Patent Application
20240150342
Estrogen-related receptor alpha (ERRα) is a 45.5 kilodalton (kDa), 423 amino acid residue protein which belongs to the nuclear receptor (NR) superfamily. This nuclear receptor family comprises 48 genes, coding for DNA-binding transcription factors that are involved in the regulation of diverse function including inter alia homeostasis, reproduction, development and metabolism. The ERR family—the NR3B subgroup—consists of ERRα, ERR-β, and ERR-γ: to date, no endogenous ligands have been identified for any of the ERR isoforms and therefore they are considered orphan receptors.
In Bookout et al. Anatomical profiling of nuclear receptor expression reveals a hierarchical transcriptional network, Cell. 126:789-99 (2006), organism-wide expression profiling of the three ERR isoforms determined that ERRα is widely distributed, with significant protein expression in most adult tissues. Knockout studies of the ERR family members have revealed that each receptor has tissue- and function-specific metabolic phenotypes that are important for adaptation to energy stress at the whole body level. Knockout studies have also indicated limited in vivo compensation among the ERR family members. The disclosure of inter alia Tremblay et al. The NR3B subgroup: an overview, Nuclear Receptor Signaling, 5:e009 (2007) may be noted in this context.
Genomic studies have indicated that ERRα regulates large numbers of genes. The following references are instructive in this regard: Puigserver et al. A cold-inducible coactivator of nuclear receptors linked to adaptive thermogenesis, Cell. 92(6):829-839 (1998); Yoon et al. Control of hepatic gluconeogenesis through the transcriptional coactivator PGC-1, Nature 413(6852):131-138 (2001); Huss et al. Estrogen-related receptor alpha directs peroxisome proliferator-activated receptor at signaling in the transcriptional control of energy metabolism in cardiac and skeletal muscle, Mol. Cell Biol. 24(20):9079-9091 (2004); and, Mootha et al. ERRα and Gabpa/b specify PGC-1alpha-dependent oxidative phosphorylation gene expression that is altered in diabetic muscle, Proc. Natl. Acad. Sci. USA, 101(17):6570-6575 (2004).
These references support a physiological model of ERRα function in regulating energy metabolism and, in particular, in the transcriptional regulation of genes required for mitochondrial biogenesis, the tricarboxylic acid cycle, oxidative phosphorylation, fatty acid oxidation and lipid metabolism. In particular, ERRα induces the expression of Nuclear Respiratory Factor 1 (NRF1), GA-binding protein alpha (GABPα), and Peroxisome Proliferator-activated Receptor alpha (PPARα). The nuclear receptor coactivators Peroxisome Proliferator-activated Receptor gamma coactivator 1-alpha (PGC-1α), PGC-1β and Peroxisome Proliferator-activated Receptor gamma Coactivator-related protein 1 (PPRC-1) are implicated in the regulation of these genes and in the auto-regulation of the expression of ERRα. PGC-1α is expressed at low basal levels but is induced by fasting and other metabolic stresses. PGC-18, a related coactivator, has similar functions, but its expression may not be regulated as acutely by variations in energy demand. Conversely, co-repressors that bind to ERRs, such as co-repressor nuclear Receptor Interacting Protein 140 (RIP140), compete with ERR co-activators to negatively regulate ERR-dependent gene expression.
The pleiotropic effect of ERRα activity on energy metabolism has interested the present inventors in the possibility that it should be a target for the discovery of new therapies for diseases in which metabolic disturbances or modifications play a central role, such as type-2 diabetes, progressive heart failure, osteoporosis and cancer. Of particular interest is ERRα as a novel target for tumor therapy, through effects on the regulation of tumor cell energy metabolism associated with energy stress within tumor microenvironments. And of specific interest, is ERRα as a novel target for therapeutic treatment of cancers with stem-like properties—Cancer Stem Cells (CSC), Tumor Initiating Cells (TIC) and Circulating Tumor Cells (CTC)—that rely on mitochondrial respiration for their energy requirements.
The initiation and development of cancer, in particular, is known to be associated with major metabolic alterations and mitochondria play a key role in tumorigenesis. A common abnormality observed in many cancer types—termed the Warburg effect—is a shift in glucose metabolism from oxidative phosphorylation to aerobic glycolysis and is characterized by a drastic increase in glucose consumption accompanied by an elevated rate of lactate excretion regardless of oxygen abundance: aerobic glycolysis meets the metabolic needs of highly proliferative cells, including providing sufficient energy and providing for the accumulation of precursors for anabolic reactions. LeBleu et al. PGC-1alpha mediates mitochondrial biogenesis and oxidative phosphorylation in cancer cells to promote metastasis, Nat. Cell Biol. 16(10):992-1 (2014) demonstrated that tumor cells display metabolic plasticity to engage either glycolysis or oxidative phosphorylation depending on the tumor environment and their proliferative or metastasizing phenotype during cancer progression. It is thus evident that the targeting of metastatic progenitors and resistant tumor cells should not only happen via the glycolytic route but also via the mitochondrial oxidative phosphorylation.
