Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
  • C07D417/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing three or more hetero rings
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
  • A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene or sparfloxacin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents

Definitions

• Cancer is caused by uncontrolled and unregulated cellular proliferation.
• the consequence of this often-rapid proliferation is a high level of oxidative stress within the tumor which damages DNA and leads to a much-increased mutation rate.
• Tumor cells therefore engage and rely heavily upon DNA damage repair mechanisms.
• Single-strand breaks are the most common type of lesion arising in cells and PARG (Poly ADP-ribose glycohydrolase) together with PARP (poly ADP -ribose polymerase) is involved along with a number of other proteins in single strand break repair (SSBR) and another repair mechanism called base excision repair (BER).
• PARG Poly ADP-ribose glycohydrolase
• PARP poly ADP -ribose polymerase
• PARP poly ADP-ribose polymerase
• PARG poly ADP-ribose
• PARG is derived from a single gene with isoforms that reside in the nucleus, mitochondria and cytosol.
• Another known protein with glycohydrolase activity is ARH3 which is localized to the mitochondria (Mashimo, Kato et al. 2014).
• PARG impacts PAR signaling in splicing, transcriptional and epigenetic pathways (Ji and Tulin 2009) (Le May, litis et al. 2012) (Dahl, Maturi et al. 2014) (Guastafierro, Catizone et al. 2013) (Caiafa, Guastafierro et al. 2009).
• Cancer cells may become reliant upon a specific DNA repair pathway when other mechanisms of DNA repair are non-functional.
• Tumors carrying mutations in proteins involved in double strand break repair are often more sensitive to PARP inhibitors of SSBR.
• PARP inhibitors of SSBR There is already some evidence that PARG depletion inhibits SSBR and reduces survival of BRCA2- deficient cells (Fathers, Drayton et al. 2012).
• BRCA-ness double strand DNA repair mechanisms
• PARG depletion sensitizes lung, cervical and pancreatic cancer cells to y- irradiation or experimental DNA damaging agents (e.g. hydrogen peroxide, Methylmethanesulfonate) (Ame, Fouquerel et al. 2009) (Nakadate, Kodera et al. 2013) (Shirai, Poetsch et al. 2013).
• DNA damaging agents e.g. hydrogen peroxide, Methylmethanesulfonate
• PARP inhibitors are currently undergoing multiple clinical trials where the concept of synthetic lethality or chemo-sensitization is being explored. Clinical resistance to PARP inhibitors has already been described (Drost and Jonkers 2014) (Barber, Sandhu et al. 2013) and therefore there is a requirement that alternative inhibitors targeting the DNA damage repair machinery are found.
• An object of this invention is to provide cell permeable inhibitors of PARG.
• a compound of Formula (I): or a pharmaceutically acceptable salt thereof is provided herein.
• provided herein is a compound of Formula (C) or a pharmaceutically acceptable salt thereof.
• a compound of Formula (C) is a compound of Formula (C)
• a pharmaceutical composition comprising a compound of Formula (I), Formula (A), Formula (B), or Formula (C), or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients.
• the cancer is a human cancer.
• a compound of Formula (I), Formula (A), Formula (B), or Formula (C), or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as defined herein in the manufacture of a medicament for use in the treatment of cancer.
• the medicament is for use in the treatment of human cancers.
• a method of inhibiting PARG in vitro or in vivo comprising contacting a cell with an effective amount of a compound of Formula (I), Formula (A), Formula (B), or Formula (C), or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as defined herein.
• a method of inhibiting cell proliferation in vitro or in vivo comprising contacting a cell with an effective amount of a compound of Formula (I), Formula (A), Formula (B), or Formula (C), or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as defined herein.
• a method of treating cancer in a patient in need of such treatment comprising administering to said patient a therapeutically effective amount of a compound of Formula (I), Formula (A), Formula (B), or Formula (C), or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as defined herein.
• a method of treating a cancer resistant to one or more platins or one or more PARP inhibitors in a patient in need thereof comprising administering to said patient an effective amount of a compound Formula (I), Formula (A), Formula (B), or Formula (C), or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as defined herein.
• a method of treating cancer in a patient in need of such treatment comprising administering to said patient a therapeutically effective amount of a compound of Formula (I), Formula (A), Formula (B), or Formula (C), or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as defined herein, wherein the patient has been previously treated for cancer with a platin.
• a method of treating cancer in a patient in need of such treatment comprising administering to said patient a therapeutically effective amount of a compound of Formula (I), Formula (A), Formula (B), or Formula (C), or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as defined herein, wherein the patient has been previously treated for cancer with a PARP inhibitor.
• a method of identifying PARG activity in a test compound of PARG inhibitory activity comprising (i) contacting the test compound with isolated PARG enzyme, a biotinylated-PARylated PARP substrate to form a PARG reaction pre-mixture; (ii) contacting the PARG reaction pre-mixture with a detection antibody and streptavidin-europium to form a PARG reaction mixture; and (iii) measuring fluorescence intensity of the PARG reaction mixture, wherein said method further comprises performing steps (i)-(iii) with a positive control sample represented by a compound of Formula (I), Formula (A), Formula (B), or Formula (C), or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as defined herein.
• the detection antibody is an anti-His monoclonal antibody-ULight.
• the streptavidin-europium binds to the biotinylated-PARylated PARP substrate.
• fluorescence is measured by providing an excitation wavelength of 317 nM and measuring emissions at 620 nM (streptavidin- europium emission) and 665 nM (ULight emission).
• a compound of Formula (I), Formula (A), Formula (B), or Formula (C), or a pharmaceutically acceptable salt obtainable by, or obtained by, or directly obtained by a method of synthesis as defined herein.
• FIG. 1 plots percent viability of PARPi resistant MDA-MB-436 cells as a function of the Log concentration of Formula A as described in Example 4.
• Parent IC50 refers to the IC50 of Formula A before the cells developed PARPi resistance
• Nonraparib resistance IC50 refers to the IC50 of Formula A after the cells developed PARPi resistance.
