Patent Application
20180265444
Members of the p160 steroid receptor coactivator (SRC) family, SRC-1, SRC-2/TIF2/GRIP1, and SRC-3/AM1/RAC3/ACTR/pCIP, interact with nuclear receptors and other transcription factors to drive target gene expression while also functioning as integrators of upstream cell signaling pathways. Although the proteins share homology with each other, they have distinct and important roles in multiple physiological processes, including growth and development, reproduction, and metabolism. All three proteins also have been found to be broadly involved in different aspects of tumorigenesis. SRC-3 is most well-known for its oncogenic role, whose gene is amplified in 9.5% of breast cancers and whose mRNA has been shown to be overexpressed in different breast cancer cohorts, often at the 50% level or greater. Clinically, SRC-3 overexpression in breast cancer correlates with larger tumor size, higher tumor grade, and poor survival rates. Direct evidence supporting SRC-3 as a bona fide oncogene comes from a MMTV-SRC-3 transgenic mouse model, in which overexpression of SRC-3 was sufficient to cause spontaneous development of malignant mammary tumors. SRC-3 overexpression also has been observed in endometrial, ovarian, prostate, colorectal, gastric, lung, pancreatic, and liver cancers. Additional in vitro and in vivo studies have bolstered the importance of SRC-3 in tumor initiation, progression, metastasis, and drug resistance. SRC-1 also is overexpressed in about 20% breast cancers and is positively correlated with ERBB2 expression, disease recurrence, and poor survival. It has been demonstrated that SRC-1 plays a critical role in cancer cell migration, invasion, and metastasis. Finally, SRC-2 has been proposed as a key oncogene in prostate cancer based on a comprehensive analysis on prostate tumors, cell lines, and xenografts, revealing that SRC-2 gene amplification and mutation specifically arise to levels of 38% in metastatic prostate tumors.
Although tumor formation is a multistage process involving activation of oncogenes and inactivation of tumor suppressors, loss of a specific oncogene can frequently reverse the malignant progression of cancer cells, suggesting that cancer cells rely on the continued activation or overexpression of an oncogene. This “oncogene addiction” theory, combined with the fact that SRC proteins integrate and promote multiple growth factor signaling pathways crucial for cancer cell growth and survival, highlights the potential value of SRC targeting drugs as future anti-cancer agents.
Steroid receptor coactivator (SRC) stimulators and methods for their use in treating and/or preventing cancer are provided. The methods include administering to a subject a compound as described herein.
A method for treating cancer in a subject includes administering to the subject an effective amount of a compound of the following formula:
or a pharmaceutically acceptable salt or prodrug thereof. In the compounds for use in this method, A1, A2, A3, A4, A5, A6, A7, A8, A9, and A10 are each independently selected from CR1 and N, wherein each R1 is hydrogen, halogen, alkoxy, cyano, trifluoromethyl, or substituted or unsubstituted C1-6 alkyl; and X is CR2R3, O, or NR4, wherein R2, R3, and R4 are each independently selected from the group consisting of hydrogen and substituted or unsubstituted C1-6 alkyl. Optionally, the compound is
A method for treating cancer in a subject includes administering to the subject an effective amount of a compound of the following formula:
or a pharmaceutically acceptable salt or prodrug thereof. In the compounds for use in this method, A1, A2, A3, A4, and A5 are each independently selected from CR1 and N, wherein each R1 is hydrogen, halogen, alkoxy, cyano, trifluoromethyl, or substituted or unsubstituted C1-6 alkyl; and X is CR2R3, O, or NR4, wherein R2, R3, and R4 are each independently selected from the group consisting of hydrogen and substituted or unsubstituted C1-6 alkyl. Optionally, the compound is selected from the group consisting of:
Optionally, the cancer is glioblastoma, such as a glioblastoma multiforme tumor. Optionally, the cancer is a pediatric glioblastoma multiforme tumor. Optionally, the cancer is breast cancer, liver cancer, lung cancer, pancreatic cancer, or prostate cancer.
Also described herein are methods of stimulating a steroid receptor coactivator protein in a cell. A method of stimulating a steroid receptor coactivator protein in a cell includes contacting a cell with an effective amount of a compound of the following formula:
or a pharmaceutically acceptable salt or prodrug thereof. In the compounds for use in this method, A1, A2, A3, A4, A5, A6, A7, A8, A9, and A10 are each independently selected from CR1 and N, wherein each R1 is hydrogen, halogen, alkoxy, cyano, trifluoromethyl, or substituted or unsubstituted C1-6 alkyl; and X is CR2R3, O, or NR4, wherein R2, R3, and R4 are each independently selected from the group consisting of hydrogen and substituted or unsubstituted C1-6 alkyl. Optionally, the compound is
A method of stimulating a steroid receptor coactivator protein in a cell includes contacting a cell with an effective amount of a compound of the following formula:
or a pharmaceutically acceptable salt or prodrug thereof. In the compounds for use in this method, A1, A2, A3, A4, and A5 are each independently selected from CR1 and N, wherein each R1 is hydrogen, halogen, alkoxy, cyano, trifluoromethyl, or substituted or unsubstituted C1-6 alkyl; and X is CR2R3, O, or NR4, wherein R2, R3, and R4 are each independently selected from the group consisting of hydrogen and substituted or unsubstituted C1-6 alkyl. Optionally, the compound is selected from the group consisting of:
Optionally, the steroid receptor coactivator protein is SRC-1, SRC-2, or SRC-3. Optionally, the method of stimulating a steroid receptor coactivator protein in a cell is performed in vitro or in vivo.
Also described herein are compounds selected from the group consisting of:
The details of one or more embodiments are set forth in the drawings and the description below. Other features, objects, and advantages will be apparent from the description and drawings, and from the claims.
Described herein are steroid receptor coactivator (SRC) stimulators and methods for their use. Specifically, the small molecules described herein are stimulators of one or more of the SRC family protein members, including SRC-1, SRC-2/TIF2/GRIP1, and SRC-3/AIB1/RAC3/ACTR/pCIP. The compounds and methods described herein are useful for treating cancer, including glioblastoma multiforme tumors. As detailed herein, acute super-activation of SRC coactivators effectively kills cancer cells by inducing aberrant cellular stress. The over-stimulation of the SRC family proteins is an unexpected but effective approach to selectively kill cancer cells whose cellular stress response pathways already are maximally engaged.
A class of SRC stimulators described herein is represented by Formula I:
and pharmaceutically acceptable salts or prodrugs thereof.
In Formula I, A1, A2, A3, A4, A5, A6, A7, A8, A9, and A10 are each independently selected from CR1 and N. Each R1 group present in Formula I is independently selected from hydrogen, halogen, alkoxy, cyano, trifluoromethyl, and substituted or unsubstituted C1-6 alkyl.
Also, in Formula I, X is CR2R3, O, or NR4. R2, R3, and R4 are each independently selected from the group consisting of hydrogen and substituted or unsubstituted C1-6 alkyl.
As used herein, the terms alkyl, alkenyl, and alkynyl include straight- and branched-chain monovalent substituents. Examples include methyl, ethyl, isobutyl, 3-butynyl, and the like. Ranges of these groups useful with the compounds and methods described herein include C1-C20 alkyl, C2-C20 alkenyl, and C2-C20 alkynyl. Additional ranges of these groups useful with the compounds and methods described herein include C1-C12 alkyl, C2-C12 alkenyl, C2-C12 alkynyl, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C4 alkyl, C2-C4 alkenyl, and C2-C4 alkynyl.
Heteroalkyl, heteroalkenyl, and heteroalkynyl are defined similarly as alkyl, alkenyl, and alkynyl, but can contain O, S, or N heteroatoms or combinations thereof within the backbone. Ranges of these groups useful with the compounds and methods described herein include C1-C20 heteroalkyl, C2-C20 heteroalkenyl, and C2-C20 heteroalkynyl. Additional ranges of these groups useful with the compounds and methods described herein include C1-C12 heteroalkyl, C2-C12 heteroalkenyl, C2-C12 heteroalkynyl, C1-C6 heteroalkyl, C2-C6 heteroalkenyl, C2-C6 heteroalkynyl, heteroalkyl, C2-C4 heteroalkenyl, and C2-C4 heteroalkynyl.
The terms cycloalkyl, cycloalkenyl, and cycloalkynyl include cyclic alkyl groups having a single cyclic ring or multiple condensed rings. Examples include cyclohexyl, cyclopentylethyl, and adamantanyl. Ranges of these groups useful with the compounds and methods described herein include C3-C20 cycloalkyl, C3-C20 cycloalkenyl, and C3-C20 cycloalkynyl. Additional ranges of these groups useful with the compounds and methods described herein include C5-C12 cycloalkyl, C5-C12 cycloalkenyl, C5-C12 cycloalkynyl, C5-C6 cycloalkyl, C5-C6 cycloalkenyl, and C5-C6 cycloalkynyl.