ERRα, together with PGC1α/β, controls the regulation of genes encoding enzymes in the tricarboxylic acid (TCA) cycle and oxidative phosphorylation. As discussed in Ariazi et al. Estrogen-related receptor alpha and estrogen-related receptor gamma associate with unfavorable and favorable biomarkers, respectively, in human breast cancer, Cancer Res. 62(22):6510-8 (2002), ERRα is expressed in a range of cancerous cells—including breast and prostate cancerous cells—and is associated with more invasive disease and a higher risk of recurrences in both these cancer types. Chang et al. The metabolic regulator ERRα, a downstream target of HER2/IGF-1R, as a therapeutic target in breast cancer, Cancer Cell 20, 500-510 (2011) and Fujimoto et al. Clinical implication of estrogen-related receptor (ERR) expression in ovarian cancers, J. Steroid Biochem. Mol. Biol. 104, 301-304 (2007) document that ERRα is expressed in most cancers and that increased activity of this receptor is associated with a negative outcome in both breast and ovarian cancers. In the first of these references, it is confirmed that the transcription factor is involved in mitochondrial biogenesis and also in the regulation of oxidative phosphorylation. This latter point is considered important as resistance to the inhibition of KRAS pathway in pancreatic cancer, BRAF inhibitors in melanoma and oxaliplatin and 5-fluorouracil in colon cancer are also associated with a shift to oxidative metabolism.
The present inventors have therefore opined that inhibition of the activity of ERRα would enable a selective disruption of mitochondrial function in cancer, in particular in cancers of the aforementioned types. For this purpose, but equally for utility in the treatment of other ERRα mediated diseases and conditions, they have developed non-covalent, non- steroidal ERRα inverse agonists.
In accordance with a first aspect of the present invention, there is provided a compound according to Formula I
or a pharmaceutically acceptable salt thereof, wherein
In an embodiment, the invention relates to a compound according to Formula I in which A1 is N, A2 is NRA and A3 is CR3.
In another embodiment, the invention relates to a compound according to Formula I in which A1 is N, A2 is NH and A3 is CH.
In another embodiment, the invention relates to a compound according to Formula I in which R5 is C(1-3)alkoxy and R4, R6 and R7 are H.
In another embodiment, the invention relates to a compound according to Formula I in which R5 is methoxy and R4, R6 and R7 are H.
In another embodiment, the invention relates to a compound according to Formula I in which A8-A12 are CR8, CR9, CR10, CR11 and CR12, respectively.
In another embodiment, the invention relates to a compound according to Formula I in which R8-R12 are independently H, C(1-4)alkyl, halogen, hydroxyl or C(1-3)alkoxy. For instance, R8-R12 may be independently H, C(1-4)alkyl or halogen. Good results have been obtained where R9 and R11 are independently C(1-4)alkyl, halogen, hydroxyl or C(1-3)alkoxy and R8, R10 and R12 are H. And, of particular note, R9 and R11 are independently C(1-4)alkyl and R8, R10 and R12 are H.
In another embodiment, the invention relates to a compound according to Formula I in which R9 and R11 are CF3 and R8, R10 and R12 are H.
In another embodiment, the invention relates to a compound according to Formula I in which R13 is H.
In another embodiment, the invention relates to a compound according to Formula I in which R14 is O.
In another embodiment, the invention relates to a compound according to Formula I in which R15 and R′15 are H.
In another embodiment, the invention relates to a compound according to Formula I in which Y is a single carbon-carbon bond.
The above embodiments, where they relate to a preferred form of different substituents of Formula (I), are not intended to be mutually exclusive of one another. Rather, combinations of these embodiments are envisaged within the scope of the present invention and, in certain circumstances, such combinations represent preferred structures for compounds of Formula I. In that regard, particular mention may be made of compounds according to Formula I in which: A1 is N, A2 is NH and A3 is CH; A4-A7 are CR4, CR5, CR6 and CR7 respectively, wherein R5 is C(1-3)alkoxy and R4, R6 and R7 are H; A8-A12 are CR8, CR9, CR10, CR11 and CR12, respectively, wherein R9 and R11 are independently C(1-4)alkyl, halogen, hydroxyl or C(1-3)alkoxy and R8, R10 and R12 are H; R13 is H; R14 is O; R15 and R′15 are both H; Y is a single carbon-carbon bond; and, R16 is H.
Mention may also be made of compounds according to Formula I in which: A1 is N, A2 is NH and A3 is CH; A4-A7 are CR4, CR5, CR6 and CR7 respectively, wherein R5 is C(1-3)alkoxy and R4, R6 and R7 are H; A8-A12 are CR8, CR9, CR10, CR11 and CR12, respectively, wherein R9 and R11 are independently C(1-4)alkyl and R8, R10 and R12 are H; R13 is H; R14 is O; R15 and R15 are both H; Y is a single carbon-carbon bond; and, R16 is H.
As used herein, the singular forms “a”, “an” and “the” include plural referents unless the context clearly dictates otherwise.
The terms “comprising”, “comprises” and “comprised of” as used herein are synonymous with “including”, “includes”, “containing” or “contains”, and are inclusive or open-ended and do not exclude additional, non-recited members, elements or method steps. If used, the phrase “consisting of” is closed and excludes all additional elements. Further, the phrase “consisting essentially of” excludes additional material elements but allows the inclusion of non-material elements that do not substantially change the nature of the invention.
When amounts, concentrations, dimensions and other parameters are expressed in the form of a range, a preferable range, an upper limit value, a lower limit value or preferable upper and lower limit values, it should be understood that any ranges obtainable by combining any upper limit or preferable value with any lower limit or preferable value are also specifically disclosed, irrespective of whether the obtained ranges are clearly mentioned in the context.