• FIG. 2 plots percent viability of PARPi resistant HCC1428 cells as a function of the Log concentration of Formula A as described in Example 4.
• Parent TC50 refers to the IC50 of Formula A before the cells developed PARPi resistance
• Nonraparib resistance IC50 refers to the IC50 of Formula A after the cells developed PARPi resistance.
• FIG. 3 plots patient derived cells (PDC) from ovarian tissue with BRCA1/2 alterations and cisplatin resistance as a function of the concentration of Formula A as described in Example 4.
• FIG. 4 Shows Patient-derived xenograft (PDX) study with HBCx-34 (Formula A).
• the compounds of the present disclosure are potent inhibitors of PARG in both cellular and in vitro assays. Kinetic solubility studies also demonstrate that these compounds are highly soluble at both pH 2.0 and pH 7.4. Collectively, the combined parameters of the compounds described herein make them ideal compounds for targeting and inhibiting PARG activity.
• salts are meant to include salts of a compound of Formula (I), Formula (A), Formula (B), or Formula (C) which are prepared with relatively nontoxic acids or bases, depending on the particular substituents found on the compounds described herein.
• Base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired base, either neat or in a suitable inert solvent.
• salts derived from pharmaceutically acceptable inorganic bases include aluminum, ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganic, manganous, potassium, sodium, zinc and the like.
• Salts derived from pharmaceutically-acceptable organic bases include salts of primary, secondary and tertiary amines, including substituted amines, cyclic amines, naturally-occurring amines and the like, such as arginine, betaine, caffeine, choline, N,N’ -dibenzyl ethylenediamine, di ethylamine, 2-di ethylaminoethanol, 2- dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purines, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine and the like.
• Acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired acid, either neat or in a suitable inert solvent.
• suitable inert solvent examples include those derived from inorganic acids like hydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic, or phosphorous acids and the like, as well as the salts derived from relatively nontoxic organic acids like acetic, propionic, isobutyric, malonic, benzoic, succinic, suberic, fumaric, mandelic, phthalic, benzenesulfonic, p- tolylsulfonic, citric, tartaric, methanesulfonic, and the like.
• salts of amino acids such as arginate and the like, and salts of organic acids like glucuronic or galactunoric acids and the like (see, for example, Berge, S.M., et al, “Pharmaceutical Salts”, Journal of Pharmaceutical Science 1977, 66, 1-19).
• Certain specific compounds of the present invention contain both basic and acidic functionalities that allow the compounds to be converted into either base or acid addition salts.
• stereochemical depiction it is meant to refer the compound in which one of the isomers is present and substantially free of the other isomer. ‘Substantially free of another isomer indicates at least an 80/20 ratio of the two isomers, more preferably 90/10, or 95/5 or more. In some embodiments, one of the isomers will be present in an amount of at least 99%.
• Formula (I), Formula (A), Formula (B), or Formula (C) may also contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute such compounds.
• Unnatural proportions of an isotope may be defined as ranging from the amount found in nature to an amount consisting of 100% of the atom in question.
• the compounds may incorporate radioactive isotopes, such as for example tritium ( 3 H), iodine-125 ( 125 I) or carbon-14 ( 14 C), or non-radioactive isotopes, such as deuterium ( 2 H) or carbon-13 ( 13 C).
• radioactive isotopes such as for example tritium ( 3 H), iodine-125 ( 125 I) or carbon-14 ( 14 C), or non-radioactive isotopes, such as deuterium ( 2 H) or carbon-13 ( 13 C).
• isotopic variations can provide additional utilities to those described elsewhere within this application.
• isotopic variants of the compounds of the invention may find additional utility, including but not limited to, as diagnostic and/or imaging reagents, or as cytotoxic/radiotoxic therapeutic agents.
• isotopic variants of Formula (I), Formula (A), Formula (B), or Formula (C) can have altered pharmacokinetic and pharmacodynamic characteristics which can contribute to enhanced safety, tolerability or efficacy during treatment. All isotopic variations of Formula (I), Formula (A), Formula (B), or Formula (C), whether radioactive or not, are intended to be encompassed within the scope of the present invention.
• patient or “subject” are used interchangeably to refer to a human or a nonhuman animal (e.g., a mammal). In one embodiment, the patient or subject is a human.
• administration refers to contact of, for example, an inhibitor of PARG, a pharmaceutical composition comprising same, or a diagnostic agent to the patient, cell, tissue, organ, or biological fluid.
• administration includes contact (e g., in vitro or ex vivo) of a reagent to the cell, as well as contact of a reagent to a fluid, where the fluid is in contact with the cell.
• treat refers to a course of action (such as administering an inhibitor of PARG or a pharmaceutical composition comprising same) initiated after a disease, disorder or condition, or a symptom thereof, has been diagnosed, observed, and the like so as to eliminate, reduce, suppress, mitigate, or ameliorate, either temporarily or permanently, at least one of the underlying causes of a disease, disorder, or condition afflicting a patient, or at least one of the symptoms associated with a disease, disorder, condition afflicting a patient.
• treatment includes inhibiting (e.g., arresting the development or further development of the disease, disorder or condition or clinical symptoms association therewith) an active disease.
• the term “in need of treatment” as used herein refers to a judgment made by a physician or other caregiver that a patient requires or will benefit from treatment. This judgment is made based on a variety of factors that are in the realm of the physician’s or caregiver’s expertise.
• prevent refers to a course of action (such as administering a PARG inhibitor or a pharmaceutical composition comprising same) initiated in a manner (e.g., prior to the onset of a disease, disorder, condition or symptom thereof) so as to prevent, suppress, inhibit or reduce, either temporarily or permanently, a patient’s risk of developing a disease, disorder, condition or the like (as determined by, for example, the absence of clinical symptoms) or delaying the onset thereof, generally in the context of a patient predisposed to having a particular disease, disorder or condition.
• the terms also refer to slowing the progression of the disease, disorder or condition or inhibiting progression thereof to a harmful or otherwise undesired state.
• in need of prevention refers to a judgment made by a physician or other caregiver that a patient requires or will benefit from preventative care. This judgment is made based on a variety of factors that are in the realm of a physician’s or caregiver’s expertise.
