Patent Application
20210299107
The present disclosure provides, inter alia, compounds having the structure:
Also provided are pharmaceutical compositions containing the compounds of the present disclosure, as well as methods of using such compounds and compositions.
Cell death is crucial for normal development, homeostasis and the prevention of hyper-proliferative diseases such as cancer (Fuchs and Steller, 2011; Thompson, 1995). It was once thought that almost all regulated cell death in mammalian cells resulted from the activation of caspase-dependent apoptosis (Fuchs and Steller, 2011; Thompson, 1995). More recently this view has been challenged by the discovery of several regulated non-apoptotic cell death pathways activated in specific disease states, including poly(ADP-ribose) polymerase-1 (PARP-1) and apoptosis inducing factor 1 (AIF1)-dependent parthanatos, caspase-1-dependent pyroptosis and receptor interacting protein kinase 1 (RIPK1)-dependent necroptosis (Bergsbaken et al., 2009; Christofferson and Yuan, 2010; Wang et al., 2009). It is believed that additional regulated forms of non-apoptotic cell death likely remain to be discovered that mediate cell death in other developmental or pathological circumstances.
The RAS family of small GTPases (HRAS, NRAS and KRAS) is mutated in about 30% of all cancers (Vigil et al., 2010). Finding compounds that are selectively lethal to RAS-mutant tumor cells is, therefore, a high priority. Two structurally unrelated small molecules, named erastin and RSL3, were previously identified. These molecules were selectively lethal to oncogenic RAS-mutant cell lines, and together, they were referred to as RAS-selective lethal (RSL) compounds (Dolma et al., 2003; Yang and Stockwell, 2008). Using affinity purification, voltage dependent anion channels 2 and 3 (VDAC2/3) were identified as direct targets of erastin (Yagoda et al., 2007), but not RSL3. ShRNA and cDNA overexpression studies demonstrated that VDAC2 and VDAC3 are necessary, but not sufficient, for erastin-induced death (Yagoda et al., 2007), indicating that additional unknown targets are required for this process.
The type of cell death activated by the RSLs has been enigmatic. Classic features of apoptosis, such as mitochondrial cytochrome c release, caspase activation and chromatin fragmentation, are not observed in RSL-treated cells (Dolma et al., 2003; Yagoda et al., 2007; Yang and Stockwell, 2008). RSL-induced death is, however, associated with increased levels of intracellular reactive oxygen species (ROS) and is prevented by iron chelation or genetic inhibition of cellular iron uptake (Yagoda et al., 2007; Yang and Stockwell, 2008). In a recent systematic study of various mechanistically unique lethal compounds, the prevention of cell death by iron chelation was a rare phenomenon (Wolpaw et al., 2011), suggesting that few triggers can access iron-dependent lethal mechanisms.
Accordingly, there is a need for the exploration of various pathways of regulated cell death, as well as for compositions and methods for preventing the occurrence of regulated cell death. This disclosure is directed to meeting these and other needs.
Without being bound to a particular theory, the inventors hypothesized that RSLs, such as erastin, activate a lethal pathway that is different from apoptosis, necrosis and other well-characterized types of regulated cell death. It was found that erastin-induced death involves a unique constellation of morphological, biochemical and genetic features, which led to the name “ferroptosis” as a description for this phenotype. Small molecule inhibitors of ferroptosis that prevent ferroptosis in cancer cells, as well as glutamate-induced cell death in postnatal rat brain slices have been identified and disclosed herein. The inventors have found an underlying similarity between diverse forms of iron-dependent, non-apoptotic death and that the manipulation of ferroptosis may be exploited to selectively destroy RAS-mutant tumor cells or to preserve neuronal cells exposed to specific oxidative conditions.
Accordingly, one embodiment of the present disclosure is a compound according to formula (1):
wherein:
R2 cannot be
Another embodiment of the present disclosure is a compound selected from the group consisting of:
and combinations thereof, or an N-oxide, crystalline form, hydrate, or a pharmaceutically acceptable salt thereof.
Another embodiment of the present disclosure is a pharmaceutical composition. This pharmaceutical composition comprises a pharmaceutically acceptable carrier or diluent and one or more compounds according to formula (1):
wherein:
R2 cannot be
A further embodiment of the present disclosure is a kit. This kit comprises a compound or a pharmaceutical composition according to the present disclosure with instructions for the use of the compound or the pharmaceutical composition, respectively.
Another embodiment of the present disclosure is a method for treating or ameliorating the effects of a disorder in a subject in need thereof. This method comprises administering to the subject an effective amount of one or more compounds having the structure of formula (1):
wherein:
R2 cannot be
An additional embodiment of the present disclosure is a method for treating or ameliorating the effects of a disorder in a subject in need thereof. This method comprises administering to the subject an effective amount of a pharmaceutical composition comprising a pharmaceutically acceptable carrier or diluent and one or more compounds having the structure of formula (1):
wherein:
R2 cannot be
Another embodiment of the present disclosure is a method of modulating ferroptosis in a subject in need thereof. This method comprises administering to the subject an effective amount of a ferroptosis inhibitor, which comprises one or more compounds having the structure of formula (1):
wherein:
R2 cannot be
A further embodiment of the present disclosure is a method of reducing reactive oxygen species (ROS) in a cell. This method comprises contacting a cell with a ferroptosis modulator, which comprises one or more compounds having the structure of formula (1):
wherein:
R2 cannot be
An additional embodiment of the present disclosure is a method for treating or ameliorating the effects of a neurodegenerative disease in a subject in need thereof. This method comprises administering to the subject an effective amount of one or more compounds having the structure of formula (1):
wherein:
R2 cannot be
A further embodiment of the present disclosure is a compound according to formula (2):
wherein:
Still another embodiment of the present disclosure is a compound according to formula (3):
wherein:
Another embodiment of the present disclosure is a compound selected from the group consisting of:
and combinations thereof, or an N-oxide, crystalline form, hydrate, or a pharmaceutically acceptable salt thereof.
Another embodiment of the present disclosure is a pharmaceutical composition. This pharmaceutical composition comprises a pharmaceutically acceptable carrier or diluent and one or more compounds according to formula (2):
wherein:
Another embodiment of the present disclosure is a pharmaceutical composition. This pharmaceutical composition comprises a pharmaceutically acceptable carrier or diluent and one or more compounds according to formula (3):
wherein:
An additional embodiment of the present disclosure is a method for treating or ameliorating the effects of a neurodegenerative disease in a subject in need thereof. This method comprises administering to the subject an effective amount of a compound having the structure selected from the group consisting of:
and combinations thereof, or an N-oxide, crystalline form, hydrate, or a pharmaceutically acceptable salt thereof.
An additional embodiment of the present disclosure is a method of modulating ferroptosis in a subject in need thereof. This method comprises administering to the subject an effective amount of a ferroptosis inhibitor, which comprises a compound having the structure selected from the group consisting of:
and combinations thereof, or an N-oxide, crystalline form, hydrate, or a pharmaceutically acceptable salt thereof.
An additional embodiment of the present disclosure is a method of reducing reactive oxygen species (ROS) in a cell. This method comprises contacting a cell with a ferroptosis modulator, which comprises a compound having the structure selected from the group consisting of:
and combinations thereof, or an N-oxide, crystalline form, hydrate, or a pharmaceutically acceptable salt thereof.
An additional embodiment of the present disclosure is a method for treating or ameliorating the effects of a neurodegenerative disease in a subject in need thereof. This method comprises administering to the subject an effective amount of a compound having the structure selected from the group consisting of:
and combinations thereof, or an N-oxide, crystalline form, hydrate, or a pharmaceutically acceptable salt thereof.
Still another embodiment of the present disclosure is a method for alleviating side effects in a subject undergoing radiotherapy and/or immunotherapy, comprising administering to the subject an effective amount of one or more compounds disclosed herein.
A further embodiment of the present disclosure is a method for treating or ameliorating the effects of an infection associated with ferroptosis in a subject, comprising administering to the subject an effective amount of one or more compounds disclosed herein.
The application file contains at least one drawing executed in color. Copies of this patent application with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
In the present disclosure, new analogs of Fer-1 are provided. Certain of the analogs have improved microsomal stability and solubility while still maintaining good inhibition potency of ferroptosis. Accordingly, one embodiment of the present disclosure is a compound according to formula (1):
wherein:
R2 cannot be
In one aspect of this embodiment, the compound has the structure of formula (1a):
In another aspect of this embodiment, the compound has the structure of formula (1b):
wherein:
In another aspect of this embodiment, the compound is selected from the group consisting of:
or an N-oxide, crystalline form, hydrate, or a pharmaceutically acceptable salt thereof.
Preferably, the compound is selected from the group consisting of:
and combinations thereof, or an N-oxide, crystalline form, hydrate, or a pharmaceutically acceptable salt thereof.
More preferably, the compound is selected from the group consisting of:
or an N-oxide, crystalline form, hydrate, or a pharmaceutically acceptable salt thereof.
