, and either before or after the predrug reaches the area of the body where administration of the drug is indicated.
The term “patient” refers to a warm-blooded animal, more preferably a human, who/which is awaiting or receiving medical care or is or will be the object of a medical procedure.
The term “human” refers to subject of both genders and at any stage of development (i.e. neonate, infant, juvenile, adolescent, adult).
The terms “treat”, “treating” and “treatment, as used herein, are meant to include alleviating or abrogating a condition or disease and/or its attendant symptoms.
The terms “prevent”, “preventing” and “prevention”, as used herein, refer to a method of delaying or precluding the onset of a condition or disease and/or its attendant symptoms, barring a patient from acquiring a condition or disease, or reducing a patient's risk of acquiring a condition or disease.
The term “therapeutically effective amount” (or more simply an “effective amount”) as used herein means the amount of active agent or active ingredient (e. g. GPR43 agonist or partial agonist) which is sufficient to achieve the desired therapeutic or prophylactic effect in the individual to which it is administered.
The term “administration”, or a variant thereof (e.g., “administering”), means providing the active agent or active ingredient (e. g. a GPR43 agonist or partial agonist), alone or as part of a pharmaceutically acceptable composition, to the patient in whom/which the condition, symptom, or disease is to be treated or prevented.
By “pharmaceutically acceptable” is meant that the ingredients of a pharmaceutical composition are compatible with each other and not deleterious to the patient thereof.
The term “agonist” as used herein means a ligand that activates an intracellular response when it binds to a receptor. An agonist according to the invention may promote internalization of a cell surface receptor such that the cell surface concentration of a receptor is decreased or remove.
The term “partial agonist” as used herein means an agonist which is unable to induce maximal activation of a receptor, regardless of the amount of compound applied on the receptor.
The term “pharmaceutical vehicle” as used herein means a carrier or inert medium used as solvent or diluent in which the pharmaceutically active agent is formulated and/or administered. Non-limiting examples of pharmaceutical vehicles include creams, gels, lotions, solutions, and liposomes.
The term “lipid disorder” as used herein means any plasma lipid disorder including but not limited to dyslipidemia such as mixed or diabetic dyslipidemia, hypercholesterolemia, low HDL cholesterol, high LDL cholesterol, hyperlipidemia and hypertriglyceridemia.
The present invention will be better understood with reference to the following examples. These examples are intended to representative of specific embodiments of the invention, and are not intended as limiting the scope of the invention.
All temperatures are expressed in ° C. and all reactions were carried out at room temperature (RT) unless otherwise stated.
Analytical thin layer chromatography (TLC) was used to monitor reactions, establish flash chromatography conditions and verify purity of intermediates or final products. TLC plates used were Merck TLC aluminium sheet silica gel 60 F254 purchased from VWR International. TLC plates were revealed using ultraviolet irradiation (wavelength=254 nm) at room temperature or bromocresol green spray reagent at 0.1% in propan-2-ol purchased from VWR International upon heating at 160° C. or KMnO4 revelator upon heating at 160° C. The KMnO4 revelator was prepared by dissolving 3 g of potassium permanganate, 20 g of sodium carbonate, 0.5 g of sodium hydroxide in 100 mL of distilled water.
HPLC-MS spectra were obtained on Agilent LCMS using Electrospray ionization (ESI). The Agilent instrument includes an Autosampler 1200, a binary pump 1100, a 5 wave length detector 1100 and a 6100 Single Quad. The column used was an XBridge C18, 4.6×50 mm, 3.5 μm.
Eluent was a mixture of solution A (0.1% TFA in H2O) and solution B (0.1% TFA in ACN). Gradient was applied at a flow rate of 2 mL min−1 as follows: gradient A: held the initial conditions of 5% solution B for 1 min, increased linearly to 95% solution B in 4 min, held at 95% during 1 min, returned to initial conditions in 0.5 min and maintained for 1 min; gradient B: held the initial conditions of 5% solution B for 1 min, increased linearly to 60% in 10 min, increased linearly to 95% in 0.5 min, held at 95% during 3 min, returned to initial conditions in 0.5 min and maintained for 1 min.
Determination of ee was performed on an Agilent 1100 (binary pump and 5 wavelengths detector) with manual or automatic (Autosampler 1100) injection. Columns used were CHIRALPAK IA CHIRALPAK IB or CHIRALPAK IC in isocratic mode. Mixtures of eluents were selected depending on the separation obtained of enantiomers or diastereosiomers. Usual mixtures were:
Hexane and Ethanol (0.1% TFA)
Hexane and Propanol (0.1% TFA)
Hexane and Ethyl acetate (0.1% TFA)
Hexane and Dichloromethane (0.1% TFA)
Hexane and tert-butyl methyl ether (0.1% TFA)
Selected specific methods A, B and C are reported below. Method A: compound was characterized on a CHIRALPAK IA column (isocratic mode) using a mixture of hexane and dichloromethane (65/35) acidified by 0.4% of TFA at a flow rate of 1.2 mL/min, and confirmed on a CHIRALPAK IC column (isocratic mode) using a mixture of heptane and Ethyl acetate (75/25) acidified by 0.1% of TFA at 1 ml/min. Method B: compound was characterized on a CHIRALPAK IC column (isocratic mode) using a mixture of heptane and ethyl acetate (70/30) acidified by 0.1% of TFA at a flow rate of lml/min. Method C: compound was characterized on a CHIRALPAK IC column (isocratic mode) using a mixture of heptane and ethanol (95/5) acidified by 0.1% of TFA at a flow rate of 1.5 ml/min.
Preparative HPLC purifications were carried out on Fractionlynx instrument, from Waters. This instrument consists of a Fraction Collector, a 2767 Sample Manager, a pump control a module II, a 515 HPLC Pump, a 2525 Binary Gradient Module, a Switching Valve, a 2996 Photodiode Array Detector and a Micromass ZQ. The column used was a Waters Sunfire C18 Eluent was a mixture of solution A (0.1% TFA in H2O) and solution B (0.1% TFA in ACN). The gradient was adapted depending on impurities present in samples, to allow sufficient separation between impurities and target compound.
Chiral preparative HPLC purification were performed on an Agilent 1100 instrument (binary pump and 5 wavelengths detector) with manual injection using a CHIRALPAK IA or a CHIRALPAK IB column in isocratic mode. Mixtures of eluents were selected depending on the separation of enantiomers or diastereosiomers obtained with the analytical method. Usual mixtures were the same as those used for the determination of ee.
1H and 13C NMR spectra were recorded on a Bruker ARX 300 MHz. Chemical shifts are expressed in parts per million, (ppm, δ units). Coupling constants are expressed in Hertz units (Hz). Splitting patterns describe apparent multiplicities and are described as s (singlet), d (doublet), t (triplet), q (quintet), m (multiplet), or br (broad).
Solvents, reagents and starting materials were purchased from well known chemical suppliers such as for example Sigma Aldrich, Acros Organics, Fluorochem, Eurisotop, VWR International, Sopachem and Polymer labs and the following abbreviations are used:
Most compounds of the invention are synthesized according to Scheme 1.
Chiral syntheses of intermediates 1 were carried out using Evans' chiral auxiliary approach (Evans et al. J. Org. Chem. 1999, 64, 6411-6417; Tararov et al. J. Chem. Soc. Perkin Trans. 1, 1997, 3101-3106) (Scheme 2).
This methodology was also used for the synthesis of (R)-cycloalkylalkylsuccinic acid, (R)-heterocyclylalkylsuccinic acid, (R)-arylalkylsuccinic acid and (R)-heteroarylalkylsuccinic acid monoester intermediates 1.
As depicted on Scheme 3, (R)-benzylsuccinic acid monoester intermediates 1 can also be made starting from maleic anhydride followed by the application of Wittig reaction, asymmetric hydrogenation (Wallace et al. Org. Proc. Res. & Dev. 2004, 8, 738-743), tBu ester protection and selective saponification of the methyl ester (Atkinson et al. J. Org. Chem. 1999, 64, 3467).
This methodology was also used for the synthesis of (R)-cycloalkylalkylsuccinic acid, (R)-heterocyclylalkylsuccinic acid, (R)-arylalkylsuccinic acid and (R)-heteroarylalkylsuccinic acid monoester intermediates 1.
4-aryl-2-amino-thiazoles can be made using Hantzsch-type synthetic methodology as shown in Scheme 4. Thus, halogenation of substituted acetophenones (Larock, R. C. Comprehensive Org Transf 2nd Ed., Wiley, 1999, pp 709-719; White et al. J. Med. Chem. 1996, 39, 4382-95) and subsequent condensation with thiourea (Swain et al. J. Med. Chem. 1991, 34, 140-151; Barton et al. J. Med. Chem. 1998, 41, 1855-68) will furnish 4-aryl-2-amino-thiazoles.
Alternatively, synthesis of N-substituted-4-aryl-2-amino-thiazoles can be achieved through the method described by Rudolph (Rudolph, J. Tetrahedron 2000, 56, 3161)
Synthetic routes for the preparation of selected bioisosteres of the carboxylic acid moiety are given hereunder. Isosterism is a concept defined by I. Langmuir in J. Am. Chem. Soc. 1919, 41, 1549 and developed by H. L. Friedman in Symposium on Chemical-Biological correlations, National Council Publication, Washington, D.C. (1951). As used herein the term “bioisosteres” refers to “groups or molecules which have chemical and physical similarities producing similar biological effects” (as defined in Chem. Soc. Rev. 1979, 8, 563). Suitable well-known bioisosteric replacements of carboxylic acid groups and synthetic routes are reported in The Practice of Medicinal Chemistry, 2nd edition, by C. G. Wermuth. It is obvious to the person skilled in the art to synthesize carboxylic acid isosteres, selected useful references are Drysdale et al. J. Med. Chem. 1992, 35, 2573-2581, Liljebris et al. J. Med. Chem. 2002, 45, 1785-1798.
The tetrazole and hydroxy-oxadiazole isosteres can be synthesized using a common nitrile intermediate (see Scheme below). (Arienti et al. J. Med. Chem. 2005, 48, 6, 1882; Rodriguez et al. Tetrahedron 1997, 38, 24, 4221; Claremon et al. Tet. Lett. 1988, 28, 2155).
Treatment of the aforesaid nitrile intermediate with sodium azide can be used to afford the tetrazole isostere (see Scheme below). (Matthews et al. J. Comb. Chem. 2000, 2, 19-23)
Treatment of the aforesaid nitrile intermediate with hydroxylamine, followed by dehydrative cyclization can be used to yield the hydroxy-oxadiazole isostere (see Scheme below) (Peretto et al. J. Med. Chem. 2005, 48, 5705-5720).
In addition, synthetic approaches to the preparation of other well-recognized carboxylic acid isosteres are outlined below.
An alternative approach towards synthesis of intermediates 1 (see Scheme 2) can be envisioned through Stobbe condensation as depicted in Scheme 6.
Synthesis of Compound no68 (Scheme 7):
As shown in Scheme 7, upon treatment of (R)-benzylsuccinic acid t-butyl ester with excess LiHMDS in the presence of MeI, the desired monomethylated intermediate was isolated as an epimeric mixture, which was used in turn to furnish the final target structure (as epimeric mixture), as per the general procedure outlined on Scheme 1.
Suzuki coupling between pyridinyl or pyrimidinyl chloride and phenylboronic acid reagents allowed synthesizing the aryl-pyridine and aryl-pyrimidines intermediates 2.
A Suggested Synthesis of Compound no74 (Scheme 9):
Suggested Syntheses of Compounds no75 and no76 (Scheme 10):
Suggested Syntheses of Compounds no79 and no80 (Scheme 11):
Suggested Syntheses of Compounds no83 to no85 (Scheme 12):
The HWE methodology as depicted in Scheme 13 is the preferred methodology of the invention for the synthesis of intermediates 1.
Synthesis of Compound no198 (Scheme 18):
General Synthetic Scheme for the Preparation of Substituted Acetophenone Reagents through Weinreb Amide Approach (Scheme 20):
Synthesis of Compound no238 (Scheme 22):
(S)-4-benzyloxazolidin-2-one (0.011 mol) was dissolved in THF (50 mL). A 1.6 M solution of n-BuLi (0.0124 mol) was added dropwise at −78° C. A solution of 2-phenylacetyl chloride (0.011 mol) in THF (20 mL) was added dropwise to the obtained dark solution at the same temperature. The reaction mixture was stirred for 1 h at −78° C. Then a saturated solution of NH4Cl (2 mL) and a solution of NaHCO3 (4 mL) were added dropwise, and the reaction mixture was warmed to RT. The organic layer was separated, and the aqueous one was extracted with diethyl ether (3×25 mL). The combined extracts were washed with water, brine, dried over Na2SO4, and evaporated. The residue was purified by chromatography (silica gel, hexane/ether, 2/1) to yield title compound. Y: 2.1 g (64.7%).
A 1M solution of NaHMDS (7.8 mmol) in THF was added to a solution of (S)-4-benzyl-3-(2-phenylacetyl)oxazolidin-2-one (7.1 mmol) in THF at −78° C. in a flow of argon. After keeping for 1.5 h at the same temperature tert-butyl bromoacetate (21.3 mmol) was added. The reaction mixture was stirred for 2 h at −78° C. and warmed to RT. A saturated solution of NH4Cl (15 mL) and ethyl acetate (12 mL) were added. The organic layer was separated, and the aqueous one was extracted with ethyl acetate (3×30 mL). The combined extracts were washed with brine, dried over Na2SO4, and evaporated to give title compound. Y: 1.64 g (57%).
(S)-Tert-butyl 4-((S)-4-benzyl-2-oxooxazolidin-3-yl)-4-oxo-3-phenylbutanoate (4 mmol) was dissolved in THF, and a 35% solution of H2O2 in water (16 mmol) was added dropwise at 0° C. Then a solution of LiOH (8 mmol) in H2O (19 mL) was added. The reaction mixture was stirred for 1.5 h at 0° C. (TLC: CCl4/ethyl acetate=7/3) indicated reaction was complete. A solution of Na2SO3 (15 mL) and NaHCO3 (15 mL) were added at 0° C. The reaction mixture was evaporated in a rotary evaporator by one half. Water (50 mL) was added to the residue, and the mixture was extracted with CH2Cl2 (3×45 mL). The aqueous layer was acidified with 6M HCl to pH=2 at 0° C. The product was extracted with ethyl acetate (3×50 mL). Combined extracts were washed with brine, dried over Na2SO4, and evaporated. The residue was recrystallized from hexane to give title compound. Y: 0.75 g (75%).
The following intermediates were synthesized or may be synthesized using general method B adapting the oxazolidinone chirality and starting materials to targeted intermediate:
A solution of maleic anhydride (105 g, 1.07 mol) was added dropwise to a solution of triphenylphosphine (270 g, 1.03 mol) in acetone (1.2 L). The reaction mixture was stirred overnight at room temperature, cooled to 5° C., and filtered. The product was washed with acetone (2×100 mL), diethyl ether (100 mL), and dried under vacuum to give title compound. Y: 360 g (97%), rt=3.21 min (gradient A), (M+H)+=379.
A solution of 3-(triphenylphosphoranylidene)dihydrofuran-2,5-dione (110 g, 0.305 mol) in methanol (600 mL) was stirred overnight at room temperature and evaporated. The residue was recrystallized from ethyl acetate (500 mL) to give title compound. Y: 98 g (81%) rt=3.32 min (gradient A), (M+H)+=393.
4-methoxy-4-oxo-3-(triphenylphosphoranylidene)butanoic acid (50 g, 0.127 mol) was suspended in benzene (100 mL). A solution of benzaldehyde (14.8 g, 0.14 mol) in a mixture of dichloromethane (30 mL) and benzene (7.5 mL) was added dropwise. The reaction mixture was stirred at RT for 20 h, diluted with diethyl ether (200 mL), and extracted with a solution of potassium bicarbonate (0.23 mol) in water (300 mL). The organic layer was discarded and the aqueous one was washed with a mixture of benzene (200 mL) and ether (100 mL). The aqueous solution was acidified with HCl (30 mL) under cooling and extracted with an ethyl acetate/benzene mixture, 1:2 (2×400 mL). The organic layer was washed with water (50 mL) and brine (50 mL), dried over sodium sulfate, and evaporated. The obtained crude product (28 g) was purified by column chromatography (silica gel, CCl4/ethyl acetate, 1:0→9:1) to give title compound. Y: 18.9 g (67.5%) rt=3.49 min (gradient A), (M+H)+=221.
A mixture of (3E)-3-(methoxycarbonyl)-4-phenylbut-3-enoic acid (10.75 g, 48.8 mmol), dicyclohexylamine (18.62 g, 102.6 mmol), water (10 mL), and dichloro((S)-(−)-2,2-bis(diphenylphosphino)-1,1-binaphthyl)ruthenium(I) (40 mg) in methanol (90 mL) was hydrogenated in a Parr apparatus at 60° C. and 60 psi for 30 h. The resulting mixture was evaporated in a rotary evaporator by ½. Acetonitrile (90 mL) was added to the residue, and the mixture was evaporated again by ½. This operation was repeated once more, and the solution was left at RT overnight. The formed precipitate was filtered off and washed with cold acetonitrile. The product (9 g) was dissolved in water (150 mL) and acidified with concentrated HCl to pH=3 under cooling. The product was extracted with an ethyl acetate/benzene 1:2 mixture (300 mL). The organic layer was washed with water, brine, dried over Na2SO4, and evaporated to give title compound. Y: 6.35 g (58.6%) P>95%, rt=3.54 min (gradient A), (M+H)+=222, ee: 96% (method C).
