Patent Application
20240315994
The invention relates to compounds, compositions, and methods for inducing ferroptosis in a cell.
Efforts to inhibit GPX4 have focused on the use of electrophiles capable of forming covalent bonds with a selenocysteine residue in the GPX4 active site. Despite its clear biological role in protecting cancer cells from ferroptosis, to date, no targeted GPX4 inhibitors have been profiled in the clinic. There is a need in the art for novel modulators of GPX4.
All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and/or take precedence over any such contradictory material.
“Isomer” means molecules with identical molecular formulas, that is the same number of atoms of each element, but distinct arrangements of atoms in space.
“Covalent inhibitor” means compounds that by design are intended to form a covalent bond with a specific molecular target.
“Pharmaceutically acceptable” means on balance, safe for use in humans or animals, without undue side effects.
“Pharmaceutically acceptable salt” means a salt of the compounds of the present invention which is pharmaceutically acceptable and which possess the desired pharmacological activity. Such salts include, for example, acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or with organic acids such as acetic acid, propionic acid, hexanoic acid, heptanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, o-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, p-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, p-toluenesulfonic acid, camphorsulfonic acid, 4-methylbicyclo[2.2.2]oct-2-ene-1-carboxylic acid, glucoheptonic acid, 4,4′-methylenebis(3-hydroxy-2-ene-1-carboxylic acid), 3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid, lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid and the like. Pharmaceutically acceptable salts also include base addition salts which may be formed when acidic protons present are capable of reacting with inorganic or organic bases. Acceptable inorganic bases include sodium hydroxide, sodium carbonate, potassium hydroxide, aluminum hydroxide and calcium hydroxide. Acceptable organic bases include ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine and the like.
“Ferroptosis” means regulated cell death that is iron-dependent. Ferroptosis is characterized by the iron-dependent accumulation of lethal lipid reactive oxygen species.
“GPX4” means the glutathione peroxidase 4, a glutathione metabolism enzyme.
“In vitro” means an artificial environment created outside a living multicellular organisms (e.g., a test tube or culture plate) used in experimental research to study a disease or process. As used herein, “in vitro” includes processes performed in intact cells growing in culture.
“In vivo” means that which takes place inside an organism and more specifically to a process performed in or on the living tissue of a whole, living multicellular organism (animal), such as a mammal, as opposed to a partial or dead one.
“Ex vivo” refers to a process performed in an artificial environment outside the organism on living cells or tissue which are removed from an organism and subsequently returned to an organism.
“Mesenchymal tumor” or “mesenchymal cancer” refers to tumors that arise from mesenchymal tissue or tumors that have undergone epithelial to mesenchymal transition. The epithelial-mesenchymal transition (EMT) is a process by which epithelial cells lose their cell polarity and cell-cell adhesion and gain migratory and invasive properties to become mesenchymal stem cells. EMT has also been shown to occur in the initiation of metastasis in cancer progression.
“Mesenchymal” refers to cells that develop into connective tissue, blood vessels, and lymphatic tissue.
The invention provides small molecule inducers of ferroptosis. In various embodiments, the invention provides compounds that target the active site of the GPX4 enzyme, wherein binding of the compound to the active site of GPX4 effectively inhibits the activity of the enzyme.
In one embodiment, the invention provides a composition comprising a compound of the invention or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier, adjuvant, or vehicle.
In one embodiment, the invention provides a method for inducing ferroptosis in a cell, the method comprising contacting the cell with an effective amount of a compound of the invention or a pharmaceutically acceptable salt thereof.
In one embodiment, the invention provides a method for decreasing GPX4 activity in a cell, the method comprising contacting the cell with an effective amount of a compound of the invention or a pharmaceutically acceptable salt thereof.
The compounds of the invention are useful for inducing ferroptosis in a cell. In one embodiment, the compounds of the invention may be used in cancer therapy to induce ferroptosis in a cancer cell, such as a mesenchymal cancer cell.
In various embodiments, the invention provides compounds that target the active site of the GPX4 enzyme, wherein binding of the compound to the active site of GPX4 effectively inhibits the activity of the enzyme.
In one embodiment, compounds of the invention, or pharmaceutically acceptable salts thereof, have the structure indicated by Formula 1:
In one embodiment, compounds of the invention, or pharmaceutically acceptable salts thereof, have the structure of a compound provided in Table 1.
In one embodiment, compounds of the invention, or pharmaceutically acceptable salts thereof, have the structure indicated by Formula 2:
In one embodiment, compounds of the invention, or pharmaceutically acceptable salts thereof, have the structure of a compound provided in Table 2.
In one embodiment, compounds of the invention have the structure indicated by Formula 3:
In one embodiment, compounds of the invention have the structure indicated by Formula 4:
In one embodiment, compounds of the invention have the structure indicated by Formula 5:
The compounds of the invention include the compounds of Formulas 1-5, and active derivatives and salts thereof.
In some embodiments, a compounds of the invention is a compound shown in Tables 1 and 2.
In some embodiments, a compounds of the invention is a compound shown in Table 3. The compounds of the invention include the compounds 1-643 and active derivatives and salts thereof.
Different versions of the compounds of the invention may be synthesized. For example, the base structure of Compound 90 (see Table 3) may be modified at one or more certain positions to include different “linker lengths” that may include different numbers of carbons at this position to result in Formula 91 of Table 3, where each n independently is 0, 1 or 2 and R is a 5, 6, 7 or 8 membered optionally substituted aromatic or non-aromatic carbon ring.
The Compounds 1-643 are either commercially available or may be synthesized using standard synthetic techniques known to those of ordinary skill in the art.
