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Definitions

• the present invention relates to fused cyclic compounds, to methods of using such compounds in the treatment of nuclear hormone receptor-associated conditions such as cancer, and to pharmaceutical compositions containing such compounds.
• Nuclear hormone receptors constitute a large super-family of ligand- dependent and sequence-specific transcription factors. Members of this family influence transcription either directly, through specific binding to the promoter target genes (Evans, in Science 240: 889-895 (1988)), or indirectly, via protein-protein interactions with other transcription factors (Jonat et al., Cell 62: 1189-1204 (1990), Schuele et al., Cell 62: 1217-1226 (1990), and Yang- Yen et al., Cell 62: 1205-1215 (1990)).
• the nuclear hormone receptor super-family (also known in the art as the "steroid/thyroid hormone receptor super-family”) includes receptors for a variety of hydrophobic ligands, including cortisol, aldosterone, estrogen, progesterone, testosterone, vitamine D3, thyroid hormone and retinoic acid (Evans, 1988, supra).
• the super-family contains a number of proteins that have no known ligands, termed orphan nuclear hormone receptors (Mangelsdorf et al, Cell 83: 835-839 (1995), O'Malley et al., Mol. Endocrinol. 10: 1293 (1996), Enmark et al., Mol.
• the conventional nuclear hormone receptors are generally transactivators in the presence of ligand, and can either be active repressors or transcriptionally inert in the absence of ligand. Some of the orphan receptors behave as if they are transcriptionally inert in the absence of ligand. Others, however, behave as either constitutive activators or repressors. These orphan nuclear hormone receptors are either under the control of ubiquitous ligands that have not been identified, or do not need to bind ligand to exert these activities.
• the nuclear hormone receptors have a modular structure, being comprised of three distinct domains: an N-terminal domain of variable size containing a transcriptional activation function AF-1, a highly conserved DNA binding domain and a moderately conserved ligand-binding domain.
• the ligand-binding domain is not only responsible for binding the specific ligand but also contains a transcriptional activation function called AF-2 and a dimerisation domain (Wurtz et al., Nature Struc. Biol. 3, 87-94 (1996), Parker et al., Nature Struc. Biol. 3, 113-115 (1996) and Kumar et al., Steroids 64, 310-319 (1999)).
• SB-NHR's The steroid binding nuclear hormone receptors (SB-NHR's) comprise a subfamily of nuclear hormone receptors. These receptors are related in that they share a stronger sequence homology to one another, particularly in the ligand binding domain (LBD), than to the other members of the NHR super-family (Evans, 1988, supra) and they all utilize steroid based ligands.
• LBD ligand binding domain
• NHR's are the androgen receptor (AR), the estrogen receptor (ER), the progesterone receptor (PR), the glucocorticoid receptor (GR), the mineralocorticoid receptor (MR), the aldosterone receptor (ALDR) and the steroid and xenobiotic receptor (SXR) (Evans et al, WO 99/35246).
• AR androgen receptor
• ER estrogen receptor
• PR progesterone receptor
• GR glucocorticoid receptor
• MR mineralocorticoid receptor
• ADR aldosterone receptor
• SXR steroid and xenobiotic receptor
• the natural ligands for each is derived from a common steroid core.
• examples of some of the steroid based ligands utilized by members of the SB-NHR's include cortisol, aldosterone, estrogen, progesterone, testosterone and dihydrotestosterone.
• Specificity of a particular steroid based ligand for one SB-NHR versus another is obtained by differential substitution about the steroid core.
• High affinity binding to a particular SB-NHR, coupled with high level specificity for that particular SB-NHR can be achieved with only minor structural changes about the steroid core (e.g., Waller et al., Toxicol. Appl. Pharmacol. 137, 219-227 (1996) and Mekenyan et al., Environ. Sci. Technol. 31, 3702-3711 (1997), binding affinity for progesterone towards the androgen receptor as compared to testosterone).
• RU486 is an example of a synthetic agonist of the PR, which is utilized as a birth control agent (Vegeto et al., Cell 69: 703-713 (1992)), and Flutamide is an example of an antagonist of the AR, which is utilized for the treatment of prostate cancer (Neri et al, Endo. 91, 427-437 (1972)).
• Tamoxifen is an example of a tissues specific modulator of the ER function, that is used in the treatment of breast cancer (Srnigel, J.
• Tamoxifen can function as an antagonist of the ER in breast tissue while acting as an agonist of the ER in bone (Grese et al., Proc. Natl. Acad. Sci. USA 94, 14105-14110 (1997)). Because of the tissue selective effects seen for Tamoxifen, this agent and agents like it are referred to as "partial-agonist" or partial-antagonist". In addition to synthetically derived non-endogenous ligands, non-endogenous ligands for NHR's can be obtained from food sources (Regal et al., Proc. Soc. Exp. Biol. Med.
• the flavanoid phytoestrogens are an example of an unnatural ligand for SB-NHR's that are readily obtained from a food source such as soy (Quella et al., J. Clin. Oncol. 18, 1068-1074 (2000) and Banz et al, J. Med. Food 2, 271-273 (1999)).
• soy Quella et al., J. Clin. Oncol. 18, 1068-1074 (2000) and Banz et al, J. Med. Food 2, 271-273 (1999)
• the ability to modulate the transcriptional activity of individual NHR by the addition of a small molecule ligand makes them ideal targets for the development of pharmaceutical agents for a variety of disease states.
• non-natural ligands can be synthetically engineered to serve as modulators of the function of NHR's.
• engineering of an unnatural ligand can include the identification of a core structure which mimics the natural steroid core system. This can be achieved by random screening against several SB-NHR's or through directed approaches using the available crystal structures of a variety of NHR ligand binding domains (Bourguet et al., Nature 375, 377-382 (1995), Brzozowski, et al., Nature 389, 753-758 (1997), Shiau et al., Cell 95, 927-937 (1998) and Tanenbaum et al., Proc. Natl. Acad. Sci.
• Differential substitution about such a steroid mimic core can provide agents with selectivity for one receptor versus another. In addition, such modifications can be employed to obtain agents with agonist or antagonist activity for a particular SB- NHR. Differential substitution about the steroid mimic core can result in the formation of a series of high affinity agonists and antagonists with specificity for, for example, ER versus PR versus AR versus GR versus MR. Such an approach of differential substitution has been reported, for example, for quinoline based modulators of steroid NHR in J. Med.
• the compounds of the present invention comprise a core which serves as a steroid mimic, and are useful as modulators of the function of steroid binding nuclear hormone receptors, as well as other NHR as described following.
• the present invention provides fused cyclic compounds of the following formula I and salts thereof, which compounds are especially useful as modulators of nuclear hormone receptor function:
• Zi is O, S, NH, or NR 6 ;
• Z2 is O, S, NH, or NR 6 ;
• W is CR 7 R 7 — CR 7 R 7'
• CR 8 CR 8'
• CR 7 R 7 ' — C O, cycloalkyl or substituted cycloalkyl, cycloalkenyl or substituted cycloalkenyl, or aryl or substituted aryl
• a 1 is CR 7 or N;
• a 2 is CR 7 or N; or alternatively
• Ai is CR 7 R 7' or NR 7 ;
• a 2 is CR 7 R 7' or NR 7 ; or alternatively
• Q 2 is H, alkyl or substituted alkyl, alkenyl or substituted alkenyl, cycloalkyl or substituted cycloalkyl, cycloalkenyl or substituted cycloalkenyl, heterocycloalkyl or substituted heterocycloalkyl, arylalkyl or substituted arylalkyl, alkynyl or substituted alkynyl, aryl or substituted aryl, heterocyclo (e.g., heteroaryl) or substituted heterocyclo (e.g., substituted heteroaryl), halo,
• R and R are each independently H, alkyl or substituted alkyl, alkenyl or substituted alkenyl, cycloalkyl or substituted cycloalkyl, cycloalkenyl or substituted cycloalkenyl, heterocyclo or substituted heterocyclo, cycloalkylalkyl or substituted cycloalkyalkyl, cycloalkenylalkyl or substituted cycloalkenylalkyl, heterocycloalkyl or substitute
• the compounds of formula I and salts thereof comprise a core which can serve as a steroid mimic (and do not require the presence of a steroid-type (e.g., cyclopentanoperhydrophenanthrene analog) structure).
• a steroid-type e.g., cyclopentanoperhydrophenanthrene analog
• alkyl and alk refers to a straight or branched chain alkane (hydrocarbon) radical containing from 1 to 12 carbon atoms, preferably 1 to 6 carbon atoms.
• exemplary such groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, n-butyl, t-butyl, isobutyl, pentyl, hexyl, isohexyl, heptyl, 4,4- dimethylpentyl, octyl, 2,2,4-trimethylpentyl, nonyl, decyl, undecyl, dodecyl, and the like.
• Substituted alkyl refers to an alkyl group substituted with one or more substituents, preferably 1 to 4 substituents, at any available point of attachment.
• substituents include but are not limited to one or more of the following groups: halo (e.g., a single halo substituent or multiple halo substitutents forming, in the latter case, groups such as a perfluoroalkyl group or an alkyl group bearing Cl 3 or CF 3 ), alkoxy, alkylthio, hydroxy, carboxy (i.e., -COOH), alkoxycarbonyl, alkylcarbonyloxy, CN, amino (i.e., -NH 2 ), alkylamino, dialkylamino, carbamoyl or substituted carbomoyl, carbamate or substituted carbamate, urea or substituted urea, amidinyl or substituted amidinyl, thiol (i.e., -SH),
• alkenyl refers to a straight or branched chain hydrocarbon radical containing from 2 to 12 carbon atoms and at least one carbon-carbon double bond. Exemplary such groups include ethenyl or allyl.
• Substituted alkenyl refers to an alkenyl group substituted with one or more substituents, preferably 1 to 4 substituents, at any available point of attachment.
• substituents include, but are not limited to, alkyl or substituted alkyl, as well as those groups recited above as exemplary alkyl substituents.
• alkynyl refers to a straight or branched chain hydrocarbon radical containing from 2 to 12 carbon atoms and at least one carbon to carbon triple bond. Exemplary such groups include ethynyl.
• “Substituted alkynyl” refers to an alkynyl group substituted with one or more substituents, preferably 1 to 4 substituents, at any available point of attachment.