Another embodiment of the present disclosure is a compound according to formula (2):
wherein:
Preferably, the compound is selected from the group consisting of:
or an N-oxide, crystalline form, hydrate, or a pharmaceutically acceptable salt thereof.
Another embodiment of the present disclosure is a compound according to formula (3):
wherein:
In one aspect of this embodiment, the compound has the structure of formula (3a):
wherein:
In another aspect of this embodiment, the compound is selected from the group consisting of:
or an N-oxide, crystalline form, hydrate, or a pharmaceutically acceptable salt thereof.
Preferably, the compound is selected from the group consisting of:
or an N-oxide, crystalline form, hydrate, or a pharmaceutically acceptable salt thereof.
Another embodiment of the present disclosure is a pharmaceutical composition. This pharmaceutical composition comprises a pharmaceutically acceptable carrier or diluent and one or more compounds according to formula (1):
wherein:
when R1 and X are both H, Y is —CH and R3 is
R2 cannot be
Another embodiment of the present disclosure is a pharmaceutical composition. This pharmaceutical composition comprises a pharmaceutically acceptable carrier or diluent and one or more compounds according to formula (2):
wherein:
Another embodiment of the present disclosure is a pharmaceutical composition. This pharmaceutical composition comprises a pharmaceutically acceptable carrier or diluent and one or more compounds according to formula (3):
wherein:
Suitable and preferred compounds that are used in the pharmaceutical compositions of the present disclosure are disclosed above in formulas (1), (1a), (1 b), (2), (3) and (3a), including the particular compounds also identified above.
A further embodiment of the present disclosure is a kit. This kit comprises a compound or a pharmaceutical composition disclosed herein with instructions for the use of the compound or the pharmaceutical composition, respectively.
The kits may also include suitable storage containers, e.g., ampules, vials, tubes, etc., for each compound of the present disclosure (which, e.g., may be in the form of pharmaceutical compositions) and other reagents, e.g., buffers, balanced salt solutions, etc., for use in administering the active agents to subjects. The compounds and/or pharmaceutical compositions of the disclosure and other reagents may be present in the kits in any convenient form, such as, e.g., in a solution or in a powder form. The kits may further include a packaging container, optionally having one or more partitions for housing the compounds and/or pharmaceutical compositions and other optional reagents.
Another embodiment of the present disclosure is a method for treating or ameliorating the effects of a disorder in a subject in need thereof. This method comprises administering to the subject an effective amount of one or more compounds having the structure of formula (1):
wherein:
R2 cannot be
As used herein, the terms “treat,” “treating,” “treatment” and grammatical variations thereof mean subjecting an individual subject to a protocol, regimen, process or remedy, in which it is desired to obtain a physiologic response or outcome in that subject, e.g., a patient. In particular, the methods and compositions of the present disclosure may be used to slow the development of disease symptoms or delay the onset of the disease or condition, or halt the progression of disease development. However, because every treated subject may not respond to a particular treatment protocol, regimen, process or remedy, treating does not require that the desired physiologic response or outcome be achieved in each and every subject or subject population, e.g., patient population. Accordingly, a given subject or subject population, e.g., patient population, may fail to respond or respond inadequately to treatment.
As used herein, the terms “ameliorate”, “ameliorating” and grammatical variations thereof mean to decrease the severity of the symptoms of a disease in a subject.
As used herein, a “subject” is a mammal, preferably, a human. In addition to humans, categories of mammals within the scope of the present disclosure 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 primates, rats, mice, rabbits, guinea pigs, etc.
Suitable and preferred compounds and pharmaceutical compositions for use in this method are as disclosed above in formulas (1), (1a), (1b), (2), (3) and (3a), including the particular compounds identified above.
In one aspect of this embodiment, the disorder is a degenerative disease that involves lipid peroxidation. As used herein, “lipid peroxidation” means the oxidative degradation of fats, oils, waxes, sterols, triglycerides, and the like. Lipid peroxidation has been linked with many degenerative diseases, such as atherosclerosis, ischemia-reperfusion, heart failure, Alzheimer's disease, rheumatic arthritis, cancer, and other immunological disorders. (Ramana et al., 2013).
In another aspect of this embodiment, the disorder is an excitotoxic disease involving oxidative cell death. As used herein, an “excitotoxic disorder” means a disease related to the death of central neurons that are mediated by excitatory amino acids (such as glutamate). Excitotoxic disorders within the scope of the present disclosure include diseases involving oxidative cell death. As used herein, “oxidative” cell death means cell death associated with increased levels of intracellular reactive oxygen species (ROS). In the present disclosure, “reactive oxygen species” means chemically reactive molecules, such as free radicals, containing oxygen. Non-limiting examples of ROS include oxygen ions and peroxides.
Non-limiting examples of disorders according to the present disclosure include epilepsy, kidney disease, stroke, myocardial infarction, type I diabetes, traumatic brain injury (TBI), periventricular leukomalacia (PVL), and neurodegenerative disease. Non-limiting examples of neurodegenerative diseases according to the present disclosure include Alzheimer's, Parkinson's, Amyotrophic lateral sclerosis, Friedreich's ataxia, Multiple sclerosis, Huntington's Disease, Transmissible spongiform encephalopathy, Charcot-Marie-Tooth disease, Dementia with Lewy bodies, Corticobasal degeneration, Progressive supranuclear palsy, Chronic Traumatic Encephalopathy (CTE), and Hereditary spastic paraparesis.
In another aspect of this embodiment, the method further comprises co-administering, together with one or more compounds or pharmaceutical compositions of the present disclosure, to the subject an effective amount of one or more of additional therapeutic agents such as 5-hydroxytryptophan, Activase, AFQ056 (Novartis Corp., New York, N.Y.), Aggrastat, Albendazole, alpha-lipoic acid/L-acetyl carnitine, Alteplase, Amantadine (Symmetrel), amlodipine, Ancrod, Apomorphine (Apokyn), Arimoclomol, Arixtra, Armodafinil, Ascorbic acid, Ascriptin, Aspirin, atenolol, Avonex, baclofen (Lioresal), Banzel, Benztropine (Cogentin), Betaseron, BGG492 (Novartis Corp., New York, N.Y.), Botulinum toxin, Bufferin, Carbatrol®, Carbidopa/levodopa immediate-release (Sinemet), Carbidopa/levodopa oral disintegrating (Parcopa), Carbidopa/levodopa/Entacapone (Stalevo), CERE-110: Adeno-Associated Virus Delivery of NGF (Ceregene, San Diego, Calif.), cerebrolysin, CinnoVex, citalopram, citicoline, Clobazam, Clonazepam, Clopidogrel, clozapine (Clozaril), Coenzyme Q, Creatine, dabigatran, dalteparin, Dapsone, Davunetide, Deferiprone, Depakene®, Depakote ER®, Depakote®, Desmoteplase, Diastat, Diazepam, Digoxin, Dilantin®, Dimebon, dipyridamole, divalproex (Depakote), Donepezil (Aricept), EGb 761, Eldepryl, ELND002 (Elan Pharmaceuticals, Dublin, Ireland), Enalapril, enoxaparin, Entacapone (Comtan), epoetin alfa, Eptifibatide, Erythropoietin, Escitalopram, Eslicarbazepine acetate, Esmolol, Ethosuximide, Ethyl-EPA (Miraxion™), Exenatide, Extavia, Ezogabine, Felbamate, Felbatol®, Fingolimod (Gilenya), fluoxetine (Prozac), fondaparinux, Fragmin, Frisium, Gabapentin, Gabitril®, Galantamine, Glatiramer (Copaxone), haloperidol (Haldol), Heparin, human chorionic gonadotropin (hCG), Idebenone, Inovelon®, insulin, Interferon beta 1a, Interferon beta 1 b, ioflupane 1231 (DATSCAN®), IPX066 (Impax Laboratories Inc., Hayward, Calif.), JNJ-26489112 (Johnson and Johnson, New Brunswick, N.J.), Keppra®, Klonopin, Lacosamide, L-Alpha glycerylphosphorylcholine, Lamictal®, Lamotrigine, Levetiracetam, liraglutide, Lisinopril, Lithium carbonate, Lopressor, Lorazepam, losartan, Lovenox, Lu AA24493, Luminal, LY450139 (Eli Lilly, Indianapolis, Ind.), Lyrica, Masitinib, Mecobalamin, Memantine, methylprednisolone, metoprolol tartrate, Minitran, Minocycline, mirtazapine, Mitoxantrone (Novantrone), Mysoline®, Natalizumab (Tysabri), Neurontin®, Niacinamide, Nitro-Bid, Nitro-Dur, nitroglycerin, Nitrolingual, Nitromist, Nitrostat, Nitro-Time, Norepinephrine (NOR), Carbamazepine, octreotide, Onfi®, Oxcarbazepine, Oxybutinin chloride, PF-04360365 (Pfizer, New York, N.Y.), Phenobarbital, Phenytek®, Phenytoin, piclozotan, Pioglitazone, Plavix, Potiga, Pram ipexole (Mirapex), pramlintide, Prednisone, Prim idone, Prinivil, probenecid, Propranolol, PRX-00023 (EPIX Pharmaceuticals Inc.), PXT3003, Quinacrine, Ramelteon, Rasagiline (Azilect), Rebif, ReciGen, remacemide, Resveratrol, Retavase, reteplase, riluzole (Rilutek), Rivastigmine (Exelon), Ropinirole (Requip), Rotigotine (Neupro), Rufinamide, Sabril, safinamide (EMD Serono, Rockland, Mass.), Salagen, Sarafem, Selegiline (1-deprenyl, Eldepryl), SEN0014196 (Siena Biotech, Siena, Italy), sertraline (Zoloft), Simvastatin, Sodium Nitroprussiate (NPS), sodium phenylbutyrate, Stanback Headache Powder, Tacrine (Cognex), Tamoxifen, tauroursodeoxycholic acid (TUDCA), Tegretol®, Tenecteplase, Tenormin, Tetrabenazine (Xenazine), THR-18 (Thrombotech Ltd.), Tiagabine, Tideglusib, tirofiban, tissue plasminogen activator (tPA), tizanidine (Zanaflex), TNKase, Tolcapone (Tasmar), Tolterodine, Topamax®, Topiramate, Trihexyphenidyl (formerly Artane), Trileptal®, ursodiol, Valproic Acid, valsartan, Varenicline (Pfizer), Vimpat, Vitamin E, Warfarin, Zarontin®, Zestril, Zonegran®, Zonisamide, Zydis selegiline HCL Oral disintegrating (Zelapar), and combinations thereof.