The terms heterocycloalkyl, heterocycloalkenyl, and heterocycloalkynyl are defined similarly as cycloalkyl, cycloalkenyl, and cycloalkynyl, but can contain O, S, or N heteroatoms or combinations thereof within the cyclic backbone. Ranges of these groups useful with the compounds and methods described herein include C3-C20 heterocycloalkyl, C3-C20 heterocycloalkenyl, and C3-C20 heterocycloalkynyl. Additional ranges of these groups useful with the compounds and methods described herein include C5-C12 heterocycloalkyl, C5-C12 heterocycloalkenyl, C5-C12 heterocycloalkynyl, C5-C6 heterocycloalkyl, C5-C6 heterocycloalkenyl, and C5-C6 heterocycloalkynyl.
Aryl molecules include, for example, cyclic hydrocarbons that incorporate one or more planar sets of, typically, six carbon atoms that are connected by delocalized electrons numbering the same as if they consisted of alternating single and double covalent bonds. An example of an aryl molecule is benzene. Heteroaryl molecules include substitutions along their main cyclic chain of atoms such as O, N, or S. When heteroatoms are introduced, a set of five atoms, e.g., four carbon and a heteroatom, can create an aromatic system. Examples of heteroaryl molecules include furan, pyrrole, thiophene, imadazole, oxazole, pyridine, and pyrazine. Aryl and heteroaryl molecules can also include additional fused rings, for example, benzofuran, indole, benzothiophene, naphthalene, anthracene, and quinoline. The aryl and heteroaryl molecules can be attached at any position on the ring, unless otherwise noted.
The term alkoxy as used herein is an alkyl group bound through a single, terminal ether linkage. Likewise, the term aryloxy as used herein is an aryl group bound through a single, terminal ether linkage.
The term hydroxyl as used herein is represented by the formula —OH.
The terms amine or amino as used herein are represented by the formula —NZ1Z2, where Z1 and Z2 can each be a substitution group as described herein, such as hydrogen, an alkyl, halogenated alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group described above.
The alkoxy, aryloxy, amino, alkyl, alkenyl, alkynyl, aryl, heteroalkyl, heteroalkenyl, heteroalkynyl, heteroaryl, cycloalkyl, or heterocycloalkyl molecules used herein can be substituted or unsubstituted. As used herein, the term substituted includes the addition of an alkoxy, aryloxy, amino, alkyl, alkenyl, alkynyl, aryl, heteroalkyl, heteroalkenyl, heteroalkynyl, heteroaryl, cycloalkyl, or heterocycloalkyl group to a position attached to the main chain of the alkoxy, aryloxy, amino, alkyl, alkenyl, alkynyl, aryl, heteroalkyl, heteroalkenyl, heteroalkynyl, heteroaryl, cycloalkyl, or heterocycloalkyl, e.g., the replacement of a hydrogen by one of these molecules. Examples of substitution groups include, but are not limited to, hydroxyl, halogen (e.g., F, Br, Cl, or I), and carboxyl groups. Conversely, as used herein, the term unsubstituted indicates the alkoxy, aryloxy, amino, alkyl, alkenyl, alkynyl, aryl, heteroalkyl, heteroalkenyl, heteroalkynyl, heteroaryl, cycloalkyl, or heterocycloalkyl has a full complement of hydrogens, i.e., commensurate with its saturation level, with no substitutions, e.g., linear decane (—(CH2)9—CH3).
In some examples, Formula I is represented by Structure I-A:
In Structure I-A, A1, A2, A3, A4, A5, A6, A7, A8, A9, A10, R2, and R3 are as defined above for Formula I. In some examples of Structure I-A, each of A1, A2, A3, A4, A5, A6, A7, A8, A9, and A10 are CR1, where each R1 is independently selected from a group as defined above for Formula I. For example, the compound of Structure I-A can be represented by Structure I-A1:
In Structure I-A1, the phenyl rings of the molecule can each independently include from one to five R1 groups. Each of the R1 groups can be independently selected from a group as defined above for Formula I.
In some examples of Structure I-A, one or more of A1, A2, A3, A4, A5, A6, A7, A8, A9, and A10 can be N. For example, the compound of Structure I-A can be represented by Structure I-A2, Structure I-A3, or Structure I-A4:
In Structure I-A2, Structure I-A3, and Structure I-A4, the phenyl rings of the molecule can each independently include from one to four R1 groups. Each of the R1 groups can be independently selected from a group as defined above for Formula I.
In some examples, Formula I is represented by Structure I-B:
In Structure I-B, A1, A2, A3, A4, A5, A6, A7, A8, A9, A10, and R4 are as defined above for Formula I. In some examples of Structure I-B, each of A1, A2, A3, A4, A5, A6, A7, A8, A9, and A10 are CR1, where each R1 is independently selected from a group as defined above for Formula I. For example, the compound of Structure I-B can be represented by Structure I-B1:
In Structure I-B1, the phenyl rings of the molecule can include from one to five R1 groups. Each of the R1 groups can be independently selected from a group as defined above for Formula I.
In some examples of Structure I-B, one or more of A1, A2, A3, A4, A5, A6, A7, A8, A9, and A10 can be N. For example, the compound of Structure I-B can be represented by Structure I-B2, Structure I-B3, or Structure I-B4:
In Structure I-B2, Structure I-B3, and Structure I-B4, the phenyl rings of the molecule can each independently include from one to four R1 groups. Each of the R1 groups can be independently selected from a group as defined above for Formula I.
In some examples, Formula I is represented by Structure I-C:
In Structure I-C, A1, A2, A3, A4, A5, A6, A7, A8, A9, and A10 are as defined above for Formula I. In some examples of Structure I-C, each of A1, A2, A3, A4, A5, A6, A7, A8, A9, and A10 are CR1, where each R1 is independently selected from a group as defined above for Formula I. For example, the compound of Structure I-C can be represented by Structure I-C1:
In Structure I-C1, the phenyl rings of the molecule can include from one to five R1 groups. Each of the R1 groups can be independently selected from a group as defined above for Formula I.
In some examples of Structure I-C, one or more of A1, A2, A3, A4, A5, A6, A7, A8, A9, and A10 can be N. For example, the compound of Structure I-C can be represented by Structure I-C2, Structure I-C3, or Structure I-C4:
In Structure I-C2, Structure I-C3, and Structure I-C4, the phenyl rings of the molecule can each independently include from one to four R1 groups. Each of the R1 groups can be independently selected from a group as defined above for Formula I.
Examples of Formula I include the following compounds:
In some embodiments, the compound is SYC-930 (Compound 1-2), SYC-936 (Compound 1-9), SYC-944 (Compound 2-8) (also referred to herein as MCB-613), SYC-946 (Compound 3-2), SYC-909 (Compound 3-8), SYC-952 (Compound 3-9), SYC-849 (Compound 4-1), SYC-850 (Compound 4-2), SYC-851 (Compound 4-8), SYC-957 (Compound 4-9), SYC-852 (Compound 5-1), SYC-853 (Compound 5-4), SYC-960 (Compound 5-7), SYC-965 (Compound 6-6), SYC-966 (Compound 6-7), SYC-854 (Compound 6-8), SYC-970 (Compound 7-6), SYC-855 (Compound 7-8), SYC-856 (Compound 8-2), or SYC-857 (Compound 8-5). In some embodiments, the compound is SYC-923 (Compound 1-8-2), SYC-925 (Compound 4-8-2), SYC-927 (Compound 7-8-2), SYC-917 (Compound 7-18), or SYC-918 (Compound 7-19). In some embodiments, the compound is not SYC-944 (Compound 2-8) (also referred to herein as MCB-613).
A class of SRC stimulators described herein is represented by Formula II:
and pharmaceutically acceptable salts or prodrugs thereof.
In Formula II, A1, A2, A3, A4, and A5 are each independently selected from CR1 and N. Each R1 group present in Formula II is independently selected from hydrogen, halogen, alkoxy, cyano, trifluoromethyl, and substituted or unsubstituted C1-6 alkyl.
Also, in Formula II, X is CR2R3, O, or NR4. R2, R3, and R4 are each independently selected from the group consisting of hydrogen and substituted or unsubstituted C1-6 alkyl.
Examples of Formula II include the following compounds:
The compounds described herein can be prepared in a variety of ways. The compounds can be synthesized using various synthetic methods. At least some of these methods are known in the art of synthetic organic chemistry. The compounds described herein can be prepared from readily available starting materials. Optimum reaction conditions can vary with the particular reactants or solvent used, but such conditions can be determined by one skilled in the art by routine optimization procedures.