• substituents include, but are not limited to, alkyl or substituted alkyl, as well as those groups recited above as exemplary alkyl substituents.
• cycloalkyl refers to a fully saturated cyclic hydrocarbon group containing from 1 to 4 rings and 3 to 8 carbons per ring. Exemplary such groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, etc.
• Substituted cycloalkyl refers to a cycloalkyl group substituted with one or more substituents, preferably 1 to 4 substituents, at any available point of attachment.
• substituents include, but are not limited to, nitro, cyano, alkyl or substituted alkyl, as well as those groups recited above as exemplary alkyl substituents, and as previously mentioned as preferred aryl substituents in the definition for G.
• substituents also include spiro-attached or fused cyclic substituents, especially cycloalkenyl or substituted cycloalkenyl.
• cycloalkenyl refers to a partially unsaturated cyclic hydrocarbon group containing 1 to 4 rings and 3 to 8 carbons per ring. Exemplary such groups include cyclobutenyl, cyclopentenyl, cyclohexenyl, etc.
• Substituted cycloalkenyl refers to a cycloalkenyl group substituted with one more substituents, preferably 1 to 4 substituents, at any available point of attachment.
• substituents include but are not limited to nitro, cyano, alkyl or substituted alkyl, as well as those groups recited above as exemplary alkyl substituents, and as previously mentioned as preferred aryl substituents in the definition for G.
• Exemplary substituents also include spiro-attached or fused cyclic substituents, especially cycloalkyl or substituted cycloalkyl, or aryl or substituted aryl.
• alkoxy or alkylthio refer to an alkyl group as described above bonded through an oxygen linkage (-O-) or a sulfur linkage (-S-), respectively.
• substituted alkoxy or “substituted alkylthio” refer to a substituted alkyl group as described above bonded through an oxygen or sulfur linkage, respectively.
• alkoxycarbonyl refers to an alkoxy group bonded through a carbonyl group.
• alkylcarbonyl refers to an alkyl group bonded through a carbonyl group.
• alkylcarbonyloxy refers to an alkylcarbonyl group bonded through an oxygen linkage.
• arylalkyl'', "substituted arylalkyl,” “cycloalkylalkyl,” “substituted cycloalkylalkyl,” “cycloalkenylalkyl”, “substituted cycloalkenylalkyl”, “heterocycloalkyl” and “substituted heterocycloalkyl” refer to aryl, cycloalkyl, cycloalkenyl and heterocyclo groups bonded through an alkyl group, substituted on the aryl, cycloalkyl, cycloalkenyl or heterocyclo and/or the alkyl group where indicated as “substituted.”
• aryl refers to cyclic, aromatic hydrocarbon groups which have 1 to 5 aromatic rings, especially monocyclic or bicyclic groups such as phenyl, biphenyl or naphthyl. Where containing two or more aromatic rings (bicyclic, etc.), the aromatic rings of the aryl group may be joined at a single point (e.g., biphenyl), or fused (e.g., naphthyl, phenanthrenyl and the like). "Substituted aryl” refers to an aryl group substituted by one or more substituents, preferably 1,2,3,4 or 5 substituents, at any point of attachment.
• substituents also include fused cyclic substituents, such as heterocyclo or cycloalkenyl, or substituted heterocyclo or cycloalkenyl, groups (e.g., thereby forming a fluoroenyl, tetrahydronapthalenyl, or dihydroindenyl group).
• fused cyclic substituents such as heterocyclo or cycloalkenyl, or substituted heterocyclo or cycloalkenyl, groups (e.g., thereby forming a fluoroenyl, tetrahydronapthalenyl, or dihydroindenyl group).
• Carbamoyl refers to the group -CONH- which is bonded on one end to the remainder of the molecule and on the other to hydrogen or an organic moiety (such as alkyl, substituted alkyl, aryl, substituted aryl, heterocycle, alkylcarbonyl, hydroxyl and substituted nitrogen).
• Carbamoyl refers to the group -CONH- which is bonded on one end to the remainder of the molecule and on the other to hydrogen or an organic moiety (such as alkyl, substituted alkyl, aryl, substituted aryl, heterocycle, alkylcarbonyl, hydroxyl and substituted nitrogen).
• “Carbamate” refers to the group -O-CO-NH- which is bonded on one end to the remainder of the molecule and on the other to hydrogen or an organic moiety (such as those listed above).
• “Urea” refers to the group -NH-CO-NH- which is bonded on one end to the remainder of the molecule and on the
• “Substituted carbamoyl,” “substituted carbamate,” “substituted urea” and “substituted amidinyl” refer to carbamoyl, carbamate, urea or amidinyl groups as described above in which one more of the hydrogen groups are replaced by an organic moiety (such as those listed above).
• heterocycle refers to fully saturated, or partially or fully unsaturated, including aromatic (i.e., “heteroaryl”) cyclic groups (for example, 3 to 7 membered monocyclic, 7 to 11 membered bicyclic, or 10 to 16 membered tricyclic ring systems) 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 atoms, oxygen atoms and/or sulfur atoms, where the nitrogen and sulfur heteroatoms may optionally be oxidized and the nitrogen heteroatoms may optionally be quaternized.
• heteroarylium refers to a heteroaryl group bearing a quaternary nitrogen atom and thus a positive charge.
• the heterocyclic group may be attached to the remainder of the molecule at any heteroatom or carbon atom of the ring or ring system.
• Exemplary monocyclic heterocyclic groups include ethylene oxide, azetidinyl, pyrrolidinyl, pyrrolyl, pyrazolyl, oxetanyl, pyrazolinyl, imidazolyl, imidazolinyl, imidazolidinyl, oxazolyl, oxazolidinyl, isoxazolinyl, isoxazolyl, thiazolyl, thiadiazolyl, thiazolidinyl, isothiazolyl, isothiazolidinyl, furyl, tetrahydrofuryl, thienyl, oxadiazolyl, piperidinyl, piperazinyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolodinyl, 2-oxoazepinyl, azepinyl, hexahydrodiazepinyl, 4-piperidonyl,
• bicyclic heterocyclic groups include indolyl, isoindolyl, benzothiazolyl, benzodioxolyl, benzoxazolyl, benzoxadiazolyl, benzothienyl, quinuclidinyl, quinolinyl, tetrahydroisoquinolinyl, isoquinolinyl, benzimidazolyl, benzopyranyl, indolizinyl, benzofuryl, benzofurazanyl, chromonyl, coumarinyl, benzopyranyl, cinnolinyl, quinoxalinyl, indazolyl, pyrrolopyridyl, furopyridinyl (such as furo[2,3-c]pyridinyl, furo[3,2-b]pyridinyl] or furo[2,3-b]pyridinyl), dihydrobenzodioxinyl, dihydrodioxidobenzo
• Exemplary tricyclic heterocyclic groups include carbazolyl, benzidolyl, phenanthrolinyl, dibenzofuranyl, acridinyl, phenanthridinyl, xanthenyl and the like.
• “Substituted heterocycle,” “substituted heterocyclic,” and “substituted heterocyclo” refer to heterocycle, heterocyclic or heterocyclo groups substituted with one or more substituents, preferably 1 to 4 substituents, at any available point of attachment.
• quaternary nitrogen refers to a tetravalent positively charged nitrogen atom including, for example, the positively charged nitrogen in a tetraalkylarrrmonium group (e.g., tetramethylammonium, N-methylpyridinium), the positively charged nitrogen in protonated ammonium species (e.g., trimethyl- hydroammonium, N-hydropyridinium), the positively charged nitrogen in amine N- oxides (e.g., N-methyl-morpholine-N-oxide, pyridine-N-oxide), and the positively charged nitrogen in an N-amino-ammonium group (e.g., N-aminopyridinium).
• hydroxylamine and “hydroxylamide” refer to the groups OH-NH- and OH-NH-CO-, respectively.
• protecting groups for the methods and compounds described herein include, without limitation, those described in standard textbooks, such as Greene, T. W. et al., Protective Groups in Organic Synthesis, Wiley, N.Y. (1991).
• (CRR)n When a term such as “(CRR)n” is used, it denotes an optionally substituted alkyl chain existing between the two fragments to which it is bonded, the length of which chain is defined by the range described for the term n.
• n 0-3, implying from zero to three (CRR) units existing between the two fragments, which are attached to the primary and terminal (CRR) units.
• CRR zero to three
• any heteroatom with unsatisfied valences is assumed to have hydrogen atoms sufficient to satisfy the valences.
• Carboxylate anion refers to a negatively charged group -COO " .
• the compounds of formula I form salts which are also within the scope of this invention. Reference to a compound of the formula I herein is understood to include reference to salts thereof, unless otherwise indicated.
• the term "salt(s)", as employed herein, denotes acidic and/or basic salts formed with inorganic and/or organic acids and bases.
• zwitterions inner salts
• Salts of the compounds of the formula I may be formed, for example, by reacting a compound I with an amount of acid or base, such as an equivalent amount, in a medium such as one in which the salt precipitates or in an aqueous medium followed by lyophilization.
• the compounds of formula I which contain a basic moiety may form salts with a variety of organic and inorganic acids.
• Exemplary acid addition salts include acetates (such as those formed with acetic acid or trihaloacetic acid, for example, trifluoroacetic acid), adipates, alginates, ascorbates, aspartates, benzoates, benzenesulfonates, bisulfates, borates, butyrates, citrates, camphorates, camphorsulfonates, cyclopentanepropionates, digluconates, dodecylsulfates, ethanesulfonates, fumarates, glucoheptanoates, glycerophosphates, hemisulfates, heptanoates, hexanoates, hydrochlorides, hydrobro ides, hydroiodides, hydroxyethane
• the compounds of formula I which contain an acidic moiety may form salts with a variety of organic and inorganic bases.
• Exemplary basic salts include ammonium salts, alkali metal salts such as sodium, lithium and potassium salts, alkaline earth metal salts such as calcium and magnesium salts, salts with organic bases (for example, organic amines) such as benzathines, dicyclohexylamines, hydrabamines (formed with N,N- bis(dehydroabietyl)ethylenediamine) , N-methyl-D-glucamines , N-methyl-D- glycamides, t-butyl amines, and salts with amino acids such as arginine, lysine and the like.