The words “preferred”, “preferably”, “desirably” and “particularly” or synonyms thereof may be used frequently herein to refer to embodiments of the disclosure that may afford particular benefits, under certain circumstances. However, the recitation of one or more preferable, preferred, desirable or particular embodiments does not imply that other embodiments are not useful and is not intended to exclude those other embodiments from the scope of the disclosure.
As used throughout this application, the word “may” is used in a permissive sense—that is meaning to have the potential to—rather than in the mandatory sense.
As used herein “room temperature” is 23° C.±2° C.
Unless otherwise stated, the terms “halo” or “halogen” or “halide”, as used herein by themselves or as part of another substituent, mean a fluorine, chlorine, bromine, or iodine atom. A preference for fluorine, chlorine or bromine is noted.
The term “heteroatom” as used herein represents nitrogen, oxygen or sulfur.
As used herein, “C(1-n)alkyl” group refers to a monovalent group that contains from 1 to n carbon atoms, that is a radical of an alkane and includes straight-chain and branched organic groups. As such, a “C1-C30alkyl” group would refer to a monovalent group that contains from 1 to 30 carbon atoms, that is a radical of an alkane and includes straight-chain and branched organic groups. In the present invention, such alkyl groups may be unsubstituted or may be substituted with the groups mentioned herein below. The halogenated derivatives of hydrocarbon radicals might, in particular, be mentioned as examples of suitable substituted alkyl groups.
The term “C(1-4)alkyl” as used herein means an alkyl group having 1-4 carbon atoms, i.e. methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl or tert-butyl. All carbon atoms may optionally be substituted with one or more halogen or hydroxyl.
The term “C(1-3)alkyl” as used herein means an alkyl group having 1-3 carbon atoms, i.e. methyl, ethyl, propyl or isopropyl. All carbon atoms may optionally be substituted with one or more halogen or hydroxyl.
The term “C(3-6)cycloalkyl” as used herein means a saturated cyclic hydrocarbon having 3-6 carbon atoms, i.e. cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl. All carbon atoms may optionally be substituted with one or more halogen or methyl.
The term “C(1-3)alkoxy” means an alkoxy group having 1-3 carbon atoms, the alkyl moiety being branched or unbranched. All carbon atoms are optionally substituted with one or more F or hydroxyl.
The term “cyano” as used herein, represents a group of formula —CN.
As used herein “nitro group” or “nitro” refers to —NO2.
The term “substituted” means that one or more hydrogens on the designated atom(s) is/are replaced by a selection from the indicated group, provided that: the designated atom's normal valency under the existing circumstances is not exceeded; and, the substitution results in a stable compound. Combinations of substituents are also permissible only if such combinations result in stable compounds. The terms “stable compound” or “stable structure” refers to a compound or structure that is sufficiently robust to survive both isolation to a useful degree of purity from a reaction mixture and formulation into an efficacious therapeutic agent.
The term “optionally substituted” means optional substitution with the specified groups, radicals or moieties.
As used herein “protecting group” refers to a moiety attached to a functional group to prevent an undesired reaction. Preferably the protecting group may be easily removed after protection of the functional group is no longer required.
The compounds of Formula I may form salts, which are also within the scope of this invention. Reference to a compound of Formula I herein is understood to include reference to salts thereof, unless otherwise indicated.
The term “pharmaceutically acceptable salt” is used in accordance with its standard definition in the art to represent those salts which are, within the scope of medical judgment, suitable for use in contact with the tissues of humans and lower animals without, in particular, undue toxicity, irritation and/or allergic response: that use must be commensurate with a reasonable benefit to risk ratio. Pharmaceutically acceptable salts are well known in the art. They may either be obtained during the final isolation and purification of the compounds of the invention, or they may be obtained separately by reacting a free base function with: a suitable mineral acid, including but not limited to hydrochloric acid, phosphoric acid or sulfuric acid; or, an organic acid, including but not limited to ascorbic acid, citric acid, tartaric acid, lactic acid, maleic acid, malonic acid, fumaric acid, glycolic acid, succinic acid, propionic acid, acetic acid or methanesulfonic acid. An acid function of compounds of the invention can be reacted with an organic or a mineral base, like sodium hydroxide, potassium hydroxide or lithium hydroxide. For completeness, organic bases include the common hydrocarbyl and heterocyclic amine salts, such as diethylamino, morpholine and piperidine salts, for example.
The compounds of Formula I may contain asymmetric or chiral centers and therefore exist in different stereoisomeric forms. It is intended that all stereoisomeric forms of the compounds of Formula I as well as mixtures thereof, including racemic mixtures, form part of the present invention. In particular, stereoisomeric forms of the compounds of Formula I which, following the Cahn-lngold-Prelog system of nomenclature, are in the S configuration on the chiral center next to the five-membered heterocyclic ring definitively form part of the present invention.
As will be understood by the skilled artisan, enantiomers can be separated by: converting the enantiomeric mixture into a diastereomeric mixture by reaction with an appropriate optically active compound, for instance a chiral auxiliary such as a chiral alcohol or Mosher's acid chloride; separating the diastereomers; and, converting—by hydrolysis for example—the individual diastereomers to the corresponding pure enantiomers. Enantiomers can also be separated by use of chiral HPLC column.