Variations on Formulas I and II include the addition, subtraction, or movement of the various constituents as described for each compound. Similarly, when one or more chiral centers are present in a molecule, all possible chiral variants are included. Additionally, compound synthesis can involve the protection and deprotection of various chemical groups. The use of protection and deprotection, and the selection of appropriate protecting groups can be determined by one skilled in the art. The chemistry of protecting groups can be found, for example, in Greene, et al., Protective Groups in Organic Synthesis, 2d. Ed., Wiley & Sons, 1991, which is incorporated herein by reference in its entirety.
Reactions to produce the compounds described herein can be carried out in solvents, which can be selected by one of skill in the art of organic synthesis. Solvents can be substantially nonreactive with the starting materials (reactants), the intermediates, or products under the conditions at which the reactions are carried out, i.e., temperature and pressure. Reactions can be carried out in one solvent or a mixture of more than one solvent. Product or intermediate formation can be monitored according to any suitable method known in the art. For example, product formation can be monitored by spectroscopic means, such as nuclear magnetic resonance spectroscopy (e.g., 1H or 13C) infrared spectroscopy, spectrophotometry (e.g., UV-visible), or mass spectrometry, or by chromatography such as high performance liquid chromatography (HPLC) or thin layer chromatography.
Exemplary methods for synthesizing compounds as described herein are provided in Example 1 below.
The compounds described herein or derivatives thereof can be provided in a pharmaceutical composition. Depending on the intended mode of administration, the pharmaceutical composition can be in the form of solid, semi-solid or liquid dosage forms, such as, for example, tablets, suppositories, pills, capsules, powders, liquids, or suspensions, preferably in unit dosage form suitable for single administration of a precise dosage. The compositions will include a therapeutically effective amount of the compound described herein or derivatives thereof in combination with a pharmaceutically acceptable carrier and, in addition, may include other medicinal agents, pharmaceutical agents, carriers, or diluents. By pharmaceutically acceptable is meant a material that is not biologically or otherwise undesirable, which can be administered to an individual along with the selected compound without causing unacceptable biological effects or interacting in a deleterious manner with the other components of the pharmaceutical composition in which it is contained.
As used herein, the term carrier encompasses any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, lipid, stabilizer, or other material well known in the art for use in pharmaceutical formulations. The choice of a carrier for use in a composition will depend upon the intended route of administration for the composition. The preparation of pharmaceutically acceptable carriers and formulations containing these materials is described in, e.g., Remington's Pharmaceutical Sciences, 21st Edition, ed. University of the Sciences in Philadelphia, Lippincott, Williams & Wilkins, Philadelphia Pa., 2005. Examples of physiologically acceptable carriers include buffers, such as phosphate buffers, citrate buffer, and buffers with other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents, such as EDTA; sugar alcohols, such as mannitol or sorbitol; salt-forming counterions, such as sodium; and/or nonionic surfactants, such as TWEEN® (ICI, Inc.; Bridgewater, N.J.), polyethylene glycol (PEG), and PLURONICS™ (BASF; Florham Park, N.J.).
Compositions containing the compound described herein or derivatives thereof suitable for parenteral injection may comprise physiologically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Examples of suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (propyleneglycol, polyethyleneglycol, glycerol, and the like), suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants.
These compositions may also contain adjuvants, such as preserving, wetting, emulsifying, and dispensing agents. Prevention of the action of microorganisms can be promoted by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. Isotonic agents, for example, sugars, sodium chloride, and the like may also be included. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.
Solid dosage forms for oral administration of the compounds described herein or derivatives thereof include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the compounds described herein or derivatives thereof is admixed with at least one inert customary excipient (or carrier), such as sodium citrate or dicalcium phosphate, or (a) fillers or extenders, as for example, starches, lactose, sucrose, glucose, mannitol, and silicic acid, (b) binders, as for example, carboxymethylcellulose, alignates, gelatin, polyvinylpyrrolidone, sucrose, and acacia, (c) humectants, as for example, glycerol, (d) disintegrating agents, as for example, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate, (e) solution retarders, as for example, paraffin, (f) absorption accelerators, as for example, quaternary ammonium compounds, (g) wetting agents, as for example, cetyl alcohol, and glycerol monostearate, (h) adsorbents, as for example, kaolin and bentonite, and (i) lubricants, as for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof. In the case of capsules, tablets, and pills, the dosage forms may also comprise buffering agents.
Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethyleneglycols, and the like.
Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells, such as enteric coatings and others known in the art. They may contain opacifying agents and can also be of such composition that they release the active compound or compounds in a certain part of the intestinal tract in a delayed manner. Examples of embedding compositions that can be used are polymeric substances and waxes. The active compounds can also be in micro-encapsulated form, if appropriate, with one or more of the above-mentioned excipients.
Liquid dosage forms for oral administration of the compounds described herein or derivatives thereof include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs. In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, solubilizing agents, and emulsifiers, as for example, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyleneglycol, 1,3-butyleneglycol, dimethylformamide, oils, in particular, cottonseed oil, groundnut oil, corn germ oil, olive oil, castor oil, sesame oil, glycerol, tetrahydrofurfuryl alcohol, polyethyleneglycols, and fatty acid esters of sorbitan, or mixtures of these substances, and the like.
Besides such inert diluents, the composition can also include additional agents, such as wetting, emulsifying, suspending, sweetening, flavoring, or perfuming agents.
Suspensions, in addition to the active compounds, may contain additional agents, as for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, or mixtures of these substances, and the like.
Compositions of the compounds described herein or derivatives thereof for rectal administrations are optionally suppositories, which can be prepared by mixing the compounds with suitable non-irritating excipients or carriers, such as cocoa butter, polyethyleneglycol or a suppository wax, which are solid at ordinary temperatures but liquid at body temperature and, therefore, melt in the rectum or vaginal cavity and release the active component.
Dosage forms for topical administration of the compounds described herein or derivatives thereof include ointments, powders, sprays, and inhalants. The compounds described herein or derivatives thereof are admixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants as may be required. Ophthalmic formulations, ointments, powders, and solutions are also contemplated as being within the scope of the compositions.
The compositions can include one or more of the compounds described herein and a pharmaceutically acceptable carrier. As used herein, the term pharmaceutically acceptable salt refers to those salts of the compound described herein or derivatives thereof that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of subjects without undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit/risk ratio, and effective for their intended use, as well as the zwitterionic forms, where possible, of the compounds described herein. The term salts refers to the relatively non-toxic, inorganic and organic acid addition salts of the compounds described herein. These salts can be prepared in situ during the isolation and purification of the compounds or by separately reacting the purified compound in its free base form with a suitable organic or inorganic acid and isolating the salt thus formed. Representative salts include the hydrobromide, hydrochloride, sulfate, bisulfate, nitrate, acetate, oxalate, valerate, oleate, palmitate, stearate, laurate, borate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, naphthylate mesylate, glucoheptonate, lactobionate, methane sulphonate, and laurylsulphonate salts, and the like. These may include cations based on the alkali and alkaline earth metals, such as sodium, lithium, potassium, calcium, magnesium, and the like, as well as non-toxic ammonium, quaternary ammonium, and amine cations including, but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like. (See S. M. Barge et al., J. Pharm. Sci. (1977) 66, 1, which is incorporated herein by reference in its entirety, at least, for compositions taught therein.)
Administration of the compounds and compositions described herein or pharmaceutically acceptable salts thereof can be carried out using therapeutically effective amounts of the compounds and compositions described herein or pharmaceutically acceptable salts thereof as described herein for periods of time effective to treat a disorder. The effective amount of the compounds and compositions described herein or pharmaceutically acceptable salts thereof as described herein may be determined by one of ordinary skill in the art and includes exemplary dosage amounts for a mammal of from about 0.5 to about 200 mg/kg of body weight of active compound per day, which may be administered in a single dose or in the form of individual divided doses, such as from 1 to 4 times per day. Alternatively, the dosage amount can be from about 0.5 to about 150 mg/kg of body weight of active compound per day, about 0.5 to 100 mg/kg of body weight of active compound per day, about 0.5 to about 75 mg/kg of body weight of active compound per day, about 0.5 to about 50 mg/kg of body weight of active compound per day, about 0.01 to about 50 mg/kg of body weight of active compound per day, about 0.05 to about 25 mg/kg of body weight of active compound per day, about 0.1 to about 25 mg/kg of body weight of active compound per day, about 0.5 to about 25 mg/kg of body weight of active compound per day, about 1 to about 20 mg/kg of body weight of active compound per day, about 1 to about 10 mg/kg of body weight of active compound per day, about 20 mg/kg of body weight of active compound per day, about 10 mg/kg of body weight of active compound per day, about 5 mg/kg of body weight of active compound per day, about 2.5 mg/kg of body weight of active compound per day, about 1.0 mg/kg of body weight of active compound per day, or about 0.5 mg/kg of body weight of active compound per day, or any range derivable therein. Optionally, the dosage amounts are from about 0.01 mg/kg to about 10 mg/kg of body weight of active compound per day. Optionally, the dosage amount is from about 0.01 mg/kg to about 5 mg/kg. Optionally, the dosage amount is from about 0.01 mg/kg to about 2.5 mg/kg.