The invention provides a composition, the composition comprising a pharmaceutically acceptable carrier, adjuvant, or vehicle, and one or more compounds having any one of the structures of Compounds 1-643 or active derivatives or a pharmaceutically acceptable salt thereof.
A composition of the present invention may be administered in any desired and effective manner: for oral ingestion, or as an ointment or drop for local administration to the eyes, or for parenteral or other administration in any appropriate manner such as intraperitoneal, subcutaneous, topical, intradermal, inhalation, intrapulmonary, rectal, vaginal, sublingual, intramuscular, intravenous, intraarterial, intrathecal, or intralymphatic. Further, a composition of the present invention may be administered in conjunction with other treatments. A composition of the present invention may be encapsulated or otherwise protected against gastric or other secretions, if desired.
The compositions of the invention are pharmaceutically acceptable and may comprise one or more active ingredients in admixture with one or more pharmaceutically-acceptable carriers and, optionally, one or more other compounds, drugs, ingredients and/or materials. Regardless of the route of administration selected, the agents/compounds of the present invention are formulated into pharmaceutically-acceptable dosage forms by conventional methods known to those of skill in the art see, e.g., Remington, The Science and Practice of Pharmacy (21st Edition, Lippincott Williams and Wilkins, Philadelphia, Pa).
Pharmaceutically acceptable carriers are well known in the art (see, e.g., Remington, The Science and Practice of Pharmacy (21st Edition, Lippincott Williams and Wilkins, Philadelphia, Pa.) and The National Formulary (American Pharmaceutical Association, Washington, D.C.)) and include sugars {e.g., lactose, sucrose, mannitol, and sorbitol), starches, cellulose preparations, calcium phosphates (e.g., dicalcium phosphate, tricalcium phosphate and calcium hydrogen phosphate), sodium citrate, water, aqueous solutions (e.g., saline, sodium chloride injection, Ringer's injection, dextrose injection, dextrose and sodium chloride injection, lactated Ringer's injection), alcohols (e.g., ethyl alcohol, propyl alcohol, and benzyl alcohol), polyols (e.g., glycerol, propylene glycol, and polyethylene glycol), organic esters (e.g., ethyl oleate and triglycerides), biodegradable polymers (e.g., polylactide-polyglycolide, poly(orthoesters), and poly(anhydrides)), elastomeric matrices, liposomes, microspheres, oils (e.g., corn, germ, olive, castor, sesame, cottonseed, and groundnut), cocoa butter, waxes (e.g., suppository waxes), paraffins, silicones, talc, silicylate, etc. Each pharmaceutically acceptable carrier used in a composition of the invention must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject. Carriers suitable for a selected dosage form and intended route of administration are well known in the art, and acceptable carriers for a chosen dosage form and method of administration can be determined using ordinary skill in the art.
The compositions of the invention may, optionally, contain additional materials commonly used in such compositions. These ingredients and materials are well known in the art. Examples of pharmaceutically acceptable materials include:
Ingredients and materials suitable for a selected dosage form and intended route of administration are well known in the art, and acceptable ingredients and materials for a chosen dosage form and method of administration may be determined using ordinary skill in the art.
Compositions suitable for oral administration may be in the form of capsules, cachets, pills, tablets, powders, granules, a solution, or a suspension in an aqueous or non-aqueous liquid, an oil-in-water or water-in-oil liquid emulsion, an elixir or syrup, a pastille, a bolus, an electuary or a paste. These formulations may be prepared by methods known in the art, e.g., by means of conventional pan-coating, mixing, granulation or lyophilization processes.
Solid dosage forms for oral administration (capsules, tablets, pills, dragées, powders, granules, and the like) may be prepared, e.g., by mixing the active ingredient(s) with one or more pharmaceutically-acceptable carriers and, optionally, one or more fillers, extenders, binders, humectants, disintegrating agents, solution retarding agents, absorption accelerators, wetting agents, absorbents, lubricants, and/or coloring agents. Solid compositions of a similar type may be employed as fillers in soft and hard-filled gelatin capsules using a suitable excipient. A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using a suitable binder, lubricant, inert diluent, preservative, disintegrant, surface-active or dispersing agent. Molded tablets may be made by molding in a suitable machine. The tablets, and other solid dosage forms, such as dragees, capsules, pills, and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein. They may be sterilized by, for example, filtration through a bacteria-retaining filter. These compositions may also optionally contain opacifying agents and may be of a composition such that they release the active ingredient only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner. The active ingredient can also be in microencapsulated form.
Liquid dosage forms for oral administration include pharmaceutically-acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs. The liquid dosage forms may contain suitable inert diluents commonly used in the art. Besides inert diluents, the oral compositions may also include adjuvants, such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents. Suspensions may contain suspending agents.
Compositions for rectal or vaginal administration may be presented as a suppository, which may be prepared by mixing one or more active ingredient(s) with one or more suitable nonirritating carriers which are solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active compound. Compositions which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams, or spray formulations containing such pharmaceutically-acceptable carriers as are known in the art to be appropriate.
Dosage forms for the topical or transdermal administration include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, drops and inhalants. The active agent(s)/compound(s) may be mixed under sterile conditions with a suitable pharmaceutically-acceptable carrier. The ointments, pastes, creams and gels may contain excipients. Powders and sprays may contain excipients and propellants.
Compositions suitable for parenteral administrations comprise one or more agent(s)/compound(s) in combination with one or more pharmaceutically-acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain suitable antioxidants, buffers, solutes which render the formulation isotonic with the blood of the intended recipient, or suspending or thickening agents. Proper fluidity can be maintained, for example, by the use of coating materials, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. These compositions may also contain suitable adjuvants, such as wetting agents, emulsifying agents, and dispersing agents. It may also be desirable to include isotonic agents. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption.