It will be recognized further that various tautomers of compounds of Formula I may be possible: it is therefore intended that all tautomeric forms of compounds of Formula I form part of the invention. For completeness, as used herein, the term “tautomer” refers to the migration of protons between adjacent single and double bonds. The tautomerization process is reversible: tautomers will generally reach an equilibrium state wherein the double bond is resonantly shared between two bond lengths.
The present invention also relates to a pharmaceutical composition comprising compounds or pharmaceutically acceptable salts thereof having the general Formula I in admixture with pharmaceutically acceptable excipients and optionally other therapeutically active agents. The excipients must be “acceptable” in the sense of being compatible with the other ingredients of the composition and not deleterious to the recipients thereof.
The invention further includes a compound of Formula I in combination with one or more other drug(s).
Compositions include, but are not limited to, those suitable for oral, sublingual, subcutaneous, intravenous, intramuscular, nasal, local, or rectal administration, all in unit dosage forms for administration. For oral administration, the active ingredient may be presented as discrete units, such as tablets, capsules, powders, granulates, solutions, suspensions and the like.
For parenteral administration, the pharmaceutical composition of the invention may be presented in unit-dose or multi- dose containers, such as injection liquids in predetermined amounts, presented for example in sealed vials and ampoules. The pharmaceutical composition may also be stored in a freeze dried (lyophilized) condition requiring only the addition of sterile liquid carrier—such as water—prior to use.
Mixed with such pharmaceutically acceptable auxiliaries, the active agent may be compressed into solid dosage units, such as pills, tablets, or be processed into capsules or suppositories. By means of pharmaceutically acceptable liquids, the active agent can be applied as a fluid composition—in the form of a solution, suspension or emulsion for instance—which may be included in an injection preparation or in a spray, such as a nasal spray.
For making solid dosage units, the use of conventional additives such as fillers, colorants, polymeric binders and the like is contemplated. In general, any pharmaceutically acceptable additive which does not interfere with the function of the active compounds can be used. Suitable carriers with which the active agent of the invention can be administered as solid compositions include lactose, starch, cellulose derivatives and the like, or mixtures thereof, when used in suitable amounts. For parenteral administration, aqueous suspensions, isotonic saline solutions and sterile injectable solutions may be used, which suspensions or solutions may contain pharmaceutically acceptable dispersing agents and/or wetting agents, such as propylene glycol or butylene glycol.
The invention further includes a pharmaceutical composition, as herein before described, in combination with packaging material suitable for said composition, said packaging material including instructions for the use of the composition for the purposes as hereinbefore described.
The exact dose and regimen of administration of the active ingredient, or a pharmaceutical composition thereof, may vary with the particular compound, the route of administration, and the age and condition of the individual subject to whom the medicament is to be administered.
In general parenteral administration requires lower dosages than other methods of administration which are more dependent upon absorption. That aside, a dosage for humans preferably contains from 0.0001 to 100 mg per kg body weight. The desired dose may be presented as one dose or as multiple sub-doses administered at appropriate intervals throughout the day.
The compounds according to the invention or a pharmaceutically acceptable salt thereof can be used as medicament in therapy.
Another aspect of the invention resides in the use of compounds having the general Formula I or a pharmaceutically acceptable salt thereof for the therapeutic and/or prophylactic treatment of ERRα-mediated diseases or ERRα mediated conditions. In particular, the invention provides for the use of compounds having the general Formula I or a pharmaceutically acceptable salt thereof for the treatment of ERRα-mediated cancer.
The compounds having the general Formula I or a pharmaceutically acceptable salt thereof can be used in therapies to treat at least one condition selected from: lung cancer; melanoma; endometrial cancer; and, acute myeloid leukemia. Without intention to limit this aspect of the present invention, the compounds having the general Formula I or a pharmaceutically acceptable salt thereof may in particular be used in therapies to treat: superficial spreading melanoma; lentigo maligna; acral lentiginous melanoma; nodular melanoma; amelanotic melanoma; ocular melanoma; melanoma of the vulva; or, vaginal melanoma.
In another aspect, the compounds having the general Formula I or a pharmaceutically acceptable salt thereof can be used in therapies to treat at least one condition selected from: breast cancer; bladder cancer; prostrate cancer;
pancreatic cancer; colorectal cancer; and, ovarian cancer.
In another aspect, the compounds having the general Formula I or a pharmaceutically acceptable salt thereof can be used to treat Type II Diabetes Mellitus.
As depicted in the Examples below, in certain exemplary embodiments compounds are prepared according to the following general procedures. It will be appreciated that, whilst the general methods depict the synthesis of certain compounds of the invention, the following general methods and other methods know to one skilled in the art, can be applied to all compounds and subclasses and species of each of these compounds, as described herein.
The compounds described herein, including compounds of general Formula I, Building Block I and Building Block II, are prepared by the reaction schemes depicted below. Furthermore, in the following schemes, where specific acids, bases, reagents, coupling agents, catalysts, solvents and the like are mentioned, it is understood that other suitable acid, bases, reagents, coupling agents, catalysts, solvents, etc. may be used and are included within the scope of the present invention. Modifications to the reaction conditions—for example, temperature, duration of the reaction or combinations thereof—are envisioned as part of the present invention.