For example, to treat or ameliorate the effects of epilepsy, a subject may be administered an effective amount of one or more compounds or pharmaceutical compositions of the present disclosure and, e.g., one or more of the following: Albendazole, Banzel, BGG492 (Novartis Corp., New York, N.Y.) Carbamazepine, Carbatrol®, Clobazam, Clonazepam, Depakene®, Depakote®, Depakote ER®, Diastat, Diazepam, Dilantin®, Eslicarbazepine acetate, Ethosuximide, Ezogabine, Felbatol®, Felbamate, Frisium, Gabapentin, Gabitril®, Inovelon®, JNJ-26489112 (Johnson and Johnson, New Brunswick, N.J.) Keppra®, Keppra XR™, Klonopin, Lacosamide, Lamictal®, Lamotrigine, Levetiracetam, Lorazepam, Luminal, Lyrica, Mysoline®, Memantine, Neurontin®, Onfi®, Oxcarbazepine, Phenobarbital, Phenytek®, Phenytoin, Potiga, Primidone, probenecid, PRX-00023 (EPIX Pharmaceuticals Inc, Lexington, Mass.), Rufinamide, Sabril, Tegretol®, Tegretol XR®, Tiagabine, Topamax®, Topiramate, Trileptal®, Valproic Acid, Vimpat, Zarontin®, Zonegran®, and Zonisamide.
To treat or ameliorate the effects of stroke, a subject may be administered an effective amount of one or more compounds or pharmaceutical compositions of the present disclosure and, e.g., one or more of the following: Aspirin, dipyridamole, Clopidogrel, tissue plasminogen activator (tPA), Warfarin, dabigatran, Heparin, Lovenox, citicoline, L-Alpha glycerylphosphorylcholine, cerebrolysin, Eptifibatide, Escitalopram, Tenecteplase, Alteplase, Minocycline, Esmolol, Sodium Nitroprussiate (NPS), Norepinephrine (NOR), Dapsone, valsartan, Simvastatin, piclozotan, Desmoteplase, losartan, amlodipine, Ancrod, human chorionic gonadotropin (hCG), epoetin alfa (EPO), Galantamine, and THR-18 (Thrombotech Ltd., Ness Ziona, Israel).
To treat or ameliorate the effects of myocardial infarction, a subject may be administered an effective amount of one or more compounds or pharmaceutical compositions of the present disclosure and, e.g., one or more of the following: lisinopril, atenolol, Plavix, metoprolol tartrate, Lovenox, Lopressor, Zestril, Tenormin, Prinivil, aspirin, Arixtra, clopidogrel, Salagen, nitroglycerin, metoprolol tartrate, heparin, Nitrostat, Nitro-Bid, Stanback Headache Powder, nitroglycerin, Activase, Nitrolingual, nitroglycerin, fondaparinux, Lopressor, heparin, nitroglycerin TL, Nitro-Time, Nitromist, Ascriptin, alteplase, Retavase, TNKase, Bufferin, Nitro-Dur, Minitran, reteplase, tenecteplase, clopidogrel, Fragmin, enoxaparin, dalteparin, tirofiban, and Aggrastat.
To treat or ameliorate the effects of type I diabetes, a subject may be administered an effective amount of one or more compounds or pharmaceutical compositions of the present disclosure and, e.g., one or more of the following: insulin, such as regular insulin (Humulin R, Novolin R, others), insulin isophane (Humulin N, Novolin N), insulin lispro (Humalog), insulin aspart (NovoLog), insulin glargine (Lantus) and insulin detemir (Levemir), octreotide, pramlintide, and liraglutide.
To treat or ameliorate the effects of Alzheimer's disease, a subject may be administered an effective amount of one or more compounds or pharmaceutical compositions of the present disclosure and, e.g., one or more of the following: Donepezil (Aricept), Rivastigmine (Exelon), Galantamine (Razadyne), Tacrine (Cognex), Memantine (Namenda), Vitamin E, CERE-110: Adeno-Associated Virus Delivery of NGF (Ceregene), LY450139 (Eli Lilly), Exenatide, Varenicline (Pfizer), PF-04360365 (Pfizer), Resveratrol, and Donepezil (Eisai Korea).
To treat or ameliorate the effects of Parkinson's disease, a subject may be administered an effective amount of one or more compounds or pharmaceutical compositions of the present disclosure and, e.g., one or more of the following: Carbidopa/levodopa immediate-release (Sinemet), Carbidopa/levodopa oral disintegrating (Parcopa), Carbidopa/levodopa/Entacapone (Stalevo), Ropinirole (Requip), Pram ipexole (Mirapex), Rotigotine (Neupro), Apomorphine (Apokyn), Selegiline (1-deprenyl, Eldepryl), Rasagiline (Azilect), Zydis selegiline HCL Oral disintegrating (Zelapar), Entacapone (Comtan), Tolcapone (Tasmar), Amantadine (Symmetrel), Trihexyphenidyl (formerly Artane), Benztropine (Cogentin), IPX066 (Impax Laboratories Inc.), Rasagiline (Teva Neuroscience, Inc.), ioflupane 1231 (DATSCAN®), safinamide (EMD Serono), and Pioglitazone.
To treat or ameliorate the effects of amyotrophic lateral sclerosis, a subject may be administered an effective amount of one or more compounds or pharmaceutical compositions of the present disclosure and, e.g., one or more of the following: riluzole (Rilutek), Lithium carbonate, Arimoclomol, Creatine, Tamoxifen, Mecobalam in, Memantine (Ebixa), and tauroursodeoxycholic acid (TUDCA).
To treat or ameliorate the effects of Friedreich's ataxia, a subject may be administered an effective amount of one or more compounds or pharmaceutical compositions of the present disclosure and, e.g., one or more of the following: Idebenone, Coenzyme Q, 5-hydroxytryptophan, Propranolol, Enalapril, Lisinopril, Digoxin, Erythropoietin, Lu AA24493, Deferiprone, Varenicline, IVIG, Pioglitazone, and EGb 761.
To treat or ameliorate the effects of multiple sclerosis, a subject may be administered an effective amount of one or more compounds or pharmaceutical compositions of the present disclosure and, e.g., one or more of the following: Avonex, Betaseron, Extavia, Rebif, Glatiramer (Copaxone), Fingolimod (Gilenya), Natalizumab (Tysabri), Mitoxantrone (Novantrone), baclofen (Lioresal), tizanidine (Zanaflex), methylprednisolone, CinnoVex, ReciGen, Masitinib, Prednisone, Interferon beta 1a, Interferon beta 1 b, and ELND002 (Elan Pharmaceuticals).
To treat or ameliorate the effects of Huntington's disease, a subject may be administered an effective amount of one or more compounds or pharmaceutical compositions of the present disclosure and, e.g., one or more of the following: Tetrabenazine (Xenazine), haloperidol (Haldol), clozapine (Clozaril), clonazepam (Klonopin), diazepam (Valium), escitalopram (Lexapro), fluoxetine (Prozac, Sarafem), sertraline (Zoloft), valproic acid (Depakene), divalproex (Depakote), lamotrigine (Lamictal), Dimebon, AFQ056 (Novartis), Ethyl-EPA (Miraxion™), SEN0014196 (Siena Biotech), sodium phenylbutyrate, citalopram, ursodiol, minocycline, remacemide, and mirtazapine.
To treat or ameliorate the effects of transmissible spongiform encephalopathy, a subject may be administered an effective amount of one or more compounds or pharmaceutical compositions of the present disclosure and e.g., Quinacrine.
