Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D209/00—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom
  • C07D209/02—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom condensed with one carbocyclic ring
  • C07D209/04—Indoles; Hydrogenated indoles
  • C07D209/30—Indoles; Hydrogenated indoles with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to carbon atoms of the hetero ring
  • C07D209/32—Oxygen atoms
  • C07D209/34—Oxygen atoms in position 2
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
  • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
  • C07D401/04—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings directly linked by a ring-member-to-ring-member bond
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/0012—Galenical forms characterised by the site of application
  • A61K9/0014—Skin, i.e. galenical aspects of topical compositions
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/0012—Galenical forms characterised by the site of application
  • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/0012—Galenical forms characterised by the site of application
  • A61K9/007—Pulmonary tract; Aromatherapy
  • A61K9/0073—Sprays or powders for inhalation; Aerolised or nebulised preparations generated by other means than thermal energy
  • A61K9/008—Sprays or powders for inhalation; Aerolised or nebulised preparations generated by other means than thermal energy comprising drug dissolved or suspended in liquid propellant for inhalation via a pressurized metered dose inhaler [MDI]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/20—Pills, tablets, discs, rods
  • A61K9/2004—Excipients; Inactive ingredients
  • A61K9/2022—Organic macromolecular compounds
  • A61K9/205—Polysaccharides, e.g. alginate, gums; Cyclodextrin
  • A61K9/2054—Cellulose; Cellulose derivatives, e.g. hydroxypropyl methylcellulose
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
  • A61K9/4841—Filling excipients; Inactive ingredients
  • A61K9/4866—Organic macromolecular compounds
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents

Definitions

• ERa directly or indirectly has been the miniaturized strategy for treating ERa+ breast cancers.
• Such strategies are termed as endocrine therapies, as they seek to block the canonical functions of ERa in induction of cell growth and proliferation.
• endocrine therapy typically with tamoxifen, fulvestrant, anastrozole, and others
• drugresistance and tumor regrowth is inevitable and represents a major clinical challenge.
• CDK4/6 or PI3KCA inhibitors drug-resistant, lethal disease is still pervasive.
• These resistant tumors typically maintain their ERa overexpression, suggesting that drugs leveraging ERa, especially those acting through new mechanisms, may provide a meaningful clinical benefit to a diverse patient population that is typically considered drug resistant and is not well-served by other therapeutic options.
• the traditional unfolded protein response is characterized by signaling in response to insufficient protein-folding capacity.
• UPR The traditional unfolded protein response
• a- UPR the anticipatory UPR
• This a-UPR is a conserved mechanism across hormone-specific receptors including ERa, androgen receptor, epidermal growth factor receptor, and others; induction of the a-UPR can be considered as a non- canonical function of ERa.
• the a-UPR is cytoprotective and has been correlated with drugresistance in a variety of settings, sustained activation is toxic to cancer cells and represents an opportunity to convert a tumor protective pathway into a potent and selective anticancer strategy.
• ErSO a compound that hyperactivates the ERa- dependent a-UPR in ERa+ cancer cells leading to eradication of ERa+ breast tumors in multiple mouse models.
• ErSO maintains activity against breast cancer cell lines that contain mutations in ERa (Y537S and D538G) representing major clinical resistance mechanisms to endocrine therapy.
• the selectivity of ErSO for ERa+ cells in culture at short incubation times (e.g. 6 or 24 hours) is impressive, with >350 fold difference in cellular IC50 between ERa+ and ERa- cancer cell lines.
• knock-in of ERa into ERa-negative MDA-MB-231 triple-negative breast cancer cells dramatically sensitizes these cells to ErSO both in cell culture and in xenograft models.
• the ability of ErSO to leverage this non-canonical activity of ERa likely enables ErSO to induce tumor regressions, contrasting the cytostatic activity typically observed with endocrine therapy.
• Estrogen receptor alpha-positive (ERa+) breast cancers are the most common type of this disease, with >200,000 new cases diagnosed annually in the United States.
• ERa drives tumor growth and disease progression, and thus targeting ERa directly with ERa antagonists and degraders (e.g., tamoxifen, fulvestrant) or indirectly with aromatase inhibitors has been a successful therapeutic strategy, with significant gains in overall survival for these patients.
• ERa antagonists and degraders e.g., tamoxifen, fulvestrant
• aromatase inhibitors has been a successful therapeutic strategy, with significant gains in overall survival for these patients.
• such treatments are rarely curative, and patients typically succumb to metastatic, drug-resistant (through ERa mutation and other mechanisms) disease.
• ErSO The small molecule, ErSO, which induces potent ERa-dependent death of ERa+ breast cancer cells through a mechanism distinct from clinically approved drugs that target ERa, namely hyperactivation of the anticipatory unfolded protein response.
• ErSO has remarkable activity in multiple mouse models of ERa+ breast cancer, in many cases inducing complete tumor eradication. Importantly, ErSO is potent and effective even when evaluated in breast cancer cell lines and preclinical tumor models that are resistant to endocrine therapy via mutated ERa. While ErSO has tremendous promise as a new drug to target ERa+ tumors, at high concentrations and long incubation times it does have some effects on ERa-negative (ERa-) cells in culture.
• ErSO-DFP a compound that maintains outstanding efficacy for inducing death of ERa+ breast cancer cells in cell culture, has markedly enhanced selectivity for ERa+ cancer cells over ERa- cancer cells, increased in vivo tolerability, and a striking antitumor effect in vivo, inducing dramatic regression of large ERa+ tumors in a murine orthotopic xenograft model.
• ErSO-DFP and related molecules represent an intriguing new class of compounds for the treatment of ERa+ cancers.
• the invention therefore provides a compound of Formula I: wherein
• X is O, S, or NR A ;
• Y is O, S, or NR A ; each R A is independently H, alkyl, or a nitrogen protecting group;
• R 1 is trifluoromethyl, trifluoromethoxy, alkyl, cycloalkyl, heterocycle, aryl, heteroaryl, halo, -OR B , -SR B , or -N(R B ) 2
• R 2 , R 3 and R 4 are each independently H, trifluoromethyl, alkyl, cycloalkyl, heterocycle, aryl, heteroaryl, halo, -OR B , -SR B , or -N(R B )2
• each R B is independently H, trifluoromethyl, alkyl, or a heteroatom protecting group
• each R c is independently H, trifluoromethyl, alkyl, or a heteroatom protecting group
• n is 0, 1, 2, 3, 4, or 5; and
• Z is a 3-8 membered nitrogen-containing heterocycle optionally substituted with one or more substituents; wherein each alkyl, cycloalkyl, heterocycle, aryl, and heteroaryl is optionally substituted with one or more substituents; or a salt thereof.
• the alkyl, cycloalkyl, heterocycle, aryl, and heteroaryl can be substituted one or more substituents selected from OH, halo, nitro, alkyl, and group of substituents as defined for substituted groups discussed herein below.
• an alkyl can be substituted with two or three halo groups to provide dihalo-alkyl or a trihalo-alkyl (such as trifluoromethyl), respectively.
• any alkyl group or substituent of Formula I can be substituted to result in a trifluoromethyl group.
• X is NH and Y is O.
• R 1 is CF3 or Me.
• R 2 , R 3 and R 4 are each independently H or halo.
• R x is OH.
• n is 1, 2, or 3.
• R x is OH and n is 1, 2, or 3.
• Various embodiments include compounds of Formula I wherein R x is OH, n is 1 or 2, and the R x groups are located at the meta or para positions of the phenyl ring to which they are attached, or wherein R x is OH, n is 1 , and the R x group is located at the para position of the phenyl ring to which it is attached.
• Z is a 3-. 4-, 5-, 6-, 7-, or 8-membered nitrogen-containing heterocycle attached to Formula I by the nitrogen atom of the heterocycle.