The compounds obtained by using the general reaction sequences may be of insufficient purity. The compounds can be purified by using any of the methods for purification of organic compounds, for example, crystallization or silica gel or alumina column chromatography using different solvents in suitable ratios. All possible stereoisomers are envisioned within the scope of the invention.
Abbreviations for the materials employed in the Reaction Schemes and Examples are as follows:
Chemical names are preferred IUPAC names, generated by using Marvin Sketch 17.24.1. If a chemical compound is referred to using both a chemical structure and a chemical name, and an ambiguity exists between the structure and the name, the structure predominates.
Depicted in scheme 1 is the general synthesis of derivatives of the invention having Formula I, wherein R14 is oxygen, R15, R′15 and R16 are H and Y is a single carbon-carbon bond. The derivatives can be prepared by methods known in the art of organic chemistry. Compounds of the invention can be obtained by a reaction between a benzaldehyde derivative of building block I, wherein A4, A5, A6, A7, A8, A9, A10, A11 and A12, have the meaning as previously described, and a derivative of building block II, wherein R13, A1, A2 and A3 have the meaning as previously described, and Meldrum's acid.
To obtain derivatives of Formula I wherein R14 is sulphur, the derivatives of Formula I wherein R14 is oxygen can be reacted with, for example, Lawesson's reagent.
To obtain derivatives of Formula I wherein R14 is nitrogen, the derivatives of Formula I wherein R14 is sulphur can be reacted with, for example, ammonia in MeOH.
Scheme 1b depicts a general route for the preparation of Formula I analogs wherein Y is a double carbon-carbon bond, R15 is H and R13, R14, A1, A2, A3, A4, A5, A6, A7, A8, A9, A10, A11 and A12 have the meaning as previously described.
Derivatives of Formula I, wherein Y is a single carbon-carbon bond and R15, R′15 and R16 are H, can be oxidized, using for example DDQ in an appropriate solvent, to obtain derivatives of Formula I, wherein Y is a double carbon-carbon bond.
Scheme 2 depicts a general method for preparing benzaldehyde building block I derivatives wherein A4, A5, A6, A7, A8, A9, A10, A11 and A12 have the meaning as previously described.
Aromatic substitution of 4-fluorobenzaldehyde 1 with phenol 2, using K2CO3 in DMF at 110° C., gives the building block I derivatives. For some substitutions on A4 to A12, it might be beneficial to use a 4-hydroxybenzaldehyde derivative with a fluorobenzene derivative to perform the aromatic substitution.
Scheme 2b depicts a general method for preparing benzaldehyde building block I derivatives, wherein A8 is C—CF2, and A4, A5, A6, A7, A9, A10, A11 and A 12 have the meaning as previously described. Aromatic substitution of fluorobenzaldehyde 4 with 4-hydroxybenzonitrile 3 under basic condition, using for example K2CO3, gives the corresponding benzonitrile 5. The aldehyde moiety can be converted to a CF2 group using a fluorinating agent, for example DAST. Via subsequent reduction of the nitrile using, for example DiBAI-H in toluene, benzaldehyde building block I derivatives can be prepared. In scheme 2b this is exemplified for R8, these conversion can also be applied for an aldehyde moiety in any of the positions R4 to R12. For some substitutions on A4 to A12, it might be beneficial to use a 4-fluorobenzonitrile derivative with a hydroxybenzaldehyde derivative to perform the aromatic substitution.
Scheme 3 depicts a general route for the preparation of Formula I analogs wherein Y is a single carbon-carbon bond, R14 is oxygen, R15 is COOH and each of R′15 and R16 is H. Compounds of the invention can be obtained by a reaction between a derivative of building block I, wherein A4, A5, A6, A7, A8, A9, A10, A11 and A12 have the meaning as previously described, a derivative of building block II, wherein R13, A1, A2, A3 have the meaning as previously described, and Meldrum's acid at room temperature.
When R15 is COOH, this carboxylic acid moiety can be functionalized towards an ester, using for example an alcohol, DIAD and PPh3 in THF. Or it can be functionalized towards an amide, using for example a primary or secondary amine, EDC and DMAP in DCM.
Scheme 4 illustrates a general route for the formation of Formula I analogs wherein A1 and A3 are CH, A2 is NH, Y is a single carbon-carbon bond, R14 is oxygen, R13 is H, each of R15, R′15 and R16 is H and A4, A5, A6, A7, A8, A9, A10, A11 and A12 have the meaning as previously described.
Intermediate 6 can be obtained via a Wittig reaction of Building block I, using for example CH3CH2OCOCH2P(Ph)3Br and tBuOK in Et2O, followed by a reaction with, for instance, vinylmagnesium bromide and PhSCu(I) in THF. The subsequent introduction of the nitro group in the E-confirmation can be achieved by using, for example, TEMPO and tBuONO in 1,4-dioxane. Pyrrole intermediate 8 was obtained via a [3+2]cycloaddition of intermediate 7, using for instance TOSMIC and tBuOK in THF. The reduction of the nitro, followed by the ring closure to obtain the Formula I analog, was performed in a single step by using, for example, zinc dust in AcOH.
All building blocks used are commercially available, known or prepared according to methods known to those skilled in the art.
Following a procedure analogous to that described for example 1, using appropriate starting materials, the following compounds have been prepared.