To treat or ameliorate the effects of Charcot-Marie-Tooth disease, a subject may be administered an effective amount of one or more compounds or pharmaceutical compositions of the present disclosure and, e.g., one or more of the following: ascorbic acid and PXT3003.
To treat or ameliorate the effects of dementia with Lewy bodies, a subject may be administered an effective amount of one or more compounds or pharmaceutical compositions of the present disclosure and, e.g., one or more of the following: Aricept, Galantamine, Memantine, Armodafinil, Donepezil, and Ramelteon.
To treat or ameliorate the effects of corticobasal degeneration, a subject may be administered an effective amount of one or more compounds or pharmaceutical compositions of the present disclosure and, e.g., one or more of the following: Davunetide and Coenzyme Q10.
To treat or ameliorate the effects of progressive supranuclear palsy, a subject may be administered an effective amount of one or more compounds or pharmaceutical compositions of the present disclosure and, e.g., one or more of the following: Tideglusib, Rasagiline, alpha-lipoic acid/L-acetyl carnitine, Riluzole, Niacinamide, and Rivastigmine.
To treat or ameliorate the effects of hereditary spastic paraparesis, a subject may be administered an effective amount of one or more compounds or pharmaceutical compositions of the present disclosure and, e.g., one or more of the following: Baclofen, Tizanidine, Oxybutinin chloride, Tolterodine, and Botulinum toxin.
In the present disclosure, one or more compounds or pharmaceutical compositions may be co-administered to a subject in need thereof together in the same composition, simultaneously in separate compositions, or as separate compositions administered at different times, as deemed most appropriate by a physician.
An additional embodiment of the present disclosure is a method for treating or ameliorating the effects of a disorder in a subject in need thereof. This method comprises administering to the subject an effective amount of a pharmaceutical composition comprising a pharmaceutically acceptable carrier or diluent and one or more compounds having the structure of formula (1):
wherein:
R2 cannot be
Suitable and preferred pharmaceutical compositions for use in this method are as disclosed above in formulas (1), (1a), (1 b), (2), (3) and (3a), including pharmaceutical compositions containing the particular compounds identified above. Suitable and preferred subjects who may be treated in accordance with this method are as disclosed above. In this embodiment, the methods may be used to treat disorders set forth above, including degenerative diseases that involve lipid peroxidation and excitotoxic diseases that involve oxidative cell death.
In another aspect of this embodiment, the method further comprises co-administering to the subject an effective amount of one or more additional therapeutic agents disclosed herein.
Another embodiment of the present disclosure is a method of modulating ferroptosis in a subject in need thereof. This method comprises administering to the subject an effective amount of a ferroptosis inhibitor, which comprises one or more compounds having the structure of formula (1):
wherein:
R2 cannot be
As used herein, “ferroptosis” means regulated cell death that is iron-dependent. Ferroptosis is characterized by the overwhelming, iron-dependent accumulation of lethal lipid reactive oxygen species. (Dixon et al., 2012) Ferroptosis is distinct from apoptosis, necrosis, and autophagy. (Id.) Assays for ferroptosis are as disclosed herein, for instance, in the Examples section.
Suitable and preferred compounds for use in this method are as disclosed above in formulas (1), (1a), (1b), (2), (3) and (3a), including the particular compounds identified above. Suitable and preferred subjects who may be treated in accordance with this method are as disclosed above. In this embodiment, the methods may be used to treat the disorders set forth above, including degenerative diseases that involve lipid peroxidation and excitotoxic diseases that involve oxidative cell death.
In another aspect of this embodiment, the method further comprises co-administering to the subject an effective amount of one or more additional therapeutic agents disclosed herein.
A further embodiment of the present disclosure is a method of reducing reactive oxygen species (ROS) in a cell. This method comprises contacting a cell with a ferroptosis modulator, which comprises one or more compounds having the structure of formula (1):
wherein:
R2 cannot be
As used herein, the terms “modulate”, “modulating”, “modulator” and grammatical variations thereof mean to change, such as decreasing or reducing the occurrence of ferroptosis. In this embodiment, “contacting” means bringing the compound and optionally one or more additional therapeutic agents into close proximity to the cells in need of such modulation. This may be accomplished using conventional techniques of drug delivery to the subject or in the in vitro situation by, e.g., providing the compound and optionally other therapeutic agents to a culture media in which the cells are located.
Suitable and preferred compounds for use in this method are as disclosed above in formulas (1), (1a), (1 b), (2), (3) and (3a), including the particular compounds identified above. In this embodiment, reducing ROS may be accomplished in cells obtained from a subject having a disorder as disclosed herein. Suitable and preferred subjects of this embodiment are as disclosed above.
In one aspect of this embodiment, the cell is a mammalian cell. Preferably, the mammalian cell is obtained from a mammal selected from the group consisting of humans, primates, farm animals, and domestic animals. More preferably, the mammalian cell is a human cancer cell.
In another aspect of this embodiment, the method further comprises contacting the cell with at least one additional therapeutic agent as disclosed herein.
An additional embodiment of the present disclosure is a method for treating or ameliorating the effects of a neurodegenerative disease in a subject in need thereof. This method comprises administering to the subject an effective amount of one or more compounds having the structure of formula (1):
wherein:
R2 cannot be
Suitable and preferred compounds for use in this method are as disclosed above in formulas (1), (1a), (1 b), (2), (3) and (3a), including the particular compounds identified above. In this embodiment, the method may be used to treat the disorders set forth above.
Suitable and preferred subjects are as disclosed herein. In this embodiment, the methods may be used to treat the neurodegenerative disorders set forth above.
In one aspect of this embodiment, the method further comprises co-administering to the subject an effective amount of one or more therapeutic agents disclosed herein.
An additional embodiment of the present disclosure is a compound having the structure selected from the group consisting of:
and combinations thereof, or an N-oxide, crystalline form, hydrate, or a pharmaceutically acceptable salt thereof.
An additional embodiment of the present disclosure is a pharmaceutical composition comprising a pharmaceutically acceptable carrier or diluent and a compound having the structure selected from the group consisting of:
and combinations thereof, or an N-oxide, crystalline form, hydrate, or a pharmaceutically acceptable salt thereof.
An additional embodiment of the present disclosure is a method for treating or ameliorating the effects of a disorder in a subject in need thereof comprising administering to the subject an effective amount of a compound having the structure selected from the group consisting of:
and combinations thereof, or an N-oxide, crystalline form, hydrate, or a pharmaceutically acceptable salt thereof.
An additional embodiment of the present disclosure is a method of modulating ferroptosis in a subject in need thereof comprising administering to the subject an effective amount of a ferroptosis inhibitor, which comprises a compound having the structure selected from the group consisting of:
and combinations thereof, or an N-oxide, crystalline form, hydrate, or a pharmaceutically acceptable salt thereof.
An additional embodiment of the present disclosure is a method of reducing reactive oxygen species (ROS) in a cell comprising contacting a cell with a ferroptosis modulator, which comprises a compound having the structure selected from the group consisting of:
and combinations thereof, or an N-oxide, crystalline form, hydrate, or a pharmaceutically acceptable salt thereof.
An additional embodiment of the present disclosure is a method for treating or ameliorating the effects of a neurodegenerative disease in a subject in need thereof comprising administering to the subject an effective amount of a compound having the structure selected from the group consisting of:
and combinations thereof, or an N-oxide, crystalline form, hydrate, or a pharmaceutically acceptable salt thereof.
An additional embodiment of the present disclosure is a compound having the structure selected from the group consisting of:
and combinations thereof, or an N-oxide, crystalline form, hydrate, or a pharmaceutically acceptable salt thereof.
An additional embodiment of the present disclosure is a pharmaceutical composition comprising a pharmaceutically acceptable carrier or diluent and a compound having the structure selected from the group consisting of:
and combinations thereof, or an N-oxide, crystalline form, hydrate, or a pharmaceutically acceptable salt thereof.
An additional embodiment of the present disclosure is a method for treating or ameliorating the effects of a disorder in a subject in need thereof comprising administering to the subject an effective amount of a compound having the structure selected from the group consisting of:
and combinations thereof, or an N-oxide, crystalline form, hydrate, or a pharmaceutically acceptable salt thereof.
An additional embodiment of the present disclosure is a method of modulating ferroptosis in a subject in need thereof comprising administering to the subject an effective amount of a ferroptosis inhibitor, which comprises a compound having the structure selected from the group consisting of:
and combinations thereof, or an N-oxide, crystalline form, hydrate, or a pharmaceutically acceptable salt thereof.
An additional embodiment of the present disclosure is a method of reducing reactive oxygen species (ROS) in a cell comprising contacting a cell with a ferroptosis modulator, which comprises a compound having the structure selected from the group consisting of:
and combinations thereof, or an N-oxide, crystalline form, hydrate, or a pharmaceutically acceptable salt thereof.
An additional embodiment of the present disclosure is a method for treating or ameliorating the effects of a neurodegenerative disease in a subject in need thereof comprising administering to the subject an effective amount of a compound having the structure selected from the group consisting of:
and combinations thereof, or an N-oxide, crystalline form, hydrate, or a pharmaceutically acceptable salt thereof.