• the heterocycle can be substituted by one or two halo groups.
• the heterocycle is piperidine substituted by one or two fluoro groups at the 3-position of the piperidine ring.
• the heterocycle is piperidine, morpholine, piperazine, pyrrolidine, azepane, aziridine, azetidine, or azocane each optionally substituted with one to six substituents.
• Z is 4,4-difluoropiperidinyl, 3,3-difluoropyrrolidinyl, or 3,3,4,4-tetrafluoropyrrolidinyl.
• the compound of Formula I is levorotatory. In alternate embodiments, the compound is of Formula I dextrorotatory.
• X is NH; Y is O; R 1 is CF3 or Me; R 2 , R 3 and R 4 are each independently H or halo; R x is OH, and n is 1, 2, or 3.
• m is 2 and each R z is halo.
• each halo is fluoro.
• the compound of Formula I, II, or III is 2, 24, or 26:
• the compound of Formula I or II is ErSO-DFP:
• the invention also provides a pharmaceutical composition comprising the compound of
• the invention further provides a method of treating an ERoc positive cancer comprising administering to subject having an ERoc positive cancer a therapeutically effective amount of a compound of Formula I or II, thereby treating the ERoc positive cancer.
• a method of treating an ERoc positive cancer comprising administering to subject having an ERoc positive cancer a therapeutically effective amount of a compound of Formula I or II, thereby treating the ERoc positive cancer.
• ERoc positive cancer is breast cancer, ovarian cancer, uterine cancer, cervical carcinoma, or endometrial cancer.
• the compound is ErSO-DFP.
• cancer can be, for example, breast cancer, ovarian cancer, uterine cancer, cervical carcinoma, endometrial cancer, lung cancer, pancreatic cancer, prostate cancer, or colon cancer.
• the invention thus provides novel compounds of Formulas I-III, intermediates for the synthesis of compounds of Formulas I-III, as well as methods of preparing compounds of Formulas I- III.
• the invention also provides compounds of Formulas I-III that are useful as intermediates for the synthesis of other useful compounds.
• the invention provides for the use of compounds of Formulas I-III for treating of cancer in a mammal, such as a human.
• the compound administered can be in the form of a composition that includes a pharmaceutically acceptable diluent, excipient, or carrier.
• ErSO-DFP is the active anticancer agent with superior LipE relative to its progenitor, ErSO.
• A Biological activity of racemic 2 (( ⁇ )-2), ErSO-DFP, and (S)-2 against MCF-7 cells incubated with compound for 24 hours. Cell viability was assessed by alamar blue fluorescence. Raptinal (100 pM) was used as the 100% dead control. Data is shown as mean ⁇ s.e.m.; n > 2 independent replicates.
• B Lipophilic efficiencies of ErSO and ErSO-DFP. Increases in LipE are often associated with more ‘drug-like’ character.
• ErSO-DFP potently kills ERa-positive cancer cell lines with a wider selectivity window than ErSO.
• To determine IC50 cells were incubated with ErSO for 24 (A) or 72 (B) hours. Viability was measured via alamar blue fluorescence, with raptinal (100 pM) used as the 100% dead control. Data is plotted/expressed as mean ⁇ s.e.m.; n > 3 independent replicates. Black double-sided arrow demonstrates the therapeutic window between IC50 MCF-7 and select ERa-negative cell lines (Table 3).
• ErSO-DFP activates the a-UPR in a manner similar to ErSO.
• MCF-7 cells were incubated with compounds as indicated, harvested, and western blot analysis of key proteins for a- UPR activation conducted. For dose-dependence (blots on the right), cells were incubated for 4 hours with compound (A). Quantification was calculated using ImageJ and the actin bands as loading controls. Data is plotted as mean ⁇ s.e.m (B). Blots are representative images of 3 independent replicates.
• FIG. 4 ErSO-DFP achieves biologically relevant concentrations when dosed intravenously in mice.
• A Summary of pharmacokinetic (PK) experiment for ErSO-DFP. Data plotted as mean ⁇ s.e.m.; n > 3 mice per time point.
• ErSO-DFP is well-tolerated in vivo and is blood brain-barrier penetrant.
• ErSO-DFP treatment leads to profound tumor regression at a low dose, which coupled with the high MTD, suggests it as a compound with a large therapeutic index.
• A ErSO-DFP regresses MCF-7 tumors in a similar manner as ErSO.
• Figure 7 Graphs showing pharmacokinetics of ErSO at 20 mg/kg I.V (A), and ErSO-DFP at 20 mg/kg I.V (B).
• ErSO appears to have a wide therapeutic window; it is tolerated at oral doses greater than 150 mg/kg in mice and canines. However, there are traits of ErSO that, if altered, might lead to an even more promising drug.
• references in the specification to "one embodiment”, “an embodiment”, etc., indicate that the embodiment described may include a particular aspect, feature, structure, moiety, or characteristic, but not every embodiment necessarily includes that aspect, feature, structure, moiety, or characteristic. Moreover, such phrases may, but do not necessarily, refer to the same embodiment referred to in other portions of the specification. Further, when a particular aspect, feature, structure, moiety, or characteristic is described in connection with an embodiment, it is within the knowledge of one skilled in the art to affect or connect such aspect, feature, structure, moiety, or characteristic with other embodiments, whether or not explicitly described.
• ranges recited herein also encompass any and all possible subranges and combinations of sub-ranges thereof, as well as the individual values making up the range, particularly integer values. It is therefore understood that each unit between two particular units are also disclosed. For example, if 10 to 15 is disclosed, then 11, 12, 13, and 14 are also disclosed, individually, and as part of a range.
• a recited range e.g., weight percentages or carbon groups
• any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, or tenths.
• each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc.
• all language such as “up to”, “at least”, “greater than”, “less than”, “more than”, “or more”, and the like include the number recited and such terms refer to ranges that can be subsequently broken down into sub-ranges as discussed above.
• all ratios recited herein also include all sub-ratios falling within the broader ratio. Accordingly, specific values recited for radicals, substituents, and ranges, are for illustration only; they do not exclude other defined values or other values within defined ranges for radicals and substituents. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint.
• contacting refers to the act of touching, making contact, or of bringing to immediate or close proximity, including at the cellular or molecular level, for example, to bring about a physiological reaction, a chemical reaction, or a physical change, e.g., in a solution, in a reaction mixture, in vitro, or in vivo.
• an “effective amount” refers to an amount effective to treat a disease, disorder, and/or condition, or to bring about a recited effect.
• an effective amount can be an amount effective to reduce the progression or severity of the condition or symptoms being treated. Determination of a therapeutically effective amount is well within the capacity of persons skilled in the art.
• the term "effective amount” is intended to include an amount of a compound described herein, or an amount of a combination of compounds described herein, e.g., that is effective to treat or prevent a disease or disorder, or to treat the symptoms of the disease or disorder, in a host.
• an “effective amount” generally means an amount that provides the desired effect.
• an “effective amount” or “therapeutically effective amount,” as used herein, refer to a sufficient amount of an agent or a composition or combination of compositions being administered which will relieve to some extent one or more of the symptoms of the disease or condition being treated. The result can be reduction and/or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system.
• an “effective amount” for therapeutic uses is the amount of the composition comprising a compound as disclosed herein required to provide a clinically significant decrease in disease symptoms.
• An appropriate "effective" amount in any individual case may be determined using techniques, such as a dose escalation study. The dose could be administered in one or more administrations.
• the precise determination of what would be considered an effective dose may be based on factors individual to each patient, including, but not limited to, the patient's age, size, type or extent of disease, stage of the disease, route of administration of the compositions, the type or extent of supplemental therapy used, ongoing disease process and type of treatment desired (e.g., aggressive vs. conventional treatment).