In some cases, in order to prolong the effect of a drug (e.g., pharmaceutical formulation), it is desirable to slow its absorption from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility.
The rate of absorption of the active agent/drug then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally-administered agent/drug may be accomplished by dissolving or suspending the active agent/drug in an oil vehicle. Injectable depot forms may be made by forming microencapsule matrices of the active ingredient in biodegradable polymers.
Depending on the ratio of the active ingredient to polymer, and the nature of the particular polymer employed, the rate of active ingredient release can be controlled. Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissue. The injectable materials can be sterilized for example, by filtration through a bacterial-retaining filter.
The formulations may be presented in unit-dose or multi-dose sealed containers, for example, ampules and vials, and may be stored in a lyophilized condition requiring only the addition of the sterile liquid carrier, for example water for injection, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the type described above.
The invention provides a method of inducing ferroptosis in a cell, the method comprising contacting the cell with an effective amount of one or more compounds according to the present invention.
In one aspect of this embodiment, the cell may be mammalian, preferably human. In other aspects of this embodiment, the cell may be from a laboratory animal. In addition to humans, categories of mammals within the scope of the present invention include, for example, agricultural animals, veterinary animals, laboratory animals, etc. Some examples of agricultural animals include cows, pigs, horses, goats, etc. Some examples of veterinary animals include dogs, cats, etc. Some examples of laboratory animals include rats, mice, rabbits, guinea pigs, etc.
In one aspect of this embodiment, the method is carried out in vitro. In other aspects of this embodiment, the method is carried out in vivo or ex vivo.
In one embodiment, the cell is a cancer cell, such as a mesenchymal cancer cell. Mesenchymal tumors (i.e., either sarcomas or tumors that have undergone epithelial to mesenchymal transition) are typically characterized by a relatively high content of polyunsaturated fatty acids and iron. Because of the relatively high polyunsaturated fatty acid and iron content, cellular lipids are subjected to relatively high levels of oxidation to produce lipid peroxides, which in the absence of GPX4 can be toxic to the cell.
The invention provides a method for decreasing GPX4 activity in a cell, the method comprising contacting the cell with an effective amount of one or more compounds of the present invention.
In one aspect of this embodiment, the cell may be mammalian, preferably human. In other aspects of this embodiment, the cell may be from a laboratory animal. In addition to humans, categories of mammals within the scope of the present invention include, for example, agricultural animals, veterinary animals, laboratory animals, etc. Some examples of agricultural animals include cows, pigs, horses, goats, etc. Some examples of veterinary animals include dogs, cats, etc. Some examples of laboratory animals include rats, mice, rabbits, guinea pigs, etc.
In one aspect of this embodiment, the method is carried out in vitro. In other aspects of this embodiment, the method is carried out in vivo or ex vivo.
The compounds of the invention may be used in cancer therapy to induce ferroptosis in a cancer cell.
In one embodiment, the invention provides a method for treating a cancer in a subject in need thereof, the method comprising, administering to the subject a pharmaceutically effective amount of a pharmaceutical composition including one or more compounds, or pharmaceutically acceptable salts thereof, of the present invention.
In one aspect, the invention provides a method for treating a mesenchymal cancer, e.g., a sarcoma, in a subject in need thereof, the method comprising, administering to the subject a pharmaceutically effective amount of a pharmaceutical composition including one or more compounds, or pharmaceutically acceptable salts thereof, of the present invention.
In one aspect, the method reduces the growth rate of a tumor, reduces the size of a tumor, eliminates a tumor, or delays progression of a cancer stage.
The following examples are included for illustrative purposes only and are not intended to limit the scope of the inventive concepts herein.
To identify potential covalent inhibitors of GPX4, we screened a panel of 294 diverse small molecules from the Enamine covalent inhibitor library (Catalog No. CSL-10480-0-Z-10, Enamine, Monmouth, NJ). Each compound contains either a chloroacetamide or acrylamide moiety theoretically capable of forming a covalent bond with a selenocysteine. The small molecule compounds were selected for a diversity of backbone structures. The screening protocol used the cell viability assay Cell Titer Glo® (available from Promega, Madison, WI). Cell Titer Glo® is a luminescent cell viability that determines the number of viable cells in culture by quantifying ATP, which indicates the presence of metabolically active cells. Briefly, HT-1080 fibrosarcoma cells were plated 24 hours prior to treatment with 10 UM of each compound in the screening panel. Cell Titer Glo® was used to read out cell viability after 48 hours of compound exposure. Data was collected in duplicate.
To determine whether the reduction in cell viability by the compounds identified in
To validate that the five ferroptosis-inducing compounds identified in
Mechanistically, ferroptosis can be induced by covalent inhibitors of GPX4 or by depletion of glutathione (GSH). Since GPX4 reduces lipid hydroperoxides using GSH as a co-substrate, both mechanisms ultimately result in loss of GPX4 activity, followed by elevated levels of reactive oxygen species which induces lipid peroxidation and subsequent cell death. In addition, changes in the levels of polyunsaturated fatty acids and/or iron in a cell in response to a potential covalent inhibitor of GPX4 activity may also induce or contribute to the ferroptotic phenotype.