Tert-butyl-2,2,2-trichloroacetimidate (9 mmol, 1.61 mL) and boron trifluoride diethyl etherate (0.675 mmol, 85 μL) was added to a solution of (3R)-3-Benzyl-4-methoxy-4-oxobutanoic acid (4.5 mmol, 1 g) in anhydrous THF (10 mL) at RT. The mixture stirred at RT under nitrogen for 3 h. TLC (cyclohexane/AcOEt=1/1) indicated reaction was complete. Reaction mixture was diluted with sat. aq. NaHCO3 (10 mL) and extracted with AcOEt (2×20 mL). Combined organic layers were washed with brine, dried over MgSO4, evaporated. Crude was purified by flash chromatography (cyclohexane/AcOEt=9/1) to give title compound as a very light yellow oil. Y: 1.25 g (62%), P>90% rt=4.65 mn (gradient A), (M+H)+=222 (−tBu) by 1H NMR.
To a solution of (R)-4-tert-butyl 1-methyl 2-benzylsuccinate (308 mg, 1.11 mmol) in THF (3 mL) was added a solution of lithium hydroxide (107 mg, 4.44 mmol) in water (3 mL). The mixture was stirred at RT overnight. TLC (cyclohexane/AcOEt=7/3) indicated reaction was complete. Reaction mixture was acidified to pH=1 with 2M HCl and extracted with DCM (2×20 mL). Combined organic layers were passed through a phase separator and evaporated. Crude was purified by flash chromatography (cyclohexane/AcOEt=9/1→7/3) (loading as solution in starting eluent) to yield title compound as a colorless oil. Y: 274 mg (94%), P>95%, rt=4.17 mn (gradient A), (M+H)+=209 (−tBu).
The following intermediates were or may be synthesized using general method B:
Thiourea (2.1 g, 27.45 mmol) was added to a solution 2-bromo-1-(2-chlorophenyl)ethanone (7 g, 27.45 mmol) in ethanol (10 mL) and reaction mixture was stirred at RT for 18 h. The solvent was evaporated and refluxed for 5 minutes in DCM. Suspension was filtered to yield 7.84 g of 4-(2-chlorophenyl)thiazol-2-amine hydrobromide as a white powder. This powder was stirred in a mixture of aq. sat. Na2CO3 and AcOEt. Phases are separated and organic layer dried over MgSO4, concentrated in vacuo to yield title compound as a yellow oil which solidifies spontaneously. Y: 5.37 g (93%), P=100%, rt=2.84 mn (gradient A), (M+H)+=211.
The following intermediates were or may be synthesized from the appropriate bromoketone (for which synthesis is described in Scheme 20) and thiourea (for which synthesis is described in Scheme 23) using general method C:
2-bromo-1-(2-chlorophenyl)ethanone (0.5 mmol, 116 mg) and dry sodium thiocyanate (0.55 mmol, 45 mg) were stirred in 1 mL ethanol for 3 h at 50° C. A solution of cyclopropane methyl amine (0.55 mmol, 39 mg) in 0.5 mL of ethanol was added at once and the reaction mixture was stirred for 12 h. The ethanol was distilled off, and ethyl acetate and water were added. The aqueous phase was extracted twice with ethyl acetate, the combined organic phases were dried over Na2SO4, and the solvent was removed in vacuo. Crude was purified by flash chromatography (cyclohexane/DCM=6/4) to give title compound as a dark yellow oil. Y: 40 mg (30%), P=100%, rt=3.5 mn (gradient A), (M+H)+=264.8.
The following intermediates were or may be synthesized from the appropriate bromoketone (for which synthesis is described in Scheme 20) and amine reagents using general method D:
To a solution of (R)-2-benzyl-4-tert-butoxy-4-oxobutanoic acid 1b (1.21 mmol, 320 mg) in anhydrous DMF (5 mL) was added HATU (1.33 mmol, 505 mg). After 5 min was added 4-(2-chlorophenyl)thiazol-2-amine 2a (1.33 mmol, 279 mg) and DIEA (1.815 mmol, 300 μL). Reaction mixture was stirred at RT for 4 days. TLC (cyclohexane/AcOEt=8/2) indicated reaction was complete. Reaction mixture (rm) was diluted with AcOEt (20 mL) and washed with sat. aq. NaHCO3 (10 mL) and water (3×10 mL). The organic phase was dried over MgSO4 and evaporated. Crude was purified by flash chromatography (cyclohexane/AcOEt=9/1) (loading onto silica) to yield title compound as a yellow gum. Y: 370 mg (67%), P>95%, rt=5.24 mn (gradient A), (M+H)+=457.1.
ACN was also used instead of DMF.
To a solution of (R)-tert-butyl 3-benzyl-4-(4-(2-chlorophenyl)thiazol-2-ylamino)-4-oxobutanoate (0.7 mmol, 320 mg) in DCM (8 mL) was added TFA (2 mL). Rm was stirred at RT overnight. TLC (cyclohexane/AcOEt=7/3) indicated reaction was complete. Reaction mixture was evaporated and residue purified using a Biotage PEAX SPE cartridge. The oil obtained was triturated in diethyl ether/pentane=2/8 to yield title compound as a colorless solid. Y: 280 mg (99%), P>99% rt=9.32 mn (gradient B), (M+H)+=401.1, ee=96% (method B), 1HNMR (CDCl3): δ=12.2 (br s, 1H), 7.39-7.33 (m, 9H), 7.14 (s, 1H), 3.36 (q, 1H), 3.14 (m, 1H), 2.89-2.77 (m, 2H), 2.57 (dd, 1H).
Examples 2 to 18 were synthesized using general method E and intermediates described above or commercially available.
compound no9: (S)-4-(4-(2-chlorophenyl)thiazol-2-ylamino)-4-oxo-3-phenylbutanoic acid was synthesized using intermediates 1a and 2a. P=99%, (M+H)+=387, ee=98% (method A), 1HNMR (DMSO-d6): δ=7.78 (d, J=2.8Hz, 1H), 7.5 (m, 2H), 7.4-7.1 (m, 9H), 4.3 (q, 1H), 3.18 (dd, J=17Hz, J=27Hz, 1H), 2.66 (dd, J=4.8Hz, J=22Hz, 1H).
compound no3: (R)-3-benzyl-4-(4-(2,4-dichlorophenyl)thiazol-2-ylamino)-4-oxobutanoic acid was synthesized using intermediate 1b and 4-(2,4-dichlorophenyl)thiazol-2-amine.
compound no4: (R)-3-benzyl-4-(4-(2-fluorophenyl)thiazol-2-ylamino)-4-oxobutanoic acid was synthesized using intermediate 1b and 4-(2-fluorophenyl)thiazol-2-amine.
compound no5: (R)-3-benzyl-4-(4-(3,4-dichlorophenyl)thiazol-2-ylamino)-4-oxobutanoic acid was synthesized using intermediate 1b and 4-(3,4-dichlorophenyl)thiazol-2-amine.
compound no8: (R)-3-benzyl-4-(4-(4-cyanophenyl)thiazol-2-ylamino)-4-oxobutanoic acid was synthesized using intermediate 1b and 4-(2-aminothiazol-4-yl)benzonitrile.
compound no12: (R)-3-benzyl-4-(4-(3-chlorophenyl)thiazol-2-ylamino)-4-oxobutanoic acid was synthesized using intermediate 1b and 4-(3-chlorophenyl)thiazol-2-amine.
compound no13: (R)-3-benzyl-4-oxo-4-(4-(3-(trifluoromethyl)phenyl)thiazol-2-ylamino)butanoic acid was synthesized using intermediate 1b and 4-(3-(trifluoromethyl)phenyl)thiazol-2-amine.
compound no14: (R)-3-benzyl-4-((4-(2-chlorophenyl)thiazol-2-yl)(methyl)amino)-4-oxobutanoic acid was synthesized using intermediates 1b and 2c. Y: 142 mg (80%), P>99% rt=10.5 mn (gradient B), (M+H)+=414.8, ee=96% (method B), 1HNMR (CDCl3): δ=7.92 (d, 1H), 7.54 (s, 1H), 7.45 (d, 1H), 7.34-7.13 (m, 7H), 3.62 (s, 3H), 3.47 (m, 1H), 3.15-3.01 (m, 2H), 2.61-2.54 (m, 1H), 2.53-2.51 (dd, 1H).
compound no17: (R)-4-(4-(2-chlorophenyl)thiazol-2-ylamino)-3-(4-fluorobenzyl)-4-oxobutanoic acid was synthesized using intermediates 1c and 2a.
compound no18: (R)-4-(4-(2-chlorophenyl)thiazol-2-ylamino)-3-(cyclohexylmethyl)-4-oxobutanoic acid was synthesized using intermediates 1d and 2a. Y: 15 mg (30%), P>90% rt=10.76 mn (gradient B), (M+H)+=401.1, ee=96% (method B), 1HNMR (CDCl3): δ=12.26 (br s, 1H), 7.35-7.45 (m, 2H), 7.15-7.30 (m, 4H), 3.15-3.25 (m, 1H), 2.7 (dd, 1H), 2.5 (dd, 1H), 1.45-1.8 (m, 6H), 1.1-1.4 (m, 5H), 0.8-1.0 (m, 2H).
compound no22: (R)-4-(allyl(4-(2-chlorophenyl)thiazol-2-yl)amino)-3-benzyl-4-oxobutanoic acid was synthesized using intermediates 1b and 2d.
compound no23: (R)-3-benzyl-4-((4-(2-chlorophenyl)thiazol-2-yl)(2-methoxy-2-oxoethyl)amino)-4-oxobutanoic acid was synthesized using intermediate 1b and 2e.
compound no11: (R)-3-benzyl-4-oxo-4-(3-phenyl-1,2,4-thiadiazol-5-ylamino)butanoic acid was synthesized using intermediate 1b and 3-phenyl-1,2,4-thiadiazol-5-amine.
compound no20: (R)-3-benzyl-4-(4-(2-chlorophenyl)-5-fluorothiazol-2-ylamino)-4-oxobutanoic acid was synthesized using intermediate 1b and 4-(2-chlorophenyl)-5-fluorothiazol-2-amine which was prepared in one step from intermediate 2a as described in Chem. Res. Toxicol. 2007, 1954-1965.
compound no21: (R)-3-benzyl-4-((5-chloro-4-(2-chlorophenyl)thiazol-2-yl)(methyl)amino)-4-oxobutanoic acid was synthesized using intermediate 1b and 5-chloro-4-(2-chlorophenyl)-N-methylthiazol-2-amine which was prepared by reacting intermediate 2c with N-chlorosuccinimide and triethylamine in chloroform.
compound no15: (R)-3-benzyl-4-(5-(2-chlorophenyl)pyridin-2-ylamino)-4-oxobutanoic acid was synthesized using intermediate 1b and 5-iodopyridin-2-amine. Amide coupling such as in general method E, subsequent Suzuki coupling with 2-chlorophenylboronic acid using PdCl2(PPh3)4 catalyst and K2CO3 in dioxane/H2O followed by tBu deprotection as described in general method E provided title compound.
compound no10: (Z)-4-(4-(2-chlorophenyl)thiazol-2-ylamino)-4-oxobut-2-enoic acid was synthesized using intermediate 2a and (Z)-4-methoxy-4-oxobut-2-enoic acid. Amide coupling such as in general method E followed by saponification such as in step 6 of general method B provided title compound.
compound no16: (R)-3-(4-(2-chlorophenyl)thiazol-2-ylcarbamoyl)heptanoic acid was synthesized using intermediate 2a and (R)-2-(2-tert-butoxy-2-oxoethyl)hexanoic acid. (R)-2-(2-tert-butoxy-2-oxoethyl)hexanoic acid was prepared from (R)-3-(methoxycarbonyl)heptanoic acid as done in steps 5 and 6 of general method B.
compound no19: (R)-3-(4-(2-chlorophenyl)thiazol-2-ylcarbamoyl)-5-methylhexanoic acid was synthesized using intermediate 2a and (R)-2-(2-tert-butoxy-2-oxoethyl)-4-methylpentanoic acid. (R)-2-(2-tert-butoxy-2-oxoethyl)-4-methylpentanoic acid was prepared from (R)-3-(methoxycarbonyl)-5-methylhexanoic acid as done in steps 5 and 6 of general method B.
compound no1: 6-(4-(2-chlorophenyl)thiazol-2-ylcarbamoyl)cyclohex-3-enecarboxylic acid was synthesized using intermediate 2a and 6-(methoxycarbonyl)cyclohex-3-enecarboxylic acid. Amide coupling such as in general method E followed by saponification such as in step 6 of general method B provided title compound.
compound no24: (R)-methyl 3-benzyl-4-(4-(2-chlorophenyl)thiazol-2-ylamino)-4-oxobutanoate may be synthesized by treating compound no2 with TMSCl in MeOH.
compound no26: (R)-3-(4-(2-chlorophenyl)thiazol-2-ylcarbamoyl)-5-phenylpentanoic acid may be synthesized from intermediates 1e and 2a using general method E.
compound no27: (S)-3-(4-(2-chlorophenyl)thiazol-2-ylcarbamoyl)-5-phenylpentanoic acid may be synthesized from intermediates 1f and 2a using general method E.
compound no28: (R)-4-(4-(2-chlorophenyl)thiazol-2-ylamino)-4-oxo-3-(4-(trifluoromethyl)benzyl)butanoic acid was synthesized from intermediates 1g and 2a using general method E.
compound no29: (R)-4-(4-(2-chlorophenyl)thiazol-2-ylamino)-4-oxo-3-(3-(trifluoromethyl)benzyl)butanoic acid was synthesized from intermediates 1h and 2a using general method E.
compound no30: (R)-4-(4-(2-chlorophenyl)thiazol-2-ylamino)-3-(2-cyanobenzyl)-4-oxobutanoic acid may be synthesized from intermediates 1i and 2a using general method E.
compound no31: (R)-4-(4-(2-chlorophenyl)thiazol-2-ylamino)-3-(3-cyanobenzyl)-4-oxobutanoic acid may be synthesized from intermediates 1j and 2a using general method E.
compound no32: (R)-4-(4-(2-chlorophenyl)thiazol-2-ylamino)-3-(4-cyanobenzyl)-4-oxobutanoic acid may be synthesized from intermediates 1k and 2a using general method E.
compound no33: (R)-4-(4-(2-chlorophenyl)thiazol-2-ylamino)-3-(4-methoxybenzyl)-4-oxobutanoic acid may be synthesized from intermediates 1l and 2a using general method E.
compound no34: (R)-4-(4-(2-chlorophenyl)thiazol-2-ylamino)-3-(3-methoxybenzyl)-4-oxobutanoic acid may be synthesized from intermediates 1m and 2a using general method E.
compound no35: (R)-4-(4-(2-chlorophenyl)thiazol-2-ylamino)-3-(2-methoxybenzyl)-4-oxobutanoic acid may be synthesized from intermediates 1n and 2a using general method E.
compound no36: (R)-3-benzyl-4-(4-(2-methoxyphenyl)thiazol-2-ylamino)-4-oxobutanoic acid was synthesized from intermediate 1b and 4-(2-methoxyphenyl)thiazol-2-amine using general method E.
compound no37: ((R)-3-benzyl-4-oxo-4-(4-(2,4,6-trichlorophenyl)thiazol-2-ylamino)butanoic acid may be synthesized from intermediates 1b and 2f using general method E.
compound no38: (R)-4-benzyl-5-((4-(2-chlorophenyl)thiazol-2-yl)(methyl)amino)-5-oxopentanoic acid may be synthesized from intermediates 1o and 2c using general method E, replacing the TFA tBu ester deprotection by a methyl ester saponification using LiOH in THF/H2O.
compound no39: (S)-4-benzyl-5-((4-(2-chlorophenyl)thiazol-2-yl)(methyl)amino)-5-oxopentanoic acid was synthesized from intermediates 1p and 2c using general method E, replacing the TFA tBu ester deprotection by a methyl ester saponification using LiOH in THF/H2O.
compound no40: (R)-methyl 4-benzyl-5-(4-(2-chlorophenyl)thiazol-2-ylamino)-5-oxopentanoate may be synthesized from intermediates 1o and 2a using general method E.
compound no41: (S)-methyl 4-benzyl-5-(4-(2-chlorophenyl)thiazol-2-ylamino)-5-oxopentanoate may be synthesized from intermediates 1p and 2a using general method E.
compound no42: (R)-3-benzyl-4-((4-(2-chlorophenyl)thiazol-2-yl)(cyclopropylmethyl)amino)-4-oxobutanoic acid was synthesized from intermediates 1b and 2b using general method E.
compound no43:(R)-3-benzyl-4-(benzyl(4-(2-chlorophenyl)thiazol-2-yl)amino)-4-oxobutanoic acid was synthesized from intermediates 1b and 2g using general method E.
compound no44: (R)-3-benzyl-4-((4-(2-chlorophenyl)thiazol-2-yl)(2,2,2-trifluoroethyl)amino)-4-oxobutanoic acid may be synthesized from intermediates 1b and 2h using general method E.
compound no45: (R)-4-((4-(2-chlorophenyl)thiazol-2-yl)(methyl)amino)-3-(4-methoxybenzyl)-4-oxobutanoic acid was synthesized from 4-(tert-butoxy)-2-(4-methoxybenzyl)-4-oxobutanoic acid and intermediate 2c using general method E and chiral preparative HPLC purification. 4-(tert-butoxy)-2-(4-methoxybenzyl)-4-oxobutanoic acid was synthesized from commercially available 4-methoxybenzaldehyde using the HWE methodology (Scheme 13).