• treating include (i) preventing a disease, pathologic or medical condition from occurring (e.g., prophylaxis); (ii) inhibiting the disease, pathologic or medical condition or arresting its development; (iii) relieving the disease, pathologic or medical condition; and/or (iv) diminishing symptoms associated with the disease, pathologic or medical condition.
• the terms “treat”, “treatment”, and “treating” can extend to prophylaxis and can include prevent, prevention, preventing, lowering, stopping or reversing the progression or severity of the condition or symptoms being treated.
• treatment can include medical, therapeutic, and/or prophylactic administration, as appropriate.
• subject or “patient” means an individual having symptoms of, or at risk for, a disease or other malignancy.
• a patient may be human or non-human and may include, for example, animal strains or species used as “model systems” for research purposes, such a mouse model as described herein.
• patient may include either adults or juveniles (e.g., children).
• patient may mean any living organism, preferably a mammal e.g., human or non-human) that may benefit from the administration of compositions contemplated herein.
• mammals include, but are not limited to, any member of the Mammalian class: humans, non-human primates such as chimpanzees, and other apes and monkey species; farm animals such as cattle, horses, sheep, goats, swine; domestic animals such as rabbits, dogs, and cats; laboratory animals including rodents, such as rats, mice and guinea pigs, and the like.
• non-mammals include, but are not limited to, birds, fish and the like.
• the mammal is a human.
• the terms “providing”, “administering,” “introducing,” are used interchangeably herein and refer to the placement of a compound of the disclosure into a subject by a method or route that results in at least partial localization of the compound to a desired site.
• the compound can be administered by any appropriate route that results in delivery to a desired location in the subject.
• the compounds and compositions described herein may be administered with additional compositions to prolong stability and activity of the compositions, or in combination with other therapeutic drugs.
• the terms “inhibit”, “inhibiting”, and “inhibition” refer to the slowing, halting, or reversing the growth or progression of a disease, infection, condition, or group of cells.
• the inhibition can be greater than about 20%, 40%, 60%, 80%, 90%, 95%, or 99%, for example, compared to the growth or progression that occurs in the absence of the treatment or contacting.
• substantially is a broad term and is used in its ordinary sense, including, without limitation, being largely but not necessarily wholly that which is specified.
• the term could refer to a numerical value that may not be 100% the full numerical value.
• the full numerical value may be less by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, or about 20%.
• halo or halide refers to fluoro, chloro, bromo, or iodo.
• halogen refers to fluorine, chlorine, bromine, and iodine.
• alkyl refers to a branched or unbranched hydrocarbon having, for example, from 1-20 carbon atoms, and often 1-12, 1-10, 1-8, 1-6, or 1-4 carbon atoms. As used herein, the term “alkyl” also encompasses a “cycloalkyl”, defined below.
• Examples include, but are not limited to, methyl, ethyl, 1-propyl, 2-propyl (Ao-propyl), 1-butyl, 2-methyl-l -propyl (isobutyl), 2-butyl (secbutyl), 2-methyl-2-propyl (t-butyl), 1-pentyl, 2-pentyl, 3-pentyl, 2-methyl-2-butyl, 3-methyl-2-butyl,
• the alkyl can be unsubstituted or substituted, for example, with a substituent described below or otherwise described herein.
• the alkyl can also be optionally partially or fully unsaturated. As such, the recitation of an alkyl group can include an alkenyl group or an alkynyl group.
• the alkyl can be a monovalent hydrocarbon radical, as described and exemplified above, or it can be a divalent hydrocarbon radical (i.e., an alkylene).
• alkylene is an alkyl group having two free valences at carbon or two different carbon atoms of a carbon chain.
• alkenylene and alkynylene are respectively an alkene and an alkyne having two free valences at two different carbon atoms.
• cycloalkyl refers to cyclic alkyl groups of, for example, from 3 to 10 carbon atoms having a single cyclic ring or multiple condensed rings. Cycloalkyl groups include, by way of example, single ring structures such as cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, and the like, or multiple ring structures such as adamantyl, and the like.
• the cycloalkyl can be unsubstituted or substituted.
• the cycloalkyl group can be monovalent or divalent and can be optionally substituted as described for alkyl groups.
• the cycloalkyl group can optionally include one or more cites of unsaturation, for example, the cycloalkyl group can include one or more carbon-carbon double bonds, such as, for example, 1 -cyclopent- 1-enyl, l-cyclopent-2-enyl, l-cyclopent-3-enyl, cyclohexyl, 1- cyclohex-l-enyl, l-cyclohex-2-enyl, l-cyclohex-3-enyl, and the like.
• heterocycloalkyl or “heterocycle” refers to a saturated or partially saturated monocyclic, bicyclic, or polycyclic ring containing at least one heteroatom selected from nitrogen, sulfur, oxygen, preferably from 1 to 3 heteroatoms in at least one ring.
• Each ring is preferably 3 to 10 membered, more preferably 4 to 7 membered.
• heterocycloalkyl groups examples include pyrrolidyl, tetrahydrofuryl, tetrahydrothiofuranyl, piperidyl, piperazyl, tetrahydropyranyl, morpholino, 1,3-diazapane, 1 ,4-diazapane, 1 ,4-oxazepane, 1 ,4-oxathiapane, and the like.
• the heterocycle can be substituted with one or more substituents.
• aromatic refers to either an aryl or heteroaryl group or substituent described herein. Additionally, an aromatic moiety may be a bisaromatic moiety, a trisaromatic moiety, and so on. A bisaromatic moiety has a single bond between two aromatic moieties such as, but not limited to, biphenyl, or bipyridine. Similarly, a trisaromatic moiety has a single bond between each aromatic moiety.
• aryl refers to an aromatic hydrocarbon group derived from the removal of at least one hydrogen atom from a single carbon atom of a parent aromatic ring system.
• the radical attachment site can be at a saturated or unsaturated carbon atom of the parent ring system.
• the aryl group can have from 6 to 30 carbon atoms, for example, about 6-10 carbon atoms.
• the aryl group can have a single ring (e.g., phenyl) or multiple condensed (fused) rings, wherein at least one ring is aromatic (e.g., naphthyl, dihydrophenanthrenyl, fluorenyl, or anthryl).
• Typical aryl groups include, but are not limited to, radicals derived from benzene, naphthalene, anthracene, biphenyl, and the like.
• the aryl can be unsubstituted or optionally substituted with a substituent described below.
• heteroaryl refers to a monocyclic, bicyclic, or tricyclic ring system containing one, two, or three aromatic rings and containing at least one nitrogen, oxygen, or sulfur atom in an aromatic ring.
• the heteroaryl can be unsubstituted or substituted, for example, with one or more, and in particular one to three, substituents, as described in the definition of "substituted”.
• Typical heteroaryl groups contain 2-20 carbon atoms in the ring skeleton in addition to the one or more heteroatoms, wherein the ring skeleton comprises a 5-membered ring, a 6-membered ring, two 5- membered rings, two 6-membered rings, or a 5-membered ring fused to a 6-membered ring.
• heteroaryl groups include, but are not limited to, 2H-pyrrolyl, 3H-indolyl, 4H- quinolizinyl, acridinyl, benzo[b]thienyl, benzothiazolyl, [3-carbolinyl, carbazolyl, chromenyl, cinnolinyl, dibenzo[b,d]furanyl, furazanyl, furyl, imidazolyl, imidizolyl, indazolyl, indolisinyl, indolyl, isobenzofuranyl, isoindolyl, isoquinolyl, isothiazolyl, isoxazolyl, naphthyridinyl, oxazolyl, perimidinyl, phenanthridinyl, phenanthrolinyl, phenarsazinyl, phenazinyl, phenothiazinyl, phenoxathiinyl
• heteroaryl denotes a monocyclic aromatic ring containing five or six ring atoms containing carbon and 1, 2, 3, or 4 heteroatoms independently selected from non-peroxide oxygen, sulfur, and N(Z) wherein Z is absent or is H, O, alkyl, aryl, or (Ci-C6)alkylaryl.