To determine whether the five ferroptosis-inducing compounds identified in
We tested each of the five ferroptosis-inducing compounds identified in
To determine whether the five ferroptosis-inducing compounds (1816, 8216, 3362, 1962, and 0973) inhibit GPX4 enzymatic activity in vitro, we used a commercially available GPX4 inhibitor screening kit (Cayman Chemical cat #701880). This assay measures a compound's ability to prevent cumene hydroperoxide reduction by recombinant GPX4 protein. Briefly, the five ferroptosis-inducing compounds (25 μM) were incubated with recombinant GPX4 protein for one hour before being run in the Cayman Chemicals GPX4 inhibitor screening assay according to manufacturer's recommendations. The assay read out is a change in absorbance between NADPH and NADP. ML162 (25 μM) and RSL3 (25 μM) were used as positive control compounds. RSL3 (available from Apex Bio) is a GPX4 inhibitor that has been shown to require an adapter protein, 14-3-3, for efficient inhibitory activity in vitro.
Referring now to
To test whether the ferroptosis-inducing compounds 8216 or 1962 display selectivity for cancer cells we performed a dose response study in HK-2 kidney epithelial cells. HK-2 kidney epithelial cells are a non-cancerous cell line that is commonly used as a “healthy” control in ferroptosis experiments. Briefly, HK-2 kidney epithelial cells were plated 24 hours prior to treatment with concentrations of the ferroptosis-inducing compounds 8216 or 1962 ranging from 10 UM to 10 nM. The ferroptosis-inducing compound RSL3 was used as a positive control. As an additional control, we included a non-specific inducer of apoptosis, staurosporine. Cell Titer Glo® was used to read out cell viability after 48 hours of compound exposure. Data was collected in triplicate.
To determine whether the reduction in viability of HK-2 renal epithelial cells exposed to compounds 8216 or 1962 is driven by induction of ferroptosis, we performed dose response curves for each compound in the presence or absence of ferrostatin-1. We also compared the EC50s for each compound between healthy cells (HK-2 renal epithelial cells) and cancer cells (HT-1080 fibrosarcoma cells) to determine a therapeutic window for healthy vs diseased cells. Briefly, HK-2 renal epithelial cells were plated 24 hours prior to treatment with concentrations of each inhibitor compound ranging from 10 UM to 10 nM in the presence or absence of 1.5 μM ferrostatin-1. The ferroptosis-inducing compound RSL3 was used as a positive control. Cell Titer Glo® was used to read out cell viability after 48 hours of compound exposure. Data was collected in triplicate.
We next examined whether the toxicity observed in HK-2 kidney epithelial cells is due to on- or off-target effects of each compound by calculating the degree of rescue by ferrostatin-1 in these cells. Referring still to
Experimental results described above suggest that five of our compounds induce ferroptosis, though likely through alternative mechanisms. This is expected as ferroptosis can be induced as a result of several mechanisms besides direct inhibition of GPX4. For example, inhibition of system Xc leads to the depletion of glutathione and subsequent inactivation of GPX4. Ferroptosis can also be induced by increasing the amount of polyunsaturated fatty acids or labile iron within a cell. Methods to distinguish these different modes (direct vs indirect) of ferroptosis-induction could be helpful in the development of ferroptosis-inducing small molecule compounds.
Expression of heme oxygenase 1 (HMOX1) mRNA may be used as a molecular response biomarker for induction of ferroptosis by direct inhibition of GPX4. To distinguish the transcriptional responses elicited by direct versus indirect inhibitors of GPX4, we profiled two direct GPX4 inhibitors, RSL3 and ML162, as well as one system Xc inhibitor, erastin, by Precision Run On followed by sequencing (PRO-seq) in IMR90 lung fibroblast cells. Briefly, IMR90 lung fibroblasts cells (a ferroptosis sensitive cell line) were treated with GPX inhibitors (RSL3 1 μM, ML162 1 μM) or system Xc inhibitor (erastin 10 UM) for one hour and samples were subjected to PRO-seq.
We tested several of our ferroptosis-inducing compounds for their ability to rapidly induce HMOX1 by RT-qPCR. Briefly, HT-1080 fibrosarcoma cells were treated with either 10 UM or 5 UM of compound RSL3, 8216, 1962, 1816, or 3362 for four hours. RNA was isolated and reverse transcribed and HMOX1 expression quantified by qPCR. Each sample was normalized to an ACTB housekeeping gene control and fold change was calculated relative to a DMSO control.
Experimental results described hereinabove suggest that compound 8216 is a potent and selective inhibitor of GPX4. To determine whether similar molecules could offer other starting points for a medicinal chemistry campaign, we screened 95 additional compounds that contain a similar base structure to that of 8216. Briefly, HT-1080 fibrosarcoma cells were treated with 10 UM of a compound for 48 hours and cell viability was measured by Cell Titer Glo®. Data was collected in duplicate. A list of the compounds tested, and a summary of the average relative cell viability data is shown in Table 5.
From the screen of 8216 derivative compounds, compound 6666 (see Table 5) was selected for further study.
An additional 34 compounds from the Enamine covalent inhibitor library with similar structures to compounds 6666 or 1816 were screened for inducing cell death. Briefly, HT-1080 fibrosarcoma cells were plated 24 hours prior to treatment on 96-well black-sided clear bottom plates. Each compound in the screening panel was tested at a 10 UM concentration for 24 hours prior to addition of Cell Titer Glo to evaluate relative cell viability.
Compounds from the Enamine covalent inhibitor library with similar structures to compounds 8216, 6666 or 1816 were further screened for inducing cell death via ferroptosis. Briefly, HT-1080 fibrosarcoma cells were plated on black-sided clear bottom plates 24 hours prior to treatment, with or without the addition of 2× ferrostatin-1, which inhibits the process of ferroptosis and rescues cells from undergoing this form of cell death. Compounds were diluted in DMSO and media then added to the cells for a total of 24 hours at which point cell viability was read out via Cell Titer Glo. Compounds were tested at 7 concentrations ranging from 50 μM to 5 nM at half log dilutions.