Still another embodiment of the present disclosure is a method for alleviating side effects in a subject undergoing radiotherapy and/or immunotherapy, comprising administering to the subject an effective amount of one or more compounds disclosed herein.
As used herein, “radiotherapy” or “radiation therapy” refers to a therapy using ionizing radiation to control or kill malignant cells. Common side effects of radiotherapy include, but are not limited to, acute side effects (such as nausea, vomiting, damage to the epithelial surfaces, mouth, throat and stomach sores, intestinal discomfort, swelling, infertility, etc.), late side effects (such as fibrosis, epilation, dryness, lymphedema, cardiovascular disorder, cognitive decline, radiation enteropathy, radiation-induced polyneuropathy), and cumulative side effects.
As used herein, “immunotherapy” refers to the treatment of disease by activating or suppressing the immune system. It can be classified as an activation immunotherapy that elicits or amplifies an immune response, or a suppression immunotherapy that reduce or suppress an immune response. Common side effects of immunotherapy include, but are not limited to, skin problems (such as pain, swelling, soreness, redness, itchiness, rash, etc.), flu-like symptoms (such as fever, chills, weakness, dizziness, nausea or vomiting, muscle or joint aches, fatigue, headache, trouble breathing, low or high blood pressure, etc.), and other symptoms such as swelling and weight gain from retaining fluid, heart palpitations, sinus congestion, diarrhea, infection, organ inflammation, etc.
A further embodiment of the present disclosure is a method for treating or ameliorating the effects of an infection associated with ferroptosis in a subject, comprising administering to the subject an effective amount of one or more compounds disclosed herein. In some embodiments, the infection is caused by Mycobacterium tuberculosis.
As used herein, a “pharmaceutically acceptable salt” means a salt of the compounds of the present disclosure which are pharmaceutically acceptable, as defined herein, and which possess the desired pharmacological activity. Such salts include 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.
In the present disclosure, an “effective amount” or “therapeutically effective amount” of a compound or pharmaceutical composition is an amount of such a compound or composition that is sufficient to effect beneficial or desired results as described herein when administered to a subject. Effective dosage forms, modes of administration, and dosage amounts may be determined empirically, and making such determinations is within the skill of the art. It is understood by those skilled in the art that the dosage amount will vary with the route of administration, the rate of excretion, the duration of the treatment, the identity of any other drugs being administered, the age, size, and species of the subject, and like factors well known in the arts of, e.g., medicine and veterinary medicine. In general, a suitable dose of a compound or pharmaceutical composition according to the disclosure will be that amount of the compound or composition, which is the lowest dose effective to produce the desired effect with no or minimal side effects. The effective dose of a compound or pharmaceutical composition according to the present disclosure may be administered as two, three, four, five, six or more sub-doses, administered separately at appropriate intervals throughout the day.
A suitable, non-limiting example of a dosage of a compound or pharmaceutical composition according to the present disclosure or a composition comprising such a compound, is from about 1 ng/kg to about 1000 mg/kg, such as from about 1 mg/kg to about 100 mg/kg, including from about 5 mg/kg to about 50 mg/kg. Other representative dosages of a compound or a pharmaceutical composition of the present disclosure include about 1 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg, 45 mg/kg, 50 mg/kg, 60 mg/kg, 70 mg/kg, 80 mg/kg, 90 mg/kg, 100 mg/kg, 125 mg/kg, 150 mg/kg, 175 mg/kg, 200 mg/kg, 250 mg/kg, 300 mg/kg, 400 mg/kg, 500 mg/kg, 600 mg/kg, 700 mg/kg, 800 mg/kg, 900 mg/kg, or 1000 mg/kg.
A compound or pharmaceutical composition of the present disclosure 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 compound or pharmaceutical composition of the present disclosure may be administered in conjunction with other treatments. A compound or pharmaceutical composition of the present disclosure may be encapsulated or otherwise protected against gastric or other secretions, if desired.
The pharmaceutical compositions of the disclosure are pharmaceutically acceptable and comprise one or more active ingredients in admixture with one or more pharmaceutically-acceptable carriers or diluents and, optionally, one or more other compounds, drugs, ingredients and/or materials. Regardless of the route of administration selected, the compounds/pharmaceutical compositions of the present disclosure 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.). More generally, “pharmaceutically acceptable” means that which is useful in preparing a composition that is generally safe, non-toxic, and neither biologically nor otherwise undesirable and includes that which is acceptable for veterinary use as well as human pharmaceutical use.
Pharmaceutically acceptable carriers and diluents 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 tryglycerides), 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 or diluent used in a composition of the disclosure must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject. Carriers or diluents suitable for a selected dosage form and intended route of administration are well known in the art, and acceptable carriers or diluents for a chosen dosage form and method of administration can be determined using ordinary skill in the art.
The pharmaceutical compositions of the disclosure may, optionally, contain additional ingredients and/or materials commonly used in such compositions. These ingredients and materials are well known in the art and include (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and silicic acid; (2) binders, such as carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, hydroxypropylmethyl cellulose, sucrose and acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, sodium starch glycolate, cross-linked sodium carboxymethyl cellulose and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as cetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, and sodium lauryl sulfate; (10) suspending agents, such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth; (11) buffering agents; (12) excipients, such as lactose, milk sugars, polyethylene glycols, animal and vegetable fats, oils, waxes, paraffins, cocoa butter, starches, tragacanth, cellulose derivatives, polyethylene glycol, silicones, bentonites, silicic acid, talc, salicylate, zinc oxide, aluminum hydroxide, calcium silicates, and polyamide powder; (13) inert diluents, such as water or other solvents; (14) preservatives; (15) surface-active agents; (16) dispersing agents; (17) control-release or absorption-delaying agents, such as hydroxypropylmethyl cellulose, other polymer matrices, biodegradable polymers, liposomes, microspheres, aluminum monosterate, gelatin, and waxes; (18) opacifying agents; (19) adjuvants; (20) wetting agents; (21) emulsifying and suspending agents; (22), solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan; (23) propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane; (24) antioxidants; (25) agents which render the formulation isotonic with the blood of the intended recipient, such as sugars and sodium chloride; (26) thickening agents; (27) coating materials, such as lecithin; and (28) sweetening, flavoring, coloring, perfuming and preservative agents. Each such ingredient or material must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject. 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.
Compounds or pharmaceutical 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, dragees, powders, granules and the like) may be prepared, e.g., by mixing the active ingredient(s) with one or more pharmaceutically-acceptable carriers or diluents 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 or diluent. 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 or diluent, 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.
In the foregoing embodiments, the following definitions apply.
The term “aliphatic”, as used herein, refers to a group composed of carbon and hydrogen that do not contain aromatic rings. Accordingly, aliphatic groups include alkyl, alkenyl, alkynyl, and carbocyclyl groups. Additionally, unless otherwise indicated, the term “aliphatic” is intended to include both “unsubstituted aliphatics” and “substituted aliphatics”, the latter of which refers to aliphatic moieties having substituents replacing a hydrogen on one or more carbons of the aliphatic group. Such substituents can include, for example, a halogen, a deuterium, a hydroxyl, a carbonyl (such as a carboxyl, an alkoxycarbonyl, a formyl, or an acyl), a thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), an alkoxyl, a phosphoryl, a phosphate, a phosphonate, a phosphinate, an amino, an amido, an amidine, an imine, a cyano, a nitro, an azido, a sulfhydryl, an alkylthio, a sulfate, a sulfonate, a sulfamoyl, a sulfonamido, a sulfonyl, a heterocyclyl, an aralkyl, an aromatic, or heteroaromatic moiety.
The term “alkyl” refers to the radical of saturated aliphatic groups that does not have a ring structure, including straight-chain alkyl groups, and branched-chain alkyl groups. In certain embodiments, a straight chain or branched chain alkyl has 6 or fewer carbon atoms in its backbone (e.g., C1-C6 for straight chains, C3-C6 for branched chains). In other embodiments, the “alkyl” may include up to twelve carbon atoms, e.g., C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11 or C12. Such substituents include all those contemplated for aliphatic groups, as discussed below, except where stability is prohibitive.
The term “alkenyl”, as used herein, refers to an aliphatic group containing at least one double bond and unless otherwise indicated, is intended to include both “unsubstituted alkenyls” and “substituted alkenyls”, the latter of which refers to alkenyl moieties having substituents replacing a hydrogen on one or more carbons of the alkenyl group. Such substituents include all those contemplated for aliphatic groups, as discussed below, except where stability is prohibitive. For example, substitution of alkenyl groups by one or more alkyl, carbocyclyl, aryl, heterocyclyl, or heteroaryl groups is contemplated.
Moreover, unless otherwise indicated, the term “alkyl” as used throughout the specification, examples, and claims is intended to include both “unsubstituted alkyls” and “substituted alkyls”, the latter of which refers to alkyl moieties having substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone. Indeed, unless otherwise indicated, all groups recited herein are intended to include both substituted and unsubstituted options.