• the terms “inhibiting” and “reducing,” or any variation of these terms in relation of PARG includes any measurable decrease or complete inhibition to achieve a desired result. For example, there may be a decrease of about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more, reduction of PARG activity compared to normal. About as used herein means within ⁇ 10%, preferably ⁇ 5% of a given value.
• the phrase “therapeutically effective amount” refers to the administration of an agent to a patient, either alone or as part of a pharmaceutical composition and either in a single dose or as part of a series of doses, in an amount capable of having any detectable, positive effect on any symptom, aspect, or characteristic of a disease, disorder or condition when administered to the patient.
• the therapeutically effective amount can be ascertained by measuring relevant physiological effects, and it can be adjusted in connection with the dosing regimen and diagnostic analysis of the patient’s condition, and the like.
• measurement of the serum level of a PARG inhibitor (or, e g., a metabolite thereof) at a particular time postadministration may be indicative of whether a therapeutically effective amount has been used.
• platinums or “platinum based-chemotherapeutics” refers to a platinum containing class of chemotherapeutic drug used for treating cancer.
• exemplary platins are cisplatin, carboplatin, satraplatin, heptaplatin, picoplatin, nedaplatin, triplatin, lipoplatin, and oxaliplatin.
• platinum-resistant cancer or “cancer resistant to one or more platins” refers to cancers that do not respond to treatment with a platin.
• Nonresponsivness can be assessed by the continued growth of a tumor when administered the agent, a tumor that does not shrink in size when administered the agent, or other known means in the art.
• Nonresponsivness of a cancer can be determined through clinical observation, diagnosed as such by a medical professional, experimentally tested with isolated cells in a laboratory setting, or by another technical means.
• PARP Poly ADP Ribose Polymerase
• PARP inhibitors include, but are not limited to, niraparib, rucaparib, olaparib, talazoparib, and veliparib.
• PARP inhibitor-resistant cancer or “cancer resistant to one or more PARP inhibitors” refers to cancers that do not respond to treatment with a PARP inhibitor.
• Nonresponsivness can be assessed by the continued growth of a tumor when administered the agent, a tumor that does not shrink in size when administered the agent, or other known means in the art.
• Nonresponsivness of a cancer can be determined through clinical observation, diagnosed as such by a medical professional, experimentally tested with isolated cells in a laboratory setting, or by another technical means.
• homologous recombination refers to the cellular process of genetic recombination in which nucleotide sequences are exchanged between two similar or identical DNA sequences.
• homologous recombination deficient (HRD) cancer refers to a cancer that is characterized by a reduction or absence of a functional HR repair pathway. HR deficiency may arise from absence or reduction of one or more HR-associated genes or presence of one or more mutations in one or more EIR-associated genes.
• HR-associated genes include BRCA1, BRCA2, RAD54, RAD51B, ATM, BARD1, CHECK!, CHECK2, CDK12, RAD51B, RAD54L, RAD51D, PPP22A, BRIP1, CtIP (CtBP-interacting protein), PALB2 (Partner and Localizer of BRCA2), XRCC2 (X-ray repair complementing defective repair in Chinese hamster cells 2), RECQL4 (RecQ Protein-Like 4), BLM (Bloom syndrome, RecQ helicase-like), WRN (Wemer syndrome , one or more HR-associated genes) Nbs 1 (Nibrin), and genes encoding Fanconi anemia (FA) proteins or FA-like genes e.g, FANCA, FANCB, FANCC, FANCDI (BRCA2), FANCD2, FANCE, FANCF, FANCG, FANCI, FANJ (BRIPI), FANCL, FANCM, FANCN (RALB2), FANC
• Formula (I) or a pharmaceutically acceptable salt thereof.
• Formula (I) is in a free-base form.
• Formula (A) is in a free-base form.
• Formula (A) is at least 70 % free of other isomers. In some embodiments, Formula (A) is at least 75 % free of other isomers. In some embodiments, Formula (A) is at least 80 % free of other isomers. In some embodiments, Formula (A) is at least 85 % free of other isomers. In some embodiments, Formula (A) is at least 90 % free of other isomers. In some embodiments, Formula (A) is at least 95 % free of other isomers. In some embodiments, Formula (A) is at least 99 % free of other isomers.
• Formula (B) or a pharmaceutically acceptable salt thereof.
• Formula (B) is in a free-base form.
• Formula (B) is at least 70 % free of other isomers.
• Formula (B) is at least 75 % free of other isomers.
• Formula (B) is at least 80 % free of other isomers.
• Formula (B) is at least 85 % free of other isomers.
• Formula (B) is at least 90 % free of other isomers.
• Formula (B) is at least 95 % free of other isomers.
• Formula (B) is at least 99 % free of other isomers.
• Formula (C) or a pharmaceutically acceptable salt thereof.
• Formula (C) is in a free-base form.
• Formula (C) is at least 70 % free of other isomers. In some embodiments, Formula (C) is at least 75 % free of other isomers. In some embodiments, Formula (C) is at least 80 % free of other isomers. In some embodiments, Formula (C) is at least 85 % free of other isomers. In some embodiments, Formula (C) is at least 90 % free of other isomers. In some embodiments, Formula (C) is at least 95 % free of other isomers. In some embodiments, Formula (C) is at least 99 % free of other isomers.
• compositions which comprise a compound of Formula (I), Formula (A), Formula (B), or Formula (C), or a pharmaceutically acceptable salt thereof, in association with a pharmaceutically acceptable diluent or carrier.
• compositions may be in a form suitable for oral use (for example as tablets, lozenges, hard or soft capsules, aqueous or oily suspensions, emulsions, dispersible powders or granules, syrups or elixirs), for topical use (for example as creams, ointments, gels, or aqueous or oily solutions or suspensions), for administration by inhalation (for example as a finely divided powder or a liquid aerosol), for administration by insufflation (for example as a finely divided powder) or for parenteral administration (for example as a sterile aqueous or oily solution for intravenous, subcutaneous, intramuscular, intraperitoneal or intramuscular dosing or as a suppository for rectal dosing).