MS(ES+) m/z 429.2 (M+H)+
MS(ES+) m/z 381.2 (M+H)+
MS(ES+) m/z 404.2 (M+H)+
MS(ES+) m/z 404.2 (M+H)+
MS(ES+) m/z 404.2 (M+H)+
MS(ES+) m/z 414.2 (M+H)+
MS(ES+) m/z 350.2 (M+H)+
MS(ES+) m/z 361.2 (M+H)+
MS(ES+) m/z 392.2 (M+H)+
MS(ES+) m/z 370.2 (M+H)+
MS(ES+) m/z 420.2 (M+H)+
MS(ES+) m/z 366.2 (M+H)+
MS(ES+) m/z 414.2 (M+H)+
MS(ES+) m/z 392.2 (M+H)+
MS(ES+) m/z 370.2 (M+H)+
MS(ES+) m/z 420.2 (M+H)+
MS(ES+) m/z 366.2 (M+H)+
MS(ES+) m/z 394.2 (M+H)+
MS(ES+) m/z 364.2 (M+H)+
MS(ES+) m/z 364.2 (M+H)+
MS(ES+) m/z 378.2 (M+H)+
MS(ES+) m/z 378.2 (M+H)+
MS(ES+) m/z 412.2 (M+H)+
MS(ES+) m/z 404.2 (M+H)+
MS(ES+) m/z 426.2 (M+H)+
MS(ES+) m/z 422.2 (M+H)+
MS(ES+) m/z 376.2 (M+H)+
MS(ES+) m/z 408.2 (M+H)+
MS(ES+) m/z 378.2 (M+H)+
MS(ES+) m/z 376.2 (M+H)+
MS(ES+) m/z 420.2 (M+H)+
MS(ES+) m/z 470.2 (M+H)+
MS(ES+) m/z 406.2 (M+H)+
MS(ES+) m/z 450.2 (M+H)+
MS(ES+) m/z 444.1 (M+H)+
MS(ES+) m/z 448.3 (M+H)+
MS(ES+) m/z 434.1 (M+H)+
MS(ES+) m/z 417.2 (M+H)+
MS(ES+) m/z 462.1 (M+H)+
MS(ES+) m/z 462.1 (M+H)+
MS(ES+) m/z 482.0 (M+H)+
MS(ES+) m/z 472.1 (M+H)+
MS(ES+) m/z 448.3 (M+H)+
MS(ES+) m/z 402.2 (M+H)+
MS(ES+) m/z 411.1 (M+H)+
MS(ES+) m/z 376.2 (M+H)+
MS(ES+) m/z 406.2 (M+H)+
MS(ES+) m/z 376.2 (M+H)+
MS(ES+) m/z 445.1 (M+H)+
MS(ES+) m/z 336.1 (M+H)+
MS(ES+) m/z 482.0 (M+H)+
MS(ES+) m/z 387.2 (M+H)+
MS(ES+) m/z 404.1 (M+H)+
MS(ES+) m/z 400.2 (M+H)+
MS(ES+) m/z 418.1 (M+H)+
MS(ES+) m/z 422.1 (M+H)+
MS(ES+) m/z 516.1 (M+H)+
MS(ES+) m/z 432.1 (M+H)+
MS(ES+) m/z 458.1 (M+H)+
MS(ES+) m/z 436.2 (M+H)+
MS(ES+) m/z 448.1 (M+H)+
MS(ES+) m/z 418.4 (M+H)+
MS(ES+) m/z 364.4 (M+H)+
MS(ES+) m/z 418.4 (M+H)+
MS(ES+) m/z 364.4 (M+H)+
Following a procedure analogous to that described for Example 67, using appropriate starting materials, the following compounds have been prepared.
MS(ES+) m/z 420.2 (M+H)+
MS(ES+) m/z 404.2 (M+H)+
MS(ES+) m/z 468.1 (M+H)+
MS(ES+) m/z 427.1 (M+H)+
Following a procedure analogous to that described for Example 72, using appropriate starting materials, the following compound has been prepared.
MS(ES+) m/z 544.4 (M+H)+
Following a procedure analogous to that described for Example 74, using appropriate starting materials, the following compound has been prepared.
MS(ES+) m/z 571.5 (M+H)+
The single enantiomers of Example 43 can be obtained by chiral separation. 2 g of racemic Example 43 was dissolved in EtOH (50 mg/mL). The solution was injected numerous times on the chiral SFC using a preparative IG column (available from Daicel, amylose tris(3-chloro-5-methylphenylcarbamate) chiral stationary phase) and an isocratic gradient of 30% EtOH and 70% liquid CO2, to obtain: 0.9 g of the (±)enantiomer (Example 79) at an Enantiomeric Excess of 99.6%; and, 0.9 g of the (−)enantiomer (Example 80) at an Enantiomeric Excess 99.0%. The optical purity of the enantiomers was determined by chiral HPLC.
The absolute configuration of the compounds of Examples 79 to 86 is not known. These compounds were dissolved in DMSO (10 mg/mL) and characterized by their optical rotation, using a Jasco P-2000 Polarimeter having the following configuration: Tungsten-Halogen (WI) light source; Glan-Taylor Prism Polarizer; Quartz Faraday Cell; and, a monitor wavelength of 589 nm.