compound no46: (R)-4-((4-(2-chlorophenyl)thiazol-2-yl)(methyl)amino)-3-(3-methoxybenzyl)-4-oxobutanoic acid may be synthesized from intermediates 1m and 2c using general method E.
compound no47: (R)-4-((4-(2-chlorophenyl)thiazol-2-yl)(methyl)amino)-3-(2-methoxybenzyl)-4-oxobutanoic acid may be synthesized from intermediates 1n and 2c using general method E.
compound no48: (R)-4-((4-(2-chlorophenyl)thiazol-2-yl)(methyl)amino)-3-(4-cyanobenzyl)-4-oxobutanoic acid was synthesized from 4-(tert-butoxy)-2-(4-cyanobenzyl)-4-oxobutanoic acid and intermediate 2c using general method E and chiral preparative HPLC purification. 4-(tert-butoxy)-2-(4-cyanobenzyl)-4-oxobutanoic acid was synthesized from commercially available 4-cyanobenzaldehyde using the HWE methodology (Scheme 13).
compound no49: (R)-4-((4-(2-chlorophenyl)thiazol-2-yl)(methyl)amino)-3-(3-cyanobenzyl)-4-oxobutanoic acid may be synthesized from intermediates 1j and 2c using general method E.
compound no50: (R)-4-((4-(2-chlorophenyl)thiazol-2-yl)(methyl)amino)-3-(2-cyanobenzyl)-4-oxobutanoic acid may be synthesized from intermediates 1i and 2c using general method E.
compound no51: (R)-3-(4-chlorobenzyl)-4-((4-(2-chlorophenyl)thiazol-2-yl)(methyl)amino)-4-oxobutanoic acid was synthesized from 4-(tert-butoxy)-2-(4-chlorobenzyl)-4-oxobutanoic acid and intermediate 2c using general method E and chiral preparative HPLC purification. 4-(tert-butoxy)-2-(4-chlorobenzyl)-4-oxobutanoic acid was synthesized from commercially available 4-chlorobenzaldehyde using the HWE methodology (Scheme 13).
compound no52: (R)-3-(3-chlorobenzyl)-4-((4-(2-chlorophenyl)thiazol-2-yl)(methyl)amino)-4-oxobutanoic acid may be synthesized from intermediates 1r and 2c using general method E.
compound no53: (R)-3-(2-chlorobenzyl)-4-((4-(2-chlorophenyl)thiazol-2-yl)(methyl)amino)-4-oxobutanoic acid may be synthesized from intermediates 1s and 2c using general method E.
compound no54: (3S)-4-((4-(2-chlorophenyl)thiazol-2-yl)(methyl)amino)-3-(2,3-dihydro-1H-inden-1-yl)-4-oxobutanoic acid may be synthesized from intermediates 1t and 2c using general method E. 1t may be synthesized using Stobbe's condensation (Scheme 6).
compound no55: (S)-4-((4-(2-chlorophenyl)thiazol-2-yl)(methyl)amino)-3-(2,3-dihydro-1H-inden-2-yl)-4-oxobutanoic acid may be synthesized from intermediates 1u and 2c using general method E. 1u may be synthesized using Stobbe's condensation (Scheme 6).
compound no56: (R)-4-(benzo[d]thiazol-2-yl(methyl)amino)-3-benzyl-4-oxobutanoic acid may be synthesized from intermediate 1b and N-methylbenzo[d]thiazol-2-amine using general method E. N-methylbenzo[d]thiazol-2-amine may be prepared by Eischweiler-Clarke methylation of benzo[d]thiazol-2-amine.
compound no57: (R)-4-(benzo[d]oxazol-2-yl(methyl)amino)-3-benzyl-4-oxobutanoic acid may be synthesized from intermediate 1b and N-methylbenzo[d]oxazol-2-amine using general method E. N-methylbenzo[d]oxazol-2-amine may be prepared by Eischweiler-Clarke methylation of benzo[d]oxazol-2-amine.
compound no58: (R)-2-((1H-tetrazol-5-yl)methyl)-N-(4-(2-chlorophenyl)thiazol-2-yl)-N-methyl-3-phenylpropanamide may be synthesized from compound no14 using methodologies described in the isosteres synthetic schemes section.
compound no59: (R)-2-benzyl-N-(4-(2-chlorophenyl)thiazol-2-yl)-N-methyl-3-(5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl)propanamide may be synthesized from compound no14 using methodologies described in the isosteres synthetic schemes section.
compound no60: (R)-3-benzyl-4-((4-(2-chlorophenyl)-5-fluorothiazol-2-yl)(methyl)amino)-4-oxobutanoic acid was synthesized using intermediate 1b and 4-(2-chlorophenyl)-5-fluoro-N-methylthiazol-2-amine which was prepared in one step from intermediate 2c as described in Chem. Res. Toxicol. 2007, 1954-1965.
compound no61: (S)-4-(4-(2-chlorophenyl)thiazol-2-ylamino)-3-cyclohexyl-4-oxobutanoic acid may be synthesized from intermediates 1v and 2a using general method E.
compound no62: (S)-4-((4-(2-chlorophenyl)thiazol-2-yl)(methyl)amino)-3-cyclohexyl-4-oxobutanoic acid was synthesized from (S)-4-(tert-butoxy)-2-cyclohexyl-4-oxobutanoic acid and intermediate 2c using general method E. (S)-4-(tert-butoxy)-2-cyclohexyl-4-oxobutanoic acid was synthesized from commercially available (S)-3-cyclohexyl-4-methoxy-4-oxobutanoic acid as described in steps 5 and 6 of general method B.
compound no63: (S)-4-((4-(2-chlorophenyl)thiazol-2-yl)(methyl)amino)-4-oxo-3-phenylbutanoic acid may be synthesized from intermediates 1a and 2c using general method E.
compound no64: (3R)-3-(4-(2-chlorophenyl)thiazol-2-ylcarbamoyl)-4-phenylpentanoic acid may be synthesized from intermediate 2a and (2R)-4-tert-butoxy-4-oxo-2-(1-phenylethyl)butanoic acid using general method E. (2R)-4-tert-butoxy-4-oxo-2-(1-phenylethyl)butanoic acid may be obtained by Stobbe condensation (Scheme 6).
compound no65: (R)-2-((1H-tetrazol-5-yl)methyl)-N-(4-(2-chlorophenyl)thiazol-2-yl)-3-phenylpropanamide was synthesized from compound no2 using methodologies described in the isosteres synthetic schemes section.
compound no66: (R)-2-benzyl-N-(4-(2-chlorophenyl)thiazol-2-yl)-3-(5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl)propanamide was synthesized from compound no2 using methodologies described in the isosteres synthetic schemes section.
compound no68: (3R)-3-benzyl-4-(4-(2-chlorophenyl)thiazol-2-ylamino)-2-methyl-4-oxobutanoic acid was synthesized as described in Scheme 7.
compound no69: (R)-2-benzyl-N-(4-(2-chlorophenyl)thiazol-2-yl)-3-(3-hydroxyisoxazol-5-yl)propanamide may be synthesized using methodologies described in the isosteres synthetic schemes section.
compound no70: (R)-3-benzyl-4-(4-(2-chlorophenyl)pyrimidin-2-ylamino)-4-oxobutanoic acid was synthesized from intermediate 1b and 4-(2-chlorophenyl)pyrimidin-2-amine using general method E. 4-(2-chlorophenyl)pyrimidin-2-amine was synthesized as described in Scheme 8.
compound no71: (R)-3-benzyl-4-(6-(2-chlorophenyl)pyridin-2-ylamino)-4-oxobutanoic acid was synthesized from intermediate 1b and 6-(2-chlorophenyl)pyridin-2-amine using general method E. 6-(2-chlorophenyl)pyridin-2-amine was synthesized as described in Scheme 8.
compound no72: (E)-3-(4-(2-chlorophenyl)thiazol-2-ylcarbamoyl)-4-phenylbut-3-enoic acid may be synthesized from (E)-2-benzylidene-4-tert-butoxy-4-oxobutanoic acid and intermediate 2a using general method E. (E)-2-benzylidene-4-tert-butoxy-4-oxobutanoic acid was synthesized from maleic anhydride following steps 1, 2, 3, 5 and 6 of general method B.
compound no74: (Z)-4-((4-(2-chlorophenyl)thiazol-2-yl)(methyl)amino)-4-oxo-3-phenylbut-2-enoic acid may be synthesized as described in Scheme 9.
compound no75: (R)-3-(N-(4-(2-chlorophenyl)thiazol-2-yl)-N-methylsulfamoyl)-4-phenylbutanoic acid may be synthesized as described in Scheme 10.
compound no76: (S)-3-(N-(4-(2-chlorophenyl)thiazol-2-yl)-N-methylsulfamoyl)-4-phenylbutanoic acid may be synthesized as described in Scheme 10.
compound no79: (R)-3-benzyl-4-(4-(2-chlorophenyl)thiazol-2-ylamino)-3-fluoro-4-oxobutanoic acid may be synthesized as described in Scheme 11.
compound no80: (R)-3-benzyl-3-(4-(2-chlorophenyl)thiazol-2-ylcarbamoyl)hex-5-enoic acid may be synthesized as described in Scheme 11.
compound no81: (E)-3-(4-(2-chlorophenyl)thiazol-2-yl)(methyl)carbamoyl)-4-phenylbut-3-enoic acid was synthesized from (E)-2-benzylidene-4-tert-butoxy-4-oxobutanoic acid and intermediate 2c using general method E. (E)-2-benzylidene-4-tert-butoxy-4-oxobutanoic acid was synthesized from maleic anhydride following steps 1, 2, 3, 5 and 6 of general method B.
compound no82: (3S)-3-((4-(2-chlorophenyl)thiazol-2-yl)(methyl)carbamoyl)-4-phenylpentanoic acid may be synthesized from intermediate 2c and (2R)-4-tert-butoxy-4-oxo-2-(1-phenylethyl)butanoic acid using general method E. (2R)-4-tert-butoxy-4-oxo-2-(1-phenylethyl)butanoic acid may be obtained by Stobbe condensation (Scheme 6).
compound no83: (R)-3-benzyl-4-((3-(2-chlorophenyl)-1,2,4-thiadiazol-5-yl)(methyl)amino)-4-oxobutanoic acid was synthesized as described in Scheme 12.
compound no84: (R)-3-benzyl-4-((3-(2-chlorophenyl)-1,2,4-oxadiazol-5-yl)(methyl)amino)-4-oxobutanoic acid may be synthesized as described in Scheme 12.
compound no85: (R)-3-benzyl-4-((1-(2-chlorophenyl)-1H-pyrazol-3-yl(methyl)amino)-4-oxobutanoic acid may be synthesized as described in Scheme 12.
compound no86: (R)-2-benzyl-N-(4-(2-chlorophenyl)thiazol-2-yl)-3-(3-hydroxyisoxazol-5-yl)-N-methylpropanamide was synthesized using methodologies described in the isosteres synthetic schemes section.
compound no89: (R)-4-((4-(2-chlorophenyl)thiazol-2-yl)(methyl)amino)-3-(cyclohexylmethyl)-4-oxobutanoic acid was synthesized from intermediates 1w and 2c using general method E. Intermediate 1w was synthesized by hydrogenation of intermediate 1b using PtO2 in MeOH.
compound no90: (R)-3-((4-(2-chlorophenyl)thiazol-2-yl)(methyl)carbamoyl)-5-methylhexanoic acid was synthesized from intermediate 2c and (R)-2-(2-tert-butoxy-2-oxoethyl)-4-methylpentanoic acid using general method E. (R)-2-(2-tert-butoxy-2-oxoethyl)-4-methylpentanoic acid was synthesized from (R)-3-(methoxycarbonyl)-5-methylhexanoic acid using methodology described in steps 5 and 6 of general method B.
compound no91: (R)-3-benzyl-4-((4-(2-cyanophenyl)thiazol-2-yl)(methyl)amino)-4-oxobutanoic acid was synthesized from intermediates 1b and 2i using general method E.
compound no92: (R)-4-(4-(2-chlorophenyl)thiazol-2-ylamino)-4-oxo-3-phenylbutanoic. Phenylacetic acid was converted to its tBu ester using tBu-TCA. Treatment of this tBu ester with LiHMDS followed by the addition of t-butyl bromoacetate provided 1-tert-butyl 4-methyl 2-phenylsuccinate. tBu deprotection with TFA yielded 4-methoxy-4-oxo-2-phenylbutanoic acid. HATU coupling of this acid with intermediate 2a and subsequent methyl ester saponification using LiOH yielded 4-(4-(2-chlorophenyl)thiazol-2-ylamino)-4-oxo-3-phenylbutanoic. Chiral preparative HPLC purification of this racemic mixture allowed isolating compound no92.
compound no93: (R)-4-(4-(2-chlorophenyl)thiazol-2-ylamino)-3-(3-fluorobenzyl)-4-oxobutanoic acid was synthesized from intermediates 1y and 2a using general method E and preparative HPLC purification.
compound no94: (S)-3-((4-(2-chlorophenyl)thiazol-2-yl)(methyl)carbamoyl)-4-methylpentanoic acid was synthesized from (S)-4-tert-butoxy-2-isopropyl-4-oxobutanoic acid and intermediate 2c using general method E. (S)-4-tert-butoxy-2-isopropyl-4-oxobutanoic acid was synthesized from commercially available (S)-3-(methoxycarbonyl)-4-methylpentanoic acid using reactions described in steps 5 and 6 of general method B.
compound no95: 4-((4-(2-chlorophenyl)thiazol-2-yl)(methyl)amino)-4-oxo-3-((tetrahydro-2H-pyran-4-yl)methyl)butanoic acid was synthesized from (R)-4-tert-butoxy-4-oxo-2-((tetrahydro-2H-pyran-4-yl)methyl)butanoic acid and intermediate 2c using general method E. (R)-4-tert-butoxy-4-oxo-2-((tetrahydro-2H-pyran-4-yl)methyl)butanoic acid was synthesized from commercially available tetrahydro-2H-pyran-4-carbaldehyde using the HWE methodology (Scheme 13).
compound no96: (R)-3-benzyl-4-((4-(2-chlorophenyl)thiazol-2-yl)(ethyl)amino)-4-oxobutanoic acid was synthesized from intermediates 1b and 2j using general method E.
compound no97: (R)-3-benzyl-4-((4-(2-chlorophenyl)thiazol-2-yl)(cyclopropyl)amino)-4-oxobutanoic acid was synthesized from intermediates 1b and 2k using general method E.
compound no98: cis-6-(4-(2-chlorophenyl)thiazol-2-ylcarbamoyl)cyclohex-3-enecarboxylic acid was synthesized from cis-3a,4,7,7a-tetrahydroisobenzofuran-1,3-dione and intermediate 2a as described in Scheme 14.
compound no99: 4-((4-(2-chlorophenyl)thiazol-2-yl)(methyl)amino)-3-(4-methoxybenzyl)-4-oxobutanoic acid was synthesized from 4-tert-butoxy-2-(4-methoxybenzyl)-4-oxobutanoic acid and intermediate 2c using general method E. 4-tert-butoxy-2-(4-methoxybenzyl)-4-oxobutanoic acid was synthesized from 4-methoxybenzaldehyde using the HWE methodology (Scheme 13).
compound no100: cis-6-((4-(2-chlorophenyl)thiazol-2-yl)(methyl)carbamoyl)cyclohex-3-enecarboxylic acid was synthesized from cis-3a,4,7,7a-tetrahydroisobenzofuran-1,3-dione and intermediate 2c as described in Scheme 14.
compound no101: cis-2-((4-(2-chlorophenyl)thiazol-2-yl)(methyl)carbamoyl)cyclohexanecarboxylic acid was synthesized from cis-hexahydroisobenzofuran-1,3-dione and intermediate 2c as described in Scheme 14.
compound no102: (R)-3-benzyl-4-(4-(2,5-dimethylthiophen-3-yl)thiazol-2-ylamino)-4-oxobutanoic acid was synthesized from intermediate 1b and commercially available 4-(2,5-dimethylthiophen-3-yl)thiazol-2-amine using general method E.
compound no103: 4-((4-(2-chlorophenyl)thiazol-2-yl)(methyl)amino)-3-(cyclohexylmethyl)-4-oxobutanoic acid was synthesized from 4-tert-butoxy-2-(cyclohexylmethyl)-4-oxobutanoic acid and intermediate 2c using general method E. 4-tert-butoxy-2-(cyclohexylmethyl)-4-oxobutanoic acid was synthesized by hydrogenation of (E)-4-tert-butyl 1-methyl 2-benzylidenesuccinate using PtO2 in MeOH.
compound no105: 4-((4-(2-chlorophenyl)thiazol-2-yl)(methyl)amino)-3-(cyclopentylmethyl)-4-oxobutanoic acid was synthesized from 4-tert-butoxy-2-(cyclopentylmethyl)-4-oxobutanoic acid and intermediate 2c using general method E. 4-tert-butoxy-2-(cyclopentylmethyl)-4-oxobutanoic acid was synthesized from commercially available cyclopentanecarbaldehyde using the HWE methodology (Scheme 13).
compound no106: (3S ,4R)-3-((4-(2-chlorophenyl)thiazol-2-yl)(methyl)carbamoyl)-4-phenylpentanoic acid from intermediates 1z and 2c using general method E.