• heteroaryl denotes an ortho-fused bicyclic heterocycle of about eight to ten ring atoms derived therefrom, particularly a benz-derivative or one derived by fusing a propylene, trimethylene, or tetramethylene diradical thereto.
• substituted or “substituent” is intended to indicate that one or more (for example, in various embodiments, 1-10; in other embodiments, 1-6; in some embodiments 1, 2, 3, 4, or 5; in certain embodiments, 1, 2, or 3; and in other embodiments, 1 or 2) hydrogens on the group indicated in the expression using “substituted” (or “substituent”) is replaced with a selection from the indicated group(s), or with a suitable group known to those of skill in the art, provided that the indicated atom’s normal valency is not exceeded, and that the substitution results in a stable compound.
• Suitable indicated groups include, e.g., alkyl, alkenyl, alkynyl, alkoxy, haloalkyl, hydroxyalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, alkanoyl, alkoxycarbonyl, amino, alkylamino, dialkylamino, carboxyalkyl, alkylthio, alkylsulfinyl, and alkylsulfonyl.
• Substituents of the indicated groups can be those recited in a specific list of substituents described herein, or as one of skill in the art would recognize, can be one or more substituents selected from alkyl, alkenyl, alkynyl, alkoxy, halo, haloalkyl, hydroxy, hydroxyalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, alkanoyl, alkoxycarbonyl, amino, alkylamino, dialkylamino, trifluoromethylthio, difluoromethyl, acylamino, nitro, trifluoromethyl, trifluoromethoxy, carboxy, carboxyalkyl, keto, thioxo, alkylthio, alkylsulfinyl, alkylsulfonyl, and cyano.
• Suitable substituents of indicated groups can be bonded to a substituted carbon atom include F, Cl, Br, I, OR’, OC(O)N(R') 2 , CN, CF 3 , OCF 3 , R’, O, S, C(O), S(O), methylenedioxy, ethylenedioxy, N(R') 2 , SR', SOR', SO2R', SO2N(R')2, SO 3 R', C(O)R', C(O)C(O)R', C(O)CH 2 C(O)R’, C(S)R', C(O)OR’, OC(O)R’, C(O)N(R') 2 , OC(O)N(R') 2 , C(S)N(R') 2 , (CH 2 )O- 2 NHC(O)R’, N(R')N(R')C(O)R', N(R')N(O)C(O
• a substituent When a substituent is monovalent, such as, for example, F or Cl, it is bonded to the atom it is substituting by a single bond.
• the compounds of the invention may contain asymmetric or chiral centers, and therefore exist in different stereoisomeric forms. It is intended that all stereoisomeric forms of the compounds of the invention, including but not limited to, diastereomers, enantiomers and atropisomers, as well as mixtures thereof, such as racemic mixtures, which form part of the present invention.
• a specific stereoisomer may also be referred to as an enantiomer, and a mixture of such isomers is often called an enantiomeric mixture.
• a 50:50 mixture of enantiomers is referred to as a racemic mixture or a racemate (defined below), which may occur where there has been no stereoselection or stereospecificity in a chemical reaction or process.
• racemic mixture and “racemate” refer to an equimolar mixture of two enantiomeric species, devoid of optical activity.
• enantiomerically enriched refers to mixtures that have one enantiomer present to a greater extent than another. Reactions that provide one enantiomer present to a greater extent than another would therefore be “enantioselective” (or demonstrate “enantioselectivity”).
• the term “enantiomerically enriched” refers to a mixture having at least about 2% ee; in another embodiment of the invention, the term “enantiomerically enriched” refers to a mixture having at least about 5% ee; in another embodiment of the invention, the term “enantiomerically enriched” refers to a mixture having at least about 20%; in another embodiment of the invention, the term “enantiomerically enriched” refers to a mixture having at least about 50%; in another embodiment of the invention, the term “enantiomerically enriched” refers to a mixture having at least about 80%; in another embodiment of the invention, the term “enantiomerically enriched” refers to a mixture having at least about 90%; in another embodiment of the invention, the term “enantiomerically enriched” refers to a mixture having at least about 95%; in another embodiment of the invention, the term “enantiomerically enriched” refers to a mixture having at least about 98%; in another embodiment of
• enantiomerically enriched includes enantiomerically pure mixtures which are mixtures that are substantially free of the species of the opposite optical activity, or one enantiomer is present in very low quantities, for example, 0.01%, 0.001% or 0.0001%.
• IC50 is generally defined as the concentration required to kill 50% of the cells in 24 hours.
• ErSO-DFP refers to the compound (7?)-3-(4,4-difluoropiperidin-l-yl)-3-(4- hydroxyphenyl)-7-(trifluoromethyl)indolin-2-one.
• variable element of Formula I or Formula II when a variable element of Formula I or Formula II is optionally substituted, it can be substituted with one or more substituents, such as one or more of the substituents described in the definition of substituents herein.
• Alkyl optionally substituted with one or more substituents can be, for example, alkyl substituted with one to six substituents, one to five substituents, one to four substituents, one to three substituents, one or two substituents, or one substituent.
• Alkyl optionally substituted with one or more substituents includes, for example, halo- substituted alkyl groups such as CF3, CHF2, CH2F, CH2CF3, CF2CH3, or CF2CF3.
• the compound of Formula I is the ( ⁇ -enantiomer. In other embodiments, the compound of Formula I is the (7?)-enantiomer. Eikewise, in some embodiments, the compound of Formula II is the (5) -enantiomer and in other embodiments, the compound of Formula II is the (2?) -enantiomer. If substituents on Formula I or Formula II result in a compound having more than one stereocenter, the (R) and (S) designations described in this paragraph refer to the location of the stereocenter of the compound corresponding to the stereocenter found in ErSO- DFP.
• the compound is (7?)-3-(4,4-difluoropiperidin-l-yl)-3-(4- hydroxyphenyl)-7-(trifluoromethyl)indolin-2-one. In some embodiments, the compound is (S)-3-(4,4- difluoropiperidin-l-yl)-3-(4-hydroxyphenyl)-7-(trifluoromethyl)indolin-2-one.
• the compound is 3-(4-hydroxyphenyl)-3-(piperidin-l-yl)-7-(trifluoromethyl)indolin-2- one; 3-(4,4-difluoro-3-methylpiperidin-l-yl)-3-(4-hydroxyphenyl)-7-(trifluoromethyl)indolin-2-one; 3-(4-hydroxyphenyl)-3-morpholino-7-(trifluoromethyl)indolin-2-one; 3-(azepan-l-yl)-3-(4- hydroxyphenyl)-7-(trifluoromethyl)indolin-2-one; 3-(4-hydroxyphenyl)-3-(piperazin-l-yl)-7- (trifluoromethyl)indolin-2-one; 3-(4-hydroxyphenyl)-3-(4-hydroxypiperidin-l-yl)-7- (trifluoromethyl)indolin-2-one; 3-(4-hydroxyphenyl)-3-(4-hydroxyphenyl)-3-
• the compound is (R)-3-(3,3-difluoropyrrolidin-l-yl)-3-(4- hydroxyphenyl)-7-(trifluoromethyl)indolin-2-one or (7?)-3-(4-hydroxyphenyl)-3-(3, 3,4,4- tetrafluoropyrrolidin-l-yl)-7-(trifluoromethyl)indolin-2-one.
• the compound of Formula I and Formula II can kill or inhibit the growth of cancer cells by hyperactivation of the unfolded protein response (UPR) in the endoplasmic reticulum.
• the cancer cells or resulting tumor can be ERoc positive.
• the compounds are cytotoxic.