The term “Cx-y” when used in conjunction with a chemical moiety, such as, alkyl and cycloalkyl, is meant to include groups that contain from x to y carbons in the chain. For example, the term “Cx-yalkyl” refers to substituted or unsubstituted saturated hydrocarbon groups, including straight-chain alkyl and branched-chain alkyl groups that contain from x to y carbons in the chain, including haloalkyl groups such as trifluoromethyl and 2,2,2-tirfluoroethyl, etc.
The term “aryl” as used herein includes substituted or unsubstituted single-ring aromatic groups in which each atom of the ring is carbon. Preferably the ring is a 3- to 8-membered ring, more preferably a 6-membered ring. The term “aryl” also includes polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings wherein at least one of the rings is aromatic, e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls. Aryl groups include benzene, naphthalene, phenanthrene, phenol, aniline, and the like.
The term “alkyl-aryl” refers to an alkyl group substituted with at least one aryl group.
The term “alkyl-heteroaryl” refers to an alkyl group substituted with at least one heteroaryl group.
The term “alkenyl-aryl” refers to an alkenyl group substituted with at least one aryl group.
The term “alkenyl-heteroaryl” refers to an alkenyl group substituted with at least one heteroaryl group.
The terms “carbocycle”, “carbocyclyl”, and “carbocyclic”, as used herein, refer to a non-aromatic saturated or unsaturated ring in which each atom of the ring is carbon. Preferably a carbocycle ring contains from 3 to 10 atoms, more preferably from 3 to 8 atoms, including 5 to 7 atoms, such as for example, 6 atoms. The term “cabocycle” also includes bicycles, tricycles and other multicyclic ring systems, including the adamantyl ring system.
The terms “halo” and “halogen” are used interchangeably herein and mean halogen and include chloro, fluoro, bromo, and iodo.
The term “heteroaryl” includes substituted or unsubstituted aromatic single ring structures, preferably 3- to 8-membered rings, more preferably 5- to 7-membered rings, even more preferably 5- to 6-membered rings, whose ring structures include at least one heteroatom, preferably one to four heteroatoms, more preferably one or two heteroatoms. The term “heteroaryl” also includes polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings wherein at least one of the rings is heteroaromatic, e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls. Heteroaryl groups include, for example, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrazine, pyridazine, and pyrimidine, and the like.
The term “heteroatom” as used herein means an atom of any element other than carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen, and sulfur; more preferably, nitrogen and oxygen.
The term “ketone” means an organic compound with the structure RC(═O)R′, wherein neither R nor R′ can be hydrogen atoms.
The term “ether” means an organic compound with the structure R—O—R′, wherein neither R nor R′ can be hydrogen atoms.
The term “ester” means an organic compound with the structure RC(═O)OR′, wherein neither R nor R′ can be hydrogen atoms.
The term “polyyne” means is an organic compound with alternating single and triple bonds; that is, a series of consecutive alkynes, (—C≡C—) n with n greater than 1.
The term “substituted” refers to moieties having substituents replacing a hydrogen on one or more carbons of the backbone. It will be understood that “substitution” or “substituted with” includes the implicit proviso that such substitution is in accordance with the permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, e.g., which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc. As used herein, the term “substituted” is contemplated to include all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic substituents of organic compounds. The permissible substituents can be one or more and the same or different for appropriate organic compounds. For purposes of this disclosure, the heteroatoms such as nitrogen may have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms. Substituents can include any substituents described herein, for example, a halogen, a hydroxyl, a carbonyl (such as a carboxyl, an alkoxycarbonyl, a formyl, or an acyl), a thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), an alkoxyl, a phosphoryl, a phosphate, a phosphonate, a phosphinate, an amino, an amido, an amidine, an imine, a cyano, a nitro, an azido, a sulfhydryl, an alkylthio, a sulfate, a sulfonate, a sulfamoyl, a sulfonamido, a sulfonyl, a heterocyclyl, an aralkyl, or an aromatic or heteroaromatic moiety. It will be understood by those skilled in the art that the moieties substituted on the hydrocarbon chain can themselves be substituted, if appropriate.
As set forth previously, unless specifically stated as “unsubstituted,” references to chemical moieties herein are understood to include substituted variants. For example, reference to an “aryl” group or moiety implicitly includes both substituted and unsubstituted variants.
As used herein, the term “oxadiazole” means any compound or chemical group containing the following structure:
As used herein, the term “oxazole” means any compound or chemical group containing the following structure:
As used herein, the term “triazole” means any compound or chemical group containing the following structure:
It is understood that the disclosure of a compound herein encompasses all stereoisomers of that compound. As used herein, the term “stereoisomer” refers to a compound made up of the same atoms bonded by the same bonds but having different three-dimensional structures which are not interchangeable. The three-dimensional structures are called configurations. Stereoisomers include enantiomers and diastereomers.
The terms “racemate” or “racemic mixture” refer to a mixture of equal parts of enantiomers. The term “chiral center” refers to a carbon atom to which four different groups are attached. The term “enantiomeric enrichment” as used herein refers to the increase in the amount of one enantiomer as compared to the other.
It is appreciated that to the extent compounds of the present disclosure have a chiral center, they may exist in and be isolated in optically active and racemic forms. Some compounds may exhibit polymorphism. It is to be understood that the present disclosure encompasses any racemic, optically-active, diastereomeric, polymorphic, or stereoisomeric form, or mixtures thereof, of a compound of the disclosure, which possess the useful properties described herein, it being well known in the art how to prepare optically active forms (for example, by resolution of the racemic form by recrystallization techniques, by synthesis from optically-active starting materials, by chiral synthesis, or by chromatographic separation using a chiral stationary phase).
Examples of methods to obtain optically active materials are known in the art, and include at least the following:
The stereoisomers may also be separated by usual techniques known to those skilled in the art including fractional crystallization of the bases or their salts or chromatographic techniques such as LC or flash chromatography. The (+) enantiomer can be separated from the (−) enantiomer using techniques and procedures well known in the art, such as that described by J. Jacques, et al., Enantiomers, Racemates, and Resolutions”, John Wiley and Sons, Inc., 1981. For example, chiral chromatography with a suitable organic solvent, such as ethanol/acetonitrile and Chiralpak AD packing, 20 micron can also be utilized to effect separation of the enantiomers.
The following examples are provided to further illustrate the methods of the present disclosure. These examples are illustrative only and are not intended to limit the scope of the disclosure in any way.
The detailed experimental procedures applied to Ferrostatin-1 and its analogs have been described previously in the International Application No. PCT/US2014/067977, filed on Dec. 1, 2014, the entirety of which is incorporated herein by reference.
Solvents, inorganic salts, and organic reagents were purchased from commercial sources such as Sigma and Fisher and used without further purification unless otherwise noted. Erastin was dissolved in DMSO to a final concentration of 73.1 mM and stored in aliquots at −20° C.
Merck pre-coated 0.25 mm silica plates containing a 254 nm fluorescence indicator were used for analytical thin-layer chromatography. Flash chromatography was performed on 230-400 mesh silica (SiliaFlash® P60) from Silicycle.
1H, 13C and 19F NMR spectra were obtained on a Bruker DPX 400 MHz spectrometer. HRMS spectra were taken on double focusing sector type mass spectrometer HX-110A. Maker JEOL Ltd. Tokyo Japan (resolution of 10,000 and 10 KV accel. Volt. Ionization method; FAB (Fast Atom Bombardment) used Xe 3Kv energy. Used Matrix, NBA (m-Nitro benzyl alcohol)).
A representative example is the esterification of the 4-chloro-3-nitrobenzoic acid with tert-butanol. 4-dimethylaminopyridine (DMAP) (2.4607 g, 20.14 mmol, 0.4 equiv) and tert-butanol (24 mL, 250.94 mmol, 5.1 equiv) were added to a solution of 4-chloro-3-nitrobenzoic acid (10.0042 g, 49.63 mmol, 1.0 equiv) dissolved in dichloromethane (350 mL) at room temperature. N, N′-dicyclohexylcarbodiimide (DCC) (13.7853 g, 66.81 mmol, 1.4 equiv) was added to the solution at 0° C. The reaction mixture was allowed to warm to room temperature and stirred overnight under nitrogen atmosphere. The white precipitate was filtered off and the solution was purified by flash-column chromatography on silica gel (hexane, ethyl acetate gradient 40% max).
A representative example is the nucleophilic aromatic substitution of tert-butyl 4-chloro-3-nitrobenzoate with 1-admantylamine. Potassium carbonate (2.1570 g, 15.61 mmol, 1.9 equiv) was added to a solution of tert-butyl 4-chloro-3-nitrobenzoate (2.0784 g, 8.07 mmol, 1.0 equiv) dissolved in DMSO (13 mL). A solution of 1-adamantylamine (1.4273 g, 9.44 mmol, 1.2 equiv) dissolved in DMSO (13 mL) was added to the reaction mixture at room temperature. The reaction mixture was heated at 75° C. and stirred overnight under nitrogen atmosphere. After the reaction mixture was cooled to room temperature, water (200 mL) was added and the aqueous layer was extracted three times with ethyl acetate (100 mL). Combined organic layers were extracted with water (30 mL), dried (MgSO4) and purified by flash-column chromatography on silica gel (hexane, ethyl acetate gradient 40% max).