compound no107: (R)-3-benzyl-4-(methyl(4-(2-(thiophen-3-yl)phenyl)thiazol-2-yl)amino)-4-oxobutanoic acid was synthesized from intermediate 1b and N-methyl-4-(2-(thiophen-3-yl)phenyl)thiazol-2-amine using general method E. N-methyl-4-(2-(thiophen-3-yl)phenyl)thiazol-2-amine was synthesized from commercially available thiophen-3-ylboronic acid using the methodology shown in Scheme 15.
compound no108: (R)-3-benzyl-4-((4-(2-(6-chloropyridin-3-yl)phenyl)thiazol-2-yl)(methyl)amino)-4-oxobutanoic acid was synthesized from intermediate 1b and 4-(2-(6-chloropyridin-3-yl)phenyl)-N-methylthiazol-2-amine using general method E. 4-(2-(6-chloropyridin-3-yl)phenyl)-N-methylthiazol-2-amine was synthesized from commercially available 6-chloropyridin-3-ylboronic acid using the methodology shown in Scheme 15.
compound no109: (R)-4-((4-(2-chlorophenyl)thiazol-2-yl)methyl)amino)-4-oxo-3-(phenylamino)butanoic acid was synthesized from (R)-4-tert-butoxy-4-oxo-2-(phenylamino)butanoic acid and intermediate 2c using general method E. (R)-4-tert-butoxy-4-oxo-2-(phenylamino)butanoic acid was synthesized from commercially available (R)-2-amino-4-tert-butoxy-4-oxobutanoic acid and iodobenzene using CuI catalyzed coupling as described in J. Am. Chem. Soc. 1998, 120, 12459.
compound no110: 4-((4-(2-chlorophenyl)thiazol-2-yl)(methyl)amino)-3-(4-methylbenzyl)-4-oxobutanoic acid was synthesized from 4-tert-butoxy-2-(4-methylbenzyl)-4-oxobutanoic acid and intermediate 2c using general method E. 4-tert-butoxy-2-(4-methylbenzyl)-4-oxobutanoic acid was synthesized from 4-methylbenzaldehyde using the HWE methodology (Scheme 13).
compound no111: (R)-4-((4-([1,1′-biphenyl]-2-yl)thiazol-2-yl)(methyl)amino)-3-benzyl-4-oxobutanoic acid was synthesized from intermediate 1b and 4-([1,1′-biphenyl]-2-yl)-N-methylthiazol-2-amine using general method E. 4-([1,1′-biphenyl]-2-yl)-N-methylthiazol-2-amine was synthesized from commercially available phenylboronic acid using the methodology shown in Scheme 15.
compound no112: (R)-3-benzyl-4-(4-(2,5-dichlorothiophen-3-yl)thiazol-2-ylamino)-4-oxobutanoic acid was synthesized from intermediate 1b and commercially available 4-(2,5-dichlorothiophen-3-yl)thiazol-2-amine using general method E.
compound no113: 4-((4-(2-chlorophenyl)thiazol-2-yl)(methyl)amino)-3-(cyclopropylmethyl)-4-oxobutanoic acid was synthesized from intermediates 1a1 (4-(tert-butoxy)-2-(cyclopropylmethyl)-4-oxobutanoic acid) and 2c using general method E. 1a1 was synthesized from cyclopropanecarbaldehyde using the HWE methodology (Scheme 13).
compound no114: 4-((4-(2-chlorophenyl)thiazol-2-yl)(methyl)amino)-4-oxo-3-(thiazol-4-ylmethyl)butanoic acid was synthesized from intermediates 1b1 (4-(tert-butoxy)-4-oxo-2-(thiazol-4-ylmethyl)butanoic acid) and 2c using general method E. 1b1 was synthesized from thiazole-4-carbaldehyde using the HWE methodology (Scheme 13).
compound no115: (R)-3-benzyl-4-((4-(2-(6-(dimethylamino)pyridin-3-yl)phenyl)thiazol-2-yl)(methyl)amino)-4-oxobutanoic acid was synthesized from intermediate 1b and 4-(2-(6-(dimethylamino)pyridin-3-yl)phenyl)-N-methylthiazol-2-amine using general method E. 4-(2-(6-(dimethylamino)pyridin-3-yl)phenyl)-N-methylthiazol-2-amine was synthesized from (6-(dimethylamino)pyridin-3-yl)boronic acid and 2l using the methodology shown in Scheme 15.
compound no116: (R)-3-benzyl-4-((4-(2-(6-methoxypyridin-3-yl)phenyl)thiazol-2-yl)(methyl)amino)-4-oxobutanoic acid was synthesized from intermediate 1b and intermediate 2m (4-(2-(6-methoxypyridin-3-yl)phenyl)-N-methylthiazol-2-amine) using general method E. Intermediate 2m was synthesized from (6-methoxypyridin-3-yl)boronic acid and 2l using the methodology shown in Scheme 15.
compound no117: (R)-3-benzyl-4-((4-(2-(2-methoxypyridin-3-yl)phenyl)thiazol-2-yl)(methyl)amino)-4-oxobutanoic acid was synthesized from intermediate 1b and (4-(2-(2-methoxypyridin-3-yl)phenyl)-N-methylthiazol-2-amine) using general method E. (4-(2-(2-methoxypyridin-3-yl)phenyl)-N-methylthiazol-2-amine) was synthesized from (2-methoxypyridin-3-yl)boronic acid and 2l using the methodology shown in Scheme 15.
compound no118: (R)-3-benzyl-4-((4-(2-((ethoxycarbonyl)amino)phenyl)thiazol-2-yl)(methyl)amino)-4-oxobutanoic acid was synthesized from intermediates 1b and 2n (ethyl (2-(2-(methylamino)thiazol-4-yl)phenyl)carbamate) using general method E. Intermediate 2n was synthesized using the methodology described in Scheme 16.
compound no119: (R)-3-benzyl-4-((4-(2-(6-fluoropyridin-3-yl)phenyl)thiazol-2-yl)(methyl)amino)-4-oxobutanoic acid was synthesized from intermediates 1b and 2p (4-(2-(6-fluoropyridin-3-yl)phenyl)-N-methylthiazol-2-amine) using general method E and preparative HPLC purification. Intermediate 2p was synthesized from (6-fluoropyridin-3-yl)boronic acid and 2l using the methodology described in Scheme 15.
compound no120: (R)-3-benzyl-4-(methyl(4-(2-(6-methylpyridin-3-yl)phenyl)thiazol-2-yl)amino)-4-oxobutanoic acid was synthesized from intermediates 1b and 2q (N-methyl-4-(2-(6-methylpyridin-3-yl)phenyl)thiazol-2-amine) using general method E and preparative HPLC purification. Intermediate 2q was synthesized from (6-methylpyridin-3-yl)boronic acid and 2l using the methodology described in Scheme 15.
compound no121: (R)-4-((2-amino-2-oxoethyl)(4-(2-chlorophenyl)thiazol-2-yl)amino)-3-benzyl-4-oxobutanoic acid was synthesized from intermediates 1b and 2r using general method E.
compound no122: (R)-3-benzyl-4-oxo-4-((4-(3-(trifluoromethoxy)phenyl)thiazol-2-yl)amino)butanoic acid was synthesized from intermediates 1b and commercially available 2s (4-(3-(trifluoromethoxy)phenyl)thiazol-2-amine) using general method E.
compound no123: (R)-3-benzyl-4-((4-(2,5-dichlorophenyl)thiazol-2-yl)amino)-4-oxobutanoic acid was synthesized from intermediates 1b and commercially available 2t (4-(2,5-dichlorophenyl)thiazol-2-amine) using general method E.
compound no124: (R)-3-benzyl-4-((4-(3-chloro-4-fluorophenyl)thiazol-2-yl)amino)-4-oxobutanoic acid was synthesized from intermediates 1b and commercially available 2u (4-(3-chloro-4-fluorophenyl)thiazol-2-amine) using general method E.
compound no125: (R)-3-benzyl-4-((4-(3-chloro-4-methoxyphenyl)thiazol-2-yl)amino)-4-oxobutanoic acid was synthesized from intermediates 1b and commercially available 2v (4-(3-chloro-4-methoxyphenyl)thiazol-2-amine) using general method E.
compound no126: (R)-3-benzyl-4-((4-(2-chlorophenyl)thiazol-2-yl)(3-methoxy-3-oxopropyl)amino)-4-oxobutanoic acid was synthesized from intermediates 1b and 2x using general method E.
compound no127: 3-(bicyclo[2.2.1]heptan-2-ylmethyl)-4-((4-(2-chlorophenyl)thiazol-2-yl)(methyl)amino)-4-oxobutanoic acid was synthesized from intermediates 1c1 (2-(bicyclo[2.2.1]heptan-2-ylmethyl)-4-(tert-butoxy)-4-oxobutanoic acid) and 2c using general method E. 1c1 was synthesized from bicyclo[2.2.1]heptane-2-carbaldehyde using the HWE methodology (Scheme 13).
compound no128: (R)-3-benzyl-4-((4-(2-(6-ethoxypyridin-3-yl)phenyl)thiazol-2-yl)(methyl)amino)-4-oxobutanoic acid was synthesized from intermediates 1b and 2y (4-(2-(6-ethoxypyridin-3-yl)phenyl)-N-methylthiazol-2-amine) using general method E. Intermediate 2y was synthesized from (6-ethoxypyridin-3-yl)boronic acid and 2l using the methodology described in Scheme 15.
compound no129: (R)-3-benzyl-4-((4-(4′-methoxy-[1,1′-biphenyl]-2-yl)thiazol-2-yl)(methyl)amino)-4-oxobutanoic acid was synthesized from intermediates 1b and 2z (4-(4′-methoxy-[1,1′-biphenyl]-2-yl)-N-methylthiazol-2-amine) using general method E and preparative HPLC purification. Intermediate 2z was synthesized from (4-methoxyphenyl)boronic acid and 2l using the methodology described in Scheme 15.
compound no130: (R)-3-benzyl-4-((4-(2,5-dichlorophenyl)thiazol-2-yl)(methyl)amino)-4-oxobutanoic acid was synthesized from intermediates 1b and 2a1 using general method E.
compound no131: (R)-1-(5-(2-(2-(2-benzyl-3-carboxy-N-methylpropanamido)thiazol-4-yl)phenyl)pyridin-2-yl)pyrrolidin-1-ium 2,2,2-trifluoroacetate was synthesized from intermediates 1b and 2b1 (N-methyl-4-(2-(6-(pyrrolidin-1-yl)pyridin-3-yl)phenyl)thiazol-2-amine) using general method E. Intermediate 2b1 was synthesized from 2-(pyrrolidin-1-yl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine and 2l by Suzuki coupling with the conditions described in Scheme 15.
compound no132: (R)-4-(2′-(2-(2-benzyl-3-carboxy-N-methylpropanamido)thiazol-4-yl)-[1,1′-biphenyl]-4-yl)morpholin-4-ium 2,2,2-trifluoroacetate was synthesized from intermediates 1b and 2c1 (N-methyl-4-(4′-morpholino-[1,1′-biphenyl]-2-yl)thiazol-2-amine) using general method E. Intermediate 2c1 was synthesized from 4-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)morpholine and 2l by Suzuki coupling with the conditions described in Scheme 15.
compound no133: (R)-3-benzyl-4-(methyl(4-(2-(6-morpholinopyridin-3-yl)phenyl)thiazol-2-yl)amino)-4-oxobutanoic acid was synthesized from intermediates 1b and 2d1 (N-methyl-4-(2-(6-morpholinopyridin-3-yl)phenyl)thiazol-2-amine) using general method E. Intermediate 2d1 was synthesized from 4-(5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2-yl)morpholine and 2l using the methodology described in Scheme 15.
compound no134: (R)-3-benzyl-4-((4-(3′-chloro-[1,1′-biphenyl]-2-yl)thiazol-2-yl)(methyl)amino)-4-oxobutanoic acid was synthesized from intermediates 1b and 2e1 (4-(3′-chloro-[1,1′-biphenyl]-2-yl)-N-methylthiazol-2-amine) using general method E and preparative HPLC purification. Intermediate 2e1 was synthesized from (3-chlorophenyl)boronic acid and 2l using the methodology described in Scheme 15.
compound no135: (R)-3-benzyl-4-((4-(2-(furan-3-yl)phenyl)thiazol-2-yl)(methyl)amino)-4-oxobutanoic acid was synthesized from intermediates 1b and 2f1 (4-(2-(furan-3-yl)phenyl)-N-methylthiazol-2-amine) using general method E. Intermediate 2f1 was synthesized from furan-3-ylboronic acid and 2l by Suzuki coupling with the conditions described in Scheme 15.
compound no136: (R)-3-benzyl-4-((4-(2-(6-(2-methoxyethoxy)pyridin-3-yl)phenyl)thiazol-2-yl)(methyl)amino)-4-oxobutanoic acid was synthesized from intermediates 1b and 2g1 (4-(2-(6-(2-methoxyethoxy)pyridin-3-yl)phenyl)-N-methylthiazol-2-amine) using general method E. Intermediate 2g1 was synthesized from 5-bromo-2-(2-methoxyethoxy)pyridine and 2l using the methodology described in Scheme 17.
compound no138: (R)-3-benzyl-4-((4-(4′-isopropyl[1,1′-biphenyl]-2-yl)thiazol-2-yl)(methyl)amino)-4-oxobutanoic acid was synthesized from intermediates 1b and 2h1 (4-(4′-isopropyl-[1,1′-biphenyl]-2-yl)-N-methylthiazol-2-amine) using general method E and preparative HPLC purification. Intermediate 2h1 was synthesized from (4-isopropylphenyl)boronic acid and 2l by Suzuki coupling with the conditions described in Scheme 15.
compound no139: (R)-3-(cyclopentylmethyl)-4-((4-(2-(6-methoxypyridin-3-yl)phenyl)thiazol-2-yl)(methyl)amino)-4-oxobutanoic acid was synthesized from (R)-4-tert-butoxy-2-(cyclopentylmethyl)-4-oxobutanoic acid (ee=50%) and intermediate 2m using general method E and chiral preparative HPLC purification. (R)-4-tert-butoxy-2-(cyclopentylmethyl)-4-oxobutanoic acid (ee=50%) was synthesized from commercially available cyclopentanecarbaldehyde using the HWE methodology as described in Scheme 13.
compound no140: (R)-3-benzyl-4-((4-(2-(5-fluoro-6-methoxypyridin-3-yl)phenyl)thiazol-2-yl)(methyl)amino)-4-oxobutanoic acid was synthesized from intermediates 1b and 2i1 (4-(2-(5-fluoro-6-methoxypyridin-3-yl)phenyl)-N-methylthiazol-2-amine) using general method E. Intermediate 2i1 was synthesized from 5-bromo-3-fluoro-2-methoxypyridine using the methodology described in Scheme 17.
compound no141: (R)-3-benzyl-4-(methyl(4-(2-(6-((tetrahydro-2H-pyran-4-yl)oxy)pyridin-3-yl)phenyl)thiazol-2-yl)amino)-4-oxobutanoic acid was synthesized from intermediates 1b and 2j1 (N-methyl-4-(2-(6-((tetrahydro-2H-pyran-4-yl)oxy)pyridin-3-yl)phenyl)thiazol-2-amine) using general method E. Intermediate 2j1 was synthesized from 5-bromo-2-((tetrahydro-2H-pyran-4-yl)oxy)pyridine and 2l using the methodology described in Scheme 17.
compound no142: (R)-3-benzyl-4-(cyclopropyl(4-(2,5-dichlorophenyl)thiazol-2-yl)amino)-4-oxobutanoic acid from intermediates 1b and 2w using general method E.
compound no143: 4-((4-(2-chlorophenyl)thiazol-2-yl)methyl)amino)-3-(furan-2-ylmethyl)-4-oxobutanoic acid was synthesized from intermediates 1d1 (4-(tert-butoxy)-2-(furan-2-ylmethyl)-4-oxobutanoic acid) and 2c using general method E. 1d1 was synthesized from furan-2-carbaldehyde using the HWE methodology described Scheme 13.
compound no144: (R)-3-benzyl-4-((4-(2-cyclopropylphenyl)thiazol-2-yl)(methyl)amino)-4-oxobutanoic acid was synthesized from intermediates 1b and 2k1 (4-(2-cyclopropylphenyl)-N-methylthiazol-2-amine) using general method E and preparative HPLC purification. Intermediate 2k1 was synthesized from cyclopropylboronic acid and 2l using the methodology described in Scheme 15.
compound no145: (R)-3-benzyl-4-((4-(4′-(dimethylamino)-[1,1′-biphenyl]-2-yl)thiazol-2-yl)(methyl)amino)-4-oxobutanoic acid was synthesized from intermediates 1b and 2l1 (4-(4′-(dimethylamino)-[1,1′-biphenyl]-2-yl)-N-methylthiazol-2-amine) using general method E. Intermediate 2l1 was synthesized from N,N-dimethyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline using the methodology described in Scheme 15.
compound no146: (R)-3-benzyl-4-((4-(3′-fluoro-[1,1′-biphenyl]-2-yl)thiazol-2-yl)(methyl)amino)-4-oxobutanoic acid was synthesized from intermediates 1b and 2m1 (4-(3′-fluoro-[1,1′-biphenyl]-2-yl)-N-methylthiazol-2-amine) using general method E and preparative HPLC purification. Intermediate 2m1 was synthesized from (3-fluorophenyl)boronic acid and 4-(2-bromophenyl)-N-methylthiazol-2-amine by Suzuki coupling with the conditions described in Scheme 15.