• Basic nitrogen-containing groups may be quaternized with agents such as lower alkyl halides (e.g. methyl, ethyl, propyl, and butyl chlorides, bromides and iodides), dialkyl sulfates (e.g. dimethyl, diethyl, dibutyl, and diamyl sulfates), long chain halides (e.g. decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides), aralkyl halides (e.g. benzyl and phenethyl bromides), and others.
• lower alkyl halides e.g. methyl, ethyl, propyl, and butyl chlorides, bromides and iodides
• dialkyl sulfates e.g. dimethyl, diethyl, dibutyl, and diamyl sulfates
• Prodrags and solvates of the compounds of the invention are also contemplated herein.
• the term "prodrug” as employed herein denotes a compound which, upon administration to a subject, undergoes chemical conversion by metabolic or chemical processes to yield a compound of the formula I, or a salt and/or solvate thereof.
• Solvates of the compounds of formula I include, for example, hydrates.
• All stereoisomers of the present compounds are contemplated within the scope of this invention.
• Individual stereoisomers of the compounds of the invention may, for example, be substantially free of other isomers (e.g., as a pure or substantially pure optical isomer having a specified activity), or may be admixed, for example, as racemates or with all other, or other selected, stereoisomers.
• the chiral centers of the present invention may have the S or R configuration as defined by the IUPAC 1974 Recommendations.
• racemic forms can be resolved by physical methods, such as, for example, fractional crystallization, separation or crystallization of diastereomeric derivatives or separation by chiral column chromatography.
• the individual optical isomers can be obtained from the racemates by any suitable method, including without limitation, conventional methods, such as, for example, salt formation with an optically active acid followed by crystallization.
• All configurational isomers of the compounds of the present invention are contemplated, either in admixture or in pure or substantially pure form.
• the definition of compounds of the present invention embraces both cis (Z) and trans (E) alkene isomers, as well as cis and trans isomers of cyclic hydrocarbon or heterocyclo rings.
• the exo or endo conformation can be preferred for the fused ring system bonded to G-L in formula I.
• the preferred configuration can be a function of the particular compound and its preferred activity. Separation of configurational isomers can be achieved by any suitable method, such as column chromatography. Throughout the specifications, groups and substituents thereof may be chosen to provide stable moieties and compounds.
• the compounds of the present invention may be prepared by methods such as those illustrated in the following Schemes I to IV. Solvents, temperatures, pressures, and other reaction conditions may readily be selected by one of ordinary skill in the art. Starting materials are commercially available or readily prepared by one of ordinary skill in the art. Combinatorial techniques may be employed in the preparation of compounds, for example, where the intermediates possess groups suitable for these techniques. See the following which describe other methods which may be employed in the preparation of compounds of the present invention: Li, et al, Eur. J. Org. Chem. 9, 1841-1850 (1998); Li, Y-Q, Synlett. 5, 461-464 (1996); Thiemann, et al, Bull Chem. Soc. Jpn.
• Japanese Patent Document JP 53086035 Kato et al, Japanese Patent Document JP 51088631; Tottori etal, Japanese Patent Document JP 49124225; Augustin etal, German Patent Document DD 101271; Title et al, French Patent Document FR 2031538; Gousse etal, Polym. Int. 48, 723-731 (1999); Padwa et al, J. Org. Chem. 62, 4088-4096 (1997); Theurillat-Moritz et al, Tetrahedron: Asymmetry 7, 3163- 3168 (1996); Mathews et al, J. Carbohydr. Chem. 14, 287-97 (1995); Srivastava et al, Natl Acad. Sci.
• a diene of formula II can be reacted with a dienophile of formula III, under conditions readily selected by one skilled in the art (such as by the addition of heat (" ⁇ ")), to obtain a compound of formula IV, which is a compound of formula I.
• An intermediate diene of formula II can be obtained from commercial sources or readily made by one skilled in the art, for example, in accordance with the following literature documents and the references found therein: Hofman et al, J. Agric. Food Chem. 45, 898-906 (1997); Baciocchi etal, J. Chem. Soc, Perkin Trans. 2 8, 821-824 (1975); Wu et al, J.
• An intermediate dieneophile of formula III can be obtained from commercial sources or readily made by one skilled in the art, for example, in accordance with the following literature references and the references found therein: Deshpande et al, Heterocycles 51, 2159-2162 (1999); Seijas et al, J. Chem. Res., Synop. 7, 420-421 (1999); Langer et ⁇ /., Eur. J. Org. Chem.
• compounds of formula I can be obtained by reaction of a primary amine of formula V with a substituted anhydride-like intermediate of formula VI, for example, in a solvent such as acetic acid with or without heating, to yield a compound of formula IV, which is a compound of formula I.
• Primary amines of formula V can be obtained from commercial sources or readily synthesized by one skilled in the art.
• Anhydride-like agents of formula VI can be obtained from commercial sources or readily synthesized by one skilled in the art.
• the documents listed following describe exemplary approaches for the synthesis of intermediates of formula VI as well as synthetic approaches which can be applied to the synthesis of compounds of formula IV (all incorporated herein by reference in their entirety): Kohler, E.
• Scheme III describes a method for preparing an intermediate compound of formula VI which can be used to synthesize a compound of formula I, as described in Scheme II.
• a diene of formula II can be reacted with a dieneophile of formula VII to yield the intermediate of formula VI.
• the methods applied to obtain such a transformation are analogous to those described in Scheme I.
• Scheme IV describes a method for preparing an intermediate compound of formula VI which can be used to synthesize a compound of formula I, as described in Scheme II.
• a diene of formula II can be reacted with a dieneophile of formula VIII to yield the intermediate of formula IX.
• the intermediate of formula IX can be dehydrated to an anhydride-like intermediate of formula VI. Dehydration of the bis-acid intermediate of formula IX to can be achieved by a variety of methods, such as those known to one skilled in the art and described in the following documents and the references embodied therein: Sprague et al, J. Med. Chem. 28, 1580-1590 (1985); and/or Retemi et al, J. Org. Chem. 61, 6296-6301 (1996).
• a preferred subgenus of the compounds of the present invention includes compounds of the formula I or salts thereof wherein one or more, preferably all, of the following substituents are as defined below:
• Ai is CR 7 ; and
• a 2 is CR ; or alternatively, (iii) Y is absent; and
• the compounds of the present invention modulate the function of nuclear hormone receptors (NHR), and include compounds which are, for example, agonists, partial agonists, antagonists or partial antagonists of the androgen receptor (AR), the estrogen receptor (ER), the progesterone receptor (PR), the glucocorticoid receptor (GR), the rnineralocorticoid receptor (MR), the steroid and xenobiotic receptor (SXR), other steroid binding NHR's, the Orphan receptors or other NHR's.
• NHR nuclear hormone receptors
• AR androgen receptor
• ER estrogen receptor
• PR progesterone receptor
• GR glucocorticoid receptor
• MR rnineralocorticoid receptor
• SXR steroid and xenobiotic receptor
• Modes includes, for example, activation (e.g., agonist activity) or inhibition (e.g., antagonist activity).
• NHR-associated condition denotes a condition or disorder which can be treated by modulating the function of a NHR in a subject, wherein treatment comprises prevention, partial alleviation or cure of the condition or disorder. Modulation may occur locally, for example, within certain tissues of the subject, or more extensively throughout a subject being treated for such a condition disorder.
• the compounds of the present invention are useful for the treatment of a variety of conditions and disorders including, but not limited to, those described following:
• Compounds of formula I can be applied as agonists, partial agonists, antagonists, or partial antagonists of the estrogen receptor, preferably selectively to that receptor, in an array of medical conditions which involve modulation of the estrogen receptor pathway.
• Applications of said compounds include but are not limited to: osteoporosis, hot flushes, vaginal dryness, prostate cancer, breast cancer, endometrial cancer, cancers expressing the estrogen receptor such as the aforementioned cancers and others, contraception, pregnancy termination, menopause, amennoreahea, and dysmennoreahea.
• Compounds of formula I can be applied as agonists, partial agonists, antagonists or partial antagonists of the progesterone receptor, preferably selectively to that receptor, in an array of medical conditions which involve modulation of the progesterone receptor pathway.
• Applications of said compounds include but are not limited to: breast cancer, other cancers containing the progesterone receptor, endometriosis, cachexia, contraception, menopause, cyclesynchrony, meniginoma, dysmennoreahea, fibroids, pregnancy termination, labor induction and osteoporosis.
• Compounds of formula I can be applied as agonists, partial agonists, antagonists or partial antagonists of the glucocorticoid receptor, preferably selectively to that receptor, in an array of medical conditions which involve modulation of the glucocorticoid receptor pathway.
• Applications of said compounds include but are not limited to: inflammatory diseases, autoimmune diseases, prostate cancer, breast cancer, Alzheimer's disease, psychotic disorders, drug dependence, non-insulin dependent Diabetes Mellitus, and as dopamine receptor blocking agents or otherwise as agents for the treatment of dopamine receptor mediated disorders.
• Glucocorticoid receptor AP-1 (“GR AP-1”) inhibitors can be used as anti-inflammatory and immunosuppressive agents, for example, to treat a wide variety of inflammatory and autoimmune diseases. These diseases include without limitation: rheumatoid arthritis, osteoarthritis, inflammatory bowel disease, asthma, chronic obstructive pulmonary disease, prevention of transplant rejection, multiple sclerosis, and psoriasis, among others.
• GR AP-1 inhibitors of the present invention can be employed together with known GR AP-1 inhibitors, such as the steroid prednisone (which is used to treat the above diseases) .
• Compounds of formula I can be applied as agonists, partial agonists, antagonists or partial antagonists of the mineralocorticoid receptor, preferably selectively to that receptor, in an array of medical conditions which involve modulation of the mineralocorticoid receptor pathway.
• Applications of said compounds include but are not limited to: drug withdrawal syndrome and inflammatory diseases.
• Compounds of formula I can be applied as agonists, partial agonists, antagonists or partial antagonists of the aldosterone receptor, preferably selectively to that receptor, in an array of medical conditions which involve modulation of the aldosterone receptor pathway.
• One application of said compounds includes but is not limited to: congestive heart failure.
• Compounds of formula I can be applied as agonists, partial agonists, antagonists or partial antagonists of the androgen receptor, preferably selectively to that receptor, in an array of medical conditions which involve modulation of the androgen receptor pathway.