• the cancer cells are breast cancer cells, ovarian cancer cells, endometrial cancer cells, or other EP positive cancers known in the art.
• the compound can be administered orally, by injection, subcutaneously, sublingually, rectally, by infusion, intravenously, or by other methods known to those of skill in the art.
• ErSO is considered quite lipophilic with a clogD7.4 of 6.44. While lipophilicity can lead to entropic and enthalpic gains for target binding, molecules that are too hydrophobic often have poor “drug-like” properties (e.g. high clearance, inhibition of off-targets, poor oral bioavailability and the propensity to inhibit key safety ion channels like hERG). Lipophilicity was envisioned as a parameter ripe for modulation through systematic changes to Ring A/B of ErSO (Chart 1).
• cLogD7.4 cellular potency and selectivity (that is, differential effects on ERa+ and ERa- cancer cells), and the lipophilic efficiencies (LipE) were prioritized for the identification of promising derivatives.
• LipE lipophilic efficiencies
• a :p a and cLogD7.4 was calculated using ChemAxon MarvinSketch.
• b p/fa is reported for Ring A acidic protons.
• c To determine IC50, MCF-7 cells were incubated with compound for 24 hours and viability measured via Alamar blue fluorescence. Raptinal (100 pM) was used as the 100% dead control. Data is shown as mean ⁇ s.e.m.; n > 2 independent replicates.
• z'Pr isopropyl.
• n-BuLi n-butyllithium
• TBS tertbutyldimethylsilyl
• SOC12 thionyl chloride
• TBAF tetrabutylammonium fluoride
• THF tetrahydrofuran
• DMF dimethylformamide
• ErSO-DFP is a potent ERa-dependent anticancer compound
• Compound 2 shows significant reduction in lipophilicity relative to ErSO (cLogD?. ⁇ 4.37 vs 6.44 respectively) while maintaining activity against MCF-7 cells, warranting in-depth evaluation.
• Enantiomers of 2 were separated via preparative chiral chromatography and their absolute configurations determined by X-ray crystallography (Chart 2).
• the (R)-enantiomer ((Zf)-2, coined ErSO-DFP) is the active chemical species, approximately twice as potent as the racemic compound; the opposite enantiomer, (S)-2, is devoid of any activity against MCF-7 cells ( Figure 2B).
• LipE can be a powerful parameter to conveniently track potency and lipophilicity during drug development campaigns.
• ErSO-DFP has >2.5 fold increase in LipE when compared to ErSO, a shift that is isopotent and a direct result of the decreased lipophilic nature of ErSO-DFP (Figure 2C). Chart 2.
• ErSO-DFP is the active anticancer agent with superior LipE relative to its progenitor, ErSO. Chemical structures of ErSO-DFP and (S)-2 as confirmed by X-Ray crystallography. Physicochemical parameters for ErSO-DFP calculated via ChemAxon MarvinSketch.
• ErSO-DFP has potent activity, similar to ErSO, against other ERa+ breast cancer cell lines, including the T47D cell line and its therapy-resistant, ERa-mutant versions, T47D-ERaY537S (TYS) and T47D-ERaD538G (TDG) (Table 3). Most critically, evaluation of these compounds against ERa- cell lines shows that ErSO-DFP has notably less ERa-independent activity as compared to ErSO; this effect can be seen in Table 3 and most starkly in the dose response curves in Figure 3.
• ErSO-DFP is inactive (ICso>25 pM) in these cell lines at 24 and 72 hours and IC50 curves bottom out near 0% cell death in most cases ( Figure 3).
• the IC50 for ErSO shifts from 11 pM to 0.26 pM and effects bottom out at -38%.
• the IC50 for ErSO-DFP is 55 pM at both 24 and 72 hours, and effects approach 0% at lower concentrations of compound.
• IC50 cancer cells were incubated with compound for 24 or 72 hours and viability measured via Alamar blue fluorescence. Raptinal (100 pM) was used as the 100% dead control. Data is shown as mean ⁇ s.e.m.; n > 3 independent replicates.
• a :cLogD7.4 was calculated using ChemAxon MarvinSketch.
• b Average Fold Change between 24/72 hr IC50 values for MCF-7 and ERa negative cell line.
• ErSO-DFP activates the anticipatory unfolded protein response (a-UPR) in a manner similar to ErSO.
• a-UPR anticipatory unfolded protein response
• western blot analysis of key proteins that are consistent with a-UPR activation (appearance of P-AMPK and P-EIF2a, cleavage of ATF6a P 9o) were conducted.
• ErSO-DFP activates the a-UPR in a time- and concentration-dependent manner similar to ErSO ( Figure 4). No activation of the a-UPR is seen with the inactive enantiomer, compound (S)-2.
• Tetrafluorinated pyrrolidine derivative (R)-26 is the most potent compound with IC50 values of 4-5 nM.
• the opposite enantiomer, compound (S)-26 is inactive against MCF-7 cells, again demonstrating that only one enantiomer in this compound class possesses anticancer activity.
• Potent derivatives ((Z?)-23, (7?)-24, (7?)-276) were further evaluated for their activity against ERa- cancer cell lines MDA-MB-231, HCT-116, and HT-29. Consistent with ErSO-DFP data, compounds (7?)-23, (7?)-24, and (R)-26 have double-digit micromolar potencies against these cell lines with 24- and 72- hour compound incubations.
• Compound (7?) -26 has the largest fold change (> 3500) and represents the most potent of the fluorinated nitrogen heterocycle containing derivatives. All compounds in this set (ErSO-DFP, (Jf)-23, (Jf)-24, and (7?) -26) have significant gains in LipE driven by their increased polarity and nanomolar potencies.
• ErSO-DFP Due to its lack of activity against ERa- cells in culture (IC50 > 25 pM in all ERa- cell lines and time points) and its readily commercially available starting material (i.e., 4,4-difluoropiperidine hydrochloride), ErSO-DFP’ s in vivo profile was further investigated. ErSO-DFP has significant alterations in hydrophilicity and LipE relative to ErSO (Chart 1, Figure 2C). The pharmacokinetic (PK) profile in mice and tolerability in mice and rats was investigated for ErSO-DFP. PK assessments revealed that ErSO-DFP (administered intravenously) achieves biologically-relevant concentrations (Figure 5B), although ErSO-DFP has poor oral availability in mice (F%: 6%).
• This low F% may be the result of limited acid stability of ErSO-DFP.
• ErSO-DFP and compounds (R)-23. (R)-24, (R)-26 had limited stability in SGF with half-lives ranging from 10-77 min. This is in contrast to ErSO’s observed ( Figure 5C, Table 5) half-life of >2 hours, consistent with ErSO’s %F of 47% in mice.
• the acid instability of ErSO-DFP may be the result of acid-promoted elimination, a reactivity that is utilized in Friedel-Craft-type reactions to construct 3,3-biaryl derivatives (Scheme 1).
• Chart 3 Summary of reaction of compound 2 with trifilic acid and phenol.
• mice Regardless of its poor oral bioavailability, a head-to-head PK experiment in mice (dosed I.V.) shows that ErSO-DFP C max is approximately double the concentration seen with ErSO, with the AUC -60% of the ErSO value (Table 6, Figure 7). ErSO-DFP is cleared faster than ErSO with higher clearance values and lower calculated mean-residence times (Table 6, Figure 7). With this PK data in hand, the maximum tolerated dose (MTD) of ErSO and ErSO-DFP were determined.
• MTD maximum tolerated dose
• ErSO- DFP is better tolerated than ErSO in both mice and rats with an almost 5-fold increase in tolerability when administered intravenously; in these experiments, the MTD for intravenously injected ErSO- DFP is 95 mg/kg in mice, and >50 mg/kg in rats (Table 7). Given the similar PK parameters (Table 6), this observed improvement in tolerability of ErSO-DFP relative to ErSO does not appear to be due to a difference in compound exposure. Table 6. Summary of ErSO and ErSO-DFP Pharmacokinetics. I. V. Dosing at 20 mg/kg.