compound no147: (R)-3-benzyl-4-((4-(3′,5′-difluoro-[1,1′-biphenyl]-2-yl)thiazol-2-yl)(methyl)amino)-4-oxobutanoic acid was synthesized from intermediates 1b and 2n1 (4-(3′,5′-difluoro-[1,1′-biphenyl]-2-yl)-N-methylthiazol-2-amine) using general method E. Intermediate 2n1 was synthesized from (3,5-difluorophenyl)boronic acid and 2l by Suzuki coupling with the conditions described in Scheme 15.
compound no148: (R)-3-benzyl-4-((4-(2-chloro-6-fluorophenyl)thiazol-2-yl)amino)-4-oxobutanoic acid was synthesized from intermediates 1b and commercially available 2o1 (4-(2-chloro-6-fluorophenyl)thiazol-2-amine) using general method E.
compound no149: (R)-3-benzyl-4-((4-(4′-chloro-[1,1′-biphenyl]-2-yl)thiazol-2-yl)(methyl)amino)-4-oxobutanoic acid was synthesized from intermediates 1b and 2p1 (4-(4′-chloro-[1,1′-biphenyl]-2-yl)-N-methylthiazol-2-amine) using general method E and preparative HPLC purification. Intermediate 2p1 was synthesized from (4-chlorophenyl)boronic acid and 2l by Suzuki coupling with the conditions described in Scheme 15.
compound no150: (R)-3-benzyl-4-(methyl(4-(2-(6-(2-oxopyrrolidin-1-yl)pyridin-3-yl)phenyl)thiazol-2-yl)amino)-4-oxobutanoic acid was synthesized from intermediates 1b and 2q1 (1-(5-(2-(2-(methylamino)thiazol-4-yl)phenyl)pyridin-2-yl)pyrrolidin-2-one) using general method E. Intermediate 2q1 was synthesized from 1-(5-bromopyridin-2-yl)pyrrolidin-2-one and 2l using the methodology described in Scheme 17.
compound no151: (R)-3-benzyl-4-((4-(4-chloro-2-(6-methoxypyridin-3-yl)phenyl)thiazol-2-yl)(methyl)amino)-4-oxobutanoic acid was synthesized from intermediates 1b and 2r1 (4-(4-chloro-2-(6-methoxypyridin-3-yl)phenyl)-N-methylthiazol-2-amine) using general method E. Intermediate 2r1 was synthesized from (6-methoxypyridin-3-yl)boronic acid and 4-(2-bromo-4-chlorophenyl)-N-methylthiazol-2-amine by Suzuki coupling with the conditions described in Scheme 15. 4-(2-bromo-4-chlorophenyl)-N-methylthiazol-2-amine was synthesized using general method C.
compound no152: (R)-3-benzyl-4-((4-(5-chloro-2-(6-methoxypyridin-3-yl)phenyl)thiazol-2-yl)(methyl)amino)-4-oxobutanoic acid was synthesized from intermediates 1b and 2s1 (4-(5-chloro-2-(6-methoxypyridin-3-yl)phenyl)-N-methylthiazol-2-amine) using general method E. Intermediate 2s1 was synthesized from (6-methoxypyridin-3-yl)boronic acid and 4-(2-bromo-5-chlorophenyl)-N-methylthiazol-2-amine by Suzuki coupling with the conditions described in Scheme 15. 4-(2-bromo-5-chlorophenyl)-N-methylthiazol-2-amine was synthesized using general method C.
compound no153: (R)-3-benzyl-4-((4-(3-fluoro-2-(6-methoxypyridin-3-yl)phenyl)thiazol-2-yl)(methyl)amino)-4-oxobutanoic acid was synthesized from intermediates 1b and 2t1 (4-(3-fluoro-2-(6-methoxypyridin-3-yl)phenyl)-N-methylthiazol-2-amine) using general method E and preparative HPLC purification. Intermediate 2t1 was synthesized from (6-methoxypyridin-3-yl)boronic acid and 4-(2-bromo-3-fluorophenyl)-N-methylthiazol-2-amine by Suzuki coupling with the conditions described in Scheme 15. 4-(2-bromo-3-fluorophenyl)-N-methylthiazol-2-amine was synthesized using general method C.
compound no154: (3R)-4-((4-(2-chlorophenyl)thiazol-2-yl)(methyl)amino)-4-oxo-3-((tetrahydrofuran-2-yl)methyl)butanoic acid was synthesized from intermediates 1e1 and 2c using general method E.
compound no155: (3R)-4-((4-(2-chlorophenyl)thiazol-2-yl)(methyl)amino)-4-oxo-3-((tetrahydrofuran-2-yl)methyl)butanoic acid was synthesized from intermediates 1b and 2u1 using general method E followed by debenzylation with FeCl3 in DCM.
compound no156: (R)-3-benzyl-4-((4-(2-chlorophenyl)thiazol-2-yl)(3-hydroxypropyl)amino)-4-oxobutanoic acid was synthesized from intermediates 1b and 2v1 using general method E followed by debenzylation with FeCl3 in DCM.
compound no157: (R)-3-benzyl-4-((4-(2-(5-chloro-6-methoxypyridin-3-yl)phenyl)thiazol-2-yl)(methyl)amino)-4-oxobutanoic acid was synthesized from intermediates 1b and 2w1 (4-(2-(5-chloro-6-methoxypyridin-3-yl)phenyl)-N-methylthiazol-2-amine) using general method E. Intermediate 2w1 was synthesized from 5-bromo-3-chloro-2-methoxypyridine and 2l using the methodology described in Scheme 17.
compound no158: (R)-3-benzyl-4-((4-(2-(6-(benzyloxy)pyridin-3-yl)phenyl)thiazol-2-yl)(methyl)amino)-4-oxobutanoic acid was synthesized from intermediates 1b and 2x1 (4-(2-(6-(benzyloxy)pyridin-3-yl)phenyl)-N-methylthiazol-2-amine) using general method E and preparative HPLC purification. Intermediate 2x1 was synthesized from (6-benzyloxypyridin-3-yl)boronic acid and 2l by Suzuki coupling with the conditions described in Scheme 15.
compound no159: (R)-3-(cyclopentylmethyl)-4-((4-(2,5-dichlorophenyl)thiazol-2-yl)(methyl)amino)-4-oxobutanoic acid was synthesized from intermediates 1f1 and 2a1 using general method E.
compound no160: (R)-4-((4-(2-(6-methoxypyridin-3-yl)phenyl)thiazol-2-yl)(methyl)amino)-4-oxo-3-((tetrahydro-2H-pyran-4-yl)methyl)butanoic acid was synthesized from intermediates 1g1 and 2c using general method E.
compound no161: (R)-3-benzyl-4-((4-(2-chloro-5-(trifluoromethyl)phenyl)thiazol-2-yl)(methyl)amino)-4-oxobutanoic acid was synthesized from intermediates 1b and 2y1 using general method E.
compound no162: (R)-3-benzyl-4-((4-(2-chloro-5-fluorophenyl)thiazol-2-yl)(methyl)amino)-4-oxobutanoic acid was synthesized from intermediates 1b and 2z1 using general method E.
compound no163: (R)-3-benzyl-4-((4-(3,5-dichlorophenyl)thiazol-2-yl)(methyl)amino)-4-oxobutanoic acid was synthesized from intermediates 1b and 2a2 using general method E.
compound no164: (R)-3-benzyl-4-((4-(3-(difluoromethoxy)phenyl)thiazol-2-yl)(methyl)amino)-4-oxobutanoic acid was synthesized from intermediates 1b and 2b2 using general method E.
compound no165: (R)-4-((4-(2-chlorophenyl)thiazol-2-yl)(methyl)amino)-3-(cyclopentylmethyl)-4-oxobutanoic acid was synthesized from intermediates 1f1 and 2c using general method E.
compound no166: (R)-3-(cyclopentylmethyl)-4-(cyclopropyl(4-(2,5-dichlorophenyl)thiazol-2-yl)amino)-4-oxobutanoic acid was synthesized from intermediates 1f1 and 2w using general method E.
compound no167: (R)-4-(cyclopropyl(4-(2,5-dichlorophenyl)thiazol-2-yl)amino)-4-oxo-3-((tetrahydro-2H-pyran-4-yl)methyl)butanoic acid was synthesized from intermediates 1g1 and 2w using general method E.
compound no168: (R)-4-((4-(2,5-dichlorophenyl)thiazol-2-yl)(methyl)amino)-4-oxo-3-((tetrahydro-2H-pyran-4-yl)methyl)butanoic acid was synthesized from intermediates 1g1 and 2a1 using general method E.
compound no169: (R)-3-(cyclopentylmethyl)-4-(cyclopropyl(4-(2-(6-methoxypyridin-3-yl)phenyl)thiazol-2-yl)amino)-4-oxobutanoic acid was synthesized from intermediates 1f1 and 2c2 (N-cyclopropyl-4-(2-(6-methoxypyridin-3-yl)phenyl)thiazol-2-amine) using general method E. Intermediate 2c2 was synthesized from (6-methoxypyridin-3-yl)boronic acid and 4-(2-bromo-4-chlorophenyl)-N-cyclopropylthiazol-2-amine by Suzuki coupling with the conditions described_in Scheme 15. 4-(2-bromo-4-chlorophenyl)-N-cyclopropylthiazol-2-amine was synthesized using general method C.
compound no170: (R)-3-benzyl-4-((2-hydroxyethyl)(4-(2-(6-methoxypyridin-3-yl)phenyl)thiazol-2-yl)amino)-4-oxobutanoic acid was synthesized from intermediates 1b and 2d2 (N-(2-(benzyloxy)ethyl)-4-(2-(6-methoxypyridin-3-yl)phenyl)thiazol-2-amine) using general method E followed by debenzylation with FeCl3 in DCM. Intermediate 2d2 was synthesized from (6-methoxypyridin-3-yl)boronic acid and N-(2-(benzyloxy)ethyl)-4-(2-bromophenyl)thiazol-2-amine by Suzuki coupling with the conditions described in Scheme 15. N-(2-(benzyloxy)ethyl)-4-(2-bromophenyl)thiazol-2-amine was synthesized using general method C.
compound no171: (R)-3-(cyclopentylmethyl)-4-(methyl(4-(2-(6-morpholinopyridin-3-yl)phenyl)thiazol-2-yl)amino)-4-oxobutanoic acid was synthesized from intermediates 1f1 and 2d1 using general method E.
compound no172: (R)-3-(cyclopentylmethyl)-4-((4-(2,5-dichlorophenyl)thiazol-2-yl)(2-hydroxyethyl)amino)-4-oxobutanoic acid was synthesized from intermediates 1f1 and 2d3 using general method E followed by debenzylation with FeCl3 in DCM.
compound no173: (R)-4-((4-(2-chlorophenyl)thiazol-2-yl)(methyl)amino)-4-oxo-3-((tetrahydro-2H-pyran-4-yl)methyl)butanoic acid was synthesized from intermediates 1g1 and 2c using general method E.
compound no174: (R)-3-benzyl-4-((4-(2-chloro-5-(trifluoromethyl)phenyl)thiazol-2-yl)(methyl)amino)-4-oxobutanoic acid was synthesized from intermediates 1b and 2e2 using general method E.
compound no175: (R)-3-benzyl-4-(methyl(4-(2,3,5-trichlorophenyl)thiazol-2-yl)amino)-4-oxobutanoic acid was synthesized from intermediates 1b and 2f2 using general method E.
compound no176: (R)-3-benzyl-4-((4-(4-chloro-[1,1′-biphenyl]-3-yl)thiazol-2-yl)(methyl)amino)-4-oxobutanoic acid was synthesized from intermediates 1b and 2g2 (4-(4-chloro-[1,1′-biphenyl]-3-yl)-N-methylthiazol-2-amine) using general method E. Intermediate 2g2 was synthesized from phenylboronic acid and 4-(5-bromo-2-chlorophenyl)-N-methylthiazol-2-amine by Suzuki coupling with the conditions described in Scheme 15. 4-(5-bromo-2-chlorophenyl)-N-methylthiazol-2-amine was synthesized using general method C.
compound no177: (R)-3-benzyl-4-((4-(2-chloro-5-(6-methoxypyridin-3-yl)phenyl)thiazol-2-yl)(methyl)amino)-4-oxobutanoic acid was synthesized from intermediates 1b and 2h2 (4-(2-chloro-5-(6-methoxypyridin-3-yl)phenyl)-N-methylthiazol-2-amine) using general method E. Intermediate 2h2 was synthesized from (6-methoxypyridin-3-yl)boronic acid and 4-(5-bromo-2-chlorophenyl)-N-methylthiazol-2-amine by Suzuki coupling with the conditions described in Scheme 15. 4-(5-bromo-2-chlorophenyl)-N-methylthiazol-2-amine was synthesized using general method C.
compound no178: (R)-3-benzyl-4-(cyclopropyl(4-(2-(6-methoxypyridin-3-yl)phenyl)thiazol-2-yl)amino)-4-oxobutanoic acid was synthesized from intermediates 1b and 2c2 using general method E.
compound no179: (R)-4-(cyclopropyl(4-(2-(6-methoxypyridin-3-yl)phenyl)thiazol-2-yl)amino)-4-oxo-3-((tetrahydro-2H-pyran-4-yl)methyl)butanoic acid was synthesized from intermediates 1g1 and 2c2 using general method E.
compound no180: (R)-3-benzyl-4-(cyclopropyl(4-(2-(6-morpholinopyridin-3-yl)phenyl)thiazol-2-yl)amino)-4-oxobutanoic acid was synthesized from intermediates 1b and 2i2 (N-cyclopropyl-4-(2-(6-morpholinopyridin-3-yl)phenyl)thiazol-2-amine) using general method E. Intermediate 2i2 was synthesized from 4-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)morpholine and 4-(2-bromophenyl)-N-cyclopropylthiazol-2-amine by Suzuki coupling with the conditions described in Scheme 15. 4-(2-bromophenyl)-N-cyclopropylthiazol-2-amine was synthesized using general method C.
compound no181: (R)-3-(cyclopentylmethyl)-4-(cyclopropyl(4-(2-(6-morpholinopyridin-3-yl)phenyl)thiazol-2-yl)amino)-4-oxobutanoic acid was synthesized from intermediates 1f1 and 2i2 using general method E.
compound no182: (R)-3-benzyl-4-(methyl(4-(2-(4-methyl-3,4-dihydro-2H-pyrido[3,2-b][1,4]oxazin-7-yl)phenyl)thiazol-2-yl)amino)-4-oxobutanoic acid was synthesized from intermediates 1b and 2j2 (N-methyl-4-(2-(4-methyl-3,4-dihydro-2H-pyrido[3,2-b][1,4]oxazin-7-yl)phenyl)thiazol-2-amine) using general method E and preparative HPLC purification. Intermediate 2j2 was synthesized from commercially available 7-bromo-4-methyl-3,4-dihydro-2H-pyrido[3,2-b][1,4]oxazine and 2l using the methodology described in Scheme 17.
compound no183: (R)-3-(cyclopentylmethyl)-4-(cyclopropyl(4-(2-(6-(2-oxopyrrolidin-1-yl)pyridin-3-yl)phenyl)thiazol-2-yl)amino)-4-oxobutanoic acid was synthesized from intermediates 1b and 2k2 (1-(5-(2-(2-(cyclopropylamino)thiazol-4-yl)phenyl)pyridin-2-yl)pyrrolidin-2-one) using general method E. Intermediate 2k2 was synthesized from 1-(5-bromopyridin-2-yl)pyrrolidin-2-one and 4-(2-bromo-4-chlorophenyl)-N-cyclopropylthiazol-2-amine using the methodology described in Scheme 17. 1-(5-bromopyridin-2-yl)pyrrolidin-2-one was synthesized by reacting 5-bromopyridin-2-amine with Na2HPO4 in CHCl3, 4-bromobutyryl chloride and NaOMe in MeOH as described in Tetrahedron 1957, 1, 9635. 4-(2-bromo-4-chlorophenyl)-N-cyclopropylthiazol-2-amine was synthesized using general method C.
compound no184: (R)-4-(cyclopropyl(4-(2-(6-morpholinopyridin-3-yl)phenyl)thiazol-2-yl)amino)-4-oxo-3-((tetrahydro-2H-pyran-4-yl)methyl)butanoic acid was synthesized from intermediates 1g1 and 2i2 using general method E.
compound no185: (R)-3-benzyl-4-(methyl(4-(2-(trifluoromethoxy)phenyl)thiazol-2-yl)amino)-4-oxobutanoic acid was synthesized from intermediates 1b and 2l2 using general method E.
compound no186: (R)-4-((4-(2-chloro-5-fluorophenyl)thiazol-2-yl)(cyclopropyl)amino)-3-(cyclopentylmethyl)-4-oxobutanoic acid was synthesized from intermediates 1b and 2m2 using general method E and preparative HPLC purification.
compound no187: (R)-3-(cyclopentylmethyl)-4-(methyl(4-(2-(6-(2-oxopyrrolidin-1-yl)pyridin-3-yl)phenyl)thiazol-2-yl)amino)-4-oxobutanoic acid was synthesized from intermediates 1f1 and 2q1 using general method E.
compound no188: (R)-3-benzyl-4-(cyclopropyl(4-(3-(difluoromethoxy)phenyl)thiazol-2-yl)amino)-4-oxobutanoic acid was synthesized from intermediates 1b and 2n2 using general method E and preparative HPLC purification.
compound no189: (R)-3-benzyl-4-((4-(2-chloro-5-fluorophenyl)thiazol-2-yl)(cyclopropyl)amino)-4-oxobutanoic acid was synthesized from intermediates 1b and 2m2 using general method E and preparative HPLC purification.