• Applications of said compounds include but are not limited to: hirsutism, acne, seborrhea, Alzheimer's disease, androgenic alopecia, hypogonadism, hyperpilosity, benign prostate hypertrophia, adenomas and neoplasies of the prostate (such as advanced metastatic prostate cancer), treatment of benign or malignant tumor cells containing the androgen receptor such as is the case for breast, brain, skin, ovarian, bladder, lymphatic, liver and kidney cancers, pancreatic cancers, modulation of NCAM expression and applications therein for the treatment of heart disease, inflammation and immune modulations, modulation of NEGF expression and the applications therein for use as antiangiogenic agents, osteoporosis, suppressing spermatogenesis, libi
• Compounds of formula I can be applied as (preferably, selective) antagonists of the mutated androgen receptor, for example, found in many tumor lines.
• mutants are those found in representative prostate tumor cell lines such as LNCap, (T877A mutation, Biophys. Acta, 187, 1052 (1990)), PCa2b, (L701H & T877A mutations, J. Urol., 162, 2192 (1999)) and CWR22, (H874Y mutation, Mol. Endo., 11, 450 (1997)).
• Applications of said compounds include but are not limited to: adenomas and neoplasies of the prostate, breast cancer and endometrial cancer.
• Compounds of formula I can be applied as agonists, partial agonists, antagonists or partial antagonists of the steroid and xenobiotic receptor, preferably selectively to that receptor, in an array of medical conditions which involve modulation of the steroid and xenobiotic receptor pathway.
• Applications of said compounds include but are not limited to: treatment of disregulation of cholesterol homeostasis, attenuation of metabolism of pharmaceutical agents by co-administration of an agent (compound of the present invention) which modulates the P450 regulator effects of SXR.
• NHR NHR due to strong sequence homology to other NHR
• Orphan receptors demonstrate strong sequence homology to other NHR
• compounds of formula I include those which serve as modulators of the function of the Orphan NHR.
• Orphan receptors which are modulated by NHR modulators such as compounds within the scope of formula I are exemplified, but not limited to, those listed in Table 1.
• Exemplary therapeutic applications of modulators of said Orphan receptors are also listed in Table 1, but are not limited to the examples therein.
• the present invention thus provides methods for the treatment of NHR- associated conditions, comprising the step of administering to a subject in need thereof at least one compound of formula I in an amount effective therefor.
• Other therapeutic agents such as those described below may be employed with the inventive compounds in the present methods (for example, separately, or formulated together as a fixed dose).
• such other therapeutic agent(s) can be administered prior to, simultaneously with or following the administration of the compound(s) of the present invention.
• the present invention also provides pharmaceutical compositions comprising at least one of the compounds of the formula I capable of treating a NHR-associated condition in an amount effective therefor, and a pharmaceutically acceptable carrier (vehicle or diluent).
• compositions of the present invention can contain other therapeutic agents as described below, and can be formulated, for example, by employing conventional solid or liquid vehicles or diluents, as well as pharmaceutical additives of a type appropriate to the mode of desired administration (for example, excipients, binders, preservatives, stabilizers, flavors, etc.) according to techniques such as those well known in the art of pharmaceutical formulation.
• the compounds of the present invention are, without limitation as to their mechanism of action, useful in treating any of the conditions or disorders listed or described herein such as inflammatory diseases or cancers, or other proliferate diseases, and in compositions for treating such conditions or disorders.
• the present compounds have therapeutic utility in the modulation of immune cell activation/proliferation, e.g., as competitive inhibitors of intercellular ligand/receptor binding reactions involving CAMs (Cellular Adhesion Molecules) and Leukointegrins.
• the present compounds modulate LFA-ICAM 1, and are particularly useful as LFA-ICAM 1 antagonists, and in the treatment of all conditions associated with LFA-ICAM 1 such as immunological disorders.
• Preferred utilities for the present compounds include, but are not limited to: inflammatory conditions such as those resulting from a response of the non-specific immune system in a mammal (e.g., adult respiratory distress syndrome, shock, oxygen toxicity, multiple organ injury syndrome secondary to septicemia, multiple organ injury syndrome secondary to trauma, reperfusion injury of tissue due to cardiopulmonary bypass, myocardial infarction or use with thrombolysis agents, acute glomerulonephritis, vasculitis, reactive arthritis, dermatosis with acute inflammatory components, stroke, thermal injury, hemodialysis, leukapheresis, ulcerative colitis, necrotizing enterocolitis and granulocyte transfusion associated syndrome) and conditions resulting from a response of the specific immune system in a mammal (e.g., psoriasis, organ/tissue transplant rejection, graft vs.
• inflammatory conditions such as those resulting from a response of the non-specific immune system in a mammal (e.g., adult
• the present compounds can be used in treating asthma or as an adjunct to minimize toxicity with cytokine therapy in the treatment of cancers.
• the present compounds can be employed in the treatment of all diseases currently treatable through steroid therapy.
• the present compounds may be employed for the treatment of these and other disorders alone or with other immunosuppressive or antiinflammatory agents.
• a compound of the formula I can be administered prior to the onset of inflammation (so as to suppress an anticipated inflammation) or after the initiation of inflammation.
• the immunosupressive compound(s) are preferably provided in advance of any inflammatory response or symptom (for example, prior to, at, or shortly after the time of an organ or tissue transplant but in advance of any symptoms or organ rejection).
• the prophylactic administration of a compound of the formula I prevents or attenuates any subsequent inflammatory response (such as, for example, rejection of a transplanted organ or tissue, etc.)
• Administration of a compound of the formula I attenuates any actual inflammation (such as, for example, the rejection of a transplanted organ or tissue).
• the compounds of the formula I can be administered for any of the uses described herein by any suitable means, for example, orally, such as in the form of tablets, capsules, granules or powders; sublingualiy; bucally; parenterally, such as by subcutaneous, intravenous, intramuscular, or intrasternal injection or infusion techniques (e.g., as sterile injectable aqueous or non-aqueous solutions or suspensions); nasally, including administration to the nasal membranes, such as by inhalation spray; topically, such as in the form of a cream or ointment; or rectally such as in the form of suppositories; in dosage unit formulations containing non-toxic, pharmaceutically acceptable vehicles or diluents.
• suitable means for example, orally, such as in the form of tablets, capsules, granules or powders; sublingualiy; bucally; parenterally, such as by subcutaneous, intravenous, intramuscular, or intrasternal injection or infusion techniques (
• the present compounds can, for example, be administered in a form suitable for immediate release or extended release. Immediate release or extended release can be achieved by the use of suitable pharmaceutical compositions comprising the present compounds, or, particularly in the case of extended release, by the use of devices such as subcutaneous implants or osmotic pumps.
• the present compounds can also be administered liposomally.
• compositions for oral administration include suspensions which can contain, for example, microcrystalline cellulose for imparting bulk, alginic acid or sodium alginate as a suspending agent, methylcellulose as a viscosity enhancer, and sweeteners or flavoring agents such as those known in the art; and immediate release tablets which can contain, for example, microcrystalline cellulose, dicalcium phosphate, starch, magnesium stearate and/or lactose and/or other excipients, binders, extenders, disintegrants, diluents and lubricants such as those known in the art.
• the compounds of formula I can also be delivered through the oral cavity by sublingual and/or buccal administration.
• Molded tablets, compressed tablets or freeze-dried tablets are exemplary forms which may be used.
• Exemplary compositions include those formulating the present compound(s) with fast dissolving diluents such as mannitol, lactose, sucrose and/or cyclodextrins. Also included in such formulations may be high molecular weight excipients such as celluloses (avicel) or polyethylene glycols (PEG).
• Such formulations can also include an excipient to aid mucosal adhesion such as hydroxy propyl cellulose (HPC), hydroxy propyl methyl cellulose (HPMC), sodium carboxy methyl cellulose (SCMC), maleic anhydride copolymer (e.g., Gantrez), and agents to control release such as polyacrylic copolymer (e.g.
• Lubricants may also be added for ease of fabrication and use.
• compositions for nasal aerosol or inhalation administration include solutions in saline which can contain, for example, benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, and/or other solubilizing or dispersing agents such as those known in the art.
• compositions for parenteral administration include injectable solutions or suspensions which can contain, for example, suitable non-toxic, parenterally acceptable diluents or solvents, such as mannitol, 1,3-butanediol, water, Ringer's solution, an isotonic sodium chloride solution, or other suitable dispersing or wetting and suspending agents, including synthetic mono- or diglycerides, and fatty acids, including oleic acid, or Cremaphor.
• suitable non-toxic, parenterally acceptable diluents or solvents such as mannitol, 1,3-butanediol, water, Ringer's solution, an isotonic sodium chloride solution, or other suitable dispersing or wetting and suspending agents, including synthetic mono- or diglycerides, and fatty acids, including oleic acid, or Cremaphor.
• compositions for rectal administration include suppositories which can contain, for example, a suitable non-irritating excipient, such as cocoa butter, synthetic glyceride esters or polyethylene glycols, which are solid at ordinary temperatures, but liquify and/or dissolve in the rectal cavity to release the drug.
• a suitable non-irritating excipient such as cocoa butter, synthetic glyceride esters or polyethylene glycols, which are solid at ordinary temperatures, but liquify and/or dissolve in the rectal cavity to release the drug.
• exemplary compositions for topical administration include a topical carrier such as Plastibase (mineral oil gelled with polyethylene).
• the effective amount of a compound of the present invention can be determined by one of ordinary skill in the art, and includes exemplary dosage amounts for a adult human of from about 1 to 100 (for example, 15) mg/kg of body weight of active compound per day, which can be administered in a single dose or in the form of individual divided doses, such as from 1 to 4 times per day. It will be understood that the specific dose level and frequency of dosage for any particular subject can be varied and will depend upon a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the species, age, body weight, general health, sex and diet of the subject, the mode and time of administration, rate of excretion, drug combination, and severity of the particular condition.
• Preferred subjects for treatment include animals, most preferably mammalian species such as humans, and domestic animals such as dogs, cats and the like, subject to NHR-associated conditions.
• the compounds of the present invention can be employed alone or in combination with each other and/or other suitable therapeutic agents useful in the treatment of NHR-associated conditions.
• the compounds of the present invention can be administered either alone or in combination with other anti- cancer and cytotoxic agents and treatments useful in the treatment of cancer or other proliferative diseases, for example, where the second drug has the same or different mechanism of action than the present compounds of formula I.