Those of skill in the art will understand that the specific dose level and frequency of dosage for any particular subject may 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.
The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each subject's circumstances. Effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test systems. Further, depending on the route of administration, one of skill in the art would know how to determine doses that result in a plasma concentration for a desired level of response in the cells, tissues and/or organs of a subject.
Provided herein are methods to treat, prevent, or ameliorate a steroid receptor coactivator-related disease in a subject. Optionally, the steroid receptor coactivator-related disease is an SRC-1 related disease. Optionally, the steroid receptor coactivator-related disease is an SRC-2 related disease. Optionally, the steroid receptor coactivator-related disease is an SRC-3 related disease. In some embodiments, the steroid receptor coactivator-related disease is cancer.
The methods include administering to a subject an effective amount of one or more of the compounds or compositions described herein, or a pharmaceutically acceptable salt or prodrug thereof. Effective amount, when used to describe an amount of compound in a method, refers to the amount of a compound that achieves the desired pharmacological effect or other biological effect. The effective amount can be, for example, the concentrations of compounds at which SRC is stimulated in vitro, as provided herein. In some embodiments, the effective amount of the compounds at which SRC is stimulated in vitro can include a concentration of from about 1 μM to about 10 μM (e.g., from about 2 μM to about 8 μM or from about 3 μM to about 6 For example, the effective amount of MCB-613 at which SRC is stimulated in vitro can be from about 4 μM to about 6 μM (e.g., about 5 μM).
Also contemplated is a method that includes administering to the subject an amount of one or more compounds described herein such that an in vivo concentration at a target cell in the subject corresponding to the concentration administered in vitro is achieved.
The compounds and compositions described herein or pharmaceutically acceptable salts thereof are useful for treating cancer in humans, including, without limitation, pediatric and geriatric populations, and in animals, e.g., veterinary applications.
Optionally, the cancer is a cancer that has an increased expression of SRC-1, SRC-2, and/or SRC-3 as compared to non-cancerous cells of the same cell type. Optionally, the cancer is bladder cancer, brain cancer, breast cancer, colorectal cancer (e.g., colon cancer, rectal cancer), cervical cancer, chondrosarcoma, endometrial cancer, gastrointestinal cancer, gastric cancer, genitourinary cancer, head and neck cancer, hepatocellular carcinoma, liver cancer, lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, renal cancer, skin cancer, or testicular cancer. Optionally, the breast cancer is triple negative breast cancer. As used herein, triple negative breast cancer (TNBC) refers to a subtype of breast cancer that lacks detectable protein expression of the estrogen receptor (ER) and progesterone receptor (PR) and the absence of HER2 protein over expression. In other words, TNBC refers to an immunophenotype of breast cancer that is immunologically negative to ER, PR, and HER2.
Optionally, the cancer is glioblastoma. In some examples, the glioblastoma is a glioblastoma multiforme tumor. Optionally, the glioblastoma multiforme tumor is a pediatric glioblastoma multiforme tumor. The methods of treating glioblastoma include administering to the subject a compound as described herein. Optionally, the methods of treating glioblastoma include methods of suppressing the growth of glioblastoma cells in the subject.
The methods of treating or preventing cancer in a subject can further comprise administering to the subject one or more additional agents. The one or more additional agents and the compounds described herein or pharmaceutically acceptable salts or prodrugs thereof can be administered in any order, including concomitant, simultaneous, or sequential administration. Sequential administration can be administration in a temporally spaced order of up to several days apart. The methods can also include more than a single administration of the one or more additional agents and/or the compounds described herein or pharmaceutically acceptable salts or prodrugs thereof. The administration of the one or more additional agents and the compounds described herein or pharmaceutically acceptable salts or prodrugs thereof can be by the same or different routes and concurrently or sequentially.
Additional therapeutic agents include, but are not limited to, chemotherapeutic agents. A chemotherapeutic agent is a compound or composition effective in inhibiting or arresting the growth of an abnormally growing cell. Thus, such an agent may be used therapeutically to treat cancer as well as other diseases marked by abnormal cell growth. Illustrative examples of chemotherapeutic compounds include, but are not limited to, bexarotene, gefitinib, erlotinib, gemcitabine, paclitaxel, docetaxel, topotecan, irinotecan, temozolomide, carmustine, vinorelbine, capecitabine, leucovorin, oxaliplatin, bevacizumab, cetuximab, panitumumab, bortezomib, oblimersen, hexamethylmelamine, ifosfamide, CPT-11, deflunomide, cycloheximide, dicarbazine, asparaginase, mitotant, vinblastine sulfate, carboplatin, colchicine, etoposide, melphalan, 6-mercaptopurine, teniposide, vinblastine, antibiotic derivatives (e.g. anthracyclines such as doxorubicin, liposomal doxorubicin, and diethylstilbestrol doxorubicin, bleomycin, daunorubicin, and dactinomycin); antiestrogens (e.g., tamoxifen); antimetabolites (e.g., fluorouracil (FU), 5-FU, methotrexate, floxuridine, interferon alpha-2B, glutamic acid, plicamycin, mercaptopurine, and 6-thioguanine); cytotoxic agents (e.g., carmustine, BCNU, lomustine, CCNU, cytosine arabinoside, cyclophosphamide, estramustine, hydroxyurea, procarbazine, mitomycin, busulfan, cisplatin, vincristine and vincristine sulfate); hormones (e.g., medroxyprogesterone, estramustine phosphate sodium, ethinyl estradiol, estradiol, megestrol acetate, methyltestosterone, diethylstilbestrol diphosphate, chlorotrianisene, and testolactone); nitrogen mustard derivatives (e.g., mephalen, chlorambucil, mechlorethamine (nitrogen mustard) and thiotepa); and steroids (e.g., bethamethasone sodium phosphate).
Any of the aforementioned therapeutic agents can be used in any combination with the compositions described herein. Combinations are administered either concomitantly (e.g., as an admixture), separately but simultaneously (e.g., via separate intravenous lines into the same subject), or sequentially (e.g., one of the compounds or agents is given first followed by the second). Thus, the term combination is used to refer to concomitant, simultaneous, or sequential administration of two or more agents.
Optionally, a compound or therapeutic agent as described herein may be administered in combination with a radiation therapy, an immunotherapy, a gene therapy, or a surgery.
The methods and compounds as described herein are useful for both prophylactic and therapeutic treatment. For prophylactic use, a therapeutically effective amount of the compounds and compositions or pharmaceutically acceptable salts thereof as described herein are administered to a subject prior to onset (e.g., before obvious signs of cancer), during early onset (e.g., upon initial signs and symptoms of cancer), or after the development of cancer. Prophylactic administration can occur for several days to years prior to the manifestation of symptoms of cancer. Therapeutic treatment involves administering to a subject a therapeutically effective amount of the compounds and compositions or pharmaceutically acceptable salts thereof as described herein after cancer is diagnosed.
The compounds described herein are also useful in stimulating a steroid receptor coactivator protein in a cell. The methods of stimulating a steroid receptor coactivator protein in a cell includes contacting a cell with an effective amount of one or more of the compounds as described herein. Optionally, the steroid receptor coactivator protein is one or more of SRC-1, SRC-2, or SRC-3. Optionally, the contacting is performed in vivo. Optionally, the contacting is performed in vitro.
The methods herein for prophylactic and therapeutic treatment optionally comprise selecting a subject with or at risk of developing cancer. A skilled artisan can make such a determination using, for example, a variety of prognostic and diagnostic methods, including, for example, a personal or family history of the disease or condition, clinical tests (e.g., imaging, biopsy, genetic tests), and the like. Optionally, the methods herein can be used for preventing relapse of cancer in a subject in remission (e.g., a subject that previously had cancer).
Also provided herein are kits for treating or preventing cancer in a subject. A kit can include any of the compounds or compositions described herein. For example, a kit can include a compound of Formula I, Formula II, or combinations thereof. A kit can further include one or more additional agents, such as one or more chemotherapeutic agents. A kit can include an oral formulation of any of the compounds or compositions described herein. A kit can include an intravenous formulation of any of the compounds or compositions described herein. A kit can additionally include directions for use of the kit (e.g., instructions for treating a subject), a container, a means for administering the compounds or compositions (e.g., a syringe), and/or a carrier.