compound no190: (R)-4-((4-(2-chloro-5-fluorophenyl)thiazol-2-yl)(cyclopropyl)amino)-4-oxo-3-((tetrahydro-2H-pyran-4-yl)methyl)butanoic acid was synthesized from intermediates 1g1 and 2m2 using general method E and preparative HPLC purification.
compound no191: (R)-3-benzyl-4-((4-(2-chloro-5-(trifluoromethyl)phenyl)thiazol-2-yl)(cyclopropyl)amino)-4-oxobutanoic acid was synthesized from intermediates 1b and 2o2 using general method E and preparative HPLC purification.
compound no192: (R)-3-benzyl-4-((4-(2-(difluoromethoxy)phenyl)thiazol-2-yl)(methyl)amino)-4-oxobutanoic acid was synthesized from intermediates 1b and 2c4 using general method E
compound no193: (R)-4-((4-(2-chloro-5-(trifluoromethyl)phenyl)thiazol-2-yl)(cyclopropyl)amino)-4-oxo-3-((tetrahydro-2H-pyran-4-yl)methyl)butanoic acid was synthesized from intermediates 1g1 and 2o2 using general method E.
compound no194: (R)-3-(cyclopentylmethyl)-4-(cyclopropyl(4-(2-(4-methyl-3,4-dihydro-2H-pyrido[3,2-b][1,4]oxazin-7-yl)phenyl)thiazol-2-yl)amino)-4-oxobutanoic acid was synthesized from intermediates 1f1 and 2p2 (N-cyclopropyl-4-(2-(4-methyl-3,4-dihydro-2H-pyrido[3,2-b][1,4]oxazin-7-yl)phenyl)thiazol-2-amine) using general method E. Intermediate 2p2 was synthesized from commercially available 7-bromo-4-methyl-3,4-dihydro-2H-pyrido[3,2-b][1,4]oxazine and 2l using the methodology described in Scheme 17. 4-(2-bromo-4-chlorophenyl)-N-cyclopropylthiazol-2-amine was synthesized using general method C.
compound no195: (3R,4S)-3-((4-(2-chlorophenyl)thiazol-2-yl)(methyl)carbamoyl)-4-phenylpentanoic acid was synthesized from intermediates 1h1 and 2c using general method E.
compound no196: (R)-2-(2-benzyl-3-carboxypropanamido)-5-(2-chlorophenyl)pyridine 1-oxide was synthesized from intermediates 1b and 2q2 (2-amino-5-(2-chlorophenyl)pyridine) using general method E followed by oxidation with MCPBA. 2q2 was made from commercially available 5-bromopyridin-2-amine and (2-chlorophenyl)boronic acid using Suzuki coupling.
compound no197: (R)-3-benzyl-4-((5-(2-chlorophenyl)pyrazin-2-yl)amino)-4-oxobutanoic acid was synthesized from intermediates 1b and 2r2 (5-(2-chlorophenyl)pyrazin-2-amine) using general method E. 2r2 was made from commercially available 5-bromopyrazin-2-amine and (2-chlorophenyl)boronic acid using Suzuki coupling.
compound no198: 4-((4-(2-chlorophenyl)thiazol-2-yl)(methyl)amino)-3-(morpholinomethyl)-4-oxobutanoic acid was synthesized as described in Scheme 18.
compound no199: (R)-3-benzyl-4-((4-(2-chlorophenyl)thiazol-2-yl)(2-methoxyethyl)amino)-4-oxobutanoic acid was synthesized from intermediates 1b and 2s2 using general method E.
compound no200: (R)-4-((4-(2-chlorophenyl)thiazol-2-yl)(methyl)amino)-3-(cyclopentylamino)-4-oxobutanoic acid was synthesized from intermediates 1j1 ((R)-4-(tert-butoxy)-2-(cyclopentylamino)-4-oxobutanoic acid) and 2c using general method E. 1j1 was made from (R)-2-amino-4-(tert-butoxy)-4-oxobutanoic acid and cyclopentanone by reductive amination using sodium cyanoborohydride in methanol.
compound no201: (R)-3-benzyl-4-((2-(benzyloxy)ethyl)(4-(2-chlorophenyl)thiazol-2-yl)amino)-4-oxobutanoic acid was synthesized from intermediates 1b and 2u1 using general method E.
compound no202: (R)-3-benzyl-4-((4-(5-methylfuran-2-yl)thiazol-2-yl)amino)-4-oxobutanoic acid was synthesized from intermediates 1b and commercially available 2u2 (4-(5-methylfuran-2-yl)thiazol-2-amine) using general method E.
compound no203: (R)-3-benzyl-4-oxo-4-((3-(3-(trifluoromethyl)phenyl)-1H-pyrazol-5-yl)amino)butanoic acid was synthesized from intermediates 1b and commercially available 2v2 (3-(3-(trifluoromethyl)phenyl)-1H-pyrazol-5-amine) using general method E.
compound no204: (R)-3-benzyl-4-((4-(5-chloro-2-methoxyphenyl)thiazol-2-yl)amino)-4-oxobutanoic acid was synthesized from intermediates 1b and commercially available 2w2 (4-(5-chloro-2-methoxyphenyl)thiazol-2-amine) using general method E.
compound no205: 4-((4-(2-chlorophenyl)thiazol-2-yl)(methyl)amino)-3-(4-hydroxybenzyl)-4-oxobutanoic acid was synthesized from from intermediates 1k1 (4-(tert-butoxy)-2-(4-(methoxymethoxy)benzyl)-4-oxobutanoic acid) and 2c using general method E, the MOM group was deprotected with TFA in DCM. 1k1 was synthesized from 4-(methoxymethoxy)benzaldehyde using the HWE methodology (Scheme 13).
compound no206: (R)-3-benzyl-4-((4-(4′-cyano-[1,1′-biphenyl]-2-yl)thiazol-2-yl)(methyl)amino)-4-oxobutanoic acid was synthesized from intermediates 1b and 2x2 (2′-(2-(methylamino)thiazol-4-yl)-[1,1′-biphenyl]-4-carbonitrile) using general method E. Intermediate 2x2 was synthesized from (4-cyanophenyl)boronic acid and 2l by Suzuki coupling with the conditions described in Scheme 15.
compound no207: (3R)-3-benzyl-4-((3-carbamoyl-4-(2,4-dichlorophenyl)-5-methylthiophen-2-yl)amino)-4-oxobutanoic acid was synthesized from intermediates 1b and commercially available 2y2 (2-amino-4-(2,4-dichlorophenyl)-5-methylthiophene-3-carbonitrile) using general method E.
compound no208: (R)-3-benzyl-4-((4-(3′-methoxy-[1,1′-biphenyl]-2-yl)thiazol-2-yl)(methyl)amino)-4-oxobutanoic acid was synthesized from intermediates 1b and 2z2 (4-(3′-methoxy-[1,1′-biphenyl]-2-yl)-N-methylthiazol-2-amine) using general method E. Intermediate 2z2 was synthesized from (3-methoxyphenyl)boronic acid and 2l by Suzuki coupling with the conditions described in Scheme 15.
compound no209: 4-((4-(2-chlorophenyl)thiazol-2-yl)(methyl)amino)-3-((2-methylthiazol-4-yl)methyl)-4-oxobutanoic acid was synthesized from intermediates 1l1 (4-(tert-butoxy)-2-((2-methylthiazol-4-yl)methyl)-4-oxobutanoic acid) and 2c using general method E. 1l1 was synthesized from 2-methylthiazole-5-carbaldehyde using the HWE methodology (Scheme 13).
compound no210: 4-((4-(2-chlorophenyl)thiazol-2-yl)(methyl)amino)-3-((5-methylisoxazol-3-yl)methyl)-4-oxobutanoic acid was synthesized from intermediates 1m1 (4-(tert-butoxy)-2-((5-methylisoxazol-3-yl)methyl)-4-oxobutanoic acid) and 2c using general method E. 1m1 was synthesized from 5-methylisoxazole-3-carbaldehyde using the HWE methodology (Scheme 13).
compound no211: (R)-3-benzyl-4-((4-(2′-chloro-[1,1′-biphenyl]-2-yl)thiazol-2-yl)(methyl)amino)-4-oxobutanoic acid was synthesized from intermediates 1b and 2a3 (4-(2′-chloro-[1,1′-biphenyl]-2-yl)-N-methylthiazol-2-amine) using general method E and preparative HPLC purification. Intermediate 2a3 was synthesized from (2-chlorophenyl)boronic acid and 2l by Suzuki coupling with the conditions described in Scheme 15.
compound no212: (R)-3-benzyl-4-((4-(2-(2-methoxypyrimidin-5-yl)phenyl)thiazol-2-yl)(methyl)amino)-4-oxobutanoic acid was synthesized from intermediates 1b and 2b3 (4-(2-(2-methoxypyrimidin-5-yl)phenyl)-N-methylthiazol-2-amine) using general method E. Intermediate 2b3 was synthesized from 5-bromo-2-methoxypyrimidine and 2l using the methodology described in Scheme 17.
compound no213: (R)-3-benzyl-4-((4-(2,5-difluorophenyl)thiazol-2-yl)amino)-4-oxobutanoic acid was synthesized from intermediates 1b and commercially available 2c3 (4-(2,5-difluorophenyl)thiazol-2-amine) using general method E.
compound no214: 4-((4-(2-chlorophenyl)thiazol-2-yl)(methyl)amino)-3-(oxazol-4-ylmethyl)-4-oxobutanoic acid was synthesized from intermediates 1n1 (4-(tert-butoxy)-2-(oxazol-4-ylmethyl)-4-oxobutanoic acid) and 2c using general method E. 1n1 was synthesized from oxazole-4-carbaldehyde using the HWE methodology (Scheme 13).
compound no215: (3R)-4-((4-(2-chlorophenyl)thiazol-2-yl)(methyl)amino)-4-oxo-3-((tetrahydrofuran-3-yl)methyl)butanoic acid was synthesized from intermediates 1o1 and 2c using general method E.
compound no216: (R)-3-benzyl-4-(methyl(4-(2-(8-methyl-7-oxo-5,6,7,8-tetrahydro-1,8-naphthyridin-3-yl)phenyl)thiazol-2-yl)amino)-4-oxobutanoic acid was synthesized from intermediates 1b and 2e3 (1-methyl-6-(2-(2-(methylamino)thiazol-4-yl)phenyl)-3,4-dihydro-1,8-naphthyridin-2(1H)-one) using general method E.
Intermediate 2e3 was synthesized from 6-bromo-1-methyl-3,4-dihydro-1,8-naphthyridin-2(1H)-one (which was obtained by treatment of 6-bromo-3,4-dihydro-1,8-naphthyridin-2(1H)-one with NaH in DMF and MeI) and 2l using the methodology described in Scheme 17. Intermediate 1b was synthesized using the HWE methodology (Scheme 13):
38.125 mmol of (E)-2-benzylidene-4-(tert-butoxy)-4-oxobutanoic acid, 75 mL of methanol and 38.125 mmol of DCA were successively introduced into a Schlenck tube under Ar. The solution was degassed using three argon/vacuum cycles, and subsequently transferred into the reaction vessel under inert atmosphere. To this degassed solution was added, under argon flow, 0.121 mmol of the RuCl2-[(S)-BINAP] catalyst. The reaction vessel was then transferred into a Parr autoclave, under Ar flow. The Parr vessel was purged 3 times with H2 with a pressure up to 20 sbars; the pressure was then adjusted to 10 bars. The Parr autoclave was put into an oil bath at 55° C. The reaction mixture was stirred at this temperature for 3 days. The reaction mixture was allowed to cool to RT and the hydrogen pressure was released carefully and the Parr vessel opened. The crude reaction mixture was concentrated to dryness using rotary evaporator to afford 16.74 g of a colored solid. An aliquot of the solid was diluted with water and acidified with HCl 6N to pH 1; then, the solution was extracted with EtOAc. The organic layer was dried over magnesium sulfate, concentrated using rotary evaporator to yield the desired intermediate (ee=82.6%, determined by chiral HPLC). Solid (16.74 g) was recrystallized from an ACN/water mixture. Recrystallized product was diluted with water and acidified with 6N HCl to pH 1, the solution was extracted with EtOAc. The organic layer was dried over magnesium sulfate, concentrated at rotavap to yield the desired intermediate 1b (ee=96.6%, determined by chiral HPLC).
compound no217: (R)-3-benzyl-4-(methyl(4-(2-(1-methyl-1H-pyrrolo[2,3-b]pyridin-5-yl)phenyl)thiazol-2-yl)amino)-4-oxobutanoic acid was synthesized from intermediates 1b and 2f3 (N-methyl-4-(2-(1-methyl-1H-pyrrolo[2,3-b]pyridin-5-yl)phenyl)thiazol-2-amine) using general method E. Intermediate 2f3 was synthesized from 5-bromo-1-methyl-1-H-pyrrolo[2,3-b]pyridine (which was obtained by treatment of 5-bromo-1H-pyrrolo[2,3-b]pyridine with NaH in DMF and MeI) and 2l using the methodology described in Scheme 17.
compound no218: (R)-3-(cyclopentylmethyl)-4-(cyclopropyl(4-(2-(6-(dimethylamino)pyridin-3-yl)phenyl)thiazol-2-yl)amino)-4-oxobutanoic acid was synthesized from intermediates 1f1 and 2g3 (N-cyclopropyl-4-(2-(6-(dimethylamino)pyridin-3-yl)phenyl)thiazol-2-amine) using general method E and preparative HPLC purification. 2g3 was synthesized from 4-(2-bromophenyl)-N-cyclopropylthiazol-2-amine and 6-(dimethylamino)pyridin-3-ylboronic acid by Suzuki coupling with the conditions described in Scheme 15. 4-(2-bromophenyl)-N-cyclopropylthiazol-2-amine was synthesized using general method C.
compound no219: (R)-4-((4-(2-(5-chloro-6-methoxypyridin-3-yl)phenyl)thiazol-2-yl)(cyclopropyl)amino)-3-(cyclopentylmethyl)-4-oxobutanoic acid was synthesized from intermediates 1f1 and 2h3 (4-(2-(5-chloro-6-methoxypyridin-3-yl)phenyl)-N-cyclopropylthiazol-2-amine) using general method E. 2g3 was synthesized from 5-bromo-3-chloro-2-methoxypyridine and 4-(2-bromophenyl)-N-cyclopropylthiazol-2-amine using the methodology described in Scheme 17. 4-(2-bromophenyl)-N-cyclopropylthiazol-2-amine was synthesized using general method C.
compound no220: (R)-3-(cyclopentylmethyl)-4-(cyclopropyl(4-(2-(5-fluoro-6-methoxypyridin-3-yl)phenyl)thiazol-2-yl)amino)-4-oxobutanoic acid was synthesized from intermediates 1f1 and 2i3 (N-cyclopropyl-4-(2-(5-fluoro-6-methoxypyridin-3-yl)phenyl)thiazol-2-amine) using general method E. 2i3 was synthesized from 5-bromo-3-fluoro-2-methoxypyridine and 4-(2-bromophenyl)-N-cyclopropylthiazol-2-amine using the methodology described in Scheme 17. 4-(2-bromophenyl)-N-cyclopropylthiazol-2-amine was synthesized using general method C.
compound no221: (R)-3-benzyl-4-((4-(2-chloro-5-(difluoromethoxy)phenyl)thiazol-2-yl)(methyl)amino)-4-oxobutanoic acid was synthesized from intermediates 1b and 2j3 using general method E.
compound no222: (R)-3-benzyl-4-((4-(5-chloro-2-(5-chloro-6-methoxypyridin-3-yl)phenyl)thiazol-2-yl)(methyl)amino)-4-oxobutanoic acid was synthesized from intermediates 1b and 2k3 (4-(5-chloro-2-(5-chloro-6-methoxypyridin-3-yl)phenyl)-N-methylthiazol-2-amine) using general method E. 2k3 was synthesized from 5-bromo-3-chloro-2-methoxypyridine and 4-(2-bromo-5-chlorophenyl)-N-methylthiazol-2-amine using the methodology described in Scheme 17. 4-(2-bromo-5-chlorophenyl)-N-methylthiazol-2-amine was synthesized using general method C.
compound no223: (R)-4-((4-(5-chloro-2-(5-chloro-6-methoxypyridin-3-yl)phenyl)thiazol-2-yl)(cyclopropyl)amino)-3-(cyclopentylmethyl)-4-oxobutanoic acid was synthesized from intermediates 1f1 and 2l3 (4-(5-chloro-2-(5-chloro-6-methoxypyridin-3-yl)phenyl)-N-cyclopropylthiazol-2-amine) using general method E. 2l3 was synthesized from 5-bromo-3-chloro-2-methoxypyridine and 4-(2-bromo-5-chlorophenyl)-N-cyclopropylthiazol-2-amine using the methodology described in Scheme 17. 4-(2-bromo-5-chlorophenyl)-N-cyclopropylthiazol-2-amine was synthesized using general method C.
compound no224: (R)-4-((4-(5-chloro-2-(5-fluoro-6-methoxypyridin-3-yl)phenyl)thiazol-2-yl)(cyclopropyl)amino)-3-(cyclopentylmethyl)-4-oxobutanoic acid was synthesized from intermediates 1f1 and 2m3 (4-(5-chloro-2-(5-fluoro-6-methoxypyridin-3-yl)phenyl)-N-cyclopropylthiazol-2-amine) using general method E. 2m3 was synthesized from 5-bromo-3-fluoro-2-methoxypyridine and 4-(2-bromo-5-chlorophenyl)-N-cyclopropylthiazol-2-amine using the methodology described in Scheme 17. 4-(2-bromo-5-chlorophenyl)-N-cyclopropylthiazol-2-amine was synthesized using general method C.