• Examples of classes of anti-cancer and cytotoxic agents useful in combination with the present compounds include but are not limited to: alkylating agents such as nitrogen mustards, alkyl sulfonates, nitrosoureas, ethylenimines, and triazenes; antimetabolites such as folate antagonists, purine analogues, and pyrimidine analogues; antibiotics such as anthracyclines, bleomycins, mitomycin, dactinomycin, and plicamycin; enzymes such as L-asparaginase; farnesyl-protein transferase inhibitors; 5 reductase inhibitors; inhibitors of 17 ⁇ -hydroxy steroid dehydrogenase type 3; hormonal agents such as glucocorticoids, estrogens/ antiestrogens, androgens/ antiandrogens, progestins, and luteinizing hormone-releasing hormone antagonists, octreotide acetate; microtubule- disruptor agents
• anti-cancer and cytotoxic agents include but are not limited to mechlorethamine hydrochloride, cyclophosphamide, chlorambucil, melphalan, ifosfamide, busulfan, carmustin, lomustine, semustine, streptozocin, thiotepa, dacarbazine, methotrexate, thioguanine, mercaptopurine, fludarabine, pentastatin, cladribin, cytarabine, fluorouracil, doxorubicin hydrochloride, daunorubicin, idarubicin, bleomycin sulfate, mitomycin C, actinomycin D, safracins, saframycins, quinocarcins, discodermolides, vincristine, vinblastine, vinorelbine tartrate, etoposide, etoposide phosphate, teni
• Preferred member of these classes include, but are not limited to, paclitaxel, cisplatin, carboplatin, doxorubicin, carminomycin, daunorubicin, aminopterin, methotrexate, methopterin, mitomycin C, ecteinascidin 743, or porfiromycin, 5- fluorouracil, 6-mercaptopurine, gemcitabine, cytosine arabinoside, podophyllotoxin or podophyllotoxin derivatives such as etoposide, etoposide phosphate or teniposide, melphalan, vinblastine, vincristine, leurosidine, vindesine and leurosine.
• anticancer and other cytotoxic agents include the following: epothilone derivatives as found in German Patent No. 4138042.8; WO 97/19086, WO 98/22461, WO 98/25929, WO 98/38192, WO 99/01124, WO 99/02224, WO 99/02514, WO 99/03848, WO 99/07692, WO 99/27890, WO 99/28324, WO 99/43653, WO 99/54330, WO 99/54318, WO 99/54319, WO 99/65913, WO 99/67252, WO 99/67253 and WO 00/00485; cyclin dependent kinase inhibitors as found in WO 99/24416 (see also U.S.
• Patent No. 6,040,321) and prenyl-protein transferase inhibitors as found in WO 97/30992 and WO 98/54966; and agents such as those described generically and specifically in U.S. Patent No. 6,011,029 (the compounds of which U.S. Patent can be employed together with any NHR modulators (including, but not limited to, those of present invention) such as AR modulators, ER modulators, with LHRH modulators, or with surgical castration, especially in the treatment of cancer) .
• NHR modulators including, but not limited to, those of present invention
• the combinations of the present invention can also be formulated or co- administered with other therapeutic agents that are selected for their particular usefulness in administering therapies associated with the aforementioned conditions.
• the compounds of the invention may be formulated with agents to prevent nausea, hypersensitivity and gastric irritation, such as antiemetics, and Hi and H 2 antihistaminics.
• the compounds of this invention are most preferably used alone or in combination with anti-cancer treatments such as radiation therapy and/or with cytostatic and/or cytotoxic agents, such as, but not limited to, DNA interactive agents, such as cisplatin or doxorubicin; inhibitors of farnesyl protein transferase, such as those described in U.S. Patent No.
• anti-cancer treatments such as radiation therapy and/or with cytostatic and/or cytotoxic agents, such as, but not limited to, DNA interactive agents, such as cisplatin or doxorubicin; inhibitors of farnesyl protein transferase, such as those described in U.S. Patent No.
• topoisomerase JJ inhibitors such as etoposide
• topoisomerase I inhibitors such as CPT-11 or topotecan
• tubulin stabilizing agents such as paclitaxel, docetaxel, other taxanes, or epothilones
• hormonal agents such as tamoxifen
• thymidilate synthase inhibitors such as 5-fluorouracil
• antimetabolites such as methoxtrexate
• antiangiogenic agents such as angiostatin, ZD6474, ZD6126 and comberstatin A2
• kinase inhibitors such as her2 specific antibodies, Iressa and CDK inhibitors
• histone deacetylase inhibitors such as CI-994 and MS-27-275.
• Such compounds may also be combined with agents which suppress the production of circulating testosterone such as LHRH agonists or antagonists or with surgical castration.
• known therapies for advanced metastatic prostate cancer include "complete androgen ablation therapy” wherein tumor growth is inhibited by controlling the supply of androgen to the prostate tissues via chemical castration (castration serves to inhibit the production of circulating testosterone (T) and dihydrotestosterone (DHT)) followed by the administration of androgen receptor (AR) antagonists (which inhibit the function T/DHT derived from the conversion of circulating androgen precursors to T/DHT by the prostate tissue).
• the compounds of the present invention can be employed as AR antagonists in complete ablation therapy, alone or in combination with other AR antagonists such as Flutamide, Casodex, Nilutamide, or Cyproterone acetate.
• the compounds of the present invention may be employed adjuvant to surgery.
• Another application of the present compounds is in combination with antibody therapy such as but not limited to antibody therapy against PSCA.
• An additional application is in concert with vaccine / immune modulating agents for the treatment of cancer.
• the above other therapeutic agents when employed in combination with the compounds of the present invention, can be used, for example, in those amounts indicated in the Physicians' Desk Reference (PDR) or as otherwise determined by one of ordinary skill in the art.
• the following assays can be employed in ascertaining the activity of a compound as a NHR modulator. Preferred are those compounds with an activity greater than 20 ⁇ m for binding or transactivation in any of these assays.
• Various compounds of the present invention were determined to have AR modulator activity utilizing the transactivation assay, and standard AR binding assays as described following. Transactivation Assays;
• transactivation assays of a transfected reporter construct and using the endogenous androgen receptor of the host cells.
• the transactivation assay provides a method for identifying functional agonists and partial agonists that mimic, or antagonists that inhibit, the effect of native hormones, in this case, dihydrotestosterone (DHT).
• DHT dihydrotestosterone
• This assay can be used to predict in vivo activity as there is a good correlation in both series of data. See, e.g. T. Berger et al., J. Steroid Biochem. Molec. Biol. 773 (1992), the disclosure of which is herein incorporated by reference.
• reporter plasmid For the transactivation assay a reporter plasmid is introduced by transfection (a procedure to induce cells to take foreign genes) into the respective cells.
• This reporter plasmid comprising the cDNA for a reporter protein, such as secreted alkaline phosphatase (SEAP), controlled by prostate specific antigen (PSA) upstream sequences containing androgen response elements (AREs).
• SEAP secreted alkaline phosphatase
• PSA prostate specific antigen
• AREs upstream sequences containing androgen response elements
• the reporter acts as a surrogate for the products (mRNA then protein) normally expressed by a gene under control of the AR and its native hormone, hi order to detect antagonists, the transactivation assay is carried out in the presence of constant concentration of the natural AR hormone (DHT) known to induce a defined reporter signal. Increasing concentrations of a suspected antagonist will decrease the reporter signal (e.g., SEAP production). On the other hand, exposing the transfected cells to increasing concentrations of a suspected agonist will increase the production of the reporter signal.
• DHT natural AR hormone
• LNCaP and MDA 453 cells were obtained from the American Type Culture Collection (Rockville, MD), and maintained in RPMI 1640 or DMEM medium supplemented with 10% fetal bovine serum (FBS; Gibco) respectively.
• the respective cells were transiently transfected by electroporation according to the optimized procedure described by Heiser, 130 Methods Mol. Biol., 117 (2000), with the pSEAP2/PS A540/Enhancer reporter plasmid.
• the reporter plasmid was constructed as follows: commercial human placental genomic DNA was used to generate by Polymerase Cycle Reaction (PCR) a fragment containing the Bgi ⁇ site (position 5284) and the Hind HI site at position 5831 of the human prostate specific antigen promoter (Accession # U37672), Schuur, et al., J. Biol. Chem., 211 (12): 7043-51 (1996). This fragment was subcloned into the pSEAP2 basic (Clontech) previously digested with BglJI and HindlH to generate the pSEAP2/PS A540 construct.
• PCR Polymerase Cycle Reaction
• LNCaP and MDA 453 cells were collected in media containing 10% charcoal stripped FBS. Each cell suspension was distributed into two Gene Pulser Cuvetts (Bio-Rad) which then received 8 ⁇ g of the reporter construct, and electoporated using a Bio-Rad Gene Pulser at 210 volts and 960 ⁇ Faraday. Following the transfections the cells were washed and incubated with media containing charcoal stripped fetal bovine serum in the absence (blank) or presence (control) of 1 nM dihydrotestosterone (DHT; Sigma Chemical) and in the presence or absence of the standard anti-androgen bicalutamide or compounds of the present invention in concentrations ranging from 10-10 to 10-5 M (sample). Duplicates were used for each sample. The compound dilutions were performed on a Biomek 2000 laboratory workstation.
• MTS Olet's reagent
• MTS is bioreduced by cells into a formazan that is soluble in tissue culture medium, and therefore its absorbance at 490nm can be measured directly from 96 well assay plates without additional processing.
• the quantity of foraiazan product as measured by the amount of 490nm absorbance is directly proportional to the number of living cells in culture. For each replicate the SEAP reading was normalized by the Abs490 value derived from the MTS assay. For the antagonist mode, the % Inhibition was calculated as:
• the reporter plasmid utilized was comprised of the cDNA for the reporter
• SEAP protein as described for the AR specific transactivation assay. Expression of the reporter SEAP protein was controlled by the mouse mammary tumor virus long terminal repeat (MMTV LTR) sequences that contains three hormone response elements (HREs) that can be regulated by both GR and PR see, e.g. G. Chalepakis et al., Cell, 53(3), 371 (1988). This plasmid was transfected into A549 cells, which expresses endogenous GR, to obtain a GR specific transactivation assay. A549 cells were obtained from the American Type Culture Collection (Rockville, MD), and maintained in RPMI 1640 supplemented with 10% fetal bovine serum (FBS; Gibco).