We tested several of the ferroptosis-inducing compounds for their ability to rapidly induce expression of HMOX1. Briefly, HT-1080 fibrosarcoma cells were plated in 6-well dishes 24 hours prior to treatment with 5 μM of 8147, 6047, 3793, Rsl3, 6666 or 8216 with and without the addition of ferrostatin-1 to the media. Cells were harvested from the plate after 4 hours of compound treatment by adding Trizol reagent directly to the plate to lyse the cells and preserve the RNA. RNA was isolated using the Direct-zol 96-well RNA kit (Zymo cat #R2054). Subsequently purity and quantity of RNA in the samples were measured using a Nanodrop (Thermo Fisher cat #ND-ONE-W). RNA samples were normalized to 2 μg and reverse transcribed to cDNA using the Multiscribe High-Capacity Reverse Transcription kit (Thermo Fisher cat #4368814) following manufacturer's instructions. Quantitative real time PCR was carried out in triplicate using SYBR Select Master Mix (Thermo Fisher cat #4472903) following manufacturer's instructions with primers designed against HMOX1 and ActB from IDT. HMOX1 expression levels were normalized to ActB for each sample then fold change was determined against the vehicle control, DMSO.
An enzymatic assay that measures the reduction of glutathione by GPX4 was used to test the ability of compounds 8216, 6666, 8147, and 3793 to inhibit the activity of GPX4. The enzymatic assay was performed using a commercially available kit (Cayman cat #701880). The compounds were tested at 8 concentrations in duplicate with the compound being diluted following manufacturer's instructions. The enzymatic reaction was allowed to continue for 1 hour prior to reading out the kinetic shift in absorbance that occurs as result of GPX4 reducing glutathione.
Compounds 6666, 6047, 3973, 8216, and 8147 were also evaluated for inducing lipid peroxidation in HT-1080 fibrosarcoma cells in the presence or absence of 1.5 UM ferrostatin-1. Briefly, HT-1080 fibrosarcoma cells were treated with 10 UM of compound with or without 1.5 μM ferrostatin-1 and incubated at 37° C. for 1 hour. Bodipy C-11 and Hoescht stain were then added to the media, the cells were allowed to incubate for an additional 30 minutes and then were imaged on the Opera Phenix confocal screening system.
To determine whether the ferroptosis-inducing compounds 6666 and 8147 could function through direct binding and inhibition of GPX4, we tested binding of each compound to recombinant GPX4 protein in vitro by intact protein mass spectrometry. Compound 8216 was used as a positive control (see
As an orthogonal approach, we used the Autogrow4 algorithm to predict molecules that could bind to the GPX4 active site. Autogrow4 is a genetic algorithm that takes in a protein structure and through a series of generations (max of 30 generations) builds molecules using commonly used small molecule fragments. Throughout each generation small fragments that bind with high predicted affinity to the target will have a chance to move forward to the next level in the algorithm and interact with other small fragments to build a larger compound. At the end of each generation filters are applied to molecules so that toxic and mutant compounds are eliminated and don't proceed to the next level. In addition, Lipinski's rule was applied in order to keep the size of the molecules below 500 Da.
We ran the algorithm independently 15 times on the active site of GPX4 and recovered several compounds predicted to interact with the site with estimated binding energies ranging from −8 to −6 kcal/mol. In one of the runs, 68 compounds contained the 8216 structure as part of the molecule. We applied a clustering analysis to these compounds to divide them into similar groups. Once the clustering was done, the centroid of the clusters was selected (n=9) and among those, 4 compounds were synthesizable. These independently-derived molecules are currently being synthesized. The chemical structures of the nine compounds identified are shown in Table 4.
22.2 g (136 mmol) of 3,5-dimethylaniline (1) was added in one portion to a solution of benzoyl isothiocyanate (2) at 20° C., prepared by dissolving 15 g (123 mmol) in 60 mL acetone. The mixture was heated to 60° C. and stirred for 1 h. LC-MS showed 3,5-dimethylaniline (1) was consumed completely and one main peak with desired MS (285.1, (M+H)+) was detected. The reaction mixture was then cooled to room temperature and poured into crushed ice, whereupon a white precipitate formed. The precipitate was collected by filtration, washed with 100 ml water and dried over air to give 28 g of crude white solid intermediate product N-[(3,5-dimethylphenyl)carbamothioyl]benzamide (3).
MS (ESI): calculated 285.1 [(M+H)+]; measured 285.1 [(M+H)+].
28 g of N-[(3,5-dimethylphenyl)carbamothioyl]benzamide (3) was added in portions to a solution of NaOH in water, prepared by dissolving 5 g NaOH (140 mmol) in 200 ml of water. The mixture was heated at 80° C. and stirred for 2 hr. LC-MS showed N-[(3,5-dimethylphenyl)carbamothioyl]benzamide (3) was consumed completely and one main peak with desired MS (181.1, [M+H]+) was detected. The mixture was then cooled to room temperature and added dropwise to 200 ml of a 1N aqueous hydrochloric acid solution. The resulting mixture was diluted with 100 ml of water and extracted with EtOAc (300 mL×3). The combined organic layers were washed with saturated brine solution (300 mL×3), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was triturated in 50 mL of methyl tert-butyl ether to yield 15 g (83.2 mmol) of the white solid intermediate product (3,5-dimethylphenyl)thiourea (4).
MS (ESI): calculated 181.1 [(M+H)+]; measured 181.1 [(M+H)+].