• ErSO-DFP has different predicted brain penetrance relative to ErSO, cLogBB of 0.03 and 0.25 respectively and CNS MPO scores of 3.44 to 2.83 respectively.
• the brain-to-serum ratio of ErSO-DFP was measured experimentally through a 10 mg/kg intravenous injection in mice. Contrary to cLogBB prediction and CNS MPO scores which predict a difference, ErSO-DFP maintains similar blood-brain partitioning ratios as ErSO ( Figure 6B), suggesting that the increased tolerability of ErSO-DFP is not the result of poor BBB penetration.
• Figure 6B blood-brain partitioning ratios as ErSO
• Table 7 Summary of maximum tolerated dose (MTD) experiments with ErSO-DFP and ErSO.
• MTD MTD
• ErSO-DFP maintains ErSO -like activity in a mouse model of ERa-positive breast cancer
• mice bearing large (average size > 300 mm 3 prior to treatment) MCF-7 tumors were treated with ErSO or ErSO-DFP once-a-week for three total doses (I.V. administration at 5 mg/kg).
• ErSO-DFP treatment leads to profound regression of MCF-7 tumors, mirroring results with the ErSO-treated mice ( Figure 7A).
• ErSO-DFP and ErSO are members of the substituted 3-(4-hydroxyphenyl)indoline-2-one class of anticancer small molecules.
• a well-known member of this class is oxyphenisatin, a laxative used for over 40 years which later was shown to have antiproliferative effects against transformed and cancer cells. Derivatives of oxyphenisatin have potent anticancer activity. For example, Andruska et al (Proc. Natl. Acad. Sci.
• BHPI an oxyphenisatin derivative that retards the growth of ERa+ cancer cells through hyperactivation of ERa-mediated a-UPR.
• Initial demonstrations of the a-UPR’ s ‘druggability’ with BHPI was the basis for the discovery of ErSO, a compound that induces stark and unique cytotoxic a-UPR hyperactivation.
• progenitor a-UPR activator BHPI is cytostatic against most ERa+ cancer cell lines, ErSO is rapidly cytotoxic and induces quantitative tumor regression in preclinical tumor models as a single agent with once-a-week dosing.
• ErSO-DFP displays a similar antitumor activity to ErSO with significant tumor regressions observed with once- a-week intravenous dosing and has an even wider therapeutic window than ErSO.
• the exact molecular underpinnings for this switch between a cytostatic or a cytotoxic a-UPR activator are yet to elucidated, but the altered phenotype and tumor regressions are clear.
• ErSO-DFP and other more polar compounds (R)-23, (R)-24, (7?)-26) maintain robust selectivity against ERa+ and ERa- cancer cell lines even with 72-hour incubations. This selectivity in cell culture appears to also translate into better tolerability in vivo, as shown with ErSO-DFP.
• ErSO ErSO-DFP and its derivatives (e.g. (7?)-26) are more ideal chemical probes for studying a-UPR activation as exemplified by their striking inactivity in ERa- cancer cell lines.
• Fluorine atoms can have profound effects on a ligand’s physiochemical and confirmational properties and contribute to a variety of interactions between ligands and their protein target. Indeed, fluorination of nitrogen heterocycles utilized herein does decrease the predicted p/fa of the corresponding conjugate acid and that p/fa perturbation may be a factor in their potent activity. Further, fluorination of piperidines (and other heterocycles) can have significant effects on compound conformation and may alter the dominate conformer in solution and/or in target binding.
• ErSO-DFP preserves many of the positive features of its progenitor ErSO, namely potent ERa WT/Mutant -dependent a-UPR activation and cancer cell death.
• ErSO-DFP still maintains BBB penetration, opening the door for future development for the treatment of drug-resistant, ERa WT/Mutant - positive, brain metastases.
• ErSO-DFP and other compounds reported herein are promising toward the development of a-UPR activators for the treatment advanced ERa+ breast cancer and other ERa+ cancers.
• the invention also relates to methods of making the compounds and compositions of the invention.
• the compounds and compositions can be prepared by any of the applicable techniques of organic synthesis, for example, the techniques described hereinbelow (Example 2). Many such techniques are well known in the art. However, many of the known techniques are elaborated in Compendium of Organic Synthetic Methods (John Wiley & Sons, New York), Vol. 1, Ian T. Harrison and Shuyen Harrison, 1971; Vol. 2, Ian T. Harrison and Shuyen Harrison, 1974; Vol. 3, Louis S. Hegedus and Leroy Wade, 1977; Vol. 4, Leroy G. Wade, Jr., 1980; Vol. 5, Leroy G. Wade, Jr., 1984; and Vol. 6, Michael B.
• reaction conditions such as temperature, reaction time, solvents, work-up procedures, and the like, will be those common in the art for the particular reaction to be performed.
• the cited reference material, together with material cited therein, contains detailed descriptions of such conditions.
• the temperatures will be -100 °C to 200 °C
• solvents will be aprotic or protic depending on the conditions required
• reaction times will be 1 minute to 2 days.
• Work-up typically consists of quenching any unreacted reagents followed by partition between a water / organic layer system (extraction) and separation of the layer containing the product.
• Oxidation and reduction reactions are typically carried out at temperatures near room temperature (about 20 °C), although for metal hydride reductions frequently the temperature is reduced to 0 °C to -100 °C. Heating can also be used when appropriate.
• Solvents are typically aprotic for reductions and may be either protic or aprotic for oxidations. Reaction times are adjusted to achieve desired conversions.
• Condensation reactions are typically carried out at temperatures near room temperature, although for non-equilibrating, kinetically controlled condensations reduced temperatures (0 °C to - 100 °C) are also common.
• Solvents can be either protic (common in equilibrating reactions) or aprotic (common in kinetically controlled reactions).
• Standard synthetic techniques such as azeotropic removal of reaction by-products and use of anhydrous reaction conditions (e.g., inert gas environments) are common in the art and will be applied when applicable.
• protecting group refers to any group which, when bound to a hydroxy or other heteroatom prevents undesired reactions from occurring at this group and which can be removed by conventional chemical or enzymatic steps to reestablish the hydroxyl group.
• removable protecting group employed is not always critical and preferred removable hydroxyl protecting groups include conventional substituents such as, for example, allyl, benzyl, acetyl, chloroacetyl, thiobenzyl, benzylidene, phenacyl, methyl methoxy, silyl ethers (e.g., trimethylsilyl (TMS), t-butyl-diphenylsilyl (TBDPS), or t-butyldimethylsilyl (TBS)) and any other group that can be introduced chemically onto a hydroxyl functionality and later selectively removed either by chemical or enzymatic methods in mild conditions compatible with the nature of the product.
• silyl ethers e.g., trimethylsilyl (TMS), t-butyl-diphenylsilyl (TBDPS), or t-butyldimethylsilyl (TBS)
• TMS trimethylsilyl
• TDPS t-butyl-
• Suitable heteroatom protecting groups are known to those skilled in the art and disclosed in more detail in T.W. Greene, Protecting Groups In Organic Synthesis', Wiley: New York, 1981 ("Greene”) and the references cited therein, and Kocienski, Philip J.; Protecting Groups (Georg Thieme Verlag Stuttgart, New York, 1994), both of which are incorporated herein by reference.
• Protecting groups are available, commonly known and used, and are optionally used to prevent side reactions with the protected group during synthetic procedures, i.e., routes or methods to prepare the compounds by the methods of the invention. For the most part the decision as to which groups to protect, when to do so, and the nature of the chemical protecting group ("PG" or "P") will be dependent upon the chemistry of the reaction to be protected against e.g., acidic, basic, oxidative, reductive, or other conditions) and the intended direction of the synthesis.