Following a procedure analogous to that described for Examples 79 and 80, using appropriate starting materials and HPLC method, the following compounds have been prepared.
This assay was based on the knowledge that nuclear receptors interact with cofactors in a ligand dependent matter. The sites of interaction have been mapped to LXXLL motifs that are present in the co-activator sequences. Short peptide sequences that contain the LXXLL motif mimic the behavior of full length co-activators.
The ERRα AlphaScreen Assay described here relies on the interaction of the co-activator peptide with purified bacterial- expressed ERRα ligand binding domain (ERRα-LBD); upon ligand binding, the ERRα protein can undergo a conformational change resulting in a loss of co-activator binding.
Compounds of the present invention were tested for their ability to disrupt binding of ERRα-LBD protein to co-activator peptide using AlphaScreen® Technology (Perkin Elmer). ERRα-LBD protein was expressed in E. coli as a 6×His Small Ubiquitin-like Modifier (SUMO) fusion. Bacterial expressed 6His-SUMO-ERRα-LBD protein was purified using affinity chromatography. All experiments were performed at room temperature in 384-well white non-binding plates (Greiner) using 50 mM Tris-HCl pH 7.5, 100 mM NaCl, 0.1% Pluronic F-127, 0.05% BSA and 5 mM TCEP as the buffer. Final DMSO concentration was 1% in the assay. Compounds were assayed in triplicate and: incubated with 0.81 nM ERRα-LBD protein and 10 μg/mL streptavidin donor beads and 10 μg/mL Ni-chelate acceptor beads for 1 hour at room temperature; followed by a 2-hour incubation with 15 nM biotin-PGC1α-3 peptide (QRRPCSELLKYLTTNDDPP) corresponding to amino acids 202 to 220.
The AlphaScreen signal was measured using an Envision Xcite plate reader (Perkin Elmer). Data was normalized, and curve fitting analysis was performed in GraphPad Prism 7 using a four-parameter dose-response fit.
All exemplified compounds of Formula I (Examples 1-86) were found to have mean pIC50 values above or equal to 5.
Examples 1, 2, 4, 5, 6, 7, 14, 17, 20, 21, 22, 24, 25, 26, 49, 59, 60, 65, 66, 71, 72, 79, 81, 83 and 85 were found to have mean pIC50 values above or equal to 6 but below 7.
Examples 10, 12, 15, 23, 28, 30, 31, 40, 41, 42, 43, 44, 45, 46, 47, 48, 52, 53, 54, 55, 56, 57, 58, 61, 64, 76, 77, 78 and 82 were found to have mean pIC50 values above or equal to 7 but below 8.
Examples 27, 29, 32, 33, 34, 35, 36, 37, 38, 39, 63, 67, 68, 69, 70, 80, 84 and 86 were found to have mean pIC50 values above or equal to 8.
Examples inhibitors 1, 2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85 and 86 were tested for their ability to inhibit ERRα activity in a full length ERRα reporter gene assay.
A method was established to quantitatively screen the potency of compounds with inverse agonistic activity on the nuclear receptor ERRα of the human species. The assay allows intra-cellular screening of ERRα inverse agonists in SK-BR-3 cells using an over-expression construct coding full length ERRα and a reporter construct containing an ERRα Response Element (RE) and a luciferase gene for read out. The activity is expressed in logIC50 values and can be used to determine SAR of compound families or to de-select compounds.
In this assay, reporter cells are obtained by transient co-transfection of two constructs in SK-BR-3 cells using standard transfection techniques. The first construct contains a response element of the nuclear receptor ERRα (Plasmid pLP2175, Reporter construct ERRα-RE/luc2P, cloned variant of ERRα_v2_synthRE, Switchgear Genomics, Catalog Number S900089). This sequence drives the transcription of the luciferase reporter gene in response to binding of an ERRα protein encoded by the second construct (Plasmid pLP2124: full length ERRα expression construct using pcDNA3.1/Hygro(+) as background, Invitrogen Catalog Number V87020). The over-expressed full length ERRα is constitutively active, hence luciferase expression is reduced by inverse agonists of the nuclear receptor ERRα.
The day after transfection, cells were plated into 96 well plates, the test compound was added and the plates were incubated overnight. Subsequently, the firefly luciferase activity was quantified using luciferase detection reagent and luminescence readout.
Transfection is performed on pre-seeded SK-BR-3 cells in a T175 flask. One transfected T175 flask is sufficient for seeding 3 to 4 MW96 plates the next day, depending on the confluency of the transfected cells.
Two different media are used in this protocol for cell treatment: 1) Culture medium, i.e. McCoy's 5a with phenol red (BioWhittaker Supplier Number 12-688F), 10% FBS and 1× Penstrep.; and, 2) Assay medium, i.e. McCoy's 5a Medium phenol red free (HyClone Product Code SH30270.01) with 2% Charcoal Stripped FBS and 1× Penstrep. Compound dilutions are prepared in assay medium.
Cells are seeded at least 2 days in advance to allow the cells to adhere well to flask before transfection. Cells should be 50-80% confluent at the day of transfection.
SKBR3 cells were transfected with the transcriptional reporter construct pLP2175 and the ERRα expression construct pLP2124 (as described above).