• FBS fetal bovine serum
• Determination of the GR specific antagonist activity of the compounds of the present invention was identical to that described for the AR specific transactivation assay, except that the DHT was replaced with 5 nM dexamethasone (Sigma Chemicals), a specific agonist for GR. Determination of the GR specific agonist activity of the compounds of the present invention was performed as described for the AR transactivation assay, wherein one measures the activation of the GR specific reporter system by the addition of a test compound, in the absence of a known GR specific agonists ligand.
• the reporter plasmid utilized was comprised of the cDNA for the reporter SEAP protein, as described for the AR specific transactivation assay. Expression of the reporter SEAP protein was controlled by the mouse mammary tumor virus long terminal repeat (MMTV LTR) sequences that contains three hormone response elements (HREs) that can be regulated by both GR and PR. This plasmid was transfected into T47D, which expresses endogenous PR, to obtain a PR specific transactivation assay. T47D cells were obtained from the American Type Culture Collection (Rockville, MD), and maintained in DMEM medium supplemented with 10% fetal bovine serum (FBS; Gibco).
• FBS fetal bovine serum
• Determination of the PR specific antagonist activity of the compounds of the present invention was identical to that described for the AR specific transactivation assay, except that the DHT was replaced with 1 nM Promegastone (NEN), a specific agonist for PR. Determination of the PR specific agonist activity of the compounds of the present invention was performed as described for the AR transactivation assay, wherein one measures the activation of the PR specific reporter system by the addition of a test compound, in the absence of a known PR specific agonists ligand.
• AR Binding Assay For the whole cell binding assay, human LNCaP cells (T877A mutant AR) or
• MDA 453 wild type AR in 96-well microtiter plates containing RPMI 1640 or DMEM supplemented with 10% charcoal stripped CA-FBS (Cocaleco Biologicals) respectively, were incubated at 37°C to remove any endogenous ligand that might be complexed with the receptor in the cells. After 48 hours, either a saturation analysis to determine the K d for tritiated dihydrotestosterone, [ 3 H]-DHT, or a competitive binding assay to evaluate the ability of test compounds to compete with [ 3 H]-DHT were performed.
• media RPMI 1640 or DMEM - 0.2% CA-FBS
• [ 3 H]-DHT in concentrations ranging from 0.1 nM to 16 nM
• an aliquot of the total binding media at each concentration of [ 3 H]-DHT was removed to estimate the amount of free [ 3 H]-DHT.
• IC 5 o values were determined.
• the IC 50 is defined as the concentration of competing ligand needed to reduce specific binding by 50%.
• the K d S for [ 3 H]-DHT for MDA 453 and LNCaP were 0.7 and 0.2 nM respectively.
• test compounds were tested ("test compounds") on the proliferation of human prostate cancer cell lines.
• MDA PCa2b cells a cell line derived from the metastasis of a patient that failed castration, Navone et al., Clin. Cancer Res., 3, 2493-500 (1997), were incubated with or without the test compounds for 72 hours and the amount of [ H]-thymidine incorporated into DNA was quantified as a way to assess number of cells and therefore proliferation.
• the MDA PCa2b cell line was maintained in BRFF-HPCl media (Biological Research Faculty & Facility Inc., MD) supplemented with 10% FBS.
• cells were plated in Biocoated 96-well microplates and incubated at 37°C in 10% FBS (charcoal-stripped)/BRFF- BMZERO (without androgens). After 24 hours, the cells were treated in the absence (blank) or presence of 1 nM DHT (control) or with test compounds (sample) of the present invention in concentrations ranging from 10 "10 to 10 "5 M. Duplicates were used for each sample. The compound dilutions were performed on a Biomek 2000 laboratory work station. Seventy two hours later 0.44 uCi.
• % Inhibition 100 x ( 1 - [average control - average b iank / average sam pie - average blank]) Data was plotted and the concentration of compound that inhibited 50% of the [ 3 H]- Thymidine incorporation was quantified (IC50).
• the first assay uses a cell line, Stable 1 (clone #72), which stably expresses the full length rat androgen receptor but requires the transient transfection of an enhancer/reporter. This cell line was derived from C2C12 mouse moyoblast cells.
• the second assay uses a cell line, Stable 2 (clone #133), derived from Stable 1 which stably expresses both rAR and the enhancer/luciferase reporter.
• the enhancer/reporter construct used in this system is pGL3 /2XDR-
• 2XDR-1 was reported to be an AR specific response element in CV-1 cells, Brown et. al. The Journal of Biological Chemisty 272, 8227-8235, (1997). It was developed by random mutagenesis of an AR/GR consensus enhancer sequence.
• Stable 1 cells are plated in 96 well format at 6,000 cells/well in high glucose DMEM without phenol red (Gibco BRL, Cat. No.: 21063-029) containing 10% charcoal and dextran treated FBS (HyClone Cat. No.: SH30068.02), 50 mM HEPES Buffer (Gibco BRL, Cat. No.: 15630-080), IX MEM Na Pyruvate (Gibco
• sperm DNA (as carrier) are diluted with 5 ⁇ l/well Opti-MEMem media (Gibco BRL, Cat. No.: 31985-070). To this, 0.5 ⁇ l/well Plus reagent is added. This mixture is incubated for 15 minutes at room temperature. In a separate vessel, 0.385 ⁇ l/well LipofectAMINE reagent is diluted with 5 ⁇ l/well Opti-MEM. The DNA mixture is then combined with the LipofectAMINE mixture and incubated for an additional 15 minutes at room temperature. During this time, the media from the cells is removed and replaced with 60 ⁇ l/well of Opti-MEM. To this is added 10 ⁇ l/well of the DNA/Lipofect AMINE transfection mixture. The cells are incubated for 4 hours. 3. The transfection mixture is removed from the cells and replaced with 90 ⁇ l of media as in #1 above.
• Stable 2 cells are plated in 96 well format at 6,000 cells/well in high glucose DMEM without phenol red (Gibco BRL, Cat. No.: 21063-029) containing 10% charcoal and dextran treated FBS (HyClone Cat. No.: SH30068.02), 50 mM HEPES Buffer (Gibco BRL, Cat. No.: 15630-080), IX MEM Na Pyruvate (Gibco
• test compounds The ability of compounds of the present invention to modulate the function of the AR was determined by testing said compounds in a proliferation assay using the androgen responsive murine breast cell line derived from the Shionogi tumor, Hiraoka et al, Cancer Res., 47, 6560-6564 (1987).
• Stable AR dependent clones of the parental Shionogi line were established by passing tumor fragments under the general procedures originally described in Tetuo, et. al, Cancer Research 25, 1168-1175 (1965). From the above procedure, one stable line, SCI 14, was isolated, characterized and utilized for the testing of example compounds.
• SCI 14 cells were incubated with or without the test compounds for 72 hours and the amount of [3H]-thymidine incorporated into DNA was quantified as a surrogate endpoint to assess the number of cells and therefore the proliferation rate as described in Suzuki et. al, J. Steroid Biochem. Mol. Biol 37, 559-567 (1990).
• the SCI 14 cell line was maintained in MEM containing 10 "8 M testosterone and 2% DCC-treated FCS.
• cells were plated in 96-well microplates in the maintenance media and incubated at 37°C.
• the medium was changed to serum free medium [Ham's F-12:MEM (1;1, v/v) containing 0.1% BSA] with (antagonist mode) or without (agonist mode) 10 "8 M testosterone and the test compounds of the present invention in concentrations ranging from 10 " to 10 " M.
• Duplicates were used for each sample. The compound dilutions were performed on a Biomek 2000 laboratory work station. Seventy two hours later 0.44uCi of [3H]-Thymidine (Amersham) was added per well and incubated for another 2 hr followed by tripsinization, and harvesting of the cells onto GF/B filters. Micro-scint PS were added to the filters before counting them on a Beckman TopCount. For the antagonist mode, the % Inhibition was calculated as:
• the AP-1 assay is a cell based luciferase reporter assay.
• A549 cells which contain endogenous glucocorticoid receptor, were stably transfected with an AP-1 DNA binding site attached to the luciferase gene. Cells are then grown in RPMI + 10% fetal calf serum (charcoal-treated) + Penicillin/Streptomycin with 0.5mg/ml geneticin. Cells are plated the day before the assay at approximately 40000 cells/well.
• test compounds dissolved in DMSO and added at varying concentrations
• dexamethasome 100 nM in DMSO, positive control
• Activity is measured by analysis in a luminometer as compared to control experiments treated with buffer or dexamethasome. Activity is designated as % inhibition of the reporter system as compared to the buffer control with 10 ng/ml PMA alone.
• the control, dexamethasone, at a concentration of ⁇ 10 ⁇ M typically suppresses activity by 65%.
• Test compounds which demonstrate an inhibition of PMA induction of 50% or greater at a concentration of test compound of ⁇ 10 ⁇ M are deemed active.
• Testosterone is converted to the more active form, dihydrotestosterone, (DHT), within the prostate by 5 -reductase.
• DHT dihydrotestosterone
• Adrenal androgens also contribute about 20% of total DHT in the rat prostate, compared to 40% of that in 65-year-old men.
• this is not a major pathway, since in both animals and humans, castration leads to almost complete involution of the prostate and seminal vesicles without concomitant adrenalectomy. Therefore, under normal conditions, the adrenals do not support significant growth of prostate tissues.
• M. C. Luke and D. S. Coffey "The Physiology of Reproduction" ed. By E.
• Testosterone Proprionate in arachis oil vehicle and anti-androgen test compounds (compounds of the present invention) were dosed orally by gavage (p.o.) in dissolved/suspensions of 80% PEG 400 and 20% Tween 80 (PEGTW). Animals were dosed (v/w) at 0.5 ml of vehicle /100g body weight.
• Experimental groups were as follows: 1. Control vehicle 2. Testosterone Propionate (TP) (3 mg/rat/day, subcutaneous)
• TP plus Casodex administered p.o. in PEGTW, QD
• a recognized antiandrogen as a reference compound.
• test compound a compound of the present invention was administered (p.o. in PEGTW, QD) with TP (s.c. as administered in group 2) in a range of doses.
• test compound a compound of the present invention was administered alone (p.o. in PEGTW, QD) in a range of doses.