A representative example is the hydrogenation of tert-butyl 4-(1-adamantylamino)-3-nitrobenzoate. Pd(OH)2 on charcoal (0.5048 g) was added to a solution of tert-butyl 4-(1-adamantylamino)-3-nitrobenzoate (1.0079 g, 2.71 mmol) dissolved in MeOH (100 mL) at room temperature. The reaction mixture was stirred at room temperature overnight under hydrogen atmosphere. The black solid was filtered out and the solution was purified by flash-column chromatography on silica gel (dichloromethane, methanol gradient).
A representative example is the imine formation reaction between tert-butyl 4-(1-adamantylamino)-3-aminobenzoate and pyrimidine-5-carboxaldehyde. Pyrimidine-5-carboxaldehyde (0.5653 g, 5.23 mmol, 2.9 equiv) and MgSO4 (0.7850 g) were added to a solution of tert-butyl 4-(1-adamantylamino)-3-aminobenzoate (0.6097 g, 1.78 mmol, 1.0 equiv) dissolved in dichloromethane (122 mL) at room temperature. The reaction mixture was purged once with nitrogen and stirred at room temperature for two overnights under nitrogen atmosphere. The solution was purified by flash-column chromatography on silica gel (hexane, ethyl acetate gradient).
A representative example is the oxidized imine formation reaction between tert-butyl 4-(1-adamantylamino)-3-aminobenzoate and pyrimidine-5-carboxaldehyde. Pyrimidine-5-carboxaldehyde (0.0415 g, 0.38 mmol, 1.3 equiv) was added to a solution of tert-butyl 4-(1-adamantylamino)-3-aminobenzoate (0.1008 g, 0.29 mmol, 1.0 equiv) dissolved in tert-butanol (6 mL). 4M HCl in dioxane (10 μL) was added to the solution at room temperature. The reaction mixture was stirred at 80° C. for 4 hours under nitrogen atmosphere. The solution was purified by flash-column chromatography on silica gel (dichloromethane, methanol gradient).
A representative example is the reductive amination reaction between tert-butyl 3-(1-adamantylamino)-4-aminobenzoate and cyclohexanone. Cyclohexanone (0.5 mL, 4.83 mmol, 6.8 equiv) was added dropwise to a solution of tert-butyl 3-(1-adamantylamino)-4-aminobenzoate (0.2416 g, 0.706 mmol, 1 equiv) dissolved in 1,2-dichloroethane (24 mL) at room temperature. Sodium triacetoxyborohydride (0.8913 g, 4.21 mmol, 5.96 mmol) and glacial acetic acid (50 μL, 0.874 mmol, 1.24 equiv) were added to the solution at room temperature. The reaction mixture was stirred at room temperature overnight under nitrogen atmosphere. The solution was purified by flash-column chromatography on silica gel (hexane, ethyl acetate gradient).
A general route to obtain the compounds of formulas (I) to (III) follows a three-step synthesis (see below). An SNAr reaction between the commercially available ethyl 4-chloro-3-nitrobenzoate and cyclohexylamine, followed by catalytic hydrogenolysis of the nitro group, provided the desired ferrostatin derivatives. The anilines of the latter were reacted through reductive amination with arylaldehydes in the presence of sodium triacetoxyborohydride or through straightforward alkylation with arylalkylhalides in the presence of Hunig's base.
Experimental data pointed to the benzylic position of ferrostatin analogs as the site of metabolic liability in microsomes, and the ester group as the target of plasma esterases. Therefore, analog synthesis focuses on modification of these positions with the goal of improving microsomal and plasma stability in vitro and with the ultimate goal of producing analogs with improved in vivo properties for use in animal models of disease. Because in silico evaluation of Fer-1 analogs' P450 stability using the Schrodinger Suite P450_SOM program showed agreement with the experimental results with liver microsomes, this computer program is used to guide prioritization of compound synthesis and testing of analogs proposed based on modifications known to inhibit metabolism.
One of the most useful methods of blocking metabolism at a specific site is to use a steric shield—a bulky group that hinders oxidation at the position by cytochrome P450. An efficient synthesis of Fer-1 analogs with bulky, blocking groups incorporated at the benzylic site of oxidation is shown in Scheme 1.
Treatment of commercially available 3-fluoro-4-nitrobenzoic acid with a benzylamine containing the desired bulky substituent at the benzylic position would displace fluoride via an SNAr reaction to give the corresponding aminonitro compound (Saitoh, et al., 2009). A wide range of benzyl amines are commercially available. Enantiomerically pure amines are important because cytochrome P450s are known to be enantioselective in their oxidations. Benzylically disubstituted amines would increase the amount of steric shielding and have the advantage of being achiral. The 2,6-dimethylbenzyl amine illustrates another mode of shielding the benzylic position.
The synthetic route shown in Scheme 1 also allows ready access to other substituted amine analogs that can be explored, and that may be more resistant to metabolism, as they do not have a benzylic position to react with P450s. Thus, aniline, cyclohexylamine, and adamantly amine may be used as starting materials to give the corresponding analogs.
The t-butyl ester is resistant to plasma esterases; however, this group may be acid labile, and may not be resistant to the acidic conditions in the stomach upon oral dosing. Bioisosteres, functionalities that are biologically equivalent to the functional group they are replacing, are commonly used to produce active analogs with improved properties, such as resistance to metabolism (Hamada, et al., 2012). A number of ester bioisosteres have been reported in the literature and can be incorporated into analogs of Fer-1. As shown in the synthetic route in Scheme 2, the acid or ester group of 3-fluoro-4-nitrobenzoic acid can be readily converted into ester bioisosteres, such as oxazoles (Wu, et al., 2004), oxadiazoles (Pipik, et al., 2004), triazoles (Passaniti, et al., 2002), or ketones (Genna, et al., 2011). These intermediates can then be used in the synthetic route outlined in Scheme 1 to produce the desired Fer-1 analogs with ester bioisosteres that are resistant to esterases.
The synthetic routes of representative Fer-1 analogs are illustrated as follow:
All analogs are tested in vitro for their ability to inhibit erastin-induced ferroptosis in cells. Those with an IC50 of <50 nM are tested for metabolic stability in mouse liver microsomes and plasma. Those analogs with T1/2>30 minutes in those assays undergo pharmacokinetic analysis in mice. Those analogs with the best in vivo PK parameters are tested in the HD mouse model (see below).
HT-1080 cells are cultured in DMEM containing 10% fetal bovine serum, 1% supplemented non-essential amino acids and 1% pen/strep mixture (Gibco) and maintained in a humidified environment at 37° C. with 5% CO2 in a tissue culture incubator. 1,000 HT-1080 cells are seeded per well in duplicate 384-well plates (Corning) using a BioMek FX liquid handling robot (Beckman Coulter). The next day, the medium is replaced with 36 μL of medium containing 10 μM erastin with 4 μL of medium containing a dilution series (previously prepared) of DMSO, Fer-1 (positive control) or Fer-1 analogs. 24 hours later, 10 μL Alamar Blue (Invitrogen) cell viability solution is added to the growth media to a final concentration of 10%. Cells are incubated a further 6 hours and then the Alamar Blue fluorescence intensity recorded using a Victor 3 platereader (PerkinElmer)(ex/em 530/590). All experiments are performed at least twice and the background (no cells)-subtracted Alamar Blue values for each combination are averaged between replicates. The same procedure was repeated by replacing erastin (10 μM) with IKE (3 μM) or RSL3 (0.2 μM). From these data, sigmoidal dose-response viability curves (
Each compound (1 μM) is incubated with mouse plasma, for 4 hours at 37° C., with shaking at 100 rpm. The concentration of compound in the buffer and plasma chambers is determined using LC-MS/MS. Metabolism of each compound is predicted using Sites of Metabolism (Schrodinger Suite), which combines intrinsic reactivity analysis (Hammett-Taft) with induced fit docking against 2C9, 2D6 and 3A4. This approach identifies 90% of known metabolism sites and has a false positive rate of 17%. The in vitro metabolic stability of each compound in mouse liver microsomes is determined. Pooled mouse liver microsomes are prepared and stored at −80° C. until needed. Compound stability in liver microsomes is measured at 0, 15, 30, 45 and 60 minutes in duplicate, using LC-MS/MS analysis.
To evaluate the PK profile of compounds, IV, IP, and PO administration of each compound is used in C57BL/6J wt mice. Mice are dosed IV at 10 mg/kg and sacrificed using Nembutal and CO2 euthanasia. Six week old mice (Charles River) that have been acclimated to their environment for 2 weeks are used. All animals are observed for morbidity, mortality, injury, availability of food and water twice per day. Animals in poor health are euthanized. Blood samples are collected via cardiac puncture at each time point (0, 30 minutes, 2, 4, 8, 24 h). In addition, brains are collected, and compound concentration determined at each time point using LCO2N MS/MS. Standard PK parameters are calculated for each route of administration, including T1/2, Cmax, AUC, clearance, Vd and % F.