• oral use for example as tablets, lozenges, hard or soft capsules, aqueous or oily suspensions, emulsions, dispersible powders or granules, syrups or elix
• compositions may be obtained by conventional procedures using conventional pharmaceutical excipients, well known in the art.
• compositions intended for oral use may contain, for example, one or more coloring, sweetening, flavoring and/or preservative agents.
• An effective amount of a compound of Formula (I), Formula (A), Formula (B), or Formula (C), or a pharmaceutically acceptable salt thereof for use in therapy is an amount sufficient to treat or prevent a proliferative condition referred to herein, slow its progression and/or reduce the symptoms associated with the condition.
• the size of the dose for therapeutic or prophylactic purposes of a compound of Formula (I), Formula (A), Formula (B), or Formula (C) will naturally vary according to the nature and severity of the conditions, the age and sex of the animal or patient and the route of administration, according to well-known principles of medicine.
• compositions of Formula (A) comprise at least 70 % of the displayed isomer. In some embodiments, compositions of Formula (A) comprise at least 75 % of the displayed isomer. In some embodiments, compositions of Formula (A) comprise at least 80 % of the displayed isomer. In some embodiments, compositions of Formula (A) comprise at least 85 % of the displayed isomer. In some embodiments, compositions of Formula (A) comprise at least 90 % of the displayed isomer. In some embodiments, compositions of Formula
• compositions of Formula (A) comprise at least 95 % of the displayed isomer. In some embodiments, compositions of Formula (A) comprise at least 99 % of the displayed isomer.
• compositions of Formula (B) comprise at least 95 % of the displayed isomer. In some embodiments, compositions of Formula (B) comprise at least 99 % of the displayed isomer.
• compositions of Formula (C) comprise at least 95 % of the displayed isomer. In some embodiments, compositions of Formula (C) comprise at least 99 % of the displayed isomer.
• the present invention therefore provides a method of inhibiting PARG enzyme activity in vitro or in vivo, said method comprising contacting a cell with an effective amount of a compound of Formula (I), Formula (A), Formula (B), or Formula (C), or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as defined herein.
• the present invention also provides a method of treating a disease or disorder in which PARG activity is implicated in a patient in need of such treatment, said method comprising administering to said patient a therapeutically effective amount of a compound of Formula (I), Formula (A), Formula (B), or Formula (C), or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as defined herein.
• the disease or disorder is an advanced or metastatic solid tumor.
• the disease or disorder is cancer.
• the cancer is ovarian, gastric, or breast cancer.
• the cancer is lung, cervical, or pancreatic cancer.
• the cancer is prostate cancer.
• the cancer is a homologous recombinant deficient (HRD) cancer.
• a method of inhibiting cell proliferation comprising contacting a cell with an effective amount of a compound of Formula (I), Formula (A), Formula (B), or Formula (C), or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as defined herein.
• the proliferative disorder is a solid tumor.
• the proliferative disorder is a metastatic solid tumor.
• the proliferative disorder is cancer.
• the cancer is ovarian, gastric, or breast cancer.
• the cancer is lung, cervical or pancreatic cancer.
• the cancer is prostate cancer.
• the cancer is a homologous recombinant deficient (HRD) cancer.
• a method of treating cancer in a patient in need of such treatment comprising administering to said patient a therapeutically effective amount of a compound of Formula (I), Formula (A), Formula (B), or Formula (C), or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as defined herein.
• the cancer is ovarian, gastric, or breast cancer.
• the cancer is lung, cervical or pancreatic cancer.
• the cancer is a homologous recombinant deficient (HRD) cancer.
• a method of treating and/or preventing a homologous recombinant deficient (HRD) cancer in a patient comprising administering to the patient a therapeutically effective amount of a compound of Formula (I), Formula (A), Formula (B), or Formula (C), or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as defined herein.
• the patient is in recognized need of such treatment.
• the homologous recombinant deficient (HRD) cancer is breast cancer, ovarian cancer, gastric cancer, prostate cancer, lung cancer, cervical cancer, or pancreatic cancer.
• a method of treating and/or preventing a cancer in a patient where the cancer is characterized by a reduction or absence of BRCA1 and/or BRCA2 gene expression, the absence or mutation of BRCA1 and/or BRCA2 genes, or reduced function of BRCA1 and/or BRCA2 proteins, comprising administering to the patient a therapeutically effective amount of a compound of Formula (I), Formula (A), Formula (B), or Formula (C), or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as defined herein.
• the cancer is ovarian, gastric, or breast cancer.
• the cancer is lung, cervical or pancreatic cancer.
• the cancer is prostate cancer.
• the cancer is PARP inhibitor-resistant.
• the PARP inhibitor-resistant cancer is resistant to any one or more of niraparib, olaparib, rucaparib, talazoparib, veliparib, AZD53O5, or AZD9574.
• the PARP inhibitorresistant cancer is resistant to niraparib.
• the PARP inhibitor-resistant cancer is resistant to olaparib.
• the PARP inhibitor-resistant cancer is ovarian cancer, breast cancer, or pancreatic cancer.
• the cancer is platin-resistant.
• the platin- resistant cancer is resistant to any one or more of cisplatin, carboplatin, satraplatin, heptaplatin, picoplatin, nedaplatin, triplatin, lipoplatin, or oxaliplatin.
• the platin- resistant cancer is resistant to cisplatin.
• the platin-resistant cancer is resistant to carboplatin.
• kits for treating cancer in a patient in need of such treatment comprising administering to said patient a therapeutically effective amount of a compound of Formula (I), Formula (A), Formula (B), or Formula (C), or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as defined herein, wherein the patient has been determined to be resistant to one or more PARP inhibitors.
• said methods comprise administering to said patient a therapeutically effective amount of a compound of Formula (I), Formula (A), Formula (B), or Formula (C), or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as defined herein, wherein said patient has been diagnosed as resistant to one or more PARP inhibitors.