As used herein the terms treatment, treat, or treating refer to a method of reducing one or more symptoms of a disease or condition. Thus in the disclosed method, treatment can refer to a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in the severity of one or more symptoms of the disease or condition. For example, a method for treating a disease is considered to be a treatment if there is a 10% reduction in one or more symptoms or signs (e.g., size of the tumor or rate of tumor growth) of the disease in a subject as compared to a control. As used herein, control refers to the untreated condition (e.g., the tumor cells not treated with the compounds and compositions described herein). Thus the reduction can be a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any percent reduction in between 10% and 100% as compared to native or control levels. It is understood that treatment does not necessarily refer to a cure or complete ablation of the disease, condition, or symptoms of the disease or condition.
As used herein, the terms prevent, preventing, and prevention of a disease or disorder refer to an action, for example, administration of a composition or therapeutic agent, that occurs before or at about the same time a subject begins to show one or more symptoms of the disease or disorder, which inhibits or delays onset or severity of one or more symptoms of the disease or disorder.
As used herein, references to decreasing, reducing, or inhibiting include a change of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater as compared to a control level. Such terms can include, but do not necessarily include, complete elimination.
As used herein, subject means both mammals and non-mammals. Mammals include, for example, humans; non-human primates, e.g., apes and monkeys; cattle; horses; sheep; rats; mice; pigs; and goats. Non-mammals include, for example, fish and birds.
Throughout this application, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application.
The examples below are intended to further illustrate certain aspects of the methods and compositions described herein, and are not intended to limit the scope of the claims.
All reagents were purchased from Alfa Aesar (Ward Hill, Mass.) or Aldrich (Milwaukee, Wis.). Compounds were characterized by 1H NMR on a Varian 400-MR spectrometer (Palo Alto, Calif.). The purities were monitored by a Shimadzu Prominence HPLC with a Zorbax C18 or C8 column (4.6×250 mm) or 1H (at 400 MHz) absolute spin-count quantitative NMR analysis with imidazole as an internal standard. The purities of the compounds were found to be >95%.
General method A. Sodium hydroxide solution (400 mg, 10 mmol, 2.5 equiv.) in water/ethanol (V/V=1/1, 8 mL) was added dropwise to a solution of 3,4,5-trimethoxybenzaldehyde (4 mmol, 1 equiv.) and dihydro-2H-pyran-4(3H)-one (2 mmol, 0.5 equiv). The mixture was stirred for 12 hours at room temperature. The precipitate was filtered and washed with water and ethanol to give a white or pale yellow powder, in 70-90% yield.
General method B. A solution of hydrogen chloride in acetic acid (20 mL) was added into cyclohexanone (2 mmol, 0.5 equiv) and 3-pyridinecarboxaldehyde (4 mmol, 1 equiv.) at room temperate. The mixture was stirred at room temperature for 12 hours. The solution was neutralized with aqueous NaHCO3 (5% w/v). The precipitate was filtered and washed with water and ethanol to give a white or pale yellow powder, in 70-90% yield.
General method C. A mixture of a cyclohexanone (0.5 mmol), benzylamine (0.5 mmol) and anhydrous MgSO4 (5.0 mmol) in anhydrous CH2Cl2 (5 mL) was stirred for 24 hours. 3-Pyridinecarboxaldehyde (0.5 mmol) was added dropwise and the solution was stirred for 12 hours. MgSO4 was filtered off and the filtrate was concentrated. The concentrated filtrate was purified using column chromatography (silica gel, CH2Cl2:MeOH=10:1) to give the mono-adduct as a yellow oil. (58 mg, 62% yield).
Compound SYC-907 was prepared from cyclohexanone and 3-pyridinecarboxaldehyde, following general methods B, as a yellow solid. 1H NMR (400 MHz, CDCl3): δ 8.71 (bs, 2H), 8.56 (d, J=4.8 Hz, 2H), 7.76-7.74 (m, 4H), 7.35-7.32 (m, 2H), 2.94-2.91 (m, 4H), 1.84-1.81 (m, 2H).
Compound SYC-908 was prepared from 4-ethylcyclohexanone and 2-pyridinecarboxaldehyde, following general methods B, as a yellow solid. 1H NMR (400 MHz, CDCl3): δ 8.71-6.93 (m, 1H), 8.51-8.50 (m, 1H), 7.71-7.68 (m, 4H), 7.47-7.41 (m, 2H), 7.20-7.16 (m, 2H), 3.63-3.59 (m, 2H), 3.09-2.71 (m, 2H), 1.69-1.42 (m, 3H), 0.94-0.72 (m, 3H).
Compound SYC-909 was prepared from 4-methylcyclohexanone and 3-pyridinecarboxaldehyde, following general methods B, as a yellow solid. 1H NMR (400 MHz, CDCl3): δ 8.71 (bs, 2H), 8.57 (d, J=4.8 Hz, 2H), 7.75-7.74 (m, 4H), 7.36-7.33 (m, 2H), 3.05 (d, J=16 Hz, 2H), 2.57-2.50 (m, 2H), 1.92-1.91 (m, 1H), 1.09-1.07 (m, 3H).
Compound SYC-910 was prepared from dihydro-2H-pyran-4(3H)-one and 3,4,5-trimethoxybenzaldehyde, following general methods A, as a yellow solid. 1H NMR (400 MHz, CDCl3): δ 7.75 (s, 2H), 6.54 (s, 4H), 4.96 (m, 4H), 3.89-3.88 (m, 18H).
Compound SYC-911 was prepared from 1-methylpiperidin-4-one and 2,3-dimethoxybenzaldehyde, following general methods B, as a yellow solid. 1H NMR (400 MHz, CDCl3): δ 8.01 (bs, 2H), 7.09-7.05 (m, 2H), 6.94 (d, J=8.4 Hz, 2H), 6.83 (d, J=7.2 Hz, 2H), 3.98-3.80 (m, 12H), 3.63 (bs, 4H), 2.37 (s, 3H).
Compound SYC-912 was prepared from 1-methylpiperidin-4-one and 3,4,5-trimethoxybenzaldehyde, following general methods B, as a yellow solid. 1H NMR (400 MHz, CDCl3): δ 7.74 (bs, 2H), 6.62 (s, 4H), 3.89-3.88 (m, 18H), 3.79 (bs, 4H), 2.47 (s, 3H).
Compound SYC-913 was prepared from 1-methylpiperidin-4-one and 2-pyridinecarboxaldehyde, following general methods B, as a yellow solid. 1H NMR (400 MHz, CDCl3): δ 8.71 (bs, 2H), 7.72-7.68 (m, 2H), 7.61 (bs, 2H), 7.46 (d, J=8.0 Hz, 2H), 7.21-7.18 (m, 2H), 4.16 (bs, 4H), 2.53 (s, 3H).
Compound SYC-914 was prepared from 1-ethylpiperidin-4-one and 3,4,5-trimethoxybenzaldehyde, following general methods B, as a yellow solid. 1H NMR (400 MHz, CDCl3): δ 7.74 (bs, 2H), 6.62 (s, 4H), 3.89-3.86 (m, 22H), 2.62-2.61 (m, 2H), 1.07 (t, J=7.2 Hz, 3H).
Compound SYC-915 was prepared from 1-ethylpiperidin-4-one and 2-pyridinecarboxaldehyde, following general methods B, as a yellow solid. 1H NMR (400 MHz, CDCl3): δ 8.71 (bs, 2H), 7.72-7.68 (m, 2H), 7.62 (bs, 2H), 7.46 (d, J=8.0 Hz, 2H), 7.21-7.17 (m, 2H), 4.23 (bs, 4H), 2.71-2.66 (m, 2H), 1.14 (t, J=7.2 Hz, 3H)
Compound SYC-916 was prepared from 1-isopropylpiperidin-4-one and 2-pyridinecarboxaldehyde, following general methods B, as a yellow solid. 1H NMR (400 MHz, CDCl3): δ 8.71 (bs, 2H), 7.71-7.67 (m, 2H), 7.60 (bs, 2H), 7.43 (d, J=8.0 Hz, 2H), 7.20-7.17 (m, 2H), 4.28 (bs, 4H), 3.00-2.97 (m, 1H), 1.13-1.12 (m, 6H).
Compound SYC-917 was prepared from 1-isopropylpiperidin-4-one and 2-bromonicotinaldehyde, following general methods B, as a yellow solid. 1H NMR (400 MHz, CDCl3): δ 8.34 (bs, 2H), 7.99-7.92 (m, 2H), 7.35-7.29 (m, 4H), 3.64 (bs, 4H), 2.96-2.92 (m, 1H), 1.05-1.02 (m, 6H).