• PG chemical protecting group
• the compounds described herein can be used to prepare therapeutic pharmaceutical compositions, for example, by combining the compounds with a pharmaceutically acceptable diluent, excipient, or carrier.
• the compounds may be added to a carrier in the form of a salt or solvate.
• a pharmaceutically acceptable salts are organic acid addition salts formed with acids that form a physiologically acceptable anion, for example, tosylate, methanesulfonate, acetate, citrate, malonate, tartrate, succinate, benzoate, ascorbate, a-ketoglutarate, and [3-glyccrophosphatc.
• Suitable inorganic salts may also be formed, including hydrochloride, halide, sulfate, nitrate, bicarbonate, and carbonate salts.
• salts may be obtained using standard procedures well known in the art, for example by reacting a sufficiently basic compound such as an amine with a suitable acid to provide a physiologically acceptable ionic compound.
• a sufficiently basic compound such as an amine
• a suitable acid for example, a sufficiently basic compound such as an amine
• Alkali metal (for example, sodium, potassium or lithium) or alkaline earth metal (for example, calcium) salts of carboxylic acids can also be prepared by analogous methods.
• the compounds of the formulas described herein can be formulated as pharmaceutical compositions and administered to a mammalian host, such as a human patient, in a variety of forms.
• the forms can be specifically adapted to a chosen route of administration, e.g., oral or parenteral administration, by intravenous, intramuscular, topical or subcutaneous routes.
• the compounds described herein may be systemically administered in combination with a pharmaceutically acceptable vehicle, such as an inert diluent or an assimilable edible carrier.
• a pharmaceutically acceptable vehicle such as an inert diluent or an assimilable edible carrier.
• compounds can be enclosed in hard or soft-shell gelatin capsules, compressed into tablets, or incorporated directly into the food of a patient's diet.
• Compounds may also be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.
• Such compositions and preparations typically contain at least 0.1 % of active compound.
• compositions and preparations can vary and may conveniently be from about 0.5% to about 60%, about 1% to about 25%, or about 2% to about 10%, of the weight of a given unit dosage form.
• amount of active compound in such therapeutically useful compositions can be such that an effective dosage level can be obtained.
• the tablets, troches, pills, capsules, and the like may also contain one or more of the following: binders such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; and a lubricant such as magnesium stearate.
• binders such as gum tragacanth, acacia, corn starch or gelatin
• excipients such as dicalcium phosphate
• a disintegrating agent such as corn starch, potato starch, alginic acid and the like
• a lubricant such as magnesium stearate.
• a sweetening agent such as sucrose, fructose, lactose or aspartame
• a flavoring agent such as peppermint, oil of Wintergreen, or cherry flavoring
• the unit dosage form When the unit dosage form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier, such as a vegetable oil or a polyethylene glycol. Various other materials may be present as coatings or to otherwise modify the physical form of the solid unit dosage form. For instance, tablets, pills, or capsules may be coated with gelatin, wax, shellac or sugar and the like.
• a syrup or elixir may contain the active compound, sucrose or fructose as a sweetening agent, methyl and propyl parabens as preservatives, a dye and flavoring such as cherry or orange flavor. Any material used in preparing any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts employed.
• the active compound may be incorporated into sustained-release preparations and devices.
• the active compound may be administered intravenously or intraperitoneally by infusion or injection.
• Solutions of the active compound or its salts can be prepared in water, optionally mixed with a nontoxic surfactant.
• Dispersions can be prepared in glycerol, liquid polyethylene glycols, triacetin, or mixtures thereof, or in a pharmaceutically acceptable oil. Under ordinary conditions of storage and use, preparations may contain a preservative to prevent the growth of microorganisms.
• compositions suitable for injection or infusion can include sterile aqueous solutions, dispersions, or sterile powders comprising the active ingredient adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes.
• the ultimate dosage form should be sterile, fluid and stable under the conditions of manufacture and storage.
• the liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof.
• the proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions, or by the use of surfactants.
• the prevention of the action of microorganisms can be brought about by various antibacterial and/or antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, buffers, or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by agents delaying absorption, for example, aluminum monostearate and/or gelatin.
• Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in the appropriate solvent with various other ingredients enumerated above, as required, optionally followed by filter sterilization.
• methods of preparation can include vacuum drying and freeze-drying techniques, which yield a powder of the active ingredient plus any additional desired ingredient present in the solution.
• Useful dosages of the compounds described herein can be determined by comparing their in vitro activity, and in vivo activity in animal models. Methods for the extrapolation of effective dosages in mice, and other animals, to humans are known to the art; for example, see U.S. Patent No. 4,938,949 (Borch et al.).
• the amount of a compound, or an active salt or derivative thereof, required for use in treatment will vary not only with the particular compound or salt selected but also with the route of administration, the nature of the condition being treated, and the age and condition of the patient, and will be ultimately at the discretion of an attendant physician or clinician.
• a suitable dose will be in the range of from about 0.5 to about 100 mg/kg, e.g., from about 10 to about 75 mg/kg of body weight per day, such as 3 to about 50 mg per kilogram body weight of the recipient per day, preferably in the range of 6 to 90 mg/kg/day, most preferably in the range of 15 to 60 mg/kg/day.
• the compound is conveniently formulated in unit dosage form; for example, containing 5 to 1000 mg, conveniently 10 to 750 mg, most conveniently, 50 to 500 mg of active ingredient per unit dosage form.
• the invention provides a composition comprising a compound of the invention formulated in such a unit dosage form.
• the compound can be conveniently administered in a unit dosage form, for example, containing 5 to 1000 mg/m 2 , conveniently 10 to 750 mg/m 2 , most conveniently, 50 to 500 mg/m 2 of active ingredient per unit dosage form.
• the desired dose may conveniently be presented in a single dose or as divided doses administered at appropriate intervals, for example, as two, three, four or more sub-doses per day.
• the sub-dose itself may be further divided, e.g., into a number of discrete loosely spaced administrations.
• the desired dose may conveniently be presented in a single dose or as divided doses administered at appropriate intervals, for example, as two, three, four or more sub-doses per day.
• the sub-dose itself may be further divided, e.g., into a number of discrete loosely spaced administrations such as multiple inhalations from an insufflator or by application of a plurality of drops into the eye.
• the compounds described herein can be effective anti-tumor agents and have higher potency and/or reduced toxicity as compared to BHPI.
• compounds of the invention are more potent and less toxic than BHPI, and/or avoid a potential site of catabolic metabolism encountered with BHPI, i.e., have a different metabolic profile than BHPI.
• the compounds described herein cause less severe ataxia than BHPI and other known compounds.
• the invention provides therapeutic methods of treating cancer in a vertebrate such as a mammal, which involve administering to a mammal having cancer an effective amount of a compound or composition described herein.
• a mammal includes a primate, human, rodent, canine, feline, bovine, ovine, equine, swine, caprine, bovine and the like.
• Cancer refers to any of the various type of malignant neoplasm, which are in general characterized by an undesirable cellular proliferation, e.g., unregulated growth, lack of differentiation, local tissue invasion, and metastasis.
• Cancers that can be treated by a compound described herein include, for example, breast cancer, cervical carcinoma, colon cancer, endometrial cancer, leukemia, lung cancer, melanoma, pancreatic cancer, prostate cancer, ovarian cancer, or uterine cancer, and in particular, any cancer that is ERoc positive.
• the ability of a compound of the invention to treat cancer may be determined by using assays well known to the art. For example, the design of treatment protocols, toxicity evaluation, data analysis, quantification of tumor cell kills, and the biological significance of the use of transplantable tumor screens are known. In addition, ability of a compound to treat cancer may be determined using the Tests as described below.
• Example 1 Biological procedures.