• kits for treating cancer in a patient in need of such treatment comprising administering to said patient a therapeutically effective amount of a compound of Formula (I), Formula (A), Formula (B), or Formula (C), or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as defined herein, wherein the patient has been determined to be resistant to platinum based chemotherapeutics.
• said methods comprise administering to said patient a therapeutically effective amount of a compound of Formula (I), Formula (A), Formula (B), or Formula (C), or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as defined herein, wherein said patient has been diagnosed as resistant to one or more platinum based chemotherapeutics.
• the cancer is human cancer.
• the cancer is ovarian, gastric, or breast cancer.
• the cancer is lung, cervical or pancreatic cancer.
• the cancer is prostate cancer.
• the cancer is a homologous recombinant deficient (HRD) cancer.
• the medicament is for use in the treatment of human cancers.
• the cancer is ovarian, gastric, or breast cancer.
• the cancer is lung, cervical or pancreatic cancer.
• the cancer is prostate cancer.
• the cancer is a homologous recombinant deficient (HRD) cancer.
• the present disclosure also contemplates the use of the compound of Formula (I), Formula (A), Formula (B), or Formula (C), or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as defined herein in combination with other therapeutically active agents or compounds as described herein in order to treat the diseases, disorders and conditions contemplated by the present disclosure.
• proliferative disorder and “proliferative condition” are used interchangeably herein and pertain to an unwanted or uncontrolled cellular proliferation of excessive or abnormal cells which is undesired, such as, neoplastic or hyperplastic growth, whether in vitro or in vivo.
• proliferative conditions include, but are not limited to, pre-malignant and malignant cellular proliferation, including but not limited to, malignant neoplasms and tumors, cancers, leukemias, psoriasis, bone diseases, fibroproliferative disorders (e.g., of connective tissues), and atherosclerosis. Any type of cell may be treated, including but not limited to, lung, colon, breast, ovarian, prostate, gastric, liver, pancreas, brain, and skin.
• Proliferative disorders also include, for example, advanced or metastatic solid tumors.
• the anti-cancer effect may arise through one or more mechanisms, including but not limited to, the regulation of cell proliferation, the inhibition of angiogenesis (the formation of new blood vessels), the inhibition of metastasis (the spread of a tumor from its origin), the inhibition of invasion (the spread of tumor cells into neighboring normal structures), or the promotion of apoptosis (programmed cell death).
• the proliferative condition to be treated is cancer.
• a compound of Formula (I), Formula (A), Formula (B), or Formula (C), or a pharmaceutically acceptable salt thereof or pharmaceutical compositions comprising this compound may be administered to a patient by any convenient route of administration, whether systemically/ peripherally or topically (i.e., at the site of desired action).
• Routes of administration include, but are not limited to, oral (e.g., by ingestion); buccal; sublingual; transdermal (including, e.g., by a patch, plaster, etc.); transmucosal (including, e.g., by a patch, plaster, etc.); intranasal (e.g., by nasal spray); ocular (e.g., by eye drops); pulmonary (e.g., by inhalation or insufflation therapy using, e.g., via an aerosol, e.g., through the mouth or nose); rectal (e.g., by suppository or enema); vaginal (e.g., by pessary); parenteral, for example, by injection, including subcutaneous, intradermal,sticular, intravenous, intra-arterial, intracardiac, intrathecal, intraspinal, intracapsular, subcapsular, intraorbital, intraperitoneal, intratracheal, sub
• Embodiment 1 A compound of Formula (I): or a pharmaceutically acceptable salt thereof.
• Embodiment 2 The compound of embodiment 1, represented by Formula (A):
• Embodiment 3 The compound of embodiment 1 that is in a free-base form.
• Embodiment 3A The compound of embodiment 2, that is in a free-base form.
• Embodiment 4 The compound of embodiments 2 or 3A, which is at least 90% free of other isomers.
• Embodiment 5 The compound of embodiments 2 or 3A, which is at least 95% free of other isomers.
• Embodiment 6 The compound of embodiments 2 or 3A, which is at least 99% free of other isomers.
• Embodiment 7 The compound of embodiment 1, represented by Formula (B): or a pharmaceutically acceptable salt thereof.
• Embodiment 8 The compound of embodiment 7 that is in a free-base form.
• Embodiment 9 The compound of embodiments 7 or 8, which is at least 90% free of other isomers.
• Embodiment 10 The compound of embodiments 7 or 8, which is at least 95% free of other isomers.
• Embodiment 11 The compound of embodiments 7 or 8, which is at least 99% free of other isomers.
• Embodiment 12 The compound of embodiment 1, represented by Formula (C): or a pharmaceutically acceptable salt thereof.
• Embodiment 13 The compound of embodiment 12 that is in a free-base form.
• Embodiment 14 The compound of embodiments 12 or 13, which is at least 90% free of other isomers.
• Embodiment 15 The compound of embodiments 12 or 13, which is at least 95% free of other isomers.
• Embodiment 16 The compound of embodiments 12 or 13, which is at least 99% free of other isomers.
• Embodiment 17 A pharmaceutical composition comprising a compound or a pharmaceutically acceptable salt thereof of any one of embodiments 1 to 16, and a pharmaceutically acceptable excipient.
• Embodiment 18A The method of embodiment 18, wherein the patient is in recognized need of such treatment and the disease or disorder is cancer.
• Embodiment 18B The method of embodiment 18, wherein the disease or disorder is cancer.
• Emodiment 18B The method of embodiment 18A or 18B, wherein the cacner is breast cancer, ovarian cancer, gastric cancer, lung cacner, cervical cancer, prostate cancer, or pancreatic cancer.
• Embodiment 19 A method of treating a cancer in a patient in need thereof, said method comprising administering to said patient an effective amount of a compound or a pharmaceutically acceptable salt thereof of any one of embodiment 1 to 16, or a pharmaceutical composition of embodiment 17.
• Embodiment 20 The method of embodiment 19, wherein said cancer is ovarian, gastric, or breast cancer.
• Embodiment 20A The method of embodiment 19, wherein said cancer is lung, cervical or pancreatic cancer.