Compound SYC-918 was prepared from 1-isopropylpiperidin-4-one and 6-(trifluoromethyl)nicotinaldehyde, following general methods B, as a yellow solid. 1H NMR (400 MHz, CDCl3): δ 8.76 (bs, 2H), 7.87 (d, J=8.4 Hz, 2H), 7.77-7.75 (m, 4H), 3.84 (bs, 4H), 3.00-2.96 (m, 1H), 1.08-1.06 (m, 6H).
Compound SYC-919 was prepared from 4-isopropylcyclohexanone and 2-pyridinecarboxaldehyde, following general methods B, as a yellow solid. 1H NMR (400 MHz, CDCl3): δ 8.71 (bs, 2H), 7.72-7.68 (m, 2H), 7.47-7.43 (m, 2H), 7.20-7.16 (m, 2H), 7.20-7.17 (m, 2H), 3.62-3.58 (m, 2H), 2.84-2.77 (m, 2H), 1.70-1.63 (m, 2H), 0.95-0.81 (m, 6H).
Compound SYC-920 was prepared from 1-isopropylpiperidin-4-one and 2,3-dimethoxybenzaldehyde, following general methods B, as a yellow solid. 1H NMR (400 MHz, CDCl3): δ 7.99 (bs, 2H), 7.09-7.07 (m, 2H), 6.94-6.92 (m, 2H), 6.85-6.83 (m, 2H), 3.88-3.76 (m, 16H), 2.87-2.84 (m, 1H), 0.98-0.96 (m, 6H).
Compound SYC-921 was prepared from 1-isopropylpiperidin-4-one and 3,4,5-trimethoxybenzaldehyde, following general methods B, as a yellow solid. 1H NMR (400 MHz, CDCl3): δ 7.71 (bs, 2H), 6.63 (s, 4H), 3.94-3.82 (m, 22H), 2.92-2.88 (m, 1H), 1.08-1.06 (m, 6H).
Compound SYC-849 was prepared from dihydro-2H-pyran-4(3H)-one and 2-methoxybenzaldehyde, following general methods A, as a yellow solid. 1H NMR (400 MHz, CDCl3): δ 8.07 (bs, 2H), 7.36-7.32 (m, 2H), 7.06-7.04 (m, 2H), 6.97-6.91 (m, 4H), 4.79 (bs, 4H), 3.86 (s, 6H).
Compound SYC-850 was prepared from dihydro-2H-pyran-4(3H)-one and 3-methoxybenzaldehyde, following general methods A, as a yellow solid. 1H NMR (400 MHz, CDCl3): δ 7.79 (bs, 2H), 7.35-7.31 (m, 2H), 6.94-6.78 (m, 6H), 4.92 (bs, 4H), 3.83-3.81 (m, 6H).
Compound SYC-851 was prepared from dihydro-2H-pyran-4(3H)-one and 3-Pyridinecarboxaldehyde, following general methods B, as a yellow solid. 1H NMR (400 MHz, CDCl3): δ 8.61-8.59 (m, 4H), 7.79 (bs, 2H), 7.63-7.61 (m, 2H), 7.39-7.36 (m, 2H), 4.92 (bs, 4H).
Compound SYC-852 was prepared from 1-methylpiperidin-4-one and 2-methoxybenzaldehyde, following general methods B, as a yellow solid. 1H NMR (400 MHz, CDCl3): δ 8.07 (bs, 2H), 7.35-7.31 (m, 2H), 7.19-7.17 (m, 2H), 6.98-6.90 (m, 4H), 3.87 (s, 6H), 3.70 (bs, 4H), 2.37 (s, 3H).
Compound SYC-853 was prepared from 1-methylpiperidin-4-one and 2,6-dimethoxybenzaldehyde, following general methods B, as a yellow solid. 1H NMR (400 MHz, CDCl3): δ 7.79 (bs, 2H), 7.29-7.27 (m, 2H), 6.56 (d, J=8.4 Hz, 4H), 3.80 (s, 12H), 3.71 (bs, 4H), 2.25 (s, 3H).
Compound SYC-854 was prepared from 1-ethylpiperidin-4-one and 3-pyridinecarboxaldehyde, following general methods B, as a yellow solid. 1H NMR (400 MHz, CDCl3): δ 8.67 (bs, 2H), 8.60-8.59 (m, 2H), 7.76 (bs, 2H), 7.71-7.69 (m, 2H), 7.38-7.35 (m, 2H), 3.81 (bs, 4H), 2.64-2.59 (m, 2H), 1.06 (t, J=7.2 Hz, 3H).
Compound SYC-855 was prepared from 1-isopropylpiperidin-4-one and 3-pyridinecarboxaldehyde, following general methods B, as a yellow solid. 1H NMR (400 MHz, CDCl3): δ 8.67 (bs, 2H), 8.59-8.58 (m, 2H), 7.72-7.69 (m, 4H), 7.38-7.35 (m, 2H), 3.84 (bs, 4H), 2.97-2.92 (m, 1H), 1.06-1.04 (m, 6H).
Compound SYC-856 was prepared from 4-isopropylcyclohexanone and 3-methoxybenzaldehyde, following general methods A, as a yellow solid. 1H NMR (400 MHz, CDCl3): δ 7.75 (bs, 2H), 7.34-7.30 (m, 2H), 7.05 (d, J=7.6 Hz, 2H), 6.98 (bs, 2H), 6.90-6.88 (m, 2H), 3.83 (bs, 6H), 3.08-3.03 (m, 2H), 2.60-2.53 (m, 2H), 1.65-1.51 (m, 2H), 0.90-0.89 (m, 6H).
Compound SYC-857 was prepared from 4-isopropylcyclohexanone and 3,4,5-trimethoxybenzaldehyde, following general methods A, as a yellow solid. 1H NMR (400 MHz, CDCl3): δ 7.71 (bs, 2H), 6.71 (bs, 4H), 3.89 (bs, 18H), 3.15-3.10 (m, 2H), 2.59-2.52 (m, 2H), 1.68-1.53 (m, 2H), 0.94-0.93 (m, 6H).
Compound SYC-922 was prepared from cyclohexanone and 3-pyridinecarboxaldehyde, following general methods C, as a yellow solid. 1H NMR (400 MHz, CDCl3): δ 8.62 (bs, 1H), 7.69-7.65 (m, 1H), 7.35-7.12 (m, 3H), 3.18-3.15 (m, 2H), 2.55-2.51 (m, 2H), 1.92-1.75 (m, 4H).
Compound SYC-923 was prepared from cyclohexanone and 3-pyridinecarboxaldehyde, following general methods C, as a yellow solid. 1H NMR (400 MHz, CDCl3): δ 8.59-8.38 (m, 3H), 7.69-7.50 (m, 3H), 7.39-7.00 (m, 4H), 3.40-3.37 (m, 4H), 1.83-1.80 (m, 2H).
The data provided herein demonstrate that MCB-613 can super-stimulate SRCs' transcriptional activity. Further investigation revealed that MCB-613 increases SRCs' interaction with other coactivators and markedly induces ER stress coupled to the generation of reactive oxygen species (ROS) and the activation of the Abl kinase-SRC axis. Since cancer cells overexpress SRCs and rely on them for growth, MCB-613 induces excessive stress selectively in cancer cells.
4-Ethyl-2,6-bis-pyridin-3-ylmethylene-cyclohexanone (MCB-613) is a potent SRC small molecule stimulator, capable of hyper-activating all three SRC proteins, leading to the dramatic exacerbation of the proteostatic and oxidative stress that already exists in cancer cells, and causing selective cancer cell death. These unexpected results show that targeted over-stimulation of the very oncogenes that cancer cells depend upon for accelerated proliferation and survival can be used against them as a novel therapeutic approach.
Chemicals.
MCB-613 (4-Ethyl-2,6-bis-pyridin-3-ylmethylene-cyclohexanone) was obtained from ChemBridge Corporation (San Diego, Calif.). N-Acetyl cysteine, cycloheximide, bufalin, chloroquine and curcumin were from Sigma (St. Louis, Mo.). MG132 and z-VAD-fmk were purchased from EMD Millipore (Billerica, Mass.). 15d-PGJ2 was from Cayman Chemical (Ann Arbor, Mich.). Kinase inhibitor library, AT9283, PHA7393598 and geldanamycin were purchased from SelleckChem (Houston, Tex.). CM-H2DCFDA was from Invitrogen (Carlsbad, Calif.). Phos-tag was purchased from Wako Chemicals (Richmond, Va.).
Fluorescence Spectrometry.