68 μL of Lipofectamine LTX transfection reagent (Invitrogen Catalog Number 15338-100) was added dropwise to 8.9 ml Opti-MEM I Reduced Serum Medium (Gibco Catalog Number 51985-026) and incubated at room temperature for 5 to 20 minutes. 8.9 ml of this reagent mixture was added to 22 μg pLP2175+22 μg pLP2124 (ratio 1:1 and total volume 10 ml), and incubated at room temperature for 25 minutes.
10 minutes before adding the transfection mix to SKBR3 cells in a T175 flask, the culture medium was refreshed with 20 mL culture medium. Subsequently, the 10 mL DNA-Opti-MEM-Lipofectamine mixture was gently added to the cells, followed by overnight (16-24 hours) incubation at 37° C. and 5% CO2.
To harvest SKBR3 cells from a T175 flask, first the medium was removed. Subsequently, cells were washed with Phosphate Buffered Saline (PBS) (Lonza), after which the PBS was removed. To dissociate the cells, 2 ml of TrypLE Express (I nvitrogen) was added to the flask, followed by incubation at 37° C. for 5 minutes. The flask was tapped until cells were detached from the bottom. Cells were collected in 8 mL of culture medium (McCoy's 5a, 10% FBS, penstrep), to achieve a single cell suspension. After cell count, cells were spun down for 5 minutes at 300 g. Next cell pellets were re-suspended to 25000 cells/80 μl assay medium (McCoy's 5a phenol red free, 2% charcoal stripped FBS, penstrep).
For compound screening, the cells were harvested (as described above). 80 μL of cell suspension (25,000 cells) was plated per well into a white, flat bottom, tissue culture treated, 96 well screening plates (Greiner).
Test compounds were diluted, starting from a 10 mM dimethylsulfoxide (DMSO) stock solution, in 3 dilution steps. The first dilution step was a 12 points serial dilution of 4-fold in DMSO. These dilutions were further diluted 10 times in phenol red free assay medium containing 2% charcoal stripped FBS and penstrep. The last step was another 20-fold dilution in assay medium to obtain a 5× concentrated dilution with a DMSO concentration of 0.5%. As a last step, the compound dilutions were diluted 5× in the cell plate.
The DMSO dilution series consisted of 12 concentrations, with a final concentration in the cell plate ranging from 10 μM to 2.4 fM.
The plates were incubated overnight (16-24 hours) at 37° C. and 5% CO2.
For the luciferase readout, the luciferase reagent (BriteLite Plus, Perkin Elmer) was brought to room temperature. To each test well of the screening plates, 100 μL of 2.5-fold diluted BriteLite Plus reagent was added, followed by incubation at room temperature for 5 minutes. The luciferase luminescence signal was measured using a Wallac Victor Microplate Reader (Perkin Elmer).
The half maximum inhibitory concentration (IC50) values for the test compounds were calculated from the luciferase signal using GraphPad Prism software (GraphPad Software).
From the exemplified compounds of Formula I, Examples 10, 29, 32, 33, 34, 35, 37, 38, 39, 40, 41, 43, 63, 67, 68, 69, 70, 84 and 86 were found to have mean pIC50 values above or equal to 6.5.
Example inhibitor 80 was tested for its ability to inhibit tumor growth in a B16F10 melanoma syngeneic mouse model. Cell line and tumor model: B16F10 melanoma cell line derived allograft model in C57BL/6 mice.
Mouse B16F10 melanoma cells were sourced from American Type Culture Collection (ATCC), USA. Cells were grown in DMEM (Invitrogen, Catalogue No. 31600-034) supplemented with 10% FBS (Invitrogen, Catalogue No. 10438-026), and 1% penicillin streptomycin (Thermo Fisher Scientific, Catalogue No. 15140-122). To establish allografts, the cells were harvested by trypsinization when they reach around 70 to 80% confluence and 0.1 million B16F10 cells were suspended in 50 μl of serum free medium and mixed at 1:1 ratio with matrigel before implanting subcutaneously into the dorsal right flank of mice using a 1 mL BD syringe attached to a 24-gauge needle.
B16F10 tumor grafts were measured after 5 days of cell inoculation once they became palpable. When the average tumor volume reached around 58 mm3, animals were dosed after randomization into different treatment groups keeping tumor volume and number of animals in such a way so that the average tumor volume of each group remained same across the groups. Dosing was performed on a per kilogram weight basis, by mouth (per os, p.o.) once a day (quaque die, q.d).
Tumor dimensions—length (I) and breadth (b)—were measured by caliper on the day of animal randomization based on tumor volume (Day 1) and thrice weekly thereafter until study termination. Tumor volumes were calculated using the formula b2*I*0.52 where I=length, b=breadth (Dusan Djokovic et al., BMC Cancer, 2010, 10:641). Tumor growth inhibition was calculated after normalizing the tumor volume on a given day to that on Day 1.
Test items administration was started when tumor volume reached an average of 58 mm3.
Administration of test items was carried out until Day 14 and measurement of tumor volume was carried out until Day 13 for computing percent tumor growth inhibition (TGI). The results of the study are listed in Table 1 herein below.
In view of the foregoing description and examples, it will be apparent to those skilled in the art that equivalent modifications thereof can be made without departing from the scope of the claims.
| Number | Date | Country | Kind |
|---|---|---|---|
| 19204189.5 | Oct 2019 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2020/079166 | 10/16/2020 | WO |