• the animals were sacrificed, and the ventral prostate weighed.
• the sexual organs weights were first standardized as mg per 100 g of body weight, and the increase in organ weight induced by TP was considered as the maximum increase (100%).
• ANONA followed by one-tailed Student or Fischer's exact test was used for statistical analysis.
• the gain and loss of sexual organ weight reflect the changes of the cell number (D ⁇ A content) and cell mass (protein content), depending upon the serum androgen concentration. See Y. Okuda et al., J. Urol, 145, 188-191 (1991), the disclosure of which is herein incorporated by reference. Therefore, measurement of organ wet weight is sufficient to indicate the bioactivity of androgens and androgen antagonist, h immature castrated rats, replacement of exogenous androgens increases seminal vesicles (SN) and the ventral prostate (NP) in a dose dependent manner.
• the maximum increase in organ weight was 4 to 5-fold when dosing 3 mg/rat/day of testosterone (T) or 1 mg/rat/day of testosterone propionate (TP) for 3 days.
• the EC 50 of T and TP were about 1 mg and 0.03 mg, respectively.
• the increase in the weight of the NP and S V also correlated with the increase in the serum T and DHT concentration.
• administration of T showed 5-times higher serum concentrations of T and DHT at 2 hours after subcutaneous injection than that of TP, thereafter, these high levels declined very rapidly.
• the serum concentrations of T and DHT in TP-treated animals were fairly consistent during the 24 hours, and therefore, TP showed about 10-30-fold higher potency than free T.
• a known AR antagonist (Casodex) was also administered simultaneously with 0.1 mg of TP (ED 80 ), inhibiting the testosterone-mediated increase in the weights of the VP and SN in a dose dependent manner.
• the antagonist effects were similar when dosing orally or subcutaneously.
• Compounds of the invention also exhibited AR antagonist activity by suppressing the testosterone-mediated increase in the weights of NP and SN.
• the basis of this assay lies in the well-defined action of androgenic agents on the maintenance and growth of muscle tissues and sexual accessory organs in animals and man.
• Androgenic steroids such as testosterone (T)
• T testosterone
• Treatment of animals or humans after castrations with an exogenous source of T results in a reversal of muscular atrophy.
• the effects of T on muscular atrophy in the rat levator ani muscle have been well characterized, M. Masuoka et al., "Constant cell population in normal, testosterone deprived and testosterone stimulated levator ani muscles" Am. J. Anat. 119, 263 (1966); Z. Gori et al., "Testosterone hypertrophy of levator ani muscle of castrated rats.
• Testosterone Propionate (TP) (3 mg/rat/day, subcutaneous)
• test compound was administered (p.o. in PEGTW, QD) with TP (s.c. as administered in group 2) in a range of doses. 5.
• test compound was administered alone (p.o. in PEGTW, QD) in a range of doses.
• the animals were sacrificed by carbon dioxide, and the levator ani, seminal vesicle and ventral prostate weighed.
• the levator ani muscle and sexual organ weights were first standardized as mg per 100 g of body weight, and the increase in organ weight induced by TP was considered as the maximum increase (100%). Super-anova (one factor) was used for statistical analysis.
• the gain and loss of sexual organ weight reflect the changes of the cell number (DNA content) and cell mass (protein content), depending upon the serum androgen concentration. See Y. Okuda et al., /. Urol, 145, 188-191 (1991), the disclosure of which is herein incorporated by reference. Therefore, measurement of organ wet weight is sufficient to indicate the bioactivity of androgens and androgen antagonist, immature castrated rats, replacement of exogenous androgens increases levator ani, seminal vesicles (SN) and prostate in a dose dependent manner.
• the maximum increase in organ weight was 4 to 5-fold when dosing 3 mg/rat/day of testosterone (T) or 1 mg/rat/day of testosterone propionate (TP) for 3 days.
• the EC 50 of T and TP were about 1 mg and 0.03 mg, respectively.
• the increase in the weight of the NP and SN also correlated with the increase in the serum T and DHT concentration.
• administration of T showed 5-times higher serum concentrations of T and DHT at 2 hours after subcutaneous injection than that of TP, thereafter, these high levels declined very rapidly, h contrast, the serum concentrations of T and DHT in TP-treated animals were fairly consistent during the 24 hours, and therefore, TP showed about 10-30-fold higher potency than free T.
• MDA-PCa-2b human prostate tumors were maintained in Balb/c nu/nu nude mice. Tumors were propagated as subcutaneous transplants in adult male nude mice (4-6 weeks old) using tumor fragments obtained from donor mice. Tumor passage occurred every 5-6 weeks. For antitumor efficacy trial, the required number of animals needed to detect a meaningful response were pooled at the start of the experiment and each was given a subcutaneous implant of a tumor fragment (-50 mg) with a 13 -gauge trocar. Tumors were allowed to grow to approx. 100-200 mg (tumors outside the range were excluded) and animals were evenly distributed to various treatment and control groups. Treatment of each animal was based on individual body weight.
• Treated animals were checked daily for treatment related toxicity/mortality. Each group of animals was weighed before the initiation of treatment (Wtl) and then again following the last treatment dose (Wt2). The difference in body weight (Wt2-Wtl) provides a measure of treatment-related toxicity.
• Tumor response was determined by measurement of tumors with a caliper twice a week, until the tumors reach a predetermined "target" size of 0.5 gm.
• Tumor response end-point was expressed in terms of tumor growth inhibition
• (%T/C) defined as the ratio of median tumor weights of the treated tumors (T) to that of the control group (C).
• TNDT Median time (days) for control tumors to reach target size -
• Dunning R3327H prostate tumor is a spontaneously derived, well differentiated androgen responsive adenocarcinoma of the prostate (Smolev JK, Heston WD, Scott WW, and Coffey DS, Cancer Treat Rep. 61, 273-287 (1977)).
• the growth of the R3327H subline has been selected for its highly androgen-dependent and reproducible growth in intact male rats. Therefore, this model and other sublines of this tumor have been widely used to evaluate in vivo antitumor activities of antiandrogens such as flutamide and bacilutamide/Casodex (Maucher A., and von Angerer, J. Cancer Res. Clin.
• the Dunning tumor pieces (about 4 x 4 mm) are transplanted subcutaneously to the flank of mature male Copenhagen rats (6-7 weeks old, Harlan-Sprague Dawley, Indianapolis, MD). About 6 weeks after the implantation, the animals with tumors of measurable size (about 80 - 120 mm 2 ) are randomized into treatment groups (8-10 rats/group) and the treatments are initiated. One group of the rats are castrated to serve as the negative control of tumor growth. Animals are treated daily with compounds of the current invention, standard antiandrogens such as bacilutamide or vehicle (control) for an average of 10 to 14 weeks. Test compounds are dissolved in a vehicle of
• Typical therapeutic experiments would include three groups of three escalating doses for each standard or test compound (in a range of 300-3 mg/kg). Tumors in the vehicle (control) group reach a size of 1500 to 2500 mm 3 , whereas the castrated animal group typically shows tumor stasis over the 14 weeks of observation. Animals treated orally with 20 mg/kg of bicalutamide or flutamide would be expected to show a 40% reduction in tumor volumes compared to control after 14 weeks of treatment.
• the activity of compounds of the present invention were investigated in a mature male rat model, which is a variation of the Levator ani & wet prostate weight assay described above.
• the above in vivo assays are recognized assays for determining the anabolic effects in muscle and sustaining effects in sex organs for a given compound, as described in L. G. Hershberger et al, 83 Proc Soc. Expt. Biol. Med., 175 (1953); B. L. Beyler et al, "Methods for evaluating anabolic and catabolic agents in laboratory animals", 23 J. Amer. Med. Women's Ass., 708 (1968); H.
• the male sexual accessory organs such as the prostate and seminal vesicles, play an important role in reproductive function. These glands are stimulated to grow and are maintained in size and secretory function by the continued presence of serum testosterone (T), which is the major serum androgen (>95%) produced by the Leydig cells in the testis under the control of the pituitary luteinizing hormone (LH) and follicle stimulating hormone (FSH). Testosterone is converted to the more active form, dihydrotestosterone, (DHT), within the prostate by 5 ⁇ -reductase. Adrenal androgens also contribute about 20% of total DHT in the rat prostate, compared to 40% of that in 65-year-old men. F. Labrie et. al.
• Testosterone production in the Leydig cells of the testis is controlled by the level of circulating LH released from the pituitary gland. LH levels are themselves controlled by the level of LHRH produced in the hypothalmic region. Testosterone levels in the blood serve to inhibit the secretion of LHRH and subsequently reduce levels of LH and ultimately the levels of circulating testosterone levels.
• test compounds By measuring blood levels of LH as they are effected by compounds of the present invention ("test compounds”), it is possible to determine the level of agonist or antagonist activity of said compounds at the hypothalamic axis of this endocrine cycle.
• Control vehicle p.o., PEGTW, QD
• the gain and loss of sexual organ and levator ani weight reflect the changes of the cell number (DNA content) and cell mass (protein content), depending upon the serum androgen concentration, see Y. Okuda etal, J. Urol, 145, 188-191 (1991), the disclosure of which i ⁇ herein incorporated by reference. Therefore, measurement of organ wet weight is sufficient to indicate the bioactivity of androgens and androgen antagonist.
• active agonist agents will have no effect or will increase the weight of one or more of the androgen responsive organs (levator ani, prostate, seminal vessicle) and will have no effect or a suppressive effect on LH secretion.
• Compounds with antagonist activity will decrease the weight of one or more of the androgen responsive organs (levator ani, prostate, seminal vesicle) and will have no effect or a reduced suppressive effect on LH secretion.
• CWR22 human prostate tumors were maintained in Balb/c nu/nu nude mice. Tumors were propagated as subcutaneous transplants in adult male nude mice (4-6 weeks old) using tumor fragments obtained from donor mice. Tumor passage occurred every 5-6 weeks.
• the required number of animals needed to detect a meaningful response were pooled at the start of the experiment and each was given a subcutaneous implant of a tumor fragment (-50 mg) with a 13-gauge trocar. Tumors were allowed to grow to approx. 100-200 mg (tumors outside the range were excluded) and animals were evenly distributed to various treatment and control groups. Treatment of each animal was based on individual body weight. Treated animals were checked daily for treatment related toxicity/mortality. Each group of animals was weighed before the initiation of treatment (Wtl) and then again following the last treatment dose (Wt2). The difference in body weight (Wt2-Wtl) provides a measure of treatment-related toxicity.