Fluorescence spectrometric measurements were performed on a SLM 48000S fluorescence spectrophotometer (SLM-Aminco, Rochester, N.Y.) and an Agilent Cary Eclipse Fluorescence spectrophotometer (Agilent Technologies, Inc., Santa Clara, Calif.) using the GST fusion proteins of different portions of SRC-3 expressed and purified. A total of 1.5 μM of GST SRC-3 RID, CID or bHLH was placed in a fluorescence cuvette and excited by UV light at a wavelength of 278 nm with a 2 nm bandwidth and the emission spectra were recorded from 290 nm to above 500 nm with a bandwidth of 4 nm. The aliquot size of test compound was maintained below 5% of the total sample volume in order to minimize the effects of dilution.
Human Stress and Toxicity PathwayFinder qPCR Array.
SRC-3 WT and KO HeLa cells were treated with MCB-613 or DMSO for 24 hours. Total RNA was extracted and subjected to reverse transcription using RT2 First Strand Kit (SA Biosciences) followed by the analyses using the Human Stress and Toxicity PathwayFinder qPCR Array (SA Biosciences) according to the manufacturer's instruction.
MCF-7 Xenograft Tumor Model.
Twenty 6-8 week-old athymic nude female mice were obtained from Harlan. Two days before cancer cell injection, one estradiol pellet was embedded under the skin for each mouse and a new pellet was added twice at a one month interval. Following this, 1×106 MCF-7 cells (504) mixed with equal volume of growth factor reduced matrigel (BD Biosciences) were injected into the fat pad of each of the 2nd pair mammary gland without clearing. The MCB-613 treatment which lasted for seven weeks was started approximately seven weeks after the cell injection when tumor sizes reached above 5 mm in diameter. Ten mice received MCB-613 (20 mg/kg) in saline by i.p. injection, whereas the control group contained 10 mice which received saline only. The compound was injected three times a week. The mice were weighed once a week and tumors were measured twice a week during the treatment period.
Statistics.
Statistical significance was determined by 2-tailed Student's t test. AP value of less than 0.05 was considered statistically significant.
Antibodies.
Antibodies against SRC-1, SRC-3, Calnexin, Abl, phospho-CrKL (Y207), phospho-eIF2alpha (S51), cleaved caspase-3, ubiquitin and spliced Xbp1 were purchased from Cell Signaling (Danvers, Mass.). Antibodies against CARM1 and SRC-2 were obtained from Bethyl Laboratories (Montgomery, Tex.). FLAG antibody was obtained from Sigma. Antibody against phosphor-IRE1alpha (S724) was purchased from Abcam (Cambridge, Mass.). Antibodies against ATF4, GAPDH and CBP were purchased from Santa Cruz Biotechnology (Santa Cruz, Calif.).
Cell Culture.
Cell lines were maintained in DMEM (HeLa, MCF-7, HepG2), RPMI 1640 (H1299), or DMEM/F12 (PC-3) supplemented with 10% fetal calf serum, penicillin and streptomycin (100 U/ml). Primary mouse hepatocytes were isolated and cultured. Mouse embryonic fibroblasts (MEF) were cultured in DMEM. All cells were maintained at 37° C. under 5% CO2.
Plasmids and Transfection.
The constructs expressing the Gal4 responsive luciferase reporter (pG5-luc), Gal4 DBD fusion with SRC-1, SRC-3 and SRC-3 (pBIND-SRC-1/-2/-3), MMP2 or MMP13 promoter driven luciferase reporter (MMP2-luc, MMP13-luc) were prepared. Twenty-four hours before transfection, HeLa cells were plated in 24-well dishes. The indicated expression plasmids were transfected into cells using Lipofectamine 2000 according to the manufacturer's instructions.
Luciferase Assays.
After various compound treatments, cells were lysed in luciferase lysis buffer and assayed for luciferase activity using the ONE-Glo luciferase assay system (Promega, Madison, Wis.). All luciferase activities were normalized to protein concentration determined by Bradford assay (Bio-Rad, Hercules, Calif.).
Western Blot and Immunoprecipitation.
For Western blotting, cells were harvested and lysed in lysis buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 10% glycerol, 0.5% NP40, protease and phosphatase inhibitor) and then centrifuged for 15 min at 14000 rpm at 4° C. After total cellular protein concentration was determined by Bradford analysis (Bio-Rad, Hercules, Calif.), protein lysates were loaded onto and resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes (BioRad) which were blocked and incubated with indicated antibodies. For immunoprecipitation, 10 μg of plasmid encoding FLAG-SRC-3 was transfected into HeLa cells with 90% confluence in one 10 cm plate with lipofectamine 2000 (Invitrogen). Twenty-four hours after transfection, the cells were treated with MCB-613 for one hour and lysed with lysis buffer (25 mM Tris-Cl pH7.5, 150 mM NaCl, 5% glycerol, 1 mM EDTA, 1% Triton X-100, protease and phosphatase inhibitor) at 4° C. After a 30-second sonication, the lysate was cleared by centrifugation at 14000 rpm at 4° C. for 15 minutes and was incubated with anti-Flag-M2 agarose affinity gel (Sigma) for four hours. The agarose gel was washed four times with washing buffer (25 mM Tris pH 8.0, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100) and resuspended with SDS loading buffer for Western blotting.
Cell Viability Assays.
Cells were seeded in 96-well plates and allowed to reach 70% to 80% confluence. After indicated compound treatments, relative numbers of viable cells were measured by MTS assay using the Cell Titer 96 Aqueous One Solution Cell Proliferation Assay (Promega).
Measurement of Reactive Oxygen Species (ROS).
Cells were loaded with 10 μM general ROS indicator CM-H2DCFDA in serum free DMEM for 30 minutes at 37° C. in the dark. After a 5-minute recovery in complete DMEM, cells were treated with indicated concentrations of MCB-613 for 30 minutes, then fixed and examined by fluorescence microscopy.
Quantitative PCR Analysis.
HeLa cell total RNA was extracted using the RNeasy mini Kit (Qiagen, Valencia, Calif.), which was then reverse transcribed into cDNA with the SuperScript VILO cDNA synthesis kit (Invitrogen). Quantitative PCR was performed using a Taqman-based assay using the ABI StepOnePlus real-time PCR system (Biosystems, Foster City, Calif.). Relative quantitation was obtained by normalizing to the internal control GAPDH.
MCB-613 is a Pan-SRC Stimulator
In a series of high throughput screens originally designed to identify SRC small molecule inhibitors, HEK293 cells transfected with a Gal4 responsive luciferase reporter (pG5-luc) and a construct encoding SRC-1, SRC-2 or SRC-3 fused with the DNA binding domain of Gal4 (pBIND-SRC-1, pBIND-SRC-2 or pBIND-SRC-3) were exposed to treatments with 359,484 compounds from a MLPCN chemical library (PubChem AID:588362, AID:652008 and AID:588354). In addition to inhibitors that were initially sought in these screens, a number of compounds were found to stimulate the activity of SRC-1 and SRC-3. Primary screens indicated that there were 106 and 28 potential SRC-1 and SRC-3 stimulators, respectively, using a 2-fold activation cutoff compared with vehicle controls. Upon further testing and verification of a subset of these top compounds, MCB-613 (
Since SRC-3 is preferentially utilized by AP-1 and PEA3 as a coactivator to drive expression of MMP2 and MMP13, cells were transfected with a MMP2 or MMP13 promoter driven luciferase reporter (MMP2-luc or MMP13-luc) and treated with MCB-613. It was found that SRC-3's coactivation of the MMP2 and MMP13 promoters was greatly enhanced by MCB-613 (
In order to characterize the underlying mechanism of MCB-613's activation of SRCs, a goal was identified to determine whether MCB-613 increases SRC activity by elevating the concentrations of SRC proteins in the cell. Instead, it was found that treatment of cells with MCB-613 for 24 hours resulted in decreases in SRC-1, SRC-2 and SRC-3 protein levels (
Next, it was assessed whether MCB-613 physically interacts with SRCs. Fluorescence spectroscopy was used to measure the intrinsic fluorescence of different SRC-3 protein domains, expressed as GST fusions in the presence of MCB-613, based on the theory that the direct SRC-3 binding with MCB-613 should quench its intrinsic fluorescence emission. As shown in
Activated SRC-3 has been shown to recruit other transcriptional coactivators including CBP and CARM1 to form a multi-coactivator complex. To test whether MCB-613 affects the ability of SRC-3 to interact with these coactivator complex members, HeLa cells overexpressing FLAG-SRC-3 were treated with MCB-613 for one hour and subjected to coimmunoprecipitation to assess SRC-3-CBP-CARM1 complex formation. As shown in
MCB-613 Selectively Kills Cancer Cells
MCB-613 is cytotoxic. It can efficiently kill a variety of cancer cell lines (
A non-apoptotic form of cell death, paraptosis, is characterized by extensive cytoplasmic vacuolization caused by the dilation of the endoplasmic reticulum (ER) and mitochondria and it does not rely upon hallmarks of apoptosis such as DNA fragmentation and caspase activation. Paraptotic-like cell death has been observed in developmental processes such as embryo development and neuronal degeneration, in several artificial or natural cellular models, and in response to treatment with some anti-cancer agents. Although the molecular details underlying paraptosis are still relatively understudied, there is evidence that the cytoplasmic vacuolization associated with it results from perturbations in ER and proteasome function. In addition to proteasome dysfunction and ER stress, paraptosis requires de novo protein synthesis and is linked to cellular redox homeostasis.