• Embodiment 20B The method of embodiment 19, wherein the cancer is breast cancer, ovarian cancer, gastric cancer, lung cancer, cervical cancer, prostate cancer, or pancreatic cancer.
• Embodiment 21 A compound or a pharmaceutically acceptable salt thereof of any one of embodiments 1 to 16, or a pharmaceutical composition of embodiment 17 for use in therapy.
• Embodiment 22 The compound or pharmaceutically acceptable salt thereof or the pharmaceutical composition of embodiment 21, wherein said therapy is the treatment of a cancer.
• Embodiment 23 The compound or the pharmaceutically acceptable salt thereof or the pharmaceutical composition of embodiment 22, wherein said cancer is ovarian, gastric, or breast cancer.
• Embodiment 23A The compound or the pharmaceutically acceptable salt thereof or the pharmaceutical composition of embodiment 22, wherein said cancer is lung, cervical or pancreatic cancer.
• Embodiment 23B The compound or the pharmaceutically acceptable salt thereof or the pharmaceutical composition of embodiment 22, wherein the cancer is breast cancer, ovarian cancer, gastric cancer, lung cancer, cervical cancer, prostate cancer, or pancreatic cancer.
• Embodiment 24 The use of a compound or a pharmaceutically acceptable salt thereof of any one of embodiments 1 to 16, or a pharmaceutical composition of embodiment 17 in the manufacture of a medicament for use in therapy.
• Embodiment 25 The use of embodiment 24, wherein said therapy is the treatment of a cancer.
• Embodiment 26 The use of embodiment 25, wherein said cancer is ovarian, gastric, or breast cancer.
• Embodiment 26A The use of embodiment 25, wherein said cancer is lung, cervical or pancreatic cancer.
• Embodiment 26B The use of embodiment 25, wherein the cancer is breast cancer, ovarian cancer, gastric cancer, lung cancer, cervical cancer, prostate cancer, or pancreatic cancer.
• Embodiment 27 A method of inhibiting PARG in vivo, said method comprising administering to a patient an effective amount of a compound or a pharmaceutically acceptable salt thereof of any one of embodiments 1 to 16, or a pharmaceutical composition of embodiment 17.
• Embodiment 27A The method of embodiment 27, wherein the patient is in recongizned need of such treatment.
• Embodiment 28 A method of inhibiting cell proliferation, in vitro or in vivo, said method comprising contacting a cell with an effective amount of a compound or a pharmaceutically acceptable salt thereof of any one of embodiments 1 to 16, or a pharmaceutical composition of embodiment 17.
• Embodiment 28A A method of inhibiting cell proliferation, in vitro or in vivo, said method comprising contacting a sample with an effective amount of a compound or a pharmaceutically acceptable salt thereof of any one of embodiments 1 to 16, or a pharmaceutical composition of embodiment 17.
• Embodiment 29 A method of treating a cancer resistant to one or more PARP inhibitors in a patient in need thereof, said method comprising administering to said patient an effective amount of a compound or a pharmaceutically acceptable salt thereof of any one of embodiment 1 to 16, or a pharmaceutical composition of embodiment 17.
• Embodiment 29A The method of embodiment 29, wherein the patient is in recongizned need of such treatment.
• Embodiment 29B The method of embodiment 29, wherein the patient has been determined to be resistant to one or more PARP inhibitors.
• Embodiment 29C The method of embodiment 29, wherein the patient has been diagnosed as resistant to one or more PARP inhibitors.
• Embodiment 29D A method of treating a cancer resistant to one or more PARP inhibitors in a patient in need thereof, said method comprising administering to said patient an effective amount of a compound or a pharmaceutically acceptable salt thereof of any one of embodiment 1 to 16, or a pharmaceutical composition of embodiment 17.
• any one of embodiments 29, 29A, 29B, and 29C, wherein the one or more PARP inhibitors are Talazoparib, Olaparib, Veliparib, Rucaparib, Niraparib, AZD53O3, AZD9574, or a pharmaceutically acceptable salt thereof.
• Embodiment 30 The method of any one of embodiments 29, 29A, 29B, 29C, and 29D, wherein said cancer is ovarian, gastric, or breast cancer.
• Embodiment 30A The method of any one of embodiments 29, 29A, 29B, 29C, and 29D, wherein said cancer is lung, cervical or pancreatic cancer.
• Embodiment 30B The method of any one of embodiments 29, 29A, 29B, 29C, and 29D, wherein the cancer is breast cancer, ovarian cancer, gastric cancer, lung cancer, cervical cancer, prostate cancer, or pancreatic cancer.
• Embodiment 31 A method of treating a cancer resistant to one or more platins in a patient in need thereof, said method comprising administering to said patient an effective amount of a compound or a pharmaceutically acceptable salt thereof of any one of embodiment 1 to 16, or a pharmaceutical composition of embodiment 17.
• Embodiment 31A The method of embodiment 31, wherein the patient is in recongizned need of such treatment.
• Embodiment 31B The method of embodiment 31, wherein the patient has been determined to be resistant to one or more platins.
• Embodiment 31C The method of embodiment 31, wherein the patient has been diagnosed as resistant to one or more platins.
• Embodiment 31D A method of treating a cancer resistant to one or more platins in a patient in need thereof, said method comprising administering to said patient an effective amount of a compound or a pharmaceutically acceptable salt thereof of any one of embodiment 1 to 16, or a pharmaceutical composition of embodiment 17.
• Embodiment 31A The method of embodiment 31, wherein the patient is in recongiz
• any one of embodiments 31, 31A, 31B, and 31C, wherein the one or more platins are cisplatin, carboplatin, satraplatin, heptaplatin, picoplatin, nedaplatin, triplatin, lipoplatin, or oxaliplatin, or a pharmaceutically acceptable salt thereof.
• Embodiment 32 The method of embodiment 31, 31A, 31B, 31B, and 31D, wherein said cancer is ovarian, gastric, or breast cancer.
• Embodiment 32A The method of embodiment 31, 31A, 31B, 31C, and 31D, wherein said cancer is lung, cervical or pancreatic cancer.