1H NMR (400 MHZ, DMSO-d6) δ 9.55 (br s, 1H), 7.94-7.08 (m, 2H), 6.96 (br s, 2H), 6.76 (br s, 1H), 2.24 (s, 6H).
15.4 g (55.5 mmol) of 2-bromo-1-(4-bromophenyl)ethanone (5) and 27.5 mL (20.0 g, 197 mmol) triethylamine was added to a 20° C. solution of (3,5-dimethylphenyl)thiourea (4), prepared by dissolving 10 g (55.5 mmol) in 100 mL ethanol. The mixture was heated to 80° C. and stirred for 2 h. LC-MS showed (3,5-dimethylphenyl)thiourea (4) was consumed completely and one main peak with desired MS (358.9 [M (79Br)+H]+, 360.9 [M (81Br)+H]+) was detected. The reaction mixture was then cooled to room temperature, quenched with 100 ml water at 20° C. and extracted with 3 aliquots of 100 mL EtOAc. The combined organic layers were washed with 3 aliquots of 100 mL saturated brine, dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by silica gel flash chromatography (300 g SepaFlash® silica column, ethyl acetate/petroleum, gradient 0%-30% ethyl acetate/petroleum ether @ 200 mL/min) to yield 19 g (52.9 mmol) of the yellow solid intermediate product 4-(4-bromophenyl)-N-(3,5-dimethylphenyl)thiazol-2-amine (6).
MS (ESI): calculated 359.0 [M (79Br)+H]+, 361.0 [M (81Br)+H]+; measured 358.9 [M (79Br)+H]+, 360.9 [M (81Br)+H]+.
1H NMR (400 MHZ, DMSO-d6) δ 10.14 (s, 1H), 7.86 (d, J=8.4 Hz, 2H), 7.62 (d, J=8.4 Hz, 2H), 7.38 (s, 1H), 7.30 (s, 1H), 7.31 (s, 1H), 6.62 (s, 1H), 2.27 (s, 6H).
84.5 mg (0.835 mmol) triethylamine was added to a solution of 4-(4-bromophenyl)-N-(3,5-dimethylphenyl)thiazol-2-amine (6), which was prepared by dissolving 100 mg (0.278 mmol) in 2 mL dichloromethane. The mixture was cooled to 0° C. and 62.9 mg 2-chloroacetyl chloride (0.557 mmol) (7) was added dropwise. The mixture was then heated to 20° C. and stirred for 4 h. LC-MS showed 4-(4-bromophenyl)-N-(3,5-dimethylphenyl)thiazol-2-amine (6) was consumed completely and desired MS (435.0 [M (79Br)+H]+, 437.0 [M (81Br)+H]+) was detected. The reaction was concentrated under reduced pressure to give a residue. The residue was purified by preparative HPLC (column: Waters Xbridge, 5 μm, 150×25 mm; mobile phase A: water containing 0.1% trifluoroacetic acid, mobile phase B: acetonitrile; gradient elution of 60%-90% B:A over 9 min) and lyophilized to give 21 mg (0.048 mmol) of the white solid final product N-[4-(4-bromophenyl) thiazol-2-yl]-2-chloro-N-(3,5-dimethylphenyl)acetamide.
MS (ESI): calculated 435.0 [M (79Br)+H]+, 437.0 [M (81Br)+H]+; measured 435.0 [M (79Br)+H]+, 437.0 [M (81Br)+H]+.
1H NMR (400 MHZ, DMSO-d6) δ 7.85 (s, 1H), 7.61-7.56 (m, 2H), 7.56-7.52 (m, 2H), 7.20 (s, 1H), 7.17 (s, 2H), 4.29 (s, 2H), 2.35 (s, 6H).
0.06 g (0.67 mmol, 1.2 eq) of compound (8) was dissolved in DCM (10 mL) under an argon atmosphere. The solution was cooled to 0° C. and the following were added sequentially 0.2 g (0.56 mmol, 1 eq) of compound (6) and 0.172 g (0.83 mmol, 1.5 eq) DCC. The reaction mixture was warmed to room temperature and stirred overnight. The resulting mixture was diluted with DCM, washed with water and brine, dried over anhydrous Na2SO4, and evaporated under reduced pressure. The residue was purified by HPLC to obtain 0.061 g (0.141 mmol, 25.5% yield) of compound 637.
0.056 g (0.67 mmol, 1.2 eq) compound (9) was dissolved in DCM (10 mL) under an argon atmosphere. The solution was cooled to 0° C. and the following were added sequentially 0.2 g (0.56 mmol, 1 eq) compound (6) and DCC (0.172 g, 0.83 mmol, 1.5 eq). The reaction mixture was warmed to room temperature and stirred overnight. The resulting mixture was diluted with DCM, washed with water and brine, dried over anhydrous Na2SO4, and evaporated under reduced pressure. The residue was purified by HPLC to obtain compound 635 (0.012 g, 0.0282 mmol, 5% yield).
0.2 g (0.56 mmol, 1 eq) of compound (6) was dissolved in DCM (10 mL) under an argon atmosphere. The solution was cooled to 0° C. and the following were added sequentially 0.36 g (2.78 mmol, 5 eq) DIPEA and 0.095 g (0.72 mmol, 1.3 eq) compound (10) dropwise. The reaction mixture was warmed to room temperature and stirred overnight. The resulting mixture was diluted with DCM, washed with water and brine, dried over anhydrous Na2SO4, and evaporated under reduced pressure. The residue was purified by HPLC to obtain compound 636 (0.094 g, 0.2 mmol, 37.2% yield).