Consistent with the involvement of paraptosis in MCB-613 treated cells, vacuoles in cells treated with MCB-613 stained positive for calnexin (
SRC Hyper-Activation is Critical for Paraptosis Induced by MCB-613
Since MCB-613 super-activates SRCs and induces paraptotic-like cell death, it was determined whether a causal link between these two events exists. SRC-3 KO HeLa cell viability was found to be less affected by MCB-613 compared with WT cells (
It was also found that oxidative stress related genes are differentially regulated in MCB-613 treated SRC-3 WT and KO cells (
To further demonstrate that SRC activation underlies MCB-613-induced paraptosis, HeLa cells were treated with both MCB-613 and the SRC SMI bufalin, which has been shown to inhibit all three SRCs. If SRCs are responsible for driving MCB-613 induced paraptosis, bufalin co-treatment should abrogate the effects of MCB-613. Vacuolization induced by MCB-613 was effectively inhibited by bufalin (
One response the cells engage to counteract ER stress is to induce molecular chaperones, such as heat shock proteins, in order to enhance the folding capacity of ER and alleviate the accumulation of misfolded/unfolded proteins. Indeed, many heat shock proteins were significantly induced by the treatment with MCB-613, showing that the cells are striving to battle the enormous stress imposed by the compound. MCB-613 and geldanamycin, an HSP90 inhibitor, synergized to greatly induce ER stress and cell death, even though suboptimal concentrations were used in the assays (
More support for the close relationship between SRCs and paraptosis is evidenced by observations that additional agents, including curcumin, 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) and MG132, robustly enhance the intrinsic transcriptional activities of SRCs (
Oxidative Stress Induced by MCB-613 Contributes to SRC Hyper-Activation
The relationship between oxidative stress and the activation of SRCs was next examined. To investigate this question, the effect of co-treatment with the antioxidant N-acetylcysteine (NAC) on MCB-613-induction of SRC intrinsic transcriptional activity was tested. It was found that NAC was able to abrogate the stimulatory effects of MCB-613 on SRC transcriptional activity (
The impact of MCB-613 treatment on SRC-3 phosphorylation was also explored. Using a phos-tag SDS-PAGE system that allows phosphorylated proteins to be characterized by differences in their migration rate, it was found that MCB-613 treatment resulted in a SRC-3 species with reduced mobility in the gel (
Abl is a non-receptor tyrosine kinase present in both the cytoplasm and nucleus that has been implicated in a variety of cellular processes such as growth, differentiation and stress response. Not to be bound by theory, Abl is a promising candidate kinase for MCB-613 mediated SRC hyper-stimulation because 1) acute hyper-activation of the oncogenic Bcr-Abl fusion protein induces severe cytoplasmic vacuolization and ER stress; 2) Abl phosphorylates and activates SRC-3; and 3) Abl is activated in response to oxidative stress. It was first tested whether Abl is activated by MCB-613 treatment by assaying CrkL (Y207) phosphorylation as a marker for Abl activation. As shown in
MCB-613 Inhibits Tumor Growth In Vivo
To further evaluate the anti-cancer potential of MCB-613, a MCF-7 breast cancer mouse xenograft model was employed to assess the tumor suppressive effects of MCB-613 in vivo. Tumors were established in athymic nude mice seven weeks after injection of MCF-7 cells into cleared mammary fat pads. An MCB-613 treated group (n=10) received i.p. injection of the compound (20 mg/kg) three times a week, while a control group (n=10) was injected with a saline vehicle. MCB-613 treatment did not lead to obvious animal toxicity as the body weights between control and treated groups were identical (
A Model for SRC Hyper-Activation by MCB-613.
A model for SRC hyper-activation by MCB-613 is depicted in
The effects of MCB-613, SYC-851 (Compound 4-8), and SYC-855 (Compound 7-8) on tumor cell viability in cell culture was determined using pediatric glioblastoma multiforme cell lines 4687, 3752, and R0315. Cells were plated as monolayer cultures and treated 24 hours later with 0.5 μM, 2 μM, 3 μM, 4 μM, 6 μM, 8 μM, and 10 μM of the compounds. After 72 hours, cells were harvested and assayed for viability using a CellTiter-Glo assay (Invitrogen). The results for pediatric glioblastoma multiforme cell lines 4687, 3752, and R0315 are shown in
The effects of MCB-613, SYC-922 (Compound 1-8-1), SYC-923 (Compound 1-8-2), SYC-924 (Compound 4-8-1), SYC-925 (Compound 4-8-2), SYC-926 (Compound 7-8-1), and SYC-927 (Compound 7-8-2) on tumor cell viability in cell culture were determined using pediatric glioblastoma multiforme cell lines 4687 and R0315. Cells were plated as monolayer cultures and treated 24 hours later with 0.5 μM, 1 μM, 2 μM, 3 μM, 4 μM, 6 μM, 8 μM, and 10 μM of the compounds. After 72 hours, cells were harvested and assayed for viability using a CellTiter-Glo assay (Invitrogen). The results for pediatric glioblastoma multiforme cell lines 4687 and R0315 are shown in
The effects of SYC-907 (Compound 1-8), SYC-908 (Compound 2-9), SYC-909 (Compound 3-8), SYC-910 (Compound 4-5), SYC-911 (Compound 5-3), SYC-912 (Compound 5-5), SYC-928 (Compound 5-8), and SYC-914 (Compound 6-5) were determined using pediatric glioblastoma multiforme cell lines 4687 and R0315 according to the procedures described above. The results are shown in
The effects of SYC-915 (Compound 6-9), SYC-916 (Compound 7-9), SYC-917 (Compound 7-18), SYC-918 (Compound 7-19), SYC-919 (Compound 8-9), SYC-920 (Compound 7-3), SYC-921 (Compound 7-5), and SYC-855 (Compound 7-8) were determined using pediatric glioblastoma multiforme cell lines 4687 and R0315 according to the procedures described above. The results are shown in
The effects of SYC-851 (Compound 4-8), SYC-852 (Compound 5-1), SYC-853 (Compound 5-4), and SYC-855 (Compound 7-8) on TM4 Sertoli cells and on GBM cancer cells were determined. Cells were plated as monolayer cultures and treated 24 hours later with the indicated concentration of compounds. After 72 hours, cells were harvested and assayed for viability using a CellTiter-Glo assay (Invitrogen). The results for TM4 Setoli cells and for pediatric glioblastoma multiforme cell lines R0315 and 4687 and are shown in
The effects of MCB-613, SYC-851 (Compound 4-8), SYC-852 (Compound 5-1), and SYC-855 (Compound 7-8) on SRC-3 instrinsic transcriptional activity was determined by measuring luciferase activity. HeLa cells were transfected with a Gal4 responsive luciferase reporter (pG5-luc) and a construct encoding SRC-1, SRC-2 or SRC-3 fused with the DNA binding domain of Gal4 (pBIND-SRC-1, pBIND-SRC-2 or pBIND-SRC-3) were treated with the indicated compounds. The results are shown in
The compounds and methods of the appended claims are not limited in scope by the specific compounds and methods described herein, which are intended as illustrations of a few aspects of the claims and any compounds and methods that are functionally equivalent are within the scope of this disclosure. Various modifications of the compounds and methods in addition to those shown and described herein are intended to fall within the scope of the appended claims. Further, while only certain representative compounds, methods, and aspects of these compounds and methods are specifically described, other compounds and methods are intended to fall within the scope of the appended claims. Thus, a combination of steps, elements, components, or constituents can be explicitly mentioned herein; however, all other combinations of steps, elements, components, and constituents are included, even though not explicitly stated.
This application claims priority to U.S. Provisional Application No. 62/097,995, filed Dec. 30, 2014, which is incorporated herein by reference in its entirety.
This invention was made with government support under Grant No. DOD BC120894, awarded by the Department of Defense, and Grant Nos. DK059820 and HD076596, awarded by the National Institutes of Health. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2015/067770 | 12/29/2015 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62097995 | Dec 2014 | US |