compound no225: (S)-3-benzyl-4-((4-(2-chlorophenyl)thiazol-2-yl)amino)-4-oxobutanoic acid was synthesized from intermediates 1p1 ((S)-2-benzyl-4-(tert-butoxy)-4-oxobutanoic acid) and 2a using general method E. 1p1 was synthesized from (S)-3-benzyl-4-methoxy-4-oxobutanoic acid using the chemistry described in steps 5 and 6 of general method B.
compound no227: (R)-3-benzyl-4-((4-benzylthiazol-2-yl)amino)-4-oxobutanoic acid was synthesized from intermediates 1b and commercially available 4-benzylthiazol-2-amine using general method E.
compound no229: (R)-3-benzyl-4-oxo-4-((5-phenyl-4H-1,2,4-triazol-3-yl)amino)butanoic acid was synthesized from intermediates 1b and commercially available 5-phenyl-4H-1,2,4-triazol-3-amine using general method E.
compound no230: 3-([1,1′-biphenyl]-4-ylmethyl)-4-((4-(2-chlorophenyl)thiazol-2-yl)(methyl)amino)-4-oxobutanoic acid was synthesized from intermediates 1q1 (2-([1,1′-biphenyl]-4-ylmethyl)-4-(tert-butoxy)-4-oxobutanoic acid) and 2c using general method E. 1q1 was synthesized from [1,1′-biphenyl]-4-carbaldehyde using the HWE methodology described in Scheme 13.
compound no231: (R)-3-benzyl-4-((4-(1-methyl-1H-pyrazol-4-yl)thiazol-2-yl)amino)-4-oxobutanoic acid was synthesized from intermediates 1b and commercially available 4-(1-methyl-1H-pyrazol-4-yl)thiazol-2-amine using general method E.
compound no232: ((R)-3-benzyl-4-((4-(4-methyl-1,2,5-oxadiazol-3-yl)thiazol-2-yl)amino)-4-oxobutanoic acid was synthesized from intermediates 1b and commercially available 4-(4-methyl-1,2,5-oxadiazol-3-yl)thiazol-2-amine using general method E.
compound no233: (R)-3-benzyl-4-(methyl(4-(2-(1-methyl-1H-pyrazol-4-yl)phenyl)thiazol-2-yl)amino)-4-oxobutanoic acid was synthesized from intermediates 1b and 2n3 (N-methyl-4-(2-(1-methyl-1H-pyrazol-4-yl)phenyl)thiazol-2-amine) using general method E. 2n3 was synthesized from commercially available 1-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole and 2l using the methodology described in Scheme 15.
compound no234: (3R)-3-benzyl-4-((4-(2-(3,5-dimethylisoxazol-4-yl)phenyl)thiazol-2-yl)(methyl)amino)-4-oxobutanoic acid was synthesized from intermediates 1b and 2o3 (4-(2-(3,5-dimethylisoxazol-4-yl)phenyl)-N-methylthiazol-2-amine) using general method E. 2o3 was synthesized from commercially available 3,5-dimethyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)isoxazole and 2l using the methodology described in Scheme 15.
compound no235: (R)-3-benzyl-4-((4-((2-chlorophenyl)carbamoyl)thiazol-2-yl)amino)-4-oxobutanoic acid was synthesized from intermediates 1b and 2p3 using general method E and preparative HPLC purification. 2p3 was synthesized as described in Scheme 21.
compound no236: (R)-3-benzyl-4-((6-(2-chlorophenyl)pyridazin-3-yl)amino)-4-oxobutanoic acid was synthesized from intermediates 1b and 2q3 (6-(2-chlorophenyl)pyridazin-3-amine) using general method E. 2q3 was synthesized from 6-bromopyridazin-3-amine and 2-chlorophenylboronic acid by Suzuki coupling with the conditions described in Scheme 8.
compound no237: (R)-3-benzyl-4-(methyl(4-(2-(2-oxopyrrolidin-1-yl)phenyl)thiazol-2-yl)amino)-4-oxobutanoic acid was synthesized from intermediates 1b and 2r3 (1-(2-(2-(methylamino)thiazol-4-yl)phenyl)pyrrolidin-2-one) using general method E. 2r3 was synthesized as described in Scheme 16.
compound no238: (S)-2-((1-((4-(2-chlorophenyl)thiazol-2-yl)(methyl)amino)-1-oxo-3-phenylpropan-2-yl)oxy)acetic acid was synthesized as described in Scheme 22.
compound no239: (R)-3-benzyl-4-((1-methyl-5-phenyl-1H-imidazol-2-yl)amino)-4-oxobutanoic acid was synthesized from intermediates 1b and commercially available 1-methyl-5-phenyl-1H-imidazol-2-amine using general method E.
compound no240: (R)-3-benzyl-4-((4-(2-(1-(2-methoxyethyl)-6-oxo-1,6-dihydropyridin-3-yl)phenyl)thiazol-2-yl)(methyl)amino)-4-oxobutanoic acid was synthesized from intermediates 1b and 2s3 (1-(2-methoxyethyl)-5-(2-(2-(methylamino)thiazol-4-yl)phenyl)pyridin-2(1H)-one) using general method E. 2s3 was synthesized from 5-bromo-1-(2-methoxyethyl)pyridin-2(1H)-one and 2l using the methodology described in Scheme 17.
compound no241: (R)-3-benzyl-4-(methyl(4-(2-(1-methyl-6-oxo-1,6-dihydropyridin-3-yl)phenyl)thiazol-2-yl)amino)-4-oxobutanoic acid was synthesized from intermediates 1b and 2t3 (1-methyl-5-(2-(2-(methylamino)thiazol-4-yl)phenyl)pyridin-2(1H)-one) using general method E. 2t3 was synthesized from 5-bromo-1-methylpyridin-2(1H)-one and 2l using the methodology described in Scheme 17.
compound no242: 4-((4-(2-chlorophenyl)thiazol-2-yl)(methyl)amino)-3-((2,5-dimethyloxazol-4-yl)methyl)-4-oxobutanoic acid was synthesized from intermediates 1r1 (tert-butyl 4-amino-3-((2,5-dimethyloxazol-4-yl)methyl)-4-oxobutanoate) and 2c using general method E. 1r1 was synthesized from 2,5-dimethyloxazole-4-carbaldehyde using the HWE methodology described in Scheme 13.
compound no243: 4-((4-(2-chlorophenyl)thiazol-2-yl)(methyl)amino)-3-((1-methyl-1H-pyrazol-5-yl)methyl)-4-oxobutanoic acid was synthesized from intermediates 1s1 (4-(tert-butoxy)-2-((1-methyl-1H-pyrazol-5-yl)methyl)-4-oxobutanoic acid) and 2c using general method E. 1s1 was synthesized from 1-methyl-1H-pyrazole-5-carbaldehyde using the HWE methodology described in Scheme 13.
compound no244: (R)-3-benzyl-4-((4-(2-(6-hydroxypyridin-3-yl)phenyl)thiazol-2-yl)(methyl)amino)-4-oxobutanoic acid was synthesized by debenzylation of compound no158 with FeCl3 in DCM and preparative HPLC purification.
compound no245: (R)-3-benzyl-4-((4-(2-chlorophenyl)thiazol-2-yl)((S)-2-hydroxypropyl)amino)-4-oxobutanoic acid was synthesized from intermediates 1b and 2v3 using general method E and preparative HPLC purification.
compound no246: (R)-3-benzyl-4-((4-(2-chlorophenyl)thiazol-2-yl)((R)-2-hydroxypropyl)amino)-4-oxobutanoic acid was synthesized from intermediates 1b and 2w3 using general method E and preparative HPLC purification.
compound no247: (R)-3-(cyclohexylmethyl)-4-(cyclopropyl(4-(2-(6-methoxypyridin-3-yl)phenyl)thiazol-2-yl)amino)-4-oxobutanoic acid was synthesized from intermediates 1w and 2c2 using general method E and preparative HPLC purification.
compound no248: (R)-3-benzyl-4-((4-(5-fluoro-2-(6-methoxypyridin-3-yl)phenyl)thiazol-2-yl)(methyl)amino)-4-oxobutanoic acid was synthesized from intermediates 1b and 2u3 (4-(5-fluoro-2-(6-methoxypyridin-3-yl)phenyl)-N-methylthiazol-2-amine) using general method E. Intermediate 2u3 was synthesized from (6-methoxypyridin-3-yl)boronic acid and 4-(2-bromo-5-fluorophenyl)-N-methylthiazol-2-amine by Suzuki coupling with the conditions described in Scheme 15. 4-(2-bromo-5-fluorophenyl)-N-methylthiazol-2-amine was synthesized using general method C.
compound no250: (R)-3-benzyl-4-((4-(4,5-difluoro-2-(6-methoxypyridin-3-yl)phenyl)thiazol-2-yl)(methyl)amino)-4-oxobutanoic acid was synthesized from intermediates 1b and 2x3 (4-(4,5-difluoro-2-(6-methoxypyridin-3-yl)phenyl)-N-methylthiazol-2-amine) using general method E. Intermediate 2x3 was synthesized from (6-methoxypyridin-3-yl)boronic acid and 4-(2-bromo-4,5-difluorophenyl)-N-methylthiazol-2-amine by Suzuki coupling with the conditions described in Scheme 15. 4-(2-bromo-4,5-difluorophenyl)-N-methylthiazol-2-amine was synthesized using general method C.
compound no251: (R)-4-((4-(2,5-dichlorophenyl)thiazol-2-yl)(methyl)amino)-3-(furan-2-ylmethyl)-4-oxobutanoic acid was synthesized from intermediates 1t1 and 2a1 using general method E.
compound no252: (R)-4-((4-(2-chloro-5-fluorophenyl)thiazol-2-yl)(methyl)amino)-3-(furan-2-ylmethyl)-4-oxobutanoic acid was synthesized from intermediates 1t1 and 2z1 using general method E.
compound no253: (R)-3-(furan-2-ylmethyl)-4-((4-(2-(6-methoxypyridin-3-yl)phenyl)thiazol-2-yl)(methyl)amino)-4-oxobutanoic acid was synthesized from intermediates 1t1 and 2m using general method E.
compound no254: (S)-4-((4-(2-chlorophenyl)thiazol-2-yl)(methyl)amino)-4-oxo-3-(thiophen-2-ylmethyl)butanoic acid was synthesized from intermediates 1u1 and 2c using general method E.
compound no255: (R)-4-((4-(5-chloro-2-(6-methoxypyridin-3-yl)phenyl)thiazol-2-yl)(cyclopropyl)amino)-3-(cyclopentylmethyl)-4-oxobutanoic acid was synthesized from intermediates 1b and 2y3 (4-(5-chloro-2-(6-methoxypyridin-3-yl)phenyl)-N-cyclopropylthiazol-2-amine) using general method E. Intermediate 2y3 was synthesized from (6-methoxypyridin-3-yl)boronic acid and 4-(2-bromo-5-chlorophenyl)-N-cyclopropylthiazol-2-amine by Suzuki coupling with the conditions described in Scheme 15. 4-(2-bromo-5-chlorophenyl)-N-cyclopropylthiazol-2-amine was synthesized using general method C.
compound no256: (R)-3-benzyl-4-(cyclopropyl(4-(2-(6-(2-oxopyrrolidin-1-yl)pyridin-3-yl)phenyl)thiazol-2-yl)amino)-4-oxobutanoic acid was synthesized from intermediates 1b and 2k2 using general method E.
compound no257: (R)-3-benzyl-4-((4-(2,3-dichlorophenyl)thiazol-2-yl)(methyl)amino)-4-oxobutanoic acid was synthesized from intermediates 1b and 2z3 using general method E.
compound no258: (R)-3-benzyl-4-(methyl(4-(3-(trifluoromethoxy)phenyl)thiazol-2-yl)amino)-4-oxobutanoic acid was synthesized from intermediates 1b and 2a4 using general method E.
compound no259: (R)-4-(cyclopropyl(4-(3-(difluoromethoxy)phenyl)thiazol-2-yl)amino)-4-oxo-3-((tetrahydro-2H-pyran-4-yl)methyl)butanoic acid was synthesized from intermediates 1g1 and 2n2 using general method E.
compound no260: (R)-4-((4-(2-chlorophenyl)thiazol-2-yl)(methyl)amino)-3-(furan-2-ylmethyl)-4-oxobutanoic acid was synthesized from intermediates 1t1 and 2c using general method E.
compound no261: (R)-4-(methyl(4-(3-(trifluoromethoxy)phenyl)thiazol-2-yl)amino)-4-oxo-3-((tetrahydro-2H-pyran-4-yl)methyl)butanoic acid was synthesized from intermediates 1g1 and 2a4 using general method E.
compound no262: (R)-3-benzyl-4-(cyclopropyl(4-(3-(trifluoromethoxy)phenyl)thiazol-2-yl)amino)-4-oxobutanoic acid was synthesized from intermediates 1b and 2b4 using general method E.
compound no263: (R)-4-(cyclopropyl(4-(3-(trifluoromethoxy)phenyl)thiazol-2-yl)amino)-4-oxo-3-((tetrahydro-2H-pyran-4-yl)methyl)butanoic acid was synthesized from intermediates 1g1 and 2b4 using general method E.
The following assay can be used for determination of GPR43 activation. When a GPCR is in its active state, either as a result of ligand binding or constitutive activation, the receptor couples to a G protein and stimulates the release of GDP and subsequent binding of GTP to the G protein. The alpha subunit of the G protein-receptor complex acts as a GTPase and slowly hydrolyses the GTP to GDP, at which point the receptor normally is deactivated. Activated receptors continue to exchange GDP for GTP. The non-hydrolysable GTP analog, [35S]GTPγS, was used to demonstrate enhance binding of [35S]GTPγS to membranes expressing receptors. The assay uses the ability of GPCR to stimulate [35S]GTPγS binding to membranes expressing the relevant receptors. The assay can, therefore, be used in the direct identification method to screen candidate compounds to endogenous or not endogenous GPCR.
Membrane extracts were prepared from cells expressing the human GPR43 receptor (hGPR43) as follows: the medium was aspirated and the cells were scraped from the plates in Ca++ and Mg++-free Phosphate-buffered saline (PBS). The cells were then centrifuged for 3 min at 1500 g and the pellets were resuspended in buffer A (15 mM Tris-HCl pH 7.5, 2 mM MgCl2, 0.3 mM EDTA, 1 mM EGTA) and homogenized in a glass homogenizer. The crude membrane fraction was collected by two consecutive centrifugation steps at 40.000×g for 25 min separated by a washing step in buffer A. The final pellet was resuspended in 500 μl of buffer B (75 mM Tris-HCl pH 7.5, 12.5 mM MgCl2, 0.3 mM EDTA, 1 mM EGTA, 250 mM sucrose) and flash frozen in liquid nitrogen. Protein content was assayed by the Folin method.
The assay was performed in the presence of SCFA, and was used to determine the activity of the compounds of the invention.
The [35S]GTPγS assay was incubated in 20 mM HEPES pH7.4, 100 mM NaCl, 10 μg/ml saponin, 30 mM of MgCl2, 10 μM of GDP, 5 μg membrane-expressing hGPR43, 250 μg of wheatgerm agglutinin beads (Amersham, ref: RPNQ001), a range concentration of compounds (from 30 μM to 1 nM) in a final volume of 100 μl for 30 min at room temperature. The SCFA propionate was used at 1 mM final concentration as positive control. The plates were then centrifuged for 10 minutes at 2000 rpm, incubated for 2 hours at room temperature and counted for 1 min in a scintillation counter (TopCount, PerkinElmer). The results of the tested compounds are reported as the concentration of the compound required to reach 50% (EC50) of the maximum level of the activation induced by these compounds.
When tested in the assay described above and by way of illustration the compounds in Table 3 activate GPR43 receptor with an EC50 ranging from 13 nM to 2910 nM.
Cell Based Assay: Calcium Flux. The Aequorin-Based Assay
The following assay can be used for determination of GPR43 activation. The aequorin assay uses the responsiveness of mitochondrial apoaequorin to intracellular calcium release induced by the activation of GPCRs (Stables et al., 1997, Anal. Biochem. 252:115-126; Detheux et al., 2000, J. Exp. Med., 192 1501-1508). Briefly, GPCR-expressing clones are transfected to coexpress mitochondrial apoaequorin and Ga16. Cells expressing GPR43 receptor are incubated with 5 μM Coelenterazine H (Molecular Probes) for 4 hours at room temperature, washed in DMEM-F12 culture medium and resuspended at a concentration of 0.5×106 cells/ml (the amount can be changed for optimization). Cells are then mixed with test compounds and light emission by the aequorin is recorded with a luminometer for 30 sec. Results are expressed as Relative Light Units (RLU). Controls include assays using cells not expressing GPR43 (mock transfected), in order to exclude possible non-specific effects of the candidate compound.
Aequorin activity or intracellular calcium levels are “changed” if light intensity increases or decreases by 10% or more in a sample of cells, expressing a GPR43 and treated with a compound of the invention, relative to a sample of cells expressing the GPR43 but not treated with the compound of the invention or relative to a sample of cells not expressing the GPR43 (mock-transfected cells) but treated with the compound of the invention.