• Embodiment 32B The method of any one of embodiments 31, 31A, 31B, 31C, and 31D, wherein the cancer is breast cancer, ovarian cancer, gastric cancer, lung cancer, cervical cancer, prostate cancer, or pancreatic cancer.
• Embodiment 33 A method of treating and/or preventing a homologous recombinant deficient (HRD) cancer in a patient comprising administering to the patient a therapeutically effective amount of a compound or a pharmaceutically acceptable salt thereof of any one of embodiment 1 to 16, or a pharmaceutical composition of embodiment 17.
• Embodiment 33A The method of embodiment 33, wherein the patient is in recongizned need of such treatment.
• Embodiment 34 The method of embodiment 33 or 33A, wherein the HRD cancer is breast cancer, ovarian cancer, gastric cancer, prostate cancer, or pancreatic cancer.
• Embodiment 34A The method of embodiment 33 or 33A, wherein the HRD cancer is breast cancer, ovarian cancer, or gastric cancer.
• Embodiment 34B The method of embodiment 33 or 33A, wherein the HRD cancer is lung, cervical, or pancreatic cancer.
• Embodiment 35 A method of treating and/or preventing a cancer in a patient, where the cancer is characterized by a reduction or absence of BRCA1 and/or BRCA2 gene expression, the absence or mutation of BRCA1 and/or BRCA2 genes, or reduced function of BRCA1 and/or BRCA2 proteins, comprising administering to the patient a therapeutically effective amount of a compound or a pharmaceutically acceptable salt thereof of any one of embodiment 1 to 16, or a pharmaceutical composition of embodiment 17.
• Embodiment 35A The method of embodiment 35, wherein the patient is in recongizned need of such treatment.
• Embodiment 36 The method of embodiment 35 or 35A, wherein the cancer is breast cancer, ovarian cancer, gastric cancer, prostate cancer, or pancreatic cancer.
• Embodiment 36A The method of embodiment 35 or 35A, wherein the HRD cancer is breast cancer, ovarian cancer, or gastric cancer.
• Embodiment 34B The method of embodiment 35 or 35A, wherein the HRD cancer is lung, cervical, or pancreatic cancer.
• Embodiment 37 A PARG inhibitor for use in the treatment of cancer, wherein the PARG inhibitor is a compound of any one of embodiments 1 to 16, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of embodiment 17.
• Embodiment 38 Use of a PARG inhibitor in the manufacture of a medicament for treating cancer, wherein the PARG inhibitor is a compound of any one of claims 1 to 16, or a pharmaceutically accpetable salt thereof, or a pharmaceutical composition of claim 17.
• reaction mixture was diluted with water (500 mL) and extracted with EtOAc (2 x 300 mL), the combined organic phases were washed with brine solution (200 mL), dried over anhydrous sodium sulphate, filtered and the filtrate was concentrated under reduced pressure to get crude product as an oil.
• the crude material was purified by column chromatography using silica gel (100-200) and eluted with 20% EtOAc/Hexane as a gradient. The product was eluted with a gradient of 30% EtOAc/Hexane.
• Step 3 Preparation of 2-(difluoromethyl)-5-(4-fluoro-6-iodo-lH-indazol-l-yl)-l,3,4- thiadiazole
• Step 4 Preparation of S-(l-(5-(difluoromethyl)-l,3,4-thiadiazol-2-yl)-4-fluoro-lH-indazol- 6-yl) benzothioate
• Step 5 Preparation of N-(l-cyanocyclopropyl)-l-(5-(difluoromethyl)-l,3?4-thiadiazol-2-yl)- 4-fluoro-lH-indazole-6-sulfonamide
• the reaction mixture was diluted with water (50 mL) and extracted with EtOAc (2x30 mL). The combined organic layer was washed with brine solution (20 mL), dried over anhydrous sodium sulphate, filtered and the filterate was concentrated under reduced pressure to obtain crude product.
• the crude product was purified by column chromatography using silica gel (100-200) and eluted with 5 to 50% EtOAc/hexane as a gradient. The product was eluted at 20% EtOAc/hexane.
• Step 6 Preparation of tert-butyl (2S,6S)-4-(l-(5-(difluoromethyl)-l,3,4-thiadiazol-2-yl)-6-
• Step 7 Preparation of l-(5-(difluoromethyl)-l,3,4-thiadiazol-2-yl)-4-((3S,5S)-3,5- dimethylpiperazin-l-yl)-N-(l-methylcyclopropyl)-lH-indazole-6-sulfonamide (Formula A)
• Step 1 Preparation of tert-butyl (2R,6R)-4-(l-(5-(difluoromethyl)-l,3,4-thiadiazol-2-yl)-6-
• Step 2 Preparation of l-(5-(difluoromethyl)-l,3,4-thiadiazol-2-yl)-4-((3R,5R)-3,5- dimethylpiperazin-l-yl)-N-(l-methylcyclopropyl)-lH-indazole-6-sulfonamide (Formula B)
• reaction mixture was cooled to RT (25 °C) and combined with another batch of reaction mixture where 1.0 g of Compound 7 was used.
• Water 260 mL was slowly added to the combined reaction mixtures below 25 °C and stirred for 2 h.
• the reaction mixture was filtered and the solid was washed with water (26 mL) to afford l-(5-(difluoromethyl)-l,3,4-thiadiazol-2-yl)-4- ((3S,5R)-3,5-dimethylpiperazin-l-yl)-N-(l-methylcyclopropyl)-lH-indazole-6-sulfonamide (Formula C) (15.6 g) as light yellow solid.
• PARG enzyme was incubated with compound or vehicle (DMSO) and the biotinylated- PARylated PARP-1 substrate in a microtiter plate. After adding detection antibody and streptavidin-europium, and then incubating, the plate was read for fluorescence intensity.
• DMSO compound or vehicle
• the low control (DMSO) with low fluorescence intensity represents no inhibition of enzymatic activity
• the high control (no enzyme) with high fluorescence intensity represents full inhibition of enzymatic activity.