The properties of Ferrostatin-1 and analogs are summarized in Table 1. CFI-A8, CFI-A9, CFI-A11, CFI-L032, CFI-L034, CFI-L047, CFI-4082 and CFI-4083 show T1/2>120 minutes in either mouse or human liver microsomes. Particularly, CFI-4082 and CFI-4083 show T1/2>120 minutes in both mouse and human liver microsomes. The microsomal stability comparison (half-life measured in mouse) of Fer-1, CFI-102 and TH-2-9-1 is also provided in
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1Hofmans et al., 2016, J. Med. Chem, 59, 2041-2053
bConcentration (nM) of ferrostatin analogue required to achieve 50% viability against HT-1080 treated with 3 μM IKE.
cConcentration (nM) of ferrostatin analogue required to achieve 50% viability against HT-1080 treated with 0.2 μM RSL3.
To determine the suitability of CFI-4082 for further in vivo applications, we administered a single dose of CFI-4082 (20 mg/kg in 50% 2-hydroxypropyl-β-cyclodextrin dissolved in 40% ethanol) to male and female C67BI/6 mice (Jackson Lab) via intraperitoneal injection over the course of eight hours, with the compound concentration in plasma and tissue determined by LC/MS-MS. CFI-4082 was found to have low in vivo plasma stability, but was found to stably accumulate in kidney over 8 hours (
Selected Fer-1 analogs containing a pyridine moiety (
TH-2-9-1 and TH-2-5 compounds were first tested at a concentration range from 20 μM-0 μM. which was too high to capture any death at the lower concentrations, as evidenced by both compounds showing almost full rescue at most concentrations within the range (
The tests were repeated at a lower concentration range from 10 μM-0 μM, which was effective in capturing some of the earlier death. No death was observed with RSL3 for Fer-1, TH-2-9-1, and TH-2-5, suggesting that lower inhibitor concentrations were still needed (
By further lowering the concentration, compounds were tested at a range from 1 μM-0 μM. Erastin was also used in the test as a ferroptosis inducer. Following the same protocol, cells were treated with 10 μM erastin. As shown in
Two more compounds, CFI-102 and TH-2-30 were also tested for their anti-ferroptosis activities, using the the same protocol as described above. Starting with a concentration range from 10 μM-0 μM, both compounds demonstrated activity against both IKE and RSL3, with CFI-102 having an IC50 of ˜10-20 nM against both IKE and RSL3. TH-2-30 was relatively less potent. At 10 μM, both compounds appeared to be toxic, as evidenced by the overall drop in viability for all treatment conditions at the concentration (
The tests were repeated at a lower concentration range from 2.5 μM-0 μM, While the toxicity issues at 10 μM was not present, it appeared that 2.5 μM was too low of a starting concentration for TH-2-30 to fully establish rescue (
Further experiments were performed with a starting concentration of 5 μM to compare the potency between different compounds. According to the results shown in
Patients receiving radiotherapy and/or immunotherapy usually suffer from various side effects including, but not limited to, skin reactions (e.g., redness, itching, peeling, blistering, and dryness) and flu-like symptoms (e.g., fatigue, fever, chills, weakness, nausea, vomiting, dizziness, body aches, and high or low blood pressure). There is evidence showing these side effects may be associated with undesired cell death through ferroptosis, which suggests therapeutic potential for molecules that inhibit/reduce ferroptosis.
To explore such applications, we will introduce the Fer-1 analogs disclosed herein into conventional radiotherapy/immunotherapy protocols. We will monitor patients' (animal and then human patient's) reaction to the combined treatment, and determine whether there is any improvement with respect to common side effects, for example, less or even no occurrence, reduced intensity, etc. We anticipate using in vitro models to inform our animal trials.
It is also believed that ferroptosis plays a critical role in bacteria-induced (e.g., Mycobacterium tuberculosis) cell death and tissue necrosis. In light of this, we expect that the Fer-1 analogs disclosed herein would have therapeutic application against various pathogens through inhibiting unwanted ferroptosis.
After synthesizing and characterizing a series of ferrostatin-1 analogs, three active compounds (TH-2-31 (i.e., CFI-102), TH-4-55-2, and TH-4-67) that meet all criteria for success were identified. Three inactive controls derived from the active compounds were also obtained for comparative studies (
Potency in Suppressing Ferroptosis Induced by RSL3 in N27 Cells (20 nM, 48 Hours of Incubation) with EC50<10 nM
As shown in
EC50 values for three separate experiment, n=3 wells/per compound/per condition, are provided in Table 2 below.
In addition to the optimized ferrostatin compounds described above, three inactive controls (TH-4-50-2, TH-4-58-2, and TH-4-46-2) were developed that are unable to suppress ferroptosis induced by RSL3 in N27 cells (20 nM, 24 hours of incubation). Their structures and representative dose-response curves are shown in
Metabolic Stability in Mouse Liver Microsomes with Half-Life >60 Min
Results from three separate mouse microsomal stability experiments each performed in triplicate demonstrated that the three optimized ferrostatins (TH-2-31, TH-4-55-2, and TH-4-67) are stable in mouse liver microsomes with half-life greater than 60 minutes, with each compound indeed having a half-life greater than two hours (
A summary of the half-lives from the three independent microsomal stability tests in mouse liver microsomes is provided below in Table 3.
For the in vivo studies, the results from which are detailed below. The optimized ferrostatins were administered to C57BL/6 mice at 8 weeks of age. Compounds were administered at a concentration of 20 mg/kg in a vehicle consisting of 1:1 (65% v/v of 25% w/v 2-hydroxypropyl-β-cyclodextrin dissolved in 20% ethanol, 30% v/v PEG-400, and 5% Tween-80): H2O via intravenous injection (IV), intraperitoneal injection (IP), or oral gavage (PO). For each time-point and route of administration, two male and two female mice were used. Mice were euthanized, and plasma and brain samples were obtained from each mouse at 0, 1, 2, 4, 8, and 24 hours after compound administration. Compounds were extracted from plasma and brain homogenate in acetonitrile and analyzed via UHPLC-MS/MS against a standard curve to quantify compound concentrations.
The concentration of each analog in plasma and brain is shown below in
BBB Permeability with Log (Brain/Plasma) Ratio >0
Calculated from the above PK data, BBB permeability was determined. As shown in
Each analog rapidly accumulated in brain following IV accumulation and decreases in concentration thereafter (
A summary with the in vivo brain half-lives is provided in Table 4.
For both TH-2-31 and TH-4-55-2, the IP and PO administrations satisfy the R33 transition criterion. For TH-4-67, the criterion is not met. However, as observed in the data provided below and the corresponding graphs, TH-4-67 accumulates in brain at orders of magnitude higher than the EC50 values at 24 hours post compound administration, and it is expected to be potent irrespective of the half-live in the brain. Indeed, this compounds exceeds its 2 nM EC50 value for the full 24 hours of treatment. Therefore, although the IP and PO half-lives are slightly under 3 hours, the compound is likely to exert a PD and therapeutic effect in mice, due to exceeding its effective concentration in the brain over a 24 hour period.
Table 5 below details the Cmax in both plasma and brain and the EC50 values for each analog and route of administration. All three of the optimized ferrostatins easily meet this criterion.
As shown in
To achieve the 20 mg/kg dose for each compound, mice were injected with a 2 mg/mL solution in the vehicle described above. For all 3 optimized compounds, no precipitation was observed in the resulting 2 mg/mL solutions, even several days after preparation. As detailed in Table 6 below, each of the compounds meet this criterion, with solubility greater than concentrations needed for in vivo injections.
Plasma Stability (Mouse) with Half-Life >120 Min
In two separate experiments, all three compounds were stable in mouse plasma with minimal-to-no degradation of the compounds after a 4 hour incubation (
All optimized compounds were synthesized on a gram scale with high purity, ready for in vivo efficacy studies. >1 gram of each compound was synthesized.
All documents cited in this application are hereby incorporated by reference as if recited in full herein.
Although illustrative embodiments of the present disclosure have been described herein, it should be understood that the disclosure is not limited to those described, and that various other changes or modifications may be made by one skilled in the art without departing from the scope or spirit of the disclosure.
The present application is a continuation-in-part of PCT international application no. PCT/US2019/063640, filed on Nov. 27, 2019, which claims benefit of U.S. Provisional Patent Application Ser. No. 62/771,841, filed on Nov. 27, 2018, which applications are incorporated by reference herein in their entireties.
This disclosure was made with government support under grant nos. CA097061, CA209896 and NS109407, awarded by National Institutes of Health. The government has certain rights in the disclosure.
| Number | Date | Country | |
|---|---|---|---|
| 62771841 | Nov 2018 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/US2019/063640 | Nov 2019 | US |
| Child | 17330386 | US |