0.2 g (0.56 mmol, 1 eq) compound (6) was dissolved in DCM (10 mL) under an argon atmosphere. The solution was cooled to 0° C. and the following were added sequentially 0.36 g (2.78 mmol, 5 eq) DIPEA and 0.092 g (0.72 mmol, 1.3 eq) compound (11) dropwise. The reaction mixture was warmed to room temperature and stirred overnight. The resulting mixture was diluted with DCM, washed with water and brine, dried over anhydrous Na2SO4, and evaporated under reduced pressure. The residue was purified by HPLC to obtain compound 634 (0.099 g, 0.22 mmol, 39.5% yield).
0.20 g (0.56 mmol, 1 eq) compound (6) was dissolved in DCM (10 mL) under an argon atmosphere. To the solution were added sequentially compound (12) 3-(trimethylsilyl)propiolic acid (0.095 g, 0.670 mmol, 1.2 eq) and DCC (0.172 g, 0.830 mmol, 1.5 eq) in a basic solution. The reaction mixture was stirred overnight at room temperature. The resulting mixture was diluted with 20 mL DCM. The DCU byproduct was filtered through a pad of celite and washed with minimal amount of DCM. The combined DCM extracts were washed with water (3×20 mL), dried over anhydrous Na2SO4, and evaporated under reduced pressure to obtain desired intermediate compound (13) (0.20 g, 0.41 mmol, 74.3% yield), which was used in the next step without further purification.
0.20 g (0.41 mmol, 1 eq) compound (13) was dissolved in THF (10 mL) under an argon atmosphere. The solution was cooled to 0° C. and TBAF (0.46 mL, 1 M in THF, 1.1 eq) was added. The reaction mixture was warmed to room temperature and stirred overnight. The resulting mixture was diluted with DCM (30 mL), washed with water (3×20 mL). The organic layer was dried over anhydrous Na2SO4 and evaporated under reduced pressure. The residue was purified by HPLC to obtain compound 634 (0.0103 g, 0.025 mmol, 6% yield).
0.50 g (1.39 mmol, 1 eq) compound (6) was dissolved in DCM (20 mL) under an argon atmosphere. To the solution were added sequentially compound (14) 3-chlorocyclobutane-1-carboxylic acid (0.225 g, 1.670 mmol, 1.2 eq) and DCC (0.431 g, 2.09 mmol, 1.5 eq) in a basic solution. The reaction mixture was stirred overnight at room temperature. The resulting mixture was diluted with DCM (30 mL). The DCU byproduct was filtered through a pad of celite and washed with minimal amount of DCM. The combined DCM extracts was washed with water (3×30 mL), dried over anhydrous Na2SO4, and evaporated under reduced pressure to obtain desired intermediate compound (15) (0.40 g, 0.84 mmol, 60.4% yield), which was used in the next step without further purification.
0.4 g (0.84 mmol, 1 eq) compound (15) was dissolved in THF (15 mL) under an argon atmosphere. The solution was cooled to −40° C. and the following were added dropwise LiHMDS (0.92 mL, 1.1 M in THF, 1.1 eq) and DMAP. The reaction mixture was slowly warmed to 0° C. and stirred for 30 min. The resulting mixture was quenched with a saturated solution of NH4Cl and extracted with EtOAc (3×30 mL). The combined organic layer was dried over anhydrous Na2SO4 and evaporated under reduced pressure. The residue was purified by HPLC to obtain compound 639 (0.0092 g, 0.02 mmol, 2.44% yield).
1 g (7.87 mmol, 1 eq) compound (16) was dissolved in 20 mL concentrated H2SO4 under an argon atmosphere. 1.19 g (11.8 mmol, 1.5 eq) KNO3 was added to the solution. The reaction mixture was stirred overnight at 50° C. The resulting mixture was poured in ice water and extracted with DCM (3×50 mL). The combined organic layer was dried over anhydrous Na2SO4 and evaporated under reduced pressure to obtain compound (17) (1.1 g, 6.3 mmol, 81.2% yield).
0.20 g (0.56 mmol, 1 eq) compound (6) was dissolved in DCM (10 mL) under an argon atmosphere. To the solution were added sequentially 0.115 g (0.670 mmol, 1.2 eq) compound (17) and 0.172 g (0.830 mmol, 1.5 eq) DCC in a basic solution. The reaction mixture was stirred overnight at room temperature. The resulting mixture was diluted with DCM (20 mL). The DCU byproduct was filtered through a pad of celite and washed with minimal amount of DCM. The combined DCM extracts was washed with water (3×20 mL), dried over anhydrous Na2SO4, and evaporated under reduced pressure. The residue was purified by HPLC to obtain compound 640 (0.0473 g, 0.092 mmol, 16.5% yield).
Compounds B1-B28 were prepared by an analogous synthetic route to compounds 90, 634, 635, 636, 637, 639, 640, and 641 as illustrated above.
1 g, 5.3 mmol, 1 eq) compound (18) was dissolved in 50 mL dioxane under an argon atmosphere. To the solution were added sequentially compound (19) (0.706 g, 5.3 mmol, 1 eq), tris(dibenzylideneacetone)dipalladium(0) (0.485 g, 0.53, mol, 0.1 eq), [5-(diphenylphosphanyl)-9,9-dimethyl-9H-xanthen-4-yl]diphenylphosphane (0.46 g, 0.80 mmol, 0.15 eq), and Cs2CO3 (3.4 g, 10.4 mol, 3 eq). The reaction mixture was stirred overnight at 100° C. The resulting mixture was diluted with EtOAc (200 mL), washed with water and brine, dried over anhydrous Na2SO4, and evaporated under reduced pressure. The residue was purified by column chromatography to obtain compound (20) (0.8 g, 2.8 mmol, 52.8% yield).