• Detection Antibody anti-His monoclonal antib ody-ULight, Perkin Elmer catalog # TRF0134-M Streptavidin-Europium: Perkin Elmer catalog # AD0062
• Assay Buffer 50mM Tris-HCL pH 7.4, 50mM KCL, 3mM EDTA, 0.4mM EGTA, ImM DTT, 0.01% Tween 20, 0.01% BSA Temperature: 23 °C
• ECso values were calculated in Collaborative Drug Discovery vault (CDD). Curves were fitted by CDD as response (%) vs compound concentration (uM) using a 4-parameter inhibition model using Formula 1.
• TR-FRET EC50 values for Formula (A), Formula (B), Formula (C), and select comparators are provided in Table 1, below.
• RMUGS-NucLight Red and SNU601-NucLight Red were generated by stably transducing parental cells (RMUGS- JCRB Cell Bank, Cat no: IF050320; SNU601-Korean Cell Line Bank, Cat no:00601) with Incucyte® NucLight Red lentivirus (Sartorius, Cat no: 4476). Described below are the protocols followed for both the cell lines.
• the cells were plated at 200 cells/well in a 384-well black plate with clear flat bottom. After 24 hours, the plates were imaged using the Incucyte® S3 Live-Cell Analysis system and the number of live cells in each well (Day 0) was counted. Test compounds were then added by Tecan digital dispenser to generate a 9 point dose curve with a 3 fold dilution and lOuM top concentration. All treatments were done in triplicates. After 7 days of incubation, the plates were imaged using the Incucyte® S3 system and the number of live cells per well (Day 7) was counted.
• Sectionl Span * Frac / (1 + 10 A ((LogECso i - X) * nHl))
• Section2 Span * (1 - Frac) / (1 + 10 A ((LogECso 2 - X) * nH2))
• the cells were plated at 1000 cells/well in a 96-well black plate with clear flat bottom. After 24 hours, the plates were imaged using the Incucyte® S3 Live-Cell Analysis system and the number of live cells in each well (Day 0) was counted. Test compounds were then added by Tecan digital dispenser to generate a 9 point dose curve with a 3 fold dilution and lOuM top concentration. All treatments were done in duplicates. After 7 days of incubation, the plates were imaged using the Incucyte® S3 system and the number of live cells per well (Day 7) was counted. For every well on the plate, the live cell counts on day 7 was normalized to the cell counts from day 0 (Day 7/Day 0).
• Control compound diclofenac sodium (LOT BCBW9128) was purchased from Sigma Chemical Co.
• Multi ScreenHTS HV (0.45 pm) 96 well plate (Millipore, MSHVN4510 or MSHVN4550)
• the Solubility Sample plate was transferred to the Eppendorf Thermomixer Comfort plate shaker and shaken at 25°C at 1100 RPM for 24 hours. After completion of the 24 hours, plugs were removed and the stir sticks were removed using a big magnet, the samples from the Solubility Sample plate were transferred into the filter plate. Using the Vacuum Manifold, all the samples were filtered. Aliquot of 10 pL was taken from the filtrate followed by addition of 990 pL of a mixture of H2O and acetonitrile (1 :1 in v/v). 200 pL of diluent was transferred to a new 96-well plate for LC-MS/MS analysis. The dilution factor was changed according to the solubility values and the LC-MS signal response.
• a panel of cell lines (as disclosed in Table 2) that show inherent resistance to PARP inhibitors were identified and the effect of Formula A was tested using the cellular proliferation assay along with the 2 acquired resistant cell lines (PARPi resistant lines: HCC1428 and MDA- MB-436).
• PARPi resistant lines HCC1428 and MDA- MB-436.
• the cell lines were plated in 96 well plates (Coming #3904) at density of 1000 cells/well. DMSO dissolved compound was added using the TECAN liquid dispenser to generate a 9-point dose curve with a 3-fold dilution and lOuM starting, top concentration. After 5 population doublings, the cell lines were treated with 5uM of Vybrant DyeCycle Green (Life Technologies #V35004) and incubated for 60 minutes.
• the DMSO and compound treated wells were then imaged using the Incucyte® S3 system to determine the nuclear counts.
• the counts were normalized to the DMSO treated wells and the IC50 was determined using the standard four parametric dose response equation in GraphPad Prism Software.
• PDC Patient Derived Cells
• ovarian tissue with BRCA1/2 alterations and cisplatin resistance were identified. These cells were processed from solid tumor samples received after surgery/resection and 2-D cultured in selective media. For each PDC, upon reaching the required number of cells for the assay, the cells were detached using TrypLE and counted. Draq7 dye was added to the cells before plating to identify dead cells. The cells were plated in a 96 well plate (Greiner #655090) and incubated at 37°C and 5% CO2 for 16-24h. A Tecan HP D300 digital dispenser was used to dispense Formula (A) in a 9-point dose curve and 3-fold dilution starting at 30uM.
• the cells were fixed with 2% formaldehyde and stained with Hoechst.
• the plates were scanned using Nexcelom Celigo high content screening and the total cell count using Hoechst labeling and cell death using Draq7 fluorescence was analyzed.
• the nuclear counts were normalized to the DMSO treated wells and the IC50 was determined using the standard four parametric dose response equation in GraphPad Prism Software. The cells generated were used for the study as disclosed in FIG. 3.
• Formula A was also tested in an ovarian Patient Derived Cell (PDC) model with BRCA1 mutation and cisplatin resistance.
• Formula A had an anti-proliferative effect on the cell line with an IC50 of 260 nM (FIG. 3).
• mice with an established growing HBCx-34 tumor between 108 and 288 mm 3 were randomized into 2 treatment groups of 8 mice each, according to the groups listed in Table 3, tumor-bearing mice receive estrogen diluted in drinking water (P- estradiol, 8.5 mg/1), from the date of tumor implant to the end of the study.
• P- estradiol 8.5 mg/1
• the abbreviation “p.o.” refers to oral administration
• the abbreviation “QD” refers to administration once a day.
• Tumors were measured and mice were weighed twice per week during the experimental period.
• TGI tumor growth inhibition
• Tumor regression represents tumor volume less than the initial tumor volume at DO.

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