• Tumor response was determined by measurement of tumors with a caliper twice a week, until the tumors reach a predetermined "target" size of 0.5 gm.
• Tumor response end-point was expressed in terms of tumor growth inhibition (%T/C), defined as the ratio of median tumor weights of the treated tumors (T) to that of the control group (C).
• %T/C tumor growth inhibition
• TNDT Median time (days) for control tumors to reach target size - Median time (days) for control tumors to reach half the target size
• Log cell kill (T-C) ⁇ (3.32 x TVDT)
• TLC thin layer chromatography
• pTSA para-toluenesulfonic acid
• TMSOTf trimethylsilyl trifluoromethane sulfonate
• TEA triethylamine
• n-BuLi n-butyllithium
• rt room temperature
• Ph phenyl
• HMPA hexamethylphosphoramide
• Methyl-TY'-nitro-TV-nitrosoguanidine (7.5 g) was added portionwise to the mixture and stirred for 5 min. The Et 2 O layer was separated and added dropwise to a stirred solution of cts-5-norbornene-e ⁇ z ⁇ io-2,3-dicarboxylic anhydride (3 g, 18.3 mmol) and Pd(OAc) 2 (30 mg) in Et 2 O (60 mL) at 0 °C.
• reaction was then diluted with methylene chloride and washed with sat NaHCO 3 .
• Compound 17A was synthesized as described in L. R. Corwin; D.
• Cis-l,2,3-propenetricarboxylic acid anhydride (5.00 g, 32.1 mmol) was suspended in dichloroethane (30 mL) and freshly cracked cyclopentadiene (4.23 g, 64.2 mmol) was added. The mixture was then heated at 80 »C for 24 h, becoming homogenous after 3 h. Upon cooling to 25 *C, the product precipitated out of solution and was filtered rinsing with 1:1 hexanes/methylene chloride. After drying in ⁇ acuo, 6.9 g of compound 22A was isolated as a white powder. NMR spectroscopy showed only the endo isomer was isolated.
• Compound 31A was synthesized as described in L. R. Corwin; D.
• the resulting white solid was recrystallized twice with a 50/50 mixture of CH 2 C1 2 / hexane and followed by a recrystallization with 100% ethyl acetate to yield 3.2 g of a final white solid which was a 1:10 mixture of compounds 34A & 6A.
• anhydride-like intermediates which can be used in replacement of compounds 1A, 2A, or 7B are, but are not limited to, compounds 3A, 4A, 5A, 8B, 9A, 10A, HA, 12A, 15A, 17A, 22A, 31A, 34A, 43A, and 50A.
• 4-Methyl-3-fluoroaniline (0.010 g, 0.08 mmols) was dissolved in acetic acid (0.400 mL) and transferred to a 1.5 mL conical vial with a septa cap.
• Stock solutions of an additional 95 amines were prepared as described above.
• To each of the above vials was added 0.4 mL (0.12 mmol) of a stock solution of compound 15A in acetic acid.
• the vials were then sealed and heated at 110°C for 10 h. Upon cooling to 25°C, the solutions were transferred by robot to filter tubes with a medium pore frit. Nitrogen was then blown into the tubes to remove the acetic acid.
• LCMS YMC S5 ODS column, 4.6 X 50 mm eluting with 10-90% MeOH/H 2 O over 4 minutes containing 0.1% TFA; 4 mL/min, monitoring at 220 nm.
• LCMS* YMC S5 ODS column, 4.6 X 50 mm eluting with 10-90% MeOH/H 2 O over 2 minutes containing 0.1% TFA; 4 mL/min, monitoring at 220 nm.
• LCMS YMC S5 ODS column, 4.6 X 50 mm eluting with 10-90% MeOH/H 2 O over 4 minutes containing 0.1 % TFA; 4 mL/min, monitoring at 220 nm.
• LCMS* YMC S5 ODS column, 4.6 X 50 mm eluting with 10-90% MeOH7H 2 O over 2 minutes containing 0.1% TFA; 4 mL/min, monitoring at 220 nm.
• LCMS* YMC S5 ODS column, 4.6 X 50 mm eluting with 10-90% MeOH/H 2 O over 2 minutes containing 0.1% TFA; 4 mL/min, monitoring at 220 nm.
• LC YMC S5 ODS column 4.6 x 50 mm eluting with 10-90% MeOH/H 2 O over 4 minutes containing 0.2% phosphoric acid, 4 mL/min, monitoring at 220 nm.
• LCMS YMC S5 ODS column, 4.6 X 50 mm eluting with 10-90% MeOH/H 2 O over 4 minutes containing 0.1% TFA; 4 mL/min, monitoring at 220 nm.
• LCMS* YMC S5 ODS column, 4.6 X 50 mm eluting with 10-90% MeOH/H 2 O over 2 minutes containing 0.1% TFA; 4 mL/min, monitoring at 220 nm.
• LC YMC S5 ODS column 4.6 x 50 mm eluting with 10-90% MeOH/H 2 O over 4 minutes containing 0.2% phosphoric acid, 4 mL/min, monitoring at 220 nm.
• Table 4 The molecular mass of the compounds listed in Table 4, where provided, were determined by MS (ES) by the formula m/z. Table 4
• LCMS* YMC S5 ODS column, 4.6 X 50 mm eluting with 10-90% MeOH/H 2 O over 2 minutes containing 0.1 % TFA; 4 mL/min, monitoring at 220 nm.
• LCMS YMC S5 ODS column, 4.6 X 50 mm eluting with 10-90% MeOH/H 2 O over 4 minutes containing 0.1% TFA; 4 mL/min, monitoring at 220 nm.
• LCMS* YMC S5 ODS column, 4.6 X 50 mm eluting with 10-90% MeOH/H 2 O over 2 minutes containing 0.1% TFA; 4 mL/min, monitoring at 220 nm.
• LCMS YMC S5 ODS column, 4.6 X 50 mm eluting with 10-90% MeOH/H 2 O over 4 minutes containing 0.1% TFA; 4 mL/min, monitoring at 220 nm.
• LCMS YMC S5 ODS column, 4.6 X 50 mm eluting with 10-90% MeOH/T ⁇ 2 O over 4 minutes containing 0.1% TFA; 4 mL/min, monitoring at 220 nm.
• *LCMS YMC S5 ODS column, 4.6 X 50 mm eluting with 10-90% MeOH/H 2 O over 2 minutes containing 0.1% TFA; 4 mL/min, monitoring at 220 nm.
• LC YMC S5 ODS column 4.6 x 50 mm eluting with 10-90% MeOH/H 2 O over 4 minutes containing 0.2% phosphoric acid, 4 mL/min, monitoring at 220 nm.
• LCMS YMC S5 ODS column, 4.6 X 50 mm eluting with 10-90% MeOH/H 2 O over 4 minutes containing 0.1% TFA; 4 mL/min, monitoring at 220 nm.
• *LCMS YMC S5 ODS column, 4.6 X 50 mm eluting with 10-90% MeOH/H 2 O over 2 minutes containing 0.1% TFA; 4 mL/min, monitoring at 220 nm.
• LC YMC S5 ODS column 4.6 x 50 mm eluting with 10-90% MeOH/H 2 O over 4 minutes containing 0.2% phosphoric acid, 4 mL/min, monitoring at 220 nm.
• LC YMC S5 ODS column 4.6 x 50 mm eluting with 10-90% MeOH/H 2 O over 4 minutes containing 0.2% phosphoric acid, 4 mL/min, monitoring at 220 nm.
• LCMS YMC S5 ODS column, 4.6 X 50 mm elutmg with 10-90% MeOH/H 2 O over
• LC YMC S5 ODS column 4.6 x 50 mm eluting with 10-90% MeOH7H 2 O over 4 minutes containing 0.2% phosphoric acid, 4 mL/min, monitoring at 220 nm.
• LCMS YMC S5 ODS column, 4.6 X 50 mm eluting with 10-90% MeOH/H 2 O over 4 minutes containing 0.1% TFA; 4 mL/min, monitoring at 220 nm.
• LCMS YMC S5 ODS column, 4.6 X 50 mm eluting with 10-90% MeOH/H 2 O over 4 minutes containing 0.1 % TFA; 4 mL/min, monitoring at 220 nm.
• *LCMS YMC S5 ODS column, 4.6 X 50 mm eluting with 10-90% MeOH/H 2 O over 2 minutes containing 0.1% TFA; 4 mL/min, monitoring at 220 nm.
• LC YMC S5 ODS column 4.6 x 50 mm eluting with 10-90% MeOH/H 2 O over 4 minutes containing 0.2% phosphoric acid, 4 mL/min, monitoring at 220 nm.
• LCMS YMC S5 ODS column, 4.6 X 50 mm eluting with 10-90% MeOH7H 2 O over 4 minutes containing 0.1 % TFA; 4 mL/min, monitoring at 220 nm.
• *LCMS YMC S5 ODS column, 4.6 X 50 mm eluting with 10-90% MeOH/H 2 O over 2 minutes containing 0.1% TFA; 4 mL/min, monitoring at 220 nm.
• LC YMC S5 ODS column 4.6 x 50 mm eluting with 10-90% MeOH/H 2 O over 4 minutes containing 0.2% phosphoric acid, 4 mL/min, monitoring at 220 nm.
• Example 756 to 763 have the following structure: where G, the compound name, retention time, and the procedure employed, are set forth in Table 15 (L is a bond for the compounds of Table 15).
• LCMS YMC S5 ODS column, 4.6 X 50 mm eluting with 10-90% MeOH/H 2 O over 4 minutes containing 0.1% TFA; 4 mL/min, monitoring at 220 nm.
• *LCMS YMC S5 ODS column, 4.6 X 50 mm eluting with 10-90% MeOH/H 2 O over 2 minutes containing 0.1% TFA; 4 mL/min, monitoring at 220 nm.
• LC YMC S5 ODS column 4.6 x 50 mm eluting with 10-90% MeOH/H 2 O over 4 minutes containing 0.2% phosphoric acid, 4 mL/min, monitoring at 220 nm.
• the molecular mass of the compounds listed in Table 15, where provided, were determined by MS (ES) by the formula m/z.

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