• Cancer Cell Lines Culturing Conditions and Validation. All cell lines were cultured at 37°C with 5% CO2. All ERa-positive cell lines were grown in media lacking phenol red. MCF-7 were grown in EMEM with 10% FBS and 1% penicillin-streptomycin (P/S). T47D were cultured in MEM with 10% FBS and 1% P/S. TYS and TDG cells were grown in MEM supplemented with 10% CD- FBS and 1% P/S. HCT-116 and MDA-MB-231 cells were cultured in RPMI-1640 supplemented with 10% FBS and 1% P/S. MDA-MB-436 cells were cultured in DMEM supplemented with 10% FBS and 1% P/S.
• HT-29 were cultured in McCoy’s media supplemented with 10% FBS and 1% P/S. All cell lines were used directly from ATCC stocks and/or have been further authenticated using PowerPlexl6HS Assay (Promega) as described previously. 54 In short, >1 million cells were harvested and lysed using the cell lysis buffer (50 mM Tris, 50 mM EDTA, 25 mM sucrose, 100 mM NaCl, 1% SDS, pH 8). DNA extraction and short tandem repeats (STRs) profiling for each cell line were carried out at the University of Arizona Genetics Core (UAGC). Autosomal STR profiles were compared to reference databases such as ATCC, DSMZ, and JCRB.
• UGC University of Arizona Genetics Core
• PBS-washed cell pellets were then lysed using RIPA buffer containing phosphatase (BioVision) and protease inhibitor cocktail (Calbiochem). Protein concentrations were determined using the BCA assay (Pierce). Lysates containing 10-15 pg of protein were loaded onto 4-20% gradient gels (BioRad), and SDS-PAGE was run. Proteins were then transferred onto membrane (PDVF Millipore) and blots blocked with BSA solution (2 g in 40 mL TBST) for one hour. After blocking, primary antibody was added and incubated overnight (using manufacturer’s recommended dilutions).
• blots were washed with TBST (5-minute incubation for each wash, washed 3 times), then incubated with HRP-linked secondary antibody for 1 hour in TBST. Blots were washed (10-minute incubation for each wash for a total of two ten-minute washes). SuperSignal West Pico Solution was then added following manufacturer’ s procedures and blots imaged with ChemiDoc.
• Antibodies used: Phospho-AMPK (Thrl72): CST-2535, AMPK: CST-5832, Phospho-EIF2a (Ser51): CST-3398, EIF2a: CST-5324, ATF6a: CST-65880, (3-actin-HRP conjugate: CST-5125, P-actin: CST-4970, Anti-rabbit IgG HRP-linked: CST-7074.
• mice Female CD-I mice (Charles River, ⁇ 25 g mice) were administered compounds (i.e., ErSO or ErSO-DFP) either intravenously (I.V. tail vein) or oral gavage (P.O.) at 20 mg/kg or 40 mg/kg as indicated. Mice were then sacrificed in cohorts of 3 at time points (#,#,#). Blood was collected, centrifuged, and plasma separated for quantification of test compound utilizing LC-MS/MS (UIUC Metabolomics Center, Urbana, IL.). Determined compound concentration was then analyzed using non-linear regression programing (WinNonlin) and pharmacokinetic parameters estimated.
• WinNonlin non-linear regression programing
• SGF Stability Assay SGF was prepared fresh by adding 3.2 g pepsin (Sigma Aldrich P7000) to 1000 mL of SGF (Fischer Scientific 7108-16) and the pH adjusted to 1.2 using a pH meter. This SGF contextith pepS in solution was then warmed to 37°C. 990 pL of warmed SGF professionith pepS in was then aliquoted into multiple Eppendorf tubes, followed by 10 pL of 10 mM DMSO stock solutions of test compounds. Erythromycin (MedChemExpress HY-B0220) is a positive control for this stability assay. Samples were incubated at 37°C for the entire experiment with vortexing every 15 minutes.
• MTD Maximum Tolerated Dose Experiments in Mice and Rats.
• Rodents were monitored for signs of distress, lethargy, neurotoxicity, and so forth for multiple days after compound administration. If a given dose was tolerated, higher doses were investigated until an intolerable dose was observed (often associated with acute signs of toxicity and/or lethality) or the solubility limit was reached with a given test compound.
• the MTD was defined as the maximum dose in which any acute side effects observed were tolerated and no lethality observed.
• tail-vein injected with vehicle (5% DMSO, 10% Tween-20, 85% PBS), ErSO (5 mg/kg), or ErSO-DFP (5 mg/kg) once-a-week for three total doses (day 0, day 7, day 14). Tumor size was measured by caliper, and mouse weights were recorded.
• the resultant slurry was centrifuged twice at 13,000 RCF for 10 minutes per run and the supernatant was collected and frozen at -80°C prior to analysis. Samples were then analyzed by LC-MS/MS (Metabolomics Laboratory of the Roy J Carver Biotechnology Center UIUC) to determine ErSO or ErSO-DFP concentration in both serum and brain. To calculate absolute braimserum ratios, an approximated mouse blood volume of 58.5 mL/kg was utilized for each mouse.
• High resolution mass spectra were obtained at the UIUC SCS Mass Spectrometry Laboratory utilizing electrospray ionization (ESI).
• X-Ray crystallography was conducted at UIUC SCS George L. Clark X-Ray Facility utilizing a Bruker D8 Venture Duo instrument which can determine absolute stereochemistry. Further crystallographic information is available upon request and has been deposited on Reciprocal Net.
• the > 95% purity of compounds utilized in biological assays was determined using HPLC at X: 254 nm. Chiral HPLC will often show two peaks for racemic mixtures, an important consideration for compounds herein since often only one enantiomer for this class of compounds herein show biological activity. For a few compounds (denoted throughout), resolution of both enantiomers was not achieved with the HPLC chiral column used.
• reaction vessel was charged with the crude mixture and dissolved in THF or DMF (3.9 mL) followed by the desired amine (3.94 mmol), and pyridine (3.94 mmol) if the desired amine is an HC1 salt.
• the reaction mixture was stirred for 2-4 hours (reaction tracked by TLC to monitor the consumption of starting material).
• the reaction was poured into a 1:1 water:brine and extracted with ethyl acetate (3 times) and the combined organic layers dried over sodium sulfate and concentrated in vacuo.
• the crude oil was then dissolved in THF (4 mL) and a TBAF solution (IM in THF, 2 mL) was added and the reaction stirred until complete consumption of starting material was observed (reaction monitored by TLC).
• reaction stirred for 6 hours while the ice was allowed to melt.
• the reaction mixture was then poured into ice-filled saturated sodium bicarbonate (aqueous, ⁇ 10 mL) and the aqueous solution was extracted with ethyl acetate (3x). The combined organic layers were dried over sodium sulfate, filtered, and concentrated in vacuo.
• compositions illustrate representative pharmaceutical dosage forms that may be used for the therapeutic or prophylactic administration of a compound of a formula described herein, a compound specifically disclosed herein, or a pharmaceutically acceptable salt or solvate thereof (hereinafter referred to as 'Compound X').
• 'Compound X' a pharmaceutically acceptable salt or solvate thereof
• a specific example of Compound X is ErSO-DFP.
• compositions may be prepared by conventional procedures well known in the pharmaceutical art. It will be appreciated that the above pharmaceutical compositions may be varied according to well-known pharmaceutical techniques to accommodate differing amounts and types of active ingredient 'Compound X'. Aerosol formulation (vi) may be used in conjunction with a standard, metered dose aerosol dispenser. Additionally, the specific ingredients and proportions are for illustrative purposes. Ingredients may be exchanged for suitable equivalents and proportions may be varied, according to the desired properties of the dosage form of interest. While specific embodiments have been described above with reference to the disclosed embodiments and examples, such embodiments are only illustrative and do not limit the scope of the invention.

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