0.1 g (0.35 mmol, 1 eq) compound (20) was dissolved in DCM (10 mL) under an argon atmosphere. The solution was cooled to 0° C. and the following were added sequentially and dropwise: DIPEA (0.045 g, 0.35 mmol, 1 eq) and 2-chloroacetyl chloride (0.04 g, 0.35 mmol, 1 eq). The reaction mixture was warmed to room temperature and stirred overnight. The resulting mixture was diluted with DCM, washed with water and brine, dried over anhydrous Na2SO4, and evaporated under reduced pressure. The residue was purified by HPLC to obtain compound 593 (0.008 g, 0.0221 mmol, 6.3% yield).
1 g (5.6 mmol, 1 eq) compound (21) was dissolved in dioxane (50 mL) under an argon atmosphere. To the solution were added sequentially compound (22) (0.723 g, 5.6 mmol, 1 eq), tris(dibenzylideneacetone)dipalladium(0) (0.513 g, 0.56, mol, 0.1 eq), [5-(diphenylphosphanyl)-9,9-dimethyl-9H-xanthen-4-yl]diphenylphosphane (0.486 g, 0.84 mmol, 0.15 eq), and Cs2CO3 (4 g, 12.2 mol, 3 eq). The reaction mixture was stirred overnight at 100° C. The resulting mixture was diluted with EtOAc (200 mL), washed with water and brine, dried over anhydrous Na2SO4, and evaporated under reduced pressure. The residue was purified by column chromatography to obtain compound (23) (0.12 g, 0.442 mmol, 7.9% yield).
0.12 g (0.44 mmol, 1 eq) Compound (23) was dissolved in DCM (10 mL) under an argon atmosphere. The solution was cooled to 0° C. and the following were added sequentially DIPEA (0.057 g, 0.44 mmol, 1 eq) and 2-chloroacetyl chloride (0.05 g, 0.44 mmol, 1 eq) dropwise. The reaction mixture was warmed to room temperature and stirred overnight. The resulting mixture was diluted with DCM, washed with water and brine, dried over anhydrous Na2SO4, and evaporated under reduced pressure. The residue was purified by HPLC to obtain compound 587 (0.0114 g, 0.032 mmol, 7.4% yield).
Compounds A1, A2, and A4-A47 were prepared by an analogous synthetic route to compound A3 as illustrated below.
To a solution of 4-fluorobenzaldehyde (2 g, 16.1 mmol, 1.6 mL) in DCM (30 mL) was added 3,5-dimethoxyaniline (2.2 g, 14.5 mmol), acetic acid (968 mg, 16.1 mmol, 0.922 mL) and NaBH(OAc)3 (4.6 g, 21.9 mmol). The mixture was stirred at 25° C. for 12 h. The reaction mixture was partitioned between aqueous 1M NaOH (20 mL) and DCM (20 mL). The aqueous phase was extracted with DCM (20 mL×3), dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue which was purified by flash silica gel chromatography (20 g silica) eluting with a gradient of 0-10% EtOAc/petroleum ether at a flow rate of 120 mL/min. N-(4-Fluorobenzyl)-3,5-dimethoxyaniline (3.1 g, 75% yield) was obtained as a yellow oil. M+H+=262.2 (LCMS); 1H NMR (400 MHZ, DMSO-d6) δ 7.36 (dd, J=5.8, 8.1 Hz, 2H), 7.13 (t, J=8.8 Hz, 2H), 6.26 (br t, J=5.9 Hz, 1H), 5.78-5.68 (m, 3H), 4.20 (br d, J=5.9 Hz, 2H), 3.61 (s, 6H).
To a solution of N-(4-fluorobenzyl)-3,5-dimethoxyaniline (200 mg, 0.765 mmol) in DCM (4 mL) was added triethylamine (155 mg, 1.5 mmol, 0.213 mL) and 2-chloroacetyl chloride (173 mg, 1.5 mmol, 0.122 mL). The mixture was stirred at 25° C. for 3 h. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was partitioned between water (10 mL) and EtOAc (10 mL). The aqueous phase was extracted with EtOAc (10 mL×3), dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue which was purified by preparative TLC (silica) eluting with 20% EtOAc/petroleum ether. The title compound (131 mg, 49% yield) was obtained as a white solid. M+H+=338.0 (LCMS); 1H NMR (400 MHZ, CD3CN) δ=7.27-7.21 (m, 2H), 7.06-7.00 (m, 2H), 6.46 (t, J=2.3 Hz, 1H), 6.30 (d, J=2.3 Hz, 2H), 4.85 (s, 2H), 4.02 (s, 2H), 3.70 (s, 6H).
Biological evaluation of working examples A1-A47 and B1-B28 was performed in HT1080 cells (ATCC, cat #CCL-121) according to the procedure provided below.
This application is a continuation application of International Application No. PCT/US2022/040818, filed Aug. 18, 2022, which claims the benefit of U.S. Provisional Application No. 63/313,000, filed Feb. 23, 2022; U.S. Provisional Application No. 63/295,007, filed Dec. 30, 2021; U.S. Provisional Application No. 63/286,022, filed Dec. 4, 2021; and U.S. Provisional Application No. 63/234,829, filed Aug. 19, 2021; each of which is incorporated by reference in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| 63234829 | Aug 2021 | US | |
| 63286022 | Dec 2021 | US | |
| 63295007 | Dec 2021 | US | |
| 63313000 | Feb 2022 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/US2022/040818 | Aug 2022 | WO |
| Child | 18441446 | US |