Cell based assay: Intracellular Inositol-Phosphate Accumulation Assay. (Gq-Associated Receptor)
The following assay can be used for determination of GPR43 activation. On day 1, GPR43-expressing cells in mid-log phase are detached with PBS-EDTA, centrifuged at 2000×g for 2 min and resuspended in medium without antibiotics. After counting, cells are resuspended at 4×105 cells/ml (the amount can be changed for optimization) in medium without antibiotics, distributed in a 96 well plate (100 μl/well) and the plate is incubated overnight at 37° C. with 5% CO2. On day 2, the medium is removed and the compounds of the invention, at increasing concentrations, are added (24 μl/well) and the plate is incubated for 30 min. at 37° C. in a humidified atmosphere of 95% air with 5% CO2. The IP1 concentrations are then estimated using the IP1-HTRF assay kit (Cisbio international, France) following the manufacturer recommendations.
Cell Based Assay: cAMP Accumulation Assay (Gi/o, Associated Receptor)
The following assay can be used for determination of GPR43 activation. Cells expressing GPR43 in mid-log phase and grown in media without antibiotics are detached with PBS-EDTA, centrifuged and resuspended in media without antibiotics. Cells are counted and resuspended in assay buffer at 4.2×105 cells/ml. 96 well plates are filled with 12 μl of cells (5×103 cells/well), 6 μl of compound of the invention at increasing concentrations and 6 μl of Forskolin (final concentration of 10 μM). The plate is then incubated for 30 min. at room temperature. After addition of the lysis buffer, cAMP concentrations are estimated, according to the manufacturer specification, with the HTRF kit from Cis-Bio International.
In vitro Assays to Assess Compound Activity in 3T3-L1 Cell Line
3T3-L1 adipocytes cell line has been described as cellular model to assess compounds mimicking insulin-mediated effect such as inhibition of lipolysis and activation of glucose uptake.
Lipolysis.
3T3-L1 cells (ATCC) are cultured in Dulbecco's modified eagle's medium (DMEM) containing 10% (v/v) bovine serum (fresh regular medium) in 24 well plate. On day 0 (2 days after 3T3-L1 preadipocytes reached confluence), cells are induced to differentiate by insulin (10 μg/ml), IBMX (0.5 mM) and dexamethasone (1 μM). On day 3 and every other 3rd day thereafter, fresh regular medium is substituted until day 14.
On day 14, the medium is removed and cells are washed twice with 1 ml of a wash buffer (Hank's balanced salt solution). The wash solution is removed and the SCFA or the compounds of the invention, or a combination of both, are added at the desired concentration in Hank's buffer supplemented with 2% BSA-FAF and incubated for 10 minutes a 37° C. Then, isoproterenol (100 nM) is added to induce lipolysis and incubate for 30 minutes at 37° C. The supernatants are collected in a glycerol-free container. 25 μl (the amount can be changed for optimization) of cell-free supernatants are dispensed in 96-well microtiter plate, 25 μl of free glycerol assay reagent (Chemicon, the amount can be changed for optimization) is added in each well and the assay plate is incubated for 15 minutes at room temperature. The absorbance is recorded with a spectrophotometer at 540 or 560 nm. Using the supernatants, the free fatty acids amount can be assessed using the NEFA assay kit (Wako) according the manufacturer's recommendations.
Glucose Uptake.
3T3-L1 cells are differentiated as described previously with or without of 30 μM of compound of the invention (the concentration can be changed for optimization) during the 14 days of differentiation. The day of the experiment, the cells are washed twice with a KREBS-Ringer bicarbonate (pH 7.3) supplemented with 2 mM sodium pyruvate and starved for 30 minutes in the same buffer at 37° C. in an atmosphere containing 5% CO2 and 95% O2. Various amount of SCFA, compounds of the invention or combination of both are then added with or without 10 nM of insulin (the amount can be changed for optimization) for 30 minutes at 37° C. in an atmosphere containing 5% CO2 and 95% O2. Then, D-(3H)-2 deoxyglucose (0.2 μCi/well) and D-2-deoxyglucose (0.1 mM) is added for 30 minutes. To stop the reaction, the cells are immersed in ice-cold saline buffer, washed for 30 min, and then dissolved in NaOH 1M at 55° C. for 60 minutes. NaOH is neutralized with HCl 1M. The 3H labeled radioactivity of an aliquot of the extract is counted in the presence of a scintillation buffer.
When tested in the glucose-uptake assay described above and by way of illustration the compound n° 9 significantly increases the glucose-uptake in response to 10 nM of insulin (
It is important to note that in the above-mentioned assay the positive allosteric modulators (PAMs) disclosed in Lee et al., (Mol. Pharmacol. 74(6) pp 1599-1609, 2008) do not increase the glucose uptake. This lack of effect on glucose uptake could be explained by the weak affinity (˜1 μM) of the PAMs disclosed by Lee et al.
In vitro Assays to Assess Compound Activity in NCI-H716 Cell Line
Human intestinal cell line NCI-H716 has been described as cellular model to assess compounds mimicking nutrient-mediated effect such as glucagon-like peptide-1 (GLP-1) secretion.
GLP-1 Release.
NCl-H716 cells (ATCC, Manassas) are cultured in Dulbecco's modified eagle's medium (DMEM) containing 10% (v/v) bovine serum, 2 mM L-glutamine, 100 IU/ml penicillin and 100 μg/ml streptomycin in 75 ml flask. Cell adhesion and endocrine differentiation is initiated by growing cells in 96-well plate coated with matrigel in High Glucose DMEM containing 10% (v/v) bovine serum, 2 mM L-glutamine, 100 IU/ml penicillin and 100 μg/ml streptomycin for 2 days. On day 2, the medium is removed and cells are washed once with a pre-warmed wash buffer (Phosphate Buffered salt solution). The wash solution is removed and the SCFA or the compounds of the invention, or a combination of both, are added at the desired concentration in High Glucose DMEM containing 0.1% (v/v) bovine serum and incubated for 2 hours at 37° C. The supernatants are collected in a container. Using the cell-free supernatants, the GLP-1 amount is assessed using a GLP-1 specific ELISA assay kit according the manufacturer's recommendations (ALPCON).
When tested in the GLP-1 release assay described above and by way of illustration the compound n° 169 significantly increases the GLP-1 secretion from NCI-H716 cells (
Ex vivo Assays to Assess Compound Activity in Adipocytes from Normal and High-Fat Diet Fed Mice
Mice C56Black6 male were housed in Makrolon type IV group housing cages (56×35×20 cm3) throughout the experimental phase. Animals' cages litters were changed once a week. They were housed in groups of 10 animals at 12 light dark (at 8 h30 pm lights off), 22+/−2° C. and 50+/−5% relative humidity. Animals were acclimated one week. During the whole phase, standard diet or diet high in energy from fat (Research Diets, New Brunswick, N.J.) and tap water were provided ad libitum. The animals were 16 weeks old at the time of the study.
For keeping only mice that have responded to the high fat diet, fasted glycemia was measured in these mice just before performing the ex-vivo study.
Glucose Uptake Assay in Isolated Adipocytes.
Animals were killed by cervical dislocation and epididymal fat pads were removed and digested in collagenase buffer at 37° C./120 rpm for approximately 50 minutes. The digest was filtered through gauze to recover the adipocytes, which were washed and resuspended in Krebs-Ringer Hepes (KRH) buffer containing 1% BSA, 200 nM adenosine and 2 mM glucose.
Isolated adipocytes were washed in glucose-free KRH-buffer and resuspended to 30%. Adipocytes were then incubated at 37° C./80 rpm with either compound of the invention (30 μM, 10 μM and 1 μM) in the presence or absence of insulin (10 nM) for 30 min. 2-deoxyglucose and 2-deoxy-D-[1-3H]-glucose (3H-2-DOG) were added and incubation continued for 10 min. The reactions were then stopped by addition of cytochalasin b followed by centrifugation through dinonylphthalate to recover the adipocytes. The uptake of 3H-2-DOG- was measured by scintillation. Each data point was investigated in triplicates in two independent experiments.
When tested in the assay described above and by way of illustration the compound n° 9 significantly increase the glucose uptake in adipocytes isolated from High-fat diet fed mice (
Lipolysis Assay in Isolated Adipocytes.
Isolated adipocytes were diluted to 5% in KRH-buffer and were pre-treated with compound of the invention (30 μM, 10 μM and 1 μM) for 30 min at 37° C./120 rpm. After the pre-treatment, Isoprenaline (1 μM) was added to the adipocytes followed by 30 min incubation at 37° C./150 rpm. The reactions were put on ice and the buffer was assayed spectrophotometrically for the production of NADH+ from glycerol breakdown in reactions catalyzed by glycerol kinase and glycerol-3-phosphate dehydrogenase and/or Non Esterified Fatty Acid (NEFA). Each data point was investigated in triplicates in two independent experiments.
According to the method described above and by way of illustration the compound n° 9 dose-dependently inhibits isoprenaline-induced lipolysis in adipocytes from high-fat diet fed mice (
Compounds n° 14, 89, 126, 139, 142, 155, 169 and 183 inhibit isoprenaline-induced lipolysis in adipocytes isolated from normal rats according to the method described above (
It is important to note that in the above-mentioned assay the positive allosteric modulators (PAMs) disclosed in Lee et al., (Mol. Pharmacol. 74(6) pp 1599-1609, 2008) do not display an anti-lipolytic effect on rat adipocytes. This lack of effect could be explained by the weak affinity (˜1 μM) of the PAMs disclosed by Lee et al.
In vivo Assay to Assess Compound Activity in Rodent Diabetes Model
Genetic Rodent Models:
Rodent models of T2D associated with obesity and insulin resistance have been developed. Genetic models such as db/db and ob/ob in mice and fa/fa in Zucker rats have been developed for understanding the pathophysiology of disease and testing candidate therapeutic compounds as compound of the invention. The homozygous animals, C57 Black/6-db/db mice developed by Jackson Laboratory are obese, hyperglycemic, hyperinsulinemic and insulin resistant (J Clin Invest, 1990, 85:962-967), whereas heterozygotes are lean and normoglycemic. In the db/db model, mice progressively develop insulinopenia with age, a feature commonly observed in late stages of human T2D when sugar levels are insufficiently controlled. Since this model resembles that of human T2D, the compounds are tested for activities including, but not limited to, lowering of plasma glucose and triglycerides. Zucker (fa/fa) rats are severely obese, hyperinsulinemic, and insulin resistant, and the fa/fa mutation may be the rat equivalent of the murine db mutation.
Genetically altered obese diabetic mice (db/db) (male, 7-9 weeks old) are housed under standard laboratory conditions at 22° C. and 50% relative humidity, and maintained on a diet of Purina rodent chow and water ad libitum. Prior to treatment, blood is collected from the tail vein of each animal and blood glucose concentrations are determined using one touch basic glucose monitor system (Lifescan). Mice that have plasma glucose levels between 250 to 500 mg/dl are used. Each treatment group consists of several mice that are distributed so that the mean of glucose levels are equivalent in each group at the start of the study. Db/db mice are dosed by micro-osmotic pumps, inserted using isoflurane anesthesia, to provide compounds of the invention, saline, or an irrelevant compound to the mice intravenously (i.v). Blood is sampled from the tail vein at intervals thereafter and analyzed for blood glucose concentrations. Significant differences between groups (comparing compounds of the invention to saline-treated) are evaluated using Student t-test.
The High-Fat Diet Fed Mouse:
This model was originally introduced by Surwit et al. in 1988. The model has shown to be accompanied by insulin resistance, as determined by intravenous glucose tolerance tests, and of insufficient islet compensation to the insulin resistance. The model has, accordingly, been used in studies on pathophysiology of impaired glucose tolerance (IGT) and type 2 diabetes and for development of new treatments.
C57BL/6J mice are maintained in a temperature-controlled room (22° C.) on a 12-h light-dark cycle. One week after arrival, mice are divided into two groups and are fed either a high-fat diet or received continuous feeding of a normal diet for up to 12 months. On caloric basis, the high-fat diet consist of 58% fat from lard, 25.6% carbohydrate, and 16.4% protein (total 23.4 kJ/g), whereas the normal diet contains 11.4% fat, 62.8% carbohydrate, and 25.8% protein (total 12.6 kJ/g). Food intake and body weight are measured once a week, and blood samples are taken at indicated time points from the intraorbital retrobulbar plexus from nonfasted anesthetized mice.
For intravenous glucose tolerance tests (IVGTTs), 4-h fasted mice are anesthetized with 7.2 mg/kg fluanison/fenlanyl and 15.3 mg/kg midazolam.
Thereafter a blood sample is taken from the retrobulbar, intraorbital, capillary plexus, after which D-glucose (1 g/kg) is injected intravenously in a tail vein (volume load 10 l/g). Additional blood samples are taken at 1, 5, 10, 20, 50, and 75 min after injection. Following immediate centrifugation at 4 C, plasma is separated and stored at −20 C until analysis. For oral glucose tolerance tests (OGTTs), 16-h fasted anesthetized mice are given 150 mg glucose by gavage through a gastric tube (outer diameter 1.2 mm), which is inserted in the stomach. Blood samples are taken at 0, 15, 30, 60, 90, and 120 min after glucose administration and handled as above.
Administration of the compounds: Five-week-old mice are fed a high-fat or a normal diet for 8 weeks. After 4 weeks, the mice are additionally given the compound of the invention in their drinking water (0.3 mg/ml, the amount can be changed for optimization. Control groups are given tap water without compound. After another 4 weeks, the mice are subjected to an OGTT as described above.
Insulin and glucose measurements: Insulin is determined enzymatically using an ELISA assay kit (Linco Research, St. Charles, Mo.). Plasma glucose is determined by the glucose oxidase method.
In vivo Assay to Assess Compound Anti-Obesity Activity in Rodent Model
Mouse Acute Food Intake and Weight Change:
Male C57BL/6N wild-type mice are weighed and vehicle or compounds of the invention are administered by oral gavage to male mice approximately 30 min prior to the onset of the dark phase of the light cycle. Mice are fed ad libitum in the dark phase following dosing. A preweighed aliquot of a highly palatable medium high fat diet is provided in the food hopper of the cage 5 min prior to the onset of the dark phase of the light cycle and weighed 2 and 18 h after the onset of the dark phase of the light cycle.
Acute Studies in Diet-Induced Obesity (DIO) Rats:
For acute experiments, male Sprague-Dawley DIO rats from Charles River Laboratories are raised from 4 weeks of age on a diet moderately high fat (32% kcal) and high in sucrose (25% kcal). Animals are used at 12 weeks of age and are maintained on a 12/12 h light dark cycle. The rats are randomized into groups (n=6/group) for compounds of the invention and vehicle dosing. Rats are weighed 17 h after dosing to determine effects on overnight body weight gain. Compounds of the invention are administered orally or s.c. at amount desired 1 h before the start of the dark cycle. Powdered food is provided in food cups which are weighed continuously at 5 min intervals over 18 h and the data are recorded using a computerized system.
Chronic Studies in Diet-Induced Obesity Rats:
For the 14-day chronic experiment, male Sprague-Dawley DIO rats are obtained as described above. Animals are used at 15 weeks of age and are maintained on a 12/12 hour light-dark cycle. Rats are conditioned to dosing for 4 days prior to baseline measurements, using an oral gavage or a s.c. route of vehicle. Thereafter, animals are dosed daily with vehicle or compound by oral gavage or s.c. Compound of the invention or vehicle is administered 1 h before the dark cycle for 14 days. Body composition is measured by dual energy X-ray densitometry (DEXAscan) 5 days prior to the study and at the end of the 14-day study. Daily endpoints included body weight and food intake.
In vivo Assay to Assess Compound Anti-Lipolytic Activity in Rodent Model
Male C57BL/6N wild-type are housed one per cage in a room maintained on a 12 h light/dark cycle under constant temperature (22-25° C.) with ad libitum access to food and water. The anti-lipolytic effects of the compounds of the invention are studied in awake mice. Animals are fasted overnight before experimental use. On the day of the experiment, animals are put in metabolic cages and left undisturbed to acclimate to the environment for 1-2 h. blood samples are taken at indicated time points from the intraorbital retrobulbar plexus. A 1% sodium citrate saline solution is used to flush the lines. A pre-treatment blood sample is obtained from each animal to determine baseline values for free fatty acids (FFA) and triglycerides (TG). Compounds of the invention are given via oral gavage, sc injection, iv injection or ip injection for each different series of experiments. Blood samples are collected into pre-cooled tubes pre-coated with heparin (200 μl blood, Li-heparin, Sarstedt) for determination triglycerides and glycerol and in tri-potassium EDTA added sodium fluoride (200 μl blood, K3-EDTA, 1.6 mg/mL+1% NaF, Sarstedt) for determination of plasma free fatty acids. The tubes are placed on wet ice pending processing. Blood samples will be centrifuged at 4000×g, at 4° C., 15 min the resulting plasma will be transferred into non-coated tubes and stored at −80° C. until analyses. The plasma is thawed at 4° C. for determinations of FFA and TG using commercial kits (Wako Chemicals).
According to the method described above and by way of illustration the compounds n° 2 and 9 administered by ip injection, inhibit, 15 minutes following the injection, in vivo FFA baseline at the concentration of 15 mg/kg from normal diet fed mice in comparison to the vehicle (
While embodiments of the invention have been illustrated and described, it is not intended that these embodiments illustrate and describe all possible forms of the invention. Rather, the words used in the specification are words of description rather than limitation ant it is understood that various changes may be made without departing from the spirit and scope of the invention.
| Number | Date | Country | Kind |
|---|---|---|---|
| 08305896.6 | Dec 2008 | EP | regional |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP2009/066536 | 12/7/2009 | WO | 00 | 5/20/2011 |
| Number | Date | Country | |
|---|---|---|---|
| 61205735 | Jan 2009 | US | |
| 61152294 | Feb 2009 | US | |
| 61168159 | Apr 2009 | US |
